lOMoARcPSD|11119751

MTLE-Hematology-1 - hematology 1 intro to hema lecture

notes by xiao
Science Technology and Society (Intro. To MedTech) (Notre Dame of Marbel University)

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MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW – CLINICAL HEMATOLOGY

HEMATOLOGY 1 – RBCs & WBCs

Lecturer: Rene Jesus Alfredo Dinglasan
Notes by: Xiao - The Conqueror of Demons, The Vigilant Yaksha, & Alatus, the Golden-Winged King

INTRODUCTION TO HEMATOLOGY COMMON SUFFIXES FROM GREEK AND LATIN USED IN THE
- Hematology: from the Greek words haima (blood) and logos VOCABULARY OF HEMATOLOGY
(study/science) SUFFIX MEANING
- Blood: red liquid circulating in the heart, veins, arteries, and capillaries -algia pain along a nerve
-ase an enzyme
TYPES OF BLOOD VESSELS -cide the killer of
1. ARTERIES - distributing blood vessels that leave the heart -crit to separate
(4 mm) - have the thickest walls of the vascular system -cyte cell
2. VEINS - collecting blood vessels that return to the heart -ectomy incision and removal
(5 mm) - largest; have a more irregular lumen than arteries -emia blood
3. CAPILLARIES - blood passes from the arterial to the venous system -itis inflammation
(8 um) via the capillaries -lysis destruction or dissolving
- capillaries are the smallest, thinnest walled, and -oma swelling or tumor
most numerous of the blood vessels. -opathy disease
- Functions of the blood: -osis (1) abnormal increase; (2) disease
o Respiratory -penia deficiency, decreased
o Nutritional -phil(ic) attracted to, affinity for
o Excretory -plasia(-plastic) cell production or repair
o Buffering action -poiesis cell production, formation, and development
o Maintenance of body temperature -poietin stimulates production
o Transport of hormones
o Defense mechanism BLOOD COLLECTION
BLOOD COMPOSITION
Liquid portion - Plasma: liquid portion of unclotted blood specimen I. PATENT IDENTIFICATION
- Serum: liquid portion of clotted blood specimen - Identify patient by asking him to state and spell his full name, age, gender
Solid portion - Red blood cells: a.k.a. erythrocytes, erythroplastids, and birthdate
(cellular elements or akaryocyte (cell without nucleus) o The patient must be the one to identify himself
hemocytes) - White blood cells: a.k.a. leukocytes, leukoplastids o If the patient cannot identify himself, the physician or nurse may identify
o Granular WBC: Basophils, Eosinophils, him/her
Neutrophils - Most critical step in blood collection
o Agranular WBC: Lymphocytes, Monocytes - “mortal sin” of a phlebotomist – mislabeling
- Platelets: a.k.a. thrombocytes, thromboplastids
Gaseous portion - Oxygen, carbon dioxide, etc. Note: A phlebotomist should maintain good health and hygiene, always have
clean clothes, clean hair, and clean, short fingernails. Standard precautions
CHARACTERISTICS OF THE BLOOD: should always be followed, with special consideration to the proper use of gloves
1. Fluid (in vivo) and hand washing.
2. Red
3. Slightly alkaline
4. Average specific gravity of 1.055 II. SOME PHYSIOLOGIC FACTORS AFFECTING TEST RESULT
5. Thick and viscous (3.5 to 4.5 times thicker than water)
6. Makes up 75 to 85 mL blood per kilogram body weight FACTORS EFFECTS
7. There are about 20 grams of solids per 100 mL of blood Posture - Shift in posture from supine (lying) to a sitting or standing
position may increase the levels of:
COMMON PREFIXES FROM GREEK AND LATIN USED IN THE o Protein
VOCABULARY OF HEMATOLOGY o Cholesterol
PREFIX MEANING o Iron
a-/an- lack, without absent, decreased - Note: Their concentration increases because these large
aniso- unequal, dissimilar molecules cannot filter into the tissues at a time when the
ante- before body water transfers from the inside of the blood vessels to
crena- wrinkled the interstitial spaces.
cyt- cell
Diurnal
dys- abnormal, difficult bad In the morning In the afternoon
rhythm
erythro- red ↑ Cortisol ↓ Cortisol
ferr- iron ↑ TSH ↓ TSH
hemo-(hemato-) pertaining to blood ↑ Iron ↓ Iron
hypo- beneath, under, deficient, decreased ↓ Eosinophil count ↑ Eosinophil count
hyper- above, beyond, extreme
iso- equal, alike, same Stress - ↑ WBCs, ↑ Fibrinogen group (a.k.a. Thrombin-sensitive
leuk(o)- white group: Factors I, V, VIII, XIII)
macro- large, long Exercise - Exercise can increase different blood constituents.
mega- large, giant Examples are:
meta- (1) after, next (2) change o Creatinine
micro- small o Total protein
myel(o)- (1) from bone marrow (2) spinal cord o Creatine kinase (CK)
pan- all, overall, all-inclusive
o Myoglobin
phleb- vein
phago- ingest o Aspartate aminotransferase (AST)
poikilo- varied, irregular o HDL – cholesterol
poly- many o Lactate dehydrogenase
pre- or pro- before o WBC count
pykno- dense o Platelet count
reticulo- netlike Diet - After a fatty meal:
schis- split o Falsely ↑ hemoglobin level
scler- hard o ↑ Alkaline phosphatase (intestinal isoenzyme)
sidero- iron Smoking - ↑ WBCs and Cortisol
splen- spleen
thromb(o)- clot, thrombus
xanth- yellow

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III. SKIN PUNCTURE IV. VENIPUNCTURE

- Technique of choice to obtain blood specimen from the following: - Most common sites for venipuncture:
o Newborns and pediatric patients o Superficial veins of the antecubital fossa (area in front of the elbow)
o Severely burned patients - Most common skin antiseptic: 70% isopropyl alcohol
o Patents whose veins are reserved for therapeutic purposes o NOTE: Clean the site using a circular motion, starting in the center and
o Extremely obese patients working outward
o Elderly patients with fragile veins - TWO anatomical patterns:
o A: “H” pattern of veins
PUNCTURE SITES: o B: “M” pattern of veins
For infants (<1 year old): For older children (>1 y.o.) and
adults:

Plantar surface of the Palmar surface of the non-dominant

medial or lateral side of the heel hand (3rd or 4th finger, distal portion)

Unshaded parts/areas are Puncture should be done across the

prohibited sites fingerprints (perpendicular to the
grooves in the whorls of the
“H” pattern “M” pattern
fingerprint)
Veins to be used Veins to be used
RECOMMENDED DEPTH OF SKIN PUNCTURE:
(in order of preference): (in order of preference):
For infants and small children For adults - Median cubital vein - Median vein
<2.0 mm 2.0 mm – 2.5 mm - Cephalic vein - Accessory cephalic vein
- Basilic vein - Basilic vein
- Avoid applying pressure/squeezing/ "milking” the site
o Possible effects of squeezing:
 Can cause hemolysis
 Can introduce excess interstitial fluid (a.k.a. tissue fluid)
- Using 70% isopropyl alcohol, the medical technologist must clean the
puncture site and allow it to air-dry.
- Discard 1st drop of blood:
o WHY?
 To discard excess tissue fluid
 To discard dead epidermal cells (skin cells)
 To facilitate free flow of blood IMPORTANT CONSIDERATIONS IN VENIPUNCTURE
- Devices for collecting blood (skin puncture): capillary tubes and 1. Angle between skin and needle: <30o
microcollection tubes 2. Tourniquet application:
- Length of time: <1 minute
ORDER OF DRAW (for SKIN PUNCTURE procedure) - Effects of prolonged tourniquet application:
Mnemonic: “T S E O S” o Hemoconcentration
o Hemolysis
1. Tube for blood gas analysis o Shortened coagulation times (ex. PT and aPTT)
o First tube collected - REMEMBER:
2. Slides o Stasis - condition in which venous flow is slowed results in the
3. EDTA microcollection tube (filled first before other microcollection tubes in local accumulation of Factor VIlI and vWF (may result in FALSE
order to guarantee adequate volume and accurate results) SHORTENING of clot-based coagulation test results)
4. Other microcollection tubes with anticoagulants - Distance: 3-4 inches (7.5 to 10 cm) above the venipuncture site
5. Serum microcollection tubes - Steps in proper (rubber) tourniquet application:
o Slide the tourniquet under the arm 3 to 4 inches above the
NOTE! intended venipuncture site. Evenly adjust both ends of the
- Warming of the puncture site can increase the blood flow sevenfold. tourniquet.
- The site can be warmed using a warm washcloth (40 to 42oC) for no o Hold both ends of the tourniquet a few inches above the patient's
longer than 3 to 5 minutes. arm. Pull up on the ends to produce tension in the tourniquet.
Cross the right side of the tourniquet over the left side. With the
index finger of the right hand make a small loop in the right side
of the tourniquet while continuing to hold tension in the tourniquet.
o Insert this small loop under the left side of the tourniquet. The
resulting application will permit easy removal of the tourniquet
with one hand after the needle has been inserted into the vein.
3. Most common needle size for adult venipuncture: 21 G (length: 1 inch)
- Gauge number = bore size
- The higher the gauge number, the smaller the bore size
- The lower the gauge number, the larger the bore size
4. A phlebotomist must NEVER puncture a patient more than twice.
5. Patient should NOT pump the fist (this may affect certain test results, for
example: elevations in the potassium concentration)
6. Some of the sites to AVOID:
- Veins in the inner wrist
- Veins in the feet
- Fistula
- Arteries
- Inflamed sites
- Edematous sites

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7. Some of the causes of specimen hemolysis COMPLICATIONS (VENIPUNCTURE)

- Prolonged tourniquet application 1. Ecchymosis
- Moisture/contamination in the blood collection tube - aka: Bruise
- Using needles with too small bores - MOST COMMON encountered complication
- Excessive agitation - Leakage of small amounts of blood into the skin
- Frothing of the blood sample - Purplish discoloration of the skin (flat)
8. Standard Precautions
- Originally called: Universal precautions 2. Hematoma
- Practices to control diseases in which all blood, body fluids, and - Leakage of large amounts of blood into the skin
unfixed issues are handled as if they were potentially infectious. - Swollen/bulging, purplish discoloration of the skin
- Apply to blood, semen, vaginal secretions, cerebrospinal fluid, 3. Fainting
synovial fluid, pleural fluid, any body fluid with visible blood, any - aka: Syncope
unidentified body fluid, unfixed slides, microhematocrit clay, and - Short lapse in consciousness
saliva from dental procedures - Ammonia inhalants
9. Hand washing o No longer recommended
- Most important practice to prevent the spread of infectious diseases o Might trigger an asthma attack
- Under running water, wet hands and wrists thoroughly.
4. Hemoconcentration
- Use germicidal soap and rub hands vigorously for at least 15
- ↑ concentration of cells, larger molecules, and analytes in the blood
seconds, including between the fingers and around and over the
because of a shift in water balance
fingernails.
- Can be caused by prolonged tourniquet application
- Rinse hands thoroughly under running water in a downward flow
- After prolonged application, allow 2 minutes before reapplying the
from wrist to fingertips.
tourniquet
- Dry hands using a paper towel.
- Use the paper towel to turn off the faucet handles. 5. Intravenous (IV) Therapy
10. Needles and other sharp objects (e.g., used lancets, broken tubes, etc.) - Draw blood from the arm opposite the IV site
contaminated with blood and other possibly infectious materials must NOT - If no alternative
be manipulated in any way. o Ask the nurse to stop the infusion for 2 minutes
- Examples of such manipulation include: re-sheathing, bending, o Draw blood from below the catheter site
clipping, or removing the sharp object o First 5 mL of blood → discarded!
- Re-sheathing or recapping is allowed only when there are no other 6. Mastectomy Patients
alternatives or when recapping is required by particular medical - Mastectomy – surgical removal of the breast
procedures. - Draw blood from the arm opposite the mastectomy side
- Recapping is allowed by-use of a method other than the traditional - CLSI requires physician consultation before blood is drawn from:
two-handed procedure. The one-handed method or a re-sheathing o the same side as a prior mastectomy
device is often used. o bilateral mastectomies
- Documentation in the exposure control plan should identify the
particular procedure in which re-sheathing is allowable. 7. Obesity
11. Check sharps containers regularly. - The use of a blood pressure cuff can help in locating a vein.
- Replace them when they are no more than three-quarters full. Failure - BP cuff must NOT be:
to do so encourages overstuffing, which sometimes leads to injury. o Inflated >40 mm Hg
Disposal procedures should aways be enforced. o Left on the arm for more than 1 minute
- The most common cause of a needle puncture or a puncture from 8. Iatrogenic Anemia
other sharp objects is improper disposal. - a.k.a. nosocomial anemia, physician-induced anemia
- anemia caused by continuous blood collection for the purpose of
PROPER TOURNIQUET APPLICATION testing
9. Failure to draw blood
10. Petechiae
11. Nerve Damage
12. Hemolysis
13. Burned, Damaged, Scarred, And Occluded Veins
14. Seizures and Tremors
15. Vomiting and Choking
16. Allergies
17. Dialysis Patients
18. Edema
ADDITIVES

ADDITIVES NOTES
Antiglycolytic Agents – inhibit the use of glucose by blood cells
1. Sodium fluoride - Preserves glucose for 3 days
- Found in: gray top tube
- No. of inversions: 8x
- Used for: blood glucose and blood alcohol level
determination
- Anticoagulant used: Potassium oxalate
2. Lithium - Preserves glucose for 1 day
iodoacetate - Found in: gray top tube
- No. of inversions: 8x
- Used for: blood glucose and blood alcohol level
determination
- Anticoagulant used: Lithium heparin
Clot Activators – quicken the clotting of the blood specimen
1. Glass or Silica - Activates factor XII
particles - Found in: red top (plastic)
- No. of inversions: 5x
- Used for: Stat serum determinations
o Stat is from the latin word Statim = immediately
2. Thrombin - Activates factor I, V, VIII, and XIII
- Found in: orange top
- No. of inversions: 8x
- Used for: Stat serum determinations
Separator Gel/Thixotropic gel – inert material that undergoes temporary change
in viscosity during the centrifugation process
- Found in: Gold top
- No. of inversions: 5x
- Used for: serum determinations

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Anticoagulants – inhibit the blood from clotting COMPLETE BLOOD COUNT (CBC)
1. EDTA - Optimal anticoagulant concentration is: 1.5 - Also called: Hemogram
(Ethylenediamine mg/mL of blood - Performed on automated hematology profiling instruments and includes:
tetraacetic acid) - Action: Chelation of calcium o RBC Parameters:
 RBC count
Important reminders in using EDTA:  Hemoglobin
- Found in: - CBC can be performed on blood stored at room
lavender/purple  Hematocrit
temperature for up to 4 hours  RBC indices
top - WBC counts, Hematocrit, and Platelet counts can  Red Cell Distribution Width (RDW)
- No. of inversions: be determined up to 24 hours after blood is  Reticulocyte count
8x collected in EDTA if it is refrigerated at 4oC. o WBC Parameters
- Used for: routine - ESR-should be set up within 2 hours of collection  WBC count
hematology if the EDTA blood was stored at room temperature  NEUT count % and absolute
determination or within 6 hours if the specimen was refrigerated  LYMPH count % and absolute
- Most commonly - Blood smears can be made from EDTA tube as  MONO count % and absolute
long as they are made within 2 hours of blood  EO and BASO counts: % and absolute
used blood collection
collection tube in o Platelet Parameters
- EDTA prevents platelet aggregation (therefore the  Platelet count
the hematology preferred anticoagulant for platelet counts)  Mean Platelet Volume (MPV)
section - Mean Platelet Volume (MPV)
o EDTA causes swelling of platelets (causes
Possible causes of approximately 20% increase in MPV during the REMINDERS!
clotted blood first hour) - Collect the blood specimen of your patent using the correct order of draw
specimens: o Should be based on EDTA specimens that are (purpose: to avoid possible test errors because of cross-contamination from
between 1 to 4 hours old tube additives)
- Difficult - Invert each tube containing additive immediately after collection.
- Insufficient EDTA
phlebotomy o Cause: Over-filled tube - Label the blood collection tubes appropriately. The minimal amount of
(redirection) o Effect: Presence of clots information that should be on each tube is as follows:
- Blood sample - Excessive EDTA o Patient's full name
was NOT inverted o Cause: Underfilled tube (“short draw”) o Patients unique identification number
in the correct o Effect: ↓ Hematocrit, ↓ ESR, degenerative o Date and time (military time) of collection
number of times changes in WBCs, ↑ MCHC, ↑ platelet count o Phlebotomists initials or code number
- Expired blood - Related terms o Compare the labeled tube with the patient’s identification bracelet or
collection tube o Delta Check - a process in which a current test have the patent confirm that the information on the blood collection tube
result is compared with the result of the same is correct (whenever possible).
- Overfilled tube
test from the previous specimen from the same
patient ORDER OF DRAW
o Chelation: formation of a ring-shaped
molecular complex in which a metal ion is
covalently bound
o Reflex test: a test being ordered automatically
based on the results of prior tests or preset
parameters
2. Heparin - Optimal anticoagulant concentration is: 15-20
units per mL of blood
- Found in: green - Action: binds antithrombin (inhibits thrombin and Blood collection Number of Color
top factor Xa) tube inversions
- This is the anticoagulant of choice for: Osmotic Blood culture tube 8 Yellow Top
- No. of inversions: fragility test (OFT) and blood gas studies
8x Citrated tube 3-4 Light Blue Top
- Three heparin formulations
- Used for: flow o Ammonium heparin Serum tube 0 Red Top (glass) (Non-additive)
cytometry, o Sodium heparin 5 Red Top (plastic) (has clot activator)
plasma o Lithium heparin Heparinized tube 8 Green Top
chemistry  causes the least interference in chemistry EDTA tube 8 Lavender/Purple/Pink Top
determinations, testing; Sodium fluoride tube 8 Gray top
osmotic fragility  most widely used anticoagulant for
test (OFT), blood plasma and whole blood chemistry tests
- Important reminders in using heparin OTHER BLOOD COLLECTION TUBES:
gas studies o Lithium heparin: must NOT be used for lithium BLOOD ANTICOAGULANT USES
level determination COLLECTION
o Sodium heparin: must NOT be used for TUBES
sodium level determination or for electrolyte YELLOW TOP SPS (Sodium Polyanethol Blood culture
panel Sulfonate)
o Ammonium heparin - must NOT be used for
ammonia level determination
Actions:
o Heparin causes cellular clumping (especially
of platelets), which leads to - prevents phagocytosis
pseudoleukocytosis (falsely ↑ WBC count) and - prevents complement
pseudothrombocytopenia (falsely ↓ platelet activation
count) on some hematology analyzers. - neutralizes some
o Not to be used for blood smear preparation antibiotics
because it causes morphologic distortion of - anticoagulant
platelets and leukocytes ACD (Acid Citrate Dextrose) Blood bank tests,
o Causes bluish coloration of the background on HLA tests, DNA
blood smears stained with a Romanowsky
stain because of is pH tests, and Paternity
o Not to be used for coagulation studies because tests
it inhibits thrombin TAN TOP K2 EDTA Lead determination
3. 3.2% Sodium - Critical ratio between anticoagulant and blood: 1:9
Citrate - Action: chelation of calcium Certified to
- Forceful mixing or an excessive number of contain less than
- Found in: light inversions can activate platelets and shorten 0.01 ug/mL of lead
clotting times ROYAL BLUE K2 EDTA Toxicology,
blue top
- No. of inversions: TOP nutritional
3-4x chemistry
- Used for: Contains only low determination,
coagulation tests levels of trace trace elements
elements determination
WHITE TOP K2 EDTA with gel Molecular
diagnostic tests
BLACK TOP 3.8% sodium citrate (4:1 blood Westergren ESR
to anticoagulant ratio)

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PINK TOP K2 EDTA Blood bank tests, LIGHT BLUE 3.2% Sodium citrate Coagulation tests
whole blood CTAD (Citrate, Theophylline, Used for platelet
Has a special hematology Adenosine, Dipyridamole) function assays (ex.
crossmatch label determinations platelet factor-4 and
for patient β-thromboglobulin
information assays)
(approved by the
AABB)

