OVERVIEW OF HEMATOLOGY LABORATORY

Rmt2023
HEMATOLOGY LECTURE WEEK 2

HEMATOLOGY
➢ comes from the Greek words “Haima” and “Logos”
o Haima-blood; logos-study
➢ discipline that studies the development of cells and diseases of blood
(anemia and leukemia)
➢ In this field, the fundamental concepts of biology and chemistry are applied
to the medical diagnosis and treatment of various disorders or diseases
related to or manifested in the blood and bone marrow
✓ Liquid portion: Serum or Plasma (55%)
➢ Physicians rely on hematology laboratory test results to select and monitor
✓ Cellular portion: Erythrocytes, Leukocytes, and Thrombocytes (45%)
therapy for these disorders; consequently, a complete blood count (CBC) is
After centrifugation-> presence of buffy coat
ordered on nearly everyone who visits a physician or is admitted to a
Buffy coat
hospital
➢ contains reticulocytes, normoblast (nRBC's), leukocytes and
o CBC: routine test
thrombocytes.
➢ Lower most layer of buffy coat is basophil, upper layer of buffy coat
FUNCTIONS OF THE HEMATOLOGY LABORATORY
is platelets
➢ Medical laboratory scientists, medical laboratory technicians, laboratory
➢ <1% of the blood volume
assistants, and phlebotomists employed in the hematology laboratory play
a major role in patient care
Layers of the centrifuged sample:
➢ The assays and examinations that are performed in the laboratory can do
1st-> fatty layer-usually not visible by the naked eye
the following:
2nd-> plasma or serum
✓ Establish a diagnosis or rule out a diagnosis
3rd-> buffy coat
✓ Confirm a physician’s clinical impression of a possible hematological
4th-> Packed RBC
disorder
✓ Detect an unsuspected disorder
GENERAL CHARACTERISTICS OF BLOOD
✓ Monitor the effects of therapy
➢ In vivo → blood is red and is in the fluid state (in vitro: coagulates in 5-10
✓ Detect minimal residual disease following therapy
minutes)
➢ Color:
HISTORY OF HEMATOLOGY
✓ Arterial blood: bright red (oxygenated-> oxyhemoglobin)
➢ 1657: Describe worms in the blood by Athanasius Kircher
✓ Venous blood: dark purplish red (deoxygenated; waste products-
➢ 1658: Discovery of erythrocytes by Swammerdam
>CO2->deoxyhemoglobin)
➢ 1674: Human erythrocytes was described by Anton Van Leeuwenhoek
➢ Thick and viscous (3.5 -4.5x more viscous tan water)
➢ 1842: Guilio Bizzozero described platelets as “petites plaques”
➢ Average specific gravity of 1.055
➢ 1846: PMN (Polymorphonuclear neutrophil) distinguished from other
➢ Average pH of 7.40 (7.35 – 7.45)
leukocytes by Wharton Jones
✓ Maintained by excretion of carbon dioxide, acid-base balance
➢ 1879: First complete classification of leukocytes by Paul Ehrlich
➢ Makes up 75-85 mL per kilogram of body weight
➢ 1902: Development of Wright’s stain by James Homer Wright
➢ Blood volume differs with age and gender:
➢ 1920: Hematology was considered separate science from clinical
✓ Adult male: 5-6 liters
pathology
✓ Adult female: 4-5 liters
✓ Newborn: 250-350 ml
THE HEMATOLOGY LABORATORY TODAY
➢ Use of automated analyzers to differentiate and enumerate RBC’s; although
FUNCTIONS OF BLOOD
Wright’s Romanowsky-type stain (polychromatic, a mixture of acidic and
➢ Respiratory-O2 and CO2
basic dyes), and refinements thereof, remains the foundation of blood cell
➢ Nutritional
identification
➢ Excretory-waste products
➢ RBC, WBC, and platelet appearance is analyzed through automation or
➢ Buffering action-pH
visually using light microscopy examination of cells fixed to a glass
➢ Maintenance of body temperature-immune substance
microscope slide and stained with Wright or Wright-Giemsa stain
➢ Transport of hormones
➢ The scientific term for cell appearance is morphology, which encompasses
➢ Defense mechanism-antibodies
cell color, size, shape, cytoplasmic inclusions, and nuclear condensation
➢ Coagulation
o if the result is normal in the automation, you don't need to make
a blood smear
PLASMA
o manual if there's a flag
➢ Liquid portion that transports and nourishes blood cells
➢ Provides coagulation enzymes that protect the blood vessel

BLOOD AND ITS COMPONENTS

➢ CHARACTERISTICS:
a specialized connective tissue; average volume is 5 liters (adult)
✓ Color: pale-yellow
➢ COMPOSITION OF BLOOD:
• Pink: slight hemolysis

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 1

 • Red: gross hemolysis • Coulter counter – based on the principle of electric impedance
• Yellow-brown: icteric plasma (due to increased bilirubin)
✓ Transparency: clear; if milky/turbid: Lipemia (increased lipids inc. 2. HEMOGLOBIN
TAG) ➢ Method is called as the cyanmethemoglobin (HiCN) method gold
✓ CONTAINS FIBRINOGEN standard for hemoglobin determination
✓ Contains 90% of water and 10% solutes (proteins, lipids, hormones, ➢ Relies on a weak solution of potassium cyanide and potassium ferricyanide,
etc.) called Drabkin reagent
➢ hemolysis (destruction of RBC) ➢ Automated analyzers - ionic surfactant (detergent) sodium lauryl sulfate to
➢ what homologically test can still be performed even if the blood is reduce environmental cyanide
hemolyzed- HEMOGLOBIN (as blood is lysed in order to measure
the hemoglobin)
➢ once that the RBC is hemolyzed, the plasma will become brown
because of bilirubin COMPONENTS OF DRABKIN'S REAGENT
➢ if it still has fibrinogen, the blood is not clotted -potassium ferricyanide
SERUM -potassium cyanide
➢ liquid portion of clotted blood -sodium carbonate/ dihydrogen potassium phosphate
➢ fluid remains after coagulation has occured -surfactant/detergent
➢ no fibrinogen-> "fibrin" clot
Major difference bet plasma and serum is FIBRINOGEN 3. HEMATOCRIT
ERYTHROCYTES ➢ Hematocrit is often referred to as the packed cell volume (PCV), volume of
➢ Shape: biconcave (discoid shape cell or discocyte) erythrocytes or reading of packed cells (pRBC)
➢ Color: Salmon pink ➢ Ratio of the volume of packed RBC’s to the volume of whole blood
➢ Average size: 6-8 um (approximate size: 7.2 um) ➢ reported as %(PCV) or L/L (Erythrocyte volume fraction/EVF)
➢ Hemoglobin ➢ TWO METHODS:
✓ Main component of RBC’s; gives its red color ✓ Macrohematocrit method
✓ Function is to transport oxygen (oxyhemoglobin) and carbon dioxide ✓ Microhematocrit method
(deoxyhemoglobin) {has an iron which oxygen is binded to}
✓ Found in the central pallor/pallor area (occupies 1/3 of the RBC:
normochromic)
• If central pallor is < 1/3 of the cell – increased hgb concentration
(hyperchromic RBC)
• If central pallor is >1/3 of the cell – decreased hgb concentration
(hypochromic RBC)
• Hemoglobin concentration is inversely proportional with the size of
the central pallor
➢ Anemia → loss of oxygen carrying capacity of the blood
✓ Due to decreased RBC count
✓ Decreased Hemoglobin concentration
➢ Polycythemia
4. RBC INDICES
✓ Increased circulating RBC’s (increased RBC count)
➢ Measures parameters of a single RBC; useful in the assessment of anemia
✓ Mean cell/corpuscular volume (MCV)
RBC PARAMETERS
• Measures the volume/size of a single RBC
• Reported in femtoliters (fL)
1. RBC COUNT
✓ Mean cell/corpuscular hemoglobin (MCH)
➢ RBC count is the number of red cells in 1 uL
• measures the weight/mass of hemoglobin in a single RBC
or 1 liter of blood
➢ Used for diagnosis of leukemia and • Reported in picograms (pg)
polycythemia ✓ Mean cell/corpuscular hemoglobin concentration (MCHC)
➢ Types: • measures Hgb concentration in a single RBC; staining capacity of
✓ Manual RBC counting – visual counting RBC’s and amount of central pallor (g/dL)
of red cells using a hemacytometer; ✓ Red cell distribution width (RDW)
requires 1:200 dilution • based on the standard deviation of RBC volume and is routinely
• RBC thoma pipet reported by automated blood cell analyzers
• Hemacytometer or counting chamber
(reported in uL or L)
• RBC diluting fluid – isotonic
solution (RBCs retain their
shape, matches osmolality of
blood) ; example is 0.85%
Normal saline solution (NSS);
lyses WBC’s
✓ Automated cell counters

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 2

 • Measures the degree of anisocytosis ➢ Stained by methylene blue dyes
✓ Nucleic acid stains or vital
stains, are used to differentiate
and count these young RBCs
✓ Vital (or “supravital”) stains
are dyes absorbed by live cells
✓ Young RBCs contain
remnants of ribonucleic acid
(RNA) in its cytoplasm

Example of Supravital stains

➢ NMB-> New Methylene Blue
➢ BCB-> Brilliant Cresyl blue
➢ Crystal violet-> detection of Heinz Stain

Wright stain should not be used in RETIC count

5. RBC MORPHOLOGY
➢ Uses a light microscope and a Wright-stained blood film/ blood smear REFERENCE RANGE FOR RBC PARAMETERS
➢ Parameters assessed are:
✓ RBC diameter
✓ Color or hemoglobinization
✓ RBC shape
✓ Cytoplasmic inclusions-> normal RBC has no inclusions
➢ Anisocytosis - general term used in hematology to denote an increased
variation in cell size
➢ Poikilocytosis - general term for mature erythrocytes that have a shape other
than the normal round, biconcave appearance (variation in cell shape)
Poikilocyte-> abnormal RBC

LEUKOCYTES
➢ Loosely related category of cell types
dedicated to protecting their host
from infection and injury
➢ are transported in the blood from
bone marrow or lymphoid tissue
➢ Nearly colorless in an unstained cell
suspension
➢ Leukocytosis – increased WBC
count
RETICULOCYTES
➢ Leukopenia – decreased WBC count
➢ In a Wright-stained blood film, 0.5% to 2.5% of RBCs exceed the 7- to 8
➢ Leukemia – uncontrolled
um average diameter and stain slightly blue-gray
proliferation of malignant WBC’s
➢ These are polychromatic (polychromatophilic) erythrocytes, newly
o Cancer involving WBC
released from the bone marrow
➢ are closely observed because they indicate the ability of the bone marrow
to increase RBC production in anemia caused by blood loss or excessive
RBC destruction

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 3

 TYPES OF LEUKOCYTES 6. MONOCYTES
1. NEUTROPHIL ➢ An immature macrophage passing
➢ A phagocytic cell whose major through the blood from its point of origin
purpose is to engulf and destroy ➢ Macrophages comprise minor component
microorganisms and foreign material of peripheral blood WBC
➢ NEUTs, segmented neutrophils, ➢ Their tasks are to identify and
SEGs, polymorphonuclear phagocytose (engulf and consume)
neutrophils, PMNs foreign particles
➢ Segmented refers to their multilobed ➢ Assist the lymphocytes in mounting an
nuclei immune response through the assembly
➢ Neutrophilia – increased neutrophils and presentation of immunogenic
➢ Neutropenia – decreased neutrophils epitopes (APC)
➢ Monocytes have a slightly larger
diameter than other WBCs, blue-gray cytoplasm with fine azure
2. BAND NEUTROPHIL granules, and a nucleus that is usually indented or folded
➢ Bands are less differentiated or less mature neutrophils ➢ Monocytosis – increase in monocytes, Found in certain infections,
➢ Pink or lavender-staining granules filled with bactericidal secretions collagen vascular diseases, or in acute and chronic leukemias
➢ LEFT SHIFT: an increase in bands; ➢ Monocytopenia – seldom used
signals bacterial infection
➢ Band neutrophil first then segmented WBC PARAMETERS
neutrophil 1. WBC COUNT
➢ Technique is the same as RBC counting
➢ Refers to the number of WBC’s in 1 ul or liter of blood
➢ Requires a dilution of 1:20; diluent used is a dilute acid solution (lyses
RBC’s) (hypotonic solution)
➢ Leukocytosis - >11,500/ uL
➢ Leukopenia - <4,500 / uL
3. EOSINOPHIL
➢ are cells with bright orange-red, regular cytoplasmic 2. WBC DIFFERENTIAL COUNT
granules filled ➢ A technique where 100 WBC’s are counted; 50 WBCs or 200 WBCs can
➢ with proteins involved in immune system regulation also be counted
➢ Eosinophilia – increased eosinophils ➢ a peripheral blood film is used; blood film is also checked for RBC, WBC,
➢ Eosinopenia – theoretically not used and platelet abnormalities
4. BASOPHIL ➢ Reported as the relative count: % of specific cell (Example: Neutrophils –
➢ Basophils are cells with dark purple, irregular cytoplasmic granules that 65%)
obscure the nucleus
➢ Basophil granules contain histamines and various other proteins THROMBOCYTES/PLATELETS
➢ Basophilia – increased basophils ➢ True blood cells that maintain blood vessel integrity by initiating vessel
➢ Basopenia – theoretical and not used wall repairs; are the major cells that control hemostasis, a series of
cellular and plasma-based mechanisms that seal wounds, repair vessel
walls, and maintain vascular patency
➢ Are 2-4 um in diameter, round to oval, anucleate and slightly granular
➢ Some hematologists prefer to
call platelets as “cell
5. LYMPHOCYTES fragments” – fragments from
➢ Lymphocytes recognize foreign antigens and mount humoral megakaryocytes
(antibodies) and cell mediated antagonistic responses ➢ Thrombocytosis – elevated
➢ Most lymphocytes are nearly round, are slightly larger than RBCs, and platelet count; signal
have round featureless nuclei and a thin rim of nongranular cytoplasm inflammation or trauma but
➢ Lymphocytosis – increased lymphocytes with variant or reactive convey modest intrinsic
lymphocytes; often associated with viral infections significance
➢ Lymphopenia – decreased lymphocytes associated with drug therapy or ➢ Essential thrombocythemia - rare malignant condition characterized by
immunodeficiency extremely high platelet counts and uncontrolled platelet production-
➢ RBC has almost the same size of a small lymphocyte (8-10um) CANCER (higher than thrombocytosis)
➢ Thrombocytopenia – low platelet count; common consequence of drug
treatment and may be life threatening
➢ Uncontrolled/excessive platelet and hemostatic activation are
responsible for deep vein thrombosis, pulmonary emboli, acute myocardial
infarctions (heart attacks), cerebrovascular accidents (strokes), peripheral
artery disease, and repeated spontaneous abortions

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 4

 PLATELET COUNT TESTS FOR HEMOSTASIS
➢ Uses pipette and hemocytometer, although a different counting area, ➢ Platelet count
diluent, and dilution is usually used ➢ Mean platelet volume (MPV)
➢ Platelets are hard to distinguish visually in a hemocytometer, and phase ➢ Prothrombin time (PT)
contrast microscopy provides for easier identification ➢ Activated partial thromboplastin time (APTT)
➢ Automated blood cell analyzers have largely replaced visual platelet ➢ Thrombin time
counting and provide greater accuracy ➢ Fibrinogen assays
➢ D-dimer assays
REFERENCE RANGE FOR WBC AND
PLATELET PARAMETERS (Adult) ADVANCED HEMATOLOGY PROCEDURES
➢ Bone marrow examinations – uses a bone marrow smear (from BM
aspirate or biopsy)
o BM Aspirate- analyzed immature precursors to blood cells
(erythroid, myeloid, megakaryocyte)
➢ Cytochemical stains-classification of leukemia
➢ Flow cytometry – immunophenotyping
➢ Cytogenetic analysis
➢ Molecular diagnostics – PCR(polymerase chain reaction), DNA
sequencing
o Reciprocal translocation of chromosomes (9&22-chronic myeloid
leukemia)(15&17- acute myeloid leukemia)

SPECIAL HEMATOLOGY PROCEDURES

➢ Glucose-6-phosphate dehydrogenase assay-> phenotypically detects an
inherited RBC enzyme defieciency= episodic hemolytic anemia
➢ Sickle cell solubility tests-> sickle cell anemia
➢ Hemoglobin electrophoresis and HPLC (High performance liquid
(x103- absolute)(%-relative) chromatography) -> sickle cell anemia
COMPLETE BLOOD COUNT ➢ Erythrocyte sedimentation rate (ESR)-> inflammation and estimates its
➢ is performed on automated intensity
blood cell analyzers and ➢ Osmotic fragility test (OFT)-> hemolytic anemia
includes the RBC, WBC, and
platelets MEDICAL TERMINOLOGIES
➢ SPECIMEN:
✓ Appropriate anticoagulant
and tube (EDTA), free
from clots and hemolysis,
sufficient volume, tested
and prepared for storage
within the appropriate
time frames
✓ ACCESSION: may be automated, relying on bar code or
radiofrequency identification technology, thus reducing instances of
identification error
➢ FLAGS
➢ An indication when one of the results from the profiling is abnormal
➢ A “reflex” blood film examination is performed

HEMOSTASIS AND COAGULATION

➢ Most hematology laboratories include a blood coagulation testing
department
➢ Primary hemostasis - It refers to the role of blood vessels and platelets in
response to a vascular injury
➢ Secondary hemostasis/Coagulation pathway - Describes the activation
of a series of coagulation proteins in the plasma
➢ Tertiary hemostasis/Fibrinolysis - Final stage of coagulation; third
system of enzymes and cofactors digests clots to restore vessel patency
➢ Excessive clot formation- no substances that can lyse the clot
➢ Coagulation System- employs a complex system of plasma proteins,
some enzymes, and some enzyme cofactors to produce clot formation after
blood vessel injury

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 5

 HEMATOPOIESIS
Rmt2023
HEMATOLOGY LECTURE WEEK 3

❖ Characterized by recognizable clusters of developing erythroblasts,

HEMATOPOIESIS granulocytes, and monocytes colonizing the fetal liver, thymus,
❖ Continuous, regulated process of renewal, proliferation, spleen, placenta, and ultimately the bone marrow
differentiation, and maturation of all blood cell lines ❖ Development of spleen, thymus, kidney and lymph
❖ Result in the formation, development, and specialization of all nodes contributes to the hematopoietic process
functional blood cells that are released from the bone marrow into (extramedullary hematopoiesis)
the circulation ➢ Thymus - first organ to be developed and
❖ Mature blood cells have a limited lifespan (120 days +/- 20 days for produces T cells (T-cell maturation)
RBCs) and a cell population capable of self-renewal that sustains the ➢ Kidney and spleen – B-cell maturation
system
❖ A hematopoietic stem cell (HSC) is capable of self-renewal and ❖ IMPORTANT NOTE: After the 2nd week of life the liver will stop
directed differentiation into all required cell lineages producing blood cells but can be activated during severe blood loss
❖ Differentiation → involves stem cells and progenitor cells; immature
(are found only in the bone marrow) BLOOD CELLS FORMED
❖ Maturation → involves precursor cells; series that leads to production
of mature cells (present in the blood) EX. RBC, WBC, platelets ➢ Definitive erythroblasts
➢ Granulocytes and megakaryocytes: 3rd month
STAGES OF HEMATOPOIESIS ➢ Lymphocytes: 4th month
❖ Pre-natal/Primitive hematopoiesis (before birth) ➢ Monocytes: 5th month
o Mesoblastic phase HEMOGLOBIN FORMED
o Hepatic phase
o Medullary/Myeloid phase – continues to adult ➢ Fetal hemoglobin (Hgb F) → 2 alpha and 2 gamma globin
hematopoiesis chains
❖ Adult/Definitive hematopoiesis (after birth) ➢ Adult hemoglobin (Hgb A1 and A2) → detectable levels
✓ Hgb A1 - 2 alpha and 2 beta globin chains
MESOBLASTIC PHASE ✓ Hgb A2 – 2 alpha and 2 delta globin chains
❖ Also called as the yolk sac phase
❖ Begins during the 19th- 20th day of gestation in the blood islands of MEDULLARY PHASE/MYELOID PHASE
the yolk sac (mesodermal extraembryonic layer → yolk sac)
❖ Hematopoietic activity is confined to erythropoiesis ❖ Termed medullary hematopoiesis because it occurs in the medulla
❖ Production of cells lasts until the: 8th to 12th week of gestation or inner part of the bone cavity
o Cells produced in the yolk sac: primitive erythroblasts ❖ Primary site of hematopoiesis is the red bone marrow starting 24
and angioblasts (form blood vessels) weeks of gestation (remains throughout life)
o Primitive erythroblasts ❖ Begins between the 4th and 5th month of fetal development
✓ important in early embryogenesis to produce ❖ HSCs and mesenchymal cells migrate into the core of the bone
embryonic hemoglobin (up to the 3rd month only) ➢ Mesenchymal cells - type of embryonic tissue which
✓ Large, nucleated, and cannot extrude its nucleus differentiate into structural elements that support
✓ Migrates to the Aorta-Gonad Mesonephros (AGM) developing hematopoietic elements
region to become definitive erythroblasts (produces ❖ Myeloid activity is apparent during this stage with a normal M:E
HSCs) ratio of 3:1 – 4:1
HEMOGLOBIN PRODUCED (EMBRYONIC HEMOGLOBIN)
➢ Gower-I (2 epsilon and 2 zeta globin chains)
➢ Gower-II (2 alpha and 2 epsilon globin chains) IMPORTANT NOTES FOR M:E RATIO
➢ Portland hemoglobin (2 zeta and 2 gamma globin chains) ➢ Infection: 6:1
✓ IMPORTANT NOTE: Embryonic hemoglobins are found up to the ➢ Leukemia: 25:1
3rd month of gestation only (blood islands disappear after 3 rd month ➢ Myeloid Hyperplasia: 20:1
of gestation) ➢ Myeloid hypoplasia: 3:20
➢ Erythroid hyperplasia: 1:20
➢ Erythroid hypoplasia: 5:1
HEPATIC PHASE
❖ After 3rd month, yolk sac stops producing blood cells and is replaced DETECTABLE CYTOKINES
by the fetal liver ➢ Erythropoietin (EPO)
❖ Starts at the 5th – 7th week of gestation (primary site until the 6th ➢ Granulocyte colony- stimulating factor (G-CSF)
month and continue to produce until 1st to 2nd week of life) ➢ Granulocyte-macrophage colony-stimulating factor (GM-
CSF)

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 6

 HEMOGLOBIN FORMED ADULTS:
➢ Fetal hemoglobin (Hgb F) ➢ 40% active marrow
➢ Adult hemoglobin (Hgb A1 and Hgb A2) ➢ Active marrow is restricted to the sternum, vertebrae,
scapulae, pelvis, ribs, skull, and proximal portion of
IMPORTANT NOTE: the long bones
➢ IN ADULTS, THE PREDOMINANT HGB IS THE
ADULT HEMOGLOBIN IMPORTANT NOTE:
➢ IN NEWBORNS, FETAL HEMOGLOBIN ✓ In ADULTS, main source of blood cell production is in the:
STERNUM
✓ Safest, most accessible site for bone marrow aspiration/biopsy:
ILIAC CREST
✓ Iron 52, Iron 59, Technetium 99m-Colloid (Dyes for identification of
active hematopoietic tissue)

❖ Retrogression → process in which the red marrow is replaced by the

yellow marrow
❖ IMPORANT NOTE:
➢ Yellow marrow is
capable of
reverting back to
active marrow in
cases of increased
demand on the bone
marrow, such as in
excessive blood loss
or hemolysis
❖ Marrow cellularity - ratio of
the red marrow to the yellow
marrow; usually decreases
ADULT HEMATOPOIEITIC TISSUE
with age
➢ Normocellular:
❖ In adults, hematopoietic tissue is located in the:
marrow has 30-70%
➢ Bone marrow - erythroid, myeloid, megakaryocytic, and
hematopoietic cells
lymphoid cells
➢ Hypocellular: marrow has <30% hematopoietic cells
➢ lymph nodes, spleen, liver, and thymus (extramedullary
➢ Hypercellular: marrow has >70% hematopoietic cells
hematopoiesis)
➢
➢ Lymphoid tissues
➢ Aplastic: marrow has few or no hematopoietic cells
➢ Primary lymphoid tissues → where T and B cells are derived
✓ Bone marrow and Thymus
COLLECTION PROCEDURES
➢ Secondary lymphoid tissues → lymphoid cells respond to
➢ Bone marrow aspirate → obtained by bone marrow
foreign antigens
aspiration
✓ lymph nodes, mucosa-associated lymphoid
➢ Core biopsy → obtained by Trephine biopsy
tissue (MALT), gut-associated lymphoid tissue
COLLECTION SITES
(GALT)
➢ Posterior superior iliac crest
❖ Gut-associated lymphoid tissue (GALT) → (small
➢ Anterior superior iliac crest
❖ intestines) Peyer’s patches
➢ Sternum
❖ MALT → tonsils (Waldeyer’s ring)
➢ Anterior medial surface of the tibia
➢ Spinous process of the vertebrae, ribs, or other red-
BONE MARROW
marrow containing bones
❖ Normal bone marrow contains two major components:
➢ Red marrow → hematopoietically active marrow (blood
EQUIPMENTS USED FOR SPECIMEN COLLECTION
cells and precursors)
➢ Surgical
➢ Yellow marrow → hematopoietically inactive marrow
gloves
(adipocytes, with undifferentiated mesenchymal cells and
➢ Shaving
macrophages)
equipment
INFANCY AND EARLY CHILDHOOD
➢ 80-90% active marrow
➢ all the bones in the body contain primarily red (active)
marrow
5 TO 7 YEARS OF AGE:
➢ 60% active marrow
➢ adipocytes become more abundant and begin to occupy
the spaces in the long bones previously dominated by
active marrow

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 7

 ➢ Antiseptic and RED MARROW
alcohol pads
➢ Drape material ❖ Composed of hematopoietic cells arranged in extravascular cords
➢ Local anesthetic ❖ Cords are located in spaces between vascular sinuses and are
injection supported by trabeculae of spongy bone
➢ No. 11 scalpel ❖ The cords are separated from the lumen of the vascular sinuses by
blade endothelial and reticular adventitial cells
➢ Jamshidi biopsy ❖ Hematopoietic cells develop in specific niches within the cords
needle or ➢ Erythroblasts - develop in small clusters, and the more
Westerman-Jensen mature forms are located adjacent to the outer surfaces of the
needle; Snarecoil vascular sinuses
biopsy needle ➢ Megakaryocytes - are located adjacent to the walls of the
➢ 14 to 18 gauge aspiration needle with obturator vascular sinuses, which facilitates the release of platelets into
the lumen of the sinus
TYPES OF BONE MARROW SPECIMEN: ➢ Immature (myeloid) granulocytic cells - are located deep
➢ Direct Aspirate Smears: wedge-shape smear; avoids within the cords
crushing the spicules ➢ Niches → aka hematopoietic microenvironment; plays an
➢ Anticoagulated Aspirate Smears: K3EDTA important role in nurturing and protecting HSCs and
➢ Crush Smears: places additional glass slide directly over regulating their quiescence, self renewal, and differentiation
the specimen
➢ Imprints: closely replicate aspirate morphology
➢ Concentrate Smears: narrow-bore glass or a plastic tube
such as Wintrobe hematocrit tube
➢ Histologic Sections: 10% formalin, Zenker glacial acetic
acid, or B5 fixative
➢ Marrow Smear Dyes: Wright or Wright-Giemsa dyes

HEMATOPOIETIC STEM CELLS (HSC)

❖ Also called has pluripotential hematopoietic stem cell (PHSC)
NORMAL MARROW CELLS: ❖ can differentiate into progenitor cells committed to either
lymphoid or myeloid lineages when stimulated by cytokines
➢ HEMATOPOIETIC CELLS – PHSC, Progenitor cells, precursor ❖ are capable of self-renewal, are pluripotent, apoptosis, and may
cells give rise to differentiated progeny
➢ STROMAL CELLS → originate from mesenchymal cells that ❖ TYPES:
migrate into the central cavity of the bone ➢ Non-committed progenitor cells or undifferentiated HSCs
✓ Endothelial cells -Regulates the flow of particles entering ➢ Committed progenitor cells / Lineage specific progenitors
and leaving hematopoietic space ✓ Common myeloid progenitor/CFU-S/CFU-GEMM →
✓ Adipocytes - Secretes steroids that influences erythropoiesis granulocytic, erythrocytic, monocytic, and
and maintains bone integrity megakaryocytic lineages
✓ Macrophages - Phagocytic cell and secretes cytokines ✓ Common lymphoid progenitor → T, B, and natural
needed for the regulation of hematopoiesis killer lymphocyte and dendritic lineages
✓ Osteoblasts - Bone forming cells; water-bug or comet ✓ Lineage-specific progenitors give rise to
appearance morphologically recognizable, lineage-specific
✓ Osteoclasts - Bone resorbing cells or destroying cells precursor cells
✓ Reticular cells/Fibroblasts - Supports vascular sinuses and ✓ Precursor cells → blast forms (ex. myeloblast,
developing hematopoietic cells megakaryoblast, erythroblast)
❖ Apoptosis refers to programmed cell death, a normal physiologic
process that eliminates unwanted, abnormal, or harmful cells
❖ NON-COMMITTED → COMMITTED → PRECURSOR CELLS
→ MATURE CELLS

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 8

 STEM CELL SURFACE RECEPTORS
❖ Used for identification and origin of HSCs can be determined by
immunophenotypic analysis using flow cytometry

STEM CELL THEORY AND KINETICS

❖ STEM CELL THEORY - Describes the origin of hematopoietic
progenitor cells
MONOPHYLETIC THEORY
✓ suggests that all blood cells cells are derived from a single
progenitor stem cell called a pluripotent hematopoietic stem
cell (PHSC)
POLYPHYLETIC THEORY
✓ suggests that each of the blood cell lineages is derived from its
own unique stem cell

STEM CELL CYCLE KINETICS

➢ Bone marrow is capable of producing the following per kg body
weight:
✓ 2.5 billion erythrocytes
✓ 2.5 billion platelets
✓ 1 billion granulocytes
➢ HSCs exist in the marrow in the ratio of 1 per 1000 nucleated
blood cells
➢ Mitotic index
✓ percentage of cells in mitosis in relation to the total number of
cells
✓ normal value is 1-2%
✓ Increased mitotic index implies increased proliferation except
in megaloblastic anemia (prolonged mitosis)

CYTOKINES AND GROWTH FACTORS

❖ regulate the proliferation differentiation, and maturation of

hematopoietic precursor cells
❖ are responsible for stimulation or inhibition of production,
differentiation, and trafficking of mature blood cells and their
precursors
❖ are a diverse group of soluble proteins that have direct and indirect
effects on hematopoietic cells
❖ EXAMPLES ARE: Interleukins (ILs), Lymphokines, Monokines,
Interferons, Chemokines, Colony-stimulating factors (CSFs)
❖ Positive influence:
➢ KIT ligand, FLT3 ligand, GM-CSF, IL-1, IL-3, IL-6, and
IL-11
❖ Negative influence:
➢ Transforming growth factor-b, tumor necrosis factor-a,
and interferons

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 9

 LINEAGE SPECIFIC HEMATOPOIESIS
❖ Erythropoiesis
❖ Leukopoiesis → Myelopoiesis, Lymphopoiesis
❖ Megakaryopoiesis

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 10

 LINEAGE SPECIFIC HEMATOPOIESIS
Rmt2023
HEMATOLOGY LECTURE WEEK 4

LINEAGE SPECIFIC HEMATOPOIESIS Polychromatic / Rubricyte Polychromatic /

➢ Erythropoiesis polychromatophilic polychromatophilic
➢ Leukopoiesis → Myelopoiesis (Granulopoiesis, Monopoiesis), normoblast erythroblast
Lymphopoiesis Orthochromic Metarubricyte Orthochromic
➢ Megakaryopoiesis normoblast erythroblast
Polychromatic / Polychromatic / Polychromatic /
ERYTHROPOIESIS polychromatophilic polychromatophilic polychromatophilic
➢ Regulated process for maintaining adequate numbers of erythrocytes in erythrocyte erythrocyte erythrocyte
the peripheral blood Erythrocyte Erythrocyte Erythrocyte
➢ Major cytokine: Erythropoietin (EPO); others: IL-3, GM-CSF * Polychromatic erythrocytes are called reticulocytes when observed with vital
➢ Up to 8-32 erythrocytes in a single rubriblast/pronormoblast: 3-5 cell stains.
divisions
Cytokines are needed in order to produce cells: regulation the ➢ As erythroid precursors mature, several general trends affect their
differentiation, maturation appearance:
✓ Overall cell size/diameter decreases
ERYTHROID PROGENITORS ✓ NUCLEUS
➢ derived from the Common myeloid progenitor/CFU-S/CFU-GEMM • Size of the nucleus decreases
➢ morphologically identifiable erythrocyte precursors developed from two • N:C ratio decreases
progenitors:
• Nuclear chromatin becomes coarser, clumped, and condensed
✓ BFU-E (Burst forming unit-erythroid)
• Nucleoli disappears
• gives rise to large colonies because they are capable of multi-subunit ✓ CYTOPLASM
colonies
• changes from blue (basophilia) to gray-blue
• Contain only a few receptors for EPO
(polychromatophilia/polychromasia) to salmon pink (acidophilia)
• takes 1 week to differentiate to CFU-E
• Basophilia correlates with the amount of ribosomal RNA
✓ CFU-E (Colony forming unit-erythroid) (decreased as the cell matures)
• Gives rise to smaller colonies • Acidophilia correlates with increased hemoglobin production
• many EPO receptors and has an absolute requirement for EPO (increased as the cell matures)
• Completes at least three to five cell divisions to differentiate to a
pronormoblast PRONORMOBLAST/RUBRIBLAST
➢ PHSC → Common myeloid progenitor (CMP) → BFU-E → CFU-E → ➢ SIZE: 12-20 um; first detectable stage under light microscopy
PRONORMOBLAST ➢ NUCLEUS:
➢ Marrow transit time: Precursor cells will then take 6-7 days to become a ✓ Takes up much of the cell; round to oval
mature RBC; shortened during increased EPO production ✓ N:C ratio: 8:1
➢ It takes approximately 18-21 days from BFU-E to mature to an erythrocyte ✓ Nucleoli: 1-2
➢ marrow transit time-time in bone marrow before it would be ✓ Chromatin: Fine
release in the peripheral blood ➢ CYTOPLASM: dark blue because of the
➢ BFU-E-CFU E: 7 days concentration of ribosomes and RNA;
➢ CFU-pronormoblast: 7 days perinuclear halo
➢ pronormoblast to rbc: 6-7 days ➢ DIVISION: Divides into two basophilic normoblasts
➢ blast-> start to mature ➢ LOCATION: bone marrow
➢ CELLULAR ACTIVITY:
ERYTHROCYTE MATURATION ✓ Begins to accumulate the components necessary for hemoglobin
➢ RBCs are formally called erythrocytes production
➢ Nucleated RBC precursors, normally restricted to the bone marrow, are ✓ The proteins and enzymes necessary for iron uptake and
called erythroblasts protoporphyrin synthesis are produced; GLOBIN PRODUCTION
➢ They also may be called normoblasts, which refers to developing nucleated BEGINS
RBC precursors with normal appearance ➢ DURATION IN THIS STAGE: More than 24 hours
➢ ERYTHROCYTE PRECUSORS NOMENCLATURE: ➢ no hgb but there is the accumulation of hgb by preparing heme and
✓ Normoblastic - United states globin
✓ Rubriblastic
✓ Erythroblastic - Europe
Table 5-1. Three Erythroid Precursor Nomenclature Systems
Normoblastic Rubriblastic Erythroblastic
Pronormoblast Rubriblast Proerythroblast
Basophilic normoblast Prorubricyte Basophilic
erythroblast

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 11

 BASOPHILIC NORMOBLAST/PRORUBRICYTE ✓ The increase in the salmon pink color of the cytoplasm reflects nearly
➢ SIZE: 10-15 um complete hemoglobin production
➢ NUCLEUS: ✓ The residual ribosomes and RNA react with the basic component of the
✓ Slightly round to oval; occupies 75% of the cell stain and contribute a slightly bluish hue to the cell, but that fades toward
✓ N:C ratio: 6:1 the end of the stage as the RNA and organelles are degraded
✓ Nucleoli: 0-1 (present early but disappear in ➢ DIVISION: NOT CAPABLE OF CELL DIVISION because of
later stages) chromatin condensation
✓ Chromatin: begins to condensed ➢ LOCATION: Bone marrow
➢ CYTOPLASM: the cytoplasm may be a deeper, richer blue than in ➢ CELLULAR ACTIVITY:
➢ the pronormoblast, hence the name basophilic ✓ Hemoglobin production continues on the remaining ribosomes using
➢ DIVISION: Divides into two polychromatic normoblasts; more than 1 messenger RNA produced earlier
division is possible ✓ Late in this stage, the NUCLEUS IS EJECTED FROM THE CELL
➢ LOCATION: bone marrow ✓ The enveloped extruded nucleus, called a pyrenocyte, is then engulfed by
➢ CELLULAR ACTIVITY: DETECTABLE HEMOGLOBIN bone marrow macrophages.
SYNTHESIS OCCURS, but the many cytoplasmic organelles, including ➢ DURATION IN THIS STAGE: Approximately 48 hours
ribosomes and a substantial amount of messenger ribonucleic acid ➢ pyknotic-maliit
completely masks the minute amount of hemoglobin pigmentation ➢ rbc should be anucleated to maximize the oxygen carrying
➢ DURATION IN THIS STAGE: More than 24 hours capacity
➢ as the cell matures, it become less basophili but in ➢ metarubricyte: metataganggal ang nucleus
basophilic=more basophilic (exception), then in later stages it
would be lessen POLYCHROMATIC ERYTHROCYTE/RETICULOCYTE
➢ hemoglobin synthesis (small amount); ribosomes and RNA is ➢ SIZE: 8-10 um
more predominant and mask the color of hgb ➢ NUCLEUS: No nucleus; has CYTOPLASMIC RNA REMNANTS
➢ CYTOPLASM:
POLYCHROMATIC NORMOBLAST/RUBRICYTE ✓ predominant color is that of hemoglobin yet with a bluish tinge due to
➢ SIZE: 10-12 um some residual ribosomes and RNA
➢ NUCLEUS: ✓ By the end of the reticulocyte stage, the cell is the same color as a mature
✓ Round RBC, salmon pink
✓ N:C ratio: 4:1 to about 1:1 ➢ DIVISION: NOT CAPABLE
✓ Nucleoli: None LOCATION:
✓ Chromatin: more condensed than previous stage ✓ Bone marrow → 1-2 days; During
➢ CYTOPLASM: the first several days after exiting
✓ first stage in which the pink color associated with stained hemoglobin the marrow, reticulocyte is retained
can be seen in the spleen for pitting of
✓ Reflects the accumulation of hemoglobin pigmentation over time and inclusions and membrane polishing
concurrent decreasing amounts of RNA by splenic macrophages, which results in the biconcave discoid mature
✓ Mixture of pink and blue, resulting in a MURKY GRAY-BLUE RBC
(combination of multiple colors, because polychromatophilic means ✓ Blood → 1 day
“many color loving.”) ; POLYCHROMASIA ✓ remnants will not be seen in wright stain but we use supravital stains
➢ DIVISION: Divides into two orthochromic normoblast; LAST STAGE to see it
TO UNDERGO MITOSIS
➢ LOCATION: Bone marrow ➢ CELLULAR ACTIVITY:
➢ CELLULAR ACTIVITY: ✓ Completes production of
✓ Hemoglobin synthesis increases, and the accumulation begins to be hemoglobin from a small
visible as a pinkish color in the cytoplasm. amount of residual messenger
✓ Cellular RNA and organelles are still present, particularly ribosomes, RNA using the remaining
which contribute a blue color to the cytoplasm ribosomes (digested by
✓ The progressive condensation of the nucleus and disappearance of endoribonuclease)
nucleoli are evidence of progressive decline in transcription of ✓ Within 24-48 hrs, the cell loses
deoxyribonucleic acid (DNA) the organelles (except
➢ DURATION IN THIS STAGE: 30 hours mitochondria and golgi apparatus)
➢ last stage to undergo mitosis: after polychromatic, orthochromatic is ✓ Presence of residual ribosomal RNA
not capable of mitosis • visualized using supravital stains (cells are stained alive in
suspension)
ORTHROCHROMIC NORMOBLAST/METARUBRICYTE • appear as a mesh of small blue strands, a reticulum, or, when more
➢ SIZE: 8-10 um fully digested, merely blue dots
➢ NUCLEUS: ➢ DURATION IN THIS STAGE: 3 days
✓ Round, PYKNOTIC, eccentric, BIZARRE-
FORM (NONFUNCTIONAL)
✓ N:C ratio: 1:2
✓ Nucleoli: none
✓ Chromatin: completely condensed
CYTOPLASM:

