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SEM100 - MTAP100 - MLSCI 100 | MEDICAL LABORATORY SCIENCE INTERNSHIP

HEMATOLOGY
Rene Jesus Alfredo R. Dinglasan, RMT November 8, 2021

OUTLINE A. HORMONES RELATED TO ERYTHROPOIESIS

I. Erythropoiesis IV. Overview of Leukemia ● Erythropoietin (EPO)
II. Overview of Anemia INDEX: APPENDIX → Chief stimulatory cytokine for RBCs
III. White Blood Cells → A major hormone for RBC production
I. ERYTHROPOIESIS → Thermostable, nondialyzable, glycoprotein hormone
→ Primary cell source: Peritubular interstitial cells of the
● Erythropoiesis
kidneys
→ Process of RBC formation
→ Primary target cells: Progenitor cells: (BFU-E & CFU-E)
→ Takes place inside the bone marrow
→ Normally, EPO is released from the kidney into the
● Red marrow consists of hematopoietic cells
blood in response to hypoxia (too low tissue oxygen)
● Yellow marrow consist of fats; not capable of
→ Three (3) major effects of EPO:
erythropoiesis
1. Allowing early release of reticulocytes from the bone
Related Terms marrow
● Erythron 2. Reducing the time needed for cells to mature in the
→ Total mass of RBCs circulating in the peripheral blood bone marrow
and the bone marrow RBC precursors 3. Preventing apoptotic cell death
→ Different from the RBC mass − Apoptosis rescue is the MAJOR way in which
● RBC mass EPO increases RBC mass
→ Total population of erythrocytes in the blood vessels, → Applications:
excludes RBCs that are found elsewhere 1. Blood doping
● Erythrokinetics − Induce RBC production using erythropoietin
→ A term that describes the dynamics of RBC creation o Athletes use EPO to increase RBC mass
and destruction − Illegal and dangerous (makes the blood too
▪ Bone marrow → birthplace of RBCs viscous; prone to thrombosis)
▪ Spleen → graveyard of RBCs ● Growth hormone
→ Process from the inception of RBCs up to culling in the → Produced by: Pituitary gland
spleen → Stimulates erythropoiesis directly
● Ineffective erythropoiesis ▪ Affects bone marrow cells
→ Production of defective erythroid precursor cells ● Testosterone
→ These defective RBCs precursors often undergo → Produced by: Testes
apoptosis in the bone marrow before they have a → Stimulated erythropoiesis indirectly
chance to mature to the reticulocyte stage ▪ Affects kidneys by stimulating kidneys to produce
→ Results in anemia more EPO
→ Example of conditions: ● Prolactin
1. Vitamin B12 deficiency - macrocytic, normochromic → Produced by: Pituitary gland
2. Folate deficiency - macrocytic, normochromic → Stimulates erythropoiesis directly
3. Thalassemia - microcytic, hypochromic ▪ Same with growth hormone
4. Sideroblastic anemia - microcytic, hypochromic ● Estrogen
● Insufficient erythropoiesis → Produced by: Ovaries
→ Decrease in the number of RBC precursors in the bone → Inhibits erythropoiesis indirectly
marrow, resulting in decreased RBC production ▪ Suppresses production of EPO from kidneys of
→ Results in anemia females, hence the reason why females have lower
→ Example of conditions: RBC count
1. Iron deficiency - microcytic, hypochromic Nota bene!
2. Renal disease - normocytic, normochromic
● Males have higher Hct, Hgb, RBC counts due to
3. Acute leukemia - normocytic, normochromic
hormonal differences
● Progenitor cells
→ Testosterone in males and estrogen in females
→ Immature hematopoietic cells that are committed to a
➢ Menstruation has little to no effect on RBC count
cell line but cannot be identified morphologically
● Trans Females
→ Examples: BFU-E, CFU-E
→ Have lower RBC count after they have undergone
● Precursor cells
procedures to transition to female compared to when
→ More mature than progenitor cells
they were previously male due to administration of
→ Immature hematopoietic cells that are morphologically
estrogen
identifiable as belonging to a given cell line
→ MTF individuals do not menstruate; lower hematocrit
→ However, both are still considered young cells
and RBC counts are due to hormonal changes in
→ Examples: Rubriblast, Prorubricyte, etc.
estrogen and testosterone
● CD71
→ Earliest marker of erythroid differentiation
B. RBC STAGES OF MATURATION
→ Transferrin receptor
● Committed erythroid progenitor cells
● CD34
→ BFU-E (Burst forming unit-erythroid)
→ Classical marker of hematopoietic stem cells
→ CFU-E (Colony-forming unit-erythroid
● It takes about 18 to 21 days for the BFU-E to mature to
an erythrocyte, of which approximately 6 days are spent
as recognizable precursors in the bone marrow

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The sequence of the stages below are sequential → If the areas occupied by the nucleus and the
RUBRIBLAST NORMOBLAST ERYTHROBLAST cytoplasm are approximately equal, the N:C ratio is 1:1
Rubriblast Pronormoblast Proerythroblast → N:C Ratio < 1
Prorubricyte Basophilic Basophilic ▪ Nucleus takes up <50% of the area of the cell
Normoblast OR Erythroblast OR ▪ E.g. 1:6, 1:4
Early Normoblast Early Erythroblast → N:C Ratio > 1
Rubricyte Polychromatophili Polychromatophili → Nucleus takes up >50% of the area of the cell
c Normoblast OR c Erythroblast OR ▪ E.g. 8:1, 6:1
Intermediate Intermediate ● Basophilia
Normoblast Erythroblast → Pertains to the blueness of a particular part of the cell
Metarubricyte Orthochromatic Orthochromatic due to the acidic components that attract the basic
Normoblast OR Erythroblast OR stain (e.g. methylene blue)
Late Normoblast Late Erythroblast → Degree of cytoplasmic basophilia correlates with the
Reticulocyte Reticulocyte Reticulocyte quantity of ribosomal RNA
● Eosinophilia or Acidophilia
Mature Mature Mature
→ Pertains to the pinkness of a particular part of the cell
erythrocyte erythrocyte erythrocyte
due to the accumulation of more basic components
✓ NOTE:
that attract the acid stain (e.g. eosin)
→ Mature RBCs are not considered precursors because
→ As the RBC matures, the eosinophilia of the cytoplasm
they are the final stage
correlates with the accumulation of hemoglobin
▪ The last precursor cell is considered to be the
reticulocyte Prorubricyte (Basophilic Normoblast/Early
→ Polychromatophilic erythrocyte and Diffusely Basophilic Normoblast)
Erythrocyte ● Each prorubricyte gives rise to 4 rubricytes
▪ When the “reticulocytes” are seen in a ➢ Last stage with a nucleolus
Wright-stained smear ➢ First stage of hemoglobinization (Hb synthesis) Rodak
▪ Reticulocytes are viewed using supravital stains: → Hemoglobin is an eosinophilic material, hence it is
1. Brilliant Cresyl Blue (BCB) colored pink
2. New Methylene Blue (NMB); highly recommended → However, prorubricyte will not manifest a pink
○ Methylene blue should not be used for staining coloration since Hb synthesis is still starting
reticulocytes; not the same as NMB ● Most helpful criteria in distinguishing the prorubricyte from
Additional info on etymology the rubriblast:
1. Coarser chromatin
● The pro- and meta- prefixes:
→ “Pro-” means before
→ “Meta-” means after
2. Absence of nucleoli
Size Nucleoli Nucleus
blue
Cytoplasm
● ‘Rubri-’system Appears more
→ Blast is first Round, thin abundant than
→ Prorubricyte - cell comes before the rubricyte hence nuclear in normoblast
10 to 15
the nomenclature 0 to 1 membrane, because of
um
→ Metarubricyte - cell stage that comes after the smaller, slightly smaller nucleus
rubricyte stage. eccentric
N:C ratio: 6:1
-