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ERRORS ENCOUNTERED IN SOME HEMATOLOGY TESTS CRITICAL VALUES THAT REQUIRE IMMEDIATE COMMUNICATIONS
PARAMETERS TYPE PROBABLE REASON/S - Critical values must be determined by the laboratory in consultation with the
AFFECTED OF users.
ERROR - When a critical value is obtained, the physician of the patient or the
responsible healthcare worker must be informed by any means of
RBC count Falsely ↑ WBC >100,000/uL, large(giant) platelets, communication.
cryoglobulin, cryofibrinogen - Any critical value obtained must be documented and referred immediately
Falsely ↓ Autoagglutination, cold agglutinins, to the pathologist. A preliminary report may be issued followed by a final
hemolysis (in vitro), microcytosis, report after review by the pathologist.
schistocytes, clotting
Mean Cell Falsely ↑ Autoagglutination, high WBC TEST CRITICAL VALUES
Volume (MCV) (>50,000/uL), cold agglutinins, old Hematocrit <21% or <0.21
specimen, hyperosmolar state >65% or >0.65
(uncontrolled diabetes mellitus), reduced Hemoglobin <70 g/L
red cell deformability >200 g/dL
Falsely ↓ Cryoglobulin, cryofibrinogen, giant Reticulocyte >20%
platelets, hemolysis (in vitro), swollen red WBC count <2,000/uL on a new patient or a 1,000 difference from
cells previous, if less than 4,000/uL
Mean Cell Falsely ↑ Lipemia, icterus, chylomicrons, high WBC >50,000/uL on a new patient
Hemoglobin (>50,000/uL), spuriously high hemoglobin, Blood smear Neutrophilic phagocytosis of microorganisms
(MCH) spuriously low RBC (indicating the presence of systemic infection)
Falsely ↓ Spuriously low hemoglobin, falsely high Shows abnormal leukemoid reaction
RBC Schistocytes (may indicate hemolytic condition)
Mean Cell Falsely ↑ Cold agglutinins, autoagglutination, Sickle cells
Hemoglobin clotting, hemolysis (in vitro), hemolysis (in Blast forms (if never or not recently reported in the
Concentration vivo), spuriously high hemoglobin, falsely patient)
(MCHC) low hematocrit Presence of intracellular organisms
Falsely ↓ High WBC (>50,000/uL), falsely low Platelets <20,000/uL and not previously reported
hemoglobin, spuriously high hematocrit >1 million/uL
Hemoglobin Falsely ↑ Lipemia, icterus, chylomicrons, lysis- Prothrombin time >40 seconds
resistant RBCs with abnormal hemoglobin,
WBC>20,000/uL (>20x109/L), platelet PERIPHERAL BLOOD SMEAR
count (>700x109/L), parenteral nutrition,
hypergammaglobulinemia, cryoglobulin, I. SOURCES OF SPECIMENS
cryofibrinogen, hemolysis (in vitro),
heparin, hyperbilirubinemia - EDTA blood
Falsely ↓ Clotting o Blood smears should be made within 2-hours after collection
Hematocrit Falsely ↑ Dehydration, hemoconcentration, o Advantages of EDTA blood smear:
(Microhematocrit) insufficient centrifugation, buffy coat  Multiple blood smears may be made.
inclusion, hyponatremia, plasma trapping  The blood smear may be prepared at a later time.
Falsely ↓ Hemolysis (in vitro), improper sealing of  EDTA prevents platelet clumping.
the capillary tube, increased anticoagulant o Disadvantages of EDTA blood smear:
concentration, introduction of excess
DISADVANTAGES EFFECTS CORRECTION
tissue fluid, hypernatremia
Platelet Satellitosis Pseudothrombocytopenia Re-collect blood
Hematocrit Falsely ↑ Cryoglobulin, cryofibrinogen, giant
(aka platelet specimen using 3.2%
(Automated) platelets, high WBC (>50,000/uL),
satellitism or platelet sodium citrate
hyperglycemia (>600 mg/dL)
rosette)
Falsely ↓ Autoagglutination, clotting, hemolysis (in Platelet count (obtained
vitro), microcytic red cells Platelets adhere on from the light blue top) x
WBC Count Falsely ↑ Lysis-resistant RBCs with abnormal the surface of WBCs 1.1 (correction factor to
hemoglobin, nucleated RBCs, (usually neutrophils) compensate for the
megakaryocyte fragments, dilution brough about by
micromegakaryoblasts, platelet clumps, the light blue top)
giant platelets, cryoglobulins, EDTA - induced Pseudothrombocytopenia Re-collect blood
cryofibrinogen, heparin, monoclonal platelet clumping Pseudoleukocytosis specimen using 3.2%
proteins sodium citrate
Falsely ↓ Leukemia (especially with chemotherapy), Platelets form large
leukoagglutination, clotting, smudge cells clumps (as large as Platelet count x 1.1
Neutrophils Falsely ↓ Neutrophil aggregation, neutrophil with WBCs)
hemosiderin granules (were counted as WBC count x 1.1
eosinophils) EDTA can both
Lymphocyte Falsely ↑ Nucleated red cells, giant platelets, platelet stimulate and
clumps, malarial parasites, hypolobated prevent platelet
neutrophils clumping
Monocyte Falsely ↑ Large reactive lymphocytes, lymphoblasts, (prevention is more
lymphoma cells, immature granulocytes common)
Eosinophils Falsely ↑ Neutrophil with hemosiderin granules
(counted as eosinophils), red cells with CASE:
malarial pigments - The patient demonstrates platelet clumping in his blood collected in a
Platelet count Falsely ↑ Leukemia (especially with chemotherapy), lavender top tube. When CBC was run, the results showed that his
fragmented red cells (in microangiopathic platelet count is low at 90,000/mm3 and his leukocyte count is high at
hemolysis), microorganisms, 15,000/mm3.
cryoglobulins, cryofibrinogen, hemolysis - The phlebotomist re-collected his blood using 3.2% sodium citrate tube
(in vitro), hemolysis (in vivo), microcytic red and another CBC was run. The new results obtained are 115,000/mm3
cells, red cell inclusions, white cell for the platelet count and 11,000/mm3 for the WBC count. What set of
fragments values should finally appear in this patient’s chart?
Falsely ↓ Platelet clumps, old specimen, partial a. Platelet count: 107,800/mm3, leukocyte count: 16,500/mm3, and all
clotting, giant platelets, platelet satellitism, the other CBC values obtained from the sodium citrate specimen.
cold agglutinins, clotting, heparin b. Platelet count: 99,000/mm3, leukocyte count: 16,500/mm3, and all
MPV (Mean Falsely ↑ Old specimen, determining the MPV too the other original CBC values obtained from the EDTA specimen.
Platelet Volume) early in an EDTA specimen c. Platelet count: 121,000/ mm3, leukocyte count: 12,100/mm3, and
all the CBC values obtained from the sodium citrate specimen.
d. Platelet count: 126,500/mm3, leukocyte count 12,100/mm3, and all
the other original CBC values obtained from the EDTA specimen.

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- Anticoagulant-free blood
o Best for the evaluation of blood cell morphology
o Advantages:
 Made at the patient’s side
 Some artifacts may be prevented
o Disadvantages:
 Platelet clumping
 Few films can be made

II. METHODS OF BLOOD FILM PREPARATION

1. TWO-GLASS SLIDE METHOD (Manual Wedge Technique)

- MOST FREQUENTLY USED
- Sides being used:
o Pusher Slide (a.k.a. SPREADER SLIDE)
o Film slide
- Angle between the spreader and film slide: 30-45o
- NOTE: If the angle of the spreader is:
o Too high: thicker smear
o Too low: thinner smear

SCANNING METHODS
LONGITUDINAL BATTLEMENT

“Tail to head” “Back and forth serpentine”’

From an area where the red cells You start at an area where the red
are near each other but not cells are near each other and are
overlapping and scan the blood not overlapping, and scan using
smear from tail to head back and forth serpentine pattern

Used in the manual wedge

technique (WBC differential count)

IMPORTANT COMMENTS Characteristics of an Ideal Blood Smear (Wedge method)

CONSIDERATIONS 1. Gradual transition from thick to thin area
1. Size of the drop Too large Too small 2. 2/3 to 3/4 the length of the film slide
of blood Thicker/longer smear Thinner/shorter smear 3. Finger-shaped
2. Speed of the Too fast Too slow 4. Visible lateral edges
spreader Thicker smear Thinner smear and poor 5. Without irregularities, holes, or streaks
WBC distribution 6. Feather edge has rainbow appearance
3. Hematocrit of If too ↑ hematocrit If too ↓ hematocrit 7. Whole drop of blood is picked up and spread
the patient Ex. Polycythemia vera Ex. Anemia
Problems encountered (Wedge method)
Angle should be lowered Angle should be raised 1. Uneven distribution of WBCs in various areas of the slide
(as low as 25o) 2. Feather edge and side edges: MORE segmented neutrophils, monocytes,
and eosinophils
3. At the center of the film: MORE small lymphocytes

2. Coverslip Technique
o Glass slide - Coverslip Method (Beacom’s method)
o Two-coverslip Method (Ehrlich's method)
- Both methods are rarely used
- Sometimes used for making bone marrow aspirate smears
preparation
- Only advantage: excellent WBC distribution

3. Automated Methods
a. Miniprep
o Semi-automatic, portable instrument
o Simulates the manual wedge technique of blood smear prep
b. Centrifugal (Spinner) Type
o Uses approximately 0.2 mL of well mixed anticoagulated blood
o Advantages:
 Evenly distributed blood cells
 Consistency of preparation
 Monolayer of cells
 Fewer smudge cells (especially in CLL patients)
c. Coulter LH (can both make and stain the smear)
d. Sysmex SP-10 (can both make and stain the smear)

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III. TECHNIQUES OF BLOOD FILM STAINING - Detect fibrin strands (if they are present, the film should
- Purpose of blood smear staining: for evaluation of cellular morphology be rejected because they indicate clotted specimen)
- Important solutions used or blood smear staining - Recognize rouleaux formation or RBC agglutination
o FIXATIVE: Methanol - Quickly detect unexpected parasites
o STAIN: Wright or Wright-Giemsa - Assess the area available for suitable examination
o BUFFER: 0.05 M sodium phosphate (pH 6.4), or aged distilled water 40x Choose areas where:
(distilled water placed in a glass bottle for at least 24 hours; pH 6.4-6.8) High-Dry or - Red cells have central pallor and are near each other
o Correct pH (for blood smear staining): pH 6.4-6.8 50x but NOT overlapping
Oil - Cells are appropriately stained (eosinophil granules:
ROMANOWSKY-TYPE STAIN Immersion bright orange, red cells: salmon-pink)
- defined as any stain which contains methylene blue (and/or its products Objective - Avoid the feathered edge (red cells appear macrocytic,
of oxidation) and a halogenated fluorescein dye (commonly eosin B or flattened, and lack central pallor & white blood cells
eosin Y) often distorted)
- METHYLENE BLUE - a basic stain, it colors the nucleus and some - Avoid thick part (red cells appear microcytic and may
cytoplasmic structures a blue or purple color (stained structures are seem to form rouleaux)
described to be basophilic [e.g., DNA or RNA]) Used also to estimate total WBC count:
- EOSIN - an acidic stain, it colors some cytoplasmic structures an - Using the 40x high-dry objective, count the leukocytes
orange-red color (stained structures are described to be acidophilic in 10 fields then find the average number of WBCs per
[e.g. proteins with amino groups]) field.
- Most commonly used type of stain in the hematology laboratory - Average no. of WBCs per hpf x 2,000 = estimated WBC
- Examples of Romanowsky-based stains: Wright stain, Giemsa stain, count per uL
and May-Grunwald slain - Alternatively, one can also use a 50x oil immersion
objective. However, one should use a different
TECHNIQUES OF STAINING multiplication factor (3,000)
MANUAL AUTOMATED QUICK - Example: lf an average of 5 WBCS were observed per
- Flood the slide - Generally, 5 to 10 - Whole process: 1 field:
with Wright’s minutes to stain a minute only o Using 40x high-dry objective, the WBC estimate is:
stain batch of slides - Uses modified Wright ܽ‫ʹݔ݁݃ܽݎ݁ݒ‬ǡͲͲͲ ൌ ݁‫ݐ݊ݑ݋ܿܥܤܹ݀݁ݐܽ݉݅ݐݏ‬
- Allow the stain to - Examples: or Wright-Giemsa stain ͷ‫ʹݔ‬ǡͲͲͲ ൌ ͳͲǡͲͲͲȀ‫ܮݑ‬
remain on the o Midas III filtered into a coplin jar
o Using 50x Oil immersion objective, the WBC
slide for 1 to 3 o Hema-Tek or a staining dish
estimate is:
minutes o Coulter LH - Aged distilled water is ܽ‫͵ݔ݁݃ܽݎ݁ݒ‬ǡͲͲͲ ൌ ݁‫ݐ݊ݑ݋ܿܥܤܹ݀݁ݐܽ݉݅ݐݏ‬
- Buffer is then o Sysmex SP-10 used as the buffer ͷ‫͵ݔ‬ǡͲͲͲ ൌ ͳͷǡͲͲͲȀ‫ܮݑ‬
added
100x - When the appropriate area of a blood smear with a
IV. CHARACTERISTICS OF A WELL-STAINED BLOOD SMEAR Oil normal RBC count is viewed, there are generally about
Immersion 200-250 RBCS per 100x OIF
MACROSCOPIC MICROSCOPIC Objective - Used to examine the nuclear details of the white blood
- RBCs: orange to salmon-pink cells
- WBC Nuclei: purple to blue - Used also for the tabulation of the actual WBC
- Neutrophil cytoplasm: pink to tan (with differential and estimation of platelet count
violet to lilac granules) - For the estimation of platelet count:
- Eosinophil granules: bright-orange o Scan ten (10) oil immersion fields for the number of
platelets
o Average number of platelets/OIF x 20,000 =
estimated platelet count per uL
o In occasions of significant anemia or erythrocytosis,
use the following formula for he platelet estimate:
‫ݐ݊ݑ݋ܿܥܤܴ݈ܽݐ݋ݐݔ݈݂݀݁݅ݎ݁݌ݏݐ݈݁݁ݐ݈ܽ݌݂݋ݎܾ݁݉ݑ݊݁݃ܽݎ݁ݒܣ‬
Pink to purple ʹͲͲܴ‫݈݂݀݁݅ݎ݁݌ݏܥܤ‬

V. PROBLEMS ENCOUNTERED IN BLOOD SMEAR STAINING *200 - average number of RBCs per oil immersion field in the
optimal assessment area
PROBLEMS USUAL CAUSES
RBCs: gray (or blue) - Stain/buffer is too basic (most common)
WBCs: too dark VII. PARASITES THAT MAY APPEAR IN THE BLOOD SMEAR
- Inadequate rinsing
Eosinophil granules: gray - Heparinized blood was used 1. Malaria
RBCs: too pale or are red - Stain/buffer is too acidic (most common) - Species that infect humans:
WBCs: barely visibly - Underbuffering o Plasmodium falciparum
- Over-rinsing  Most pathologic
 Sickle cell trait persons = seem to be resistant to P.
VI. EVALUATION OF PERIPHERAL BLOOD SMEAR falciparum infections
o Plasmodium vivax
Macroscopic Examination  Most prevalent
UNUSUAL PROBABLE REASON  Fy (a-b-) = resistant to P. vivax and P. knowlesi
FINDINGS o Plasmodium ovale
Blood film bluer than Patient has increased blood proteins o Plasmodium malariae
normal (as in plasma cell myeloma) o Plasmodium knowlesi
Grainy appearance RBC agglutination 2. Filaria
(as seen in cold hemagglutinin diseases) - Some of the species that infect humans:
Holes all over the film Patient has increased lipid levels o Wuchereria bancrofti (can cause elephantiasis)
Blue specks Markedly increased WBC counts and platelet o Brugia malayi (can cause elephantiasis)
(out at the feather edge) counts
o Loa loa (can cause calabar swellings)
Microscopic Examination 3. Trypanosomes
OBJECTIVE COMMENTS - Some of the species that infect humans
10x Used to: o Trypanosoma brucei rhodesiense
Objective - Assess overall film quality, color, and distribution of cells  East African Sleeping Sickness
Examination - Locate rare abnormal WBCs (examples: blasts, reactive  Transmitted by Tsetse flies
lymphocytes) or other cells which may be examined o Trypanosoma brucei gambiense
more closely under higher magnification  West African Sleeping Sickness
- Detect “snowplow” effect which will make the blood  Transmitted by Tsetse flies
smear unacceptable o Trypanosoma cruzi
o Snowplow effect: presence of more than four times  Causes Chagas’ disease
the number of WBCs per field at the lateral edges or  Transmitted by Reduviid bugs
feather edge compared with the monolayer area

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IMPORTANT REMINDERS! - Hb F (Fetal hemoglobin)

- Malarial parasites metabolize hemoglobin, making hemozoin or malaria o The predominant hemoglobin during
pigment Fetal liver this phase
- Infections caused by malarial parasites can be detected microscopically - Thymus

HEPATIC
by demonstration of the parasites in the PBS. With some o First fully developed organ in the
o At LEAST 2 THICK AND 2 THIN BLOOD FILMS must be made contributions fetus
ASAP after collection of venous blood (EDTA) by: spleen, o Size of the thymus increases during
o Blood films may also be made directly from a capillary puncture. thymus, lymph fetal development
o For the visualization of the parasites, Wright-Giemsa stain is used. nodes o Major site of T cell production
o Thick blood films - Kidneys and spleen
 ideal for initial screening of blood o Produce B cells
 more parasites are seen in each field - Hematopoiesis starts in the bone marrow

INTRAMEDULLARY/
 stained with a water-based Wright-Giemsa (without methanol cavity before the 5th month of fetal

MEDULLARY/
fixation) to lyse the red cells development

MYELOID
o Thin blood films - The bone marrow becomes the chief site
Bone marrow of hematopoiesis by the end of 24 weeks’
 used for species identification and determination of percent
parasitemia gestation.
 stained after methanol fixation
- Percent parasitemia - determined by counting the number of parasitized
RBCs (asexual sages) among 500 to 2,000 RBCs on a thin blood film
and converting to a percentage.
- At least 300 fields on the thick and thin blood films must be examined
(100x objective) before a negative result is reported.
- Microscopy can detect 5 to 20 parasites per microliter of blood, or
0.0001% parasitemia.
- A negative result for a single set of thick and thin peripheral blood films
does NOT rule out a diagnosis of malaria.
- A platelet lying on top of an RBC in a thin blood film may be confused
with a malarial parasite by an inexperienced observer.

THICK BLOOD FILM PREPARATION (MALARIA) ADULT HEMATOPOIESIS

- Place 3 small drops of blood close together near one end of the slide. - Hematopoietic tissues of adults are located NOT ONLY in the bone marrow,
- Using one corner of a clean slide, stir the drops of blood for about 30 but also in the lymph nodes, spleen, liver and thymus.
seconds to mix the 3 drops over an area approximately 1 to 2 cm. in - Bone marrow
diameter. o contains developing erythroid, myeloid, lymphoid, and megakaryocytic
- Let the film to dry thoroughly then stain it using a water-based Wright- cells
Giemsa. o considered to be a primary lymphoid organ with functions equivalent to
that of Bursa of Fabricius (birds)
VIII. STORAGE OF BLOOD SMEAR SLIDES o is estimated to be capable of generating around (per kilogram of body
weight daily):
- at least 7 DAYS before proper disposal
 2.5 billion RBCs
HEMATOPOIESIS  1 billion granulocytes
- aka: Hemopoiesis  2.5 billion platelets
- process of blood cell formation
- Classical marker of hematopoietic stem cells: CD 34 - RETROGRESSION
- Considered to start around the nineteenth day of embryonic development o process of replacing red marrow by yellow marrow during development
after fertilization o between 5 and 7 years of age, adipocytes become more abundant and
start to occupy the spaces in the long bones previously dominated by
Two Related Theories (Origin of Hematopoietic Progenitor Cells) active marrow
1. Polyphyletic Theory o eventually results in restriction of the red marrow in the adult to the:
- Suggests that each of the blood cell lineages is derived from its own  R – Ribs
unique stem cell  S – Sternum, Skull, Shoulder blades
2. Monophyletic Theory  V – Vertebrae
- Suggests that all blood cells are derived from a single progenitor stem  P – Pelvis and proximal ends of the long bones
cell called a pluripotential stem cell o Red marrow
- Most widely accepted theory among experimental hematologists  Hematopoietically active marrow
o Yellow marrow
Chief site of Remarks  Hematopoietically inactive marrow composed primarily of fat cells
hematopoiesis (adipocytes)
- 1st blood cells: primitive erythroblasts  It is dispersed throughout the red marrow (in adults, there is
(formed during the first 2-8 weeks of life) approximately equal amounts of red marrow (50%) and yellow
- Primitive Erythroblasts (PE) marrow (50%) in these areas).
o Important in early embryogenesis to  It has the ability of reverting back to active marrow in cases of
MEGALOBLASTIC
MESOBLASTIC/

produce hemoglobin (embryonic increased demand on the bone marrow (examples: excessive
hemoglobins = Gower-1, Gower-2,
blood loss and hemolysis)
Yolk sac and Portland) necessary for delivery
of oxygen to the embryonic tissues
- Yolk sac hematopoiesis differs from
hematopoiesis that occurs later (fetal
and adult stage) in that it occurs
intravascularly (within developing blood
vessels)

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BONE MARROW COLLECTION SITES

- Posterior superior iliac crest (preferred site for BMA in adults)
- Anterior superior iliac crest (preferred site for BMA in adults)
- Sternum (preferred site for BMA in children <2 years old)
- Anterior medial surface of the tibia
- Spinous process of the vertebrae, ribs, or other red marrow-containing
bones

INDICATIONS FOR BONE MARROW EXAMINATION

INDICATION EXAMPLES
Neoplasia - Acute and chronic leukemias
diagnosis and - Hodgkin and non-Hodgkin lymphomas
staging - Metastatic tumors
Marrow failure: - Aplastic anemia
cytopenias - Pure red cell aplasia
- Sickle cell crisis
Metabolic - Gaucher disease
disorders - Mast cell disease
Infections - Miliary tuberculosis
- Fungal infections
Monitoring of - After chemotherapy or radiation therapy to assess
treatment minimal residual disease
- After stem cell transplantation to evaluate engraftment

- Lymphoid development occurs in: NORMAL MARROW CELLS

o PRIMARY LYMPHOID ORGANS
 Consist of the bone marrow and thymus NORMAL CELL MATURATION
 Where T and B lymphocytes are derived - Blood cells undergo several stages of development
 Size of the thymus increases during fetal development (growth of - Development from one stage to the next is NOT Sudden, so commonly the
this gland continues until puberty then undergoes atrophy with cell being studied may be between stages (when this occurs, the cell is
aging) usually given the name of the more mature stage.)
 Lymphoid cells fated to become B cells undergo their early stages
of differentiation within the bone marrow 1. Developing hematopoietic cells
o SECONDARY (PERIPHERAL) LYMPHOID ORGANS - Examples:
 Major secondary lymphoid organs: spleen and lymph nodes o Megakaryocytes (30-45 µm)
 2 major functions:  Largest cell in a normal marrow
 Trapping and concentration of foreign substances 2. Macrophages
 Main sites of production of antibodies and the induction of - Diameter: 40 to 50 µm
antigen-specific T lymphocytes - (+) Nonspecific (alpha naphthyl acetate or alpha naphthyl butyrate)
3. Mast cells
SECONDARY LYMPHOID ORGANS - Diameter: 12 to 25 µm
- SPLEEN - (+) Chloroacetate esterase
o LARGEST of the secondary lymphoid organs 4. Osteoblasts
o Major organ in the body in which antibodies are synthesized and from - synthesize new bone matrix
which they are released into the circulation - Waterbug/comet appearance
o Effects of splenectomy: 5. Osteoclasts
 In children: often leads to an increased incidence of bacterial sepsis - Large, multinucleated
caused primarily by S. pneumoniae, N. meningitidis, and H. - 100 µm or greater
influenzae
 In adults - less adverse effects, although it leads to some increase Marrow differential
in blood-borne bacterial infections or bacteremia - Recommended: at least 500 cells (preferably 1,000 cells) be counted
o Related terms: - 500 cells on each of 2 slides
 Splenectomy: removal of the spleen - M:E ratio in adults: 2:1 to 4:1 (NORMAL)
 Splenomegaly: enlargement of the spleen o “M” – myeloid – will become WBCs
 Hypersplenism: increased hemolytic activity of the spleen caused o “E” – erythroid – will become RBCs
by splenomegaly - In leukemia, the M:E ratio is 10:1
Bone marrow aspirate
- LYMPH NODES
o Small ovoid, bean-shaped structures (normally <1 cm in diameter) - Disturbs bone marrow architecture
found in different areas throughout the body - Spread as a smear on a slide, stained, and examined for hematologic or
o Related terms systemic disease
 Lymphadenopathy: any disorder characterized by a - Used for analysis of individual cell morphology
localized/generalized enlargement of the lymph nodes/lymph - Bone marrow smears - should be retained for 10 years
vessels Bone marrow biopsy
 Lymphoma: solid tumor neoplasm of lymphoid tissue - Bone marrow removed intact without disturbing the bone architecture
- A cylinder of gelatinous red marrow (fixed in formalin, sectioned, stained,
MINOR SECONDARY LYMPHOID ORGANS and studied for hematologic or systemic disease)
- Tonsils - Advantage: gives a better picture of the real structure of bone marrow
o to detect and respond to antigens in the respiratory and alimentary - Used for analysis of bone marrow architecture
secretions
- Appendix Extramedullary hematopoiesis
- Peyer's patches - Blood cell production outside the bone marrow
o clusters of lymphocytes distributed in the lining of the small intestine; - Occurs MAINLY in the liver and spleen (hepatomegaly and/or splenomegaly
detect substances that diffuse across the intestinal epithelium often accompany this)
- MALT (mucosa-associated lymphoid issue) - Occurs under the following conditions:
o general term for the unencapsulated lymphoid tissues present in regions o When bone marrow becomes dysfunctional such as in aplastic anemia,
underlying the mucosal areas infiltration by malignant cells, or overproliferation of a cell line (for
example, leukemia)
- LIVER o When bone marrow is incapable of meeting the demands placed on it
o Can maintain hematopoietic stem cells and progenitor cells to generate (as observed in hemolytic anemias)
various blood cells (a process called extramedullary hematopoiesis) as
a response to infectious agents or in pathologic myelofibrosis of the
bone marrow