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 12

 ERYTHROCYTE ✓ hypoxia- low oxygen in tissue which increases EPO production to
➢ SIZE: 6-8 um; approximate size: 7.2 produce rbc=more oxygen will be delivered to compensate hypoxia
um; Thickness: 1.5-2.5 um
➢ NUCLEUS: ANUCLEATED
➢ CYTOPLASM: EFFECTS OF ERYTHROPOIETIN
✓ salmon-pink stained cell with a 1. Early release of reticulocytes from the bone marrow:
central pale area that corresponds to ➢ Incudes change in the adventitial cell layer of the bone marrow; increased
the concavity; no presence of width leads to RBC precursor release
inclusions ➢ Downregulates the expression of fibronectin (adhesion molecule) causing
✓ Central pallor is about one-third the diameter of the cell; associated with earlier release of RBC precursors
hemoglobin concentration ➢ Presence of shift/stress reticulocytes → prematurely released
➢ DIVISION: NOT CAPABLE reticulocytes as a result of increased RBC production
➢ CELLULAR ACTIVITY: ➢ it is normally produced
✓ Delivers oxygen to tissues, releases it, and returns to the lung to be ➢ increase hepcidin=prevents iron from being absorbed by
reoxygenated intestine thus making the rbc absorbed more iron to produced
✓ Interior of the contains mostly hemoglobin, the oxygen- carrying hemoglobin
component. It has a surface area-to-volume ratio and shape that enable ➢ shift/stress retics-larger
optimal gas exchange to occur 2. Inhibition of apoptosis
✓ Oxygen delivery throughout the body requires a membrane that is flexible ➢ Early release of FasL bearing erythroid cells will indirectly allow more
and deformable, that is, able to flex but return to its original shape younger RBC precursors to mature leading to increased RBC
➢ DURATION IN THIS STAGE: 120 days (+/- 20 days) ; removed by the production
spleen (old/senescent) or liver (severely damaged) ✓ FasL → ligand for the FasR (death receptor) found in more mature RBC
Table 5-2. Normoblastic Series: Summary of Stage Morphology precursors
Cell/Stage Diamete N:C Nucleol % of Bone ➢ Direct EPO rescue from apoptosis
r (um) Ratio i Nucleate Marrow ✓ Effector of EPO is mediated by the transcription factor GATA-1
d Cells in Transmi ✓ EPO binds to its receptor on the CFU-E reducing production of Fas
Bone t Time ligand
Marrow ✓ Stimulate production of various anti-apoptotic molecules
• Binding of EPO to its receptor (EPOR) activates JAK2 protein →
Pronormoblas 12-20 8:1 1-2 1% 24 hrs.
activates the STAT5 pathway leading to production of anti-apoptotic
t
molecule Bcl-XL (now called as Bcl-2-like protein 1)
Basophilic 10-15 6:1 0-1 1%-4% 24 hr.
• Protein will then prevent release of cytochrome c which is an
normoblast
apoptotic initiator
Polychromati 10-12 4:1 0 10%-20% 30 hrs. ➢ if FasL is combined with is receptor, FasR= it will undergo
c normoblast apoptosis or programmed cell death that's why EPO will eraly
Orthochromic 8-10 1:2 0 5%-10% 48 hrs. release the FasL to inhibit the binding of it to FasR and prevents
normoblast apoptosis
Bone marrow 8-10 No 0 1% 24-48 ➢ limits the production of FasL- as it kills rbc
polychromati nucleu hrs. ➢ JAK2= Janus activated tyrosinase kinase 2
c erythrocyte s 3. Reduced marrow transit time → increase the rate at which the surviving
precursors can enter the circulation
*Also called reticulocyte ➢ Increased rate of cellular processes
if there are remnants=abnormal ✓ Accelerated hemoglobin production
✓ Erythroid precursors secrete erythroferrone → acts on hepatocytes to
ERYTHROKINETICS decreased hepcidin production allowing iron absorption for accelerated
➢ term describing the dynamics of RBC production and destruction hemoglobin synthesis
➢ Erythron → the name given to the collection of all stages of erythrocytes ✓ Hepcidin → regulates iron release; high levels prevent intestinal iron
throughout the body absorption
✓ The developing precursors in the bone marrow ➢ Decreased cell cycle times
✓ Circulating erythrocytes in the peripheral blood ✓ Cells spend less time in the bone marrow and are larger because of fewer
✓ Vascular spaces within organs, such as the spleen mitotic divisions
➢ RBC mass → refers to RBCs in the circulatio ✓ Leads to a 20% reduction in marrow transit time → from 6 days to 4
days (can be fewer than 4 days)
ERYTHROPOIETIN (EPO) ✓ Presence of shift/stress reticulocytes
➢ Thermostable, non-dialyzable, glycoprotein hormone with a molecular
weight of 34 Kd LEUKOPOIESIS
➢ Produced by the renal peritubular interstitial cells/fibroblasts and ➢ Production of leukocytes
LIVER ✓ Myelopoiesis–Granulopoiesis, Monopoiesis
➢ Production is stimulated by tissue hypoxia: ✓ Lymphoiesis
✓ detected by the renal peritubular fibroblasts; regulated by hypoxia agranulocytes-cannot see the granules but it is present
✓ Hypoxia-inducible factors (HIFs) which binds to the EPO gene causing
increased EPO production

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 13

 LEUKOCYTES GRANULOCYTE FORMATION
➢ Colorless nucleated cells that circulate in the peripheral blood MYELOBLAST
➢ main line of defense against foreign invaders such as bacteria, viruses, and ➢ Cell size: 14 to 20 um in diameter
other foreign antigens ➢ TYPES:
✓ Innate or non-specific ✓ Type I – no visible granules
✓ Adaptive or specific ✓ Type II - primary (azurophilic) granules (does not exceed 20 per cell)
➢ found in the: bone marrow, peripheral blood (transport of leukocytes), ✓ Type III – Rare; darker chromatin and a more purple cytoplasm, and
tissues they contain more than 20 granules
➢ Production: ➢ Nucleus: Round to oval; reddish purple; occupies most of the cell
✓ Granulocytes: bone marrow ✓ Chromatin: Fine
✓ Lymphocytes: bone marrow and lymphoid tissues ✓ Nucleoli: 2-4
Abundacy ➢ Cytoplasm: Slightly basophilic
✓ Never let monkeys eat bananas ➢ Granules: PRIMARY/AZUROPHILIC GRANULES
Phagocytic cells ➢ N:C ratio: 8:1 to 4:1 (the nucleus occupies most of the cell, with very little
✓ -neutrophil, mono/macrophages, eo/baso cytoplasm)
➢ Percentage in the bone marrow: 0% to 3%

CLASSIFICATION OF LEUKOCYTES
PRESENCE OF Granulocytes: neutrophils, eosinophils, basophils
GRANULES Agranulocytes: monocytes and lymphocytes
NUCLEAR Polymorphonuclear: multi-lobed or segmented
SEGMENTATION nucleus in mature form (neutrophils, eosinophils,
basophils)
Mononuclear: single mass of unsegmented
nucleus (monocytes and lymphocytes) PROMYELOCYTE
FUNCTION Phagocytes: neutrophils, monocytes, eosinophils, ➢ Cell size: 16 to 25 um in diameter
basophils (phagocytize, engulf, and destroy cells) ➢ Nucleus: round to oval and is often
Immunocytes: lymphocytes (antibody production) eccentric; paranuclear halo or “hof ”
✓ Chromatin: slightly coarse than
GRANULOCYTE MATURATION previous stage
➢ Myeloblast ✓ Nucleoli: 1-3
➢ Promyelocyte ➢ Cytoplasm: evenly basophilic and full
➢ Myelocyte (also apply to eo and baso) of primary (azurophilic) granules
➢ Metamyelocyte(also apply to eo and baso) Band Neutrophil(also ➢ Granules:
apply to eo and baso) PRIMARY/AZUROPHILIC
➢ Neutrophil, Eosinophil, Basophil GRANULES
➢ N:C ratio: 3:1 to 2:1
GRANULOCYTE and erythrocyte maturation is the same as nucleus ➢ Percentage in the bone marrow: 1% to 5%
disappear etc ➢ the production of azurophilic granules increases
GRANULOPOIESIS
NEUTROPHIL DEVELOPMENT MYELOCYTE
➢ Neutrophils are present in the peripheral blood in two forms according to ➢ Cell size:
whether the nucleus is segmented or still in a band shape ✓ Early myelocyte: 16-25 um
➢ Segmented neutrophils make up the vast majority of circulating leukocytes ✓ Late myelocyte: 15-18 um
➢ Major cytokine: CSF-G ➢ Nucleus: Round to oval
➢ PHSC → Common myeloid progenitor → CFU-GM → CFU-G → ✓ Chromatin: Coarser and more condensed
MYELOBLAST ✓ Nucleoli: Not visible
➢ There are three pools of developing neutrophils in the bone marrow: ➢ Cytoplasm: lightly basophilic to creamed-colored
✓ Stem cell pool – capable of self-renewal and differentiation ➢ Granules:
✓ Proliferation/Mitotic pool ✓ Primary granules → decreased; CONTINUES TO DECREASE AS
✓ Maturation pool/Storage – consisting of cells undergoing nuclear THE CELL MATURES
maturation that form the marrow reserve and are available for release ✓ Cell begins to manufacture SECONDARY (SPECIFIC) neutrophil
granules
➢ N:C ratio: 1:1
➢ Percentage in the bone marrow: 6% to 17%
➢ LAST STAGE CAPABLE OF MITOSIS

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 14

 METAMYELOCYTE ➢ Percentage in the bone marrow: 7%-30%
➢ Cell size: 14 to 16 um ➢ Percentage in the peripheral blood: 50%-70%
➢ Nucleus: nucleus is indented ➢ neutro-neutral; phil -love
(kidney bean shaped or peanut ➢ neutral affinity to both acid basic dyes
shaped)
✓ Chromatin: Moderately once the peripheral blood, neutrophils are divided randomly into:
clumped; coarser ➢ circulating neutrophil pool (CNP)
✓ Nucleoli: Absent ➢ marginated neutrophil pool (CMP)- localized to the walls of
➢ Cytoplasm: lightly basophilic to capillaries/ blood vessels.
creamed-colored; contains residual DIAPEDESIS-movement of neutrophils through capillary walls to their
RNA target tissue chemotaxis- signal mediated movement of neutrophils through
➢ Granules: release of chemotactic factor
✓ Primary granules - Few
✓ Secondary granules - Abundant EOSINOPHIL DEVELOPMENT
✓ Synthesis of TERTIARY GRANULES ➢ Similar to neutrophils, arise from the common myeloid progenitor (CMP)
➢ N:C ratio: 1:1 ➢ Cytokines: IL-3, IL-5, IL-33, GM-CSF
➢ Percentage in the bone marrow: 3% to 20% ➢ Eosinophilic promyelocytes can be identified cytochemical because of the
➢ nucleus is indented as it is being prepared for segmentation presence of Charcot-Leyden crystal protein in their primary granules
➢ The first maturation phase that can be identified as eosinophilic using light
BAND NEUTROPHIL microscopy and Romanowsky staining is the early myelocyte
➢ Cell size: 14 to 16 um ➢ PHSC → Common myeloid progenitor → CFU-Eo → MYELOBLAST
➢ Nucleus: Elongated, curved, sausage shaped (S OR C
SHAPED) ; Constricted but NO THREADLIKE EOSINOPHIL MYELOCYTE AND BAND
FILAMENTS; indentation exceeds half of the ➢ characterized by the presence of large pale, reddish-orange secondary
diameter granules, along with azure granules in blue cytoplasm
✓ Chromatin: Coarse and clump ➢ The nucleus is similar to that described for neutrophil myelocytes
✓ Nucleoli: Absent ➢ Eosinophil metamyelocytes and bands resemble their neutrophil
➢ Cytoplasm: Pale blue to pink counterparts with respect to their nuclear shape
➢ Granules: ➢ Secondary granules increase in number, and a third type of granule is
✓ Primary, Secondary and generated called the secretory granule or secretory vesicle
Tertiary granules
✓ Synthesis of SECONDARY EOSINOPHIL
GRANULES/VESICLES ➢ Cell size: 12 to 17 um
➢ N:C ratio: 1:1 to 1:2 ➢ Nucleus: BILOBED; connected by thin filaments
➢ Percentage in the bone ✓ Chromatin: Coarse and clump
marrow: 5% (Only WBC ✓ Nucleoli: Not visible
precursor normally seen in the ➢ Cytoplasm: Cream-colored; with characteristic
peripheral blood) refractile ORANGE-RED SECONDARY
GRANULES
NEUTROPHIL ➢ Granules: Primary, Secondary (reddish-orange color due to MBP),
➢ Cell size: 14 to 16 um Small lysosomal granules, Lipid bodies, Storage vesicles
➢ Nucleus: 2-5 LOBES connected by thin filaments ➢ N:C ratio: 1:1 to 1:2
✓ Chromatin: Coarse and clump ➢ Percentage in the bone marrow: 1%-3%
✓ Nucleoli: Absent ➢ Percentage in the peripheral blood: 1%-3%
➢ Cytoplasm: Pale-pink, cream-colored or colorless ➢ use of cytochemical stain to detect the presence of charcot leyden crystal
➢ Granules: in promyelocyte
✓ Presence of primary (rare), secondary (abundant), ➢ eo-red (acid); phil- love = affinity to acid
tertiary and continuous production of secretory
granules
✓ Synthesis of PINK TO ROSE VIOLET colored secondary granules
➢ N:C ratio: 1:1 to 1:2

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 15

 MAST CELLS
➢ Tissue effector cells of allergic responses and
inflammatory reactions
➢ Major cytokine: KIT ligand
➢ also known as Tissue basophils
➢ Size: larger than a basophil
➢ Nucleus: small and round
➢ Cytoplasm: same with basophil
➢ Granules: purple and less soluble than basophils

NEUTROPHIL FUNCTION
➢ Phagocytosis and destruction of foreign material and microorganisms
(PRIMARY PHAGOCYTIZING CELL)
➢ Generation of neutrophil extracellular traps, or NETs
✓ NETs → have enzymes from neutrophil granules attached to them and
have been shown to be able to trap and kill gram-positive and gram-
negative bacteria as well as fungi
➢ Secretory function → source of transcobalamin I or R binder protein,
necessary for the proper absorption of vitamin B12
➢ PHAGOCYTOSIS
➢ oxygen dependent-respiratory burst- the generation of H2O2
➢ non-oxidative burst-action of enzyme present in the different
granules of neutrophils
BASOPHIL AND MAST CELL DEVELOPMENT
➢ Basophils and mast cells are two cells with morphologic and functional
similarities; however, basophils are true leukocytes because they mature
in the bone marrow and circulate in the blood as mature cells with granules,
whereas mast cell precursors leave the bone marrow and use the blood as
a transit system to gain access to the tissues where they mature
➢ Major cytokines: IL-3 and TSLP (thymic stromal lymphopoietin)
➢ TYPES of basophils:
✓ IgE dependent → IL-3 elicited
✓ Non-IgE dependent → TSLP elicited
➢ PHSC → Common myeloid progenitor (CMP) → CFU-Baso →
MYELOBLAST
➢ Recognizable maturation stages include the basophilic myelocyte,
basophilic metamyelocyte, basophilic band, and basophil (segmented form)
EOSINOPHIL FUNCTION
BASOPHIL ➢ Degranulation during an inflammatory reaction (anti-inflammatory cell)
➢ Cell size: 10 to 14 um ➢ Immune regulation
➢ Nucleus: unsegmented, bi-lobed, connected by thin ✓ Acts as antigen presenting cells and promoting the proliferation of T-cells
filaments without visible chromatin; RARELY ✓ Regulate mast cell function through the release of major basic protein
HAS 3 OR FOUR LOBES (MBP), which causes mast cell degranulation as well as cytokine
✓ Chromatin: Coarse and clump production
✓ Nucleoli: Absent ✓ Produce nerve growth factor that promotes mast cell survival and
➢ Cytoplasm: colorless and activation
contains large numbers of the ➢ Parasitic infections
characteristic large blue-black ✓ Express receptors for IgE
granules ✓ Capable of destroying tissue-invading helminths through the secretion of
➢ Granules: BLUE-BLACK Major Basic protein (MBP) and eosinophil cationic protein as well as the
SECONDARY GRANULES; production of reactive oxygen species
water soluble and therefore may ➢ Allergic reactions
be dissolved if the blood film is ✓ Presence Charcot-Leyden crystals in secretions (sputum, stool, etc.)
washed too much during the ✓ Causes of airway inflammation and mucosal cell damage through
staining process secretion or production of a combination of basic proteins, lipid
➢ Percentage in the bone mediators, reactive oxygen species, and cytokines such as IL-5
marrow: 1% ✓ IgE will bind to parasite then bring it to the eosinophil, eosniphil
➢ Percentage in the peripheral blood: 0%-2% will now bind to IgE as it is the receptor then eosinophil will
phagocytize the parasite and release MBP and eosinophilic
cationic protein
✓ Eosinophil releases charcot leyden crytsal (disintegrated eo)
when it went to certain tissues

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 16

 BASOPHIL FUNCTION
➢ Allergic and inflammatory reactions (pro-inflammatory cell) MACROPHAGE
✓ Have IgE receptors on their surface membranes that, when cross-linked ➢ SIZE: 15-80 um; usually 40-50 um
by antigen, result in degranulation of histamine ➢ NUCLEUS: Eccentric, kidney or egg-
✓ Function as initiators of the allergic inflammation through the release of shaped, indented, or elongated
preformed cytokines ✓ Nucleoli: not visible
➢ Are capable of releasing large quantities of subtype 2 helper T cell cytokines ✓ Chromatin: net-like (reticulated
such as IL-4 and IL-13 that regulate immune response; Induces B-cell patterm)
synthesis of IgE ➢ CYTOPLASM: pale, often vacuolated,
➢ Incudes eosinophilia; release a chemotactic factor that attracts eosinophils and often filled with debris of phagocytized cells or organisms
to the site (parasitic infections) ➢ GRANULES: Many fine azurophilic granules
➢ Play a role in angiogenesis through the expression of vascular endothelial
growth factor (VEGF) and its receptors Kidneys: mesanglial cells
➢ IgE will bring the shrimp (allergic) to basophil then it will release Spleen: Littoral cells
histamine Placenta: Hofbauer cells
➢ angiogenesis-formation of new blood vessel Dendritic cells-most potent APC (lymph nodes)
➢ TISSUE MONOCYTE
MONOPOIESIS
MONOCYTE DEVELOPMENT
➢ Monocyte development is similar to neutrophil development because both
cell types are derived from the Granulocyte-Monocyte progenitor (CFU-
GM)
➢ Major cytokine: M-CSF
➢ PHSC → Common myeloid progenitor (CMP) → CFU-GM → CFU-M
→ MONOBLAST

MONOCYTE MATURATION
MONOBLAST
➢ SIZE: 12-20 um
➢ NUCLEUS: Round to oval; may be irregularly
shaped
✓ Nucleoli: 1-2; may not be visible
✓ Chromatin: Fine
➢ CYTOPLASM: Light blue to gray
➢ GRANULES: None
➢ N:C RATIO: 4:1 – 3:1 MONOCYTE / MACROPHAGE FUNCTION
➢ Innate immunity:
PROMONOCYTE ✓ recognize a wide range of bacterial pathogens by means of pattern
➢ SIZE: 12-18 um recognition receptors (Toll-like receptors) that stimulate inflammatory
➢ NUCLEUS: slightly indented or folded; cytokine production and phagocytosis
Irregularly shaped; may have brain like ✓ have Fc receptors for IgG and complement receptors; can phagocytize
convolutions foreign organisms or materials that have been coated with antibodies or
✓ Nucleoli: at least 1 complement components
✓ Chromatin: Fine to lacy ➢ Adaptive immunity:
➢ CYTOPLASM: Light blue to gray ✓ macrophages and dendritic cells degrade antigen and present antigen
➢ GRANULES: Primary/Azurophilic granules fragments on their surfaces (antigen presenting cells); activate both T and
(Burgundy color) B lymphocytes to initiate adaptive response
➢ N:C RATIO: 3:1 – 2:1 ✓ Dendritic cells are the most efficient and potent of the antigen-presenting
cells
➢ Housekeeping function:
MONOCYTE ✓ Blood monocytes ingest antigen-antibody complexes and activated
➢ SIZE: 15-20 um; irregularly shaped clotting factors, limiting the coagulation response
➢ NUCLEUS: round, oval, or kidney shaped but ✓ Splenic macrophages remove old/damaged RBCs and conserve iron for
more often is deeply indented (horseshoe shaped) recycling
or folded on itself ✓ Liver macrophages remove fibrin degradation products
✓ Nucleoli: not visible ✓ Bone marrow macrophages remove abnormal RBCs, ingest bare
✓ Chromatin: lace-like or stringy megakaryocyte nuclei or extruded RBC nuclei, and store and supply iron
➢ CYTOPLASM: blue-gray, with fine azure for hemoglobin synthesis
granules often referred to as AZURE DUST OR ➢ Synthesis of substances → coagulation factors, complement components,
A GROUND-GLASS/FROSTED interleukins, growth factors, and enzymes
APPEARANCE; with cytoplasmic blebs and vacuoles
➢ GRANULES: Many fine azurophilic granules
➢ LARGEST CELL IN PERIPHERAL BLOOD

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 17

 LYMPHOIESIS SMALL LYMPHOCYTE
LYMPHOCYTE ➢ CELL SIZE: 8-10 um
➢ divided into three major groups: T cells, B cells, and natural killer (NK) ➢ NUCLEUS: Round to oval; slightly indented
cells ✓ Nucleoli: not visible
➢ T and B cells are major players in adaptive immunity; NK cells make up a ✓ Chromatin: dense and clumped than lymphoblast
small percentage of lymphocytes and are part of innate immunity ➢ CYTOPLASM: moderate to dark blue, thin rim
➢ Can also be divided according to function: around the nucleus, ROBIN’S-EGG BLUE
✓ Humoral immunity → antibody production (B lymphocytes plasma CYTOPLASM
cells) ➢ GRANULES: absent or few azurophilic granules
✓ Cellular immunity → attacks foreign organisms or cells directly (T
lymphocytes and NK cells)
✓ T&B cells- adaptive immunity MEDIUM LYMPHOCYTE
✓ NK cells- innate immunity ➢ CELL SIZE: 10-12 um
✓ B cells, T cells, NK cells cannot be identified unless we used ➢ NUCLEUS: Round to oval; slightly indented
immunophenotyping (CD) ✓ Nucleoli: not visible
✓ Lymphocytes needs to be activated to mature and differentiate ✓ Chromatin: clumped but not as dense as a small
into B,T,NK cells lymmphocyte
➢ CYTOPLASM: pale to moderate blue
LYMPHOCYTE DEVELOPMENT ➢ GRANULES: absent or few azurophilic granules
➢ For both B and T cells, development can be subdivided into:
✓ Antigen-independent LARGE LYMPHOCYTE
• occurs in the bone marrow and thymus (sometimes referred to as ➢ CELL SIZE: 12-16 um
central or primary lymphatic organs) ➢ NUCLEUS: Round to oval; slightly
✓ Antigen-dependent indented, may be eccentrically located
• development occurs in the spleen, lymph nodes, tonsils, and mucosa- ✓ Nucleoli: not visible
associated lymphoid tissue such as the Peyer’s patches in the intestinal ✓ Chromatin: coarse
wall (sometimes referred to as peripheral or secondary lymphatic ➢ CYTOPLASM: abundant, clear and very
organs) pale blue
➢ PHSC → CFU-L → PRO-T/PRE-B/PRO-NK → LYMPHOBLAST ➢ GRANULES: absent or few azurophilic granules

LYMPHOCYTE MATURATION
➢ LYMPHOBLAST
➢ PROLYMPHOCYTE
➢ MATURE LYMPHOCYTE:
✓ SMALL LYMPHOCYTE
✓ MEDIUM LYMPHOCYTE
✓ LARGE LYMPHOCYTE
Small-resting
Medium and large lymphocytes- stimulated by antigen (activated)
Large lymphocyte-can be an effector or memory cells

LYMPHOBLAST
➢ CELL SIZE: 10-18 um
➢ NUCLEUS: Round to oval
✓ Nucleoli: 1-2
✓ Chromatin: Coarse and clumped
➢ CYTOPLASM: moderate to dark blue, may
stain DEEPER BLUE at the periphery and a
LIGHTER BLUE near the nucleus
➢ GRANULES: NONE
➢ N:C RATIO: 4:1 PLASMA CELL / PLASMACYOID LYMPHOCYTE
➢ End stage of B lymphocyte; dominant in lymph
PROLYMPHOCYTE nodes; not normally seen in circulation;
➢ CELL SIZE: 9-18 um responsible for antibody production
➢ NUCLEUS: Round to oval; slightly indented ➢ SIZE: 10-20 um
✓ Nucleoli: 0-1 ➢ NUCLEUS: Eccentric nucleus with a very coarse,
✓ Chromatin: Coarse and more clumped than clumped chromatin pattern; CARTWHEEL
lymphoblast APPEARANCE
➢ CYTOPLASM: moderate to dark blue ➢ CYTOPLASM: Abundant blue cytoplasm with
➢ GRANULES: usually non granular but may prominent perinuclear (golgi) zone
contains occasional azurophilic granules
➢ N:C RATIO: 3:1

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 18

 REACTIVE LYMPHOCYTE B-LYMPHOCYTES
➢ Associated with lymphocytosis ➢ Become immunocompetent in the secondary lymphoid tissue; dependent on
➢ SIZE: Large than a normal lymphocyte antigenic stimulation.
➢ NUCLEUS: Irregularly shaped ✓ Acquire specific receptors for antigens
➢ Chromatin: Fine with nucleoli ✓ Make up 20% of the peripheral blood lymphocytes
➢ CYTOPLASM: dark blue; tags, sharp ridges; ✓ Identified by membrane markers CD19, CD20, and others
indented by red cells ➢ FUNCTIONS:
✓ Contact with foreign antigens stimulates B lymphocytes to become
TYPES OF LYMPHOCYTES reactive lymphocytes, with the characteristic morphology associated with
T-LYMPHOCYTES reactivity.
➢ Become immunocompetent in the secondary lymphoid tissue; dependent ✓ Reactive lymphocytes transform into immunoblasts, and then plasma cells
on antigenic stimulation that produce antibodies to provide humoral immunity
✓ Acquire specific receptors for antigens
✓ Make up 80% of the peripheral blood lymphocytes NATURAL KILLER CELLS
➢ They are identified by membrane markers CD2, CD3, and others. The ➢ Large cells with low N:C ratio, large cytoplasmic granules, and pale blue
markers appear, disappear, and then reappear throughout cell development cytoplasm
➢ Lack B cell or T cell membrane markers; are CD16, CD56, and CD94
➢ FUNCTIONS: positive
✓ T cells provide cellular immunity. They are responsible for graft ➢ FUNCTIONS:
rejections and lysis of neoplastic cells, and they attack/destroy viral and ✓ Responsible for surveillance of cells for surface alterations such as tumor
fungal organisms. cells or virus infected cells
✓ Obtain antigenic information from monocytes; this information is passed ✓ Activated by IL-2 to express nonspecific cytotoxic functions
to other T cells and B cells ✓ modulate the functions of other cells, including macrophages and T cells
✓ Regulate humoral response by helping antigens activate B cells ✓ Attack antigens with attached IgG; called antibody-dependent cytotoxic
✓ End products of activation are cytokines/lymphokines/interleukins cells

REFERENCE RANGE FOR LEUKOCYTES

➢ RELATIVE COUNT → used for differential count; reported in %
➢ ABSOLUTE COUNT → number of cells in a given blood volume (uL or
L); reported in conventional (x103) or SI units (x109)

REACTIVE STATE ASSOCIATED CONDITIONS

NEUTROPHILIA ➢ Acute inflammatory — collagen
Absolute count: >7-8 x vascular, vasculitis
10 9/L ➢ Acute infectious — bacterial, some
viral, fungal, parasitic
➢ Drugs, toxins, metabolic —
corticosteroids, growth factors, uremia,
Ketoacidosis
➢ Tissue necrosis — burns, trauma, MI,
RBC hemolysis
➢ Physiologic — stress, exercise,
T-LYMPHOCYTE SUBSETS smoking, pregnancy
➢ Three T cell subsets are involved in the immune response and are ➢ Neoplastic —carcinomas, sarcomas,
differentiated by cluster designation (CD) markers myeloproliferative disorders
✓ T helper/inducer cell (T-h, T4) NEUTROPENIA ➢ Drugs — cancer chemotherapy,
• Identified by CD4 membrane marker Absolute count: <1.75 – chloramphenicol, sulfas/other
• Promotes activation of B cells by antigens 1.80 x 10 9/L antibiotics, phenothiazines,
✓ T suppressor cell (T-s, T8) Most common type of benzodiazepine, antithyroid,
• Identified by CDS membrane marker leukopenia anticonvulsants, quinine, quinidine,
• Suppresses activation of B cells by antigens indomethacin, procainamide, thiazides,
✓ Cytotoxic T cell (T-c, T8) AGRANULOCYTOSIS Radiation
Extreme neutropenia ➢ Toxins — alcohol, benzene compounds
• Identified by CDS membrane marker
(<0.5) ➢ Intrinsic defects — Fanconi’s,
• Functions in viral infections and organ rejections
Infantile: Kostmann’s Kostmann’s, cyclic neutropenia,
➢ The normal T4:T8 ratio in circulating blood is 2:1. This ratio must be
syndrome Chédiak-Higashi
maintained for proper immune response. It is used to monitor HIV patients.
➢ Immune-mediated — collagen
T helper (CD4) cells are destroyed by the HIV virus, which decreases the
vascular disorders, RA, AIDS
ratio as the infection spreads
➢ Hematologic — megaloblastic anemia,
myelodysplasia, marrow failure,
marrow replacement

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 19

 ➢ Infectious — any overwhelming
infection
➢ Others — starvation, hypersplenism
EOSINOPHILIA ➢ Allergic — urticaria, hay fever, asthma
Absolute count: >0.7 x 10 ➢ Inflammatory — eosinophilic fasciitis,
9/L Churg-Strauss syndrome
**eosinopenia: ACTH ➢ Parasitic — trichinosis, filariasis,
administration (Thorn’s schistosomiasis
Test) ➢ Nonparasitic infections — systemic
Churg-strauss fungal, scarlet fever, chlamydial
syndrome —Increased pneumonia of infancy
➢ Respiratory — pulmonary eosinophilic
syndromes (Löffler’s, tropical
pulmonary eosinophilia), Churg-Strauss
syndrome
➢ Neoplastic — CML, Hodgkin
lymphoma, T cell lymphomas
➢ diopathic hyper eosinophilic
syndromes — affecting heart, liver,
spleen, CNS, other organs
➢ Others — certain drugs, hematologic
and visceral malignancies, GI
inflammatory diseases, sarcoidosis,
Wiskott-Aldrich syndrome
BASOPHILIA ➢ Myeloproliferative disease
Absolute count: ➢ Allergic — food, drugs, foreign
>0.3x109/L proteins
➢ Infectious — variola, varicella
➢ Chronic hemolytic anemia —
especially post splenectomy
➢ Inflammatory — collagen vascular
disease, ulcerative colitis
MONOCYTOSIS ➢ Infectious — tuberculosis, subacute
Absolute count: bacterial endocarditis, syphilis,
>0.9x109 /L protozoan, rickettsial
➢ Recovery from neutropenia
➢ Hematologic — leukemias,
myeloproliferative disorders,
lymphomas, multiple myeloma
➢ Inflammatory — collagen vascular
disease, chronic ulcerative colitis, sprue,
myositis, polyarteritis, temporal arteritis
➢ Others — solid tumor, immune
thrombocytopenic purpura, sarcoidosis
LYMPHOCYTOSIS Infectious — many viral, pertussis,
Absolute lymphocyte tuberculosis, toxoplasmosis, rickettsial
count in: Chronic inflammatory — ulcerative colitis,
Adults: >4.0x109/L Crohn’s
infants and young Immune mediated — drug sensitivity,
children: >9.0x109/L vasculitis, graft rejection, Graves’, Sjögren’s
**CMV- resembles RS Hematologic — ALL, CLL, lymphoma
cells Stress — acute, transient

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 20

 ERYTHROCYTE MEMBRANE STRUCTURE AND FUNCTION,
METABOLISM, AND DESTRUCTION
Rmt2023
HEMATOLOGY LECTURE WEEK 5

ERYTHROCYTES ERYTHROCYTE METABOLISM

➢ Approximately 5 million per uL ERYTHROCYTE MATABOLIC PATHWAYS
➢ Average volume of 90 fL (measured through MCV) ➢ EMBDEN-MEYERHOF PATHWAY (ANAEROBIC
➢ Anucleated (contain no organelles and no synthesis of GLYCOLYSIS)
hemoglobin) ✓ End product will be ATP and pyruvate
➢ Biconcave shape (discoid or discocyte in PBS) ✓ Glycolysis diversion pathways/shunts (diversion pathways
✓ facilitates transport of oxygen and carbon dioxide of EMP)
(maximizes the ratio of the surface area to the volume) • Hexose monophosphate shunt pathway/Pentose
✓ allow flexibility and deformability (allows the cell to adjust to phosphate shunt
small vessels and still maintain a constant surface area: • Methemoglobin reductase/Cytochrome B5 reductase
volume ratio) pathway
✓ facilitate the transport O2 and CO2 • Rapoport-Luebering pathway

➢ Cytoplasm contains HEMOGLOBIN ➢ OTHERS:

✓ Hemoglobin ✓ Intracellular cationic gradient maintenance
• Contributes to the color of the cell ✓ Maintenance of membrane phospholipid distribution
• Complex of Heme (4 protoporphyrin IX molecules and 4 ✓ Maintenance of skeletal protein deformability
ferrous iron) and globin (4 globin chains) (starts in ✓ Nucleotide salvage reactions
pronormoblast)
• Each chain contains a heme molecule with an iron in the
ferrous state; this allows each hemoglobin molecule to
carry four O2 molecules
• Amount in the RBC cytoplasm correlates with the central
pallor/pallor area which occupies 1/3 of the cell

➢ ENERGY PRODUCTION: anaerobic glycolysis (Embden-

Meyerhof pathway, EMP)
✓ Without mitochondria for aerobic respiration via oxidative
phosphorylation, adenosine triphosphate (ATP) is produced
within the cytoplasm through EMP
o Glucose is needed
o ATP is the source of energy in RBC as without
ATP, RBC will die
✓ ATP → slow the oxidation of proteins and iron by
environmental peroxides and superoxide anions,
maintaining hemoglobin’s function and membrane integrity
➢ Life span: Lasts for 120 days (+/- 20 days)
✓ Engulfed by macrophages in the spleen: old/senescent
RBC and liver: severely damaged RBC (extravascular
hemolysis)
✓ Disassembled into its reusable components: globin chains
and iron from hemoglobin, and phospholipids and proteins
from the cell membrane
✓ The protoporphyrin ring of hemoglobin is not reusable and
is excreted as bilirubin
➢ Major function: transport and oxygen to the tissues and G-6-PD and pyruvate kinase-important
carbon dioxide and bicarbonates (HCO3) to the lungs
➢ Production is controlled by the hormone: Erythropoietin
(production is stimulated by hypoxia and decreased renal
oxygen tension)

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 21

 EMBDEN MEYERHOF PATHWAY ➢ Pointed arrows shows a
➢ Main metabolic pathway of RBC responsible for ATP degmacyte/bite
production cell/bronze cell with
➢ Glucose undergoes ANAEROBIC GLYCOLYSIS to produce Heinz bodies
ATP ✓ Degmacyte → an RBC
➢ Requires glucose to generate ATP, a high-energy phosphate with a remove inclusion
source; RBCs lack internal energy stores (mitochondria) and by pitting
rely on plasma glucose to enter the cell to generate ATP ✓ Pitting → removal of
➢ Provides 90-95% of ATP needed by RBCs RBC inclusions
➢ Deficiency in ATP production can result to:
✓ Premature cell death due to inherited defects in glycolysis METHEMOGLOBIN REDUCTASE PATHWAY
✓ Loss of viability during the storage of blood for transfusion ➢ Heme iron is constantly exposed to oxygen and peroxide;
✓ Pk deficiency (Pyruvate kinase) → decreased ATP Peroxide oxidizes heme iron from the ferrous (Fe2+) to the
(echinocytes) ferric state (Fe3+)
✓ Loss of ability to deform/flexible (ATP is required to maintain ➢ When the iron is in the ferric state, the affected hemoglobin
the structure of the RBC membrane) molecule is called methemoglobin
➢ Accounts for more than 65% of the methemoglobin-
HEXOSE MONOPHOSPHATE SHUNT PATHWAY reducing capacity within the RBC
➢ Also known as the Pentose phosphate shunt; occurs ➢ Although the HMP prevents hemoglobin oxidation by reducing
through a diversion of glucose catabolism peroxide, it is not able to reduce methemoglobin once it
➢ AEROBIC/OXIDATIVE GLYCOLYSIS; 5% to 10% of forms; Reduction of methemoglobin by NADPH is rendered
glucose-6-phosphate is diverted to the HMP (produces 5-10% more efficient in the presence of methemoglobin
of ATP needed by RBCs) reductase, also called cytochrome b5 reductase
➢ Requires the enzyme glucose-6-phosphate ➢ Cytochrome b5 reductase acts as an intermediate electron
dehydrogenase (G-6-PD-important to maintain the carrier, returning the oxidized ferric iron to its ferrous,
hemoglobin) oxygen-carrying state
➢ Maintaining and preventing methemoglobin
RAPOPORT-LUEBERING PATHWAY
➢ Pathway is responsible for the production of: ➢ Generates 2,3-bisphosphoglycerate (2,3- BPG; also called
✓ Ribulose-5-phosphate → required for synthesis of RNA 2,3-diphosphoglycerate or 2,3-DPG)
✓ Reduced Nicotamide adenine dinucleotide phosphate ➢ 2,3-BPG binds between the globin chains in the interior
(NADPH) cavity of the hemoglobin tetramer to stabilize it in the
• Reduces oxidized glutathione (GSSG) to REDUCED deoxygenated state/deoxyhemoglobin (tense or low
GLUTHATHIONE (GSH) in the presence of the enzyme oxygen affinity state)
glutathione reductase o If there’s no O2 in hemoglobin, 2, BPG will
• Comes from NADP and once it reduced it becomes bind to hgb
NADPH ✓ binding shifts the hemoglobin-oxygen dissociation curve
to the right, which enhances delivery of oxygen to the
✓ REDUCED GLUTATHIONE (GSH) tissues
• Extends the functional life span of the RBC by ✓ Increased 2,3-BPG → decreased Hgb-O2 affinity; oxygen
MAINTAINING membrane proteins and lipids, enzymes, IS DELIVERED to the tissues
and hemoglobin iron in the functional, reduced, ferrous ✓ Decreased 2,3-BPG → increased Hgb-O2 affinity; oxygen
state (prevents oxidation of Hgb to methemoglobin) IS NOT DELIVERED to the tissues
• Hemoglobin is ferrous when it becomes ferric=oxidized
• Prevents hemoglobin from being ERYTHROCYTE MEMBRANE STRUCTURE
oxidized=methemoglobin as it cannot bind oxygen and ➢ Bilayer of phospholipids, cholesterol, and glycolipids as well
transport it=anemia, hypoxia as associated proteins (phospholipid bilayer)
➢ Constantly changes as it moves through circulation; has the
➢ G-6-PD deficiency: ability to flex and deform to due to its biconcave shape; soft
✓ Most common inherited RBC enzyme deficiency worldwide; and pliable (elastic)
deficiency will lead to deficiency in both NAPDH and GSH ➢ Consists of approximately 8% carbohydrates(glycolipids and
making RBCs vulnerable to oxidative damage (will cause glycoproteins), 52% proteins (peripheral and integral) and
hemoglobin oxidation/denaturation)-> hgb to methgb 40% lipids (phospholipids, cholesterol)
✓ Oxidized/denatured hemoglobin will lead to formation of
Heinz bodies (pitted golf ball appearance) in the RBC
cytoplasm