Rubricyte (Polychromatic Normoblast/Intermediate

Normoblast)
● Each of this cell (rubricyte) gives rise to 2 metarubricytes
● Last stage capable of mitosis -manifests
->
● First stage in which the cytoplasm becomes PINK
→ Recall that the previous stage is when - the Agb's
hemoglobinization begins pinkness
→ By the time the cell reaches the rubricyte stage, there
will be considerable amount of hemoglobin that is
Rubriblast (Pronormoblast) eosinophilic
● This cell gives rise to 2 prorubricytes → The eosinophilic hemoglobin mixes with the basophilic
→ Capable of mitosis cytoplasm, hence the grayish color
+ blueness
● Earliest recognizable erythroid precursor using the light ● May be confused with a lymphocyte pink
microscope basophilial → Lymphocyte

S
● The more nucleoli, the younger the cell ▪ Nucleus: Crushed velvet
p dark blue
▪ Cytoplasm: Sky-blue or “Robin Egg” blue
Size Nucleoli Nucleus Cytoplasm
→ Rubricyte
Round or Small in ▪ Nucleus: Checkerboard
slightly oval, amount,
Present ▪ Cytoplasm: Muddy or gray
thin nuclear moderately
12 to 20 (1 to 2) − Gray color is attributed to the mixture of colors in
membrane, basophilic,
um usually its cytoplasm
central or grou !
very faint
slightly
homogenous
Size Nucleoli Nucleus eCytoplasm
eccentric N:C ratio = 8:1 Basophilic to
-
Round and
smaller, thick diffusely lilac in
Additional Information
10 to 12 nuclear color, depending
● N:C Ratio (Nucleus-to-Cytoplasm ratio) None on hemoglobin
→ A morphological feature used to identify and stage um membrane,
eccentric content
RBC and WBC precursors
→ A visual estimate of what area of the cell is occupied nucleus NC ratio = 4:1
by the nucleus compared with the cytoplasm -

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Metarubricyte (Orthochromatic Normoblast/late Size Nucleoli Nucleus Cytoplasm

Normoblast) Cytoplasm still with small
● Has the same color (salmon-pink) as the mature amounts of RNA =
erythrocyte polychromasia (mixed pink
→ Ortho- → the same and blue staining)
→ Chroma- → color 8 to 10
None None With Golgi apparatus
● Last stage with a nucleus um remnants and residual
→ Nucleus is extruded at this stage, and the cell becomes
mitochondria that allows
a reticulocyte after
continues aerobic
● Other names:
metabolism and hemoglobin
1. Nucleated RBC
production
2. Pyknotic erythroblast
3. Acidophilic normoblast (attributed to pink cytoplasm) Mature Erythrocyte
● Pyrenocyte ● Shape: biconcave disk; Average life span: 120 days
→ Enveloped extruded nucleus
- -
- ● Thickness: 1.5 to 2.5 um
→ Engulfed by bone marrow macrophages ● Number of erythrocytes produced from each rubriblast: 16
→ Frequently, small fragments of the nucleus are left → 1 rubriblast produces 2 prorubricytes 8 to 32
behind if the projection is pinched off before the entire → 1 prorubricyte produces 4 rubricytes
nucleus is enveloped → 1 rubricyte produces 2 metarubricytes
▪ The fragments are called Howell-Jolly bodies when ● Normal ratio of RBCs to WBCs is approximately 600:1
seen in the RBCs in the circulation ● Normal ratio of RBCs to Platelets is approximately 15:1
→ Howell-Jolly bodies are typically removed from the ● Adult RBC contains no mitochondria (no protein or Hb

they enter the circulation

O
RBCs by the splenic macrophage pitting process once
-
-
synthesis)
splenectory #T bodies
:
Size Nucleoli Nucleus Cytoplasm
Nota bene! can still be seen Salmon-pink
● Post-splenectomy: 7 to 8 (with a central pallor
→ May increase Howell-Jolly bodies in the PBS because None None
um occupying ⅓ of the cell’s
the spleen is responsible for the removal of Howell diameter)
Jolly Bodies
● According to a reference material, hemoglobin is first Recap
synthesized in the rubricyte stage ● Earliest recognizable erythroid precursor → Rubriblast
→ Other sources state that hemoglobin is synthesized in ● Last stage capable of mitosis → Rubricyte
the rubriblast stage ● First stage of hemoglobinization → Prorubricyte
→ There are multiple sources that claim different facts: ● Last stage of hemoglobin synthesis → Reticulocyte
→ Rodaks: Prorubricyte stage ● First stage in which the cytoplasm becomes pink →
★ Best answer to choose in exams Rubricyte
● Last stage with a nucleolus → Prorubricyte
Size Nucleoli Nucleus Cytoplasm ● Last stage with a nucleus → Metarubricyte
Pyknotic malit
· ● Salmon pink colored → Metarubricyte, Mature
Salmon-pink erythrocyte
8 to 10 (dense mass of
None
um degenerated
N:C ratio: 1:2 II. OVERVIEW OF ANEMIA
chromatin) -

● Anemia
→ Is not a disease; a consequence of having a disease
→ Defined as the decrease below normal of one or more
of the following:
1. Number of RBCs
2. Hemoglobin
3. Volume of packed RBCs (hematocrit)
Mechanisms of Anemia
1. Hemorrhage
→ First consideration in any case of anemia
→ Loss of erythrocyte through bleeding must always be
the first focus in any patient with anemia and must
prompt an evaluation of the hemostatic system
→ Bleeding: may be secondary to trauma, surgery or a
disease
Reticulocyte → Gastrointestinal tract: a common site for clinically
● Young RBCs containing residual RNA (last immature significant bleeding
erythrocyte stage) → Menstruation: a significant source of blood loss in
● Spends 2 to 3 days in the bone marrow and 1 day in the women
peripheral blood before developing into a mature RBC 2. Hemolysis
● Last stage of hemoglobin synthesis → Shortened erythrocyte survival time NOT explained by
● Types of reticulocytes: bleeding
1. Shift Cells → Destruction of erythrocytes prior to 120 days or before
▪ Polychromatophilic macrocyte their expected lifespan
▪ Seen in cases of increased RBC production → Can occur in the blood vessels (intravascular) or in the
2. Stress Reticulocytes spleen (extravascular)
▪ Macroreticulocytes ▪ In certain disorders, the spleen has increased
▪ Seen in more severe conditions (ex. hemolytic hemolytic function
anemia)