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ERYTHROPOIESIS - Normally, EPO is released from the kidney into the

- Process of red blood cell formation blood in response to hypoxia (too little tissue
- Takes place inside the bone marrow oxygen).
o Red bone marrow: source of RBCs, hematopoietic cells - 3 major effects
o Yellow bone marrow: consists of fats o Allowing early release of reticulocytes from
the bone marrow
RELATED TERMS o Reducing the time needed for cells to mature
- Erythron: total mass of RBCs circulating in the peripheral blood and the in the bone marrow
bone marrow RBC precursors o Preventing apoptotic cell death
(note: apoptosis rescue is the MAJOR way in
- RBC mass: erythrocytes in the circulation; total population of all RBCs in the
which EPO increases RBC mass)
blood vessels
- Current/potential therapeutic applications:
- Erythrokinetics: a term that describes the dynamics of RBC creation and
o Anemia of chronic renal disease
destruction
o Autologous pre-donation blood collection
- Bone marrow: Birthplace of erythrocytes o Anemia in HIV infection to permit use of
- Spleen: Graveyard of RBCs zidovudine (AZT)
- “blood doping”: some athletes illegally use EPO
TYPES OF ERYTHROPOIESIS injections to increase the oxygen-carrying capacity
of their blood (to enhance endurance and stamina)
1. INEFFECTIVE ERYTHROPOIESIS (can lead to deadly arterial and venous
o Production of defective erythroid precursors thrombosis)
o These defective RBC precursors often undergo apoptosis in the bone GROWTH - Produced by the pituitary
marrow before they have a chance to mature to the reticulocyte stage HORMONE - Stimulates erythropoiesis directly
o Defective RBC precursors often undergo apoptosis o Affects the bone marrow cells themselves
 Apoptosis = programmed cell death TESTOSTERONE - Produced by testes
 Low RBC count; leads to anemia
- Stimulates erythropoiesis indirectly
o Examples of conditions:
o Stimulates not the bone marrow itself but the
 Macrocytic, normochromic anemia
kidney to produce more erythropoietin
 Vitamin B12 deficiency
PROLACTIN Produced by the pituitary gland
 Folate deficiency -
 Microcytic, hypochromic anemia - Stimulates erythropoiesis directly
 Thalassemia ESTROGEN - Produced by ovaries
 Sideroblastic anemia - Inhibits erythropoiesis indirectly
- Males have higher RBC count than females
2. INSUFFICIENT ERYTHROPOIESIS because males have testosterone and females
o Decrease in the number of erythroid precursors in the bone marrow have estrogen
(resulting in decreased RBC production)
o Examples of conditions STAGES OF RBC MATURATION
 Microcytic, hypochromic anemia - Committed erythroid progenitor cells
 Iron deficiency o BFU-E (Burst forming unit-erythroid)
 Normocytic, normochromic anemia o CFU-E (Colony forming unit-erythroid)
 Renal disease & Acute leukemia - NOTE: 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
RBC SURVIVAL precursors in the bone marrow.
- Determined by extracting a blood sample, labeling the RBCs with
chromium-51 (51Cr), inactivating the excess 51Cr remaining in the plasma, RUBRIBLASTIC NORMOBLASTIC ERYTHROBLASTIC
and reinjecting the labeled RBCs into the patient Rubriblast Pronormoblast Proerythroblast
- Measurements of radioactivity in the RBCs are being made at specific Prorubricyte Basophilic normoblast/ Basophilic erythroblast/
times until over 50% of the activity has disappeared. Early normoblast Early erythroblast
- The results are commonly expressed as the 51Cr half-survival time. Rubricyte Polychromatophilic Polychromatophilic
- Normal range: normoblast/ erythroblast/
o 28 to 38 days Intermediate normoblast Intermediate erythroblast
Metarubricyte Orthochromatic normoblast/ Orthochromatic erythroblast/
PROGENITOR VS PRECURSOR CELLS Late normoblast Late erythroblast
Reticulocyte Reticulocyte Reticulocyte
PROGENITOR CELLS PRECURSOR CELLS Mature Mature erythrocyte Mature erythrocyte
- Immature hematopoietic cell that is - Relatively more mature than erythrocyte
committed to a cell line but cannot be progenitor cells (but still *Mature erythrocytes are no longer precursors
identified morphologically considered immature)
o You cannot distinguish as to what cell - Immature hematopoietic cell - Reticulocytes are called as such only when they are stained with
line do they belong that is morphologically supravital stains such as:
identifiable as belonging to a o Brilliant Cresyl Blue
given cell line o New Methylene Blue
- Examples: BFU-E, CFU-E - Examples: - When reticulocytes are found in a Wright-stained smear, they are referred
o BFU-E: Burst forming unit-erythroid o Rubriblast to as:
o CFU-E: Colony forming unit-erythroid o Prorubricyte o Polychromatophilic erythrocytes
CD71 o Diffusely basophilic erythrocytes
- Earliest marker or erythroid differentiation
- Transferrin receptor
CD34 - Classical marker of hematopoietic stem cells

HORMONES RELATED TO ERYTHROPOIESIS

HORMONE REMARKS
ERYTHROPOIETIN - Chief stimulatory cytokine for RBCs
(EPO) - Major hormone (production of RBCs)
- Thermostable, nondialyzable, glycoprotein
hormone
- Primary cell source:
o Peritubular interstitial cells of the kidneys
- Primary target cells:
o Progenitors: BFU-E & CFU-E

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STAGE 1 – RUBRIBLAST (PRONORMOBLAST) STAGE 4 – METARUBRICYTE

Size Nucleoli Nucleus Cytoplasm (ORTHOCHROMATIC NORMOBLAST/ LATE NORMOBLAST)
12-20 1 to 2, round or slightly oval, thin small in amount, moderately Size Nucleoli Nucleus Cytoplasm
um usually nuclear membrane, basophilic, homogenous 8- None pyknotic (dense mass of Salmon-pink
very faint central or slightly eccentric N:C ratio = 8:1 10um degenerated chromatin) N:C ratio = 1:2
- This cell (rubriblast) gives rise to 2 prorubricytes - Nucleus is extruded at this stage, and the cell becomes a reticulocyte.
- Earliest recognizable erythroid precursor using the light microscope - Orthochromatic: ortho = same, chroma = color. This cell has the same color
- Always contain nucleoli as the mature erythrocyte (salmon pink)
- The more the nucleoli = the younger the cell is - Other names:
- N:C ratio: Nucleus to Cytoplasm Ratio o Nucleated RBC
o a morphologic feature used to identify and stage RBC and WBC o Pyknotic erythroblast
precursors o Acidophilic normoblast
o a visual estimate of what area of the cell is occupied by the nucleus - Last stage with a nucleus
compared with the cytoplasm - Pyrenocyte
o if the areas occupied by the nucleus and the cytoplasm are o enveloped extruded nucleus
approximately equal, the N:C ratio is 1:1 o engulfed by bone marrow macrophages
o if the nucleus takes up < 50% of the area of the cell (low N:C ratio), the o Frequently, small fragments of the nucleus are left behind if the
ratio is lower (e.g., 1:5 or less than 1) projection is pinched off before the entire nucleus is enveloped (the
o if the nucleus takes up > 50% of the area of the cell (high N:C ratio), fragments are called Howell-Jolly bodies when seen in the RBCs in
the ratio is higher (e.g., 3:1 or 3) the circulation).
- Basophilia – pertains to the blueness of a particular part of the cell and is o Howell-Jolly bodies are typically removed from the RBCs by the splenic
due to the acidic components that attract the basic stain (ex.: methylene blue) macrophage pitting process once they enter the circulation.
o degree of cytoplasmic basophilia correlates with the quantity of  Howell-Jolly bodies are made of DNA
ribosomal RNA  Howell-Jolly bodies appear in numerous amounts in patients
- Eosinophilia or Acidophilia – pertains to the pinkness of a particular part which underwent splenectomy
of the cell and is due to the accumulation of more basic components that
attract the acid stain eosin STAGE 5 – RETICULOCYTE
o As the RBC matures, the eosinophilia of the cytoplasm correlates with Size Nucleoli Nucleus Cytoplasm
the accumulation of hemoglobin. 8- None None Cytoplasm still with small amounts of RNA =
10um polychromasia (mixed pink and blue
STAGE 2 – PRORUBRICYTE staining) with Golgi apparatus remnants and
(BASOPHILIC NORMOBLAST/EARLY NORMOBLAST) residual mitochondria that allows continued
Size Nucleoli Nucleus Cytoplasm aerobic metabolism and hemoglobin
10-15 0 to 1 round, thin nuclear Appears more abundant than in production
um membrane, smaller, normoblast because of smaller - Young RBCs containing residual RNA (last immature erythrocyte stage)
slightly eccentric nucleus - Spends 2 to 3 days in the bone marrow and 1 day in the peripheral blood
N:C ratio = 6:1 before developing into a mature RBC
- The Prorubricyte gives rise to 4 rubricytes - Shift Cells
- May or may not contain a nucleolus o Polychromatophilic macrocyte
- Most helpful criteria in distinguishing the Prorubricyte from the Rubriblast: o Seen in cases of increased RBC production
o Coarser chromatin - Stress Reticulocytes
o Absence of nucleoli o Macroreticulocytes
- Last stage with a nucleolus o Seen in more severe conditions (ex. Hemolytic anemia)
- First stage of hemoglobinization (Hemoglobin synthesis) but will not - Last stage of hemoglobin synthesis
demonstrate pinkness yet because it is still beginning to manufacture hgb
- Hemoglobin is eosinophilic (pink) STAGE 6 – MATURE ERYTHROCYTE
Size Nucleoli Nucleus Cytoplasm
STAGE 3 – RUBRICYTE 7-8um None None Salmon-pink
(POLYCHROMATIC NORMOBLAST/ INTERMEDIATE NORMOBLAST) (with a central pallor occupying 1/3 of the
Size Nucleoli Nucleus Cytoplasm cell’s diameter)
10- None round and smaller, basophilic to diffusely lilac in Notes:
12um thick nuclear color (muddy or gray), depending on - Shape: biconcave disk
membrane, hemoglobin content - Thickness: 1.5 to 2.5 μm
eccentric nucleus N:C ratio = 4:1 - Average life span: 120 days
- Each of this cell (rubricyte) gives rise to 2 metarubricytes. - Number of erythrocytes produced from each rubriblast: 16
- May be confused with a lymphocyte: - Normal ratio of RBCs to WBCs is approximately 600:1
o Lymphocyte - Normal ratio of RBCs to Platelets is approx. 15:1.
 Nucleus – crushed velvet - Adult RBC contains no mitochondria (no protein or Hb synthesis)
 Cytoplasm – sky blue or “robin egg” blue
o Rubricyte
 Nucleus – checkerboard
 Cytoplasm – muddy or gray
- Last stage capable of mitosis
- First stage in which the cytoplasm becomes pink; hemoglobin manifests as
pink but the cytoplasm as a whole is color gray

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RBC MEMBRANE CONSTITUENTS SKELETAL PROTEINS

- 8% carbohydrates, 40% lipids, and 52% proteins (integral and peripheral) - Provide the horizontal or the lateral support for the membrane
- TRANSMEMBRANE PROTEINS - The shape and flexibility or the erythrocyte depend on the cytoskeleton
o Channel ions, water, and glucose and anchor cell membrane - Examples are listed below:
receptors
o also provide the vertical support connecting the lipid bilayer to the Skeletal (Cytoskeletal or Function
underlying cytoskeleton to maintain membrane integrity Peripheral) Proteins
o examples are listed below: α-spectrin Primary cytoskeletal proteins
β-spectrin
Transmembrane Function Adducin Caps actin filament
(integral) Proteins Ankyrin Anchors band 3 and protein 4.2
Aquaporin 1 Water transporter Dematin Actin bundling protein
Band 3 Anion transporter, supports ABH antigens F-actin Binds β-spectrin
Ca2+-ATPase Ca2+-transporter Protein 4.1 Anchors 4.1 complex
Duffy Supports Duffy antigen Protein 4.2 Anchors Ankyrin complex
Glut-1 Glucose transporter, supports ABH antigens Tropomodulin Caps actin filament
Glycophorin A Transports negatively charged sialic acid, supports Tropomyosin Regulates actin polymerization
MN determinants
Glycophorin B Transports negatively charged sialic acid, supports
Ss determinants
Glycophorin C Transports negatively charged sialic acid, supports
Gerbich system determinants
ICAM-4 Integrin adhesion
Kell Zn2+-binding endopeptidase, Kell antigens
Kidd Urea transporter
Rh D and CcEe antigens
RhAG Necessary for expression of D and CcEe antigens,
Gas transporter, probably of CO2

HEREDITARY RBC MEMBRANE DEFECTS

CONDITION INHERITANCE DEFICIENT REMARKS
PATTERN PROTEIN
Hereditary Autosomal Ankyrin NOTE: HS patients have an increase in the MCHC value (between 35 and
spherocytosis dominant (75%) Band-3 38 g/dL) because of the spherocytosis.
α-spectrin
β-spectrin The normal range for the MCHC is: 31 to 37 g/dL
Protein 4.2
Defect in protein(s) that disturbs vertical membrane interactions between
transmembrane proteins and underlying cytoskeleton; loss of membrane
and decreased surface area-to-volume ratio

The only disease characterized by a high MCHC

REMEMBER!
- In patients with HS, the RBC membrane is stretchable, but is less elastic.
- The spleen is particularly a caustic environment for spherocytes, with its ↓ pH, ↓ ATP, and ↓ glucose.
- Spherocytic RBCs also show problems with membrane diffusion.
- Spherocytes gather sodium at a higher rate than for normal RBCS in the microenvironment of the spleen. These abnormal red cells are less able to tolerate
changes in their osmotic environment before they swell then lyse.
- Once an HS patient has been splenectomized, RBC survival is more normal, giving fewer complications from chronic hemolysis.
- EMA binding test – a flow cytometry-based test; most sensitive and specific test to confirm the diagnosis of HS
Hereditary Autosomal α-spectrin Defect in proteins that disrupt the horizontal linkages in the protein
elliptocytosis dominant β-spectrin cytoskeleton; loss of mechanical stability of membrane
Protein 4.1

Hereditary Autosomal α-spectrin Severe defect in spectrin that disrupts horizontal linkages in protein
pyropoikilocytosis recessive β-spectrin cytoskeleton; severe RBC fragmentation
(rare subtype of
hereditary
elliptocytosis)

Southeast Asian Autosomal Band 3 Defect in band 3 causing increased membrane rigidity; resistant to malaria;
ovalocytosis dominant prevalent in some areas of Southeast asia
(Hereditary
ovalocytosis)

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Overhydrated Autosomal Rh- Increased membrane permeability to sodium and potassium; increased
hereditary dominant associated intracellular sodium causing influx of water, increase in cell volume, and
stomatocytosis protein decreased cytoplasmic viscosity; typical RBC morphology: stomatocytes (5-
(RHAG) 50%) to macrocytes
Others
unknown

Dehydrated Autosomal Piezo-type Increased membrane permeability to potassium; decreased intracellular

hereditary dominant mechanosen potassium, resulting in loss of water from cell, decrease in cell volume, and
stomatocytosis sitive ion increased cytoplasmic viscosity; typical RBC morphology: target cells, burr
(Hereditary channel cells, stomatocytes (<10%), RBCs with “puddled” hemoglobin at periphery,
Xerocytosis) component 1 desiccated cells with spicules
Note: this is the most
common form of
stomatocytosis
NOTE!
- Mutations that change the membrane structure:
o Hereditary spherocytosis
o Hereditary elliptocytosis/pyropoikilocytosis
o Hereditary ovalocytosis (Southeast Asian ovalocytosis)
- Mutations that change the membrane transport proteins:
o Overhydrated hereditary stomatocytosis
o Dehydrated hereditary stomatocytosis or Hereditary Xerocytosis

RETICULOCYTES
- immature, non-nucleated RBC which contains >2 blue-stained,
granulofilamentous materials (RETICULUM) after staining (Supravital
stains)
- continues to generate hemoglobin (last stage)
- normal maturation time for reticulocytes in blood: 1 DAY
- production of reticulocytes: 50 x 109/L/day

RETICULOCYTE COUNT
- permits effective assessment of RBC production by the bone marrow
- a measure of EFFECTIVE ERYTHROPOIESIS
- Reference ranges:
o Adults: 0.5 to 1.5%
o Newborns: 1.8 to 5.8% (by 1 to 2 weeks of age, reference values are METHODS OF COUNTING RETICULOCYTES
the same as for adults)
o Increased (↑) reticulocyte counts (reticulocytosis) A. ROUTINE LIGHT MICROSCOPE METHOD
 aka: polychromasia or polychromatophilia - Combine equal amounts of blood and supravital stain (2 to 3 drops, or
 considered as the first sign of accelerated erythropoiesis and around 50 uL each), and allow to incubate at room temperature for 3 to 10
observed in hemolytic anemias, individuals with iron deficiency minutes.
anemia receiving iron therapy, thalassemia, sideroblastic - Remix the preparation.
anemia, and in acute and chronic blood loss - Prepare two blood smears.
o Decreased (↓) reticulocyte counts (reticulocytopenia) - In the region in which cells are near each other but NOT touching, count
 Observed in aplastic anemia and in conditions in which the bone 1000 RBCS under the oil immersion objective lens (1000x total
marrow is not producing RBCs magnification). Note: Reticulocytes are included in the total RBC count (i.e.,
reticulocytes are counted as both an RBC and a reticulocyte).
MATERIALS
SUPRAVITAL STAINS MILLER DISK - To increase accuracy, have another medtech count the other blood smear;
counts should agree within 20%.
1. New Methylene Blue
o More preferred for - Calculation:
ܰ‫݀݁ݒݎ݁ݏܾ݋ݏ݁ݐݕܿ݋݈ݑܿ݅ݐ݁ݎ݂݋ݎܾ݁݉ݑ‬
staining reticulocytes ܴ݁‫݁ݐݕܿ݋݈ݑܿ݅ݐ‬ሺΨሻ ൌ  ‫ͲͲͳݔ‬
ͳǡͲͲͲܴ‫݀݁ݒݎ݁ݏܾ݋ݏܥܤ‬
o Composed of:
 Sodium oxalate B. CALIBRATED MILLER DISK METHOD
(prevents
coagulation) - Count a minimum of 112 RBCs in small square (B).
 Sodium chloride - A reticulocyte in square B is counted as both an erythrocyte and a
(provides reticulocyte.
isotonicity) - At this point, theoretically, the number of reticulocytes in 1,008 RBCs has
been counted.
- Calibrated disk placed in the
2. Brilliant Cresyl Blue - Computation:
ocular of the microscope ܶ‫ܣ݁ݎܽݑݍݏ݊݅ݏ݁ݐݕܿ݋݈ݑܿ݅ݐ݁ݎ݈ܽݐ݋‬
o Provides inconsistent
Large Square (A) is used for: ܴ݁‫݁ݐݕܿ݋݈ݑܿ݅ݐ‬ሺΨሻ ൌ  ‫ͲͲͳݔ‬
ܶ‫ͻݔܤ݁ݎܽݑݍݏ݊݅ݏܥܤܴ݈ܽݐ݋‬
-
staining results
o Composed of: counting reticulocytes
 Sodium citrate - Small Square (B) is used for: C. FLOW CYTOMETRY
(prevents counting RBCs; 1/9th of square A
- Most rapid, accurate, and precise method for reticulocyte count
coagulation) - In this method, the reticulocytes are counted on the basis of optical scatter
 Sodium chloride (minimum no. of cells that should be
or fluorescence after treatment with fluorescent dyes or nucleic acid stains
(provides counted: 200)
- The test values in this method are reported in absolute and in relative
isotonicity) terms.
- Example: Sysmex R-3500

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SYSMEX R-3500 3. RETICULOCYTE PRODUCTION INDEX

- Uses Auramine O (a supravital fluorescent dye) - RPI (also known as shift correction) provides a further refinement of the
- Reticulocytes fall into low-fluorescence, middle-fluorescence, or high- CRC.
fluorescence regions, with the less mature reticulocytes showing higher - A general indicator of the rate of erythrocyte production increase above
fluorescence. normal in anemias
- Immature Reticulocyte Formation (IRF): sum of the middle-fluorescence - Calculation:
and high-fluorescence ratios ܿ‫ݐ݊ݑ݋ܿ݁ݐݕܿ݋݈ݑܿ݅ݐ݁ݎ݀݁ݐܿ݁ݎݎ݋‬
o Demonstrates an early indication of erythropoiesis (sensitive ܴܲ‫ ܫ‬ൌ  
݉ܽ‫݀݋݋݈ܾ݈ܽݎ݄݁݌݅ݎ݁݌݄݁ݐ݊݅݁݉݅ݐ݊݋݅ݐܽݎݑݐ‬
index of bone marrow erythropoietic activity)
o The quantity of reticulocytes with the highest content of RNA
HEMATOCRIT (%) MATURATION TIME (Days)
o Indicates the ratio of immature reticulocytes to total reticulocytes in
a blood sample 40 to 45 1.0
o Used together with ARC to distinguish types of anemia 35 to 39 1.5
25 to 34 2.0
15 to 24 2.5
IMPORTANT REMINDERS: <15 3
1. New methylene blue is chemically different from methylene blue. - RPI >3 = generally indicates adequate bone marrow response
2. Brilliant Cresyl blue also stains reticulocytes but shows too much - RPI <2 = generally indicates inadequate bone marrow response
unpredictability in staining for routine use.
3. If a patient is very anemic or polycythemic, the proportion of dye to blood
should be adjusted accordingly. For best results, a larger proportion of CASE
blood should be added to the stain when the patient’s hematocrit is low. 29-year-old male patient | Hematocrit = 28% (0.28 L/L) | RC = 5.0%
Add a smaller amount of blood to the stain when the patient has a very high What is the corrected reticulocyte count?
hematocrit. ͲǤʹͺ‫ܮ‬Ȁ‫ܮ‬
4. The time allowed for staining of the reticulocyte is NOT critical. It should ‫ ܥܴܥ‬ൌ ͷǤͲሺΨሻ‫ݔ‬ ൌ ૜Ǥ ૚૚Ψ
ͲǤͶͷ‫ܮ‬Ȁ‫ܮ‬
not however, be less than 10 minutes.
5. Increased blood glucose or the use of heparin as anticoagulant may cause What is the reticulocyte production index?
the reticulocytes to show pale staining. ͵Ǥͳͳ
6. The blood and stain should be mixed well prior to making smears. The ܴܲ‫ ܫ‬ൌ   ൌ ૚Ǥ ૟
ʹ
reticulocytes have a lower specific gravity than mature erythrocytes and,
therefore settle on top of the red blood cells in the mixture.
7. Other RBC inclusions stained by NMB: ERYTHROCYTES