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 22

 ERYTHROCYTE MEMBRANE LIPIDS IMPORTANT NOTES FOR CHOLESTEROL
CHOLESTEROL AND PHOSPHOLIPIDS
➢ Esterified and largely hydrophobic, resides parallel to the ➢ When phospholipid distribution is disrupted, as in sickle cell
acyl tails of the phospholipids anemia and thalassemia or in aging RBCs, Phosphatidyl
➢ Equally distributed between the outer and inner layers of the serine PS, the only negatively charged phospholipid,
phospholipid bilayer redistributes to the outer layer
o PS is in inner, will go outside layer=will be
✓ FUNCTIONS: targeted by macrophage
• confers tensile strength to the lipid bilayer ➢ Splenic macrophages possess receptors that bind to the PS
• Regulates membrane fluidity and permeability displayed on senescent and damaged RBCs and remove
• Important determinant of membrane surface area and them from circulation
fluidity ➢ C-reactive protein and inflammatory conditions increase the
• In liver diseases, membrane cholesterol increases due to PS distribution in the outer layer of the RBC membrane
increased plasma bile salt concentration resulting to leading to increased RBC death (also called eryptosis)
increased surface area-to-volume ratio and decreased
deformability (will lead to hemolysis) GLYCOLIPIDS
• High cholesterol in RBC= RBC will destroyed ➢ Make up 5% of the external half of the RBC membrane
➢ Bear copies of carbohydrate-based blood group antigens,
➢ Membrane enzymes maintain the cholesterol such as antigens of the ABH and the Lewis blood group
concentration by regularly exchanging membrane and systems
plasma cholesterol (LCAT)-> Lecithin-chol ➢ Associate in clumps or rafts and support carbohydrate
acyltransferase=acanthocytes side chains that extend into the aqueous plasma to anchor
➢ Deficiencies in these enzymes are associated with RBC the glycocalyx
membrane abnormalities such as acanthocytes and target
cells (codocytes) ➢ GLYCOCALYX
✓ layer of carbohydrates whose net negative charge prevents
PHOSPHOLIPIDS microbial attack and mechanical damage caused by
➢ asymmetrically distributed; form an impenetrable fluid adhesion to neighboring RBCs or to the endothelium
barrier ERYTHROCYTE MEMBRANE PROTEINS
✓ Hydrophilic polar head groups are arrayed on the ➢ Although cholesterol and phospholipids constitute the
membrane’s surfaces, oriented toward both the aqueous principal RBC membrane structure, proteins make up 52%
plasma and the cytoplasm of the membrane structure
• Composed of Phosphatidylcholine and sphingomyelin; ✓ Transmembrane/Integral proteins
glycolipids are also present ✓ Cytoskeletal/Skeletal/Peripheral proteins
✓ Hydrophobic nonpolar acyl tails arrange themselves to ➢ At least 300 RBC membrane proteins, including 105
form a central layer sequestered (hidden) from the aqueous transmembrane proteins (50 have been characterized
plasma and cytoplasm and named)
• Composed of phosphatidylserine (PS) and
phosphatidylethanolamine TRANSMEMBRANE / INTEGRAL PROTEINS
➢ Phosphatidylinositol → present in both the inner and outer ➢ Penetrates the lipid bilayer and are firmly anchored within it
layers ➢ Assemble into one of two major macromolecular complexes
named by their respective cytoskeletal anchorages:
✓ Ankyrin complex
✓ Actin junctional complex, also called protein 4.1 complex
➢ The anchoring of these transmembrane complexes to
cytoskeletal proteins (adjacent to the inner or cytoplasmic
side of the membrane) prevents loss of the lipid bilayer
✓ Linking of cytoskeletal proteins by the actin junctional
complex provides membrane structural integrity
because the cell relies on an intact cytoskeleton to
maintain its biconcave shape despite deformability
✓ Binds to integral proteins to penetrate into the membrane

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 23

 SIALIC ACID-> responsible for ZETA POTENTIAL which
causes RBC to repel one another

PERIPHERAL / CYTOSKELETAL PROTEINS

➢ Stabilizes the plasma membrane; forms the membrane
cytoskeleton
OTHER FUNCTIONS OF INTEGRAL PROTEINS ➢ principal cytoskeletal proteins are the filamentous a-spectrin
➢ Transport sites and b-spectrin (which assemble to form an antiparallel
✓ Disruption in transport protein function changes the osmotic heterodimer held together with a series of lateral bonds)
tension of the cytoplasm, which leads to a rise in viscosity ➢ Protein responsible for RBC deformability: SPECTRIN
and loss of deformability ➢ Important integral proteins in maintaining cytoskeleton is
BAND 3
➢ Adhesion sites
✓ change affecting adhesion proteins permits RBCs to adhere ➢ integral: vertical
to one another and to the vessel walls, promoting ➢ Cytoskeleton-horizontal
fragmentation (vesiculation), reducing membrane flexibility,
and shortening the RBC life span
➢ Signaling receptors
✓ bind plasma ligands and trigger activation of intracellular
signaling proteins, which then initiate various energy-
dependent cellular activities, a process called signal
transduction
➢ Support surface carbohydrates
✓ Through glycosylation; join with glycolipids to make up the
protective glycocalyx
➢ Provide vertical membrane structure

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 24

 ➢ Cytoplasmic hemoglobin viscosity
✓ As MCHC rises, internal viscosity rises; MCHCs greater
than 36% compromise deformability and shorten the
RBC life span because viscous cells become damaged as
they stretch to pass through narrow capillaries or splenic
ERYTHROCYTE MEMBRANE DEFORMABILITY pores
AND PERMEABILITY ✓ As RBCs age, they lose membrane surface area, while
➢ RBCs surface area is 140 um2, a 40% excess of surface retaining hemoglobin. As the MCHC rises, the RBC, unable
area compared with a sphere of 6 to 8 um in diameter (due to pass through the splenic pores, is phagocytized and
to its biconcave shape) destroyed by splenic macrophages
➢ RBC plasma membrane is 100 times more elastic than a
comparable latex membrane, yet it has tensile (lateral) ➢ Membrane elasticity (pliancy)-function of lipids
strength greater than that of steel ✓ ratio of cholesterol to phospholipids remains relatively
➢ DEFORMABLE RBC MEMBRANE: constant to maintain the balance of deformability or
✓ provides the broad surface area and close tissue contact elasticity and strength
necessary to support the delivery of O2 from the lungs to ✓ Membrane phospholipids and cholesterol redistribute
body tissues and to transport CO2 from body tissues to the laterally so that the RBC membrane may respond to
lungs stresses and deform within 100 milliseconds of being
✓ Enables RBCs to stretch undamaged up to 2.5 times their challenged by the presence of a narrow passage
resting diameter as they pass through narrow capillaries (capillaries)
and through splenic pores 2 um in diameter
ERYTHROCYTE MEBRANE PERMEABILITY
FACTORS THAT INFLUENCE ➢ The erythrocyte membrane also regulates osmotic balance
MEMBRANE DEFORMABILITY by being semi-permeable
➢ Membrane geometry ➢ Permeability
✓ interaction of RBC membrane proteins; vertical and ✓ Ability of the RBC Membrane to allow substances to pass
horizontal through in and out of the membrane
✓ Spectrin dimer bonds that appear along the length of the ✓ Prevents colloid hemolysis and controls RBC volume
✓ These flexible interactions plus the spectrin-protein 4.1 o Colloid hemolysis-present intra/extravascular
junctions in the actin junctional complexes between the hemolysis
tetramers are key regulators of membrane elasticity and
mechanical stability ➢ PERMEABLE: water and negatively charged anions
(bicarbonate (HCO3) and chloride (Cl-); freely exchange
✓ HEREDITARY SPHEROCYTOSIS between plasma and RBC cytoplasm
• quantitative deficiency of spectrin, ankyrin, band 3, or o RBC is negatively charged
protein 4.2 (vertical interactions) ➢ IMPERMEABLE: positively charged cations such as Sodium,
Potassium, and Calcium (Na+, K+, Ca2+)
✓ HEREDITARY ELLIPTOCYTOSIS o If it is positively charge, it needs ATP to pass
• Affects spectrin dimer-to-dimer lateral bonds/spectrin- through
protein 4.1 junction (horizontal interactions)

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 25

➢ OSMOTIC BALANCE IS MAINTAINED BY: ERYTHROCYTE DESTRUCTION
✓ Aquaporin 1 ➢ All cells experience deterioration of their enzymes over
• Transmembrane protein that forms pores or channels time because of natural catabolism
whose surface charges create inward water flow in ➢ Most cells are able to replenish needed enzymes and
response to internal osmotic changes continue their cellular processes
o No need for ATP ➢ As a non-nucleated cell, however, the mature erythrocyte is
• Decreases in aquaporin 1 expression has been linked with unable to generate new proteins, such as enzymes, so as its
hereditary spherocytosis cellular functions decline, the cell ultimately approaches death
➢ Loss of glycolytic enzymes is central to this process of
✓ Cation pumps/ATPase systems cellular aging, called senescence, which culminates in
• Sodium and Potassium ATPase phagocytosis by macrophages
o ATP dependent cation pumps ➢ Aside from loss of ATP and enzymes, other factors contribute
o regulate the concentrations of sodium and to erythrocyte destruction such as:
potassium, maintaining intracellular-to-extracellular ✓ Loss of sialic acid and lipids
ratio ✓ Decreased size
Sodium → 1:12 (outside-inside) ✓ Increased density
Potassium → 25:1 (inside-outside) ✓ Increased calcium
o Increase sodium without loss of potassium: RBC will ✓ Loss of selective membrane permeability (increased sodium
gain water causing cell lysis and decreased potassium inside RBCs)
o Increase potassium inside the cell: cell
shrinkage/crenated RBC TYPES OF DESTRUCTION
EXTRACASCULAR / MACROPHAGE-MEDIATED
• Calcium ATPase HEMOLYSIS
o ATP dependent cation pump ➢ Lysis of erythrocytes outside the circulation
o expels calcium from the cell, maintaining low intracellular ➢ Approximately 1% of RBC’s leave the circulation each day
levels of 30 to 60 nM compared with 1.8 mM in the and are broken down by the mononuclear phagocytic
plasma system (liver, spleen and bone marrow)
o Calmodulin, a cytoplasmic calcium binding protein, ✓ Iron is removed from the heme; stored in the macrophage
controls the function of Calcium-ATPase as ferritin/hemosiderin until transported out
o INCREASED INTRACELLULAR CALCIUM: decrease ✓ Globin is deranged and returned to the metabolic amino
deformability causing abnormality in shape which can acid pool
lead to cell lysis ✓ Protoporphyrin component of heme is degraded to
bilirubin (released into the blood and excreted by the liver in
➢ Cation pumps consume a significant portion of RBC ATP bile
production
➢ ATP loss or pump damage permits calcium and sodium influx, ➢ No complement activation
with water following osmotically; the cell swells, becomes ➢ RBC destruction: 90%
spheroid, and eventually rupture; this phenomenon is called ➢ Process is called: Culling (removal of senescent/old RBC’s)
colloid osmotic hemolysis ➢ Increased unconjugated bilirubin; urine and fecal
o Where sodium goes, water follows=sphere- urobilinogen/stercobilinogen
rupture=colloid osmotic hemolysis
➢ Defects in the ion channels result in red cell volume 1. When an erythrocyte is phagocytized and digested by the
disorders, such as overhydrated stomatocytosis macrophages of the reticuloendothelial system, the hemoglobin
(hereditary hydrocytosis) and dehydrated stomatocytosis molecule is disassembled components are iron,
(hereditary xerocytosis) protoporphyrin, and globin
➢ Sickle cell disease also provides an example of increased ✓ Iron is transported in the plasma by transferrin to be
cation permeability. When hemoglobin S polymerizes on recycled by the red bone marrow in the manufacture of new
deoxygenation, the cell deforms into a sickle shape and the hemoglobin (stored as ferritin or hemosiderin)
membrane becomes more permeable to calcium; this ✓ Globin is catabolized in the liver into its constituent amino
causes a downstream effect of increased sodium and acids and enters the circulating amino acid pool
potassium, resulting in hemolysis ✓ The porphyrin ring is broken at the alpha methene bridge by
the heme oxidase enzyme
• The alpha carbon leaves as carbon monoxide
• Biliverdin converts the tetrapyrrole to unconjugated
bilirubin (B1) and is carried by plasma albumin to the

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 26

 liver, where it is conjugated to glucuronide/conjugated 2. The formation of this large molecular HAPTOGLOBIN-
bilirubin (B2) and excreted in the bile HEMOGLOBIN COMPLEX prevents urinary excretion of plasma
2. Both unconjugated (pre-hepatic) and conjugated (post- hemoglobin. This stable complex is removed from the circulation
hepatic) bilirubin is present in the plasma by the hepatocytes, where it is processed by the cells in a manner
3. Conjugated bilirubin/Bilirubin diglucuronide is excreted similar to normal intact erythrocyte breakdown
into the gut, converted by bacterial action to urobilinogen, and ✓ Once plasma haptoglobin is depleted in the blood
excreted in the feces as stercobilinogen circulation, unbound hemoglobin alpha and beta dimers are
4. A small amount of urobilinogen is reabsorbed into the blood rapidly filtered by the glomeruli in the kidneys, reabsorbed
circulation and excreted in the urine (urine urobilinogen) by the renal tubular cells, and converted to hemosiderin

3.Hemoglobin that is neither bound by haptoglobin nor directly

excreted in the urine is oxidized to methemoglobin; the heme
groups in methemoglobin are released and taken up by another
transport protein, HEMOPEXIN

4. HEMOPEXIN-HEME COMPLEXES is cleared from the

circulation by the low-density lipoprotein receptor– related
protein (LRP/CD91) a multifunctional scavenger found in the
liver (also expressed in the brain, macrophage/monocytes, and
placenta)

5. Heme groups in excess of the hemopexin-binding capacity

combine with albumin to form methemalbumin until more
hemopexin is available. Once it is needed, hemopexin becomes
available, and the complex is subsequently phagocytized by
hepatocytes
✓ IMPORANT NOTE: The combined depletion of
haptoglobin and hemopexin and the presence of
methemalbuminemia and hemosiderinuria can be seen
in cases of intravascular hemolytic anemia and in
o When unconjugated bilirubin went to liver, it will become
intramarrow destruction of erythrocyte precursors
b2
INTRAVASCULAR / MECHANICAL /
FRAGMENTATION HEMOLYSIS
➢ Although most natural RBC deaths occur in the spleen, a
small portion of RBCs rupture intravascularly (within the
lumen of blood vessels)
➢ Intravascular rupture of RBCs from purely mechanical or
traumatic stress results in fragmentation and release of the
cell contents into the blood; this is called fragmentation or
intravascular hemolysis
➢ Destruction of RBC’s within blood vessels with complement
activation
➢ Hemoglobin is broken down and released as free
hemoglobin into the plasma
➢ RBC destruction: 10%
✓ Haptoglobin: transports hemoglobin alpha-beta dimers
into the liver (haptoglobin-hemoglobin complex)
✓ Hemopexin: transports heme to the liver (hemopexin-heme
complex)
o Catabolized the same way as extravascular hemolysis
o If there’s a presence of hemosiderin in urine=hemolytic
1. As the result of intravascular destruction, hemoglobin
episodes in the body
is released directly into the bloodstream (free
o Hemopexin and haptoglobin decreases once it binds to
hemoglobin) and undergoes dissociation into alpha
heme and alpha and beta dimers, respectively
and beta dimers, which are quickly bound to the plasma
o Hemopexin will bring heme to liver then it will be
globulin HAPTOGLOBIN
catabolized

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 27

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 28
 HEMOGLOBIN METABOLISM
Rmt2023
HEMATOLOGY LECTURE WEEK 8

HEMOGLOBIN
➢ Is one of the most studied proteins in the body because of
the ability to easily isolate it from red blood cells (RBCs)
➢ 95% of cytoplasmic content of RBCs
➢ When released into the plasma, it is rapidly salvaged to
preserve its iron and amino acid components; when salvage
capacity is exceeded, it is excreted by the kidneys
➢ Concentration: 34 g/dL (measured by MCHC: normal)→ 1/3
central pallor
➢ Molecular weight: 64,000 Daltons
➢ FUNCTION:
✓ Transport oxygen to the tissues and carbon dioxide from GLOBIN STRUCTURE
the tissues to the lungs ➢ The four globin chains comprising each hemoglobin
✓ Contributes to acid-base balance by binding and releasing molecule consist of two identical pairs of unlike polypeptide
hydrogen ions (Bohr effect) chains (Ex. Hgb A1 → 2alpha and 2beta globin chains)
✓ Transports nitric oxide (induces relaxation of vascular wall ➢ Variations in amino acid sequences give rise to different
of smooth muscle vasodilation) types of polypeptide chains
➢ Each chain is designated by a Greek letter
HEMOGLOBIN STRUCTURE Table 7.1 Globin Chains
➢ Spherical, has four heme SYMBOL NAME NO. OF AMINO ACIDS
groups attached to four α Alpha 141
polypeptide chains, and may β Beta 146
carry up to four molecules of γA Gamma A 146 (position 136; alanine)
oxygen γG Gamma G 146 (position 136; glycine)
δ Delta 146
➢ CONJUGATED GLOBULAR ε Epsilon 146
PROTEIN CONSISTING OF: ζ Zeta 141
(TETRAMER) θ Theta Unknown
✓ Globin → two different
pairs of polypeptide chains (4 globin chains) GLOBIN BIOSYNTHESIS
✓ Heme → four heme groups, with one heme group ➢ Six structural genes code for the six globin chains; are on
imbedded in each of the four polypeptide chains the short arms (p arm) of chromosomes
✓ 2,3-diphosphoglycerate (2,3-DPG) ✓ Chromosome 16 – alpha and zeta
• Produced in the anaerobic glycolytic pathway ✓ Chromosome 11 – beta, delta, gamma, and epsilon
(Luebering-Rapoport pathway) ➢ Production of globin chains takes place in the nucleus and
• Bonded in the center of beta chains ribosomes of erythroid precursors from the pronormoblast
• Inversely related to the hemoglobin-oxygen affinity through the circulating polychromatic erythrocyte
(reticulocyte), but not in mature erythrocytes
➢ Transcription of the globin genes to messenger ribonucleic
acid (mRNA) occurs in the nucleus, and translation of
mRNA to the globin polypeptide chain occurs on ribosomes
in the cytoplasm
JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 29
➢ Although transcription of a-globin genes produces more
mRNA than the b-globin gene, there is less efficient
translation of the a-globin mRNA; Therefore, a and b chains IMPORANT NOTES:
are produced in approximately equal amounts ✓ Transferrin, a plasma protein, carries iron in the ferric
➢ After translation is complete, chains are released from the (Fe3+) form to developing erythroid cells
ribosomes in the cytoplasm ✓ Transferrin binds to transferrin receptors on erythroid
➢ Production in hemoglobin starts in Basophilic normoblast precursor cell membranes and the receptors and
transferrin (with bound iron) are brought into the cell in an
HEME STRUCTURE endosome
➢ Also called as Ferroprotoporphyrin IX ✓ Acidification of the endosome releases iron from
➢ Heme consists of: transferrin
✓ Protoporphyrin IX → ring of carbon, hydrogen, and ✓ Iron is transported out of the endosome and into the
nitrogen atoms (pyrrole ringe) mitochondria, where it is reduced to the ferrous state and
✓ Central atom of divalent ferrous iron (Fe2+): reduced is united with protoporphyrin IX to make heme
iron ✓ Heme leaves the mitochondria and is joined to the globin
o Divalent-2+ chains in the cytoplasm
➢ Each of the four heme groups is positioned in a pocket of
the polypeptide
chain near the
surface of the
hemoglobin
molecule
➢ The ferrous iron
in each heme
molecule
reversibly
combines with one oxygen molecule
➢ When the ferrous irons are oxidized to the ferric state
(Fe3+), they no longer can bind oxygen (methemoglobin)
➢ Ferrous iron/reduced iron→ metabolically active iron; binds
oxygen; used for heme synthesis
➢ Ferric iron/ oxidized iron→Transported by transferrin;
stored form of iron (ferritin or hemosiderin)
o Ferritin→ if the protein apoferritin is present
(ferric iron + apoferritin=ferritin)
o Hemosiderin→ If ferric iron only

HEME BIOSYNTHESIS
➢ Heme biosynthesis occurs in the mitochondria and
cytoplasm of bone marrow erythroid precursors, beginning
with the pronormoblast through the circulating
polychromatic erythrocyte (reticulocyte)(starts at basophilic
normoblast/ prorubricyte)
➢ As they lose their ribosomes and mitochondria, mature
erythrocytes can no longer make hemoglobin
➢ Porphyrias → disorders in the heme biosynthesis pathway

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 30

 ➢ The hemoglobin molecule can be described by its primary,
secondary, tertiary, and quaternary protein structures
✓ Primary
• refers to the amino acid sequence of the polypeptide
chains
✓ Secondary
• refers to chain arrangements in helices and non-helices
✓ Tertiary
➢ Cofactor of succinyl CoA is glycine to produce ALA
• refers to the arrangement of the helices into a pretzel-
➢ Ferrochelatase- also known as heme synthase
like configuration
✓ Quaternary
HEMOGLOBIN ASSEMBLY
• also called a tetramer, describes the complete
➢ After their release from ribosomes, each globin chain binds
hemoglobin molecule
to a heme molecule, then forms a heterodimer
• Hb A, is composed of two a-globin chains and two b-
➢ Two heterodimers then combine to form a tetramer; this
globin chains.; strong a1-b1 and a2-b2 bonds hold the
completes the hemoglobin molecule
dimers in a stable form
➢ In the native configuration of the hemoglobin molecule, the
• The a1-b2 and a2-b1 bonds are important for the
four hemes and four polypeptide chains are assembled in a
stability of the quaternary structure in the oxygenated
very specific spatial configuration
and deoxygenated forms
➢ Each of the four chains in the molecule coils into eight
helices, forming an egg-shaped molecule with a central
NORMAL HEMOGLOBINS
cavity
➢ Embryonic hemoglobin → present up to the 3rd month of
➢ In the process of the binding of the first heme group to a
fetal life
molecule of oxygen, a change in the overall configuration of
➢ Fetal hemoglobin → predominant during second and third
the hemoglobin molecule occurs
trimester of fetal life and at birth (up to 6 months)
➢ This altered configuration of the molecule favors the
➢ Adult hemoglobin (HgbA1) → predominant by 6 months of
additional binding of oxygen to the remaining heme groups,
age until adulthood with small amounts of HgbA2
if sufficient oxygen pressure is present.
➢ Hemoglobin variants → genetic abnormalities in the
hemoglobin molecule (normal is 6 Hgb) (abnormal: more
than 350 Hgb)
➢ Hemoglobin electrophoresis and HPLC
✓ Are used for fractionation, presumptive identification, and
quantification of normal hemoglobins and hemoglobin variants
✓ Hemoglobin ontogeny-which Hgb are produced by erythroid
precursor cells from the fetal period through birth to adulthood
HGB MOLECULAR STAGE OF NEWBOR ADULTS
STRUCTURE LIFE N
Portlan 2 zeta, 2 Embryonic 0% 0%
d gamma
Gower I 2 zeta, 2 Embryonic 0% 0%
epsilon
Gower 2 alpha, 2 Embryonic 0% 0%
II epsilon
Fetal 2 alpha, 2 Newborn, 60%-90% 1%-2%
COMPLETE HEMOGLOBIN MOLECULE gamma Adult

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 31

 A1 2 alpha, 2 Newborn, 10%-40% >95% o Anemia
beta Adult
A2 2 alpha, 2 Newborn, <0.5% 3.5%
beta Adult

HEMOGLOBIN OXYGEN DISSOCIATION CURVE

➢ The function of hemoglobin is to readily bind oxygen
molecules in the lungs
FACTORS AFFECTING HGB-O2 DISSOCIATIO CURVE
✓ High oxygen affinity to transport oxygen
➢ 2, 3-DPG
✓ Low oxygen affinity to efficiently unload oxygen to the
➢ Body Temperature
tissues
➢ Blood pH → Bohr effect
➢ During oxygenation, each of the four heme iron atoms in a
➢ Carbon dioxide → Haldane effect
hemoglobin molecule can reversibly bind one oxygen
➢ Fetal hemoglobin → Causes left shift
molecule
o If predominant, gamma chain cannot release
➢ Approximately 1.34 mL of oxygen is bound by each gram of
oxygen
hemoglobin (3.47 mg of iron)
➢ Abnormal hemoglobin variants → causes left shift
➢ The affinity of hemoglobin for oxygen relates to the partial
pressure of oxygen (PO2), often defined in terms of the
amount of oxygen needed to saturate 50% of hemoglobin,
called P50 value
✓ Normal value: 27 mm/Hg
✓ Shift to the left: <27 mm/Hg to saturate 50% of Hgb
✓ Shift to the right: >27 mm/Hg to saturate 50% of Hgb
➢ The relationship is described by the oxygen dissociation
curve of hemoglobin, which plots the percent oxygen
saturation of
hemoglobin versus
the PO2
➢ The curve is
sigmoidal, which
indicates low FACTORS SHIFT TO LEFT SHIFT TO RIGHT
hemoglobin affinity for (Low except pH) (Rise except pH)
oxygen at low oxygen pH Increased (basic) Decreased (acidic)
tension and high Temperature Decreased Increased
affinity for oxygen at high oxygen tension Carbon Decreased Increased (Lungs)
➢ SHIFT TO THE LEFT → Increased oxygen affinity to dioxide (tissues)
hemoglobin: OXYGEN IS NOT DELIVERED TO THE 2,3-DPG Decreased Increased
TISSUES ASSOCIATED Lowered Body High Body
➢ SHIFT TO THE RIGHT → Decreased oxygen affinity to CONDITIONS temperature temperature
hemoglobin: OXYGEN IS DELIVERED TO THE TISSUES Alkalosis Acidosis
o Hypoxia- as it needs oxygen to be released Blood transfusions Conditions that
Increased produce hypoxia
carboxyhemoglobi (high altitude,
n pulmonary
Methemoglobinem insufficiency,
ia congestive heart

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 32

 Presence of Hgb F failure, severe ✓ METHEMOGLOBIN
Some Hb variants anemia) ✓ SULFHEMOGLOBIN
with high affinity Some Hb variants ✓ CARBOXYHEMOGLOBIN
for oxygen with low affinity for
oxygen METHEMOGLOBIN (MetHb or Hi)
SHIFT TO THE LEFT: HGB LOVES OXYGEN: LOW EXCEPT ➢ formed by the reversible oxidation of heme iron to the ferric
pH state (Fe3+)
SHIFT TO THE RIGHT: RESISTS/ REJECTS OXYGEN : RISE ➢ small amount of methemoglobin is continuously formed by
EXCEPT pH oxidation of iron during normal oxygenation and
deoxygenation of hemoglobin (1% of total hemoglobin)
ROLE OF 2,3-DPG ➢ COLOR OF BLOOD: CHOCOLATE BROWN
➢ DEOXYHEMOGLOBIN ➢ Cannot carry oxygen because oxidized ferric iron cannot
✓ Deoxygenated or bind it; an increase in methemoglobin level results in
Tense state decreased delivery of oxygen to the tissues
✓ Binding of 2,3-BPG ➢ TOXIC LEVELS:
between the beta- ✓ <25% - asymptomatic
globin chains; the ✓ >30% - cyanosis and symptoms of hypoxia
formation of salt ✓ 50% - coma or death
bridges between the ➢ is assayed by spectral absorption analysis instruments such
phosphates of 2,3-BPG and positively charged groups on as the CO-oximeter (absorption peak at 630 nm)
the globin chains further stabilizes the tetramer in the T ➢ METHEMOGLOBINEMIA
conformation ✓ Increased in methemoglobin; can be acquired or
✓ The binding of 2,3-BPG shifts the oxygen dissociation hereditary
curve to the right, OXYGEN IS RELEASED TO THE
TISSUES ✓ Acquired
• Aka toxic methemoglobinemia; occurs in normal
➢ OXYHEMOGLOBIN individuals after exposure to an exogenous oxidant,
✓ Oxygenated or Relaxed state such as nitrites, primaquine, dapsone, or benzocaine
✓ As hemoglobin binds oxygen molecules, a change in • Treatment → removal of offending oxidant; >30% is
conformation of the hemoglobin tetramer occurs with a treated with methylene blue or exchange transfusion
change in hydrophobic interactions at the a1b2 contact ✓ Hereditary
point, a disruption of the salt bridges, and release of 2,3- ➢ Mutations in the gene (CYB5R3) → required for NADH-
BPG cytochrome b5 reductase 3
✓ A 15-degree rotation of the a1b1dimer, relative to the o Autosomal dominant
a2b2 dimer, occurs along the a1b2 contact point o Hemoglobin is called as Hemoglobin M → 30-50% of
✓ OXYGEN IS NOT RELEASED TO THE TISSUES total hemoglobin
o No effective treatment
DYSHEMOGLOBINS ➢ Cytochrome b5 reductase deficiency
➢ Hemoglobin variants: genetic change o Autosomal recessive
➢ Dysfunctional hemoglobin that are unable to transport o <50% of total hemoglobin
oxygen
➢ Form and may accumulate to toxic levels, after exposure to SULFHEMOGLOBIN (HgbS)
certain drugs or environmental chemicals or gasses., the ➢ Formed by the addition of a sulfur atom to the pyrrole ring
offending agent modifies the structure of the hemoglobin of heme; In vitro and in the presence of oxygen, hemoglobin
molecule, preventing it from binding oxygen reacts with hydrogen sulfide
➢ EXAMPLES:

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 33

➢ Produces an IRREVERSIBLE CHANGE in the polypeptide ✓ 20-30% - headache, dizziness, disorientation
chains of the hemoglobin molecule due to oxidant stress, ✓ >40% - coma, seizure, hypotension, cardiac arrhythmias,
and further change can result in denaturation and the pulmonary edema, and death
precipitation of hemoglobin as Heinz bodies ➢ CAUSES:
➢ Can combine with carbon monoxide to form ✓ Exhaust of automobiles
carboxysulfhemoglobin ✓ Tobacco smoke – in smokers, carboxyhemoglobin levels
➢ Sulfhemoglobin has a similar peak to methemoglobin (630 may be as high as 15% (higher hematocrit and
nm) on a spectral absorption instrument; cannot be polycythemia to compensate for hypoxia)
converted to cyanmethemoglobin ✓ Industrial pollutants (coal, coal gas, charcoal burning)
➢ COLOR OF BLOOD: greenish pigment (mauve-lavender in ➢ Diagnosis of carbon monoxide poisoning is made if the level
sulfhemoglobinemia) is >3% in nonsmokers and >10% in smokers
➢ Treatment:
➢ CAUSES: ✓ Removing the carbon monoxide source and
✓ Drugs (such as sulfanilamides, phenacetin, nitrites, and administration of 100% oxygen
phenylhydrazine) ✓ Use of hyperbaric oxygen therapy is controversial; it is
✓ Exposure to sulfur chemicals in industrial or primarily used to prevent neurologic and cognitive
environmental settings impairment after acute carbon monoxide exposure in
✓ Patients with severe constipation, in cases of bacteremia patients whose level exceeds 25%
due to Clostridium perfringens and Clostridium welchii,
and in a condition known enterogenous cyanosis
➢ Ineffective for oxygen transport, and patients with elevated
levels present with cyanosis
➢ Cannot be converted to normal Hb A; it persists for the life
of the cell
➢ Treatment consists of prevention by avoidance of the
offending agent

CARBOXYHEMOGLOBIN (HgbCO)
➢ Results from the combination of carbon monoxide
(CO) with heme iron
➢ Carbon monoxide has 210-240 more affinity of carbon
monoxide for hemoglobin compared to oxygen
➢ CO shifts the hemoglobin-oxygen dissociation curve to
the left (shift to the left) further increasing its affinity
and severely impairing release of oxygen to the
tissues
➢ Some carboxyhemoglobin is produced endogenously,
but it normally comprises less than 2% of total
➢ hemoglobin
➢ Has been termed the silent killer because it is an
odorless and colorless gas, and victims may quickly
➢ become hypoxic
➢ May be detected by spectral absorption instruments at
540 nm
➢ COLOR OF BLOOD: CHERRY RED
➢ TOXIC LEVELS:

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 34

 ERYTHTOCYTE ABNORMALITIES
Rmt2023
HEMATOLOGY LECTURE WEEK 9

NORMAL ERYTHROCYTE MEAN CELL/CORPUSCULAR HEMOGLOBIN (MCH)

➢ Shape: Biconcave shape (discocyte) ➢ measures the weight/mass of hemoglobin in a single RBC
➢ Color: Light to red pink on wright’s stain (salmon pink) ➢ Reported in picograms (pg)
➢ Size: 6-8 um in diameter (normocytic); approximately 7.2 um ➢ Reference range: 26-34 pg (normal)
(normal MCV) ✓ <26 pg: decreased
➢ Hemoglobin content: Correlates with pallor area/central pallor ✓ >34 pg: increased
occupying 1/3 of the RBC (normochromic → normal MCHC)
➢ IN VITRO (slides): flattened, round appearance with a central
pallor
MEAN CELL/CORPUSCULAR HEMOGLOBIN
➢ NO PRESENCE OF INCLUSIONS
CONCENTRATION (MCHC)
➢ measures Hgb concentration in a single RBC; staining capacity
of RBC’s and amount of central pallor (g/dL)
➢ Reference range: 31-37 g/dL (normochromic)
✓ <31 g/dL: hypochromic

✓ >37 g/dL: hyperchromic (de facto) → not reported

MCHC: B muna bago C(hemoglobin/hematocrit)(100)

RBC INDICES
➢ Measures parameters of a single RBC; useful in the
assessment of anemia

MEAN CELL/CORPUSCULAR VOLUME (MCV)

➢ Measures the volume/size of a single RBC
➢ Reported in femtoliters (fL)
➢ Reference range: 80-100 fL (normocytic) RED CELL DISTRIBUTION WIDTH
✓ Microcytic: <80 fL ➢ based on the standard deviation of RBC volume/size and
✓ Macrocytic: >100 fL is routinely reported by
➢ automated blood cell analyzers
➢ Measures the degree of anisocytosis
ABNORMAL ERYTHROCYTES
➢ VARIATIONS ARE EXAMINED BASED ON:
➢ Size→anisocytosis
➢ Shape→ poikilocytosis
➢ Color or hemoglobinization
➢ Presence of inclusions
➢ Alteration in the distribution of cells on a blood smear→
check in an area where cells do not overlap

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 35

 ✓ Normochromic: central pallor/pallor area occupies 1/3 of the
cell
VARIATION IN CELL SIZE ✓ Hypochromic: central pallor/pallor area is >1/3 of cell;
➢ Normocyte: 6-8um in diameter (MCV = 80-100fL) decreased hemoglobin concentration
➢ Macrocyte: >8um in diameter (MCV = >100fL) ✓ Hyperchromic: central pallor/pallor area is <1/3 of the cell;
✓ result of a defect in either nuclear maturation or stimulated increased hemoglobin concentration
erythropoiesis; vitamin B12 or folate (B9) deficiency→ ➢ Anisochromia: variation in the normal coloration; presence of
Megaloblastic anemia both hypochromic and normochromic cells (dimorphic anemia
➢ Microcyte: <6um in diameter (MCV = <80fL)
✓ Decreased hgb synthesis (iron deficiency, impaired globulin
synthesis, defect in heme synthesis)
➢ Anisocytosis: abnormal/increased variation in cell size
➢ Red Cell Distribution Width (RDW)
✓ variation in red cell population size or diameter (degree of
anisocytosis)
✓ Helps to determine the cause of an anemia
seen in sideroblastic anemia, iron therapy for IDA, and
✓ Normal range: 11.5 – 14.5%
transfusion)
• Increased: heterogenous cell size
➢ Polychromatophilia or Polychromasia
o RBC has different sizes
✓ blue-gray coloration indicates immature erythrocytes
• Decreased: homogenous cell size (normal)
(reticulocytes); is a combination of the affinity of Hb for acid
stains and the affinity of RNA for basic stains
MICROCYTIC HYPOCHROMIC RBC

POLYCHROMASIA GRADING
MACROCYTES/MACROCYTIC RBC
Slight 1%
1+ 3%
2+ 5%
3+ 10%
4+ >11%

ANISOCYTOSIS AND DIMORPHIC ANEMIA

VARIATION IN HEMOGLOBIN CONTENT

➢ normal erythrocyte has a moderately pinkish-red appearance
(salmon pink) with a lighter-colored center (central pallor) when
stained with blood stains
➢ Depth of staining serves as a rough guide to the amount of
hemoglobin in red cells

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 36

 VARIATION IN SHAPE
➢ Poikilocytosis: variation in cell shape
➢ Greek origin is used to identify poikilocytes → abnormal shaped POIKILOCYTES CAUSED BY MEMBRANE ABNORMALITIES
RBC’s ➢ deficiency or dysfunction in any one of these membrane
➢ CAUSED BY: proteins can weaken or destabilize the membrane, ultimately
✓ Developmental macrocytosis resulting in reduced life span leading to hemolysis
✓ RBC Membrane abnormalities → intrinsic defect ➢ Abnormal lipid membrane concentration, osmotic gradient
✓ Abnormal hemoglobin content imbalance, and hemoglobin concentration can also destabilize
✓ Trauma/Physical damage → extrinsic defect (coming from the the membrane
outside) ➢ Spherocytes, Elliptocytes And Ovalocytes,
Echinocytes/Burr Cells crenated RBC, Pyknocytes,
Acanthocytes/Spur Cells/Thorn Cells, Stomatocytes,
Target Cells/Target Cells/Mexican Hat Cell

SPHEROCYTES
➢ SMALL, ROUND, RBC with no central pallor (increased
MCHC); may appear as artifacts if a slide is examined at the
thin end of a normal blood smear→ perfectly round
➢ Hereditary spherocytosis → Deficiency in either akyrin, band 3,
Spectrin and Protein 4.2 (horizontal interaction)

➢ Laboratory findings:
✓ MCHC >37g/dL; can be microcytic RBCs (microcytic and
hyperchromic)
✓ Increased OFT
✓ Decreased surface area: volume ratio
➢ CLINICAL SIGNIFICANCE:
✓ HEREDITARY SPHEROCYTOSIS
✓ Pre & Post splenectomy→ spleen cannot fix the rbc
POIKILOCYTES CAUSED BY DEVELOPMENTAL ✓ HDN→ Hemolytic disease of the newborn
MACROCYTOSIS ✓ WAIHA→ Warm autoimmune hemolytic anemia
➢ Macrocytes: large erythrocytes, mostly oval in shape (MCV= ✓ MAHA→ Microangiopathic hemolytic anemia
>100 fL) ✓ Severe burns or thermal injury
➢ Seen in patients with Megaloblastic ✓ Jaundice
anemia (macroovalocytes, oval
macrocytes)

ELLIPTOCYTES
➢ Have a cigar, elliptical, pencil, egg shape
➢ Hemoglobin is concentrated at the two ends of the cell with
normal pallor area

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 37

➢ Hereditary elliptocytosis → Due to deficiency in either Spectrin
and/or Protein 4.1 (vertical interaction)
➢ CLINICAL FINDINGS:
✓ HEREDITARY ELLIPTOCYTOSIS
✓ Iron deficiency anemia
o Pencil shape RBC→ Common in IDA (Iron deficiency
anemia)
✓ Thalassemia major ➢ CLINICAL SIGNIFICANCE:
✓ Sickle cell anemia ✓ Acute, severe hemolytic anemia
✓ Pernicious anemia ✓ Glucose-6-phosphate dehydrogenase (G6PD) deficiency
✓ Myelofibosis ✓ Hereditary lipoprotein deficiency
➢ LEACH PHENOTYPE→ deficiency in Gerbich Antigens
(associated with elliptocytosis)
OVALOCYTES
➢ Seen in cases of MEGALOBLASTIC ANEMIA (Oval
macrocytes), IDA and Myelopthisic anemia (cancer)
➢ Southeast Asian ovalocytes
ECHINOCYTES/CRENATED RBC/BURR CELLS
➢ Echinocytes: evenly distributed blunt serrated edges/short
projections
➢ Burr cells: not evenly distributed blunt serrated edges/short ARTIFACTS
projections
➢ CAUSES:
✓ IN-VIVO: Due to plasma abnormalities, osmotic changes
✓ IN-VITRO: prolonged standing of blood film with AC, moist
slide, and stored blood
o Stored blood causes depletion of ATP (also
associated with PK deficiency)
✓ Can be an artifact
o Artifact-no disease
➢ CLINICAL SIGNIFICANCE: ACANTHOCYTES/SPUR CELLS/THORN CELLS
✓ Renal insufficiency ➢ Contains uneven spaced, pointed projections without central
✓ Liver disease pallor
✓ Uremia ➢ Fewer projections than echinocytes
✓ Pyruvate kinase deficiency ➢ Due to the changes in the ratio of plasma lipids; decreased
plasma and RBC membrane lipids (lecithin and sphingomyelin)
o Alteration in lipids→ acanthocytes
➢ CLINICAL SIGNIFICANCE:
✓ ABETALIPOPROTENEMIA
✓ Alcoholic cirrhosis w/HA
✓ Malabsorption states
✓ Post splenectomy
✓ Hepatitis of newborn
PYKNOCYTES
✓ Pyruvate kinase deficiency
➢ distorted, contracted erythrocytes that are similar to burr cells
✓ Lipid metabolism disorders
➢ may be seen in small numbers during the first 2 to 3 months of
life as infantile pyknocytes