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of
bone marrow problem
3. Decreased production of erythrocytes
→ RBCs usually arise from bone marrow
−
J
FA is NOT the same as Fanconi Syndrome
=
o Fanconi Syndrome is a defect involving the
▪ Issues with the red marrow can lead to decreased proximal convoluted tubules of the kidneys
production of erythrocytes, and thus, anemia Fanconi Anemia is a bone marrow disorder
o
→ Kidney disease may be associated with decreased ★ According to a source, Fanconi Anemia is characterized
production of EPO by macrocytic anemia;
→ Nutritional deficiencies (iron, vitamin B12, or folic acid) → Macrocytosis and reticulocytopenia
are common and readily correctable causes of 2. Paroxysmal Nocturnal Hemoglobinuria
hypoproliferative anemia
● a.k.a. Marchiafava-Micheli Syndrome
A. MORPHOLOGIC CLASSIFICATION OF ANEMIAS ● Hemolytic anemia from uncontrolled complement
● Two methods of classifying anemia: activation, bone marrow failure, and a propensity for
→ Morphological thrombosis
-▪ Basis of classification are MCV and MCHC ● Caused by deficiency of:
▪ Groups: 1. DAF (decay-accelerating factor or CD55)
1. Normocytic, normochromic 2. MIRL (membrane inhibitor of reactive lysis or CD59)
2. Microcytic, hypochromic → Both enzymes are complement-regulatory proteins
3. Macrocytic, normochromic ● Tests for PNH:
→ Pathophysiologic (not discussed; less common) → Ham’s Acidified Serum Test
▪ Basis of classification are mechanisms → Sugar Water Test (A.K.A Sucrose Hemolysis Test)
→ Flow Cytometry (confirmatory test)
NORMOCYTIC NORMOCHROMIC
● MCV and MCHC are both normal MICROCYTIC HYPOCHROMIC
Normal or Decreased Increased ● With small cells that have increased central pallor on
Reticulocyte Count Reticulocyte Count the smear
Aplastic Anemia Paroxysmal Nocturnal ● MCV and MCHC are both low
Hemoglobinuria ● Microcytic anemia results from an iron level insufficient

Kinneycute
Renal Disease
for maintaining normal erythropoiesis and is
Paroxysmal Cold
blood loss Hemoglobinuria
characterized by abnormal results of iron studies
● Early development of a microcytic anemia may reveal
(Donath-Landsteiner
reduced iron stores, but an obvious anemia has not
Hemolytic Anemia)
developed
Sickle Cell Disease ASSOCIATED CONDITIONS
Enzyme Deficiencies Thalassemia
(G6PD def., PK def.) Anemia of Chronic Inflammation (ACI)
Other hemolytic anemias Iron Deficiency anemia
Lead poisoning (A.K.A. plumbism)
1. Aplastic Anemia Sideroblastic anemia
● Rare, potentially deadly bone marrow failure syndrome
● Characteristic features: 1. Anemia of Chronic Inflammation
● Was originally called ACD (Anemia of Chronic Disease)
alls
1. Pancytopenia (marked decrease in the number of
RBCs, WBCs, and platelets in the blood) ● Most common anemia among hospitalized patients
2. Reticulocytopenia ● Associated with chronic infectious such as tuberculosis,
3. Bone marrow hypocellularity chronic inflammatory conditions such as rheumatoid
4. Depletion of hematopoietic stem cells arthritis and tumors
● May be classified as: ● Body stores have abundant iron; red cells are deficient in
1. Acquired Aplastic Anemia -
majority iron
▪ Approx. 80 to 85% of aplastic anemia cases ● Central feature of ACI: sideropenia (decreased serum
▪ Two (2) categories: iron) despite abundant iron stores
1. Idiopathic AAA – no known cause → When iron serum levels are low, the body naturally
2. Secondary AAA – associated with an identified respond by releasing iron from the storage sites
Benzene
-

cause → But due to chronic inflammation, macrophages keep

clearing o Some of the causes include: chemicals (e.g. the iron and thus iron stores are abundant in ACI
insecticides, benzene), viruses (e.g. EBV), → Quick pathophysiology:
agent =
▪ Chronic inflammation results to protracted increase
drugs (e.g. chloramphenicol)
o Chloramphenicol: drug most frequently in APR
implicated in acquired aplastic anemia ▪ Hepcidin inhibits iron transcytosis from cells to the
2. Inherited Aplastic Anemia blood because hepcidin inhibits ferroportein activity.
▪ Approx. 15 to 20% of AA cases ● Usually PBS shows normocytic, normochromic
▪ Associated diseases: → Advanced states: microcytic, hypochromic
− Dyskeratosis congenita ● Acute phase reactants (APRs) that contribute to ACI
− Shwachman-Bodian-Diamond Syndrome → Ferritin
− Fanconi Anemia → Lactoferrin
o Most common of the inherited aplastic → Hepcidin
anemia ▪ Master regulatory hormone for systemic iron
o A chromosome instability disorder metabolism
o Characterized by: aplastic anemia, cancer ▪ Inactivates ferroportin (a protein present inside
susceptibility, and physical abnormalities tissues that transports iron from the tissues to the
o Skeletal abnormalities (thumb malformation, blood)
microcephaly, scoliosis)
o Skin pigmentation (hyperpigmentation,
hypopigmentation, cafe-au-lait lesions)
o Short stature
o Abnormalities of the eyes, kidneys and genitals
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2. Sideroblastic Anemias MACROCYTIC NORMOCHROMIC