INCLUSIONS COLOR REMARKS RBC COUNT

(using NMB) - Manual RBC counts:
Howell-Jolly Deep purple - Nuclear fragments (DNA) o This is rarely done because of its inaccuracy and questionable
bodies necessity
Heinz bodies Light blue - Usually present at the peripheral o The use of other, more accurate manual RBC tests, such as the
green edge of the red cell microhematocrit and hemoglobin concentration, is desirable when
- Denatured and precipitated automation is not available.
hemoglobin o The RBC count, hemoglobin, and hematocrit values are ELEVATED
Pappenheimer Purple - Generally, appear as several in people living at a HIGHER ALTITUDE over what they would be at
bodies granules in a small cluster sea level. The difference is around 1 g Hb/dL at 2 km altitude and 2g
- A wright-stained or a Prussian blue- Hb/dL at 3 km.
stained smear may be examined to - RBC count reference range:
confirm their presence
- Hemosiderin in the mitochondria AGE GROUP CONVENTIONAL UNITS S.I. UNITS
Hemoglobin H Greenish- Children (8 to 13) 4.00 to 5.40 X 106/uL 4.00 to 5.40 X 1012/L
- Multiple small dots
bodies blue Adult (male) 4.60 to 6.00 X 106/uL 4.60 to 6.00 X 1012/L
- Seen in a form of alpha-thalassemia Adult (female) 4.00 to 5.40 X 106/uL 4.00 to 5.40 X 1012/L

3 METHODS OF REPORTING THE RETICULOCYTE COUNTS RBC METABOLIC PATHWAYS

1. ABSOLUTE RETICULOCYTE COUNT Embden-Meyerhof pathway
- Actual number of reticulocytes in 1 liter of whole blood - Glucose penetrates the red blood cell with no energy expenditure via
- Computation: GLUT-1 (a transmembrane protein)
- Major glycolytic pathway
‫ݏ݁ݐݕܿ݋݈ݑܿ݅ݐ݁ݎ‬ሺΨሻ‫ݐ݊ݑ݋ܿܥܤܴݔ‬ሺ‫Ͳͳݔ‬ଵଶ ‫ܮݎ݁݌‬ሻ
‫ ܥܴܣ‬ൌ  ‫ͲͲͲͳݔ‬ - Shunts:
ͳͲͲ o Hexose-monophosphate shunt
- Reference range: 25-75 x 109/L o Methemoglobin reductase shunt
- Used together with IRF to distinguish types of anemia: o Rapoport-Luebering Shunt
CONDITION ARC IRF - Handles 90% of glucose utilization in the RBCs
Hemolytic anemia ↑ ↑ - Non-oxidative, anaerobic pathway
(an anemia with ↑ marrow erythropoiesis) - Produces 2 molecules of ATP
Hemorrhage ↑ ↑
Chronic renal disease ↓ ↓ ATP
Early response to therapy in nutritional anemias N to ↓ ↑ Used by the RBCs in the following ways:
- Maintenance of RBC shape and deformability
2. CORRECTED RETICULOCYTE COUNT - Gives energy for the active transport of cations
- Helps in modulating the amount of 2,3-BPG (2,3-Bisphosphoglycerate)
- Sometimes referred to as: Reticulocyte Index, Hematocrit Correction, or
Poor Man’s Bone Marrow Aspirate generated
- Percentage of reticulocytes may appear increased because of early
reticulocyte release into the circulation or because of a decrease in the Pyruvate kinase (PK) deficiency
number of mature cells in circulation (anemia may cause falsely ↑
- Most common enzyme deficiency of the EMP and is the most common form
reticulocyte count).
of HNSHA (Hereditary Nonspherocytic Hemolytic Anemia)
- The CRC corrects the observed reticulocyte count to a normal hematocrit
- Possible PBS findings include: Acanthocytes & Burr Cells
of 0.45 L/L (average normal hematocrit for both genders) to allow
correction for the degree of patient’s anemia. - Tests
o PK fluorescent spot test
Formula:
 recommended screening test for PK deficiency
-
݄݁݉ܽ‫ܮ݊݅ݐ݅ݎܿ݋ݐ‬Ȁ‫ܮ‬
‫ ܥܴܥ‬ൌ ܴ݁‫ݏ݁ݐݕܿ݋݈ݑܿ݅ݐ‬ሺΨሻ‫ݔ‬  o Autohemolysis test
ͲǤͶͷ‫ܮ‬Ȁ‫ܮ‬
 A screening test for PK deficiency
 Pattern of autohemolysis associated with PK deficiency:
autohemolysis is greatly increased and glucose has no effect (but
ATP corrects the hemolysis [Type II])
o Quantitative PK assay
 Confirmatory test

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III Mild to Episodic, acute hemolytic G6PD-A-

moderately anemia associated with G6PD-Canton
deficient infections and certain drugs;
10-60% activity self-limited
IV Mild deficient to None G6PD-B
normal (Wildtype)
60-150%
activity G6PD-A+
(may also
manifest as class
III)
V Increased None
>150% activity

Some of the examples of drugs, chemicals, and food that may cause hemolysis
in G6PD deficiency patients (MUST BE AVOIDED):

- Dapsone - Legumes (ex. garbanzos,

- Methylthioninium chloride kadyos, munggo)
(methylene blue) - Blueberry
- Nitrofurantoin - Soya food (ex. taho, tokwa,
- Phenazopyridine soy sauce)
- Primaquine - Ampalaya (bitter gourd or
- Rasburicase bitter melon)
- Tolonium chloride (toluidine - Menthol (ex. efficasent oil,
blue) listerine mouthwash)
- Aniline dyes - Cotrimoxazole
- Fava beans - Quinolones
- Red wine - Sulfadiazine
- Naphthalene (mothballs)
- Some herbal supplements
2. Methemoglobin Reductase Pathway
- Methemoglobin reductase is a.k.a. Cytochrome b5 reductase
- Maintains iron in the heme (Hb) in its reduced state (ferrous)
Heme iron is constantly exposed to oxygen and peroxide. Peroxide oxidizes heme
iron from the ferrous (2+) to the ferric (3+) state. When the iron state is ferric, the
affected hemoglobin molecule is called methemoglobin. Although the HMP prevents
hemoglobin oxidation by reducing peroxide, it is not able to reduce methemoglobin
once it forms. NADPH is able to do so, but only slowly. The reduction of
GLYCOLYSIS DIVERSION PATHWAYS (SHUNTS) methemoglobin by NADPH is rendered more efficient in the presence of
- These are the three alternate pathways that branch from the glycolytic methemoglobin reductase, also called cytochrome b5 reductase. Using H+ from NADH
pathway: formed when G3P is converted to 1,3-BPG, cytochrome b5 reductase acts as an
intermediate electron carrier, returning the oxidized ferric iron to its ferrous, oxygen-
carrying state. This enzyme accounts for more than 65% of the methemoglobin-
1. Hexose Monophosphate Shunt (Aerobic Glycolysis) reducing capacity within the RBC. (Rodak’s)
- A.k.a. pentose phosphate pathway, phosphogluconate pathway
- aerobically converts glucose to pentose and produces NADPH
(Nicotinamide adenine dinucleotide phosphate [reduced] = NADPH) 3. Rapoport-Luebering Shunt
- NADPH reduces glutathione (reduced glutathione reduces peroxides and - For the production of 2,3-BPG
guards: proteins (globin portion of hemoglobin), lipids, and heme iron from
- 2,3-BPG: binds to the hemoglobin and decreases the oxygen affinity of
oxidation
hemoglobin
- Functionality dependent on G6PD
- Prevents the denaturation of the globin by oxidation - Two variables affecting the degree of association or dissociation between
oxygen and hemoglobin
Glucose-6-phosphate dehydrogenase (G6PD) deficiency o partial pressure of oxygen
- Most common enzyme deficiency in the pentose phosphate pathway and o affinity of hemoglobin for oxygen
is also the most common RBC enzyme defect (prevalence of 5% of the - The affinity of hemoglobin for oxygen is dependent on 5 factors:
global population, or approximately 400 million people worldwide) o pH
- Possible PBS findings include: Heinz bodies and Bite cells o Partial pressure of carbon dioxide
- Tests o Concentration of 2,3-bisphosphoglycerate (2,3-BPG)
o G6PD fluorescent spot test o Temperature
 Recommended screening test for G6PD deficiency o Presence of other hemoglobin species that are non-functional
o Autohemolysis test
 A screening test for G6PD deficiency
 Pattern of autohemolysis associated with G6PD deficiency: - OXYGEN DISSOCIATION CURVE
autohemolysis is slightly to moderately increased but is partially o The curve produced when the 2 variables (partial pressure of oxygen
corrected by glucose (Type I) and affinity of hemoglobin for oxygen) are plotted on a graph (oxygen
o Quantitative G6PD assay saturation of hemoglobin versus the partial pressure of oxygen)
 Confirmatory test o BOHR EFFECT
 a shift in the curve due to an alteration in pH (or hydrogen ion
CLASSIFICATION OF G6PD DEFICIENCY VARIANTS BY THE WHO concentration)
Class G6PD Enzyme Clinical manifestations Examples of  effect of hydrogen ions and CO2 on the affinity of hemoglobin for
Activity Variants oxygen
I Severely Chronic, hereditary non- G6PD-Serres o HALDANE EFFECT
deficient spherocytic hemolytic G6PD-Madrid  depicts the occurrence by which the binding of O2 to the
<1% activity or anemia (HNSHA); severity is hemoglobin promotes the release of CO2
not detectable variable; rare
II Severely Severe, episodic acute G6PD- SHIFT TO THE LEFT SHIFT TO THE RIGHT
deficient hemolytic anemia associated Meditteranean
<10% activity with infections, certain drugs, G6PD- ↑ pH ↓ pH
and fava beans; not self- Chatham ↓ pCO2 ↑ pCO2
limited and may require ↓ 2,3-BPG ↑ 2,3-BPG
transfusions during hemolytic ↓ Temperature ↑ Temperature
episodes INCREASED (↑) OXYGEN DECREASED (↓) OXYGEN
AFFINITY AFFINITY
Only a small group of G6PD-
deficient individuals
demonstrate favism (unusual
sensitivity to fava beans), and
most of these have the
G6PD-Meditteranean variant
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RED BLOOD CELL ANOMALIES - Reference range for newborns: 14.2% to 19.9%
o RDW is markedly increased in newborns but gradually, the value will
ANISOCYTOSIS decrease until it reaches adult levels by 6 months of age
- Increased number of red cells with variation in size - If the RBC histogram is wider than normal, the RDW would be abnormal
- Normal RBCs (normocytes): 7-8 um in diameter (usually seen when MCV - Examples of conditions with their MCV and RDW values:
is 80-100 fL)
RBC Size (as defined by MCV)
Red Cell Decreased (↓) Normal MCV Increased (↑)
Distribution MCV (Normocytic) MCV
Width (RDW) (microcytic) (Macrocytic)
NORMAL Anemia of G6PD Liver disease
(little or no Chronic Deficiency
RELATED TERMS anisocytosis) Disease (ACD)
MACROCYTES MICROCYTES INCREASED Iron Deficiency Sickle cell Megaloblastic
- Larger than normal RBCS - Smaller than normal RBCs (anisocytosis) Anemia anemia anemia
(diameter > 8.0 um) (diameter <7.0 um)
- Usually seen when the MCV is - Usually seen when he MCV is 4. Using the RBC histogram
> 100 fL < 80 fL o Blood cell histogram - A visual display of cell size (X-axis) and cell
- Associated with impaired DNA - Associated with defective frequency or the number of cells (Y-axis)
synthesis hemoglobin formation o provided by many high-volume instruments to provide size distribution
of the different cell populations
o automated hematology analyzers produce histograms for RBCs,
WBCs, and platelets
o two parameters calculated from RBC histogram MCV and RDW
o instruments being used count those cells with volume sizes between
36 fL and 360 fL as RBCs
o RBC histogram can measure cells as small as 24 fL. (However, cells
that are counted in the 24 to 36 fL range are not included in the RBC
count)
Four ways to detect anisocytosis: o Leukocytes are present in the diluted fluid containing RBCs, but their
1. Using the nucleus of a small lymphocyte in a peripheral blood smear (PBS) numbers are statistically insignificant in the count
o Macrocytes: RBCs larger than the nucleus of the small lymphocyte o The instrument computer can be calibrated to compensate for the
o Microcytes: RBCs smaller than the nucleus of the small lymphocyte presence of leukocytes.
2. Using the MCV value o If the leukocyte count is significantly elevated, the RBC histogram will
o Average volume of individual RBCs be affected
o Formula: o If the RBCs are macrocytic, the curve will shift to the right (refer to
݄݁݉ܽ‫ݐ݅ݎܿ݋ݐ‬
‫ ܸܥܯ‬ൌ  ‫Ͳͳݔ‬ Figure C)
ܴ‫ݐ݊ݑ݋ܿܥܤ‬ o If the RBCs are microcytic, the curve will shift to the left (refer to Figure
o Cases: B)
 ↓ MCV: microcyte o If the RBC histogram curve is bimodal, then there are two populations
 Normal MCV: normocyte of RBCs in the sample (refer to figure D). Examples of cases that may
 ↑ MCV: macrocyte cause a bimodal distribution curve include the following:
o Reference range: 80-100 fL  blood transfusion (When normocytic donor erythrocytes are
3. Using the RDW value transfused to a recipient with microcytic red cells)
o Measures the degree of anisocytosis  cold agglutinin disease
o Red Cell Distribution Width: a calculated index (from the RBC  hemolytic anemia with schistocytes present
histogram) given by hematology analyzers to help identify o A wider or flattened curve on a histogram indicates more variation in
anisocytosis and provide information about its degree the size of the cells (The cell population is NOT homogeneous.)
o Reference range:
 Adults: 11.5% to 14.5% RED BLOOD CELL
 Newborns: 14.2% to 19.9% HISTOGRAMS
o Most recent hematology analyzers have provided two methods to (A), Normocytic red blood
calculate the RDW: cell population with MCV of
96.8 fL and RDW-CV of
RDW-CV RDW-SD 14.1%
(Coefficient of Variation) (Standard deviation)
Based on.. - Both the width of the - The actual measurement of (B), microcytosis with MCV
RBC distribution curve the width of the RBC of 54.6 fL and RDW-CV of
and the mean RBC size distribution curve in fL 13.2%
(femtoliters)
(C), macrocytosis and
Reference - 11.5% to 14.5% - 39-46 fL anisocytosis with MCV of
ranges 119.2 fL and RDW-CV of
(adults) 23.9%
Remarks - Earliest method - Width of the curve is
provided by the measured at the point that is (D), dimorphic red blood
cells with MCV of 80.2 fL and
hematology analyzers to 20% above the baseline
RDW-CV of 37.2%.
measure red cell - Not influenced by the MCV
variations - Better and more reliable Note the microcytic and the
- Dependent on the width measure of erythrocyte normocytic red blood cell
of the distribution curve variability, specifically in populations.
and the MCV highly abnormal conditions
Reference intervals: MCV,
80 to 100 fL; RDW-CV,
11.5% to 14.5%.
DERIVATION OF RDW-CV
- A: Beckman Coulter, Inc.
- B: Sysmex Corporation

DERIVATION OF RDW-SD

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ANISOCHROMIA
- General term for a variation in the normal coloration of RBCs
- Normally, RBCS have a central area of pallor approximately 1/3 the
diameter
- Normal color of RBCs: orange to salmon-pink
- “Anisochromia”
o may also mean the occurrence of hypochromic cells and
normochromic cells in the same blood smear
o may be found in sideroblastic anemias, also in a hypochromic
anemia after transfusion with normal cells and some weeks after iron
therapy for iron deficiency anemia

Hypochromic cells
- central pallor >1/3 of diameter
- usually microcytic

GRADING OF HYPOCHROMIA
1+ Area of central pallor = 1/2 of diameter
2+ Area of central pallor = 2/3 of diameter
3+ Area of central pallor = ¾ of diameter
4+ Thin rim of hemoglobin Polychromatophilic erythrocytes
- Anulocyte - A.k.a. Diffusely basophilic erythrocytes
o a.k.a. Pessary cell, Ghost cell - Larger than normal red cells with bluish tinge (Wright’s stain)
o RBC with a thin rim of hemoglobin and a large, clear center
- Bluish tinge - caused by the presence of residual RNA
o May be observed in iron deficiency anemia
o Graded as 4+ - Large numbers: associated with decreased RBC survival, hemorrhage, or
erythroid hyperplasic marrow

GRADING OF POLYCHROMASIA
Grade Percentage of Polychromatophilic RBCs
Slight 1%
1+ 3%
2+ 5%
3+ 10%
4+ >11%

Hyperchromic cells
- RBCS that lack central pallor even though they lie in a desirable area for
evaluation
- These RBCS are actually caused by a shape change (such as that found
in SPHEROCYTES)
- True hyperchromia occurs when MCHC is HIGH
- Hereditary Spherocytosis
o Basically the only disease in which the MCHC is high (above the
reference range)
 Normal MCHC = 31-37 g/dL
 HS = 35-38 g/dL
o Some of the findings:
 Symptomatic (HS has 3 key clinical manifestations)
 Splenomegaly
 Anemia
 Jaundice
 DAT (direct antiglobulin test): Negative
 Remember: the immune disorders that have spherocytes
also are usually characterized by a positive (+) result on the
DAT
 MCV: Normal to low
 MCH: Normal
 MCHC: Slightly increased
o Some of the tests include:
 Autohemolysis test
 Greatly increased (but can be corrected with either glucose
or ATP)
 OFT
 Increased osmotic fragility
 Not diagnostic of HS
 EMA (Eosin-5’-maleimide) Binding test
 ↓ fluorescence (flow cytometry)
 EMA binding test has been proposed as a more sensitive -
alternative test for confirmation of HS

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POIKILOCYTOSIS
(increased number of red cells with variation in SHAPE)
RED CELL DESCRIPTION EXAMPLES OF ASSOCIATED CONDITIONS IMAGE
SPHEROCYTE - Almost - Hereditary spherocytosis, autoimmune hemolytic anemia, burns, ABO HDN, and
spherical in following transfusion of stored blood
shape
- Lacks the Remember:
central pallor - Spherocytes may be wrongly reported if one examines the feathered edge of the
blood film because the red blood cells in the said area lack central pallor
- Natural RBC death can result also to spherocytic red cells

STOMATOCYTE - Elongated - Rh deficiency syndrome, alcoholism, electrolyte imbalance, severe liver diseases,
a.k.a. “Mouth RBCs with a overhydrated hereditary stomatocytosis
cell” slit-like central - Dehydrated hereditary stomatocytosis (also known as: Hereditary Xerocytosis)
pallor (may be o Most common form of stomatocytosis
considered as o Characterized by the presence of xerocytes
an artifact) o Xerocyte
 A dehydrated form of a stomatocyte
 Appears to have puddled at one end (half-light, half-dark)

ACANTHOCYTE - RBCs with - Abetalipoproteinemia (also known as: Bassen-Kornzweig Syndrome and Hereditary
a.k.a. Thorn cell irregularly Acanthocytosis)
or Spur Cell spiculated o Characterized by defective apo B synthesis
surface o VLDL, LDL, and Chylomicron: not found in plasma
- McLeod syndrome, Pyruvate kinase deficiency, Hepatic hemangioma, Neonatal
hepatitis, After heparin administration, Post-splenectomy, cirrhosis of the liver with
associated hemolytic anemia
BURR CELL - RBCs with - Uremia
a.k.a. regularly o Characterized by: marked ↑ in plasma urea and other nitrogenous waste
Echinocytes spiculated products
surface - Acidemia, electrolyte imbalance (K+ elevation), normocytic, normochromic anemia,
uremic frost (dirty skin), generalized edema, foul breath, urine-like sweat
- Pyruvate kinase deficiency

OVALOCYTE - Oval shaped - Hereditary ovalocytosis (a.k.a. Southeast-Asian Ovalocytosis)

RBCs

ELLIPTOCYTE - Elliptical - Hereditary elliptocytosis

(cigar- - Thalassemia (a.k.a. Mediterranean anemia, Hereditary Leptocytosis)
shaped) RBC

DACRYOCYTE - Pear-shaped - Primary myelofibrosis (PMF)

a.k.a. Teardrop or teardrop o Previously known as myelofibrosis with myeloid metaplasia, chronic idiopathic
cells shaped RBCs myelofibrosis, agnogenic myelofibrosis
o An example of an MPN (Myeloproliferative Neoplasm)
o Some of the characteristics
 Splenomegaly
 In the marrow: hypercellularity, increased megakaryocytes, and fibrosis
 In the PBS: immature granulocytes, normoblasts, dacryocytes, and other
bizarre RBC shapes
- Megaloblastic anemia, myelophthisic anemia, after Heinz body formation induced
by drug ingestion, tuberculosis, tumor metastasized to the bone marrow,
thalassemia, and some hemolytic anemias
SCHISTOCYTE - Fragmented - Patients with artificial heart valves, uremia, severe burns, MAHAs (microangiopathic
a.k.a. Schizocyte RBCs hemolytic anemias):
o Group of disorders characterized by RBC fragmentation and thrombocytopenia
o Major MAHAs: TTP, HELLP, HUS, and DIC
o Characterized by narrowing or obstruction of small blood vessels by fibrin or
platelet aggregates resulting in the fragmentation of RBCs
o Helmet cell: helmet-shaped schistocytes that may appear in MAHA
 TTP (Thrombotic thrombocytopenic purpura)
 HELLP Syndrome (Hemolysis, elevated liver enzymes, low platelet count)
 HUS (Hemolytic Uremic Syndrome)
 Associated with Escherichia coli serotype O157:H7 infection
 Characterized by: renal failure, thrombocytopenia, schistocytes (PBS),
and severe mucocutaneous hemorrhage
 DIC (Disseminated intravascular coagulation)
 It is also known as defibrination syndrome, consumption coagulopathy
 Generalized over-activation of the coagulation and fibrinolytic systems
 Some of the tests:
o D-dimer test, PF 1+2 (Prothrombin fragment)
 Related conditions (TOMASA): Tissue trauma, Obstetric
complications, Mucus-secreting tumors, Acute infections (Ex. Malaria,
Gram-negative septicemia), Snake bites, Acute promyelocytic
leukemia