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 38

✓ Mcleod phenotype→ absence of Kx gene; absence of Kell ➢ Due to increased membrane surface after loading of the
antigens membrane with cholesterol and phospholipids
(phosphatidylcholine)
➢ In some instances, such as abnormal hemoglobins, the defect
is related to a maldistribution of hemoglobin
➢ LEPTOCYTE: THINNER VARIANT OF A TARGET CELL
➢ CLINICAL SIGNIFICANCE:
✓ Hemoglobinopathies (Hb C disease, S-C and S-S disease,
sickle cell thalassemia, and thalassemia)
✓ Hemolytic anemias
✓ Hepatic disease with or without jaundice
STOMATOCYTES
✓ Iron deficiency anemia
➢ Characterized by elongated or slit like pallor area instead of
✓ Post-Splenectomy
circular pallor (mouth like central pallor)
➢ Stoma Greek word means mouth
➢ Can appear as an artifact in blood film
➢ Overhydrated stomatocytosis: increased membrane
permeability to sodium and potassium leading to water influx
➢ Overhydrated is related to RHAG protein deficiency
associated with Rh (RH NULL DISEASE)
➢ Dehydrated stomatocytosis: increased membrane permeability
to potassium leading to loss of water from cell
POIKILOCYTES CAUSED BY TRAUMA OR
➢ Piezo type→ increased intracellular potassium
PHYSICAL DAMAGE
➢ Can be seen with puddled hemoglobin at the periphery of cells
➢ RBC may fragment and lyse when subjected to excessive
with spicules
physical trauma in cardiovascular system (CVS)
➢ CLINICAL SIGNIFICANCE:
➢ Schistocyte/Schizocytes, Keratocytes/Helmet Cells/Horn Cells,
✓ RH NULL DISEASE
Blister Cell, Knizocyte, Microspherocytes/Pyropoikilocytes,
✓ Acute alcoholism, alcoholic cirrhosis
Dacryocyte, Semilunar Bodies
✓ Glutathione deficiency
✓ Hereditary spherocytosis
SCHISTOCYTE/SCHIZOCYTE/FRAGMENTED RBC
✓ Infectious mononucleosis
➢ Characterized by cleaved, fragmented RBC
✓ Lead poisoning
➢ fragments of erythrocytes that are small and irregularly shaped;
✓ Malignancies
these cells are produced as the result of the breaking apart of
✓ Thalassemia minor
an erythrocyte, the schistocyte is about half the size of a normal
✓ Transiently accompanying hemolytic anemia
erythrocyte and may have a deeper red appearance
➢ Includes helmet, triangular, variety of small, irregular shapes
with few pointed extremities
➢ FRAGMENTATION IS
CAUSED BY:
✓ Altered vessel walls
✓ Presence of fibrin
TARGET CELLS/ CODOCYTES/MEXICAN HAT CELL (CLOTS)
➢ target cells are in one portion of the film- artifact (fixed with ✓ Prosthetic heart
methanol to avoid) valves
➢ Central area of hemoglobin surrounded by colorless ring and a ✓ Renal transplant
peripheral ring of hemoglobin, resembles a target rejections

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 39

➢ CLINICAL SIGNIFICANCE:
✓ Severe burns
✓ Glomerulonephritis KNIZOCYTE
✓ MAHA ➢ Resemble a pinched bottle;
✓ TTP→ Thrombotic thrombocytopenic purpura can be triangular in shape
✓ DIC→ Disseminated intravascular coagulation ➢ This abnormality is associated
with hemolytic anemias,
KERATOCYTES/HELMET CELLS including hereditary
➢ Type of schistocyte with one or more hornlike projections, spherocytosis
hollow interior portion of cell; resembling helmet or horn ➢ RBC has 2 pallor area
➢ larger scooped out part of the cell that remains after the
rupturing of a blister cell and are formed as a result of the MICROSPHEROCYTES/PYROPOIKILOCYTES
physical process of fragmentation ➢ Disk shaped cells with smaller volume
➢ Formed in the spleen and intravascular fibrin clots ➢ Due to thermal damage to cell membrane
protein spectrin, no pallor area
➢ CLINICAL SIGNIFICANCE: ➢ Laboratory:
✓ Severe burns ✓ 2-3um in diameter
✓ Glomerulonephritis ✓ MCV = <60fL
✓ MAHA
✓ TTP→ Thrombotic thrombocytopenic purpura ➢ CLINICAL SIGNIFICANCE:
✓ DIC→ Disseminated intravascular coagulation ✓ Severe burns
✓ Hereditary pyropoikilocytosis →
Fragmented cells increases when
heated in vitro at 45C (normal
fragmentation: 49C)

DACRYOCYTE
➢ Tear drop, pear drop shaped with blunt pointed projection
➢ Due to squeezing of red cells through small openings or splenic
BLISTER CELLS sinuses and remains behind
➢ RBC containing one or more vacuoles that resemble a blister ➢ CLINICAL SIGNIFICANCE:
on the skin ✓ Myeloid metaplasia→problems in the spleen
➢ Has a significantly thinned area at the periphery or outer border ✓ Primary myelofibrosis
of the cell membrane ✓ Myelopthisic anemia
➢ The vacuoles may rupture. If rupturing does occur, distorted ✓ Pernicious anemia
cells (keratocytes) and cell ✓ Beta thalassemia
fragments (schistocytes) are ✓ Tuberculosis
produced ✓ Heinz body formation
➢ CLINICAL SIGNIFICANCE:
✓ Pulmonary emboli in sickle
cell anemia
✓ Microangiopathic hemolytic anemia
✓ Heinz body hemolytic anemia

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 40

 SEMILUNAR BODIES ➢ CLINICAL SIGNIFICANCE: HGB CC DISEASE
➢ Half-moon cell, half crescent cell
➢ Large, pale pink staining ghost of HEMOGLOBIN SC CRYSTALS
red cell ➢ Dark-hued crystals of condensed
➢ Causing overt hemolysis hemoglobin
➢ Associated with malaria ➢ Crystals appear straight with parallel
sides and one blunt, point, protruding
end, fingerlike
➢ WASHINGTON’S MONUMENT
POIKILOCYTES CAUSED BY ABNORMAL APPEARANCE
HEMOGLOBIN CONTENT ➢ Confirmed by hemoglobin electrophoresis
➢ Sickle Cells/Drepanocytes/Menisocytes, Hemoglobin CC ➢ CLINICAL SIGNIFICANCE: HGB SC DISEASE
Crystals, Hemoglobin SC Crystals
SICKLE CELLS/DREPANOCYTES/MENISOCYTES ERYTHROCYTE INCLUSIONS
➢ Long, rod, crescent shaped, with thin and elongated with DEVELOPMENTAL ORGANELLES
pointed ends; holly-leaf like app ➢ Howell-Jolly bodies
➢ Contains polymers of abnormal hemoglobin ➢ Basophilic stippling/Punctuate basophilia
➢ Fully hemoglobinized, no pallor area ➢ Pappenheimer bodies
➢ Polymerization of Hb S is influenced by lowered oxygen levels ➢ Ringed sideroblast
and decreased blood pH as well as influx of sodium ions and ➢ Diffuse basophilia
increased intracellular calcium ➢ Reticulocytes in Supravital stains
➢ Can be due to amino acid substitution resulting to cell ➢ Cabot rings
membrane alterations ABNORMAL HEMOGLOBIN PRECIPITATION
➢ Heinz bodies
➢ CLINICAL SIGNIFICANCE: ➢ Hemoglobin H inclusions
✓ SICKLE CELL ANEMIA/Hgb S PROTOZOAN PARASITES
✓ Hgb SC disorder ➢ Plasmodium species
✓ Hgb C-Harlem disorder ➢ Babesia microti
✓ Hgb S-Memphis disorder ➢ Leishmania species
DEVELOPMENTAL ORGANELLES
HOWELL-JOLLY BODIES
➢ Small, 1-2 um in size; nuclear fragments of DNA
➢ Normally pitted by splenic macrophages and are not seen in
normal RBCs
➢ Develop in periods of accelerated or abnormal erythropoiesis
because the spleen can’t keep up with pitting of the remnants
➢ Reddish blue; Dark blue-purple with Wright’s stain
HEMOGLOBIN CC CRYSTALS
➢ Positive in Feulgen reaction (DNA stain)
➢ Homogeneous:
➢ CLINICAL SIGNIFICANCE:
hexagonal with blunt
✓ Post splenectomy
ends; BAR OF GOLD
✓ Congenital absence of spleen
APPEARANCE
✓ Splenic atrophy
➢ Leaves the hemoglobin
✓ Sickle cell anemia
free and colorless
✓ Alcoholism
➢ Cell membrane is not
✓ Megaloblastic anemia
usually visible
➢ Confirmed with hemoglobin electrophoresis

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 41

 BASOPHILIC STIPPLING/PUNCTUATE BASOPHILIA
➢ Multiple, tiny, fine or coarse rRNA inclusions (precipitated RNA)
➢ rRNA inclusions aggregates in drying & staining
➢ Hemoglobin appears homogeneous
➢ Blueberry gel appearance
➢ Dark blue to purple with Wright’s stain
➢ CLINICAL SIGNIFICANCE:
✓ Megaloblastic anemia
✓ Alcoholism RINGED SIDEROBLAST
✓ Thalassemia ➢ Numerous siderotic granules
✓ Lead & arsenic toxicity found within mitochondria
✓ Pyrimidine-5’-nucleotidase deficiency and forms a ring around the
nucleus
➢ CLINICAL SIGNIFICANCE:
Sideroblastic anemia

CABOT RINGS
➢ Thin ring like structure, circular and
may form figure of eight, incomplete
PAPPENHEIMER BODIES/SIDEROTIC GRANULES rings
➢ Aggregates of mitochondria, ribosomes, and IRON ➢ Represent remnants of
PARTICLES microtubules from the mitotic
➢ Appears in the periphery of the erythrocytes spindle
➢ Small, irregular, light blue to purple staining IRON GRANULES ➢ Stains reddish violet in Wright’s
with Wright’s stain stain
➢ SIDEROTIC GRANULES: iron (ferritin) aggregates ➢ Associated with Howell jolly bodies
➢ Pers Prussian blue → iron stain in the same RBC
➢ Siderocyte: RBC with siderotic granules ➢ CLINICAL SIGNIFICANCE:
➢ Sideroblast: Normoblast with siderotic granules ✓ Megaloblastic anemia
✓ Myelodysplastic syndromes
➢ CLINICAL SIGNIFICANCE: DIFFUSE BASOPHILIA
✓ Sideroblastic anemia ➢ Young cells with no nucleus but contains RNA remnants
✓ Hemoglobinopathies ➢ WRIGHT’S STAIN: Bluish tinge throughout cytoplasm also
✓ Thalassemias called polychromasia/polychromatophilia (cannot see the
✓ Megaloblastic anemia remnants)
✓ Myelodysplastic syndromes
✓ Hyposplenism, Post-splenectomy

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 42

➢ SUPRAVITAL STAINS: Dark
blue RNA remnants in th
cytoplasm (called
reticulocytes)
➢ Supravital stain:
✓ New Methylene blue → CLSI
recommended
✓ Brilliant Cresyl blue
✓ Crystal violet → for Heinz bodies preparation
➢ CLINICAL SIGNIFICANCE: (Increased reticulocytes)
✓ Increased RBC production
✓ Increased EPO stimulation of the marrow
ABNORMAL HEMOGLOBIN CONCENTRATION PROTOZOAN PARASITES
HEINZ BODIES ➢ Plasmodium species
➢ Precipitated/denatured globin due to ✓ Causative agent of Malaria; Vector: Anopheles mosquito
destruction of Hgb ✓ Rings, trophozoite, schizonts, gametocytes
➢ 0.2 to 2.0 um in size, that can be seen ✓ Includes: Plasmodium vivax, P. falciparum, P. malariae, P.
with a stain such as crystal violet or ovale, and P. knowlesi
brilliant cresyl blue ➢ Babesiosis
➢ Pitted golf ball appearance ✓ Causative agent is called Babesia microti (Maltese-cross
➢ BITE CELL/DEGMACYTE → RBC with pitted Heinz bodies
➢ NOT VISIBLE ON WRIGHT’S STAIN

HEMOGLOBIN H INCLUSIONS
➢ Small, dark blue or greenish
granules; Precipitated beta-globin
chains of hemoglobin
appearance)
➢ Failure of synthesizing three alpha
➢ Leishmania species → hemoflagellates; found in mononuclear
globin chains (Hgb H disease)
cells
➢ Results to unstable, easily oxidized
and easily precipitated hemoglobin
➢ Can be mistake as reticulocytes
✓ Retics: granulofilamentous
pattern
✓ Hb H inclusions: single body
➢ Seen after 4 drops of blood
incubated with 0.5ml 1% BCB for 20 minutes at 37C
➢ CLINICAL SIGNIFICANCE: HEMOGLOBIN H DISEASE

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 43

 ➢ Can be an artifact (thicker parts)
➢ True rouleaux formation
(pathologic rouleaux) is
determine in the thin parts
of blood films
➢ CLINICAL
SIGNIFICANCE:
✓ Hyperproteinemia
✓ Plasma cell dyscrasias
• Multiple Myeloma
• Waldenstrom’s Macroglobulinemia
✓ Increased fibrinogen (Hyperfibrinogenemia)
AGGLUTINATION
➢ Erythrocytes are exposed to antibodies
➢ AUTOAGGLUTINATION: red cells agglutination in his own
serum/plasma with no known agglutinins
➢ Cold agglutinin disease (CAD):
✓ RBCs agglutinates <25C: specimen should be warmed before
testing to correct the
false decrease of
hematocrit and false
increase of MCHC
✓ Associated with IgM
antibodies and
complement proteins
✓ IgM-malamig; IgM cold
reacting antibody
✓ IgG-grabe ang init- Warm reacting antibody
➢ CLINICAL SIGNIFICANCE:
✓ Primary Atypical pneumonia (PAP)→ Mycoplasma
pneumoniae infection
✓ Staphylococcal infections
✓ Trypanosomiasis
✓ Cold agglutinin disease

ABNORMAL ERYTHROCYTE DISTRIBUTION

ROULEAUX FORMATION
➢ Appears short or long stacks of coins (stacking or RBC)
➢ IN-VIVO → Due to abnormal or increased plasma proteins
(decreases zeta potential); associated with increased ESR
➢ IN-VITRO → thick moist blood film under coverslip

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 44

BLOOD SMEAR GRADING OF POIKILOCYTES, INCLUSION
AND ROULEAUX
MORPHOLOGY GRADING
Polychromatophilic RBC 1+= 1 to 5/field
Keratocytes/Helmet cells 2+= 6 to 10/field
Dacryocytes/Teardrop RBC 3+= >10/field
Acanthocytes
Schistocytes/Schizocytes
Spherocytes

Poikilocytes 1+= 3 to 10/field

Ovalocytes 2+= 11 to 20/field
Elliptocytes 3+= >20/field
Echinocytes/Burr cells
Bizarre-shaped RBC
Codocytes/Target cells
Stomatocytes
Rouleaux 1+= aggregates of 3 to 4 RBC
2+= aggregates of 5 to 10 RBC
3+= numerous aggregates with
only few free RBC

Sickle cells Graded as positive only

Basophilic stippling
Pappenheimer bodies
Howell-Jolly bodies

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 45

 Introduction and classification of
Rmt2023

anemia
HEMATOLOGY LECTURE WEEK 10

ANEMIA PHYSIOLOGIC ADAPTATIONS TO ANEMIA

➢ ETIOLOGY: Derived from the Greek word anaimia, MECHANISMS OF ANEMIA
meaning “without blood”
➢ Decrease in hemoglobin concentration or number of
RBCs results in decreased oxygen delivery to tissue,
resulting in tissue hypoxia
o Oxygen binds to iron in hgb
➢ Defined operationally as reduction in the hemoglobin
content of blood that can be caused by a decrease in the
RBC count, hemoglobin concentration, and hematocrit
below the reference interval for healthy individuals
➢ Anemia should not be thought of as a disease but rather as
manifestation of an underlying condition or deficiency
➢ Rbc count- number of rbc in a specific amount of blood
➢ Rbc mass: number of rbc in whole blood

➢ RELATIVE ANEMIA:
✓ RBC mass is normal; increased plasma volume
✓ Secondary to an unrelated condition and can be transient INEFFECTIVE ERYTHROPOIESIS
in nature causes include conditions that result in ➢ Refers to the defective production of erythroid precursor
hemodilution, such as pregnancy and volume cells
overload ➢ These defective precursors often undergo apoptosis
✓ Reticulocyte count normal; normocytic/normochromic (programmed cell death) in the bone marrow before they
anemia have a chance to mature to the reticulocyte stage and be
released into the peripheral circulation
➢ ABSOLUTE ANEMIA: ➢ Peripheral blood hemoglobin concentration is low, which
✓ RBC mass is decreased, but plasma volume is normal triggers an increase in erythropoietin production leading
✓ This is indicative of a true decrease in erythrocytes and to increased erythropoietic activity
hemoglobin ➢ Although the RBC production rate is high, it is ineffective
in that many of the defective erythroid precursors
IMPORTANCE OF PATIENT HISTORY undergo destruction in the bone marrow. The end result is
AND CLINICAL FINDINGS a decreased number of circulating RBCs resulting in
➢ To elucidate the reason for a patient’s anemia, one starts anemia
by obtaining a detailed history
➢ ASSOCIATED WITH:
➢ PHYSICAL EXAMINATION OF PATIENTS WITH ✓ Megaloblastic anemia → impaired DNA synthesis due to
ANEMIA: Vitamin B12 or folate (B9) deficiency
✓ Skin (for petechiae) ✓ Thalassemia → deficient globin chain synthesis
✓ Eyes (for pallor, jaundice, and hemorrhage) ✓ Sideroblastic anemia → deficient protoporphyrin
✓ Mouth (for mucosal bleeding) synthesis
✓ The examination should also search for sternal
tenderness, lymphadenopathy, cardiac murmurs or INSUFFICIENT ERYTHROPOEISIS
arrhythmias, splenomegaly, and hepatomegaly ➢ Refers to a decrease in the number of erythroid
✓ Jaundice is important for the assessment of anemia, precursors in the bone marrow, resulting in decreased
because it may be due to increased RBC destruction, RBC production and anemia
which suggests a hemolytic component to the anemia
o Hemoglobin is converted to bilirubin→ ➢ ASSOCIATED WITH:
hemolytic anemia ✓ Iron deficiency → inadequate intake, malabsorption,
✓ Measuring vital signs is also a crucial component of the excessive loss from chronic bleeding
physical evaluation ✓ EPO deficiency → renal disease
✓ Loss of erythroid precursors → aplastic anemia, pure
➢ CLASSIC SYMPTOMS red cell aplasia, Parvovirus B19
✓ Fatigue and shortness of breath ✓ Infiltration of bone marrow space with leukemic cells
and non-hematopoietic cells → metastatic tumors,
➢ MODERATE ANEMIA (Hgb 7-10 g/dL) granulomas, fibrosis, myelophthisic anemia (cancer)
✓ pallor of conjunctivae and nail beds, dyspnea, vertigo,
headache, muscle weakness, and lethargy can occur BLOOD LOSS AND HEMOLYSIS
➢ Anemia can also develop as a result of acute blood loss
➢ SEVERE ANEMIA (Hgb <7 g/dL) or chronic blood loss
✓ Symptoms of moderate anemia plus tachycardia,
hypotension, and other symptoms of volume loss ➢ ACUTE
✓ Characterized by sudden loss of blood resulting from
trauma or other severe forms of injury

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 46

➢ CHRONIC ✓ Detect occult blood and blood parasites
✓ Characterized by gradual, long-term loss of blood; often
caused by gastrointestinal bleeding
➢ Increased hemolysis results in a shortened RBC life span,
thus increasing the risk for anemia, may be due to: ➢ CHEMISTRY STUDIES
✓ INTRINSIC DEFECT → RBC membrane, enzyme ✓ Measure serum haptoglobin, lactate dehydrogenase, and
systems, hemoglobin unconjugated bilirubin (to detect excessive hemolysis);
✓ EXTRINSIC DEFECT → Immune (antibody mediated) or renal and hepatic function tests
non-immune (mechanical fragmentation or infection- ✓ Serum Vitamin B12 and folate assays for macrocytic
related destruction) anemia (megaloblastic anemia)
➢ DIRECT ANTIGLOBULIN TEST (DAT) → differentiate
LABORATORY DIAGNOSIS OF ANEMIA autoimmune anemias for other hemolytic anemia
➢ To detect the presence of anemia, the medical laboratory
professional performs a COMPLETE BLOOD COUNT CLASSIFICATION OF ANEMIA
(CBC) using an automated blood cell analyzer, the CBC MORPHOLOGIC: BASED ON MCV
includes RBC count, Hemoglobin and Hematocrit, RBC ➢ The MCV is an extremely important tool and is key in the
indices → MCV, MCH, MCHC (computed values), and RDW morphologic classification of anemia
(provided by blood cell analyzers), White blood cell count, ➢ Microcytic anemia characterized by an MCV of less than
Platelet count 80 fL with small RBCs (less than 6 um in diameter)
➢ RBC count is normal=hgb is defective=anemia ➢ Macrocytic anemias are characterized by an MCV greater
➢ RBC is low=hemolytic anemia (high retics) than 100 fL with large RBCs (greater than 8 um in diameter)
➢ Normocytic anemias are characterized by an MCV in the
RETICULOCYTE COUNT
➢ assess the bone marrow’s ability to increase RBC
production in response to an anemia (hemolytic anemia)
➢ Includes:
✓ Reticulocyte count
✓ Absolute reticulocyte count (ARC)
✓ Corrected reticulocyte count (CRC)
✓ Reticulocyte production index (RPI)
✓ Immature reticulocyte fraction (IRF)

PERIPHERAL BLOOD FILM EXAMINATION

➢ Assessment of RBC diameter, shape, color/amount of
hemoglobin, inclusions

BONE MARROW EXAMINATION

➢ indicated for a patient with an unexplained anemia
associated with or without other cytopenias, fever of
unknown origin, or suspected hematologic neoplasm
➢ Important findings in bone marrow that can point to the
underlying cause of the anemia include:
✓ Abnormal cellularity (e.g., hypocellularity in aplastic
anemia)
o Aplastic- no RBC precursors= BM shut down
✓ Evidence of ineffective erythropoiesis and megaloblastic
changes (e.g., folate/vitamin B12 deficiency or
myelodysplastic syndromes)
✓ Lack of iron-on-iron stains of bone marrow (the gold
standard for diagnosis of iron deficiency)
✓ Presence of granulomata, fibrosis, infectious agents, and
tumor cells that may be inhibiting normal erythropoiesis

OTHER LABORATORY TESTS

➢ IRON STUDIES → useful for proper diagnosis of microcytic,
hypochromic anemia
✓ Serum Iron→ measures serum iron
✓ Serum ferritin→ indicator of iron storage status
✓ Total iron-binding capacity (TIBC)→ measures the
ability of transferrin to bind iron
✓ Transferrin saturation→ serum iron/ TIBC x 100
✓ Free erythrocyte protoporphyrin (FEP)→ excess
porphyrins; helpful in differentiating thalassemia from IDA
and anemia of chronic disease; levels build up during iron
deficiency
➢ URINALYSIS
✓ detect hemoglobinuria or an increase in urobilinogen;
also, to detect hematuria and hemosiderin range of 80 to 100 fL (RBCs are 6-8 um in diameter)
➢ FECALYSIS

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 47

 MORPHOLOGIC: BASED ON RETIC COUNT
➢ The absolute reticulocyte count is useful in initially
classifying anemias into the categories of:
➢ Decreased or ineffective RBC production (decreased
reticulocyte count)
✓ When the reticulocyte count is decreased, the MCV can
further classify the anemia into three subgroups:
normocytic anemias, microcytic anemias, and macrocytic
anemias
➢ Excessive RBC loss (increased reticulocyte count)

MORPHOLOGIC: BASED ON RDW

➢ The RDW can help determine the cause of an anemia when
used in conjunction with the MCV
➢ Each of the three MCV categories mentioned previously
(normocytic, microcytic, macrocytic) can also be
subclassified by the RDW:
✓ Homogeneous (normal RDW)
✓ Heterogeneous (increased or high RDW)

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 48

 ✓ Chronic blood loss: gastrointestinal bleeding in males
and excessive menstrual bleeding in females, regular
blood donation (every 3 months) , chronic hemolysis
PATHOPHYSIOLOGIC CLASSIFICATIONS

STAGES OF IDA
➢ STAGE 1
✓ characterized by a progressive loss of storage iron
✓ Serum ferritin levels drop over time, which indicates the
decline in stored iron, and this also could be detected in
an iron stain of the bone marrow

➢ STAGE 2
✓ defined by the exhaustion of the storage pool of iron
and depleted iron transport

➢ STAGE 3
✓ Frank anemia
✓ Hemoglobin concentration and hematocrit are low
relative to the reference intervals; depletion of storage
iron and diminished levels of transport iron prevent
normal development of RBC precursors
✓ RBCs become microcytic hypochromic → because of
the restriction in Hgb synthesis

CLINICAL FINDINGS
➢ Fatigue, weakness, shortness of breath pallor
➢ Sore tongue (glossitis)
➢ Cracks at the corners of the mouth (angular cheilosis)
➢ Spooning or clubbing of the fingernails (Koilonychias)
➢ Pica/Pica syndrome → severe iron deficiency, neurologic
problems
✓ Perversion in the appetite; craving for non-food items
• Aka Patterson Kelly / Plummer-vinson syndrome

LABORATORY FINDINGS
MICROCYTIC HYPOCHROMIC ANEMIA ➢ Microcytic, hypochromic RBCs; blood smear shows
➢ IDA, sideroblastic anemia, Anemia of chronic ovalocytes/pencil forms
inflammation, porphyrias (associated with ➢ Decreased: Hgb, Hct, MCV, MCH, MCHC, RPI, reticulocyte
normocytic, normochromic RBC) count
➢ Microcytic= low hgb (hypochromic) ➢ Decreased: Transferrin saturation, serum ferritin, serum
➢ Defective heme synthesis: IDA, anemia of chrionic iron
inflammation ➢ Increased: TIBC, FEP, RDW
➢ Defective protoporphyrin: sideroblastic anemia,
thalassemia, porphyrias 1st pic: Koilonychias 2nd pic: Angular cheilosis
IRON DEFICIENCY ANEMIA (IDA)
➢ Most common type of anemia worldwide
CAUSES
✓ Inadequate intake: pure milk fed infants (red meat is rich
in iron) (iron is not supplemented by the body)
✓ Increased demand for iron supply: periods of rapid
growth, pregnancy
✓ Impaired absorption (Duodenum or jejunum): Chron’s
disease, celiac disease, matriptase-2 protein mutation,
disease that decrease stomach acidity, drugs

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 49

 ANEMIA OF CHRONIC DISEASE/INFLAMMATION
➢ Second common type of anemia
➢ Inability to use available iron for hemoglobin production
➢ Found in infections (Tuberculosis and HIV infections),
inflammatory (rheumatoid arthritis) or malignant disease
➢ Anemia of chronic disease is more correctly termed anemia
of chronic inflammation, because inflammation is the
unifying factor among the three mentioned general types of
conditions in which this anemia is seen

CAUSES
✓ IMPAIRED FERROKINETICS→ hepcidin
✓ DIMINISHED ERYTHROPOIESIS
✓ SHORTENED RBC LIFE SPAN

IMPAIRED FERROKINETICS LABORATORY FINDINGS

HEPCIDIN ➢ Microcytic, Hypochromic RBCs
➢ During inflammation, the liver increases the synthesis of ➢ Increased: FEP, Serum ferritin (iron is not released by
hepcidin in response to interleukin-6 produced by macrophages)
activated macrophages ➢ Decreased: Hemoglobin, Hematocrit, Serum iron, TIBC,
➢ The increase occurs regardless of systemic iron levels in Transferrin saturation
the body. As a result, during inflammation, there is a ➢ Prussian blue: shows stored iron in macrophages
decrease in iron absorption from the intestine and iron ➢ RPI: <2.0 (BONE MARROW)(ineffective/ insufficient
release from macrophages and hepatocytes erythropoiesis)
➢ Although there is plenty of iron in the body, it is
unavailable to developing RBCs because it is SIDEROBLASTIC ANEMIA
sequestered in the macrophages and hepatocytes ➢ Caused by blocks in the protoporphyrin pathway (no
➢ Hepcidin→ regulates iron release; high levels prevent protoporphyrin IX) resulting in defective hemoglobin
intestinal iron absorption synthesis and iron overload
LACTOFERRIN ➢ Excess iron accumulates in the mitochondrial region of the
➢ During infection and inflammation, neutrophil lactoferrin mature erythrocyte in circulation and these cells are called
is released into the blood and extracellular spaces with the as siderocytes; inclusions are called siderotic granules/
death of neutrophils Pappenheimer bodies
➢ When it is carrying iron, lactoferrin binds to macrophages ➢ Siderocytes are best demonstrated using Perl's
and liver cells that take up and salvage the iron Prussian blue stain (iron stain)
➢ Because of high hepcidin, however, the macrophages ➢ diverse group of diseases → hereditary/acquired conditions
and hepatocytes cannot export iron and it remains
sequestered away from erythroblasts; Erythroblasts cannot
acquire iron salvaged by lactoferrin directly because
they do not have lactoferrin receptors

DIMINISHED ERYTHROPOEISIS
➢ Production of inflammatory cytokines (such as tumor
necrosis factor-a and interleukin-1 from activated
macrophages and interferon-g from activated T cells) also
impairs proliferation of erythroid progenitor cells, diminishes
their response to erythropoietin, and decreases production
of erythropoietin by the kidney LABORATORY FINDINGS
SHORTENED LIFE SPAN ➢ Increased: Serum iron, Serum ferritin, Transferrin saturation
➢ Inflammation appears to induce increased production of ➢ Decreased: Hemoglobin, hematocrit, TIBC
hemophagocytic macrophages ➢ Inclusions: Basophilic stippling, ringed sideroblast,
➢ Although inflammatory suppression and shortened RBC life pappenheimer bodies
span contribute to the anemia of chronic inflammation, the ➢ Poikilocytosis with anisocytosis: dimorphic RBC
impaired ferrokinetics is the most significant cause of (dimorphism), target cells
the anemia
LEAD AND ARSENIC POISONING
➢ Lead interferes with porphyrin synthesis at several steps
including:
✓ The conversion of aminolevulinic acid (ALA) to
porphobilinogen (PBG) by ALA dehydratase (also called
PBG synthase); the result is the accumulation of
aminolevulinic acid
✓ The incorporation of iron into protoporphyrin IX by
ferrochelatase (heme synthase); the result is
accumulation of iron and protoporphyrin in the
mitochondria
➢ Anemia, when present in lead poisoning, is most often
normocytic and normochromic; however, with chronic

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 50

 exposure to lead, a microcytic, hypochromic blood PORPHYRIAS
picture may be seen ➢ The porphyrias are diseases characterized by impaired
➢ Seen mostly in children exposed to lead-based paints; In production of the porphyrin component of heme; the
the case of lead poisoning, calcium disodium edetate impairments to heme synthesis may be acquired, as with
(CaNa2EDTA) and/or dimercaprol are often used to lead poisoning, or hereditary
chelate the lead present in the body so it can be excreted in ➢ The term porphyria is most often used to refer to the
urine hereditary conditions that impair production of
protoporphyrin
CLINICAL SYMPTOMS ➢ Among the inherited disorders, single deficiencies of most
➢ Abdominal pain enzymes in the synthetic pathway for heme have been
➢ Muscle weakness identified an
➢ Gum lead line that forms from blue/black deposits of lead ➢ Lead poisoning: acquired sideroblastic anemia and
sulfate acquired porphyria

LABORATORY FINDINGS ➢ ASSOCIATED WITH NORMOCYTIC RBCs

➢ Increased: FEP, Transferrin saturation ✓ ACQUIRED PORPHYRIAS:
➢ Decreased: Hemoglobin, Hematocrit • Lead poisoning
➢ Normal: Serum ferritin, Serum iron, TIBC • Porphyria cutanea Tarda
➢ Inclusions: Basophilic stippling (Pyrimidine-5’-nucleotidase
deficiency)
MICROCYTIC LABORATORY FINDINGS
HYPOCHROMIC ANEMIA ✓ HEREDITARY PORPHYRIAS:
IRON DEFICIENCY ➢ Decreased: Hgb, Hct, MCV, • Acute intermittent porphyria
ANEMIA (IDA) MCH, MCHC, RPI, • Variegate porphyria
reticulocyte count • Hereditary coproporphyria
➢ Decreased: Transferrin • Porphyria cutanea tarda
saturation, serum ferritin, • Congenital erythropoietic Protoporphyria
serum iron • Erythropoietic protoporphyria
➢ Increased: TIBC, FEP, RDW
➢ When an enzyme in heme synthesis is missing, the
ANEMIA OF CHRONIC ➢ Increased: FEP, products from earlier stages in the pathway accumulate
DISEASE/INFLAMMATION Serum ferritin in cells that actively produce heme, such as erythrocytes
➢ Decreased: and hepatocytes
Hemoglobin, ➢ The excess porphyrins leak from the cells as they age or
Hematocrit, Serum die and may be excreted in urine or feces, which allows
iron, TIBC, Transferrin diagnosis (port wine color of urine)
saturation ➢ The accumulated products also deposit in body tissues;
➢ Prussian blue: shows some of the accumulated products are fluorescent. Their
stored iron in deposition in skin can lead to photosensitivity with
macrophages severe burns on exposure to sunlight
➢ RPI: <2.0 (BONE
MARROW)

SIDEROBLASTIC ANEMIA ➢ Increased: Serum iron,

Serum ferritin,
Transferrin saturation
➢ Decreased:
Hemoglobin, hematocrit,
TIBC
➢ Inclusions: Basophilic
stippling, ringed
sideroblast
➢ Poikilocytosis with
anisocytosis: dimorphic
RBC (dimorphism),
target cells

LEAD/ARSENIC ➢ Increased: FEP,

POISONING Transferrin saturation
➢ Decreased:
Hemoglobin, Hematocrit
➢ Normal: Serum ferritin,
Serum iron, TIBC
➢ Inclusions: Basophilic
stippling (Pyrimidine-
5’-nucleotidase
deficiency

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 51

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 52
 PORPHYRIAS BRIEF INFORMATION Inheritance Enzyme deficiency CLINICAL FINDINGS:
(NORMOCYTIC, NORMOCHROMIC
RBCs)
ACUTE INTERMITTENT PORPHYRIA (AIP) ➢ Most common acute and autosomal dominant PBG deaminase ✓ Increased urine ALA and PBG
probably the most common ✓ urine turns to dark red upon exposure to air
inherited porphyria and light (due to oxidation of PBG)
✓ abdominal pain, nausea and vomiting, mental
confusion, tachycardia, neuropathy

CONGENITAL ERYTHROPOIETIC ➢ distinct from the other Autosomal recessive Uroporphyrinogen III synthase ✓ Increased urine urophophyrin and
PORPHYRIA (CEP) porphyrias because of its coproporphyrin
inheritance ✓ Red pigmented urine, hemolytic anemia,
➢ Prognosis for CEP is severe cutaneous photosensitivity,
significantly worse than Erythrodontia
for the other porphyrias,
with death occurring at an
early age in many cases

PORPHYRIA CUTANEA TARDA (PCT) ➢ most common of the Type I (acquired), Types II and III Uroporphyrinogen decarboxylase (UROD) ✓ Type I: only hepatic UROD is decreased;
porphyrias in the United (autosomal dominant) erythrocyte UROD is normal
States ✓ Type II: both hepatic and erythrocyte UROD
➢ Has three types: Types I, II, is decreased
and III ✓ Type III: hepatic UROD decreased
✓ Increased urine uroporphyrin I and
uroporphyrin III
➢ Mild trauma to sun-exposed areas
➢ Exacerbating factors are alcohol and
estrogens
➢ patients with hemochromatosis (iron
overload) are at increased risk for acquired
PCT
HEREDITARY COPROPORPHYRIA (HCP) ➢ Less common than AIP autosomal dominant Coproporphyrinogen oxidase ✓ increased urine ALA and PBG
✓ increased urine and fecal
Coproporphyrinogen III
✓ resembles a milder form of AIP with its
neurovisceral attacks
✓ cutaneous manifestations are seen in
roughly a third of patients
✓ Red, fluorescent pigment in the urine

VARIEGATE PORPHYRIA (VP) ➢ sometimes referred to as autosomal dominant Protoporphyrinogen oxidase ✓ Increased urine ALA and PBG
South African porphyria ✓ Increased fecal and urine uroporphyrin and
(common in Caucasians in coproporphyrin
the region) ✓ Cutaneous manifestations in the form of
erosions or bullae following trauma (sun-
exposed skin)
✓ Red, fluorescent pigment in the urine

ERYTHROPOIETIC PROTOPORPHYRIA Autosomal recessive partially deficiency heme synthase; ferrochelatase (last ✓ Clinical symptoms begin in childhood,
(EPP) enzyme needed to produce heme) earlier than in other porphyrias
✓ painful, itching erythema occurs within a
short time (minutes) of sun exposure
✓ EPP has a normal urine profile, and RBC
testing must be performed to render a
diagnosis

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 53

 THALASSEMIA BETA THALASSEMIA MAJOR/COOLEY’S ANEMIA
➢ Reduced or absent synthesis of one or more of the globin ➢ Characterized by a severe anemia that requires regular
chains of hemoglobin (quantitative defect of globin transfusion therapy
chains) ➢ It is usually diagnosed between 6 months and 2 years of
➢ diverse group of inherited disorders caused by genetic age (after completion of the gamma to beta switch) when
mutations affecting the globin chain component of the the child’s Hb A level does not increase as expected
hemoglobin (Hb) tetramer ➢ Homozygous in nature; Markedly decreased rate of
➢ named according to the chain with reduced or absent synthesis or absence of both beta chains results in an
synthesis; mutations affecting the alpha or beta globin excess of alpha chains; no Hgb A can be produced;
gene are most clinically significant because Hb A (alpha compensate with up to 90% Hgb F
and beta) is the major adult hemoglobin ➢ Excess alpha chains precipitate on the RBC membrane,
➢ Severity varies from no clinical abnormalities to form HEINZ BODIES, and cause rigidity; destroyed in the
transfusion-dependent to fatal bone marrow or removed by the spleen
➢ PATHOPHYSIOLOGY: ➢ CLINICAL SYMPTOMS:
✓ Reduced or absent production of a particular globin ✓ Symptoms are usually manifested in early life after
chain, which diminishes hemoglobin synthesis and hemoglobin synthesis switches from gamma-chain to beta
produces microcytic, hypochromic RBCs chain synthesis several months after birth
✓ Unequal production of the alpha- or beta-globin chains, ✓ Infants fail to grow
causing an imbalance in the a/b chain ratio; this leads ✓ Splenomegaly is common
to a markedly decreased survival of RBCs and their ✓ Severe anemia → most outstanding feature of the
precursors; the alpha/beta chain imbalance is more disorder and is responsible for many related problems
significant and determines the clinical severity of the ✓ Patients are transfusion dependent → transfusion related
thalassemia complications (iron overload, alloimmunization, viral
➢ Inheritance of thalassemia is autosomal; whether it is infections)
autosomal dominant or recessive ➢ LABORATORY FINDINGS:
➢ Thalassemia major: Severe anemia; either no alpha or no ✓ Decreased hemoglobin (2-3 g/dL), hematocrit, and RBC
beta chains produced count; SEVERE MICROCYTIC HYPOCHROMIC RBCs
➢ Thalassemia minor/trait: Mild anemia; sufficient alpha ✓ Decreased MCV, MCH, MCHC
and beta chains produced to make normal hemoglobins A, ✓ Blood smears: reveal anisocytosis, poikilocytosis,
A2, and F, but may be in abnormal amounts hypochromia, target cells, teardrop RBCs, Basophilic
➢ Found in: Mediterranean (beta), Asian (alpha), and African stippling, Howell-Jolly bodies, Pappenheimer bodies,
(alpha and beta) populations Heinz bodies polychromatophilia, and few to many
➢ CATEGORIES: nucleated red cells
✓ Beta-Thalassemia ✓ Increased RDW and TIBC
✓ Alpha-Thalassemia ✓ Decreased OFT
✓ Hemoglobin electrophoresis: Increased Hgb F and
BETA-THALASSEMIA decreased Hgb A
➢ In beta-thalassemia, UNPAIRED, EXCESS ALPHA
CHAINS PRECIPITATE in developing erythroid precursors BETA-THALASSEMIA MINOR/TRAIT
forming inclusion bodies; this causes oxidative stress ➢ Results when one beta-globin gene is affected by a
and damage to cellular membranes mutation that decreases or abolishes its expression,
➢ This underproduction of beta chains contributes to a whereas the other beta-globin gene is normal
decrease in the total erythrocyte hemoglobin (heterozygous state)
production, INEFFECTIVE ERYTHROPOIESIS, and a ➢ LABORATORY FINDINGS:
chronic hemolytic process ✓ MILD MICROCYTIC HYPOCHROMIC RBCs
➢ Marrow macrophages destroy precipitate-filled erythrocytes ✓ Hemoglobin level can range from approximately 11 to 15
in the bone marrow, precipitate-filled circulating g/dL in affected men and 10 to 13 g/dL in affected women
erythrocytes are destroyed prematurely in the spleen; iron ✓ RBC count with a normal or slightly elevated RBC count;
accumulation in erythroid precursors target cells, basophilic stippling
TYPES OF BETA- ASSOCIATED FINDINGS: ✓ Hemoglobin electrophoresis: Hgb A is slightly decreased,
THALASSEMIA: but Hgb A2 is slightly increased to compensate
Beta-thalassemia silent No hematologic
carrier (Heterozygous abnormalities or clinical
state) symptoms
Beta-thalassemia minor Mild hemolytic anemia,
(heterozygous state) microcytic and hypochromic
RBCs, and no clinical
symptoms
Beta-thalassemia Mild to moderate hemolytic
intermedia anemia, microcytic and
hypochromic RBCs,
moderate clinical symptoms,
and non-transfusion
dependence
Beta-thalassemia Severe hemolytic anemia,
major/Cooley’s Anemia microcytic and
(homozygous or hypochromic RBCs, severe
compound heterozygous clinical symptoms, and
state) transfusion dependence