● Develop when the production of protoporphyrin or the ● High MCV, Normal MCHC
incorporation of iron into protoporphyrin is prevented Differentiati Megaloblastic Non Megaloblastic
● Sideroblast ng Factors Anemia Anemia
→ Nucleated RBC precursor with cytoplasmic iron Some of ● Vit. B12 deficiency ● Liver disease
granules the Causes ● Folate deficiency ● Alcoholism
● Siderocyte ● Acute (chronic)
→ Anucleated RBC with iron granules erythroleukemia ● Bone marrow
● Iron is trapped in the mitochondria (cannot be fully utilized failure
in Hb synthesis) Presence
● Ring sideroblasts of
→ Hallmark of the sideroblastic anemias hypersegm PRESENT ABSENT
→ The “rings” in ring sideroblastic: mitochondria loaded ented
with iron neutrophils
● Dimorphic peripheral blood picture (normochromic and NOTE:
hypochromic RBCs) ● Normal neutrophil have 3 to 5 nuclear lobes
3. Iron Deficiency Anemia ● Hypersegmented neutrophils
● Most common anemia → With ≥ 6 lobes
● Possible causes: → Essentially pathognomonic for megaloblastic anemia
1. Blood loss (especially menstruating women) Shape =
OVAL ROUND
2. Nutritional deficiency (infants) macrocytes
3. Increased iron demand (pregnancy, lactation, Megaloblas
adolescence) ts in the
PRESENT ABSENT
4. Malignancies of gastrointestinal tract bone
5. Hookworm infections marrow
● Clinical features: 1. Megaloblastic: Vitamin Deficiency
→ Fatigue, weakness, irritability, headache, palpitations, ● Vitamin B12 Deficiency
loss of concentration → May be caused by:
→ Shortness of breath 1. Inadequate intake
→ Brittle hair 2. Increased need (pregnancy, lactation, growth)
→ Pallor 3. Competition (e.g. D. latum infection)
→ Koilonychia (spooning of the fingernails) 4. Lack of intrinsic factor (e.g. gastrectomy,
→ Glossitis (inflammation of the tongue) Helicobacter pylori infection, pernicious anemia)
→ Angular cheilitis (lesion of the corners of the mouth) → Note: Only natural source for vitB12 are the animals
→ Pica or cravings for non-food items (e.g. dirt, clay, ▪ Strict vegetarians (do not eat animal products) are
laundry, starch, or ice) prone to develop vitamin B12 deficiency
▪ Pagophagia (compulsive ice chewing, most common) → H. pylori infection
● Ferritin test → most sensitive test for IDA ▪ The bacteria colonize the parietal cells and
→ Ferritin → storage form of iron eventually destroy the cells
DIFFERENTIATION OF → Gastrectomy
MICROCYTIC, HYPOCHROMIC ANEMIAS ▪ Surgical removal of the stomach
Serum Serum − Removes parietal cells
TIBC FEP
Iron Ferritin → Pernicious Anemia
Thalassemias ↑ N ↑ N ▪ Pernicious → a.k.a dangerous
ACI ↓ ↓ ↑ ↑ ▪ Characterized by the presence of antibodies against
IDA ↓ ↑ ↓ ↑ parietal cells and antibodies against intrinsic factor
Lead N to ↑ ▪ Parietal cells (found in the stomach) produce intrinsic
Poisoning (adults) factor
N N ↑ ▪ Intrinsic factor is important for vit. B12 absorption
N to ↓
(children) ● Folate Deficiency
Sideroblastic ↓ and ↑ → May be caused by:
↑ N ↑ 1. Inadequate intake
Anemia (mixed)
2. Increased need
STAGES OF IRON DEFICIENCY 3. Renal dialysis
TIBC Ferritin
Serum Total Additional Information
Hb Most
Iron Iron-Binding Sensitive
Test for IDA
● Impaired DNA synthesis: considered as root cause of
Capacity
megaloblastic anemia
Normal Iron
N N N N ● DNA synthesis is dependent on an important structure:
Status
thymidine triphosphate (TTP)
Stage 1: → This structure cannot be made unless it receives a
Storage Iron N N N ↓ methyl group from methyltetrahydrofolate or folic acid
Depletion → Vit. B12 is the cofactor responsible for transferring the
Stage 2: methyl group to methyl tetrahydrofolate
Transport Iron N ↓ ↑ ↓ → Adequate amounts of vit. B12 and folic acid are
Depletion important to the formation of TTP
Stage 3: → If TTP cannot be made, then it is replaced by
Functional Iron ↓ ↓ ↑ ↓ deoxyuridine triphosphate
Depletion* ▪ The synthesis of this structure results in nuclear
*Characterized by Frank Iron Deficiency Anemia fragmentation and destruction of cells and impaired
cell division

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● Vit. B12 a.k.a. Cobalamin Serum

● Folate is the general term used for any form of the folic Lactate
acid Dehydrog ↑ ↑
● Cyanocobalamin – synthetic form of vitamin B12 (found enase
only in supplements) Serum
● Methylcobalamin – naturally occurring form that may be Vit. B12 N ↓
obtained from either food sources or supplements Serum
● In either folate or vitamin B12 deficiency, patients may Folate ↓ N or ↑
experience: RBC
→ Fatigue, weakness, and shortness of breath Folate ↓ N or ↓
→ Glossitis (loss of epithelium on the tongue results in a Serum
smooth surface and soreness) Methylma N ↑
→ Gastritis, nausea, or constipation Specific
lonic Acid
● Blood pictures seen in folate deficiency and vitamin B12 Diagnostic
Test Serum/Pl
deficiency are indistinguishable. asma
→ However, their clinical presentations differ. Homocyst ↑ ↑
● In vitamin B12 deficiency, neurologic symptoms may be eine
noticeable and neuropsychiatric symptoms may also be holoTC
present Assay
→ Neurologic symptoms include:
▪ Memory loss
(holotrans N ↓
cobalami
▪ Numbness n)
▪ Tingling in toes and fingers
● Mean Cell Hemoglobin (MCH) – elevated by the increased
▪ Loss of balance
volume of the cells
▪ Further impairment of walking by loss vibratory
● Mean Cell Hemoglobin Concentration (MCHC) – usually
sense, especially in the lower limbs
within the reference interval (Hb production is unaffected)
→ Neuropsychiatric symptoms include:
● Other possible findings in Megaloblastic Anemia:
▪ Personality changes
1. Teardrop cells
▪ Psychosis
2. Nucleated RBCs
● Dietary deficiency of vitamin B12 is infrequent.
3. Howell-Jolly bodies
→ However, this condition is possible for vegans (strict
4. Basophilic stippling
vegetarians) who do not eat meat, eggs or dairy
5. Cabot rings
products
● Once in the enterocyte, the vitamin B12 is then liberated
● Sources of Vit. B12:
from IF and bound to transcobalamin (previously called
→ Liver, meat, oyster and clams, fish, eggs, cheese and
transcobalamin II) and released into the blood.
other dairy products
→ In the plasma, 10 to 30% of the vitamin B12 is bound to
● Vitamin B12 is NOT destroyed by cooking
transcobalamin (75% is bound to transcobalamin II and
● Pregnancy needs a significant increase in folate to fulfill
III, referred to as the haptocorrin)
the requirements related to rapid fetal growth, uterine
→ The vitamin B12-transcobalamin complex, called
expansion, placental maturation, and expanded blood
holotranscobalamin (holoTC), is the metabolically
volume. Folate deficiency during pregnancy can lead to
active form of Vitamin B12
impared formation of the fetal nervous system, resulting
in neural tube defects (like spina bifida) B. PATHOPHYSIOLOGIC CLASSIFICATION OF
● Sources of Folate: ANEMIAS
→ Leafy green vegetables, dried beans, liver, beef, ● Anemias caused by decreased production of RBCs
fortified breakfast cereals ● Anemias caused by increased destruction
→ Broccoli, dairy products whole grains, some fruits ● Blood loss (hemorrhage)
especially oranges
● Folates are heat labile (overcooking of foods can Anemias caused by Decreased Production of RBCs
decrease their nutritional value) ● Aplastic anemia
● Megaloblastic anemia
LABORATORY TESTS USED TO DIAGNOSE VITAMIN ● Iron deficiency anemia
B12 AND FOLATE DEFICIENCY ● Thalassemia
Folate Vit. B12 ● Anemia of Chronic Renal failure
Deficiency Deficiency ● Anemia of Endocrine Disorders
CBC ↓Hb, Hct, RBCs, ● Anemia of Chronic Inflammation
WBCs, PLTs ● Anemia associated with marrow infiltration
● Sideroblastic anemia
↑MCV, MCH
Manual Hypersegmented Same as Anemia cause Increased Destruction of RBCs
Diff. NEU, oval Folate ● Intracorpuscular abnormality
Count macrocytes, deficiency → Membrane defect
anisocytosis, ▪ Hereditary spherocytosis
Screening poikilocytosis, ▪ Hereditary elliptocytosis
Tests RBC inclusions ▪ Pyropoikilocytosis
Abs. → Enzyme deficiency
Retic. ↓ ↓ ▪ G6PD
Count ▪ Pyruvate kinase
Serum ▪ Porphyria
Total and → Globin abnormality
Indirect ↑ ↑ ▪ Hemoglobinopathies (e.g. Hb SS, CC, SC)
Bilirubin → Paroxysmal Nocturnal Hemoglobinuria