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DREPANOCYTE - Sickle or - Two forms:

a.k.a. Sickle cell, crescent- o ISC (Irreversible Sickle Cells)
Meniscocyte shaped RBCs  Crescent-shaped with long projections
 When reoxygenated: may undergo fragmentation
o Oat-shaped Cells
 Less pronounced projections
 When reoxygenated, they return to the original biconcave disk shape
- Sickle cell anemia, Hemoglobin SC disease, etc.
LEPTOCYTE - RBCs which - Liver disease
a.k.a. Codocyte, show a - Certain hemoglobinopathies
Platycyte, Greek centrally - Thalassemia (a.k.a. Hereditary Leptocytosis and Mediterranean Anemia)
helmet cell, stained area - LCAT deficiency (Lecithin cholesterol acyltransferase)
Mexican hat cell, with a thin
Bull’s eye cell, outer rim of
Target cell hemoglobin

BITE CELL - Demonstrate - Glucose-6-phosphate dehydrogenase (G6PD) deficiency

a.k.a. a semicircular
Degmacyte defect in their
edge
(resembles a
bite mark)

BISCUIT CELL - Folded RBCs - Hemoglobin SC disease

BRONZE - Bipolar or central distribution of hemoglobin - Sickle cell anemia
ELLIPTOCYTE
SEMILUNAR - As large as leukocytes - Frequently seen in malaria and in other
BODY - Pale-pink staining ghost of the red cell (the membrane conditions causing overt hemolysis
remaining after the contents have been released)

RBC INCLUSION BODIES

INCLUSION CONTENT VISUALIZATION ASSOCIATED CONDITIONS IMAGES
BASOPHILIC STIPPLING Aggregated RNA Wright stain (deep blue to - Lead poisoning (aka: Plumbism)
a.k.a. Punctate basophilia purple) - Arsenic poisoning
- Pyrimidine-5-nucleotidase deficiency
(described as irregular, dark Supravital stain
- Anemias with impaired hemoglobin
blue to purple granules
synthesis (ex. thalassemia)
evenly distributed within an
RBC) - Refractory anemias
- Alcoholism
- Megaloblastic anemias
Two forms of Basophilic stippling:
a. Fine stippling usually observed when there is ↑ polychromatophilia
b. Coarse stippling may be observed in plumbism or other disorders with impaired Hb synthesis, in megaloblastic anemia, and in other forms of severe anemia

SIDEROTIC GRANULES Intraerythrocytic Siderotic granules: - Sideroblastic anemias: characterized by

a.k.a. Pappenheimer bodies collections of iron - Iron stains (ex. Perls’ a dimorphic peripheral blood picture
reagent through (presence of both normochromic and
described as multiple dark Prussian Blue hypochromic RBCs in the same blood
blue irregular granules (in Reaction) smear [anisochromia])
Prussian blue iron staining) - Thalassemia
Pappenheimer bodies: - Hemochromatosis or Hemosiderosis
described as pale blue - New methylene blue
clusters (in Wright staining) - Wright stain
HOWELL-JOLLY BODIES Round remnants of Wright stain - Megaloblastic anemias
nuclear chromatin New Methylene blue - After splenectomy (no spleen to remove
Description: (DNA) Howell-Jolly bodies)
Frequently appears singly in Feulgen reaction (+) – - Thalassemia
a cell (only one per cell), histochemical staining
usually round, and <1 um in reaction for DNA
diameter, blue to purple in
color

CABOT RINGS Mitotic spindle Wright stain - Megaloblastic anemias

remains - Refractory anemias
Described as threadlike - Lead poisoning
structures that appear as
purple-blue loops or rings

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HEINZ BODIES Denatured and Not demonstrated using - G6PD deficiency

precipitated Wright’s stain o Favism = an inherited condition
Most of the time appear hemoglobin resulting from sensitivity to the fava
eccentrically along the inner Supravital stains: beans
RBC membrane, large, - Brilliant Cresyl Blue o Occurs mainly in people of
round, blue to purple (BCB) Mediterranean origin and in the
materials - New Methylene Blue Chinese who have G6PD deficiency
(NMB) o May result in a severe hemolytic
- Methyl Violet episode
- Crystal Violet - Drug-induced hemolytic anemias (DIHA)
- Unstable hemoglobin disease (congenital
Heinz body hemolytic anemia)
o Examples of unstable hemoglobin
variants:
 Hb Koln  Hb M-Saskatoon
 Hb Casper/  Hb Bristol
Southhampton  Hb Torino
 Hb Genova  Hb Seattle
 Hb Gun Hill
Hb H INCLUSION BODIES Precipitated Not demonstrated using - Hb H disease (a subtype of alpha
Hemoglobin H Wright’s stain thalassemia)
Described as small, multiple,
evenly distributed throughout (Hb H – an abnormal Supravital stains:
the red cell, granular, Hb composed of 4 β - New Methylene Blue
greenish-blue bodies (beta) globin chains) - Brilliant Cresyl Blue
(denatured β globin
chains) Note:
Appearance of RBCs with
Hb H bodies: “Pitted golf
ball”
PARASITES Protozoa/protozoans Wright stain - Parasitic infections
Ex.
- Malaria Giemsa stain
(Schuffner’s
dots, a.k.a.
Eosinophilic
stippling)
- Babesia spp.
(Maltese cross)

THE RED CELL MORPHOLOGY GRADING CHART BTTSO

MORPHOLOGY GRADE AS:
Polychromatophilia 1+ = 1 to 5 per field
Helmet cells (helmet-shaped 2+ = 6 to 10 per field
schistocytes) 3+ = greater than 10 per field
Dacrocytes (teardrop cells)
Acanthocytes (thorn or spur cells)
Schistocytes (fragmented RBCs)
Spherocytes
Poikilocytosis 1+ = 3 to 10 per field
Ovalocytes 2+ = 11 to 20 per field
Elliptocytes 3+ = greater than 20 per field
Burr cells
Bizarre-shaped RBC
Target cells
(codocytes/leptocytes/bull's eye
cell/Mexican hat cells/Greek helmet
cells/platycytes)
Stomatocytes mouth cells)
Rouleaux Formation 1+ = aggregates of 3 to 4 RBCs
(a.k.a. Pseudoagglutination) 2+ = aggregates of 5 to 10 RBCs
3+ = many aggregates with only a few
“STACKS OF COINS” appearance of free RBCs
red cells

REMEMBER
- Cased by a serum protein
abnormality (either increased
globulin or fibrinogen)
- May be observed in:
o Multiple Myeloma (now
called: Plasma Cell
Myeloma)
- Rouleaux maybe wrongly
reported if one examines the
thick part of the blood film
because the red blood cells in
the said area are overlapping
Sickle cells Whenever present, grade as positive
Basophilic stippling only
Pappenheimer bodies
Howell-Jolly bodies

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HEMOGLOBIN HEMOGLOBINOMETRY (HEMOGLOBIN DETERMINATION)

- Reference method: Cyanmethemoglobin (HiCN) Method
HEMOGLOBIN REFERENCE RANGE o Best and most widely used
AGE GROUP CONVENTIONAL UNITS S.I. UNITS o Principle:
Children (8-13) 12 to 15 g/dL 120 to 150 g/L
Adult (male) 14 to 18 g/dL 140 to 180 g/L Hemoglobin (Fe2+) + K3Fe (CN)6 → methemoglobin (Fe3+) + KCN → cyanmethemoglobin
Adult (female) 12 to 15 g/dL 120 to 150 g/L
o Uses Drabkin’s reagent – major components:
 Potassium ferricyanide (K3Fe (CN)6)
HEMOGLOBIN
 Converts the hemoglobins into methemoglobin
- Main component of the red blood cell (approximately 95% of the  Potassium cyanide (KCN)
cytoplasmic content of RBCs)  Provides cyanide ions
- also known as the: respiratory pigment o HiCN is measured at 540nm using a spectrophotometer
- Concentration of Hb within RBCs: approximately 34 g/dL o All types of Hb may be measured through this method, EXCEPT:
- Molecular weight: approximately 64,000 daltons Sulfhemoglobin; because it cannot be converted to
- Felix Seyler identified the respiratory protein hemoglobin (in 1862) cyanmethemoglobin
- About 65% of cytoplasmic hemoglobin is produced before the nucleus is
POSSIBLE SOURCES OF ERROR (TURBIDITY)
extruded, and the remaining 35% is synthesized in the early reticulocyte
SOURCES OF ERROR CORRECTION
- Single most common complex organic molecule in vertebrates (Falsely Elevated Hb)
- 1 gram of hemoglobin can carry 1.34 mL of O2 Turbidity brought by: Centrifuge reagent-sample solution, then the
- 1 gram of hemoglobin can carry a constant 3.47 mg iron supernatant is measured
↑ WBC count (>20 x
STRUCTURE OF HEMOGLOBIN 109/L) & ↑ platelet count
Complete adult hemoglobin molecule is composed of 4 different constituents: (>700 x 109/L)
Turbidity brought by: Add 0.01mL of the patient’s plasma to 5mL of the
1. A protein component (globin) composed of two sets of two different cyanmethemoglobin reagent and using this
polypeptide chains. Lipemia solution as the reagent blank
2. Four molecules of the nitrogenous protoporphyrin IX.
3. Four iron atoms in the ferrous form (Fe2+) that combine with protoporphyrin Note: fasting is not required
IX to create the four heme molecules. Cells containing Hb S Make a 1:2 dilution with distilled water (1 part
4. One 2,3-BPG molecule as a sometime (transient) resident in the center of and Hb C diluted sample plus 1 part water) and multiplying
the hemoglobin unit. (resistant to lysis) the results from the standard curve by 2
Abnormal globulins Add 0.1 gram of potassium carbonate to the
(such as those found in cyanmethemoglobin reagent. Commercially
patients with plasma cell available cyanmethemoglobin reagent has been
myeloma or Waldenstrom modified to contain KH2PO4 salt, so this problem
macroglobulinemia)
is not likely to occur.

REMINDERS
- Cyanmethemoglobin reagent is sensitive to light (should be stored in a
brown bottle or in a dark place)
- Another technique that has been used in some automated instruments
involves the use of sodium lauryl sulfate (SLS) to transform hemoglobin
to SLS-methemoglobin. This method does not produce toxic wastes.
- Hemocue
o An example of commercially available handheld system to measure
the hemoglobin concentration
o In here, hemoglobin is converted to azidemethemoglobin and is ready
photometrically at two wavelengths (570 nm and 880 nm).

HEMOGLOBIN ELECTROPHORESIS
- Electrophoresis: movement of charged particles in an electric field
- CELLULOSE ACETATE (pH 8.4-8.6)
o Considered as the primary screening procedure to detect variant
(abnormal) hemoglobins
o In an alkaline buffer (8.4 to 8.6) hemoglobin is a negatively charge
molecule
o During electrophoresis, the hemoglobin molecules travel toward the
PROTEIN STRUCTURES OF HEMOGLOBIN anode (+) because of their net negative charge
Protein Remarks o The difference in the net charge of the hemoglobin molecules defines
Structure its mobility and reveals itself by the speed with which it migrates t the
Primary describes the amino acid sequence of the polypeptide chains positive pole
Secondary describes the chain arrangements in helices and non-helices o Fastest = Hb H
Tertiary describes the arrangement of the helices into a pretzel-like  an abnormal hemoglobin; 4 beta globin chains
configuration or formation  in normal individuals, the fastest is Hemoglobin A1
Quaternary describes the complete hemoglobin molecule (complete o Slowest = Hb C, HbA2, Hb E, Hb CHarlem, Hb OArab
(Tetramer) hemoglobin molecule is spherical, has four heme groups o Note: Hb S, Hb D, and Hb G migrate to the same area at the cellulose
attached to four polypeptide chains, and may carry up to four acetate electrophoresis
molecules of oxygen) o Screening test for Hemoglobin S

PRIMARY FUNCTIONS OF HEMOGLOBIN

1. Delivery of oxygen to the tissues

2. To carry waste product CO2 away to the lungs
o Oxygen must bind to hemoglobin to release carbon dioxide (Haldane
effect)
3. Binding, inactivation, and transport of nitric oxide (NO)

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- CITRATE AGAR (pH 6.0-6.2)

o Complementary procedure to cellulose acetate hemoglobin
electrophoresis
o Used to confirm variant hemoglobins and further differentiates
hemoglobin S from D and G, and hemoglobin C from hemoglobins E,
Oarab, Charlem
o Confirmatory test for hemoglobin test

HEPCIDIN
- Master regulatory hormone of systemic iron metabolism
HEMOGLOBIN SYNTHESIS - Produced by the liver
- Ferroportin: transports iron from the tissues into the blood by increasing
HEME SYNTHESIS
iron absorption in the intestines and iron recycling
- Heme, a.k.a. Ferroprotoporphyrin IX
o Ferrous form of iron + protoporphyrin IX PROTEINS INVOLVED IN BODY IRON SENSING
- Belongs to class of pigments known as porphyrins AND HEPCIDIN PRODUCTION
- Site of heme synthesis: Mitochondrion PROTEIN LOCATION
- Heme biosynthesis Hemochromatosis protein (HFE) Hepatocyte membrane
o Occurs in all metabolically active cells containing mitochondria Transferrin receptor 2 (TfR2) Hepatocyte membrane
o Most prominent in bone marrow and liver Bone morphogenic protein (BMP) Secreted product of macrophages
o Erythroid marrow is the major heme-forming tissue, generating 85% Bone morphogenic protein Hepatocyte (and other cells) membrane
of the daily heme requirement receptor (BMPR)
- Ferrochelatase Hemojuvelin (HJV) Hepatocyte membrane
o A.k.a. heme synthetase SMAD (sons of mothers against Hepatocyte (and other cells) cytoplasm
o Enzyme needed to insert the ferrous form of iron to the Protoporphyrin decapentaplegic)
IX ring
PROCESSES INVOLVED IN IRON METABOLISM

1. ABSORPTION
- Duodenum and upper jejunum (sites of maximal absorption of iron)
- Foods containing ↑ levels of iron include:
o Red meats, legumes, and dark green leafy vegetables
- Iron can be absorbed as either ionic iron (should be in the Fe2+ [ferrous]
form) or heme (nonionic iron).
o Heme is more readily absorbed than ionic iron
- Unfortunately, most dietary iron is Fe3+ [ferric] form, especially from plant
sources (therefore, NOT readily absorbed).
- Also, other dietary compounds (e.g., oxalates, phytates, phosphates, and
calcium) can bind iron and prevent its absorption.
- Enhance the reduction of ferric (Fe+3) form to the ferrous (Fe+2) form and
release of iron from those that bind it:
o Gastric acid
o Acidic foods such as citrus
o Intestinal component known as DcytB (duodenal cytochrome B)
- Meat (with heme in the myoglobin of muscle and hemoglobin of blood) –
most bioavailable source of dietary iron
- The process by which heme is absorbed by enterocytes is NOT entirely
clear.
- Ferrous iron (Fe2+) – carried across the luminal side of the enterocyte by
DMT1 (divalent metal transporter 1).
- Once absorbed into enterocytes, ferrous iron (Fe2+) requires ferroportin to
deliver it into the blood.
IRON METABOLISM o Hephaestin – reoxidizes ferrous (Fe2+) into ferric (Fe3+) form as it exits
for transport into the blood.
Iron stores
decrease (↓) 2. TRANSPORTATION
- Hephaestin - a protein that is able to oxidize iron as it exits the enterocyte
Ferroportin in the enterocyte and Plasma iron
decreases (↓) - Once oxidized, iron is ready for plasma transport (carried by
macrophage membranes is
inactivated by hepcidin (↓ iron apotransferrin).
intestinal absorption and ↓ iron - Apotransferrin with bound ferric (Fe3+) form of iron is called Transferrin.
recycling)
3. UTILIZATION
Liver is alerted REGULATION OF IRON Liver is alerted and
stops hepcidin - Cell membranes have a receptor for transferrin called transferrin receptor
and produces METABOLISM
hepcidin production 1 (TfR1).
- Some of the iron → transferred into the mitochondria of the cell
(incorporated into cytochromes or incorporated into heme for Hb production
Ferroportin in the enterocyte
and macrophage membranes [in the case of RBCs])
activate again and will transport - Some of the other iron → stored (ferritin)
Iron stores iron into the blood (↑ iron
increase (↑) intestinal absorption and ↑ iron
recycling)
Plasma iron
increases (↑)

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4. STORAGE 6. IRON RECYCLING

- All cells store iron as ferritin when they have an excess, although - The iron of the cells is recycled when cells die.
macrophages and hepatocytes contain the most - Most of the recycled iron comes from RBCs. Aging erythrocytes are
- FERRITIN ingested by splenic macrophages and their hemoglobin is degraded. The
o an intracellular protein secreted into the plasma by macrophages in macrophages will hold the iron from the hemoglobin as ferritin.
proportion to the amount of stored iron - Similar to enterocytes, macrophages have ferroportin in their membranes.
o an acute phase reactant (APR) and is elevated in inflammation This allows them to be iron exporters so that saved iron can be used by
(unrelated to the amounts of stored iron). other cells.
- When needed, iron can be released from ferritin by degradation of the - Haptoglobin is a plasma protein that is able to save free hemoglobin.
protein by lysosomes. Likewise, hemopexin is also a plasma protein but is able to save heme.
- HEMOSIDERIN These proteins prevent the urinary loss of free hemoglobin and heme and
o partially degraded ferritin return the iron to the liver.
o appears in cases of iron overload - Hepatocytes are also important to iron salvage (like the macrophages).
o may be seen microscopically using the Prussian blue iron stain These cells also have ferroportin so that the saved iron can be exported to
transferrin and ultimately to other body cells.
5. EXCRETION
- Normally, there is only minimal daily loss of iron with exfoliated skin and IRON COMPARTMENTS IN NORMAL HUMANS
hair and intestinal epithelia. COMPARTMENT FORM AND ANATOMICAL TOTAL
- Iron is so vital to the body that there is NO mechanism for active excretion. LOCATION BODY IRON
FUNCTIONAL Hemoglobin iron in the blood ~68%
- It is very important to remember that iron is significant to energy production
in all cells, being at the center of the cytochromes of mitochondria. Myoglobin iron in muscles ~10%
Peroxidase, catalase, cytochromes, ~3%
- Also, the oxygen needed for energy production is carried bound to iron in
riboflavin enzymes in all cells
the hemoglobin of the RBCS
STORAGE Ferritin and hemosiderin mostly in ~18%
- Iron is even recycled to save as much as possible inside the body. macrophages and hepatocytes; small
- The body also stores iron to insure against times when iron may be scarce amounts in all cells EXCEPT mature
in the diet RBCS
TRANSPORT Transferrin in plasma <1%

ASSESSMENT OF BODY IRON STATUS

TESTS REFERENCE RANGE COMMENTS
Serum Iron 50 to 160 ug/dL - Used for the differential diagnosis of disorders of iron metabolism
- In IDA: ↓
- Specimen:
o Non-hemolyzed serum
o Should be obtained in the morning (serum iron levels may be approximately 25% lower in the evening)
o 12-hour fasting required
Total Iron Binding 250 to 400 ug/dL - Indirectly measures the concentration of transferrin by measuring its ability to bind iron
Capacity (TIBC) - In IDA: ↑
- Specimen:
o Non-hemolyzed serum
o 12-hour fasting required
o TIBC values are independent on the time of day the sample is drawn.
Transferrin 20% to 55% Serum iron/TIBC x 100 = Percent Transferrin Saturation
Saturation
Serum Ferritin 40 to 400 ng/mL (men) - Reveals the body's tissue iron stores
- Good indicator of iron storage status
12 to 160 ng/mL (women) - Useful in diagnosing iron deficiency (generally the first laboratory test to become abnormal when iron stores
begin to decline)
- In IDA: ↓
- Measured using radioimmunoassay
Prussian Blue Normal iron stores - Visual qualitative assessment of tissue iron stores
Staining visualized - Staining process uses acidic potassium ferrocyanide as reagent/stain (ferric iron in tissue reacts with the
(bone marrow or liver reagent forming the Fe7(CN)18 [Prussian blue compound] that is readily seen microscopically as dark blue dots.
biopsy) - Prussian blue stain is considered the gold standard for assessment of body iron.
- Although ferric iron reacts with the reagent, ferritin is NOT detected, likely due to the intact protein cage.
However, hemosiderin stains readily.
Soluble transferrin 1.15 to 2.75 mg/L - Indicator of functional iron available in cells
receptor (sTfR) level
sTfR/log ferritin 0.63 to 1.8 - Indicator of functional iron available in cells
index
RBC zinc <80 ug/dL - Indicator of functional iron available in red blood cells
protoporphyrin level
Hemoglobin content 27 to 34 pg/cell - Indicator of functional iron available in red blood cells
of reticulocytes
Free erythrocyte Depends on the method - RBCs produce slightly more protoporphyrin than is necessary, but in iron deficiency, protoporphyrin levels
protoporphyrin being used but an build up in RBCs to several times the normal level.
approximate reference- May be measured directly by a hematofluorometer or by an extraction and fluorescence method
value is < 50 ug/dL of RBC - Useful in differentiating thalassemia from iron deficiency and anemia of chronic disorders
- In IDA: ↑
*NOTE: Reference ranges stated above are for adult males, unless otherwise specified

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DIFFERENTIATION OF SOME DISORDERS OF IRON METABOLISM

(MICROCYTIC, HYPOCHROMIC ANEMIAS)
SERUM IRON TIBC SERUM FERRITIN FEP
Thalassemias ↑ N ↑ N
Anemia of Chronic Inflammation ↓ ↓ ↑ ↑
Iron deficiency anemia ↓ ↑ ↓ ↑
Lead poisoning Normal to ↑ (adults) N N ↑
Normal to ↓ (children)
Sideroblastic anemias ↑ N ↑ Mixed (↑↓)