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 54

 ALPHA THALASSEMIA ascites, the fetus has gross hepatosplenomegaly and
➢ In contrast to beta-thalassemia, with most cases caused by cardiomegaly
point mutations, the major cause of alpha-thalassemia is IRON STUDIES FOR MACROCYTIC ANEMIA
deletions that remove one or both a-globulin genes from the
affected chromosome 16
➢ TYPES:
ALPHA- GENOTYPE: DELETED
THALASSEMIA: GENE:
Silent carrier (- α / α α) One alpha gene
deletion
Alpha-thalassemia Homo: (- α / - α) Two alpha gene
minor/trait Hetero: (- - / α α) deletion
Hgb H (- - / - α) Three alpha
disease/Alpha- gene deletion
thalassemia
intermedia
Alpha-thalassemia (--/--) Four alpha
major gene deletion

SILENT CARRIER
➢ One alpha gene is deleted; the three remaining a gene
direct the synthesis of an adequate number of alpha
chains for normal hemoglobin synthesis
➢ No clinical manifestation of hemoglobinopathy
➢ Hemoglobin electrophoresis, red blood cell measurements,
and peripheral blood smears are ESSENTIALLY NORMAL

ALPHA THALASSEMIA MINOR/TRAIT

➢ Two alpha genes is deleted; can be homozygous ( - α / -
α ) or heterozygous ( - - / α α )
➢ Asymptomatic and characterized by a mild microcytic
anemia with High RBC count and target cells
➢ Up to 6% Hgb Bart's (γ4) in newborns may be helpful in
diagnosis; absent by 3 months of age

HGB H DISEASE/ALPHA-THALASSEMIA INTERMEDIA

➢ Common in Southeast Asia
➢ Three alpha genes are deleted; decrease in alpha chains
leads to beta chain excess called (Hgb H → β4)
➢ In the newborn Hb Bart comprises 10% to 40% of the
hemoglobin, with the remainder being Hb F and Hb A
➢ Bone marrow exhibits erythroid hyperplasia, and the spleen
is usually enlarged
➢ LABORATORY FINDINGS:
✓ Characterized by a mild to moderate, chronic hemolytic
anemia
✓ Hemoglobin: 7 to 10 g/Dl
✓ Reticulocyte count: 3% to 10%
✓ Decreased MCV, MCH, MCHC
✓ Blood smears: MICROCYTIC HYPOCHROMIC RBCs,
marked poikilocytosis, target cells, bizarre cells, Hgb H
inclusions (stained by supravital stains)

ALPHA-THALASSEMIA MAJOR/HYDROPS FETALIS

➢ Homozygous Alpha Thalassemia
➢ All four alpha genes are deleted; no normal hemoglobins
are produced
➢ Usually results in death in utero or shortly after birth,
although a small number survive with aggressive
transfusion therapy, including intrauterine transfusions
➢ Without intrauterine transfusion, the fetus becomes
severely anemic, which leads to cardiac failure and
edema in fetal subcutaneous tissues (hydrops fetalis);
Hb Bart’s is the predominant hemoglobin, along with a
small amount of Hb Portland and traces of Hb H
➢ Fetus is delivered prematurely and is usually stillborn or
dies shortly after birth; In addition to anemia, edema, and

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 55

 MACROCYTIC ANEMIA ➢ IMPAIRED USE OF FOLATE
➢ Characterized by an MCV of >100fL with large RBCs (>8um) ✓ Drugs
➢ Arise from conditions that result in megaloblastic or non- ➢ EXCESSIVE LOSS OF FOLATE
megaloblastic red cell development in the bone marrow ✓ occurs through the kidney (dialysis patients)

➢ ASSOCIATED CONDITIONS:
✓ MEGALOBLASTIC ANEMIA → impaired DNA synthesis
leading to abnormal nuclear maturation
• Vitamin B12 (Cobalamin) deficiency
• Vitamin B9 (Folate) deficiency
• Myelodysplasia
• Erythroleukemia

✓ NON-MEGALOBLASTIC ANEMIA → due to disruption of

the cholesterol-to-phospholipid ratio
• Aplastic anemia
• Chronic liver disease
• Alcoholism

MEGALOBLASTIC ANEMIA
➢ Defective DNA synthesis causes abnormal nuclear
maturation; RNA synthesis is normal, so the cytoplasm is not
affected (megaloblastic: RBC precursors; macrocytic: mature
RBC in circulation)
VITAMIN B12 DEFICIENCY
➢ Associated with Vitamin B12 and folate deficiency (leads ➢ INADEQUATE INTAKE
to impaired thymidine production) ✓ Rare in strict vegetarians who do not eat meat, eggs, and
✓ Folate deficiency → preventing methylation of dUMP dairy products
(deoxyuridine monophosphate) ✓ Best sources are liver, dairy products, fish, shellfish, and
✓ Vitamin B12 deficiency → preventing production of THF eggs
from 5-methyl THF (5-methyltetrahydrofolate) ➢ INCREASED DEMAND
✓ Absorption of VIT B12 and B9 is absorbed in Ileum ✓ Pregnancy, lactation, and periods of growth
➢ IMPAIRED ABSORPTION
➢ PATHOPHYSIOLOGY: ✓ Failure to separate vitamin B12 from food proteins
✓ Thymidine deficiency leads to the production of non- ✓ Failure to separate vitamin B12 from haptocorrin
functional and impaired DNA replication ✓ Malabsorption
✓ Cell division is halted which leads to lysis or apoptosis ✓ Inherited disorders of vitamin B12 absorption and transport
of many erythroid precursors (ineffective ✓ Competition for vitamin B12
erythropoiesis= RBC precursors are destroyed) ✓ Lack of intrinsic factor (IF)
✓ Red blood cells have an abnormal nuclear maturation
and imbalance between nuclear and cytoplasmic IMPAIRED ABSORPTION
maturation; the pre-mitotic interval is prolonged; results in a ➢ Failure to separate vitamin B12 from food proteins
large nucleus, increased cytoplasmic RNA, and early ✓ Known as food-cobalamin malabsorption is
synthesis of hemoglobin (INEFFECTIVE characterized by hypochlorhydria (high pH in stomach)
ERYTHROPOIESIS) and the resulting inability of the body to release vitamin B12
✓ Also associated with impaired maturation of other myeloid from food or intestinal transport proteins for subsequent
cells; ASSOCIATED WITH PANCYTOPENIA binding to intrinsic factor
• Leukopenia with hypersegmented neutrophils
• Thrombocytopenia with giant platelets ➢ Failure to separate vitamin B12 from haptocorrin
(transporter)
VITAMIN B9 (FOLATE) DEFICIENCY ✓ Due to chronic pancreatitic disease; lack of gastric
➢ INADEQUATE INTAKE acidity or lack of trypsin
✓ synthesized by microorganisms and higher plants; good
sources are leafy green vegetables, dried beans, liver, beef, ➢ Malabsorption
fortified breakfast cereals, and some fruits, especially ✓ Same conditions interfering with folate absorption, such as
oranges celiac disease, tropical sprue, and inflammatory
➢ INCREASED DEMAND bowel disease
✓ During pregnancy and lactation; periods of growth

➢ IMPAIRED ABSORPTION ➢ Inherited errors of vitamin B12 absorption and transport

✓ Deficiency in folate transporter protein (PCFT) ✓ Imerslund-Gräsbeck syndrome and Transcobalamin
✓ Intestinal diseases deficiency
• Sprue/Tropical sprue and Celiac disease (gluten-induced
enteropathy) ➢ Competition for vitamin B12

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 56

✓ Fish tapeworm disease (Diphyllobothrium latum • Pronounced multinuclearity: up to 12 nuclei per cell
infections) • Affects 30% of RBC precursors, gigantoblasts: (50 to 60
✓ Blind-loop syndrome - portions of the intestines that are
stenotic as a result of surgery or Inflammation

➢ Lack of intrinsic factor (IF)

✓ Lack of intrinsic factor constitutes a significant cause of
impaired vitamin B12 absorption
✓ Associated with Pernicious anemia, Helicobacter pylori
(most common cause of ulcer) infection, total or partial
gastrectomy

PERNICIOUS ANEMIA
• an autoimmune disorder characterized by impaired
absorption of vitamin B12 because of an intrinsic
factor deficiency
• Associated with Lymphocyte-mediated destruction of
gastric parietal cells and autoantibodies to parietal
cells (anti-parietal cell antibodies); also anti-IF
• PARIETAL CELLS → cells that secrete intrinsic factor

OTHER CAUSES OF MEGALOBLASTIC ANEMIA

➢ Myelodysplastic syndrome (MDS)
➢ Acute erythroid leukemia (AML) → FAB M6

➢ Congenital dyserythropoietic anemia (CDA)

✓ Normoblasts in the bone marrow show mutinuclearity,
karyorrhexis (impaired DNA) causing dyserythropoiesis
(asynchrony of nuclear and cytoplasmic
maturation=Megaloblastic)
✓ Associated with refractory anemia and ineffective
erythropoiesis
✓ 3 types: Type I, Type II, Type III (based on cellular
morphology)

TYPE I CDA
• Associated with neonatal jaundice and splenomegaly
LABORATORY FINDINGS:
✓ Basophilic stippling and cabot rings
✓ Mild macrocytosis, anisocytosis, poikilocytosis
✓ Binucleated cells, cells with incompletely
separated or multi-lobulated nuclei
✓ Feulgen (+): presence of internuclear chromatin
bridges joining two normoblasts

TYPE II CDA
• Most common CDA; aka as HEMPAS (Hereditary
Erythroblast multinuclearity with positive acidified serum
test) um in diameter)
• presence of the HEMPAS antigen hepatosplenomegaly
and jaundice
TYPES OF CONGENITAL DYSERYTHROPOIETIC ANEMIA
LABORATORY FINDINGS:
✓ Anisocytosis, poikilocytosis, basophilic stippling MEGALOBLASTIC ANEMIA
✓ Ham’s test positive (Ham’s acidified serum test), ➢ CLINICAL FINDINGS (COMMON IN PERNICIOUS
sugar water test negative (sucrose hemolysis test) ANEMIA)
✓ Paroxysmal Nocturnal hemoglobinuria→ both ✓ Weakness, shortness of breath
positive in ham’s and sugar water test ✓ Gastrointestinal symptoms
✓ Paresthesias (numbness of the fingers in the extremities)
✓ Megaloblastic madness (severe vitamin B12 def)
✓ Achlorhydia (lack trypsin; not produce enough pH)

➢ LABORATORY FINDINGS:
✓ TYPE III CDA

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 57

✓ Complete blood count: Slight macrocytosis, giant platelets,
decreased hemoglobin (7-8 g/dL) and hematocrit,
pancytopenia, reticulocytopenia, increased MCV (>120 fL),
increased MCH, normal MCHC, oval macrocytes,
hypersegmented neutrophils, low reticulocyte count,
teardrop cells, fragmented RBC, microspherocytes,
nucleated RBC, Howell-Jolly bodies (DNA), basophilic
stippling, and cabot rings (mitotic spindle)
✓ Increased bilirubin and LDH→ indication of hemolysis
✓ Bone marrow examination → confirmatory test to identify
the megaloblastic appearance (characterized by a
nuclear-cytoplasmic asynchrony) of the developing
erythroid precursors especially in the polychromatic
normoblast

➢ SPECIAL DIAGNOSTIC TESTS:

✓ Schilling’s test → measures radioactive vitamin B12 in the
urine
✓ Homocysteine and Methylmalonic acid (MMA) assays
✓ Gastric analysis → to confirm achlorhydria in pernicious
anemia
✓ Serum gastrin → increased as a result to gastric
achlorhydria
✓ Antibody assays → antibodies to intrinsic factor and
parietal cells
✓ Holotranscobalamin assay → decreased in Vitamin B12
deficiency
✓ Stool analysis for parasites → for D. latum

NORMOCYTIC ANEMIA
➢ MCV is 80-100 fL (RBCs are 6-8 um)
➢ Characterized by normocytic, normochromic RBCs

➢ ASSOCIATED CONDITIONS:

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 58

✓ NORMAL OR DECREASED RETICULOCYTE COUNT:
• Aplastic anemia
• Myelophthisic anemia
• Anemia of renal disease
• Parvovirus B19 infections
• Anemia of chronic inflammation

✓ INCREASED RETICULOCYTE COUNT:

• HEMOLYTIC ANEMIAS
INTRINSIC
o RBC membrane, enzyme defects
o Hemoglobinopathies
EXTRINSIC
o Immune or antibody mediated
o Non-immune RBC injury (mechanical fragmentations) →
Microangiopathic hemolytic anemia, Infectious agents,
drugs, etc

APLASTIC ANEMIA
➢ Associated with bone marrow failure; is one of a group of
disorders, known as hypoproliferative disorders, that are
characterized by reduced growth or production of blood cells,
ALSO ASSOCIATED WITH MACROCYTIC RBCs

➢ BONE MARROW FAILURE:

✓ Destruction of hematopoietic stem cells as a result of injury
by drugs, chemicals, radiation, viruses, or autoimmune
mechanisms
✓ Premature senescence and apoptosis of hematopoietic
Stem cells as a result of genetic mutations
✓ Disruption of the bone marrow microenvironment that
supports hematopoiesis
✓ Decreased production of hematopoietic growth factors or
related hormones
ACQUIRED APLASTIC ANEMIA
✓ Loss of normal hematopoietic tissue as a result of infiltration
➢ Quantitative and qualitative deficiency of hematopoietic
of the marrow space with abnormal cells
stem cells
➢ TYPES:
➢ Classified into two major categories: idiopathic and
✓ Inherited → 15-20%
secondary
✓ Acquired → 80-85%
✓ Idiopathic: No known cause
Exposure to benzene and chloramphenicol→ cause aplastic
✓ Secondary: with identified cause
anemia
➢ CLINICAL FINDINGS:
✓ Pallor, fatigue, and weakness
✓ Severe and prolonged anemia can result in serious
cardiovascular complications, including tachycardia,
hypotension, cardiac failure, and death
✓ Symptoms of thrombocytopenia are also varied and
include petechiae, bruising, epistaxis, mucosal bleeding,
menorrhagia, retinal hemorrhages, intestinal bleeding, and
intracranial hemorrhage
✓ Fever and bacterial or fungal infections are unusual at initial
presentation but may occur after prolonged periods of
neutropenia

➢ LABORATORY FINDINGS:
✓ Pancytopenia (assocaiated with megaloblastic anemia and
aplastic anemia)
✓ Hemoglobin: <10 g/Dl
✓ MCV is decreased or normal
PHASES OF APLASTIC ANEMIA ✓ Decreased reticulocyte count

INHERITED APLASTIC ANEMIA

➢ Compared with acquired aplastic anemia, patients with
inherited/ congenital bone marrow failure syndromes present
JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 59
 at an earlier age and may have characteristic physical distinguishes DBA from the hypocellular marrow in aplastic
stigmata anemia
➢ Blood cell transfusion dependent ✓ Marked decreased in red cell precursors; normal WBC and
➢ TYPES: platelet count
✓ Fanconi’s anemia (FA) ✓ Increased EPO and Decrease CFU-E
✓ Pure red cell aplasia (PRCA)
✓ Dyskeratosis congenita (DC)
✓ Shwachman-Bodian-Diamond syndrome (SBDS)

FANCONI’S ANEMIA
➢ Aka Congenital aplastic anemia; chromosome instability
disorder characterized by aplastic anemia, physical
abnormalities, and cancer susceptibility

➢ CLINICAL FINDINGS:
✓ Physical malformations at birth: skeletal abnormalities,
thumb malformations, skin pigmentations, short stature,
abnormalities in the eyes, kidneys, and genitalia, low birth
weight, developmental delay
✓ Increased risk for cancer

➢ LABORATORY FINDINGS:
✓ Pancytopenia, reticulocytopenia, and a hypocellular bone
marrow
✓ Increased Fetal hemoglobin (Hb F) and alpha-fetoprotein
✓ Chromosome breakage analysis→ diagnostic test for FA
• Breakage with DEB (diepoxybutane) and MMC
(Mitomycin C)

PURE RED CELL APLASIA (PRCA)

➢ rare disorder of erythropoiesis characterized by a
selective and severe decrease in erythroid precursors in
an otherwise normal bone marrow
➢ Patients have severe anemia (usually normocytic),
reticulocytopenia, and normal WBC and platelet counts
➢ PRCA may be acquired or congenital
✓ Acquired:
• Primary PRCA may be idiopathic or autoimmune related;
in children called as transient erythroblastopenia of
childhood (TEC)
• Secondary PRCA occur in association with an underlying
thymoma, hematologic malignancy, solid tumor, infection,
chronic hemolytic anemia, collagen vascular disease, or
exposure to drugs or chemicals
✓ Congenital:
• Diamond-Blackfan anemia

DIAMOND-BLACKFAN ANEMIA
➢ a congenital erythroid hypoplastic disorder of early
infancy with an estimated incidence of 7 to 10 cases per
million live births

➢ CLINICAL FINDINGS:
✓ Physical anomalies, including craniofacial dysmorphisms,
short stature, and neck and thumb malformations

➢ LABORATORY FINDINGS:
✓ Severe macrocytic anemia with reticulocytopenia; WBC
count is normal or slightly decreased, and the platelet count
is normal or slightly increased. Bone marrow examination

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 60

 FANCONI’S VS. DIAMOND-BLACKFAN

MYELOPHTHISIC ANEMIA
➢ Myelophthisic anemia is due to the infiltration of abnormal
cells into the bone marrow and subsequent destruction and
replacement of normal hematopoietic cells
➢ Metastatic solid tumor cells (particularly from lung, breast,
and prostate), fibroblasts, and inflammatory cells (such as
those found in military tuberculosis and fungal infections)
have been implicated
➢ Cytopenia results from the release of substances such as
cytokines and growth factors that suppress hematopoiesis
and destroy stem, progenitor, and stromal cells

INHERITED APLASTIC ANEMIA

producing a decrease in the normal average life span of

HEMOLYTIC ANEMIA the erythrocyte
➢ Increase in erythrocyte destruction initiated primarily by ➢ Associated with increased bone marrow activity (increased
trapping of cells in sinuses of the spleen or liver and reticulocyte count)
➢ ASSOCIATED CONDITIONS:
✓ INTRINSIC (INHERITED)
JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 61
 • RBC membrane, enzyme defects OVERHYDRATED HEREDITARY
• Hemoglobinopathies STOMATOCYTOSIS/HEREDITARY HYROCYTOSIS
✓ EXTRINSIC (ACQUIRED) ➢ A very rare hemolytic anemia that results from a defect in
• Immune or antibody mediated membrane cation permeability that causes the RBCs to be
• Non-immune RBC injury (mechanical fragmentations) → overhydrated; it is inherited in an autosomal dominant pattern
Microangiopathic hemolytic anemia, Infectious agents, ➢ RBC membrane is excessively permeable to sodium and
drugs, etc potassium at 37° C; there is an influx of sodium into the cell
that exceeds the loss of potassium, which results in a net
HEREDITARY/INTRINSIC HEMOLYTIC ANEMIA: increase in the intracellular cation concentration
ERYTHROCYTE MEMBRANE DEFECTS ➢ As a result, more water enters the cell, and the cell swells and
HEREDITARY SPHEROCYTOSIS becomes stomatocytic
➢ heterogeneous group of hemolytic anemias caused by
defects in proteins that disrupt the vertical interactions ➢ LABORATORY FINDINGS:
between transmembrane proteins and the underlying protein ✓ Moderate to severe hemolytic anemia
cytoskeleton ✓ 5-50% stomatocytes
➢ Deficiency in spectrin, ankyrin, band 3, protein 4.2 ✓ Increased MCV and decreased MCHC
➢ Most common membrane defect; autosomal dominant; ✓ Increased OFT→ decreased surface:area/volume ratio
characterized by splenomegaly, variable degree of anemia,
spherocytes on the peripheral blood smear DEHYDRATED HEREDITARY
➢ LABORATORY FINDINGS: STOMATOCYTOSIS/HEREDITARY XEROCYTOSIS
✓ Decreased MCV (severely decreased if microspherocytes), ➢ Autosomal dominant hemolytic anemia as a result of a defect
hemoglobin, retics count (aplastic crisis) in membrane cation permeability that causes the RBCs to
✓ Increased MCHC be dehydrated; it is the most common form of stomatocytosis
✓ Positive autohemolysis test ➢ RBC membrane is excessively permeable to potassium,
✓ Normal RDW the potassium leaks out of the cell, but this is not balanced by
✓ Microspherocytes an increase in sodium; because of the reduced intracellular
✓ INCREASED OFT cation concentration, water is lost from the cell
✓ Negative DAT (Direct autoglobin test) DAT + if antibody
is involved ➢ LABORATORY FINDINGS:
✓ mild to moderate anemia, reticulocytosis, jaundice, and mild
HEREDITARY ELLIPTOCYTOSIS to moderate splenomegaly
➢ Heterogeneous group of hemolytic anemias caused by ✓ Crenated RBC
defects in proteins that disrupt the horizontal or lateral ✓ Decreased MCV
interactions in the protein cytoskeleton ✓ Increased MCHC
➢ Due to defective spectrin dimer-dimer interaction and ✓ Decreased OFT→ increased surface:area/volume ratio
protein 4.1 ✓ Target cells, burr cells
➢ Membrane defect is caused by polarization of cholesterol at
the ends of the cell rather than around pallor area OTHER CONDITIONS ASSOCIATED W/ STOMATOCYTES
➢ Familial pseudohyperkalemia
HEREDITARY PYROPOIKILOCYTOSIS ✓ excessive potassium leaks out of the RBCs at room
➢ Due to defective spectrin dimer-dimer interaction, temperature
increased calcium ✓ Mutations in the ABCB6 gene
➢ CLINICAL FINDINGS: Severe transfusion related hemolytic ➢ Cryohydrocytosis
anemia, jaundice, splenomegaly, gallbladder disease ✓ cold-induced leakage of sodium and potassium from the
➢ LABORATORY FINDINGS: RBCs
✓ Extreme microcytosis (MCV 25-55 fL) ✓ RBCs have marked increase in cation permeability, cell
✓ Increased: retics count swelling, and hemolysis when stored at 4° C for 24 to 48
✓ Presence of micro spherocytes, fragmented cells, hours
elliptocytes, bizarre forms ➢ Rh null disease/Rh deficiency syndrome→ defect in RhAg
protein (synthesis of Rh antigens)
HEREDITARY OVALOCYTOSIS
➢ Hereditary ovalocytosis or Southeast Asian ovalocytosis HEREDITARY ACANTHOCYTOSIS
(SAO) is a condition caused by a mutation in the gene for ➢ Associated with neuroacanthocytosis and
band 3 that results in increased rigidity of the membrane and abetalipoproteinemia; mutations in the MTP (microsomal
resistance to invasion by malaria triglyceride transfer protein)
➢ It is common in the malaria belt of Southeast Asia, where its ✓ Autosomal recessive; mild anemia associated with
prevalence can reach 30% steatorrhea, neurological and retinal abnormalities; 50-
➢ The inheritance pattern is autosomal dominant, and all 100% of erythrocytes are acanthocytes
patients identified are heterozygous ✓ Increased lecithin:sphingomyelin ratio in the membrane
➢ LABORATORY FINDINGS: due to abnormal plasma lipid concentrations; absence of
✓ Mild or absent hemolysis serum β-lipoprotein needed for lipid transport
✓ 30% ovalocytes ➢ OTHER CONDITIONS WITH NEUROACANTHOCYTOSIS:
✓ Mcleod syndrome → absence of the KX gene (required to
produce Kell Ag)

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 62

✓ Chorea acanthocytosis (ChAc) PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH)
• mutations in the VPS13A gene (codes for the protein ➢ Rare chronic intravascular hemolytic anemia caused by an
chorein) acquired clonal hematopoietic stem cell mutation that results
• Chorein deficiency - lead to abnormal membrane protein in circulating blood cells that lack
structure and acanthocyte formation) glycosylphosphatidylinositol (GPI)-anchored proteins on
their surfaces, such as CD55 (DAF or Decay accelerating
factor) and CD59 (MIRL or membrane inhibitor of reactive
lysis)
➢ Mutations in the PIGA gene → codes for
phosphatidylinositol N-acetylglucosaminyltransferase subunit
A
➢ Absence of CD55 and CD59 on the surface of the RBCs
renders them susceptible to spontaneous lysis by
complement
➢ Also found in platelets, granulocytes, monocytes, and
lymphocytes

➢ TYPES:
✓ Classic PNH
✓ Subclinical PNH
✓ Hypoplastic PNH

➢ CLINICAL FINDINGS:
✓ Hemolytic anemia
✓ Thrombosis → leading cause of death
✓ Smooth muscle dystonia
✓ Hemoglobinuria and jaundice
✓ Chronic renal failure

➢ LABORATORY FINDINGS:
✓ Decreased: Hemoglobin (<6 g/dL) serum haptoglobin
✓ Increased: plasma hemoglobin, serum indirect bilirubin and
lactate dehydrogenase (LDH)
✓ Hemoglobinuria, and hemosiderinuria
✓ Reticulocyte count mildly to moderate increased
✓ Increased MCV
✓ DAT negative

✓ SCREENING TEST: Sucrose hemolysis test/Sugar water

screening test, Ham’s acidified serum test

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 63

✓ CONFIRMATORY TEST: Flow cytometry using anti- ✓ AUTOIMMUNE → associated with autoantibodies
CD59

HEREDITARY/INTRINSIC HEMOLYTIC ANEMIA:

ERYTHROCYTE MEMBRANE DEFECTS
G-6-PD DEFICIENCY
➢ most common RBC enzyme defect, with a prevalence of
5% of the global population, or approximately 400 million
people worldwide
➢ Some variants confer resistance to Malaria
➢ Defect in the hexose-monosphosphate shunt pathway;
Reduced glutathione levels are not maintained because of • Warm autoimmune hemolytic anemia (WAIHA)
decreased NADPH generation • Cold autimmune hemolytic anemia (CAIHA)
➢ Results in oxidation of hemoglobin to methemoglobin; • Paroxysmal cold hemoglobinuria (PCH)
denatures to form Heinz bodies • Mixed type autoimmune hemolytic anemia
• Drug induced hemolytic anemia
➢ CLINICAL FINDINGS:
✓ ISOIMMUNE/ALLOIMMUNE → associated with
✓ are asymptomatic throughout their lives
isoantibodies/alloantibodies
✓ However, some patients have clinical manifestations. The
• Hemolytic disease of the newborn (HDN)
clinical syndromes are acute hemolytic anemia, neonatal
• Hemolytic transfusion reaction (HTR)
jaundice (hyperbilirubinemia), and chronic hereditary
nonspherocytic hemolytic anemia (HNSHA) ➢ NON-IMMUNE HEMOLYTIC ANEMIAS
✓ Microangiopathic hemolytic anemia
✓ Macroangiopathic hemolytic anemia
G-6-PD VARIANTS
Fava beans→ G6PD Mediterranean variant (Favism) ✓ Infectious agents
✓ Caused by other red blood cell injury
PYRUVATE KINASE DEFICIENCY
➢ Autosomal recessive; most common enzyme deficiency in
Embden-Meyerhof pathway AUTOIMMUNE HEMOLYTIC ANEMIA
➢ Lack of ATP causes impairment of the cation pump that WARM AUTOIMMUNE HEMOLYTIC ANEMIA
controls intracellular sodium and potassium levels ➢ RBCs are coated with IgG and/or complement
➢ Decreased erythrocyte deformability reduces their life ➢ 60% of cases are idiopathic; other cases are secondary to
diseases that alter the immune response; can also be drug
span
➢ Severe hemolytic anemia with reticulocytosis and induced
echinocytes ➢ LABORATORY FINDINGS:
✓ Progressive weakness, Occasional acute fever, Pain,
Hemoglobinuria, Mild jaundice, Splenomegaly,
Hepatomegaly,
ACQUIRED/EXTRINSIC HEMOLYTIC ANEMIA
➢ IMMUNE HEMOLYTIC ANEMIAS ✓ Lymphadenopathy

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 64

➢ CLINICAL FINDINGS:
✓ Spherocytes
✓ MCHC may be >37 g/dL
✓ increased osmotic fragility, bilirubin, reticulocyte count
✓ occasional nRBCs present; positive DAT helpful in
differentiating from hereditary spherocytosis

COLD AUTOIMMUNE HEMOLYTIC ANEMIA

➢ RBCs are coated with IgM and complement at temperatures
below 37°C. Antibody is usually anti-I but can be anti-i
➢ Can be idiopathic, or secondary to Mycoplasma pneumoniae
(anti-I), lymphoma, or infectious mononucleosis (anti-i)

➢ CLINICAL FINDINGS:
✓ Seasonal symptoms; RBC clumping can be seen both
macroscopically and microscopically
✓ MCHC >37 g/dL; increased bilirubin, reticulocyte count
✓ Positive DAT detects complement-coated RBCs

PAROXYSMAL COLD HEMOGLOBINURIA

➢ Caused by binding of Donath Landsteiner antibodies (IgG)
to red cells
➢ Donath Landsteiner antibody/autoanti-P→ biphasic
hemolysin; binds at cold temperature and lyse at warm
temperature
➢ Antibodies binds to red cell antigen in the presence of
complement at 150C and shows specificity for the Pp blood
group system (autoanti-P)
➢ CLINICAL FINDINGS:
✓ headache, vomiting, pain in the abdomen, hemoglobinuria
➢ LABORATORY FINDINGS:
✓ Spherocytes, fragmented red cells, polychromasia
✓ DAT (+), Donath Landsteiner test (+)

ALLOIMMUNE HEMOLYTIC ANEMIA

HEMOLYTIC TRANSFUSION REACTIONS
➢ One of the most severe and potentially life-threatening
complications of blood transfusion is a hemolytic transfusion
reaction (HTR) caused by immune-mediated destruction of
donor cells by an antibody in the recipient
➢ The offending antibody in the recipient may be IgM or IgG,
complement may be partially or fully activated or not activated
at all, and hemolysis may be intravascular or extravascular
➢ Can be acute HTR or delayed HTR

➢ ACUTE HTR
✓ associated with ABO antibodies (IgM) with complement
activation, intravascular hemolysis
➢ DELAYED HTR
✓ associated with Rh (Big D Ag), Kidd (Big K Ag), Duffy
antibodies (IgG); with or without complement activation ; ✓ Group O woman develops IgG antibody that crosses the
extravascular placenta and coats fetal RBCs when fetus is group A or; the
coated RBCs are phagocytized
HEMOLYTIC DISEASE OF NEWBORN ✓ LABORATORY FINDINGS: Mild or no anemia, few
➢ Also called as Hemolytic disease of the fetus and newborn spherocytes, weakly positive DAT, slightly increased
(HDFN) bilirubin
➢ May be due to ABO or Rh incompatibility
➢ Rh incompatibility
➢ ABO incompatibility ✓ Rh negative woman is exposed to Rh antigen (D antigen)
from fetus and forms IgG antibody (anti-D); this antibody will
JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 65
 cross the placenta and destroy RBCs of the next fetus that
is Rh positive (D positive)
✓ Exchange transfusions in utero or shortly after birth
✓ No longer a common problem with use of Rh
immunoglobulin (RhoGam)
✓ LABORATORY FINDINGS: Severe anemia, nRBCs,
positive DAT; very high bilirubin levels cause kernicterus
leading to brain damage

✓ Hemolytic Uremic Syndrome

✓ HELLP Syndrome
✓ Disseminated Intravascular Coagulation

➢ LABORATORY FINDINGS:
✓ Decreased: hemoglobin, decreased serum haptoglobin
NON-IMMUNE HEMOLYTIC ANEMIA ✓ Increased: Reticulocyte count, indirect bilirubin, serum LDH,
➢ A common feature in the nonimmune extrinsic hemolytic urine urobilinogen
anemias is the presence of a condition that causes physical ✓ Hemoglobinuria
or mechanical injury to the RBCs ✓ Presence of schistocytes, helmet cells, microspherocytes,
➢ Mechanical fragmentation; associated with schistocytes Polychromasia and nucleated RBC
(fragmented RBC) ✓ THROMBOCYTOPENIA → due to the consumption of
➢ This injury can be caused by: platelets in thrombi that form in the microvasculature
✓ Abnormalities in the microvasculature (microangiopathic)
✓ The heart and large blood vessels (macroangiopathic) THROMBOCYTIC THROMBOCYTOPENIC PURPURA (TTP)
✓ Infectious agents ➢ rare, life-threatening disorder characterized by the abrupt
✓ Chemicals appearance of microangiopathic hemolytic anemia, severe
✓ Drugs thrombocytopenia, and markedly elevated serum LD activity
✓ Venoms ➢ caused by a deficiency of the von Willebrand factor-
✓ Extensive burns cleaving protease known as a disintegrin and
metalloprotease with a thrombospondin type 1 motif,
member 13 (ADAMTS13)
➢ ADAMTS13 → regulates the size of circulating von
Willebrand factor (VWF) by cleaving ultralong VWF multimers
(ULVWF) into shorter segments; preventing VWF from
excessively binding and activating platelets

➢ TYPES:
✓ Idiopathic → autoantibodies (IgG, IgM, IgA) to ADAMTS13
✓ Secondary → infections, pregnancy, surgery, trauma,
MICROANGIOPATHIC HEMOLYTIC ANEMIA inflammation, and disseminated malignancy
➢ group of potentially life-threatening disorders characterized ✓ Inherited → also called Upshaw-Schülman syndrome, is a
by RBC fragmentation and thrombocytopenia severe ADAMTS13 deficiency caused by mutations in the
➢ RBC fragmentation occurs intravascularly by the ADAMTS13 gene
mechanical shearing of RBC membranes as the cells
rapidly pass-through turbulent areas of small blood vessels HEMOLYTIC UREMIC SYNDROME (HUS)
that are partially blocked by microthrombi or damaged ➢ Characterized by microangiopathic hemolytic anemia,
endothelium thrombocytopenia, and acute renal failure from damage to
➢ Upon shearing, RBC membranes quickly reseal with minimal endothelial cells in the glomerular microvasculature
escape of hemoglobin, but the resulting fragments (called
schistocytes) are distorted and become rigid ➢ TYPES:
➢ The spleen clears the rigid RBC fragments from the ✓ Typical/Shiga toxin-associated HUS, or Stx-HUS
circulation through the extravascular hemolytic process • 90% HUS
• caused by E.coli (serotype O157:H7) and Shigella
➢ ASSOCIATED CONDITIONS: strains that produce Shiga toxin and is preceded by an
✓ Thrombotic Thrombocytopenic Purpura episode of acute gastroenteritis, often with bloody diarrhea
JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 66
 ✓ Can cause direct damage to the RBC membrane, producing
✓ Atypical HUS acute hemolysis, which is characterized by severe anemia
• 10% HUS with many schistocytes and micro spherocytes
✓ uncontrolled activation of the alternative complement
system, which causes endothelial cell injury, activation of
platelets and coagulation factors, and formation of platelet-
fibrin thrombi that obstruct the microvasculature in the
glomerulus and other organs

HELLP SYNDROME
➢ serious complication in pregnancy, is named for its
characteristic presentation of hemolysis, elevated liver
enzymes, and low platelet count (HELLP)
➢ It occurs in approximately 0.5% of all pregnancies but
develops in approximately 4% to 12% of pregnancies with
preeclampsia and 30% to 50% of pregnancies with
eclampsia, most often in the third trimester

DISSEMINATED INTRAVASCULAR COAGULATION

➢ Characterized by the widespread activation of the
hemostatic system, resulting in fibrin thrombi formation
throughout the microvasculature
➢ Major clinical manifestations are organ damage due to
obstruction of the microvasculature and bleeding due to the
consumption of platelets and coagulation factors and
secondary activation of fibrinolysis
➢ DIC is a complication of many disorders’ metastatic
cancers, acute leukemias, infections, obstetric complications,
crush or brain injuries, acute hemolytic transfusion reactions,
extensive burns, snake or spider envenomation, and chronic
inflammation
➢ Thrombocytopenia with prolonged PT and APTT,
decreased fibrinogen levels

MACROANGIOPATHIC HEMOLYTIC ANEMIA

➢ TRAUMATIC CARDIAC HEMOLYTIC ANEMIA
✓ Can occur in patients with prosthetic cardiac valves due to
turbulent blood flow through and around the implanted
devices
➢ EXERCISED-INDUCED HEMOGLOBINURIA
✓ Also known as MARCH HEMOGLOBINURIA
✓ Transient hemolytic anemia that occurs after forceful
repeated impact of the feet or hands on hard surfaces

OTHER CAUSES OF HEMOLYTIC ANEMIA

➢ INFECTIOUS AGENTS
✓ Malaria, Babesiosis, Clostridial sepsis, Bartenellosis
➢ DRUGS AND CHEMICALS
✓ Causes oxidative denaturation of hemoglobin leading to
formation of methemoglobin and Heinz bodies
✓ Dapsone, Naphthalene, Primaquine, Arsine hydride,
Copper and Lead exposure
➢ VENOM
✓ Snakes, spiders, bees, or wasps
✓ Causes hemolysis by direct disruption of the RBC
membrane, alteration of the RBC membrane that results in
complement-mediated lysis, and initiation of DIC
➢ EXTENSIVE BURNS/THERMAL INJURY

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 67

 ➢ Represents the 2nd X chromosome in
WEEK 14: LEUKOCYTE females
MORPHOLOGICAL ABNORMALITIES ➢ Seen in 2-3% of neutrophils
➢ Characteristic “DRUMSTICK” appearance
AND NON-MALIGNANT LEUKOCYTE of neutrophils
DISORDERS
• Non-malignat→ not cancer