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● Extracorpuscular abnormality 2. Type II Myeloblasts:

→ Mechanical ▪ Shows the presence of dispersed primary
▪ Microangiopathic hemolytic anemia, etc. (azurophilic) granules in the cytoplasm
→ Infection ▪ Other names of granules in type II
▪ Malaria, Bartonella, Ehrlichia, Babesia − Nonspecific granules
→ Chemical and physical agents − Azurophilic granules
▪ Drugs, toxins, burns − Primary granules
→ Antibody-mediated ▪ Number of granules does not exceed 20 per cell
▪ Acquired hemolytic anemia due to warm-reacting 3. Type III Myeloblasts:
antibodies ▪ Have a darker chromatin and a more purple
III. WHITE BLOOD CELLS cytoplasm, and they contain more than 20 granules
● Nucleated cells that function in body defense that do not obscure the nucleus
● Reference ranges: ▪ Rare in normal bone marrow, but they can be seen
→ Adult both males and females: 4.5 - 11x 109/L (SI) or in certain types of acute myeloid leukemias
4,500 - 11,000/mm3 (conventional) A2. Promyelocyte
→ Newborn 13.5 - 38 x109/L SI or 13,500 - 38,000/mm 3 ● Size: 16-25um
conventional ● Slightly bigger than myeloblast
● NOTE: The difference between mm3 and uL is → Only exemption in the cell size rule
INSIGNIFICANT; can be used interchangeably ● 1-5% of the nucleated cells in the bone marrow
Additional Information ● Nucleus round to oval, often eccentric
● How do you convert the WBC count from conventional ● Hof: indicator of normal promyelocytes
units to SI? → Usually seen in normal promyelocytes but not in the
→ Move the decimal point three decimal places to the left malignant promyelocytes of acute promyelocytic
● Predominant WBC in adult: neutrophil leukemia
● Predominant WBC in children <4 years of age: → The hollow in the cytoplasm of a cell that lodges the
lymphocyte nucleus
● On determining WBC maturity, most valuable and ● Cytoplasm is evenly basophilic and full of primary
reliable criterion is: nuclear chromatin pattern (azurophilic/nonspecific) granules
→ Better demonstrates the primary granules
WBC Classification ● One to three nucleoli can be seen but may be obscured by
the granules
GRANULOCYTES AGRANULOCYTES
Basophils Lymphocytes A3. Myelocyte
Eosinophils Monocytes ● Size: 15-18um
Neutrophils ● 6-17% of the nucleated cells in the bone marrow
● Last stage capable of mitosis
POLYMORPHONUCLEAR MONONUCLEAR ● Stage of synthesis of secondary granules (aka. specific
Basophils Lymphocytes granules)
Eosinophils Monocytes ● Rubricyte is the RBC precursor counterpart
Neutrophils → 3: third stage of granulocyte and the erythroid series
PHAGOCYTES IMMUNOCYTES are the last stages capable of mitosis
Basophils Lymphocytes ● Two types:
Eosinophils 1. Early myelocytes
Neutrophils ▪ May look very similar to the promyelocyte, EXCEPT
Monocytes that patches of grainy pale pink cytoplasm
representing secondary granules start to become
A. GRANULOCYTIC SERIES obvious in the area of the Golgi apparatus called
● In general, granulocytes mature: ‘Dawn of neutrophilia’
1. Nuclear chromatin becomes more condensed − A phenomenon that describes the appearance of
2. Nucleoli disappear secondary granules near the area of golgi
3. Abundant basophilic cytoplasm with nonspecific apparatus
granulation progresses to more scant cytoplasm 2. Late myelocytes
containing specific granules ▪ Smaller than promyelocytes (15-18um)
4. The nucleus indents and becomes segmented ▪ The nucleus has considerably more
5. Overall cell size decreases heterochromatin, nucleoli are difficult to see by light
microscopy
A1. Myeloblast
● Size: 14-20 um A4. Metamyelocyte/Juvenile Cell
● Earliest recognizable granulocytic precursor using the light ● Size:14-16 um
microscope ● Production of tertiary granules/gelatinase granules
● 0-3% of the nucleated cells in the bone marrow ● First stage of nuclear indentation (curve in the nucleus of
● Three types: the cell)
1. Type I Myeloblasts: ● 3-20% of the nucleated cells in the bone marrow
▪ Does not contain or show any granules yet ● Nucleoli are absent
▪ Nucleus occupies most of the cell, with very little ● Synthesis of tertiary granules (also known as gelatinase
cytoplasm granules) may begin at this stage
▪ Slightly basophilic cytoplasm, fine nuclear chromatin, ● May sometimes be mistaken as band cells due to the
and two to four visible nucleoli, no visible granules indentation
when observed under light microscopy with → Metamyelocyte (peanut or kidney bean-shaped)
Romanowsky stains ▪ Indentation will only be shallow, did not exceed ½ of
the width of the nucleus
→ Band cell
▪ Indentation is more than ½ of the nuclear width
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● Appearance in peripheral blood smear B. Eosinophil