INCREASED IRON ACCUMULATION (BLOOD AND TISSUES) HEMOGLOBIN DERIVATIVES

HEMOSIDEROSIS HEMOCHROMATOSIS
- Increased tissue - Characterized by increased iron accumulation in FUNCTIONAL HEMOGLOBINS
iron stores without body tissues leading to tissue damage OXYHEMOGLOBIN DEOXYGENATED HEMOGLOBIN
accompanying - Example: Hereditary Hemochromatosis - Symbol: HbO2 - Hemoglobin with Fe2+, not
tissue damage o a.k.a. Bronze Diabetes - Hemoglobin with Fe2+ oxygen bound to oxygen
- May progress to o Most common form of iron overload disease - Found in arterial blood - Found in venous blood
hemochromatosis o Autosomal recessive disorder that causes (bright red) (dark red)
body to absorb and store too much iron - Conformation of hemoglobin in - Conformation of hemoglobin in
o Caused by mutations in the HFE gene the oxygenated state is: the deoxygenated state is:
(regulates the amount of iron absorbed from R state (Relaxed) T state (Tense)
food)
o Arthritis, liver cirrhosis, congestive heart DYSHEMOGLOBINS
failure, impotence, bronze skin, diabetes,
- Symbol: HbCO

CARBOXYHEMOGLOBIN
thyroid deficiency
o Treatment - Hemoglobin with Fe2+, bound to CO (carbon monoxide)
 Avoiding foods that contain iron - Carbon monoxide gas
 Phlebotomy o Has 210 times greater affinity to hemoglobin than O2
 Deferoxamine (Desferal) o A tasteless, colorless, and odorless gas
o Source: automobile exhaust
GLOBIN SYNTHESIS - Color of blood and skin in HbCO poisoning: cherry red
- Site: ribosomes in the normoblast cytoplasm
- Chromosome 16 = dictates the production of A and Z
Chromosome 11 = dictates the production of B, E, D, and G globin chains Symbol: Hi
METHEMOGLOBIN

- -
- Other names: ferrihemoglobin, hemiglobin
FORMS OF HEMOGLOBIN - Hemoglobin with Fe3+, not bound to O2
(ACCORDING TO STAGE OF LIFE)
- Color of blood (methemoglobinemia): chocolate brown
PROPORTION
PROPORTION
MOLECULAR (%) IN ADULTS
HEMOGLOBIN (%) IN
STRUCTURE (OLDER THAN 1
NEWBORNS
YEAR)
Portland ζ2 γ 2 0 0 - Symbol: SHb
Embryonic hemoglobins

(2 zeta + 2 - Mixture of oxidized, partially denatured forms of hemoglobin

gamma)
Some of the causes of SHb formation:
ζ2 ε 2
-
SULFHEMOGLOBIN

Gower I 0 0
(2 zeta + 2
o Prolonged constipation, enterogenous cyanosis,
epsilon) bacteremia (caused by C. perfringens)
Gower II α2ε2 0 0 - Color of blood (sulfhemoglobinemia): mauve-lavender
(2 alpha + 2
epsilon) NOTE:
In-vitro, sulfhemoglobin forms when hydrogen sulfide is added
α2γ2
-
F 80 <1
(2 alpha + 2
to hemoglobin, thus the name Sulfhemoglobin
In-vivo, sulfhemoglobin forms in the occasional patient as a
Adult hemoglobins

gamma) -
result of hemoglobin oxidation by certain drugs and chemicals
A1 α2β2 20 97
(2 alpha + 2 beta) HEMATOCRIT, ESR & OFT

A2 α2δ2 <0.5 2.5 HEMATOCRIT DETERMINATION

(2 alpha + 2
delta) REFERENCE RANGES
CONVENTIONAL UNITS S.I. UNITS
Hemoglobin F Adult Males 40 to 54% 0.40 to 0.54 L/L
- Called the fetal hemoglobin Adult Females 35 to 49% 0.35 to 0.49 L/L
- Predominant form of hemoglobin in the fetus and newborn Newborn 53 to 65% 0.53 to 0.65 L/L
Hemoglobin A1
- Predominant form of adult hemoglobin - "Hematocrit” – actually pertains to the
instrument used to determine packed cell Top layer
volume (PCV) (Fatty Layer)

- After centrifugation of an anticoagulated whole

blood specimen, the red blood cells along with
other formed elements (white blood cells and
platelets) will settle at the bottom of the tube.
- The volume of the red blood cells that have
settled is called the packed cell volume
otherwise known as the hematocrit.
- Parallax object being seen in a different position by changing the position
of the head of the observer, or as seen by one eye versus the other eye.
- Micro-Hematocrit Tube:
o Approx. 75 mm long
o Inner bore: 1.2 mm
o Can hold 0.05 mL of blood

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o Plug: 4 to 6 mm long (seal the capillary tubes at the end of the tube ERYTHROCYTE INDICES
with the colored ring)
o 2 TYPES: - Indices: plural for index; Index = Guide
 With red band - Mean Cell Volume (MCV)
 Has an anticoagulant (heparin) o Average volume of an individual RBC
 Used for the collection of non-anticoagulated blood o Formula:
 With blue band ݄݁݉ܽ‫ݐ݅ݎܿ݋ݐ‬
‫ ܸܥܯ‬ൌ  ‫Ͳͳݔ‬
 Has no anticoagulant ܴ‫ݐ݊ݑ݋ܿܥܤ‬
 Used for the collection of anticoagulated-blood (ex. EDTA o Cases:
blood)  ↓ MCV - microcytic
 Normal MCV - normocytic
Summarized procedure (using non-anticoagulated whole blood):
 ↑ MCV
-
- macrocytic
1.) Perform skin puncture.
o Reference range: 80 to 100 femtoliters
2.) Wipe off the first drop of blood.
3.) Fill two heparinized capillary tubes two-thirds with blood.
o Note: Air bubbles denote poor skills but do not actually affect the - Mean Cell Hemoglobin (MCH)
test results. o Average weight or amount of hemoglobin in an individual RBC
o Tubes with a colored ring at one end are filled from opposite end. o MCH follows the MCV (smaller RBCs necessarily hold less
4.) In a vertical position, carefully seal the dry end of the heparinized hemoglobin: larger RBCs can hold more hemoglobin)
capillary tubes with the sealing day and the plug should be 4 to 6 mm o Formula:
݄݁݉‫ܾ݊݅݋݈݃݋‬
long. ‫ ܪܥܯ‬ൌ  ‫Ͳͳݔ‬
ܴ‫ݐ݊ݑ݋ܿܥܤ‬
5.) Place the two heparinized capillary tubes in the radial grooves of the
microcentrifuge with their heads exactly opposite each other. The o Not used in the classification of anemia (because it just parallels the
sealed end should be away from the center of the centrifuge. MCV value)
6.) Spin for 5 minutes at 10,000 RPM (revolutions per minute). (Note: o Normal MCV, ↑ MCH = Falsely ↑ hemoglobin
RPM must be checked periodically with a tachometer.) o Reference range: 26 to 32 picograms
7.) After centrifugation, read the hematocrit (the buffy coat layer should
NOT be included). - Mean Cell Hemoglobin Concentration (MCHC)
o Note: Results should agree within +0.02 L/L for the 2 patient o Average amount or mean concentration of hemoglobin in the average
samples run. RBCs
o Formula:
REMINDERS:
Trapped plasma may cause the hematocrit to be falsely increased by as ݄݁݉‫ܾ݊݅݋݈݃݋‬
- ‫ ܥܪܥܯ‬ൌ  ‫ͲͲͳݔ‬
much as 0.02 L/L. ݄݁݉ܽ‫ݐ݅ݎܿ݋ݐ‬
o Encountered in manual methods o Cases:
- When determined by fully automated methods, the hematocrit may be 0.01  ↓ MCHC - hypochromic
to 0.03 L/L lower than the microhematocrit method because it is  Normal MCHC - normochromic
electronically calculated and therefore is unaffected by trapped plasma.  ↑ MCHC - hyperchromic
The difference in the hematocrit results is usually insignificant unless there o Reference range: 31 to 37 g/dL or %
is a more severe case of poikilocytosis and anisocytosis.
- Automated hematocrit - a calculated value from RBC and MCV REMINDERS:
- Trapped plasma - small amount of plasma that remains in the erythrocyte - RBCs cannot accommodate more hemoglobin than 37 g/dL; therefore, a
portion of the spun hematocrit even when proper centrifugation is used. result greater than 37 g/dL should be recomputed, making sure that all
- More trapped plasma in the following: values were accurately measured and no interfering substances are
o Sickle cell anemia present
o Hypochromic anemia - MCH and MCHC have lost some clinical value (however, all RBC indices
o Spherocytosis are valuable quality control tools and help in the recognition of instrument
o Macrocytosis malfunctions).
o Thalassemia
- Certain abnormal RBC shapes (for example: spherocytes and sickle cells) POLYCYTHEMIA
inhibit complete packing.
- Immediately after a blood loss, hematocrit is not a reliable estimate of the - CLASSICALLY defined as increased hematocrit level above normal
degree of anemia because plasma volume is replaced faster than RBC - In the clinical setting: polycythemia occurs when hemoglobin and RBC
volume, therefore causing a temporarily lower hematocrit count are elevated (reflecting an elevation of the total erythrocyte volume).
- Two general kinds:
POTENTIAL CAUSES OF ERRORS o Absolute polycythemia: elevated total red cell mass
Some of the causes of Falsely Some of the causes of Falsely o Relative polycythemia: normal total red cell mass, but hematocrit is
INCREASED Hematocrit DECREASED Hematocrit increased because plasma volume is decreased
- Dehydration - Hemolysis
- Hemoconcentration - Improper sealing POLYCYTHEMIA
- Insufficient centrifugation - Increased anticoagulant ABSOLUTE POLYCYTHEMIA RELATIVE
POLYCYTHEMIA
Buffy coat inclusion in the concentration
- Secondary polycythemia with appropriately ↑
-
reading - Introduction of excess - Diminished
interstitial fluid EPO production plasma volume:
o Decreased oxygen LOADING: hypoxia, high dehydration;
altitude; pulmonary disease, cyanotic heart shock
RELATED TOPICS:
disease, carboxyhemoglobinemia; - Spurious
methemoglobinemia; Нb М polycythemia
RULE OF THREE
o Decreased oxygen UNLOADING: high oxygen (stress
- Used for checking validity of test results affinity hemoglobinopathy, polycythemia;
- Works only on normocytic, normochromic specimens biphosphoglycerate deficiency Gaisböck's
RBC count x 3 = Hemoglobin - Secondary polycythemia with inappropriately ↑ syndrome)
Hemoglobin x 3 = Hematocrit (+3%) EPO production
o Neoplasms: Wilms' tumor, renal carcinoma;
- Results from an adult male: cerebellar hemangioma; hepatoma
o Localized tissue hypoxia: polycystic kidney;
Case 1: Case 2: renal artery stenosis
(ACCURATE SPECIMEN) (INACCURATE SPECIMEN) o Post-renal transplant
Results from an adult male: Results from an adult male: o Acute hepatitis
- Genetic polycythemia
RBC ct.: 5.4 x 1012/L RBC ct.: 4.0 X 1012/L o Primary familial congenital polycythemia
Hb: 16.4 g/dL [3 x 5.4 = 16.2] Hb: 3.2 g/dL [3 x 4.0 = 12] (mutated Epo receptor)
Hct: 49% (0.49 L/L) [3 x 16.4 = 49.2] Hct: 37% (0.37 L/L) [3 x 3.2 = 9.6] o Chuvash polycythemia (mutated VHL gene)

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- Primary marrow disorders o VES-MATIC 20 Instrument

POLYCYTHEMIA VERA  a bench top analyzer designed to measure ESR in 20 samples
Absolute Polycythemia (for Polycythemia Vera)  blood is obtained in special cuvets, and mixed by the instrument
 samples are then left to sediment for a specific period
o PV-Polycythemia vera (aka: Polycythemia
 18-degree slant of the tubes with respect to the vertical axis:
Rubra Vera) causes acceleration in sedimentation, allowing results
o It is a panmyelosis (a case in which excessive comparable to those of Westergren, at the 1st hour to be obtained
proliferation occurs in RBCs, megakaryocytes in only 25 minutes, while those comparable to Westergren at the
and granulocytes). 2nd hour require only 45 minutes
o Manifested by erythrocytosis, leukocytosis,  The optoelectric sensors automatically read the RBC
and thrombocytosis of varying degree sedimentation level.
o Most patients (90%-97%) with PV has JAK2
V617F mutation REFERENCE RANGES
MODIFIED WESTERGREN WINTROBE
o Hallmark: PLETHORA
Adult Males 0 to 10 mm/hour 0 to 9 mm/hour
 A condition characterized by an excess of Adult Females 0 to 15 mm/hour 0 to 20 mm/hr
blood and marked by turgescence and a
florid complexion STAGES OF RBC SEDIMENTATION
 Turgescent – becoming turgid, distended, First 10 minutes Next 40 minutes Last 10 minutes
or swollen - Lag phase - Decantation phase - Final settling phase
o Treatment of choice: therapeutic phlebotomy - Rouleaux formation - More rapid and - RBC sedimentation
(at a frequency necessary to maintain the occurs (↑ fibrinogen constant RBC is slow
hematocrit at < 45%) or ↑ globulins) sedimentation
SOME FACTORS THAT INFLUENCE RBC SEDIMENTATION
ERYTHROCYTE SEDIMENTATION RATE FACTORS ESR INCREASED ESR DECREASED
Proteins  ↑ cholesterol  ↑ albumin
- Rate of fall of RBCs settling to the bottom of the tube (distance in mm. that and lipids in  ↑ fibrinogen  ↑ glucose
the RBCs fall in 1 hour) the blood  ↑ gamma globulins  ↑ bile salts
- It is ordered with other tests to detect and monitor the course of  ↑ phospholipids

inflammatory conditions (such as rheumatoid arthritis, an example of a  ↓ albumin  ↓ fibrinogen

 ↓ gamma globulins
chronic inflammatory condition).
Red blood  Anemia  Polycythemia
- ESR in certain disorders:  Macrocytosis  Anisocytosis (marked)
cells
o In sickle cell disease (SCD): low value in absence of painful crisis,  Microcytosis
moderately increased 1 week into crisis  Acanthocytosis
o In osteomyelitis: elevated, helpful in following therapy  Hemoglobin C
o In stroke: ESR of >28 mm/hr has poorer prognosis  Sickle cells
o In prostate cancer: ESR > 37 mm/hr has higher incidence of disease  Spherocytosis
progression and death  Thalassemia

o In Coronary artery disease: ESR >22 mm/hr in white men had high White blood  Leukemia  Leukocytosis (marked)

risk of CAD cells

o In patients with known cancer: when value exceeds 100 mm/h, Drugs  Dextran  Adrenocorticotropic
 Heparin hormone (Corticotropin)
metastases are usually present
 Penicillamine  Cortisone
o ESR - indicated in establishing the diagnosis and in monitoring
 Procainamide  Ethambutol
polymyalgia rheumatica and temporal arteritis (rate typically exceeds  Theophylline  Quinine
90 mm/hr)  Vitamin A  Salicylates
o Moderate elevations - common in active inflammatory disease like Clinical  Inflammatory conditions  Cachexia
rheumatoid arthritis, chronic infections, collagen disease, and conditions (associated with increased  Congestive heart failure
neoplastic disease (NOTE: ESR has little diagnostic value in these plasma proteins, particularly,  Newborn status
fibrinogen, alpha globulins,
disorders, but can be useful in monitoring disease activity) and beta globulins)
- Reminders:  Infections (acute and
o ESR is prone to technical errors. chronic)
o ESR has low specificity and sensitivity. Therefore, it is NOT  Subacute bacterial
recommended as a screening test to detect inflammatory conditions endocarditis
in asymptomatic individuals.  Acute heavy metal
- Methods of ESR Determination: poisoning
 Collagen vascular
o Westergren diseases
 Results are determined after 1 hr. & after 2 hrs.  Diabetes mellitus
 Anticoagulant used: 3.8% sodium citrate (black top tube)  End-stage renal failure
 Hepatitis
o Modified Westergren  Gout
 Results are determined after 1 hr.  Malignancy
 Most commonly used method  Menstruation
 Recommended by the ICSH and CLSI  Multiple Myeloma
 Waldenstrom's
 ICSH - International Council for Standardization in macroglobulinemia
Hematology  Myocardial infarction
 CLSI - Clinical and Laboratory Standards Institute  Rheumatic fever
 Anticoagulant used: Ethylenediaminetetraacetic acid (EDTA)  Rheumatoid arthritis
 Diluents: 0.85% Sodium chloride or 3.8% sodium citrate  Syphilis
 Diluted sample is placed in a 200-mm column with an internal  Temporal arteritis
diameter of 2.55 mm or more.  Nephrosis
 Tuberculosis
 Adults over 60 years of age
o Wintrobe
(frequently have a slightly
 results are determined after 1 hr. higher ESR value due
 Anticoagulant used: double oxalate or EDTA primarily to decreased
 Wintrobe tube concentrations of plasma
 Left side (red) → 0 (top) and 10 cm (bottom) (for ESR) albumin)
 Right side (white) → 10 cm (top) and 0 (bottom) (for Macro-  Pregnancy
hematocrit). Technique  Refrigerated sample NOT  Clotted blood sample
o Macrohematocrit determination is an obsolete manual returned to room  Delay in testing
procedure temperature  Bubbles in ESR column
 High room temperature  Low room temperature
 Vibration  Narrow ESR column
 Tilted ESR tube diameter
o "A tilt of as little as 3o
from the vertical can
cause error of up to
30%."

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CASE STUDY GENERAL OVERVIEW OF ANEMIA

- Because of lack of space in the laboratory: the rack of sedimentation rate - ANEMIA – defined as the decrease below normal of one or more of the
tubes was placed directly on top of a small refrigerator. What are the three following:
possible effects on the ESR results? o Number of red blood cells
1. A falsely decreased (↓) ESR - because of lower temperatures from o Hemoglobin
air rushing out on opening the refrigerator or freezer o Volume of packed red cells (hematocrit)
2. A falsely increased (↑) ESR - because of vibrations from opening and - Anemia is not a disease but the consequence of having one or a
closing the refrigerator and freezer doors manifestation of disease
3. A falsely increased (↑) ESR - because of heat released from the
refrigerator motor MECHANISMS OF ANEMIA
OSMOTIC FRAGILITY TEST (OFT) - HEMORRHAGE
o Loss of erythrocytes through bleeding must always be the
- Principle: Red Blood cells are diluted in 0 to 0.85% saline solutions and the FIRST/INITIAL FOCUS in any patient with anemia and must prompt
amount of hemolysis at each concentration is determined by measuring the an evaluation of the hemostatic system.
absorbance of the supernatant at 540nm. o Bleeding - may be secondary to trauma, surgery, or a disease
Anticoagulant to be used: HEPARIN (avoids adding more salts to the o Gastrointestinal tract - a common site for clinically significant bleeding
o Menstruation – a significant source of blood loss in women
-
blood)
- Normal values: Hemolysis begins (initial hemolysis) at 0.45% and ends
(complete hemolysis) at 0.30% - HEMOLYSIS
o Destruction of erythrocytes prior to the end of their normal lifespan
- Examples of Initial and complete hemolysis in different conditions:
(abnormal hemolysis)
CONDITIONS INITIAL HEMOLYSIS COMPLETE HEMOLYSIS o Shortened erythrocyte survival time NOT explained by bleeding
(% NaCl) (% NaCl) o Average survival time of RBCs after leaving bone marrow: 120 days
Normal 0.45 0.30 o May occur inside the blood vessel or inside the spleen
o Normal hemolysis: destruction of erythrocytes (in the spleen) who has
Hereditary 0.65 0.45
already consumed their lifespan
spherocytosis
Thalassemia 0.35 0.20
(characterized by - DECREASED PRODUCTION OF ERYTHROCYTES
numerous target o Nutritional deficiencies (iron, vitamin B12, or folic acid) are common
cells) and readily correctible causes of hypoproliferative anemia.
Sickle cell anemia 0.35 0.20 o Kidney disease may be associated with decreased production of EPO
- Increased (↑) OF seen in: Hereditary Spherocytosis and acquired
hemolytic anemias with spherocytes CLASSIFICATION OF ANEMIAS
- Decreased (↓) OF seen in: anemias where there are many target cells - Morphologic classification (basis: MCV and MCHC)
(these cells are thin and are able to expand when water enters) - Physiologic classification (less commonly used; basis: mechanism of
anemia)
INTRAVASCULAR & EXTRAVASCULAR HEMOLYSIS
MORPHOLOGIC CLASSIFICATION
NORMAL HEMOLYSIS
- Normocytic, normochromic
- RBCs live approximately 120 days.
- Microcytic, hypochromic
- Normal catabolism of aged (senescent) RBCs is MOSTLY Extravascular.
- Macrocytic, normochromic
- Intravascular hemolysis accounts for a minor component of normal RBC
destruction.
NORMOCYTIC, NORMOCHROMIC ANEMIAS
EXTRAVASCULAR HEMOLYSIS - Aplastic anemia
- A.k.a. "Macrophage-mediated Hemolysis" - Renal disease
- Macrophages involved: - Paroxysmal nocturnal hemoglobinuria
- Paroxysmal cold hemoglobinuria
Kupffer cells Splenic macrophages/ *MCV and MCHC are both normal
(liver) Littoral cells (spleen)
- Spleen: "graveyard" of RBCS Normal (N) or decreased (↓) Increased (↑) reticulocyte count
reticulocyte count
Process: Process: - Aplastic anemia - Paroxysmal nocturnal hemoglobinuria
- RBCs with: C3b on surface - RBCs with: inclusion bodies - Anemia of renal disease - Paroxysmal cold hemoglobinuria
- RBCs which are: severely damaged - RBCs which are: senescent - Sickle cell disease
- Hemolytic anemia
- Lab findings in excessive extravascular hemolysis: o Intrinsic
Urine Serum Peripheral Blood Smear  Membrane defects
 ↑ Urobilinogen  ↑ Total Bilirubin  Spherocytes  Hemoglobinopathies
 ↑ B1  Enzyme deficiencies (ex.
 Normal B2 G6PD, PK def.)
o Extrinsic
INTRAVASCULAR HEMOLYSIS  Immune causes
- A.k.a. "Fragmentation Hemolysis”  Non-immune RBC injury
 Microangiopathic
- Lab findings:
 Macroangiopathic
Urine Serum Peripheral Blood Smear  Infectious agents
 ↑ Urobilinogen  ↑ Total bilirubin  Schistocytes  Other injury: drugs,
 Hemoglobinuria  ↑ B1  Chemicals, venoms,
 (+) Prussian blue  Normal B2  Extensive burns
reaction (urine  ↓ Haptoglobin
sediments)  ↓ Hemopexin