LEUKOCYTE MORPHOLOGICAL ABNORMALITIES

GRANULOCYTE CYTOPLASMIC ABNORMALITIES
➢ Granulocytes
➢ Alder-reilly granules
✓ Nuclear abnormalities ➢ May-hegglin granules
✓ Cytoplasmic abnormalities ➢ Chediak-higashi granules
➢ Lymphocytes ➢ Auer rods
➢ Monocytes/macrophages ➢ Reactive changes/secondary morphological changes
➢ Most affected is neutrophils→ most abundant o Toxic granulation
o Dohle-amato bodies/dohle bodies
GRANULOCYTE NUCLEAR ABNORMALITIES
o Cytoplasmic vacoules
➢ Hypersegmentation/hypersegmented neutrophil
o Pyknotic and necrotic cells
➢ Hyposegmented neutrophils
o Cytoplasmic swelling
➢ Le cell o Reactive eosinophils and basophils
➢ Barr body (sex chromatin) ➢ Abnormal inclusions
HYPERSEGMENTATION/HYPERSEGMENTED NEUTROPHILS o Erhlichia and anaplasma
➢ Normal neutrophils contain three to five lobes that are o Histoplasma
separated by filaments
➢ Hypersegmented neutrophils have more
ALDER-REILLY GRANULES
than five lobes and are usually larger
➢ Darkly staining metachromatic (purple-red) cytoplasmic
than normal neutrophils
granules in granulocytes (less often in
➢ CLINICAL SIGNIFICANCE:
monocytes and lymphocytes
✓ Megaloblastic anemia→ can be acquired or hereditary ➢ Abnormal large primary granules
✓ Undritz anomaly → Hereditary neutrophil ➢ Granules contain degraded
hypersegmentation mucopolysaccharides due to an enzyme
✓ Myelodysplasia defect
➢ May resemble toxic granules (seen in infections and toxic
HYPOSEGMENTED NEUTROPHILS
states)
➢ Hyposegmentation of granulocyte nucleus
➢ All neutrophils are affected
➢ Nuclei may appear:
✓ round, ovoid, or peanut shaped (Homozygous Pelger-Huet)
➢ CLINICAL SIGNIFICANCE:
✓ Bilobed forms the characteristic spectacle-like (“pince- ✓ Alder-Reilly anomaly
nez”) morphology with the nuclei ✓ Mucopolysaccharidoses (MPSs’)
attached by a thin filament
(Heterozyous Pelger-Huet)
MAY-HEGGLIN GRANULES
➢ CLINICAL SIGNIFICANCE:
➢ Gray-blue spindle shaped inclusions in the cytoplasm
✓ Pelger-Huet anomaly
➢ Large Dohle body-like inclusions in neutrophils, eosinophils,
✓ Pseudo or Acquired Pelger-Huet anomaly (associated with
basophils, and monocytes
cancer)
➢ The basophilic Dohle body-like leukocyte
o Myelodysplastic syndrome
inclusions are composed of precipitated
o Acute myeloid leukemia myosin heavy chains (mRNA in older
o Myeloproliferative neoplasms references)
✓ Bilobed→ pince nez, dumbell, peanut appearance ➢ True Dohle bodies consist of lamellar rows of rough
endoplasmic reticulum (rRNA)
➢ Mostly seen in neutrophils but can also be seen to other
LE CELL
leukocytes
➢ Usually a neutrophil that has ingested the antibody-coated
➢ PAS-→ may hegglin
nucleus of another neutrophil or has engulf
➢ PAS +→ Dohle bodies
the homogenous, globular nuclear mass of
➢ CLINICAL SIGNIFICANCE:
destroyed cell ✓ May-Hegglin anomaly
➢ Presence of: ANTI-NUCLEAR ANTIBODIES, cell
nuclei, phagocytes with ingested material
➢ Usually mistaken with a tart cell (monocyte CHEDIAK-HIGASHI GRANULES
with an ingested lymphocyte)
➢ A neutrophil phagocytized with another cell
➢ Giant red, blue, to grayish round inclusions in the cytoplasm
➢ CLINICAL SIGNIFICANCE:
➢ Giant lysosomal granules in granulocytes, monocytes, and
✓ Systemic Lupus Erythematosus
lymphocytes
✓ Connective tissue disorders
➢ Cells in the body are affected and exhibit
abnormally large lysosomes, which
BARR BODY contain fused dysfunctional granules
➢ Can be seen in all leukocytes but mostly
affected are neutrophils
➢ CLINICAL SIGNIFICANCE:
✓ Chediak-Higashi syndrome

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 68

 ➢ A delay in preparing the blood film after collection may
➢ Pseudo-Chediak-Higashi granules are cytoplasmic inclusions affect Dohle body appearance in that they are more grey
that resemble the fused lysosomal granules in Chediak- than blue or in some cases may not be visible
Higashi syndrome ➢ Confused with May-Hegglin granules
➢ CLINICAL SIGNIFICANCE:
➢ CLINICAL SIGNIFICANCE: ✓ Infections
✓ Acute myeloid leukemia ✓ Pregnancy
✓ Chronic myeloid leukemia ✓ Burns
✓ Myelodysplastic syndrome (MDS) ✓ Toxic states
PAS RNA
May hegglin - mRNA
Granules
Dohle Amato Bodies + rRNA

AUER RODS
➢ Pink or red shaped cytoplasmic granules; found in myeloid
and monocytic series only CYTOPLASMIC VACUOLATIONS
➢ Fused primary granules (peroxidase stain ➢ Less encountered than toxic granules and Dohle bodies
positive) ➢ Reflect phagocytosis, either of self (autophagocytosis) or of
➢ Faggot cells: bundle of auer rods (seen in extracellular material
acute promyelocytic leukemia; M3; also ➢ TYPES:
associated with DIC) ✓ AUTOPHAGOCYTIC VACUOLES
➢ Clinical significance: • Tend to be small
✓ Acute myelogenous leukemia (AML, M1 and M2) (approximately 2 um)
✓ Acute promyelocytic leukemia (APL, M3) and distributed
✓ Acute myelomonocytic leukemia (AMML, M4) throughout the
cytoplasm
REACTIVE CHANGES / SECONDARY MORPHOLOGICAL CHANGES • Autophagocytosis can
be induced by
TOXIC GRANULATION specimen storage in
➢ Appears as dark, blue-black granules in the cytoplasm of ethylene
neutrophils, usually in segmented and band forms diaminetetraacetic
➢ Granulation may represent the precipitation of ribosomal (EDTA) for more than 2
protein (RNA) caused by metabolic toxicity within the cells hours, autoantibodies,
➢ Are peroxidase positive and reflect an increase in acid acute alcoholism, and exposure to high doses of
mucosubstance within primary, azurophilic granules that radiation
may enhance bactericidal activity ✓ PHAGOCYTIC VACUOLES
➢ The extent of toxic granulation • Tend to be large (up to 6 um) and often accompanied
is usually graded on a scale of by toxic granulation induced by either bacteria or fungi
1+ to 4+, with 4+ being the most are suggestive of sepsis
severe. Grading of the • When phagocytic vacuoles are seen, a careful
granulation is dependent on examination sometimes reveals organisms within the
the coarseness and amount of vacuoles
granulation within the cellular
cytoplasm. Table 26.7 Reactive Morphologic Changes in Neutrophils
➢ Primary granules are larger
than secondary granules CHANGE APPEARANCE ASSOCIATED W/
➢ Toxic granulation can mimic Toxic Dark, blue-black Inflammation,
granulation found in Alder- granulation cytoplasmic infection, G-CSF
Reilly anomaly; one helpful defining characteristic of toxic granules
granulation is that in most cases, not all neutrophils are Dohle Intracytoplasmic Infection, G-CSF,
equally affected bodies pale blue round or pregnancy, burns
➢ CLINICAL SIGNIFICANCE: elongated bodies
✓ Inflammations bet. 1 and 5 um in
✓ Infections diameter, usually
✓ Toxic states adjacent to cellular
✓ Burns membranes
✓ Malignant disorders Cytoplasmic Small-large circular Bacterial infection,
vacuoles clear areas in autophagocytosis
DOHLE BODIES / DOHLE AMATO BODIES cytoplasm, rarely secondary to drug
➢ Are light/pale blue round or elongated cytoplasmic many contain ingestion, acute
inclusions between 1-5 um consisting of remnants of organism alcoholism or excess
ribosomal ribonucleic acid (rRNA) arranged in parallel row; storage of sample
close to cellular membranes before making blood
➢ Are typically found in band and segmented neutrophils and film
can appear together with toxic granulations;
can be seen in eosinophils, basophils,
PYKNOTIC AND NECROTIC CELLS
monocytes, and lymphocytes
➢ Pyknotic nuclei in neutrophils generally indicate imminent
➢ Localized failure of cytoplasmic maturation
cell death
➢ PAS reaction positive
➢ In a pyknotic nucleus, water has been lost and the chromatin
o Periodic Acid Schiff (PAS)→ stains the glucose
becomes dense and dark; however, chromatin or filaments

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 69

 can still be seen between nuclear lobes (depending on ➢ H. capsulatum is a fungus; this
whether the cell is a band or segmented form) organism lives intracellularly in cells
➢ Necrotic nuclei are found in dead neutrophils/necrobiotic of the mononuclear phagocyte
neutrophils; they are rounded nuclear fragments with no system, cells of the bone marrow,
filaments and no chromatin pattern or cells from sputum or effusion
➢ Increased numbers of pyknotic or necrotic cells suggest that specimens
an extended amount of time has elapsed between blood ➢ The fungus appears as a tiny oval
collection and blood film preparation body with a clear halo surrounding
➢ CLINICAL SIGNIFICANCE: a small nucleus
✓ Bacterial infection
✓ Drug intoxication

LYMPHOCYTE AND MONOCYTE /MACROPHAGE

ABNORMAALITIES

LYMPHOCYTE ABNORMALITIES
➢ Basket/smudge cell
➢ Hairy cell
➢ Sezary cell
➢ Reed-sternberg cell
➢ Rieder cell
➢ Reactive lymphocyte
CYTOPLASMIC SWELLING ➢ Hand-mirror lymphocyte
➢ result of osmotic swelling of the cytoplasm or by increased ➢ Abnormal plasma cells
adhesion to the glass slide in stimulated neutrophils o Flame cell
➢ Regardless of the cause, the result is a variation in neutrophil o Grape cell
size or neutrophil anisocytosis BASKET / SMUDGE CELL
➢ Associated with Degenerated nucleus or
ruptured cell in form or basket/smudge
REACTIVE EOSINOPHILS AND BASOPHILS ➢ THUMBPRINT APPEARANCE
➢ Hypogranular eosinophils have been associated with acute ➢ FRAGILE LYMPHOCYTES that appear during
lymphoblastic leukemia and hypereosinophilic syndrome smear preparation; ARTIFACT → appear at
➢ In-vitro disruption of the cellular membrane may occur the end of smears
during the process of making the blood film as eosinophils ➢ Clinical significance: Chronic Lymphocytic
are fragile. To promote leukemia
accuracy, it is recommended
that fractured eosinophils be HAIRY CELL
counted as eosinophils in the ➢ Gray-blue hair like cytoplasmic projections
manual WBC differential surrounding the nucleus (fried-egg
➢ Basophil granules are water appearance)
soluble, and it is not ➢ Nearly all blood cells contain 7 non-erythroid
uncommon for them to be isoenzymes of ACP (0, 1, 2, 3b, 4 & 5)
partially or completely washed ➢ Isoenzyme 5 (tartrate resistant is produced in
away during staining ABUNDANCE in hairy cell leukemia)
➢ Sometimes few or no granules ➢ TRAP (Tartrate-resistant acid phosphatase stain) positive
remain; only a pinkish tinge in or around the cell is seen. This ➢ Clinical significance: Hairy cell leukemia
can hamper proper identification of basophils when
performing manual WBC differentials
SEZARY CELL
EHRLICHIA AND ANAPLASMA ➢ Lymphocyte with convoluted
➢ Are small, obligate, intracellular bacteria transmitted by ticks nucleus/brain-like nucleus
to humans and other vertebrate hosts ➢ Presence indicates LEUKEMIC PHASE of
➢ These organisms grow as acluster (morulae) in neutrophils mycosis fungoides (sezary syndrome)
and monocytes ➢ Clinical significance:
➢ Morulae can be mistaken ✓ Mycosis fungoides (cutaneous T-cell
for Dohle bodies in lymphoma)
neutrophils ✓ Sezary syndrome (Variant of mycoses
✓ Neutrophils → Anaplasma fungoides)
phagocytophilum and
rarely in Ehrlichia ewingii
✓ Monocytes → Ehrlichia
chaffeensis
➢ Human granulocytic erlichiosis is transmitted by the black-
legged tick (Ixodes scapularis) and the western black-
legged tick (I. pacificus)

HISTOPLASMA

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 70

REED-STERNBERG CELL
➢ Abnormal lymphocyte with an “owl’s eye MONOCYTE / MACROPHAGE ABNORMALITIES
appearance”
➢ Pathognomonic sign for Hodgkin’s GAUCHER CELL
lymphoma ➢ are distinctive macrophages, single or in
➢ Clinical significance: Hodgkin’s Lymphoma clusters
➢ abundant fibrillar blue-gray cytoplasm
with a striated or wrinkled appearance
RIEDER CELL (sometimes described as onion skin-like)
➢ Lymphocyte with a clover leaf like nucleus ➢ Positive with trichrome, aldehyde
➢ Clinical significance: Chronic lymphocytic fuchsin, periodic acid-Schiff (PAS) and acid phosphatase
leukemia ➢ CLINICAL SIGNIFICANCE: Gaucher’s
disease

FOAM CELL
REACTIVE LYMPHOCYTE ➢ macrophages with cytoplasm
➢ Reactive changes in lymphocyte packed with lipid-filled lysosomes
morphology occur as lymphocytes are that appear as small vacuoles (foam)
stimulated when interacting with antigens after staining
in peripheral lymphoid organs ➢ CLINICAL SIGNIFICANCE: Niemann-Pick’s
➢ AKA variant, atypical, transformed, disease
effector, plasmacytoid, Turk cells,
Downey, and immunoblasts SEA-BLUE HISTIOCYTES
➢ B and T lymphocyte activation results in ➢ Are macrophages with
the transformation of small, resting lymphocytes into lipofuscin, glycophospholipid,
proliferating larger cells and sphingomyelin contained
➢ Reactive lymphocytes often present as a in cytoplasmic granules, 1 to 3
heterogeneous population of various um in diameter, that appear
shapes and sizes blue with Wright stain
➢ A plasmacytoid lymphocyte is a type of
reactive lymphocyte that has some morphologic features of
plasma cells NON-MALIGNANT LEUKOCYTE DISORDERS
➢ TYPES: ➢ Not caused by clonal or neoplastic changes in
➢ Type I: turk’s irration plasma cytoid; lymphocyte with a hematopoietic precursor cells
large block of chromatin ➢ Causes can be genetic or acquired and involve one or more
➢ Type II: also known as Infectious mononucleosis (IM) cells; lineages: neutrophil, lymphocyte, monocyte, eosinophil, and
round mass of chromatin (ballerina skirt appearance) basophil, affecting the number of circulating cells,
➢ Type III: vacuolated (swiss cheese appearance) morphology, or both
➢ CLINICAL SIGNIFICANCE: ➢ Many of these disorders are associated with significant
✓ Infectious mononucleosis clinical manifestations, although some are benign in nature
✓ Leukemias ➢ TYPES:
✓ Viral infections ✓ QUALITATIVE NON-MALIGNANT
✓ QUANTITATIVE NON-MALIGNANT/REACTIVE STATES

ABNORMAL PLASMA CELLS QUALITATIVE NON-MALIGNANT LEUKOCYTE DISORDERS

➢ Morphological abnormalities involving neutrophils
FLAME CELL ➢ Defective leukocyte motility/movement
➢ Abnormal plasma cell with red to ➢ Defective respiratory burst
pink cytoplasm ➢ Lysosomal storage disorders
➢ Associated with increased ➢ Inherited disorders of lymphocytes
Immunoglobulins (usually IgA);
➢ Inclusion: Russel bodies (individual
bodies of Immunoglobulin) MORPHOLOGICAL ABNORMALITIES INVOLVING NEUTROPHILS
➢ Clinical significance: ➢ HYPERSEGMENTATION
✓ Multiple myeloma ➢ ALDER-REILLY ANOMALY
✓ Waldenstrom’s macroglobulinemia ➢ MAY-HEGGLIN ANOMALY
➢ CHEDIAK-HIGASHI SYNDROME
GRAPE CELL ➢ PELGER-HUET ANOMALY
➢ Abnormal plasma cell with small colorless vacuoles ➢ PSEUDO/ACQUIRED PELGER-HUET
➢ AKA Berry, Mott, Morula Cell
➢ Inclusions: Dutcher’s bodies (intranuclear PELGER-HUET ANOMALY
protein inclusions) ➢ autosomal dominant disorder characterized by decreased
➢ Large protein globules giving nuclear segmentation and distinctive coarse chromatin
appearance of grapes: “Honeycomb clumping pattern
appearance” stain pink, colorless or blue ➢ affects all leukocytes, although morphologic changes are
➢ Clinical significance: most obvious in mature neutrophils
✓ Multiple myeloma ➢ Mutations in the lamin B-receptor gene
✓ Reactive states ➢ lamin B receptor→ inner nuclear membrane protein that
combines B-type lamins and heterochromatin and plays a

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 71

 major role in leukocyte nuclear shape changes that occur granules in
during normal maturation platelets
➢ TYPES: MAY HEGGLIN Thrombocytopeni Usually
✓ HETEROZYGOUS PHA → normal individuals, pince-nez ANOMALY a, giant platelets, asymptomatic,
appearance of the nucleus large dohle body sometimes mild
✓ HOMOZYGOUS PHA → cognitive impairment, heart like inclusions in bleeding related
defects, and skeletal abnormalities may occur; single neutrophils, to
nuclei eosinophils, thrombocytopen
basophils and ia
ALDER-REILLY ANOMALY monocytes
➢ is a rare inherited disorder characterized by granulocytes
(monocytes and lymphocytes less often) with large, darkly DEFECTIVE LEUKOCYTE MOTILITY/MOVEMENT
staining metachromatic cytoplasmic granules
➢ AR anomaly was initially reported in patients with gargoylism; JOB’S SYNDROME
however, it can be seen in otherwise healthy individuals ➢ Normal random movement; abnormal
➢ Granulations are also seen in MPSs CHEMOTACTIC/DIRECTIONAL MOTILITY
➢ Patient suffer from PERSISTENT BOILS and recurrent “cold”
staphylococcal abscesses
➢ Associated with increased IgE
MAY-HEGGLIN ANOMALY
➢ A rare, autosomal dominant disorder characterized by LAZY LEUKOCYTE SYNDROME
variable thrombocytopenia, giant platelets, and large Dohle ➢ Abnormal random and chemotactic movement
body-like inclusions in neutrophils, eosinophils, basophils, and ➢ Cells failed to respond to inflammatory stimuli but have
monocytes normal phagocytic and bactericidal activity
➢ Caused by a mutation in the MYH9 gene with disordered
production of myosin heavy chain type IIA, which affects LEUKOCYTE ADHESION DISORDERS (LADs)
megakaryocyte maturation and platelet fragmentation ➢ Are rare autosomal recessive inherited conditions resulting in
the inability of neutrophils and monocytes to move from
CHEDIAK-HIGASHI SYNDROME circulation to the site of inflammation (called extravasation)
➢ A rare autosomal recessive disease of immune dysregulation ➢ Consequences of these disorders are recurrent severe
➢ Mutation in the CHS1 LYST gene bacterial and fungal infections
➢ Many types of cells in the body are affected and exhibit ➢ Hematopoietic stem cell transplant is the only curative
abnormally large lysosomes, which contain fused treatment
dysfunctional granules ➢ LAD: Problem in recognizing and adhering to Ag→
➢ Clinical manifestations begin in infancy with partial albinism recurrent infections
and severe recurrent life-threatening bacterial infections ➢ TYPES: LAD I, II, III, others (Shwachman-Bodian Diamond
➢ Patients often have bleeding issues as a result of abnormal syndrome)
dense granules in platelets; death occurs before the age of
10 years LAD I
MORPHOLOGIC ABNORMALITIES OF NEUTROPHILS WITH AND ➢ Mutation in the ITGB2 gene; gene that encodes CD18 subunit
WITHOUT FUNCTIONAL DEFECTS of b2 integrins, resulting in either a decreased or truncated
MORPHOLOGIC MORPHOLOGIC CLINICAL form of the b2integrin, which is necessary for adhesion to
ABNORMALITIES CHANGES FINDINGS endothelial cells, recognition of bacteria, and outside-in
PELGER HUET Decreased Asymptomatic signaling
ANOMALY nuclear ➢ Shortly after birth, patients suffer from recurrent infections,
segmentation in often affecting skin and mucosal infections
neutrophils ➢ Lymphadenopathy, splenomegaly, and neutrophilia are
sometimes also common findings
affects other
WBCs LAD II
PSEUDO PELGER Decreased Depends on ➢ Mutation in the SLC35C1 gene; leukocytes have normal b2
HUET ANOMALY nuclear underlying integrins
segmentation in condition ➢ Defective fucose transporter and selectin synthesis
neutrophils ➢ Without fucose transporter, there would be no
NETROPHIL >5 nuclear lobes Depends on selectin
HYPERSEGMENTATI in neutrophils underlying cause ➢ Patients have recurring infections, neutrophilia, growth
ON retardation, a coarse face, and other physical deformities
ALDER REILLY Granulocytes Normal
ANOMALY contain large, neutrophil LAD III
darkly staining function. Clinical ➢ Caused by mutations in Kindlin-3 ; Kindlin-3 protein along with
metachromatic findings, if talin are required for activation of b integrin and leukocyte
cytoplasmic present, rolling
granules Depends on ➢ Leukocytes and platelets have normal expression of integrins;
underlying however, there is failure in response to external signals that
condition normally results in leukocyte activation
CHEDIAK HIGASHI Giant lysosomal Leukocyte ➢ LAD III patients experience a mild LAD I-like
DISEASE granules in dysfunction and immunodeficiency with recurrent infections
granulocytes, recurrent ➢ Additionally, there is decreased platelet glycoprotein
monocytes and pyogenic IIb/IIIa), resulting in bleeding similar to that seen in
lymphocytes infection. Glanzmann’s thrombasthenia
Bleeding due to
abnormal dense WHIM SYNDROME

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 72

 ➢ Warts, hypogammaglobulinemia, infections, and ➢ Absence of MPO slows down bactericidal killing
myelokathexis syndrome
➢ Defect in intrinsic and innate immunity LYSOSOMAL STORAGE DISORDERS
➢ Mutations in the CXCR4 gene ➢ Are a group of more than 50 inherited enzyme deficiencies
➢ CXCR4 protein regulates movement of white blood resulting from mutations in genes that code for the
cells (WBC) between the bone marrow and peripheral production of lysosomal enzymes
blood ➢ The result is flawed degradation of phagocytized material
➢ Neutrophils accumulate in the bone marrow and buildup of undigested substrates within lysosomes
(myelokathexis), which results in low numbers of ➢ This causes cell dysfunction, cell death, and a range of
circulating neutrophils clinical symptoms; all cells containing lysosomes can be
➢ In addition to neutropenia, lymphopenia, affected
monocytopenia, and hypogammaglobulinemia are ➢ LSDs are classified according to the undegraded
present; as a result, patients experience recurrent macromolecule that accumulates in the cell
bacterial infections and are highly susceptible to ➢ EXAMPLES:
human papillomavirus (HPV) infection, which leads to ✓ LIPID STORAGE DISEASE/SPHINGOLIPIDOSES
warts ✓ MUCOPOLYSACCHARIDOSES
➢ HPV→ causes warts
➢ WHIM→ lymphocytes cannot escape the BM LIPID STORAGE DISEASES / SPHINGOLIPIDOSES
➢ The macrophages are particularly prone to accumulate
undegraded lipid products, which subsequently leads to an
expansion of the reticuloendothelial tissue
DEFECTIVE RESPIRATORY BURST ➢ qualitative disorders involving monocytes and macrophages
➢ Chronic granulomatous disease (CGD) ➢ EXAMPLES:
➢ Congenital c3 deficiency ✓ GAUCHER’S DISEASE
➢ G-6pd deficiency ✓ NIEMANN-PICK’S DISEASE
➢ Myeloperoxidase (MPO) deficiency
GAUCHER’S DISEASE
CHRONIC GRANULOMATOUS DISEASE (CGD) ➢ most common of the lysosomal lipid storage diseases ; at
➢ A rare condition caused by the decreased ability of least 1 in 17 Ashkenazi Jews are carriers
neutrophils to undergo a respiratory burst after phagocytosis ➢ It is an autosomal recessive disorder caused by a defect or
of foreign organisms deficiency in the catabolic enzyme beta-
➢ Can be X-linked recessive (60%) or autosomal recessive (40%) glucocerebrosidase
➢ Caused by mutations in genes responsible for proteins that ➢ Accumulation in sphingolipid glucocerebroside in
make up the reduced form of nicotinamide adenine macrophages throughout the body, including osteoclasts in
dinucleotide phosphate (NADPH) oxidase bone and microglia in the brain
➢ Patients experience life-threatening catalase-positive ➢ Bone marrow replacement by Gaucher cells contribute to
bacterial and fungal infections anemia and thrombocytopenia
➢ Pseudo-Gaucher cells can be found in bone marrow of some
➢ DETECTION OF RESPIRATORY BURST patients with thalassemia, chronic myeloid leukemia, acute
✓ Chemiluminescence → uses dihydrorhodamine to lymphoblastic leukemia, non-Hodgkin lymphoma, and
measure intracellular production of reactive oxygen plasma cell neoplasms
species CLINICAL SUBTYPES OF GAUCHER DISEASE
✓ NITROBLUE TETRAZOLIUM (NBT) TEST TYPE I: NON TYPE II: TYPE III:
• NBT is a yellow, water-soluble dye NEURONOPAT ACUTE SUBACUTE
• Normal: REDUCED NBT because of the generation of HIC NEURONO NEURONOPAT
respiratory burst (+BLUE FORMAZAN) PATHIC HIC
• PATIENTS WITH CGD: UNREDUCED (COLORLESS-YELLOW) AGE AT Childhood/a Infancy Childhood/a
❖ X-linked→ males are affected while females are the PRESENTATION dulthood dulthood
only carrier HEPATOSPLEN +→+++ + +→+++
❖ If NBT dye test is reduced→ it would be color blue→ OMEGALY
patient is normal SKELETAL -→+++ - ++→+++
❖ Unreduced→ remain yellow→ CGD ABNORMALITY
CNS DISEASE - +++ +→+++
CONGENITAL C3 DEFICIENCY LIFE SPAN 6-80 yrs <2 yrs 2-60 yrs
➢ Autosomal recessive ETHNICITY Ashkenazi Panethnic Panethnic,
➢ Heterozygous: carriers have half the normal C3 activity Jews Swedes
(adequate for disease resistance)
➢ Homozygous: repeated severe infections with encapsulated
NIEMANN-PICK’S DISEASE
bacteria which are poorly recognized and inefficiently
➢ Characterized by accumulation of sphingomyelin in cellular
phagocytized because of failure of opsonization by C3
lysosomes in the liver, spleen, and lungs
➢ Deficiency in the enzyme acid sphingomyelinase (ASM)
G-6PD DEFICIENCY
➢ Associated with foam cells and sea-blue histiocytes in the
➢ Absence affects the: HEXOSE MONOPHOSPHATE SHUNT
bone marrow
➢ Leukocytes are unable to produce a respiratory burst,
➢ TYPES:
resulting in a DEFECTIVE BACTERICIDAL ACTIVITY
NEURONOPAT MUTATION DISEASE
➢ G6PD is needed for the production of NADPH oxidase
HIC FORM
TYP Acute <5% normal Present in
MYELOPEROXIDASE (MPO) DEFICIENCY
E A neuronopathic sphingomyelinase infancy and is
➢ Autosomal recessive; also known as ALIUS-GRIGNASHI
form; affects activity; mutation associated with
ANOMALY
mostly Eastern in the SMPD1 failure to thrive,
➢ MPO is low or absent in neutrophils and monocytes but not
Europhean gene lymphadenopa
in eosinophils
Jews thy,

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 73

 hepatosplenom Sea-blue Unknown
egaly, vision Histiocytes
problems, and
rapid MUCOPOLYSACCHARIDES
neurodegenera ➢ family of inherited disorders of mucopolysaccharide or
tive decline that glycoaminoglycan (GAG) degradation
results in death, ➢ Each MPS is caused by deficient activity of an enzyme
usually by 4 necessary for the degradation of dermatan sulfate, heparan
years of age sulfate, keratan sulfate, and/or chondroitin sulfate
TYPE B Non- 10-20% Presents in the first ➢ The partially degraded material builds up in lysosomes and
neuronopathic normal decade to results in serious physical and cognitive problems and
form; more enzyme adulthood with a shortened survival
common in activity; variable clinical ➢ Presence of Alder-Reilly bodies in neutrophils, monocytes,
individuals of mutation in course and lymphocytes; macrophages in the bone marrow can
Northern the SMPD1 also demonstrate cytoplasmic metachromatic material
African gene Although there is
descent no neurocognitive MUCOPOLYSACCHARIDOSES DISORDERS
impairment, NA SUBTYP ENZYME AS GENE CLINICAL
patients ME E DEFIECIENCY TICS FEATURES
experience ENZYM
massive E
hepatosplenomeg DEFICI
aly, heart disease, ENCY
and pulmonary MP Hurler a-I- DS IDUA Coarse
insufficiency SI syndro iduronidase +HS 4P16. facies, short
TYPE Mutations in Causes impaired me 3 stature,
C the NPC1 or cellular trafficking corneal
NP2 gene and homeostasis clouding
of cholesterol; MP Scheie a-I- DS IDUA Hepatosplen
buildup of SI syndro iduronidase +HS 4P16. omgealy,
unesterified me 3 joint
cholesterol in contractures
lysosomes MP Hunter Iduronate DS IDS Coarse facial
S II syndro sulfatase +HS Xq28 features,
Clinical me short stature
presentation in MP Sanfilip Heparan N- HS SGSH Hirsutism,
type C NP is S III po sulfatase 17q2 coarse facial
heterogeneous A syndro 5.3 features
with regard to age me
of onset and type MP Sanfilip a-N- HS NAG Cardiomegal
and severity of S III po acetylglucos LU y, coarse
neurologic and B syndro aminidase 17q2 facial
psychiatric me 1.2 features
symptoms, as well MP Sanfilip Heparan HS HGS Delayed
as visceral S III po acetyl-CoA: NAT psychomotor
involvement C syndro a- 8P11. developmen
me glucosaminid 21 t
Prognosis in type C ase N-
NP is poor, with acetyltransfer
most patients ase
dying before the MP Morqui Galactose-6- KS GAL Muscoskelet
age of 25 years S IV o sulfatase + NS al
A syndro CS 16q2 abnormalitie
OTHER LIPID STORAGE DISEASES me 4.3 s
DISEASES DEFICIENT ACCUMULATED MP Morqui B- KS GLB1 Muscoskelet
ENZYME SUBSTANCE S IV o galactosidas 3P22. al
Tay-Sach’s Hexosaminidase GM2-gangliosides B syndro e 3 abnormalitie
Disease A me s
Sandhoff’s
Disease
Sandhoff’s Hexosaminidase Globosides INHERITED DISORDERS OF LYMPHOCYTES
Disease B ➢ B-Cell Deficiencies
GM1- GM1-Beta- GM1-gangliosides o Bruton-Tyrosine Kinase Deficiency
Gangliosidosis Galactosidase o Common Variable Hypogammaglobulinemia
Fabry’s Disease Alpha- Ceramide ➢ T-Cell Deficiencies
Galactosidase Trihexoside o 22q11 Syndromes
Metachromatic Arylsulfatase A Sulfatide ➢ Severe Combined Immunodeficiency Disease
Leukodystrophy o X-Linked Scid
Krabbe’s Disease Beta- Galactocerebroside o Ada Deficiency
Galactosidase o Wiskott-Aldrich Syndrome
Farber’s Disease Ceramidase Ceramides ➢ Others

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 74

B-Cell Deficiencies
ADENOSINE DEAMINASE DEFICIENCY (ADA) DEFICIENCY
BRUTON-TYROSINE KINASE (BTK) DEFICIENCY ➢ Autosomal recessive adenosine deaminase deficiency
➢ Also called as X-linked agammaglobulinemia, Bruton’s represents 10% to 20% of SCID cases and is caused by one of
agammaglobulinemia many mutations in the ADA gene
➢ Is a primary immunodeficiency disease characterized by ➢ ADA deficiency results in an intra- and extracellular
reductions in all serum immunoglobulin isotypes and accumulation of adenosine, which is lymphotoxic, leading to
profoundly decreased or absent B cells profound decreases in T, B, and NK cells
➢ BTK deficiency is caused by a mutation in the gene encoding ➢ Patients experience a range of recurring, life-threatening
Bruton tyrosine kinase, resulting in decreased production of bacterial, viral, and fungal infections beginning early in life.
BTK, which is important for B cell development, In addition, there are skeletal abnormalities, neurologic
differentiation, and signaling deficits, and skin rashes
➢ Without BTK, lymphocytes fail to fully mature, leading to
severe hypogammaglobulinemia and an inability to WISKOTT-ALDRICH SYNDROME
produce specific antibodies. ➢ Rare X-linked disease caused by one of more than 400
➢ Infants with BTK deficiency display symptoms between 4 and mutations in the WAS gene, which results in decreased levels
6 months, once maternal antibodies have cleared.; recurring of WASp protein
life-threatening bacterial infections ensue ➢ WASp is important in cytoskeletal remodeling and nuclear
➢ Risk of fungal and viral (except enterovirus) infection is low transcription in hematopoietic cells
because of normal T cell function ➢ T cells are decreased; B cells, T cells and NK cells, neutrophils
and monocytes are dysfunctional which leads to bacterial,
COMMON VARIABLE HYPOGAMMAGLOBULINEMIA viral and fungal infections
➢ One or a combination of immunoglobulins is either missing ➢ Risk of bleeding due to thrombocytopenia and small
entirely or is synthesized in small quantities abnormal platelets
➢ Inability of B-cells to mature to plasma cells which can be
excessive production of T-suppressor cells OTHERS
SEX-LINKED AGAMMAGLOBULINEMIA
T-CELL DEFICIENCIES ➢ A defect in the helper cellular immune mechanism leading
to agammaglobulinemia
22q11 SYNDROMES ➢ Defect is seen only in boys
➢ All the disorders within the 22q11 deletion syndrome (mostly
associated with the TBX1 gene) have variable degrees of SWISS-TYPE AGAMMAGLOBULINEMIA
immunodeficiency because of the absence or decreased ➢ Autosomal recessive; involves loss of both T-cell and B-cell
size of the thymus and low numbers of T lymphocytes functions
➢ Associated with a broad range of problems such as cardiac ➢ Little or no immunoglobulin is found in the blood; thymus is
defects, palatal abnormalities, distinctive facial features, present but lacks lymphoid elements
developmental delays, psychiatric disorders, short stature,
kidney disease, and hypocalcemia ATAXIA TELANGIECTASIA
➢ Hematologic issues include thrombocytopenia and large ➢ Autosomal recessive
platelets, autoimmune cytopenia’s, and increased risk of ➢ Ataxia: progressive loss of muscle coordination
malignancy ➢ Telangiectasia: dilation of the small blood vessels
➢ Death rate is high usually before 1 year of age ➢ Associated with peripheral blood lymphopenia, aplastic or
➢ EXAMPLES: hypoplastic thymus with minimal T-cell population
✓ Nezelof’s syndrome ➢ Decreased IgA and IgE, normal to increased IgG
✓ DiGeorge syndrome
✓ Autosomal dominant Opitz GBBB QUANTATIVE NON-MALIGNANT LEUKOCYTE DISORDERS
✓ Sedlackova syndrome REACTIVE STATE ASSOCIATED CONDITION
✓ Caylor cardiofacial syndrome NEUTROPHILIA Acute inflammatory—collagen
✓ Shprintzen syndrome Absolute count: >7-8 x vascular, vasculitis
✓ Conotruncal anomaly face syndrome 10 9/L Acute infectious—bacterial, some
viral, fungal, parasitic
SEVERE COMBINED IMMUNODEFICIENCY DISEASE Drugs, toxins, metabolic—
corticosteroids, growth factors,
X-LINKED SCID uremia, Ketoacidosis
➢ Gamma chain deficiency, or X-linked SCID, is the most Tissue necrosis—burns, trauma, MI,
common form of SCID and is caused by mutations in the RBC hemolysis
IL2RG gene Physiologic—stress, exercise,
➢ IL2RG normally codes for the common gamma chain in smoking, pregnancy
leukocyte receptors that bind with interleukins 2, 4, 7, 9, 15 Neoplastic—carcinomas,
and 21 (provide growth, differentiation, and survival signals sarcomas, myeloproliferative
for B, T, and NK cells) disorders
➢ Patients become symptomatic between 3 to 6 months of NEUTROPENIA Drugs—cancer chemotherapy,
age as protective maternal immunoglobulins are depleted, Absolute count: <1.75 chloramphenicol, sulfas/other
presenting without tonsils or lymph nodes along with severe – 1.80 x 10 9/L antibiotics, phenothiazines,
life-threatening recurring infections Most common type of benzodiazepine, antithyroids,
➢ Circulating T and natural killer (NK) lymphocytes are nearly leukopenia anticonvulsants, quinine, quinidine,
absent; B cells are adequate in number but are dysfunctional indomethacin, procainamide,
➢ Children with gamma chain deficiency fail to thrive and AGRANULOCYTOSIS thiazides, Radiation
death usually occurs before age 2 unless treatment with • Extreme Toxins—alcohol, benzene
hematopoietic stem cell transplant is successful neutropenia (<0.5) compounds

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 75

• Infantile: Intrinsic defects—Fanconi’s,
Kostmann’s Kostmann’s, cyclic neutropenia,
syndrome Chédiak-Higashi
Immune-mediated—collagen
vascular disorders, RA, AIDS
Hematologic—megaloblastic
anemia, myelodysplasia, marrow
failure,marrow replacement
Infectious—any overwhelming
infection
Others—starvation, hypersplenism
EOSINOPHILIA Allergic—urticaria, hay fever,
Absolute count: >0.7 x asthma
10 9/L Inflammatory—eosinophilic fasciitis,
Churg-Strauss syndrome
Parasitic—trichinosis, filariasis,
**eosinopenia: ACTH schistosomiasis
administration (Thorn’s Nonparasitic infections—systemic
Test) fungal, scarlet fever, chlamydial
Churg-strauss pneumonia of infancy
syndrome—Increased Respiratory—pulmonary
eosinophilic syndromes (Löffler’s,
tropical pulmonary eosinophilia),
Churg-Strauss syndrome
Neoplastic—CML, Hodgkin
lymphoma, T cell lymphomas
diopathic hypereosinophilic
syndromes—affecting heart, liver,
spleen, CNS, other organs
Others—certain drugs, hematologic
and visceral malignancies, GI
inflammatory diseases, sarcoidosis,
Wiskott-Aldrich syndrome
BASOPHILIA Myeloproliferative disease
Absolute count: Allergic—food, drugs, foreign
>0.3x109/L proteins
Infectious—variola, varicella
Chronic hemolytic anemia—
especially post splenectomy
Inflammatory—collagen vascular
disease, ulcerative colitis
MONOCYTOSIS Infectious—tuberculosis, subacute
Absolute count: bacterial endocarditis, syphilis,
>0.9x109/L protozoan, rickettsial
Recovery from neutropenia
Hematologic—leukemias,
myeloproliferative disorders,
lymphomas, multiplemyeloma
Inflammatory—collagen vascular
disease, chronic ulcerative colitis,
sprue,
myositis, polyarteritis, temporal
arteritis
Others—solid tumor, immune
thrombocytopenic purpura,
sarcoidosis
LYMPHOCYTOSIS Infectious—many viral, pertussis,
Absolute lymphocyte tuberculosis, toxoplasmosis,
count in: rickettsial
• Adults: >4.0x109/L Chronic inflammatory—ulcerative
• infants and young colitis, Crohn’s
children: Immune mediated—drug sensitivity,
>9.0x109/L vasculitis, graft rejection, Graves’,
**CMV- resembles RS Sjögren’s
cells Hematologic—ALL, CLL, lymphoma
Stress—acute, transient

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 76

 WBC ANOMALIES APPEARANCE AFFECTED CELLS ASSOCIATED CONDITIONS MISCELLANEOUS
INVOLVED
(size, color, shape,
inclusions)

GRANULOCYTES:

CYTOPLASMIC ABNORMALITIES

• Inflammations, Infections,
TOXIC GRANULES • Appears as dark, blue- Neutrophils Toxic states, Burns, Malignant • primary/azurophilic granules are larger
black granules in the disorders than secondary granules
cytoplasm of neutrophils,
usually in segmented and Associated with toxic vacuoles (can be
band forms associated. with auto phagocytosis)

•Granulation may •peroxidase positive, and reflect an

represent the precipitation increase in acid mucosubstance within
of ribosomal protein (RNA) primary, azurophilic granules that may
caused by metabolic enhance bactericidal activity
toxicity within the cells
•Toxic granulation can mimic granulation
found in Alder-Reilly anomaly; one helpful
defining characteristic of toxic
granulation is that in most cases, not all
neutrophils are equally affected

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 77

DOHLE-BODIES/ DOHLE-AMATO •light/pale blue round or •Typically found in •severe infections, toxic states, •Periodic acid schiff positive
BODIES elongated cytoplasmic segmented/band burns, pregnancy
inclusions between 1-5 um neutrophils •Confused with May-Hegglin bodies (PAS
consisting of remnants of negative)
ribosomal ribonucleic acid •Can also be seen in
(rRNA) arranged in parallel eosinophils, •A delay in preparing the blood film after
row; close to cellular basophils, collection may affect Dohle body
membranes monocytes, and appearance in that they are more grey
lymphocytes than blue or in some cases may not be
visible

Can be seen in all

CHEDIAK-HIGASHI GRANULES •Giant red, blue, to grayish leukocytes but Chediak-Higashi syndrome •Poor chemotactic activity (poor
round inclusions in the mostly affected are (autosomal recessive), Albinism, directional motility)
cytoplasm (giant lysosomal neutrophils Severe recurrent life-threatening
granules) bacterial infections •Deficiency in enzymes in the lysosomes
required for phagocytsosis (lysosomal
defect)

MAY-HEGGLIN GRANULES • Gray-blue spindle shaped Neutrophils Infections, Thrombocytopenia, • The basophilic Dohle body-like
inclusions in the cytoplasm leukopenia, May-Hegglin leukocyte inclusions are composed of
anomaly precipitated myosin heavy chains (mRNA
•Composed of mRNA in older references)

• Presence of giant • True Dohle bodies consist of lamellar

platelets (abnormal rows of rough endoplasmic reticulum
platelets= lack dense (rRNA)
granules)
•PAS negative
• Resembles Dohle bodies
(smaller).