→ Metamyelocyte → always pathologic
● Size: 9-15um
→ Band cells → may be normal
● Increased level in blood: EOSINOPHILIA
A5. Band Cell/ Stab Cell/ Staff Cell ● Decreased level in blood: EOSINOPENIA
● Size: 9-15um ASSOCIATED CONDITIONS
● 9-32% of the nucleated cells in the bone marrow
EOSINOPHILIA EOSINOPENIA
● Youngest granulocytic precursor to normally appear in the
1. Asthma 1. ACTH administration
peripheral blood
2. Hay fever 2. Autoimmune disorders
→ Any younger stages that appear in the PBS will be
3. Psoriasis 3. Steroid therapy
considered pathologic
4. Eczema 4. Stress
→ The younger the stage that is present in the PBS, the
5. Scarlet fever 5. Sepsis
more alarming the condition will be.
6. Eosinophilic leukemia 6. Acute inflammatory
● Secretory granules (aka secretory vesicles) may begin to
7. Parasitic infections states
be formed during this stage
● Nucleus: elongated, curved, or sausage-shaped with ● Parasitic infections
rounded ends (filaments not present) → Most common cause of severe and moderate
● CLSI recommends that bands should be included within eosinophilia
the neutrophil counts and not reported as separate ▪ Parasites include: helminthes, nematodes, cestodes,
category due to the difficulty in distinguishing bands from trematodes (excludes protozoans; usually do not
segmented neutrophils increase eosinophils)
→ Trichinosis caused by T. spiralis may possibly produce
Recap
the highest eosinophil count
● Primary (nonspecific) granules → Type II Myeloblast ▪ Acquired via eating improperly cooked pork with
● Secondary (specific) granules → Myelocyte encysted larvae
● Tertiary (gelatinase) granules → Metamyelocyte
● Secretory granules (Secretory vesicles) → Band cell C. Basophil
● Size: 10-15um
A6. Segmented Neutrophil/Mature Granulocytes ● Increased level in the blood is called: BASOPHILIA
● Size: 9-15um ● Decreased level in the blood is called: BASOPENIA
● 7-30% of the nucleated cells in the bone marrow ASSOCIATED CONDITIONS
● Normal value of neutrophil is 1.6 -7.2 x 109/L BASOPHILIA BASOPENIA
● Increased levels: NEUTROPHILIA
1. Immediate 1. Acute infections
→ Appendicitis, bacterial infections, parasitic infections
hypersensitivity 2. Stress
particularly liver flukes and malaria, pseudo neutrophilia
reactions 3. Hyperthyroidism
or physiologic neutrophilia
2. Hypothyroidism 4. Increase levels of
→ Physiologic neutrophilia
3. Ulcerative colitis glucocorticoids
▪ Shift of marginated cells to the circulatory pool
4. Estrogen therapy 5. Chronic urticaria
▪ Occur in exercise, excessive temperature changes,
nausea, vomiting, pregnancy and labor, rage, panic, B. AGRANULOCYTIC SERIES
stress
● Decreased levels: NEUTROPENIA A. Lymphocytic Series
→ Agranulocytosis is < 0.5 x 109/L also known as extreme ● Stages:
neutropenia 1. Lymphoblast
▪ Associated drugs: amidopyrine, cephalosporin 2. Prolymphocyte
3. Lymphocyte
ASSOCIATED CONDITIONS ● Sizes:
NEUTROPHILIA NEUTROPENIA → 7-10um: Small lymphocyte
1. Bacterial infections 1. Overwhelming infections ▪ Round nucleus comprises majority of the cell with
2. Appendicitis 2. Splenomegaly scanty cytoplasm
3. Rheumatoid arthritis 3. Hemodialysis ▪ Predominant form of lymphocyte in the normal adult
4. Acute destruction 4. Copper deficiency blood
represented by 5. Alcoholism → 10-12um: Medium/intermediate lymphocyte
pancreatitis, colitis, 6. Babies dorm from → 11-25um: Large lymphocyte
myocardial infarction, hypertensive mothers ▪ Has abundant cytoplasm
severe hemolysis, 7. Chemical toxicity ● Increased level: LYMPHOCYTOSIS
surgical or traumatic (benzene) → B. pertussi (bacteria); T. gondii (parasite)
wounds, thermal injury 8. Marrow replacement ● Decreased level: LYMPHOPENIA/ LYMPHOCYTOPENIA
5. Parasites (malaria, liver 9. Nutritional deficiencies
flukes) 10. Cytotoxic drugs ASSOCIATED CONDITIONS
6. Lithium LYMPHOCYTOSIS LYMPHOPENIA
7. Chemicals (lead, 1. Infectious 1. Aplastic anemia
mercury) mononucleosis 2. AIDS
8. Drugs (digitalis, 2. Infectious lymphocytosis 3. SARS
phenacetin) 3. Cytomegalovirus 4. Ethanol abuse
9. Corticosteroids infection 5. Zinc deficiency
10. Myelogenous leukemia 4. Acute viral hepatitis
11. Venoms (Spiders, bees, 5. Bordetella pertussis
wasps) infection
12. Actinomyces fungi 6. Brucellosis
13. Response to therapy 7. Toxoplasmosis
14. Physiologic neutrophilia 8. Acute HIV infection
(pseudo neutrophilia)

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● Plasma Cells Peritoneum Peritoneal macrophages