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APLASTIC ANEMIA - Associated with chronic infections such as tuberculosis, chronic

- Rare but potentially deadly bone marrow failure syndrome inflammatory conditions such as rheumatoid arthritis, and tumors
- Characteristic features: - Central feature of ACI:
o pancytopenia (marked ↓ in the number of RBCs, WBCs, and platelets o sideropenia (↓ serum iron) despite abundant iron stores
in the blood) - Usually PBS shows normocytic, normochromic
o reticulocytopenia o advanced states: microcytic, hypochromic
o bone marrow hypocellularity - Acute phase reactants (APRs – increase during inflammation) that
o depletion of hematopoietic stem cells contribute to ACI:
o Ferritin
May be classified as: o Lactoferrin
o Hepcidin
- Acquired Aplastic Anemia  Master regulatory hormone for systemic iron metabolism
o approximately 80 to 85% of aplastic anemia cases  Inactivates ferroportin
o 2 categories:  Ferroportin: protein in tissues which transports iron from
 Idiopathic acquired aplastic anemia – no known cause tissues towards the blood
 Secondary acquired aplastic anemia – associated with an  The reason why tissues are able to release iron into the blood
identified cause  Prevents the release of iron into the blood
 some of the causes include:
o chemicals (ex.: insecticides, benzene), viruses (ex.:
Epstein-Barr virus), drugs (ex.: chloramphenicol)
o Chloramphenicol = drug most frequently implicated in
acquired aplastic anemia

- Inherited Aplastic Anemia

o approximately 15% to 20% of aplastic anemia cases
o associated diseases:
 Dyskeratosis congenital
 Shwachman-Bodian-Diamond syndrome
 Fanconi Anemia (FA) IRON DEFICIENCY ANEMIA (IDA)
 It is characterized by macrocytosis and reticulocytopenia
Iron deficiency – the most common anemia
 most common of the inherited aplastic anemias
-
 a chromosome instability disorder - Possible causes:
 characterized by: aplastic anemia, cancer susceptibility, and o Blood loss (especially menstruating women)
physical abnormalities  Menstruating women should compensate iron loss by intake of
iron supplements or iron rich foods such as legumes, beans,
 physical abnormalities may include:
dark green leafy vegetables, red meat
o skeletal abnormalities (ex.: thumb malformations,
o Nutritional deficiency (infants)
microcephaly, scoliosis)
o ↑iron demand (pregnancy, lactation, adolescence)
o skin pigmentation (ex.: hyperpigmentation,
o Malignancies of gastrointestinal tract
hypopigmentation, café-au-lait lesions)
o Hookworm infections
o short stature
o abnormalities of the eyes, kidneys, and genitals - Clinical features:
o Fatigue, weakness, irritability, headache, palpitations, loss of
concentration
- Fanconi anemia is NOT the same as Fanconi Syndrome.
o Shortness of breath
o Fanconi syndrome: defect involving the proximal convoluted tubule of
o Brittle hair (craving for ice is called pagophagia)
kidneys
o Pallor
PAROXYSMAL NOCTURNAL HEMOGLOBINURIA o Koilonychia (spooning of the fingernails)
o Angular cheilosis/chelitis (inflammation of the sides of the mouth)
- A.k.a. Marchiafava-Micheli Syndrome o Pica – unusual craving for non-food items such as dirt, clay, laundry
- caused by deficiency of complement regulatory proteins, namely: starch, or ice (most common)
o DAF (decay-accelerating factor, or CD55) o Glossitis (inflammation of the tongue)
o MIRL (membrane inhibitor of reactive lysis, or CD59)
- Tests for PNH: STAGES OF IRON DEFICIENCY
o Ham’s Acidified Serum Test
o Sugar Water Test Hemoglobin Serum TIBC Ferritin
 A.k.a. Sucrose hemolysis test; sugar used: sucrose Iron
o Flow Cytometry (confirmatory test) Normal Iron Status N N N N
Stage 1 N N N ↓
PAROXYSMAL COLD HEMOGLOBINURIA (Storage Iron Depletion)
Stage 2 N ↓ ↑ ↓
- A.k.a. Donath-Landsteiner Hemolytic Anemia
(Transport Iron Depletion)
- Caused by the presence of autoantibodies Stage 3 ↓ ↓ ↑ ↓
(Functional Iron depletion)
MICROCYTIC, HYPOCHROMIC ANEMIAS
- Thalassemia *characterized by frank iron
- Anemia of Chronic inflammation deficiency anemia
- Iron deficiency anemia - TIBC: Total Iron Binding Capacity
- Lead poisoning (plumbism) - Ferritin: Most sensitive test for IDA
- Sideroblastic anemia o Ferritin is the storage form of iron
o The only test which becomes abnormal in stage 1 of Iron deficiency
- With small cells that have increased central pallor on the smear
- MCV and MCHC = both ↓ DIFFERENTIATION OF MICROCYTIC, HYPOCHROMIC ANEMIAS
- Microcytic anemia results from an iron level insufficient for maintaining
normal erythropoiesis and is characterized by abnormal results of iron Serum Iron TIBC Serum FEP
studies. ferritin
- Early development of a microcytic anemia may reveal reduced iron stores, Thalassemias ↑ N ↑ N
but an obvious anemia has not developed. Anemia of Chronic ↓ ↓ ↑ ↑
inflammation
ANEMIA OF CHRONIC INFLAMMATION (ACI) Iron deficiency ↓ ↑ ↓ ↑
anemia
- Was originally called ACD (anemia of chronic disease) Lead poisoning N to ↑ (Adults) N N ↑
- Most common anemia among hospitalized patients
- Normal person: Low iron levels cause the release of iron from storage N to ↓ (Children)
Patients with ACI: body stores have abundant iron; but iron from storage is Sideroblastic ↑ N ↑ Mixed
(↑ and ↓)
-
anemias
not released and red cells are deficient in iron

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SIDEROBLASTIC ANEMIAS NOTES

- develop when the production of protoporphyrin or the incorporation of iron - Impaired DNA synthesis - considered as root cause of megaloblastic
into protoporphyrin is prevented anemia
- SIDEROBLAST – nucleated RBC precursor with cytoplasmic iron granules - DNA synthesis is dependent on an important structure: thymidine
- SIDEROCYTE – anucleate RBC w/ iron granules triphosphate (TTP). This structure cannot be made unless it receives a
- Iron is trapped in the mitochondria (cannot be fully utilized in Hb synthesis) methyl group from methyl tetrahydrofolate or folic acid. Vitamin B12 is the
cofactor responsible for transferring the methyl group to methyl
- Ring sideroblasts – hallmark of the sideroblastic anemias
tetrahydrofolate. Adequate amounts of vitamin B12 and folic acid are
- The “rings” in ring sideroblasts = mitochondria loaded with iron important to the formation of TTP. If TTP cannot be made, then it is
- Dimorphic peripheral blood picture (normochromic and hypochromic replaced by deoxyuridine triphosphate. The synthesis of this structure
RBCs) results in nuclear fragmentation and destruction of cells and impaired cell
division.
MACROCYTIC, NORMOCHROMIC ANEMIAS
- Vitamin B12 is also known as Cobalamin.
- Megaloblastic anemia - Folate is the general term used for any form of the folic acid.
- Non-megaloblastic anemia - Cyanocobalamin - synthetic form of vitamin B12 (found only in
*MCV = High, MCHC = Normal supplements)
- Methylcobalamin - naturally occurring form that may be obtained from
MEGALOBLASTIC ANEMIA NON-MEGALOBLASTIC ANEMIA either food sources or supplements
- Vitamin B12 deficiency - Aplastic anemia - In either folate or vitamin B12 deficiency, patients may experience:
- Folate deficiency - Chronic liver disease o fatigue, weakness, and shortness of breath
- Myelodysplasia - Alcoholism o glossitis (loss of epithelium on the tongue results in a smooth surface
- Erythroleukemia - Bone marrow failure and soreness)
- Some drugs o gastritis, nausea, or constipation
- Blood pictures seen in folate deficiency and vitamin B12 deficiency are
DIFFERENTIATING MEGALOBLASTIC NON- indistinguishable. However, their clinical presentations differ.
FACTORS ANEMIA MEGALOBLASTIC - In vitamin B12 deficiency, neurologic symptoms may be noticeable and
ANEMIA neuropsychiatric symptoms may also be present.
1. Some of the Vitamin B12 deficiency – may be o Liver disease
causes caused by: o Alcoholism Neurologic symptoms: Neuropsychiatric
(chronic) o memory loss symptoms:
- inadequate intake (dietary
o Bone marrow o numbness o personality
deficiency is infrequent)
failure o tingling in toes and fingers changes
- increased need (occurs during o loss of balance o psychosis
pregnancy, lactation and o further impairment of walking by loss of
growth) vibratory sense, especially in the lower
- competition (ex. limbs
Diphyllobothrium latum)
- lack of intrinsic factor (IF) - Dietary deficiency of vitamin B12 is infrequent. However, this condition is
o ex. Gastrectomy [surgical possible for vegans (strict vegetarians) who do NOT eat meat, eggs, or
removal of the stomach], dairy products.
Helicobacter pylori
- Sources of Vitamin B12:
infection, Pernicious
anemia
o liver o meat o oysters and clams
- The only natural source of o fish o eggs o cheese and other dairy products
vitamin B12 are animals, not
plants
- Vitamin B12 is NOT destroyed by cooking.
- Strict vegetarians are prone to
- Pregnancy needs a significant increase in folate to fulfill the requirements
Vitamin B12 deficiency
related to rapid fetal growth, uterine expansion, placental maturation, and
expanded blood volume. Folate deficiency during pregnancy can lead to
PA (Pernicious Anemia)
impaired formation of the fetal nervous system, resulting in neural tube
- characterized by the presence defects (like spina bifida).
of antibodies against parietal - Sources of Folate:
cells and antibodies against
IF.
o leafy green o liver o fortified breakfast
- Parietal cells are cells in the vegetables o whole grains o oranges
stomach which produce o broccoli o beef o cereals
intrinsic factor and o dried beans o some fruits,
hydrochloric acid o dairy products especially
- Intrinsic factor is important for
vitamin B12 absorption - Folates - heat labile (overcooking of foods can ↓ their nutritional value)
- Pernicious means dangerous
LABORATORY TESTS USED TO DIAGNOSE VITAMIN B12 DEFICIENCY
Folate deficiency – may be caused AND FOLATE DEFICIENCY
by:
- inadequate intake SCREENING TESTS Folate deficiency Vitamin B12
- increased need (No difference) deficiency
- renal dialysis Complete blood count ↓Hb, Hct, RBCs, ↓Hb, Hct, RBCs,
WBCs, PLTS WBCs, PLTS
Acute erythroleukemia
MA NMA ↑MCV, MCH ↑MCV, MCH
2. Presence of hypersegmented neutrophils Present Absent Manual differential count Hypersegmented Hypersegmented
(with >6 nuclear lobes) neutrophils, neutrophils,
REMEMBER: Oval macrocytes, Oval macrocytes,
o Normal neutrophils have 3 to 5 nuclear lobes. Anisocytosis, Anisocytosis,
o Hypersegmented neutrophils - essentially pathognomonic for megaloblastic Poikilocytosis Poikilocytosis
anemia RBC inclusions RBC inclusions
3. Shape of the macrocytes Oval Round Absolute reticulocyte ↓ ↓
4. Presence of megaloblasts in the bone marrow Present Absent count
Serum total and indirect ↑ ↑
- Megaloblasts: giant RBC precursors in the bone
bilirubin
marrow
Serum lactate ↑ ↑
dehydrogenase

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Folate Vitamin B12  Some of the findings include:

SPECIFIC DIAGNOSTIC TESTS o Hypochromic RBCs with basophilic stippling
deficiency deficiency
Serum Vitamin B12 N ↓ o Toxic granulation in neutrophils
Serum folate ↓ N or ↑ o Elevated Zinc Erythrocyte Protoporphyrin test
RBC folate ↓ N or ↓
Serum methylmalonic acid N ↑ 2.) HEREDITARY PORPHYRIAS
Serum/plasma homocysteine ↑ ↑ o examples include the:
holoTC assay (Holotranscobalamin) N ↓
THREE HEMATOLOGICALLY SIGNIFICANT PORPHYRIAS
- Mean cell hemoglobin (MCH) - elevated by the ↑volume of the cells (ERYTHROPOIETIC PORPHYRIAS):
Mean cell hemoglobin concentration (MCHC) - usually within the reference Congenital Erythropoietic X-linked

Differentiating
-
interval (Hb production is unaffected.) Erythropoietic Protoporphyria Erythropoietic

factors
Other possible findings in megaloblastic anemia: teardrop cells, nucleated Porphyria Protoporphyria
Gunther’s
-
RBCs, Howell-Jolly bodies, basophilic stippling, and Cabot rings (a.k.a.
- Once in the enterocyte, the vitamin B12 is then liberated from IF and bound disease)
to transcobalamin (previously called transcobalamin II) and released into
the blood. In the plasma, 10 to 30% of the vitamin B12 is bound to Uroporphyrinogen III Ferrochelatase ALA-synthase 2

affected
Enzyme
transcobalamin (75% is bound to transcobalamin I and III, referred to as synthase deficiency deficiency (gain of function)
the haptocorrins).
- The vitamin B12-transcobalamin complex, called holotranscobalamin
(holoTC), is the metabolically active form of Vitamin B12.
Autosomal recessive Autosomal X-linked dominant

Inheritance
PATHOPHYSIOLOGIC CLASSIFICATION dominant

1.) ANEMIAS CAUSED BY DECREASED PRODUCTION OF RBCs

- Aplastic anemia
- Megaloblastic anemia
- Photosensitivity, - Photosensitivity; - Photosensitivity;
- Iron deficiency anemia
hemolytic anemia, anemia is mild if mild microcytic,
- Thalassemia red-pigmented present hypochromic
- Anemia of chronic renal failure urine, erythrodontia anemia with
- Anemia of endocrine disorders - Prognosis for this reticulocyte
- Anemia of chronic inflammation (ACI) disease is response is
- Anemia associated w/ marrow infiltration significantly possible
- Sideroblastic anemia WORSE than for
the other
Clinical features

2.) ANEMIAS CAUSED BY INCREASED DESTRUCTION porphyrias, with

- Intracorpuscular abnormality death occurring at a
o Membrane defect: -hereditary spherocytosis young age in many
 hereditary elliptocytosis cases.
 pyropoikilocytosis - Some of the other
o Enzyme deficiency clinical features:
 G-6-PD o Excessive hair
 pyruvate kinase growth
 porphyria
o Deformed
 globin abnormality – hemoglobinopathies (e.g. Hb SS, CC, SC)
 paroxysmal nocturnal hemoglobinuria fingers and
fingernails
- Extracorpuscular abnormality
o mechanical: microangiopathic hemolytic anemia, etc. o Scars
o infection: malaria, Bartonella, Ehrlichia, Babesia o Port wine red
o chemical and physical agents: drugs, toxins, burns urine
o antibody-mediated: acquired hemolytic anemia due to warm-reacting - Associated with the - -
Additional

antibodies legend of the

Notes

werewolves
3.) BLOOD LOSS (HEMORRHAGE)

PORPHYRIAS
- Diseases characterized by impaired production of HEME
- Heme biosynthesis:
o occurs in all metabolically active cells containing mitochondria
o most prominent in bone marrow and liver.
o Erythroid marrow is the MAJOR heme-forming tissue, generating
85% of the daily heme requirement
- “Porphyria” - derived from the Greek word “porphyra”, which means purple
- Primary cause: specific enzyme deficiencies in the heme biosynthetic
pathway
- Products from earlier stages of the pathway accumulate in cells that
actively produce heme (like the RBCs and hepatocytes).
- Excess porphyrins leak from cells as they age or die (may be excreted in
urine or feces).
- Accumulated products deposit in tissues as well.
- Some of the accumulated products are fluorescent (deposition in skin can
lead to photosensitivity with severe burns upon exposure to sunlight).
- Only 3 porphyrias have hematologic manifestations (others have a greater
effect on the liver). Even in those with hematologic effects, the hematologic
impact is relatively minimal, and photosensitivity is a greater clinical
problem.
- May be ACQUIRED or HEREDITARY

1.) ACQUIRED PORPHYRIAS

o an example is:
 Lead poisoning
 Lead is able to inhibit some enzymes like pyrimidine
5'-nucleotidase and ferrochelatase
 Pyrimidine-5-nucleotidase: enzyme needed to
remove the RNA remnants from the reticulocytes
 Ferrochelatase: a.k.a. heme synthetase; enzyme
needed to insert the Fe+2 at the center of the
protoporphyrin ring

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LABORATORY FEATURES OF THE 3 HEMATOLOGICALLY SIGNIFICANT PORPHYRIAS

Laboratory Features Congenital Erythropoietic Porphyria Erythropoietic Protoporphyria X-linked Erythropoietic Protoporphyria
(CEP) (EPP) (XLEPP)
Red blood Protoporphyrin Normal ↑↑↑ ↑
cells Uroporphyrin ↑↑↑ Normal Normal
Coproporphyrin ↑↑ Normal Normal
Urine Porphobilinogen Normal Normal Normal
Uroporphyrin ↑↑↑ Normal Normal
Coproporphyrin ↑↑ Normal Normal
Feces Protoporphyrin Normal ↑↑ Normal or ↑
Coproporphyrin ↑ Normal Normal
Confirmatory ↓↓ Uroporphyrinogen III synthase ↓↓ Ferrochelatase activity ↑↑ ALA-synthase activity
tests activity
Genetic testing ↑↑ free erythrocyte protoporphyrin and zinc
Genetic testing protoporphyrin
- Single arrow – minimal increase or decrease
- Double arrows – moderate increase or decrease
- Triple arrows – marked increase or decrease

HEMOGLOBINOPATHIES Associated with decreased oxygen affinity:

- Qualitative globin synthesis defect 10. Hb Agenogi
11. Hb Beth Israel
- Due to differences in the arrangement of amino acids in the polypeptide
12. Hb Yoshizuka
chain
- Major Groups of Hemoglobinopathies:
HEMOGLOBIN S
o Alpha-hemoglobinopathies
o Beta-hemoglobinopathies (most frequently encountered) - Glutamic acid on the 6th position of the B chain is replaced by valine
o Gamma-hemoglobinopathies - Defined by the structural formula:
o Delta-hemoglobinopathies ߙଶ ߚଶ଺ீ௟௨՜௏௔௟
- The name of the hemoglobinopathy depends on the affected globin chain - When oxygenated, Hb S is fully soluble (sickling happens when oxygen
- Of the three adult hemoglobins (A1, A2, F), only hemoglobin A2 is affected drops at the issue level). When oxygen is released from the molecule, a
by beta hemoglobinopathies since Hb A2 is made up of α2β2 (2 alpha + 2 conformational change happens which results in polymerization of
beta) hemoglobin molecules leading to the formation of tactoids or crystals (in
turn, this causes the cells to become rigid).
β-hemoglobinopathies - Degree of sickling depends on the concentration of hemoglobin S in the
Homozygous β- Heterozygous β- RBC:
hemoglobinopathies hemoglobinopathies o If the concentration of Hemoglobin S is 80 to 100% (as in sickle cell
- Both β genes: mutated - One of the β genes: normal anemia), sickling of the red blood cells occur readily at only slightly
- Hb A1 is absent - Other β gene: mutated decreased oxygen concentrations
- Abnormal hemoglobin: - Hemoglobin A1 is greater than or o If the concentration of Hemoglobin S is only 20 to 40% (as in the sickle
becomes the predominant equal to the amount of abnormal cell trait), oxygen concentrations must be much lower before sickling
hemoglobin type hemoglobin occurs.
- Examples: - Examples: - Sickle cells obstruct blood low to tissues and organs (resulting in tissue
o Sickle cell disease o Sickle cell trait death organ infarction, and pain).
 Genotype: Hb SS  Genotype: Hb AS
 S gene is the  A is the normal beta
mutated beta gene
gene  S is the mutated beta
 S gene is gene
inherited  Resistant to P.
o Hb C disease falciparum
 Genotype: Hb CC o Hb C trait
 C gene is the  Genotype: Hb AC
mutated beta  A is the normal beta
gene gene
 C gene is  C is the mutated beta
inherited gene

Examples of abnormal hemoglobins present in certain

β-hemoglobinopathies:
1. Hb S
- Most common
- Most severe TESTS FOR HEMOGLOBIN S
Structural formula: ߙଶ ߚଶ଺ீ௟௨՜௏௔௟
SCREENING

-
TESTS

- At the 6th position of the beta chain, the glutamic acid is replaced by
valine PRINCIPLE POSSIBLE RESULTS
2. Hb C
- 2nd most common - Whole blood is POSITIVE (+)
଺ீ௟௨՜௅௬௦
- ߙଶ ߚଶ mixed with sodium - presence of either sickle cells or "holly-
- At the 6th position of the beta chain, the glutamic acid is replaced by metabisulfite (a leaf” form of the RBCs
lysine reducing agent - Note: "Holly-leaf” form of RBCs is
3. Hb E which frequently found in the sickle cell trait.
Sodium metabisulfite method

- 3rd most common deoxygenates Hb) Bear in mind, however, that using this
ଶ଺ீ௟௨՜௅௬௦
- ߙଶ ߚଶ o Reducing method, it is NOT possible to
- At the 26th position of the beta chain, the glutamic acid is replaced by agent removes differentiate sickle cell trait from sickle
lysine oxygen from cell anemia. Although in sickle cell
M hemoglobins (associated with methemoglobinemia and cyanosis): the test anemia, the sickling reaction happens
4. Hb M-Saskatoon environment more rapidly than in sickle cell trait, this
5. Hb M-Milwaukee-1 - In such conditions, observation must NOT be relied upon
6. Hb M-Milwaukee-2 (Hyde-Park) Hb S existing in the to distinguish between the two
RBC causes the conditions.
Associated with increased oxygen affinity: formation of sickle
7. Hb Hiroshima shaped RBCs NEGATIVE (-)
8. Hb Rainier - Normal looking or slightly crenated
9. Hb Bethesda RBCS

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- When RBCs are added POSITIVE (+) HEMOGLOBIN C

to the working solution - Turbid solution (black lines on the Glutamic acid on the 6th position of the beta chain is replaced by lysine.
Sodium dithionite tube test (Solubility test)
-
containing sodium reader scale are NOT visible through - Defined by the structural formula:
dithionite (reducing the test solution)
agent) and saponin ଺ீ௟௨՜௅௬௦
ߙଶ ߚଶ
(hemolytic agent), the NEGATIVE (-)
red cells immediately - Clear solution (black lines on the
lyse. reader scale are visible through the - Two crystals related to Hb C:
- Hb S (and sickling test solution) 1. Hb SC crystals
hemoglobins), in the o Characteristic appearance:
- Note: If this test is positive, a Hb
reduced state (in a “Washington monument”
electrophoresis should be performed
concentrated buffer o Found protruding the RBC membrane
on the specimen
solution), forms liquid - On the other hand, Hb
crystals and produces a 2. Hb CC crystals:
electrophoresis results showing the o Characteristic appearance:
turbid appearance presence of Hb S should be “Bar of gold”
confirmed by a positive sodium o Found within the RBC membrane
dithionite tube test (solubility test)

THALASSEMIAS
- Quantitative globin synthesis defect
- Initially called “Thalassic (Greek for "great sea") Anemia”
- Other names:
o Hereditary Leptocytosis
o Mediterranean Anemia
- Reduction or total absence of synthesis of one or more of the globin chains
- Thalassemias – named according to the chain with reduced or absent
synthesis
- Mutations affecting the α- or β-globin gene – most clinically significant (the
reason: Hb A [a2β2] is the major adult hemoglobin)
- Thalassemia occurs in all parts of the world. However, its distribution is
concentrated in the "thalassemia belt".
- Thalassemia Belt
o extends from the Mediterranean east through the Middle East and
India to Southeast Asia and South to Northern Africa
o Its geographic location coincides with areas in which malaria is
prevalent
o Thalassemia minor (heterozygous thalassemia) seems to impart
resistance to malaria.
- Individual and family histories are important in thalassemia diagnosis. The
Hemoglobin - If performed alone, it serves as a screening test ethnic background of the person should be investigated because of the
Electrophoresis for hemoglobin S increased prevalence of particular gene mutations in specific populations.
(Cellulose - If performed along with sodium dithionite tube test, - Clinical findings that suggest thalassemia include (these are particularly
Acetate) it becomes a confirmatory test for hemoglobin S prominent in untreated or partially treated β-thalassemia major):
- In an alkaline buffer (8.4 to 8.6) hemoglobin is a o pallor (due to the anemia)
Note: Considered negatively charged molecule o jaundice (due to hemolysis)
as the primary o splenomegaly (due to sequestration of abnormal red cells, too much
- During electrophoresis, the Hb molecules travel
screening extravascular hemolysis, and some extramedullary erythropoiesis)
toward the anode (+) because of their net negative
procedure to o skeletal deformities (due to the massive expansion of the bone
charge
detect variant marrow cavities)
(abnormal) - The difference in the net charge of the Hb
molecule defines its mobility and reveals itself by - Clinical manifestations of thalassemia arise from:
hemoglobins o Decreased or absent production of a specific globin chain, which
the speed with which it migrates to the positive
pole. reduces hemoglobin synthesis and generates microcytic,
hypochromic RBCs; and
o Unequal production of the α- or β-globin chains causing an imbalance
in the α/β chain ratio (leads to a markedly decreased survival of
erythrocytes and their precursors)
- REMEMBER: The α/β chain imbalance is more significant and determines
the clinical severity of the thalassemia. The mechanism and the degree of
shortened red cell survival are dissimilar for the β-thalassemias and α-
thalassemias.