ALDER-REILLY GRANULES/BODIES Darkly staining • May be found in • Alder Reilly anomaly • May resemble toxic granules (seen in
metachromatic (purple- all leukocytes with (autosomal recessive) infections and toxic states)
red) cytoplasmic granules dense granules
in granulocytes (less often in • Mucopolysaccharidoses, • Accumulation of: degraded
monocytes and hunter’s syndrome, Hurler’s mucopolysaccharides due to an enzyme
lymphocytes syndrome defect

• Abnormal large primary

granules

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 78

AUER RODS • Pink or red shaped • found in myeloid • Acute myelogenous • Fused primary granules (peroxidase
cytoplasmic granules and monocytic leukemia (AML, M1 and M2), stain positive)
series only Acute promyelocytic leukemia
(APL, M3), acute Acute • Faggot cells: bundle of auer rods (seen
myelomonocytic leukemia in acute promyelocytic leukemia; M3;
(AMML, M4) also associated with DIC)

•Bacterial infection
PYKNOTIC CELLS • In a pyknotic nucleus, •Neutrophils •Pyknotic nuclei in neutrophils generally
water has been lost and indicate imminent cell death
•Drug intoxication
the chromatin becomes
dense and dark; however,
chromatin or filaments can
still be seen between
nuclear lobes (depending
on whether the cell is a
band or segmented form)

•Bacterial infection
NECROTIC CELLS •rounded nuclear •Neutrophils •Necrotic nuclei are found in dead
fragments with no filaments neutrophils/necrobiotic neutrophils
•Drug intoxication
and no chromatin pattern

CLEAR ABNORMALITIES APPEARANCE AFFECTED CELLS ASSOCIATED CONDITIONS MISCELLANEOUS

INVOLVED
(size, color, shape,
inclusions)

HYPERSEGMENTED NEUTROPHIL • Neutrophil has more than Neutrophil •Megaloblastic anemia • Abnormal DNA synthesis
5 lobes (acquired)

•Undritz anomaly →Hereditary

neutrophil hypersegmentation

• Myelodysplasia

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 79

 Nuclei may appear:
PELGER-HUET All leukocytes but •Preleukemia/ Myelodysplastic • Hyposegmentation can also be
mostly affected are syndromes, Pseudo pelger-Huet caused by shift to the left
• round, ovoid, or peanut
Neutrophils (seen in myeloproliferative
shaped (Homozygous
disorders; CML), Pelger-Huet
Pelger-Huet)
anomaly

• Bilobed forms the

characteristic spectacle-
like (“pince-nez”)
morphology with the nuclei
attached by a thin filament
(Heterozyous Pelger-Huet)

LE CELL • Neutrophil with large Neutrophil •Systemic Lupus Erythematosus • Usually mistaken with a tart cell
purple homogenous round (monocyte with an ingested
inclusion with engulfed lymphocyte)
nucleus of another cell
(usually another neutrophil)

• Presence of ANTI-
NUCLEAR ANTIBODIES, cell
nuclei, phagocytes with
ingested material

LYMPHOCYTES APPEARANCE AFFECTED CELLS ASSOCIATED CONDITIONS MISCELLANEOUS

INVOLVED
(size, color, shape,
inclusions)

BASKET CELL/SMUDGE CELL • Degenerated nucleus or Lymphocytes Chronic Lymphocytic leukemia • Fragile lymphocytes that appear during
ruptured cell in form or smear preparation (appear at the end of
basket/smudge smears)

• Thumbprint appearance

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 80

 Lymphocytes • Isoenzyme 5 (tartrate resistant is
HAIRY CELL • Hair like cytoplasmic Hairy cell leukemia produced in abundance in hairy cell
projections surrounding the leukemia)
nucleus (fried-egg
appearance)
• TRAP (Tartrate-resistant acid
phosphatase stain) positive

Lymphocytes
SEZARY CELL • Round lymph cell with • Mycosis fungoides (cutaneous • Presence indicates leukemic phase of
convoluted nucleus (brain- T-cell lymphoma), Sezary mycosis fungoides
like nucleus) syndrome (variant of mycoses
fungoides)

Lymphocytes
FLAME CELL • Plasma cell with red to • Multiple myeloma, • Associated with increased
pink cytoplasm Waldenstrom’s Immunoglobulins (usually IgA)
macroglobulinemia
•Inclusion: Russel bodies
(individual bodies of
Immunoglobulin)

•Lymphocytes-
GRAPE CELL • Plasma cell with small Plasma cells •Multiple myeloma; reactive •It is also known as Berry, Mott and
colorless vacuoles states Morula Cell

•Large protein globules

giving appearance of
grapes: “Honeycomb
appearance

•Inclusions:”Dutcher’s
bodies (intranuclear protein
inclusions)

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 81

 Lymphocytes
REED-STERNGBERG CELLS • “owl’s eye appearance” Hodgkin’s Lymphoma • Pathognomonic sign for Hodgkin
Lymphoma

Lymphocytes
RIEDER CELL • Lymphocyte with a clover Chronic lymphocytic leukemia Abnormal myeloblasts in which the
leaf like nucleus nucleus may be widely and deeply
indented or may actually be a bilobate
or multilobate structure

REACTIVE/VARIANT/ATYPICAL • Type I: turk’s irration Lymphocytes infectious mononucleosis, • T-cells reacting to B-cells infected with
LYMPHOCYTE/ DOWNEY CELLS plasma cytoid; lymphocyte Leukemia, viral infections Epstein-Barr virus (EBV)
with a large block of
chromatin •Reactive changes in lymphocyte
morphology occur as lymphocytes are
• Type II: also known as IM stimulated when interacting with antigens
cells; round mass of in peripheral lymphoid organs
chromatin (ballerina skirt
appearance)

• Type III: vacuolated (swiss

cheese appearance)

MACROPHAGES APPEARANCE AFFECTED CELLS ASSOCIATED CONDITIONS MISCELLANEOUS

INVOLVED
(size, color, shape,
inclusions)

GAUCHER CELLS Within the BM; large Macrophages Gaucher’s disease (def. in the Positive with trichrome, aldehyde fuchsin,
macrophages with enzyme B glucocerebrosidase) periodic acid-Schiff (PAS) and acid
crumpled tissue paper phosphatase
appearance

abundant fibrillar blue-gray

cytoplasm with a striated or
wrinkled appearance

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 82

 (sometimes described as
onion skin-like)

FOAM CELLS •Macrophages whose Macrophages Niemman Pick’s disease (def. in The small vacuoles or foam seen in the
cytoplasm is filled with small the enzyme acid cytoplasm after staining are lipid filled
lipid droplets sphingomyelinase) lysosomes.

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 83

WEEK 15: INTRODUCTION TO PROGRESSION
WITHOUT
Rapid; weeks to
months
Slower; months
to years
HEMATOLOGIC NEOPLASMS TREATMENT
HISTORY OF HEMATOLOGIC NEOPLASMS
• Hematologic neoplasms were the first human cancers
in which a consistent genetic defect was identified LYMPHOMA
• Started during the time of Hippocrates • solid tumors of lymphoid cells that usually originate in
• 1839-1845 - Virchow was the first to recognize the lymphatic system and proliferate in lymph nodes
leukemia as a distinct clinical disorder. He named this and other lymphoid organs and tissues
disorder leukemia because of the white appearance
MYELOMA (PLASMA CELL NEOPLASMS)
of the blood from patients with fever, weakness, and • Cancer of the plasma cells; In myeloma, the cells
lymphadenopathy overgrow, forming a mass or tumor that is located in
• 1960 - Nowell and Hungerford published an abstract the bone marrow
that described a consistent shortened chromosome in
seven patients with chronic myeloid/myelogenous
leukemia (CML); referred to as the Philadelphia
chromosome
• 1973 – Rowley reported the t(9;22) translocation in MYELODYSPLASTIC SYNDROMES (MDS)
CML • group of acquired clonal hematologic disorders
• 1982 –Taub and colleagues reported the t(8;14) characterized by progressive cytopenias in the
translocation in Burkitt lymphoma peripheral blood, reflecting defects in erythroid,
myeloid, and/or megakaryocytic maturation

HEMATOLOGIC NEOPLASMS BY DEFINITION COMPARATIVE FEATURES OF LEUKEMIAS AND LYMPHOMAS

• The term leukemia is derived from the ancient Greek Leukemias Lymphoma Myelomas
words leukos (leykóç), meaning “white,” and haima s
(aἷma), meaning “blood” Basic Overproductio Solid Overproducti
• include leukemias, lymphomas, myelomas (plasma characteris n of various malignant on of plasma
cell neoplasms) and myelodysplastic syndromes tic types tumors of cells in the
(previously called preleukemias) immature/mat the lymph bone marrow
• Terms that refer to large heterogeneous groups of ure leukocytes nodes with
in the bone concurrent
disorders; they initiate in a hematopoietic cell as a
marrow/ or production of
result of ACQUISITION OF ONE OR MORE MUTATIONS IN
peripheral abnormal
KEY GENES that regulate cell growth (proliferation), blood, in most proteins
survival, differentiation, or maturation types of
• Occur in hematopoietic cells of all lineages and at leukemias
various stages of their development Cell type Usually involves Lymphocyt Plasma cells
• Most hematologic neoplasms are not localized but leukocytes of e is the
rather are systemic at initiation of the malignant the distinctive
process myelogenous cell type.
or lymphocytic Reed
LEUKEMIA cell types Sternberg
• originate in bone marrow, and leukemia cells readily cells are
pass into peripheral blood, but they can also infiltrate diagnostic
lymphoid tissues (spleen, liver, lymph nodes) as well as type
other organs and tissues of the body lymphoma
• are divided into lymphoid and myeloid lineages, and Site of Malignant cells Malignant Plasma cells
further into acute (precursor cell) and chronic (mature malignant freely trespass cells are form a mass
cell) categories cells the blood initially or tumor that
brain barrier confined to is located in
the organs the bone
containing marrow
mononucle
ar
GENERAL CHARACTERISTICS OF ACUTE AND CHRONIC phagocyte
LEUKEMIA cells such
ACUTE ELEUKEMIA CHRONIC as the
LEUKEMIA lymph
PREDOMINANT Precursor cell or Mature nodes,
CELL TYPE blast spleen,
ONSET Sudden Insidious liver, and
SYMPTOMS AT Fever (neutropenia), Variable, non- bone
PRESENTATION mucocutaneous specific; some marrow
bleeding asymptomatic Notes Lymphoma
(thrombocytopenia), s can spill
fatigue (anemia) over into
WBC COUNT Variable Increased the
circulating

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 84

 blood and ✓ Predominance of blasts cells (>20% for WHO;
present a >30% for FAB) in about 50% of patients
leukemic- (lymphoblasts with lymphocytes and smudge
appearing cells)
picture on ✓ Granulocytopenia
a PBS ✓ IMMUNOPHENOTYPING:
▪ B-CELL ALL: CD 10, 19, 22, 34, TdT
▪ T-CELL ALL: CD2, 3, 4, 5, 7, 8, TdT
ACUTE LEUKEMIAS
• Refers to the rapid, clonal proliferation in the bone • CLINICAL FINDINGS:
marrow of lymphoid or myeloid progenitor cells known ✓ Present with fatigue (caused by anemia),
as lymphoblasts and myeloblasts fever (caused by neutropenia and infection),
• When proliferation of blasts overwhelms the bone and mucocutaneous bleeding (caused by
marrow, blasts are seen in the peripheral blood and thrombocytopenia)
the patient’s symptoms reflect suppression of normal ✓ Lymphadenopathy, including enlargement, is
hematopoiesis often a symptom
• Onset is sudden, progression is rapid, and the outcome ✓ Splenomegaly and hepatomegaly
is fatal in weeks or months if left untreated ✓ Bone pain often results from intramedullary
• TYPES: growth of leukemic cells
✓ Acute Myeloid / Myelogenous/ ✓ Eventual infiltration of malignant cells into the
Myeloblastic Leukemia (AML) Or ANLL (Acute meninges, testes, or ovaries occurs frequently,
Non-Lymphoblastic Leukemia) and lymphoblasts can be found in the
✓ Acute Lymphoblastic Leukemia (ALL) cerebrospinal fluid

• CLASSIFICATION SCHEMES FOR ACUTE LEUKEMIAS: WHO CLASSIFICATION OF ALL

✓ FRENCH-AMERICAN-BRITISH (FAB) • B-lymphoblastic leukemia/lymphoma (B-ALL) is
Classification subdivided into nine subtypes that are associated with
✓ WORLD HEALTH ORGANIZATION (WHO) recurrent cytogenetic abnormalities
Classification • T-lymphoblastic leukemia/lymphoma (T-ALL) have
abnormal gene rearrangements, none of the
FAB CLASSIFICATION abnormalities is clearly associated with specific
• Devised in the 1970’s biologic features
• Based on: bone marrow morphology, cytochemical
reactions, cytogenetics, T and B cell markers
(immunophenotyping)
• CRITERIA FOR DIAGNOSIS OF ACUTE LEUKEMIA: >30% of
blasts in the blood

WHO CLASSIFICATION
• GOLD STANDARD in classifying leukemias
• Based on: bone marrow morphology, cytochemical
reactions, immunologic probes of cell markers, clinical
manifestations, cytogenetics
• CRITERIA FOR DIAGNOSIS OF ACUTE LEUKEMIA: >20% of
blasts in the blood is associated with leukemia

ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)

• Is primarily a disease of childhood and adolescence,
accounting for 25% of childhood cancers and up to
75% of childhood leukemia (most common type of
leukemia in children) • The three subtypes are differentiated based on
• Peak incidence of ALL in children is between 2 and 5 morphology, including cell size, prominence of
years of age.; although ALL is rare in adults, risk nucleoli, and the amount and appearance of
increases with age; most adult patients are older than cytoplasm
50 years of age • TYPES:
• The subtype of ALL is an important prognostic indicator ✓ FAB L1 (children)
for survival; adults have a poorer outlook: 80% to 90% ✓ FAB L2 (older children and adults)
experience complete remission, but the cure rate is ✓ FAB L3 (patients with leukemia secondary to
<40% Burkitt’s lymphoma; t(8;14)
• Lymphoblasts stain PAS positive ; Sudan Black B (SBB)
and Myeloperoxidase (MPO) negative
MORPHOLOGICAL CLASSIFICATION AND CHARACTERISTICS
OF ALL
FAB SIZE OF NUCLEAR NUCLEOLI CYTOPLAS
• LABORATORY FINDINGS: TYP BLASTS SHAPE M
✓ WBC count of >50 x 109/ L; <15% of patients E
have extreme leukocytosis (WBC count of L1 Small Indistinct Scant Invinsible
>100 x 109/L)

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 85

 L2 Large, Indented, Large, Moderately o most often in teenaged males with a
heterogenou prominen abundant clefted mediastinal mass, elevated peripheral
s t blast counts, meningeal involvement,
L3 Large Regular Prominent Prominent, and infiltration of extra marrow sites
oval to , vacuoles
round basophilic IMMUNOPHENOTYPIC CHARACTERISTIC OF ALL
MORPHOLOGY OF ABNORMAL LYMPHOBLASTS SUBTYPE IMMUNOPHENOTYPE
• Lymphoblasts vary in size but fall into two morphologic EARLY (PRO/PRE B CELL) CD34, 19, cytoplasmic CD22,
types TdT
• The most common type seen is a small lymphoblast INTERMEDIATE(COMMON) B CD34, 19, 10 cytoplasmic
(1.0 to 2.5 times the size of a normal lymphocyte) with ALL CD22, TdT
scant blue cytoplasm and indistinct nucleoli PRE B ALL CD34, 19, cytoplasmic CD22,
• The second type of lymphoblast is larger (two to three TdT, cytoplasmic u
times the size of a lymphocyte) with prominent nucleoli T-ALL CD2,3,4,5,7,8,TdT
and nuclear membrane irregularities; these cells may
be confused with the blasts of AML

ACUTE MYELOID LEUKEMIA (AML)

• AML is the most common type of leukemia in adults,
and the incidence increases with age
• AML is less common in children
• The FAB classification of AML was based on
morphology and cytochemistry; the WHO
classification relies heavily on molecular
characterization and cytogenetics
• CLINICAL FINDINGS:
✓ Infiltration of malignant cells into the gums
and other mucosal sites and skin
✓ Splenomegaly is seen in half of AML patients,
but lymph node enlargement is rare
✓ Hyperuricemia (caused by increased cellular
turnover), hyperphosphatemia (due to cell
lysis), and hypocalcemia (the latter two are
also involved in progressive bone destruction)
✓ Hypokalemia is also common at presentation
✓ During induction chemotherapy, especially
when the WBC count is quite elevated, tumor
lysis syndrome may occur

• LABORATORY FINDINGS:
✓ Decreased production of normal bone
marrow elements
✓ WBC count: 5-30 x 109/L
IMMUNOPHENOTYPING ✓ Myeloblasts (>20% for WHO ; >30% for FAB)
• Although morphology is the first tool used to distinguish are present in the peripheral blood in 90% of
ALL from AML, immunophenotyping and genetic patients
analysis are the most reliable indicators of a cell’s ✓ Anemia, thrombocytopenia, and
origin neutropenia
• Because both B and T cells are derived from lymphoid ✓ IMMUNOPHENOTYPING: CD13, CD33, CD117,
progenitors, both usually express CD34, terminal CD14/CD64
deoxynucleotidyl transferase (TdT), and human
leukocyte antigen, DR subregion (HLA-DR) Clinical and Laboratory Features of Acute Myeloid Leukemia
• Affects all ages, but increases with older age (>60
• TYPES OF ALL BASED ON IMMUNOLOGIC METHODS: years)
✓ Early B cell ALL (pro-B or pre B cell ALL) • May resemble acute infection at presentation
o 5% in children and 11% in adults • Requires 20% blasts in blood or marrow for diagnosis
✓ Intermediate (common) B cell ALL • Key myeloid antigens myeloperoxidase, CD13,
CD33, CD 117, and CD14/CD64
o t(4;11)
• Recurrent cytogenetic abnormalities that
✓ Pre-B cell ALL
characterize defined subtypes
o t(9;22) • Classification made by morphology, cytogenetics,
o Most mature B cell ALL; TdT negative, flow cytometry, cytochemistry
CD34 variable
o 15% of childhood and 10% of adult B cell
ALL WHO CLASSIFICATION OF AML
• The 2017 WHO classification for myeloid malignancies
has categorized AMLs with recurrent cytogenetic
✓ T cell ALL
o t(7;11)
JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 86
 abnormalities into subgroups based on the primary
cytogenetic aberrations along with a few new entities

WHO Classification of Myeloid Leukemia and Related

Precursor Neoplasms
• Acute myeloid leukemia with recurrent genetic
abnormalities
• Acute myeloid leukemia with myelodysplasia-
related changes
• Therapy-related myeloid neoplasms
• Acute myeloid leukemia, not otherwise specified
• Myeloid sarcoma
• Myeloid proliferations associated with Down
syndrome
• Blastic plasmacytoid dendritic cell neoplasm
• Acute leukemias of ambiguous lineage
ACUTE MYELOID LEUKEMIA, WITHOUT MATURATION (FAB M1)
• May comprise >90% myeloblasts, and <10% of the
leukocytes show maturation to the promyelocyte
stage or beyond
• At least 3% of blasts give positive results with
myeloperoxidase or Sudan black B stains ; also stain
with CAE
• Markers present: CD13, CD33, CD117, CD34
• Presence of chloroma → green appearance of tissue
using MPO
• Auer rods may be present

FAB CLASSIFICATION OF AML

• FAB classification is synonymous with WHO
classification of “AML not otherwise specified”
• Based on:
✓ Bone marrow morphology
✓ Cytochemical reactions ACUTE MYELOID LEUKEMIA, WITH MATURATION (FAB M2)
✓ Cytogenetics • Similar to t(8;21)(q22;q22.1); RUNX1/RUNX1T1 mutation
✓ T and B cell markers (immunophenotyping) in WHO classification
• A common variant that presents with <90%,
myeloblasts and >10% maturing cells of neutrophil
ACUTE MYELOID LEUKEMIA, MINIMALLY DIFFERENTIATED (FAB M0) lineage, and <20% precursors with monocytic lineage
• Account for <5% of AML, and patients are generally • Auer rods are often present
either infants or older adults • Most blasts stain with MPO, SBB, and CAE
• Auer rods typically are absent, and there is no clear • Markers present: CD13, CD33, CD117, CD34
evidence of cellular maturation • FAB M1 and M2 account for 50% of AML cases
• Markers present are CD13, CD33, CD34, CD117
• Cells yield negative results with the cytochemical
stains myeloperoxidase and Sudan black B

ACUTE PROMYELOCYTIC LEUKEMIA (FAB M3)

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 • Comprises 5% to 10% of AML cases ; occurs in all age
groups but is seen most commonly in young adults
• Characterized by a differentiation block at the
promyelocytic stage
• The presence of >30% abnormal promyelocytes with
bundles of auer rods (faggot cells)
• Abnormal promyelocytes can be:
✓ Hypergranular → associated with DIC and
absence of HLA
✓ Hypogranular/Microgranular → aPML-M3v ;
20-30% of APL cases; presence of “butterfly”
or coin-on-coin nucleus
✓ Promyelocytes stain positive with SBB, MPO,
and CAE
• Cytogenetic abnormality involves balanced
translocation between chromosomes 15 and 17 ACUTE MONOCYTIC LEUKEMIA (FAB M5)
t(15;17) ; fusion of PML gene and retinotic acid • Characterized by >20% (WHO) or >30% (FAB) marrow
receptor alpha (RARa) or the PML-RARa gene monoblasts ; account for 10% of AML cases
• 95% of APL cases have the PML-RARa protein • In these leukemias, which are divided into monoblastic
• Treated with all-trans-retinoic acid (ATRA) and arsenic (M5a) and monocytic (M5b) based on the degree of
trioxide maturity of the monocytic cells present
• SUBTYPES:
✓ FAB M5a/Schilling’s type/AML poorly
differentiated/Acute monoblastic
leukemia/AML without maturation
▪ Seen in children ; >80% monoblasts in
the bone marrow
✓ FAB M5b/AML well differentiated/AML with
maturation
▪ Seen in middle-aged adults ; <80%
monoblasts
• Cytogenetic abnormality: t(8;16)(p11;p13) has been
associated with M5 (also M4)
• Markers present: CD14, CD4, CD11b, CD11c, CD 64
• Cells stain positive with NSE

ACUTE MYELOMONOCYTIC LEUKEMIA (FAB M4)

• characterized by a significantly elevated WBC count
and the presence of myeloid and monocytic cells in
the peripheral blood and bone marrow
• Characterized by >20% (WHO) or >30% (FAB) marrow
myeloblasts with >20%-80% cells of monocytic origin
(monoblast, promonocyte, monocyte); may have
Auer rods
• Also called as NAEGELI’S TYPE ; accounts for 30% of
AML cases
• FAB M4Eo → variant of FAB M4 with myeloblasts and
monoblasts along with abnormal eosinophils
• Markers present:
✓ Myeloid: CD13, CD33, CD34, CD117
✓ Monocytic: CD14, CD4, CD11b, CD11c,
CD64, CD36, CD68, and lysozyme
ACUTE ERYTHROLEUKEMIA (FAB M6)
• Myeloid cells stain with SBB, MPO, and CAE , • One of the major changes in the 2017 WHO
monocytic cells with NSE
classification is the removal of acute erythroleukemia
(erythroid/myeloid type) – most of these cases will now
be classified as MDS with excess blasts
• ACUTE ERYTHROLEUKEMIA
✓ Aka known as Erythemic myelosis, Di
Guglielmo’s syndrome

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 ✓ Characterized by >20% (WHO) or >30% (FAB)
marrow myeloblasts and >50% dysplastic
marrow normoblasts
✓ Markers present:
▪ Erythroblasts: CD45, Glycophorin A
(CD71)
▪ Myeloblasts: CD13, CD15, CD33,
CD117
✓ Myeloblasts stain positive with SBB, MPO, CAE
; erythroid cells are PAS-POSITIVE
• PURE ERYTHROID LEUKEMIA
• Remains as M6; characterized by >80% erythroid cells ;
>30% proerythroblasts
• RBC precursors have significant dysplastic features ;
presence of ringed sideroblasts, Howell-Jolly bodies
and other inclusions

ACUTE MEGAKARYOBLASTIC LEUKEMIA (FAB M7)

• Chacterized by the presence of at least >20% (WHO)
or >30% (FAB) blasts, of which at least 50% must be of
megakaryocyte origin ; Rarest type of AML
• Cells are stained with Alpha Naphthyl acetate
esterase (ANAE) and PAS
• Markers present: von Willebrand factor, CD41 (gp IIb) ,
CD42b (gp Ib), CD61 (gp IIIa)

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ACUTE OCCURRENCE PAS MPO WBC COUNT B CELL T CELL CLINICAL FINDINGS WHO FAB IMMUNOLOGIC
LEUKEMIAS & CLASSIFICATION CLASSIFICATION METHODS
SBB
ACUTE childhood + - >50 x 109/ L CD 10, CD2, 3, 4, Fatigue, fever, • B-ALL→ • FAB L1 • PRE/PRO B
LYMPHOBLASTIC and 19, 22, 5, 7, 8, TdT, mucocutaneous recurrent (children) CELL→ 5% in
LEUKEMIA (ALL) adolescence GRANULOCYTOPENIA 34, TdT, HLA, HLA- bleeding, cytogenetic • FAB L2 children and
25% of HLA, DR. CD34 Lymphadenopathy, abnormalities (older 11% in adults
childhood HLA-DR splenomegaly, • T-ALL→ children • INTERMEDIATE→
cancers hepatomegaly, abnormal gene and adults) t(4:11)
Bone pain, infiltration rearrangements • Mature B
• FAB L3
of malignant cells cells→ TdT (-),
(patients
into the meninges, CD34 variable
with
testes, or ovaries • T cells→ t(7:11)
leukemia
secondary
to Burkitt’s
lymphoma;
t(8;14)

ACUTE Adults 5-30 x 109/L IMMUNOPHENOTYPING Infiltration of recurrent ➢ M0-M7

MYELOBLASTIC malignant cells into cytogenetic
LEUKEMIA Decreased CD13, CD33, CD117, the gums and other abnormalities
(NALL) production of normal CD14/CD64 mucosal sites and
bone marrow skin, splenomegaly,
elements Hyperuricemia,
hyperphosphatemia,
hypocalcemia,
Hypokalemia, tumor
lysis syndrome

Anemia,
thrombocytopenia,
and neutropenia

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 ACUTE MYELOBLASTIC LEUKEMIA (NALL)
% OF AML & AUER RODS CELLULAR MATURATION MARKERS MPO AND SBB CAE STAIN CYTOGENETIC
PATIENTS STAIN ABNORMALITY
ACUTE MYELOID <5%, infants or ABSENT NOT EVIDENT CD13, CD33, - -
LEUKEMIA, MINIMALLY older adults CD34, CD117
DIFFERENTIATED (FAB
M0)

ACUTE MYELOID >90% myeloblasts, may be present <10% of the leukocytes CD13, CD33, 3% of blasts + +
LEUKEMIA, WITHOUT show maturation CD117, CD34 MPO→ chloroma
MATURATION (FAB M1) 50% of AML cases (green
appearance)
ACUTE MYELOID <90%, myeloblasts often present >10% maturing cells of CD13, CD33, + +
LEUKEMIA, WITH neutrophil lineage, and CD117, CD34
MATURATION (FAB M2) 50% of AML cases <20% precursors with
monocytic lineage
ACUTE 5% to 10% of AML bundles of auer >30% abnormal + + chromosomes
PROMYELOCYTIC cases, young adults rods (faggot promyelocytes 15 and 17
LEUKEMIA (FAB M3) cells) Hypergranular- DIC, t(15;17) ; fusion
ABSENCE HLA of PML gene
HYPOGRANULAR→ aPML- and retinotic
M3v, “butterfly” or coin- acid receptor
on-coin nucleus alpha (RARa)
or the PML-
RARa gene
TREATED WITH
all-trans-
retinoic acid
(ATRA) and
arsenic trioxide
ACUTE may have Auer significantly elevated WBC Myeloid: CD13, Myeloid: +
MYELOMONOCYTIC rods count CD33, CD34, Monocytic→ NSE only
LEUKEMIA (FAB M4) >20%-80% cells of CD117
/ NAEGELI’S TYPE monocytic origin
Monocytic: CD14,
64, 4, 11b, 11c, 36,
68, lysozyme
ACUTE MONOCYTIC 10% of AML cases ✓ AB M5a/Schilling’s CD 14, 64, 4, 11b NSE→ + t(8;16)(p11;p13)
LEUKEMIA (FAB M5) type/AML poorly and 11c
differentiated/Acute
monoblastic
leukemia/AML
without maturation/
monoblast
▪ Seen in
children ; >80%
monoblasts in

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 91

 the bone
marrow
✓ FAB M5b/AML well
differentiated/AML
with maturation.
Monocyte
▪ Seen in middle-
aged adults ;
<80%
monoblasts

ACUTE >50% dysplastic marrow Erythroblasts: Myeloblast→ + MPO, SBB, CAE

ERYTHROLEUKEMIA normoblasts→ MATURE CD45, ERYTHROBLAST→ PAS +
(FAB M6)/ Di RBC Glycophorin A
Guglielmo’s >80% erythroid cells ; >30% (CD71)
syndrome/ MDS proerythroblasts
presence of ringed Myeloblasts:
sideroblasts, Howell-Jolly CD13, CD33,
bodies and other CD117, CD15
inclusions
ACUTE at least 50% must be of von Willebrand Alpha Naphthyl acetate esterase
MEGAKARYOBLASTIC megakaryocyte origin factor, CD41 (gp (ANAE) and PAS +
LEUKEMIA (FAB M7) IIb) , CD42b (gp
RAREST TYPE Ib), CD61 (gp IIIa)

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 92

 ➢ Overproduction of one or more types of blood
WEEK 16: CHRONIC LEUKEMIAS cells with dominance of a transformed clone
CHRONIC LEUKEMIAS
➢ Hypercellular marrow or marrow fibrosis
❖ Abnormal mature cells are present (abnormal ➢ Thrombotic and/or hemorrhagic bleeding
progenitor cells)
➢ Extramedullary hematopoiesis
❖ Increased mature rather than immature cells in the
lymphoid or myeloid lines ➢ Transformation to acute leukemia

❖ The basic defect in growth control presumably affects ❖ Clonal→ genetic mutations→ neoplastic
progenitor cells, but in these disorders most neoplastic ❖ Chronic to acute→ the disease is progressive;
cells differentiate medication is not effective
❖ Progression is slower compared to acute leukemias ; ❖ Myelofibrosis→ scarring of bone marrow
can be months up to years
❖ Thrombotic→ excessive clot formation
TYPES:
❖ Extramedullary hematopoiesis→ can go to different
➢ MYELOPROLIFERATIVE NEOPLASMS (MPN’s) tissues in the body (outside the bm)
➢ MATURE LYMPHOID
NEOPLASMS/LYMPHOPROLIFERATIVE A. WHO CLASSIFICATION OF MPNs
DISORDERS
❖ BCR-ABL1 MUTATION
lymphoma,myeloma
➢ CHRONIC MYELOID LEUKEMIA (CML)
❖ Chapter 32 and 34
❖ JAK2V617F MUTATION
❖ Chronic leukemia→ lasts for months to years
➢ POLYCYTHEMIA VERA (PV)
❖ Leukemia→ abnormal cells are in the blood
➢ ESSENTIAL (PRIMARY) THROMBOCYTHEMIA (ET)
❖ Lymphoma→abnormal cells are in the lymphoid
tissues ➢ PRIMARY MYELOFIBROSIS (PMF)
❖ Myeloma→ abnormal cells are in the bone
marrow (plasma cells) CHRONIC MYELOID LEUKEMIA (CML)

❖ IN ACUTE→ MATURATION BLOCK→ BLAST IN BM ❖ CML is an MPN arising from a single genetic
AND BLOOD translocation in a pluripotential HSC producing a clonal
overproduction of the myeloid cell line
❖ CHRONIC→ BLAST WILL MATURE→ MANY Myeloid- refers to granulocytes only
ABNORMAL MATURE CELLS CML is also known as CHRONIC GRANULOCYTIC
❖ MATURE LYMPHOID NEOPLASMS→ LYMPHOMA LEUKEMIA
(TUMOR IN LYMPH NODES) AND MYELOMA (TUMOR ❖ CML occurs at all ages but is seen predominantly in
IN PLASMA CELLS) those aged 46 to 53 years; it represents about 20% of all
cases of leukemia, is slightly more common in men than
in women
❖ CML begins with a chronic clinical phase and, if
MYELOPROLIFERATIVE NEOPLASMS (MPNs) untreated, progresses to an accelerated phase in 3 to
4 years and often terminates as an acute leukemia
❖ are clonal mutated hematopoietic disorders caused by (blast crisis phase)
genetic mutations in the hematopoietic stem cells
(HSCs) that result in expansion, excessive production,
❖ Chronic granulocytic leukemia/ chronic myeloid
and accumulation of mature erythrocytes,
leukemia
granulocytes, platelets, and mast cells
❖ Myeloid→ referring only to granulocytes
❖ Each has a chronic course that may terminate as acute
leukemia (means progressive, worsen,ineffective
meds), myelofibrosis (scarring of BM), or a
CYTOGENETIC ABNORMALITY:
coagulopathy
➢ Presence of the BCR-ABL1 gene (Philadelphia
❖ Molecular analysis is now incorporated into the
chromosome/Ph’) BEQ
diagnosis workup of MPNs, research molecular
advances were capped by the discovery of the JAK2, ✓ Reciprocal
Janus kinase 2 (JAK2V617F mutation). This discovery translocation between the
allowed for refinement of the classification of MPNs long arms (q arm) of
chromosomes 9 and 22 → t
❖ Myeloproliferation largely is due to hypersensitivity or
(9;22) BEQ
independence of normal cytokine regulation resulting
from genetic mutations that reduces cytokine levels ✓ This acquired
through negative feedback systems normally induced somatic mutation
by mature cells specifically reflects the
translocation of an ABL1
❖ All of the MPNs involve dysregulation at the multipotent
proto-oncogene from
hematopoietic stem cell (CD34), with one or more of
band q34 of chromosome
❖ the following shared features: 9 to the breakpoint cluster region (BCR) of band
q11 of chromosome 22 (q34; q11)
➢ Cytogenetic abnormalities

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 93

 ✓ The BCR-ABL protein has constitutive kinase ✓ Based on AL definitions, blasts constitute more
activity that deregulates signal transduction than 20% of total BM cellularity, and the
pathways, causing abnormal cell cycling, peripheral blood exhibits increased blasts
inhibition of apoptosis, and increased
✓ Extramedullary growth may occur as
proliferation of cells
lymphocytic or myeloid cell proliferations; the
✓ Also found in FAB M1 (3-5%) and FAB L1 and L2 latter are often referred to as granulocytic
(10-25%)
sarcoma
✓ Clinical symptoms of blast crisis mimic those of
AL, including severe anemia, leukopenia of all
WBCs except blasts, and thrombocytopenia

B. PHILADELPHIA CHROMOSOME

C. CLINICAL FINDING S: D. LABORATORY FINDINGS:

➢ CHRONIC PHASE: ➢ BLOOD:

✓ Most cases (85%) are diagnosed in this phase ✓ Marked expansion of the granulocyte pool
✓ Anemia and thrombocytopenia (variable)
✓ Frequent infection, anemia secondary to
✓ Left shift with few blasts in the peripheral blood
massive pathologic accumulation of myeloid left shift: increased blast in blood
progenitor cells in bone marrow, peripheral ✓ Extramedullary hematopoiesis (spleen, liver)
blood, and extramedullary tissues ➢ BONE MARROW:
✓ NEUTROPHILIA WITH ALL MATURATIONAL ✓ intense hypercellularity
STAGES PRESENT, BASOPHILIA, EOSINOPHILIA ✓ Reticulin fibers are increased in
✓ Progressive fatigue and malaise, low-grade approximately 20% of patients
✓ Increased megakaryocyte density is
fever, anorexia, weight loss, and bone pain
associated with an increase in
✓ Night sweats and fever, associated with an myelofibrosis
increased metabolism caused by ✓ Presence of pseudo-Gaucher cells
granulocytic cell turnover, may occur
❖ OTHER LABORATORY FINDINGS:
✓ Splenomegaly: splenic infarction is common
because of the abnormal overproduction and ➢ Hyperuricemia and uricosuria (from cell
turnover)
accumulation of granulocyte precursors in the
bone marrow, spleen, and blood ➢ Detection of t (9;22) translocation
✓ On fresh incision, extramyeloid masses appear (cytogenetic analysis, FISH, RT-PCR)
green (Chloromas), presumably because of ➢ DECREASED LAP SCORE BEQ (LAP is used to
the presence of the myeloid enzyme differentiate CML from leukemoid reaction )
myeloperoxidase (MPO) ➢ Hyperuricemia and uricosuria (from cell
turnover)
✓ Infection→ increased granulocytes in the
➢ Detection of t (9;22) translocation
blood are cancer cells (cytogenetic analysis, FISH, RT-PCR)
✓ More granulocytes→ more MPO→ Chloromas
➢ DECREASED LAP SCORE BEQ (LAP is used to
(tumors appears green)
differentiate CML from leukemoid reaction )
➢ ACCELERATED PHASE: ➢ Presence of Philadelphia chromosomes→ 80-
✓ Increased splenomegaly 90% CML
✓ Worsening anemia and thrombocytopenia ➢ LEUKOCYTE ALKALINE PHOSPHATASE (LAP)→
✓ Gradual failure of response to treatment an enzyme normally present in neutrophils as
✓ Additional cytogenetic abnormalities NETs are cancer cells
✓ 10-19% blasts ➢ Can be found on secretory vesicles of
✓ neutrophils
➢ BLAST CRISIS (ACUTE):
➢ Leukomoid reaction→ exaggerated response
✓ involves the peripheral blood, bone marrow, to an infection
and extramedullary tissues
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 POLYCYTHEMIA VERA (PV)
❖ Is a clonal stem cell proliferation affecting primarily the
erythroid series, characterized by excessive
proliferation of erythroid and also usually granulocytic
and megakaryocytic elements in the marrow
❖ The very slow evolution of the malignant erythroid
clone leads to overexpansion of the red cell mass,
hypervolemia, and splenomegalic red cell pooling
❖ These consequences eventually cause generalized
marrow hyperplasia with subsequent increases in the
quantity of all three cell lines
❖ Associated with the JAK2 mutation, JAK2 V617F, is
detected in more than 95% of patients with PV and is
found on chromosome band 9p24 ; also associated
with JAK2 exon 12 mutation
❖ JAK 2 protein
➢ Tyrosine kinase phosphorylates signal
transducers and activators of transcription
(STAT) proteins, eventually generating
transcription proteins that bind promotor
regions and signal gene expression
➢ Controls transphosphorylation through
conformational inhibition
➢ Constitutive tyrosine kinase activity of the
mutated JAK2 protein causes continuous
activation of several signal transduction
pathways that are normally activated after
erythropoietin stimulation via the
erythropoietic receptor
➢ Mutated JAK2 is active and will phosphorylate
STAT proteins in the absence of erythropoietin
or will over phosphorylate in its presence
➢ Mutated JAK2 is active and will phosphorylate
STAT proteins in the absence of erythropoietin
E. CML VS LEUKEMOID REACTION or will over phosphorylate in its presence
➢ JAK2→ phosphorylates STAT→ initiates
hematopoiesis
➢ Mutated jak2 protein→ need not EPO

TYPES POLYCYTHEMIA VERA (PV)

❖ ABSOLUTE POLYCYTHEMIA VERA
➢ Increased Hct: increase BM production
➢ Types: Primary, secondary with appropriate
EPO production, inappropriate production of
EPO

➢ PRIMARY
❖ Panmyelosis
CHRONIC MYELOID LEUKEMIA (CML) ❖ Increased BM production of
erythrocytes, leukocytes, and
thrombocytes
❖ EPO: decreased
➢ SECONDARY WITH APPOPRIATE EPO
PRODUCTION
❖ Occurs in response to hypoxia
❖ Patients with pulmonary or cardiac
disease

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 95

 ❖ Increased BM production of ➢ BM normoblasts may collect in large clusters,
erythrocytes, leukocytes, and megakaryocytes are enlarged and exhibit lobulated
thrombocytes nuclei, and BM sinuses are enlarged without fibrosis
❖ EPO: increased
➢ Pseudo-Gaucher cells are rare
➢ SECONDARY WITH INAPPROPRIATE EPO ➢ 80% of patients manifest BM panmyelosis
PRODUCTION
❖ Tumors of kidneys, liver, brain,
adrenals
❖ Increased erythrocytes, leukocytes,
and thrombocytes
❖ EPO: Increased

❖ RELATIVE POLYCYTHEMIA VERA

➢ Increase hematocrit; decrease plasma
volume
➢ Dehydration, stress, spurious polycythemia,
anxiety
➢ Gaisbock’s syndrome: spurious polycythemia
PRIMARY→ associated with JAK2
• Panmyelosis→ increased RBC (most affected), WBC,
Platelets counts
• Increased BM production bec. Of JAK2
• RELATIVE ANEMIA→ decrease rbc, increase plasma
volume
• RELATIVE POLYCYTHEMIA VERA→ increase rbc, increase
plasma volume

F. CLINICAL FINDINGS:
➢ PV often presents with a history of mild symptoms POLYCYTHEMIA VERA (PV)
occurring for several years
✓ Increased RCM produces blood
hyperviscosity, often resulting in
cardiovascular disease
✓ In the early stages of the disease, before
treatment, extended periods of high HCT (60%)
and hyperviscosity produce hypertension in
about 50% of patients with PV
✓ Hyperviscosity and hyperproliferation and
include headache, weakness, pruritis, weight
loss, and fatigue
✓ About half of PV patients have thrombocytosis
and 1/3 experience thrombotic or
hemorrhagic episodes

➢ PV patients older than 60 years of age or those

associated with a history of thrombosis are considered
high risk for thrombotic or hemorrhagic events
➢ The stable phase of PV can progress to a spent phase
in a few patients, usually within 10 years from diagnosis
✓ Splenomegaly (palpable spleen)
✓ Hypersplenism
✓ BM hyperplasia
✓ Cytopenias
✓ Triad of myelofibrosis, splenomegaly, and
anemia with teardrop-shaped poikilocytes
called as postpolycythemic myeloid H. DIAGNOSIS OF POLYCYTHEMIA VERA (PV)
metaplasia and develops in about 30% of PV
patients within 20 years

G. LABORATORY FINDINDS:
➢ Common morphologic patterns in peripheral blood and
BM morphologic and cellular changes

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 96

 ➢ Arterial thrombi can cause myocardial infarction, transient
ischemic attack, and cerebral vascular accident

➢ Bleeding occurs most often from mucous membranes in the

gastrointestinal, skin, urinary, and upper respiratory tracts.