→ Size: 10-28um Synovial tissues Type A cells
→ Final maturation stage of B lymphocyte Reproductive organ Reproductive organ MACs
→ Nucleus: small, oval and eccentric Bone Osteoclasts
▪ Appearance: Placenta Hofbauer Cells
− Tortoise shell
− Cartwheel REFERENCE RANGES
− Clock face WBC Type Relative Count Absolute Count
→ Cytoplasm is basophilic, dark blue/sea blue/ cornflower Neutrophils 51 to 67% 1,600 to 7,260/uL
in color; ovoid (egg-like), nongranular Lymphocytes 25 to 33% 960 to 4,400/uL
→ May contain round, discrete globules called Russell Monocytes 2 to 6% 180 to 880/uL
bodies that contain immunoglobulins Eosinophils 1 to 4% 45 to 440/uL
→ Plasma cell myeloma Basophils 0 to 1% 45 to 110/uL
▪ aka. Multiple Myeloma _ Most abundant __ Least abundant
▪ Most common malignant disease of plasma cells ● How do we determine basopenia if the lower limit in the
▪ Elderly patients are more affected relative count is 0%?
▪ Individuals with MM will excrete Bence Jones → Automated machines can now perform absolute count.
protein in their urine → Base the assessment depending on the absolute count
− Will precipitate if the urine is heated but dissolve
in higher temperatures IV. OVERVIEW OF LEUKEMIA
B. Monocytic Series ● Malignant neoplasm of the blood-forming tissues of the
bone marrow, spleen, and lymph system
● Size: 15-20 um; largest leukocyte
● Stages General characteristics
1. Monoblast ● More blasts: shorter, more fatal course of disease
2. Promonocyte ● Increased WBC count with shift to the left
3. Monocyte → Shift to the left → increased forms of younger forms of
WBCs in the PBS
ASSOCIATED CONDITIONS
● Higher M:E ratio
MONOCYTOSIS MONOCYTOPENIA
(increased level) (decreased level) → Myeloid:Erythroid ratio → 10:1
1. Tuberculosis 1. Aplastic anemia → Normal: M:E ratio → 2:1 - 4:1
2. Subacute Bacterial 2. Overwhelming ● Type of anemia usually present in cases of acute
Endocarditis infections in leukemia: NORMOCYTIC, NORMOCHROMIC
3. Syphilis immunocompromised → Expected most of the time but not all the time
4. Protozoal and patients ACUTE LEUKEMIAS CHRONIC LEUKEMIAS
rickettsial infections 3. Hemodialysis 1. Described by symptoms 1. Described by symptoms
(e.g. malaria, typhus) 4. Epstein-Barr virus of short duration of long duration
5. Brucellosis infection 2. Numerous immature cell 2. Mostly mature cell forms
6. Typhoid 5. Steroid therapy forms in the bone marrow in the bone marrow
7. Gaucher disease and/or peripheral blood and/or peripheral blood
8. Hodgkin’s disease 3. Increased total WBC 3. Total WBC range from
9. Collagen vascular count extremely elevated to
diseases (e.g. lupus lower than normal
erythematosus)
10. Gastrointestinal FAB Classification of Leukemias
disease ● According to French-American-British (FAB) Cooperative
11. Surgical trauma Group 1970s
● OLD WAY of classifying leukemia
● Macrophages → WHO Criteria is the modern classification system
→ Size: 40-50 um ● Classification:
→ Considered to be monocytes → Based on morphology of cells in
▪ Monocytes → if found in blood Romanowsky-stained smear
▪ Macrophages → if found extravascularly ▪ Romanowsky stains
▪ Most abundant cell in the body − Group of stains that contain methylene blue and a
− More abundant than all the RBCs or skin cells halogenated fluorescein dye such as Eosin Y and
→ Functions: Eosin B
1. Phagocytosis − E.g. Wright’s, Giemsa, May-Grunwald Stain
2. Synthesize nitric oxide (cytotoxic against viruses, → Based on cytologic and histochemical
bacteria, fungi, protozoa, helminths, and tumor cells) characteristics of cells involved
3. Release interleukin 1 (stimulates T-lymphocytes) ▪ E.g. of cytochemical stains: Myeloperoxidase (MPO)
4. Produce transcobalamin II and Sudan Black B (SBB)
− Transcobalamin II is now called transcobalamin
− Primary transport factor of Vitamin B12 A. LYMPHOCYTIC LEUKEMIA
LOCATION SPECIFIC NAMES OF Acute Lymphocytic Leukemia (ALL)
MACROPHAGES ● Generally:
Liver Kupffer cells → Myeloperoxidase negative
Lungs Dust Cells → Sudan Black B negative
Kidneys Renal Mesangial Cells ● ALL is the most common form of childhood leukemia
Brain Microglial Cells ● ALL can be subclassified under the following parameters:
Skin Langerhans Cells 1. FAB (ALL L1, ALL L2, ALL L3)
Spleen Littoral cells 2. Use of Immunologic Markers (T-ALL, B-ALL,
Intestines Intestinal macrophages Null-ALL, Common ALL)

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FAB CLASSIFICATION OF ALL B. NON-LYMPHOCYTIC LEUKEMIAS OR

Differentiat L3 MYELOGENOUS LEUKEMIAS
L1 L2
ing Factors Burkitt-type ● Generally:
Rare in → Myeloperoxidase positive
70% of 70% of adult
Patients children and → Sudan Black B positive
childhood ALL ALL
adults
slg Acute Myelogenous Leukemia (AML)
CALLA (CD10)
CD19
Immunologi TdT
TdT CD20
GRP ORIGIN REMARKS
c markers CD19 AML; with minimal differentiation
CD22
CD20 M0 Myelocytic MPO: negative
CD24
Homogenous SBB: negative
population of AML; without maturation
Homogenous Heterogeneous large blasts May demonstrate Auer rods (Linear
Cell size population of population of (with nuclear projections of primary granules)
small blasts large blasts and M1 Myelocytic
Auer rods may be found in AML but
cytoplasmic is not pathognomonic and unique
vacuoles) for AML
Uniformly AML; with maturation
Nucleus Irregular Round to oval
round, small
M2 Myelocytic Most common subtype of AML
Single to
Nucleolus Single 2-5 May also demonstrate Auer rods
several
Slightly
Acute Promyelocytic Leukemia
Coarse with (APL)
reticulated with
Chromatin Fine clear
perinucleolar Associated with DIC and Faggot
parachromatin
clumping cells (abnormal WBC with bundles
Moderate, of Auer rods)
blue, DIC (disseminated intravascular
Cytoplasm Scant, blue Moderate, pale
prominently M3 Myelocytic coagulation) aka. Defibrination
vacuolated Syndrome; Consumption
ALL L3 → also known as Burkitt-type of leukemia Coagulopathy (DIC is not a disease
CYTOCHEMISTRY but a consequence of a disease)
Periodic
M3V: APL, microgranular variant;
Acid-Schiff + + - cells have characteristic “butterfly”,
(PAS)
“bowtie”, or “apple core” nuclei
Methyl
Green ● DIC is a generalized overactivation of the coagulation
Pyronine - - + and fibrinolytic system of the body
(MGP) → There is clot formation and bleeding
Oil Red O Acute Myelomonocytic Leukemia
(ORO) + + + (AMML)
Sometimes observed in L1 and L2 Naegeli Monocytic Leukemia
Myelocytic
M4 Monocytic
2nd most common subtype of AML
BY USE OF IMMUNOLOGIC MARKERS May demonstrate Auer rods
Serum
Classification E Rosettes Surface Ig M4E: AMML with increased marrow
Anti-ALL
eosinophils
T-ALL + - -
Acute Monocytic Leukemia
B-ALL - + - (AMoL) a.k.a. Schilling Leukemia
Common
- - + M5a: AMoL, poorly differentiated
ALL
Null ALL - - - Seen in children
M5 Monocytic
> 80% monoblasts in BM
Chronic Lymphocytic Leukemia (CLL)
M5b: AMoL, well differentiated
● Most common type of leukemia in the elderly Seen in middle-aged adults
● Characterized by: < 80% monoblasts in BM
1. Persistent lymphocytosis Acute Erythroleukemia
2. Presence of increased number of smudge cells and DiGuglielmo’s Syndrome
rieder cells in the PBS
▪ Smudge cells → nuclear remnants of the May demonstrate Auer rods
Erythrocytic
lymphocytes; “thumbprint” appearance M6 Myelocytic
Type of anemia is macrocytic and
▪ Rieder cells → lymphocytes with notched, lobulated normochromic
or cloverleaf-like nucleus Erythroid cells in AML M6 is
● Examples of clinical variations of CLL: strongly positive in PAS
1. Hairy-cell leukemia Acute Megakaryocytic Leukemia
▪ A lymphocyte with little cytoplasmic projections
M7 Megakaryocytic Requires immunocytochemistry
▪ TRAP (+): Tartrate-Resistant Acid Phosphatase
staining for accurate diagnosis
2. Lymphosarcoma cell leukemia
Positive in Factor VIII stain
3. Prolymphocytic leukemia
● AML types that show Auer Rods include:
→ M1, M2, M3, M4, M6

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CYTOCHEMICAL REACTIONS ● Leukocyte (Neutrophil) Alkaline Phosphatase

M1 (LAP/NAP) Test
M2 M4 M5 M6 M7 → Neutrophil is the only leukocyte that has alkaline
M3 phosphatase activity
Myeloperoxidase → Generally used to distinguish LR from CML
MPO
+ + - +/- - → Principle: Increased LAP activity can be observed in
Sudan Black B neutrophils that have undergone normal growth
SBB
+ + +/- +/- - → Kaplow’s Method
Naphthol AS-D ▪ Principle: Hydrolysis of sodium alpha naphthyl
chloroacetate + + - +/- - phosphate by alkaline phosphatase produces a
(Specific Esterase) colored precipitate with a diazotized amine
α-naphthyl
FUNCTION REAGENTS
butyrate esterase - + + - -
(Nonspecific Esterase Fixative Methanol and formalin
α-naphthyl Buffer Propanediol
Localized
acetate esterase - + + - Substrate Sodium alpha naphthyl
positivity
(Nonspecific Esterase) phosphate
Factor VIII Stain - - - - + Initial stain Brentamine-fast gamet salt
“Myelocytic” = (+) → MPO, SBB, SE Counterstain Aqueous Mayer’s
“Monocytic” = (+) → NSE (butyrate), NSE (acetate) hematoxylin
Chronic Myelogenous Leukemia Procedure
● Also known as: Chronic Granulocytic Leukemia 1. Immerse dry blood smear in fixative for 30 seconds
● Characterized by Philadelphia Chromosome (Ph1) 2. Pour onto smear the working substrate (made up of
→ 90% of patients with CML have Ph 1 buffer, substrate, and initial stain) and allow to stand
→ Presence of Ph1 = good prognosis for at least 10 minutes
● First described in 1960 by Peter C. Nowell, a faculty 3. Rinse with distilled water and dry
member of the University of Pennsylvania, Philadelphia, 4. Counterstain for 10-15 minutes
as an unfamiliar small chromosome present in the WBCs 5. Rinse with distilled water and mount in mounting
of patients with CML solution like glycerol
● Due to reciprocal translocation involving the long arms of 6. Examine immediately under the microscope and look
chromosome 9 and 22 for the presence of reddish-brown to black precipitate
1. A piece of chromosome 9 and a piece of C22 break off of alkaline phosphatase activity in the cytoplasm of
and trade places neutrophils
2. The BCR-ABL gene is formed on chromosome 22 7. Count 100 segmented neutrophils and bands and
where the piece of chromosome 9 attaches. score each of these cells**
3. The changed chromosome 22 is called the Philadelphia 8. After which, total the individual scores of the 100
chromosome neutrophils
4. Results in the formation of BCR-ABL1 fusion gene ▪ Normal Kaplow’s score: 20-100
SCORE** DESCRIPTION
0 No reddish-brown to black precipitate
1+ Slightly diffused reddish brown-black
precipitate
2+ Moderately diffused reddish-brown to black
precipitate
3+ Heavily diffused reddish-brown to black
precipitate
4+ Very heavily diffused reddish-brown to black
precipitate
● Increased LAP Score ⇒ leukemoid reaction
● Decreased LAP Score ⇒ chronic myelogenous leukemia
→ Recall that abnormal neutrophils have little to no
alkaline phosphatase activity
● Three (3) clinical phases:
1. Chronic phase DISORDERS WITH DISORDERS WITH
2. Accelerated phase INCREASED KAPLOW’S DECREASED KAPLOW’S
3. Blast crisis (LAP) SCORE (LAP) SCORE
● Must be differentiated from Leukemoid Reaction (LR) Third trimester of Paroxysmal Nocturnal
→ A clinical syndrome resembling leukemia pregnancy Hemoglobinuria
▪ Confused with CML Polycythemia vera CML
→ Not a disease; it is a description only Infections Sideroblastic Anemia
→ Excessive leukocytic response against a specific Intoxication Myelodysplastic syndrome
stimulus
→ WBC count: > 50 x 109/L
▪ With neutrophilia and a marked left shift (presence of
immature neutrophilic forms)
▪ LR most frequently refers to neutrophils, but the
increased count may be due to an increase in other
types of WBCs

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C. SAMPLE PROBLEM
SCORE # OF LAP SCORE
NEUTROPHILS
0 32 0
1+ 24 24
2+ 21 42
3+ 15 45
4+ 8 32
TOTAL: 143
● Interpretation: Leukemoid Reaction
D. FACTORS THAT DIFFERENTIATE CML FROM LR

CML LEUKEMOID
REACTION
Leukocyte in the Blasts/Promyeloc Usually
peripheral blood ytes myelocytes
Toxic granulation Absent Present
Eosinophils/Baso ↑ ↓
phils
LAP ↓ ↑
Philadelphia Usually present Absent
Chromosome
(Ph1)
Splenomegaly Usually prominent Mild (if present)
Platelet Count >600 or <50 x Normal
109/L

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INDEX: APPENDIX
Stage Size Nucleoli Nucleus Cytoplasm
Small in amount, moderately
Rubriblast Round or slightly oval, thin
Present (1 to 2) basophilic, homogenous
12 to 20 um nuclear membrane, central or
Pronormoblast usually very faint
slightly eccentric
NC ratio = 8:1
Appears more abundant than
Prorubricyte Round, thin nuclear in normoblast because of
Basophilic Normoblast 10 to 15 um 0 to 1 membrane, smaller, slightly smaller nucleus
Early Normoblast eccentric
N:C ratio: 6:1
Rubricyte
Basophilic to diffusely lilac in
Polychromatic Round and smaller, thick color, depending on
Normoblast 10 to 12 um None nuclear membrane, eccentric hemoglobin content
Intermediate nucleus
NC ratio = 4:1
Normoblast
Metarubricyte
Pyknotic Salmon-pink
Orthochromatic
8 to 10 um None (dense mass of degenerated
Normoblast
chromatin) N:C ratio: 1:2
Late Normoblast
Cytoplasm still with small
amounts of RNA =
polychromasia (mixed pink
and blue staining)
Reticulocyte 8 to 10 um None None With Golgi apparatus
remnants and residual
mitochondria that allows
continues aerobic metabolism
and hemoglobin production
Salmon-pink
(with a central pallor
Mature Erythrocyte 7 to 8 um None None
occupying ⅓ of the cell’s
diameter)

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