J. LABORATORY FINDINGS:
➢ Most consistent finding in the peripheral blood is
thrombocytosis that must exceed 400 x 10^9/L to meet
WHO criteria but often ranges from 1000 to 5000 x 10^9/L

➢ Platelets often appear normal, but giant bizarre platelets,

platelet aggregates, micromegakaryocytes, and
megakaryocyte fragments can also be observed

➢ Platelets are present in clusters and tend to accumulate

near the thin edge of the blood film; abnormal platelet
function testing can occur

➢ BM has marked megakaryocytic hypercellularity, clustering

of megakaryocytes, and increased megakaryocyte
diameter with nuclear hyper lobulation and density

ESSENTIAL THROMBOCYTHEMIA (ET)

❖ a clonal MPN with increased megakaryopoiesis and
thrombocytosis, usually with a count greater than 600 x
109/L ; sometimes with a count greater than 1000 x 109/L
abnormal megakaryocytes in BM → abnormal platelets
in platelets → increased clot production

❖ However, WHO criteria require a sustained

thrombocytosis with a platelet count of 400 3 109/L

❖ AKA as primary thrombocytosis, idiopathic

thrombocytosis, and hemorrhagic thrombocythaemia

❖ Associated with mutations in:

➢ JAK2 (64.1%)
➢ MPL (4.3%) myeloproliferative leukemia gene
➢ CALR (15.5) Cal-reticulin gene
K. DIAGNOSIS OF ESSENTIAL THROMBOCYTHEMIA (ET)
❖ MPL→ Myeloproliferative Leukemia Gene L. WHO CRITERIA FOR DIAGNOSIS OF ET:
❖ CALR→ Calreticulin ➢ MAJOR CRITERIA:
❖ Ischemic→ no oxygen in heart ✓ Megakaryocyte proliferation with large and
mature morphology, little to no granulocyte or
erythroid proliferation
I. CLINICAL FINDINGS:
✓ Minor increase in reticulin fibers
➢ Vascular occlusions are often the result of microvascular ✓ Must not meet any criteria for BCR-ABL1-
thromboses in the digits or thromboses in major arteries and positive CML, PV, PMF, MDS or other myeloid
veins that occur in a variety of organ systems, including
neoplasms
splenic or hepatic veins, as in Budd-Chiari syndrome
✓ Demonstrate JAK2 V617F (most common),
➢ Repeated splenic infarcts can result in splenic atrophy CALR, or MPL mutations
➢ MINOR CRITERIA: presence of a clonal marker or
➢ Thrombosis can result in pulmonary emboli and neurologic absence of evidence of reactive thrombocytosis
complications like headache, paresthesis of the
extremities, visual impairments, and tinnitus

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 97

 ➢ Based on WHO standards, a diagnosis of ET requires demonstrate hemosiderinuria and decreased blood
meeting all four major criteria or the first three major haptoglobin levels suggesting intravascular hemolysis
criteria and one minor criteria
❖ Some patients develop bleeding diathesis (damaged
blood vessels) resulting from a combination of
PRIMARY MYELOFIBROSIS (PMF) thrombocytopenia, abnormal platelet function, and
❖ A clonal hematopoietic stem cell MPN in with hemostatic abnormalities suggestive of chronic
splenomegaly and ineffective hematopoiesis disseminated intravascular coagulation (DIC)
associated with areas of marrow hypercellularity
(leukoerythroblastosis – increased WBC and many N. LABORATORY FINDINGS:
immature RBCs), extramedullary hematopoiesis, fibrosis,
❖ In the early stages, anemia, leukocytosis with a left shift,
and increased megakaryocyte; it is the least common
and thrombocytosis are identified that are consistent with
but most aggressive form of MPN
a MPN
❖ PMF can form de novo or as an evolutionary
❖ As the fibrosis develops in the bone marrow, blood cell
consequence of PV or ET
counts fall and pancytopenia eventually develops, along
with leukoerythroblastosis, anisocytosis, and poikilocytosis
❖ AKA chronic idiopathic myelofibrosis, agnogenic
myelofibrosis, and myelofibrosis with myeloid
❖ Examination of the BM biopsy specimen provides most of
metaplasia
the information for diagnosis
❖ Chronic idiopathic myelofibrosis (CIM) is an uncommon
❖ Left shift→ presence of abundant immature WBC in the
disease with an incidence one-third that of CML
blood Dacryocytes→ squeezing through spleen
(splenomegaly)
❖ ASSOCIATED MUTATIONS: JAK2 (V617F) – 60% ; CALR -
30% ; MPL – 5%

❖ Leukoerythroblastosis→ increased WBC, and many

immature rbc

❖ Diathesis→ damaged blood vessels

❖ MYELOFIBROSIS

❖ Fibroblasts → produce collagen to provide

structural support for HSCs

❖ In PMF a reactive process causes

overproduction of collagen that eventually
disrupts the normal architecture of the BM and
replaces hematopoietic tissue resulting in
pancytopenia

❖ EXTRAMEDULLARY HEMATOPOIESIS

❖ recognized as hepatomegaly or
splenomegaly, seems to originate from release
of clonal stem cells into the circulation

❖ The cells accumulate in the spleen, liver, or

other organs, including adrenals, kidneys,
lymph nodes, bowel, breasts, lungs,
mediastinum, mesentery, skin, synovium,
thymus, and lower urinary tract

M. CLINICAL FINDINGS:
❖ PMF occurs in patients older than age 60 and equally in
both genders

❖ The disease may be asymptomatic and usually progresses

as a slow, chronic condition

❖ Symptoms result from anemia, myeloproliferation, or

splenomegaly and include fatigue, weakness, shortness of
breath, palpitations, loss of appetite, weight loss, night
sweats, pruritis, pain in the extremities and bones, bleeding,
and discomfort or pain in the left upper quadrant
associated with splenomegaly

❖ Major hemolytic episodes occur in 15% of PMF patients

during the course of their disease and 10% of patients

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 98

 O. OTHER MYELOPROLIFERATIVE NEOPLASMS
❖ CHRONIC NEUTROPHILIC LEUKEMIA
❖ CHRONIC EOSINOPHILIC LEUKEMIA
❖ MYELOID/LYMPHOID NEOPLASMS WITH EOSINOPHILIA
AND REARRANGEMENT OF PDGFRA, PDGFRB, OR FGFR1
OR WITH PCM1-JAK2
❖ MASTOCYTOSIS
❖ MYELOPROLIFERATIVE NEOPLASM, UNCLASSIFIABLE

MATURE LYMPHOID NEOPLASMS

❖ Are a diverse collection of disease entities with varying
clinical presentations and natural histories; however,

common to all is identification with a particular lineage

❖ Disease states in large part can be identified with a
normal counterpart in the blood, bone marrow or lymph
nodes. Neoplastic transformation of these cells results in
abnormal changes in growth and differentiation
patterns, resulting in disease
❖ Lymphoproliferative disorders in which the primary site
of disease is the blood or bone marrow are classified as
leukemias; disorders in which the localization of disease
is in the lymph nodes and spleen are considered
lymphomas
BEQ yung nakacheck
CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)
❖ Fibrosis→ collagen and megakaryocytes
❖ Increased LDH bec of hemolysis ❖ CLL is the most common leukemia in adults in Western
countries counterpart of ALL
❖ Clonal proliferation of small B lymphocytes involving
BM, blood, and lymph nodes (LYMPH NODE
INVOLVEMENT IS CALLED SMALL LYMPHOCYTIC
LYMPHOMA)
❖ CLL is generally a disease of the older adult; median
age of diagnosis is approximately 72 years which is
common in males
❖ Most patients are asymptomatic on presentation, and
the disease is often detected by an abnormality in a
routine CBC
❖ The International Workshop on Chronic Lymphoid
Leukemia (IWCLL) requires the presence of at least 5 x
109 cells/L of circulating B lymphocytes for more than 3

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 99

 months to establish the diagnosis of CLL, with ➢ Hepatosplenomegaly
confirmation of clonality performed by flow cytometry
❖ CYTOGENETIC ABNORMALITIES:

➢ 13q14.3 del → DLEU2/MIR15A/MIR16A

➢ 6q21 del
➢ Trisomy 12
➢ ATM gene →11q23 del
➢ TP53 gene → 17p13 del worst prognosis
❖ Typical CLL presents with >85% of the lymphocytes
appearing small and mature with scant cytoplasm and
a dense nucleus with a condensed chromatin pattern
without a defined nucleolus
❖ This characteristic chromatin pattern has been labeled
as “cobblestone” or likened to a soccer ball
❖ ATYPICAL CLL

➢ Seen in 20% of patients

➢ Prolymphocytes or atypical lymphoid cells
represent less than 10% or less than 15% of
circulating lymphocytes
➢ Associated with biologic markers indicating
more aggressive disease
➢ Subtypes:
❖ CLL/PLL → >10% but <55% of total
lymphocytes (>55% is associated with
Prolymphocytic leukemia)
❖ Mixed-type/atypical CLL → >15%
large atypical lymphoid cells
Q. IMMUNOPHENOTYPING
❖ Smudged lymphocytes are a common finding on
❖ MARKERS PRESENT:
peripheral blood film review in CLL
P. CLINICAL FINDINGS: ➢ Positive:

➢ The typical patient with CLL is asymptomatic, ✓ CD19, CD20, CD5, CD23, CD79a
and the disease is usually discovered at the ✓ dim surface Ig (single light chain with
time of a routine physical examination IgM or IgM and IgD)
➢ Common symptoms include malaise, low- ➢ Negative:
grade fever, and night sweats
✓ CD10, BCL-6, and cyclin D1
➢ Weakness, fatigue, anorexia, and weight loss
❖ This pattern is very useful in distinguishing CLL/SLL from
➢ Physical examination usually reveals cervical mantle cell, follicular, and marginal zone lymphomas.
and supraclavicular adenopathy
❖ Antigen expression is sometimes variable; not all cases
fit into a classic CLL immunophenotypic profile, to
account for this variability and differentiate CLL from
related B cell neoplasms, a scoring system has been
developed
❖ Each criterion is given 1 point, and the total score is
cumulative:
➢ 4 or more: Chronic lymphocytic leukemia
➢ 1 or 2: Non-Hodgkin’s lymphoma
➢ 3 (Intermediate): remains problematic, and these
cases may require biopsy of a node or molecular
data to establish the diagnosis requires lymph node
biopsy or cytogenetics

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 100

 ❖ More than 55% of blood leukemic cells are
prolymphocytes
❖ IMMUNOPHENOTYPING:
➢ Positive: CD19, CD20, FMC-7, bright surface Ig
➢ Negative: CD5, CD23
❖ CLL→ dim surface, positive in CD5, 23
❖ PLL→ bright surface, negative in CD5, 23
HAIRY CELL LEUKEMIA
R. STAGING AND PROGNOSIS
❖ Hairy cell leukemia (HCL) is an indolent disease of B cell
❖ Staging systems are important prognostic determinants
lineage most commonly found in middle age (median
for CLL and are used to guide clinical management
age, 50 years); more common in males
❖ The Rai and Binet staging systems have been used for
❖ Major focus of disease is in the spleen, blood, and bone
this purpose
marrow
❖ Both systems divide patients into risk categories
❖ HAIRY CELLS:
reflecting the degree of organomegaly and
lymphadenopathy or compromise of bone marrow ➢ Have round to ovoid nuclei, lack nucleoli, and
function have relatively abundant cytoplasm with
ragged projections that extend
❖ PROGNOSTIC INDICATORS: presence indicate worse
circumferentially around the entire cell
prognosis
➢ TRAP stain positive *BEQ
➢ del(17q)(TP53)
❖ CYTOGENETICS: BRAF (V600e) mutation
➢ IGHv mutational status
❖ IMMUNOPHENOTYPING:
➢ Testing for CD38 and ZAP (zeta-associated
protein of 70 kDa) ➢ CD 20, CD22, CD79a, CD103, CD25, CD11c
❖ Annexin A is specific to HCL and helps to differentiate
HCL from related B cell disorders
S. CLINICAL FINDINGS:
➢ Fatigue
➢ Anemia, leukocytopenia, thrombocytopenia,
splenomegaly (most predominant finding),
and marrow fibrosis (bone marrow aspirates
frequently are unsuccessful)
➢ Bleeding and infection can be present

PROLYMPHOCYTIC LEUKEMIA (PLL)

❖ Originally described as a variant of CLL
❖ Is characterized by
a very marked
lymphocytosis
(usually greater
than 100 × 103/μL),
massive
splenomegaly,
moderate
hepatomegaly,
and inconspicuous
lymphadenopathy
❖ The malignant lymphoid cells have a large vesicular
nucleolus, condensed nuclear chromatin, and a
moderate amount of cytoplasm

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 101

 II. PAUTRIER’S MICROABCESS
MYCOSIS FUNGOIDES AND SEZARY SYNDROME
❖ Is the most familiar form of cutaneous T cell lymphoma
abnormal T cells
❖ MF is the more common disorder, making up
approximately 60% to 70% of the cutaneous T cell
lymphoma cases
❖ MF is largely confined to the skin, although in the later
stages of the disease, dissemination to lymph nodes,
organs, and blood can be seen
❖ MF is generally an indolent disease with a slow
progressive course; SS is defined as a systemic disorder
with peripheral blood involvement (leukemic phase of
MF) and a worse prognosis than MF if peripheral blood
is involved, MF → SS which is the leukemic phase
❖ SEZARY CELLS:
➢ Abnormal appearance with scant cytoplasm III. MYCOSIS FUNGOIDES AND SEZARY SYNDROME
and a cerebriform, folded nucleus, variably U. CLINICAL FINDINGS:
condensed chromatin, and inconspicuous
➢ MYCOSIS FUNGIODES
nucleoli
✓ Psoriatic-like skin lesions requiring repeated
❖ IMMNUNOPHENOTYPING: biopsy before a definitive diagnosis is made.
➢ CD2, CD3, CD4, and CD5 ✓ Skin biopsy can show a characteristic
localization of atypical cells in Pautrier micro
abscesses in the epidermis, but this occurs in
only a minority of cases
T. LABORATORY FINDINGS:
➢ SEZARY SYNDROME
➢ Biopsies of the skin reveal bandlike
lymphocytic infiltrates in the dermis often with ✓ Characterized by erythroderma, generalized
admixed histiocytes and occasional lymphadenopathy, and the presence of
eosinophils clonal T cells in skin, lymph nodes, and
peripheral blood
➢ T cells with irregular nuclei show single-cell
✓ An absolute Sézary cell count greater than 1
infiltration of the epidermis and often form
X109/L is required to make a diagnosis of SS
clusters known as Pautrier’s abscesses *BEQ
➢ Presence of Sezary cells (Sezary syndrome)

PLASMA CELL NEOPLASMS/MYELOMAS

❖ Also known as Plasma cell dyscrasias; tumor consisting
of plasma cells usually originating in the BM
I. SEZARY CELLS ❖ are malignant disorders of terminally differentiated B
cells
❖ Although they constitute approximately 1% to 2% of
cancers, PCNs are the second most common
hematologic malignancy

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 102

 ❖ Plasma cells to secrete monoclonal immunoglobulin ➢ overproduction of IgM; “M-spike”
(paraproteins) is an essential characteristic of the PCNs ❖ Presence of monoclonal serum protein (mostly IgG, can
be IgA or IgD)
❖ Associated with rouleaux formation and elevated ESR
❖ Presence Bence Jones proteins (free light chains—
❖ TYPES: kappa or lambda) found in the urine
➢ Monoclonal Gammopathy of Undetermined ❖ Paraproteins→ increases plasma volume→ causes
Significance (MGUS)
hypervolemia
➢ Multiple (Plasma cell) myeloma
❖ M/Monoclonal Spike→ increased production of
❖ Waldenstrom’s Macroglobulinemia monoclonal immunoglobulins (mostly IgM)
Ig/Ab→ polyclonal
❖ Bence jones proteins→ IgG light chains
➢ Plasma cells are abnormal→ monoclonal
immunoglobulins→ elevated plasma
proteins→ elevated ESR
MONOCLONAL GAMMOPATHY OF UNCERTAIN SIGNIFICANCE
(MGUS)
❖ Benign monoclonal proliferation of plasma cells that is
usually considered with the plasma cell neoplasms
because it represents a precursor state for myeloma
❖ Presence of circulating monoclonal protein WITHOUT
ASSOCIATED PLASMA CELL DYSCASIA or other
neoplasm
❖ Detections relies on detection of paraproteins in serum
or urine
MULTIPLE (PLASMA) CELL MYELOMA
❖ Malignant bone marrow–based, plasma cell neoplasm
associated with abnormal protein production
❖ Plasma cell leukemia is an increased number of plasma
cells in the peripheral blood and should be considered
a form of multiple myeloma and not a separate entity
❖ IMMUNOPHENOTYPING:
➢ CD38, CD138 (Kappa or lambda clonal
excess)
❖ SOLITARY PLASMA CYTOID OF THE BONE→ only present
in BM
❖ MULTIPLE MYELOMA→ present of multiple tissues IV. WALDENSTROM’S MACROGLOBULINEMIA
V. CLINICAL FINDINGS: ❖ AKA as LYMPHOPLASMACYTIC LYMPHOMA
➢ Include bone pain (typically in the back or chest) ❖ B-cell neoplasm characterized by
➢ Weight loss and night sweats are not prominent until the lymphoplasmoproliferative disorder with infiltration of
disease is advanced the bone excessive IgM (macroglobulin) and
➢ Abnormal bleeding may be a prominent feature decreased production of the other immunoglobulins
➢ In some patients, the major symptoms result from acute
❖ High levels of IgM can result in a hyperviscosity
infection, renal insufficiency, hypercalcemia, or
syndrome
amyloidosis
➢ Approximately 90% of patients suffer from broadly ❖ CYTOGENETIC ABNORMALITY: mutation in the Myeloid
disseminated destruction of the skeleton; bone marrow differentiation factor 88 (MYD88) gene
failure X. CLINICAL FINDINGS:
➢ Major cause of death are infection and renal ➢ Symptoms of WM are due to the extent of tumor infiltration
insufficiency and to elevated
➢ Weakness, fatigue attributable to anemia, and bleeding
➢ Bone pain is virtually nonexistent; HYPERVISCOCITY
SYNDROME
W. LABORATORY FINDINGS: ➢ Lymphadenopathy and Hepatosplenomegaly
❖ Bone marrow plasma cells >30% Y. LABORATORY FINDINGS:
❖ Increased plasma volume caused by monoclonal ➢ Pleomorphic B-lineage cells at different stages of
protein commonly produces hypervolemia maturation, such as small lymphocytes,
❖ Rouleaux formation and elevated ESR lymphoplasmacytoid cells (abundant basophilic
❖ Serum electrophoresis: cytoplasm but lymphocyte-like nuclei), and plasma cells

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 103

➢ IMMUNOPHENOTYPING: CD154 ✓ SUBTYPES: Nodulary sclerosis, Mixed cellularity,
➢ Absolute lymphocytosis Lymphocyte depleted, Lymphocyte-rich
➢ IgM levels in the blood circulation (IgM >3 g/dL); “M-spike”
serum electrophoresis REED-STERNBERG CELL
➢ Rouleaux and elevated ESR

Z. VARIANTS OF RS CELLS:
❖ HODGKIN CELLS
V. HODGKIN’S LYMPHOMA ➢ Large mononuclear (reed-sternberg is
❖ Hodgkin lymphoma (HL) is primarily a lymph node- binuclear) lymphoid cells with an oval nucleus,
based disease, distinct from NHL that involves the lymph thick nuclear membrane, distinct eosinophilic
nodes nucleolus, and abundant cytoplasm

❖ HL originates in lymph nodes while non-HL are tumors ❖ MUMMIFIED CELLS

involving lymph nodes ➢ Degenerated or apoptotic cells with a
❖ The hallmark of classical HL is the Reed Sternberg cell, pyknotic nucleus and condensed cytoplasm
which is a large binucleated, multinucleated, or
mononuclear (Hodgkin) cell with each nucleus bearing ❖ LACUNAR CELLS
a very large inclusion-like nucleolus ➢ Lobated nucleus and artifactual retraction of
cytoplasm secondary to formalin fixation
❖ DIAGNOSIS involves tissue biopsy, Immunophenotyping,
➢ Occurs predominantly in the nodular sclerosis
cytogenetics, DNA analysis
variant of Classical Hodgkin’s Lymphoma
❖ Epstein Barr Virus→ infectious mononucleosis ➢ Appear to be situated in a clear space
(“LACUNA”)
CATEGORIES:
➢ NODULAR LYMPHOCYTE-PREDOMINANT
HODGKIN’S LYMPHOMA
✓ Is a B-cell neoplasm composed relatively rare
neoplastic cells called “popcorn cells” or
lymphocytic histiocytic cell (L&H cell)
✓ Popcorn cells- large lymphoid cells with
abundant cytoplasm and vesicular multi-
lobated nuclei (popcorn nuclei)
✓ IMMUNOPHENOTYPING: CD 45, CD20, BCL-6
➢ CLASSICAL HODGKIN’S LYMPHOMA
✓ Comprises a heterogenous group of lymphoid
neoplasm derived from the germinal center;
can be associated with EBV infections ex:
infectious mononucleosis
✓ Presence of Reed-Sternberg cells (a usual
pathognomonic sign for Classical Hodgkin’s
Lymphoma)
✓ IMMUNOPHENOTYPING: CD15, CD30, CD45

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 104

HODGKIN’S LYMPHOMA • NLPHL
• Classical
• Nodular sclerosis
• Lymphocyte depletion
• Mixed cellularity
• Lymphocyte rich classical

AA. CLINICAL FINDINGS:

➢ Painless enlargement of the lymph node (usually in the
neck)
➢ Mediastinal mass
➢ Enlarged cervical nodes
➢ Fever (over 100.4oF)
➢ Night sweats
➢ Weight loss (>10%)
BB. LABORATORY FINDINGS:
➢ Peripheral blood:
✓ Increase in monocytes and eosinophils
✓ As disease progresses, there is often
leukocytosis
✓ Transient lymphocytosis; lymphocytopenia is a
sign of poor prognosis associated with
advanced stages with histologic subtypes that
have poor prognosis

➢ Bone marrow:
✓ Stage IV disease
✓ Mostly aspirates are negative, seldom positive
in bone marrow biopsies
✓ If positive, histologic class can’t be determined
STAGING FOR HODGKIN’S LYMPHOMA

NON-HODGKIN’S LYMPHOMA
HRS→ presence of reed sternbeg cells and Hodgkin
cells
Types/categories of hodgkin
JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 105
 ❖ The most frequent type of NHL is diffuse large B-cell
Siderocytes/Sideroblas + + +/- - +/-
lymphoma, which accounts for approximately 40% of
ts
new cases of lymphoma
❖ More than half of patients with diffuse large B-cell
Ringed Sideroblasts +/- >15 +/- - +/-
lymphoma are older than 60 years of age
%
❖ Three different types of diffuse large B-cell lymphomas
can be defined based on gene expression subgroups:
CC. RELATIONSHIP OF LEUKEMIAS AND LYMPHOMAS
➢ Germinal center, B-cell–like lymphoma that
expresses high levels of genes characteristic of
germinal center, B-cell–like lymal germinal
center B cells
➢ Activated B-cell–like lymphoma, which
expresses genes characteristic of
mitogenically activated blood B cells
➢ New subgroup, type 3 diffuse large B-cell
lymphoma, which has a heterogeneous gene
expression that suggests it includes more than
one subtype of lymphoma
MYELODYSPLASTIC SYNDROMES (MDS)
➢ Characterized by a decreased in one or more types of
peripheral blood cells due to abnormal maturation in
the bone marrow
➢ Clonal stem cell disorders are characterized by:
✓ Maturation defects
✓ Ineffective erythropoiesis
✓ Peripheral pancytopenia in spite of marrow
cellularity
➢ FAB classification of myelodysplastic syndromes based
on the presence of dysmyelopoiesis and the
quantification of myeloblasts (Type I and Type II) or
erythroblasts:
✓ REFRACTORY ANEMIA (RA)
✓ REFRACTORY ANEMIA WITH RINGED SIDEROBLASTS (RARS)
✓ Refractory Anemia with Excess Blasts (RAEB)
✓ CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML)
✓ REFRACTORY ANEMIA WITH EXCESS BLASTS IN
TRANSFORMATION (RAEB-T)

RA RAR RAE CMM RAEB

S B L -t

% of blasts in <1 <1% <5% <5% >5%

peripheral blood %

% of blasts in bone <5 <5% 5- 5-20% 20-

marrow % 20% 30%

Dyserythropoiesis ++ ++ +/- +/- +/-

+

Dysgranulopoiesis - - ++ ++ +/-

Dysmegakarypoiesis - - +/- +/- +/-

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 106

WEEK 16: CYTOCHEMISTRY

CYTOCHEMISTRY
❖ Techniques such as flow cytometry, cytogenetic
analysis, and molecular testing are now commonly
used in the diagnosis of acute and chronic leukemias;
However, older techniques, such as cytochemical
stains, still retain their importance
❖ Cytochemistry involves the use of cytochemical stains
❖ The GENERAL PRINCIPLE of most cytochemical stains is SUDAN BLACK B
to incubate cells on the blood smear with the substrate ❖ is a fat-soluble dye
that reacts with the intracellular marker that stains
❖ CLASSIFICATION OF CYTOCHEMICAL STAINS: phospholipids neutral
➢ Enzymatic fats and sterols ; Can
❖ Myeloperoxidase (MPO) be done on stored
❖ Naphthol AS-D Chloroacetate specimens
Esterase (Specific esterase) ❖ Useful for
❖ Alpha-Naphthyl acetate esterase differentiation of AML
(Non-specific esterase) (POSITIVE) from ALL
❖ Alpha-Naphthyl Butyrate Esterase (NEGATIVE);
(Non-specific esterase) PARALLELS MPO FOR
❖ Acid Phosphatase (ACP) ; with INTERPRETATION
tartrate inhibition (TRAP) ❖ POSITIVE RESULT: DARK PURPLE-BLACK GRANULES in
❖ Leukocyte Alkaline Phosphatase neutrophil precursors
(LAP)
➢ Non-enzymatic
❖ Periodic Acid Schiff (PAS)
❖ Toluidine blue
❖ Prussian blue
➢ Immunocytochemical
❖ Terminal Deoxyribonucleotidyl
Transferase (TdT)

MYELOPEROXIDASE (MPO)
❖ MPO is present in the primary granules of most
granulocytic cells, beginning at the promyelocyte
ESTERASES
stage and continuing throughout maturation;
❖ are used to differentiate myeloblasts and neutrophilic
leukemic myeloblasts are usually positive for MPO
granulocytes from cells of monocytic origin
❖ LYMHPOCYTES DO NOT EXHIBIT MPO ACTIVITY
❖ Nine isoenzymes are present in leukocytes
❖ Stain should be DONE ONLY ON FRESH SPECIMENS
❖ Esterase with low substrate specificity is known as non-
because it deteriorates
specific esterase (Alpha-Naphthyl acetate and
❖ Used to differentiate acute myelogenous leukemia
butyrate) ; called non-specific because these stains
(POSITIVE) from acute lymphoblastic leukemia
may also produce a positive result with other cells
(NEGATIVE)
❖ Esterase with high specificity to substrate is known as
❖ In many cases of the AMLs (without maturation, with
specific esterase (Naphthol AS-D Chloroacetate
maturation, and promyelocytic leukemia), it has been
Esterase) ; called specific because granulocytic cells
found that more than 80% of the blasts show MPO
show staining
activity; Auer rods found in leukemic blasts and
❖ Esterase activity is inhibited by varying degrees of
promyelocytes test strongly MPO positive
mercury, acid solutions, heat and iodine (false
❖ Lymphoblasts in ALL and lymphoid cells are MPO
negative results)→ MAHI
negative
❖ POSITIVE RESULT:
❖ POSITIVE RESULT: Peroxidase activity produces RED
➢ SPECIFIC ESTERASE (Chloroacetate esterase):
BROWN GRANULES in cytoplasm of granulocytes and
✓ BRIGHT RED GRANULES in cytoplasm
monocytes
of neutrophils, neutrophil precursors
and mast cells
➢ NON-SPECIFIC ESTERASE (acetate and
butyrate):
✓ DARK BROWN GRANULATION in the
cytoplasm of Monocytes,
Megakaryocytes, erythroblasts,
plasma cells
✓ IMPORTANT NOTE: alpha-naphthyl
esterase is inhibited by sodium
fluoride

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 107

 ❖ Acid phosphatase hydrolyzes the naphthol AS-B1
phosphoric acid in the incubation mixture releasing
insoluble naphthol, which then couples with
pararosanilin, giving a red-colored precipitate
❖ ACP→ Naphthol AS-B1 phosphoric acid→ releasing
insoluble naphthol + pararosanilin→ red color
❖ Most leukocytes exhibit a positive reaction in varying
degrees to this test; however, monocytes demonstrate
a more intense positive reaction than do neutrophils
❖ Although lymphocytes display little activity, T cells do
exhibit intense positivity in the Golgi region, whereas B
cells may be positive or negative. For this reason, the
procedure is useful in differentiating subgroups of ALL
SPECIFIC ESTERASE ❖ Tartrate resistant acid phosphatase (TRAP STAIN)
➢ In the presence of L(+) tartaric acid (Tartrate
resistant acid phosphatase), the activity of
most acid phosphatase is inhibited, the
exception is ACP Isoenzyme 5 (present in
Hairy cell leukemia), which is resistant to L(+)
tartaric acid
➢ POSITIVE RESULT: RED COLOR PRECIPITATE

NON-SPECIFIC ESTERASE

TRAP STAIN

LEUKOCYTE ALKALINE PHOSPHATASE (LAP)

❖ Alkaline phosphatase is present in the cytoplasm of
neutrophils in varying degrees and referred to as
leukocyte alkaline phosphatase (LAP) or Neutrophil
alkaline phosphatase (NAP)
❖ Eosinophils are rarely positive and B lymphocytes; all
other leukocytes are negative
❖ Leukocyte alkaline phosphatase (LAP) activity can be
increased, normal, or decreased in a variety of
conditions
❖ This procedure is frequently used to distinguish
between leukemoid reactions (INCREASED) and
chronic myelogenous leukemia (DECREASED)
❖ IMPORTANT NOTE: Capillary blood is preferred to
PHOSPHATASES anticoagulated whole blood; if anticoagulated whole
❖ are widely distributed in mammalian tissue blood must be used, heparin is preferred and EDTA
❖ The two major classifications, based on pH, are must be avoided
alkaline and acid phosphatase ❖ POSITIVE RESULT: BLUE (Naphthol AS-MX + fast blue RR
❖ Microscopic determinations of these enzymes are salt) or VIOLET (Naphthol AS-MX + fast violet B salt)
based on methods that produce a visible precipitate pigment
of the organic phosphates hydrolyzed by these
LAP SCORE/GRADING
enzymes
❖ Also called as KAPLOW’S COUNT
ACID PHOSPHATASE (ACP) ❖ The deposits of blue or violet pigment viewed
❖ Almost all blood cells contain the acid phosphatase microscopically reflect the sites of granulocytic
enzyme and show positivity with acid phosphatase alkaline phosphatase activity, The granulocyte
stain; is one of many acid hydrolases that have been population is rated on the basis of the number of cells
demonstrated in lysosomes stained and the intensity of the pigment deposits

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 108

 ❖ A 100 cell count (band and segmented neutrophils) is
done and is rated on a scale of 0 to 4+ based on the
quantity and intensity of the precipitated dye within
the cytoplasm of cells
LAP GRADING
GRADING AMOUNT (%) SIZE OF INTERPRETATION
GRANULE
0 None - No staining
1+ 50 Small Faint and
diffuse staining
2+ 50-80 Small Pale with a
moderate
amount of blue
staining
3+ 80-100 Medium to Strong blue
Large precipitated
staining
4+ 100 Medium to Deep blue or
Large brilliant staining
with no visible
cytoplasm
Normal value for LAP Score: 30 to 185
4+ = 50 (4 x 50) = 200
3+ = 30 (3 x 30) = 90
2+ = 20 (2 x 20) = 40
1+ = 0
0 =0
= 100 CELLS
LAP SCORE = 330 (INCREASED)

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 109

 ENZYMATIC METHOD COLOR AND INCLUSIONS SUBSTRATE POSITIVE NEGATIVE MISCELLANEOUS
(+)
MYELOPEROXIDASE (MPO) Insoluble red brown H2O2 + 3 amino-9 ethyl Eosinophil, Neutrophil Lymphocytes/ plasma Fresh specimens only
granules in cytoplasm carbazole except blast forms, cell series, RBC
Phi bodies (rod/dot Monocytes except blast
shaped ppt) forms
ALL
AML
SUDAN BLACK B Dark purple black SBB Eosinophil, Neutrophil, Lymphocytes, RBC, Can used stored spx
granules in Neutrophil Monocytes, Basophil Megakaryocytes,
precursors Thrombocytes
AML ALL
ESTERASES FALSE NEGATIVE:
• Specific Bright Red Granules Naphthol AS-D Neutrophil, Neutrophil mercury, acid solutions,
Chloroacetate Esterase Precursors, Mast Cells heat and iodine
(Isoenzymes 1, 2, 7, 8, 9)

Monocytes,
• Non-specific Dark Brown Granules Alpha-Naphthyl acetate Megakaryocytes, alpha-naphthyl
and butyrate Plasma Cells (Isoenzymes esterase is inhibited
3, 4, 5, 6) by sodium fluoride
RBC
PHOSPHATASES All blood cells→ +
Monocytes→ strongly
ACID PHOSPHATASE RED GRANULES Naphthol AS-B1 Neutrophils, Monocytes + > neutrophils
Phosphoric acid (Isoenzyme 2, 4)
Lymphocytes: T cells
Lymphocytes, Platelets in golgi region→
(Isoenzyme 3) strongly +, B cells→
+/-
Hairy Cell (Isoenzyme ALL can be
5)→ TRAP (+) determined

LEUKOCYTE ALKALINE BLUE (Naphthol AS-MX + Neutrophils All other leukocytes Capillary blood is
PHOSPHATASE (LAP) fast blue RR salt) or preferred to
VIOLET (Naphthol AS-MX anticoagulated
+ fast violet B salt) Eosinophil, B whole blood; if
pigment lymphocytes rarely anticoagulated
positive whole blood must be
used, heparin is

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 110

 preferred and EDTA
must be avoided

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 111

NON-ENZYMATIC STAINING IMMUNOCYTOCHEMICAL STAIN

PERIODIC ACID-SHIFF (PAS) TERMINAL DEOXYRIBONUCLEOTIDYL TRANSFERASE (TdT)

❖ The PAS reaction is important in carbohydrate ❖ TdT is an intranuclear
histochemistry (PA is used as an oxidizing agent and enzyme that catalyzes the
Schiff is used as the irreversible addition of
stain) deoxynucleotides to the 3-
❖ Positive staining prime region on the end of
reactions indicate the DNA
presence of ❖ Present in almost 90% of ALL
glycogen, a polymer cases ; MARKER FOR
of glucose, and other LYMPHOID PRECUSORS
1,2-glycol–containing ❖ Used to differentiate ALL (POSITIVE) from AML
carbohydrates (NEGATIVE)
❖ Lymphoblasts,
malignant erythroblasts, and megakaryocytic cells STAIN
POSITIVE with this stain; myeloblasts and normal
erythrocytic cells are NEGATIVE with this stain
❖ Useful in diagnosis of erythroleukemia (FAB M6) and
acute lymphoblastic leukemia
❖ POSITIVE RESULT: BRIGHT FUCHSIA PINK, MAGENTA TO
PURPLE pattern of staining which varies with each cell
type

AML M4
SBB/MPO: +
PER’S PRUSSIAN BLUE SE: +
❖ Free iron precipitates into small blue/green granules in NSE: +
mature erythrocytes; cells are called siderocytes
❖ Iron inclusions are called siderotic granules (Prussian AML M1-M3
blue) or Pappenheimer bodies (Wright's stain) SBB/MPO: +
❖ Sideroblasts are nucleated RBCs in bone marrow that SE: +
contain iron granules NSE: -
(normal)
❖ Ringed sideroblasts AML M5
contain iron that SBB/MPO: -
encircles the nucleus SE: -
(abnormal) NSE: +
❖ Increased percentage
of siderocytes is seen ALL
in severe hemolytic SBB/MPO: -
anemias (e.g., beta- TdT: +
thalassemia major), iron overload, sideroblastic anemia,
and post-splenectomy; ringed sideroblasts are seen in
bone marrow of myelodysplastic syndrome (refractory
anemia with ringed sideroblasts) and sideroblastic
anemias

TOLUIDINE BLUE
❖ Binds with acid
mucopolysaccharides in
blood cells
❖ It is used for recognition
of mast cells and
basophils (purplish red)
❖ Cooper-cruickshank
stain – stain for basophils

JOVERLYN S. TOLENTINO / BSMD 3Y-5/ MEDICAL TECHNOLOGY/ HEMATOLOGY 112