SCREENING TESTS
TESTS EXPECTED FINDINGS
CONFIRMATORY PRINCIPLE Complete Blood ↓ Hb, ↓ Hct, ↓ MCV, ↓ MCH, ↓ MCHC, Slight to moderate
TESTS Count (CBC) ↑ Reticulocyte count
Hemoglobin - Migration distances of the different hemoglobins Peripheral Varying degrees of microcytosis, hypochromia, target
Electrophoresis are based on the electrophoretic charge of the Blood Smear cells, anisocytosis, NRBCs (nucleated red blood cells),
(Citrate Agar) molecules and their adsorption to the agar poikilocytosis and RBC inclusions (examples include:
compound basophilic stipplings, Howell-Jolly bodies, Pappenheimer
Note: Used to bodies, Hb H inclusion bodies)
confirm variant Iron studies Serum ferritin and serum iron: Normal or ↑ TIBC: Normal
hemoglobins and (to rule out IDA)
further
differentiates - Confirmatory test: Molecular Genetic Tests
hemoglobin S
from D and G, and
hemoglobin C
from hemoglobins
E,
OArab, CHarlem

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ALPHA THALASSEMIA β-thalassemia βsilent/βsilent - Moderate clinical symptoms,

intermedia β+/βsilent transfusion-independent
- typically caused by a deletion of one, two, three or all four of the α-globin β0/βsilent - Mild to moderate hemolytic
genes (results in reduced or absent production of a chains anemia, microcytic, hypochromic
- Genotype in normal persons: αα/αα (4 α-genes) RBCs

Clinical No. of Genotype Remarks

syndromes Deleted NOTE
Gene Note: The following are examples of β-thalassemic genes and their
Silent carrier One of (-α/αα) - Asymptomatic definitions:
state four - β0 = β-globin gene mutation in which no β chains are produced
(α-thalassemia) genes - β+ = β-globin gene mutation that results in 5% to 30% reduction in β
α-thalassemia Two of (--/αα) - Mild microcytic chain production
trait four or hypochromic anemia βsilent = β-globin gene mutation that results in mildly decreased β chain
α-
-
(aka: genes (-α/-α) production
thalassemia
minor)
Hb H disease Three of (--/-α) - Mild microcytic WHITE BLOOD CELLS
four hypochromic anemia
genes - Nucleated cells that function in body defense.
- Hb H found in adults
- Hb Bart found in neonates - RBC vs WBC: RBCs are anucleate while WBCs are nucleated
- Reference ranges
- Hb H may coexist with Hb
Constant Spring (a more o Adult (males and females): 4.5 to 11.0 x 109/L (SI)
 4,500 to 11,000 /mm3 (conventional)
severe disease than Hb H)
o Newborn: 13.5 to 38.0 x 109/L (SI)
Hb Bart Four of (--/--) - Hb Bart’s Disease  13,500 to 38,000/mm3 (conventional)
hydrops fetalis four - MOST SEVERE FORM OF
syndrome genes - How to convert WBC count from conventional units to SI?
ALPHA THALASSEMIA o Ex. 4,200/mm3 (conventional) to SI
(a.k.a. Hb Bart - Most severe form may
Syndrome, α- o Move three decimal points to the left
cause stillbirth/hydrops o Answer: 4.2 x 109/L
Thalassemia fetalis
Major) o Move three decimal points to the right if converting from SI to
- NOTE: Fetus commonly conventional
cannot survive gestation, - NOTE: The difference between mm3 and µL is INSIGNIFICANT (either of
causing stillbirth with the two may be utilized in reporting the WBC count using the conventional
hydrops fetalis. units)
- However, presently, with
the help of intrauterine NORMAL WBC COUNTS AND DIFFERENTIAL COUNTS
transfusion and the (In different ages)
neonatal intensive care AGE TOTAL WBC (x109/L)

LYMPHOCYTES (%)
NEUTROPHILS (%)
unit, survival may be (in range)

EOSINOPHILS (%)
MONOCYTES (%)
possible (survivors will
have severe transfusion-
dependent anemia like
patents suffering from β-
thalassemia major).
- Bone marrow transplant or
cord blood transplant may 6 months 6.0 to 17.5 32 61 5 3
be helpful. 2 years 6.0 to 17.0 33 59 5 3
4 years 5.5 to 15.5 42 50 5 3
BETA THALASSEMIA 6 years 5.0 to 14.5 51 42 5 3
8 years 4.5 to 13.5 53 39 4 2
- caused by mutations that affect the B-globin gene complex 16 years 4.5 to 13.0 57 35 5 3
- no deletion in the beta gene 21 years 4.5 to 11.0 59 34 4 3
- Normal persons: 2 beta genes only (1 from the mother, 1 from the father)
- Predominant WBC in an adult: neutrophils
Clinical Examples Remarks - Predominant WBC in children <4 years of age: lymphocytes
syndromes of - On determining WBC maturity, MOST valuable and reliable criterion is:
Genotypes nuclear chromatin pattern
β-thalassemia βsilent/β - asymptomatic; normal hematologic
silent carrier parameters WBC Classifications:
state
β-thalassemia β+/β - asymptomatic; mild hemolytic Granulocytes Agranulocytes
trait (aka: β- β0/β anemia; microcytic, hypochromic o Neutrophils o Monocytes
thalassemia RBCs o Eosinophils o Lymphocytes
minor) o Basophils
β-thalassemia β+/β+ - MOST SEVERE FORM OF BETA Polymorphonuclears Mononuclears
major β+/β0 THALASSEMIA
(aka: Cooley’s β0/β0 o Neutrophils o Monocytes
- Severe hemolytic anemia; o Eosinophils o Lymphocytes
Anemia) microcytic, hypochromic RBCs; o Basophils
transfusion-dependent
- Possible PBS findings: target cells, Phagocytes Immunocytes
teardrop cells, elliptocytes, nucleated o Neutrophils o Lymphocytes
red cells, polychromasia, basophilic o Eosinophils
stippling, Howell-Jolly bodies, and o Basophils
Pappenheimer bodies o Monocytes
- Some of the β-thalassemia major - All WBCs have phagocytic function except lymphocytes
patients' characteristics include: - Lymphocytes are the only type of WBCs that have immunocytic function
o frontal bossing
o prominence of the cheekbones GRANULOCYTIC SERIES
and upper jaw
o skull radiographs may exhibit a - In general, as granulocytes mature:
typical “hair on end" appearance 1. Nuclear chromatin becomes more condensed
o iron accumulation in various 2. Nucleoli disappear
organs (mainly due to the 3. Abundant basophilic cytoplasm with nonspecific granulation
regular RBC transfusions progresses to more scant cytoplasm containing specific granules
required in β-thalassemia major) 4. The nucleus indents and becomes segmented
5. Overall cell size decreases

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NEUTROPHIL PRECURSORS Stage:

MYELOCYTE
Size: 15 to 18 um
Comments:
- 6 to 17% of the nucleated cells in the bone marrow
- Last stage capable of mitosis
- Stage of synthesis of secondary granules (specific granules)
- Two types:
Stage:  Early myelocytes
MYELOBLAST o may look very similar to the
promyelocytes (EXCEPT that
Size: 14 to 20 um
patches of grainy pale pink cytoplasm
Comments:
representing secondary granules
- Earliest recognizable granulocytic precursor using light microscope start to become obvious in the area of
- 0 to 3% of the nucleated cells in the bone marrow the Golgi apparatus called: dawn of
- Three types: neutrophilia)
 Type I myeloblasts
o Nucleus occupies most of the cell, with very little cytoplasm  Late myelocytes
o Slightly basophilic cytoplasm, fine nuclear chromatin, and two to o smaller than promyelocytes (15 to 18
four visible nucleoli um), and the nucleus has
o No visible granules when observed under light microcopy with considerably more heterochromatin,
Romanowsky stains nucleoli are difficult to see by light
microscopy

Stage:
METAMYELOCYTE
Size: 14 to 16 um
Comments:
- 3 to 20% of the nucleated cells in the
bone marrow
 Type II myeloblasts
o Shows the presence of dispersed primary (azurophilic) - Nucleoli are absent
granules or nonspecific granules in the cytoplasm - Synthesis of tertiary granules (also
o The number of granules does NOT exceed 20 per cell known as gelatinase granules) may
begin at this stage
- Also known as “juvenile cell”
- First stage of nuclear indentation
(curve in the nucleus of the cell)
- May sometimes be mistaken as the
band cell and the band cell is
sometimes mistaken as the
metamyelocyte
o How to differentiate?
 Type III myeloblasts  Metamyelocyte has shallow indentation and does not exceed ½ of
o Have a darker chromatin and a more purple cytoplasm nuclear width
o Contain more than 20 granules that do NOT obscure the nucleus  Metamyelocytes have kidney bean shaped or peanut shaped
o Rare in normal bone marrows nucleus
o They can be seen in certain types of acute myeloid leukemias  Band cells have deeper nuclear indentation and exceeds ½ of
nuclear width
Stage:  Band cells are described to have sausage shaped nucleus
PROMYELOCYTE
Size: 16 to 25 μm
Comments:
- 1 to 5% of the nucleated cells in the bone marrow
- Relatively larger than the myeloblast (this is an exception to the normal
phenomena where, as precursors mature, they become smaller)
- Nucleus round to oval, often eccentric
- “hof” is usually seen in normal promyelocytes but NOT in the malignant
promyelocytes of acute promyelocytic leukemia
o Hof: the hollow in the cytoplasm of a cell that lodges the nucleus
o Malignant promyelocytes do not demonstrate hof - Normally, metamyelocytes are only found inside the bone marrow, not in the
- Cytoplasm is evenly basophilic and full of primary circulation; if present in the circulation, it is a sign of an abnormality
(azurophilic/nonspecific) granules
- 1-3 nucleoli can be seen but may be obscured by the granules
Stage:
BAND CELLS
Size: 9 to 15 um
Comments:
- 9 to 32% of the nucleated cells in
the bone marrow
- Also known as stab cell or staff
cell
- Youngest granulocytic precursor to
normally appear in the peripheral
blood (no precursor cells younger
than the band cell should appear in
PROBABLE QUESTIONS the bone marrow)
- Secretory granules (also known as secretory vesicles) may begin to be
The synthesis of primary granules The synthesis of primary granules formed during this stage
begins in the: begins in the:
- Nucleus: elongated, curved, or sausage-shaped with rounded ends (filaments
a. Myeloblast a. Type II Myeloblast
NOT present)
b. Promyelocyte b. Promyelocyte
- CLSI recommends that bands should be included within the neutrophil counts
c. Myelocyte c. Myelocyte
and NOT reported as a separate category (due to the difficulty in reliably
d. Metamyelocyte d. Metamyelocyte distinguishing bands from segmented neutrophils)

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MATURE GRANULOCYTES Stage:

BASOPHIL
Stage: Size: 10 to 16 um
SEGMENTED NEUTROPHIL
Size: 9 to 15 um
Comments:
- 7 to 30% of the nucleated cells in the bone marrow

Basophilia Basopenia
- Increased (↑) level in the blood is - Decreased (↓) level in the
called: Basophilia blood is called: Basopenia
- Associated conditions: - Associated condition:
o Immediate hypersensitivity o Acute infections
Neutrophilia Neutropenia reactions o Stress
- Increased (↑) level in the blood is - Decreased (↓) level in the o Hypothyroidism o Hyperthyroidism
called: neutrophilia blood is called: o Ulcerative colitis o Increased levels of
- Some associated conditions: neutropenia o Estrogen therapy glucocorticoids
o Bacterial infections - Some associated o Chronic urticaria
(generally) conditions:
o Appendicitis o Overwhelming
o Rheumatoid arthritis infections AGRANULOCYTIC SERIES
o Acute destruction o Splenomegaly
represented by pancreatitis, o Hemodialysis LYMPHOCYTIC SERIES
colitis, myocardial infarction, o Copper deficiency
severe hemolysis, surgical or o Alcoholism - Lymphoblast
traumatic wounds, thermal o Babies born from - Prolymphocyte
injury hypertensive - Lymphocyte
o Parasites (malaria, liver mothers - Sizes:
flukes) o Chemical toxicity o 7 to 10 μm – Small lymphocyte
o Lithium (benzene)  Predominant type of lymphocyte
o Chemicals (lead, mercury) o Marrow replacement (normal adult blood)
o Drugs (digitalis, phenacetin) o Nutritional  Composed mostly of nucleus
o Corticosteroids deficiencies  Scanty cytoplasm
o Myelogenous leukemia o Cytotoxic drugs o 10 to 12 μm – Medium or Intermediate Lymphocyte
o Venoms (spiders, bees, - Agranulocytosis is
wasps) neutrophil count: <0.5 x o 11 to 25 μm –Large Lymphocyte
o Actinomyces fungi 109/L (extreme  Has abundant cytoplasm
o Response to therapy neutropenia)
o Physiologic neutrophilia o Associated drugs:
(“Pseudoneutrophilia”) –  amidopyrine
usually caused by a shift of  cephalosporin
marginated cells to the
circulatory pool (common
reasons for this are: exercise,
excessive temperature
Lymphocytosis Lymphopenia/
changes, nausea and
Lymphocytopenia
vomiting, pregnancy and
labor, rage, panic, and stress) Some associated conditions: Some associated
- Infectious mononucleosis conditions:
Infectious lymphocytosis - Aplastic anemia
Stage: -
EOSINOPHIL - Cytomegalovirus infection - AIDS
Size: 9 to 15 um - Acute viral hepatitis - SARS
- Bordetella pertussis infection - Ethanol abuse
- Brucellosis - Zinc deficiency
- Toxoplasmosis (Toxoplasma gondii)
- Acute HIV infection
- Viral infections are commonly accompanied by lymphocytosis but not
always

PLASMA CELLS
- 10 to 28 µm
Eosinophilia Eosinopenia
- Final maturation stage of B lymphocytes
- Increased (↑) level in the blood is called: - Decreased (↓) level - Nucleus: small, oval and eccentric
eosinophilia in the blood is called: o Eccentric: not in the middle
- Some associated conditions: eosinopenia o “tortoise shell”, “cartwheel” or “clock
o Asthma - Associated face” in appearance
o Hay fever condition: - Cytoplasm: ovoid, dark blue/ sea blue /
o Psoriasis o ACTH cornflower in color, nongranular
o Eczema administration o Basophilic cytoplasm
o Scarlet fever o Autoimmune o may contain round, discrete globules
o Eosinophilic leukemia disorders (called: Russell bodies) that contain
o Parasitic infections o Steroid immunoglobulins
- Trichinosis therapy
o Stress - Most common malignant disease of plasma
o Caused by Trichinella spiralis cells: Plasma Cell Myeloma (former name:
o May possibly produce the highest o Sepsis
o Acute Multiple Myeloma)
eosinophil count o Involves elderly patients; excrete
Moderate to severe eosinophilia: inflammatory
-
states Bence-Jones proteins in urine
o Most commonly associated with o Bence-Jones proteins precipitate
helminthic infections (parasitic upon heating but dissolve at higher
worms include nematodes, temperatures
trematodes, cestodes)

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MONOCYTIC SERIES NEUTROPHILS

- Monoblast NUCLEUS CYTOPLASM
- Promonocyte - contain 3 to 5 lobes - Pink to tan with violet or lilac granules
(separated by filaments)
- Monocyte
o Largest WBC in the peripheral blood - MOST COMMON WBC in the normal peripheral blood
o Size: 15 to 20 μm - Neutrophils are commonly the first phagocytes to reach the infected areas and
are then followed by the monocyte.
PROMONOCYTE MONOCYTE - Neutrophils move in a zigzag manner, but their movement changes into a
straight line path if a chemotactic attractant or factor (e.g., a bacterium coated
with particular components of complement) is within a particular distance
- Neutrophils share a common progenitor with monocytes: GMP (Granulocyte-
Monocyte Progenitor)
- Major stimulatory cytokine for neutrophil production: G-CSF (Granulocyte
colony-stimulating factor)
- Half-life of neutrophils in the blood is relatively short (approximately 7 hours)
- 3 major functions:
(1) Phagocytosis and destruction of foreign material and microorganisms
(2) Generation of NETS (Neutrophil extracellular traps)
o NETS - extracellular threadlike structures thought to represent chains
of nucleosomes from unfolded nuclear chromatin material (DNA)
o Have enzymes from neutrophil granules attached to them
Monocytosis Monocytopenia o Able to trap and kill gram-positive and gram-negative bacteria as well
Some associated conditions: Some associated conditions: as fungi
(3) Secretory function (neutrophils are a source of a variety of cytokines and
- Tuberculosis - Aplastic anemia also a source transcobalamin 1 [necessary for the proper absorption of
- Subacute Bacterial Endocarditis - Overwhelming infections in Vitamin B12])
- Syphilis immunocompromised NEUTROPHIL GRANULES
- Protozoal and rickettsial patients - Order of formation: Primary → Secondary → Tertiary → Secretory
infections (e.g., malaria, typhus) - Hemodialysis - Order of degranulation: Secretory → Tertiary → Secondary → Primary
- Brucellosis - Epstein-Barr virus infection - Size (largest to smallest):
- Typhoid - Steroid therapy - TYPES of NEUTROPHIL GRANULES:
- Gaucher disease
- Hodgkin’s disease 1.) PRIMARY (AZUROPHILIC) 3.) TERTIARY (GELATINASE
- Collagen vascular diseases GRANULES GRANULES)
(e.g., lupus erythematosus) o Formed during the o Formed during the
- Gastrointestinal disease promyelocyte stage metamyelocyte and band
o Last to be released stages
- Surgical trauma (exocytosis) o Second to be released
o Contain: o Contain:
MACROPHAGES  Myeloperoxidase (MPO)  Gelatinase
- Size: 40 to 50 μm  An antimicrobial  Collagenase
“Monocytes” found in tissues enzyme  Lysozyme
 Acid β-glycerophosphatase
-
 Acetyltransferase
Most abundant cell in the body (more abundant than skin cells and RBCs)
 Lysozyme  β2-microglobulin
-
- Some of the functions of macrophages:  A.k.a. muramidase;
o Phagocytosis an antimicrobial 4.)
SECRETORY GRANULES
o Synthesize nitric oxide (cytotoxic against viruses, bacteria, fungi, enzyme (SECRETORY VESICLES)
protozoa, helminths, and tumor cells)  Cathepsins o Formed during the band
o Release interleukin 1 (stimulates T lymphocytes)  Defensins and segmented neutrophil
o Produce transcobalamin II  Elastase stages
 The primary transport factor for vitamin B12  Proteinase-3 o First to be released (fuse
 New name of Transcobalamin II: Transcobalamin  Others to plasma membrane)
o Contain (attached to
2.) SECONDARY (SPECIFIC) membrane):
GRANULES  CD11b/CD18
o Formed during the myelocyte  Contribute to tight
and metamyelocyte stages stationary binding
o Third to be released between
o Contain: neutrophils and
 β2-microglobulin endothelial cells
 Collagenase  Vesicle-associated
 Gelatinase membrane-2
 Lactoferrin  CD10, CD13, CD14,
 An iron-binding CD16
protein that competes  Cytochrome b558
with bacteria for iron  Complement 1q
 Neutrophil gelatinase- receptor
associated lipocalin  Alkaline phosphatase
Location Specific name of macrophages  Transcobalamin I  The only WBC
Liver Kupffer cells  Lysozyme with ALP activity
Lungs Alveolar macrophage/Dust cells  Others is neutrophil
Kidneys Renal macrophage/Mesangial cells  Complement receptor-
Brain Microglial cells 1
Skin Langerhans cells NEUTROPHIL POOLS
Spleen Splenic macrophages/Littoral cells A. In the BONE MARROW: B. In the PERIPHERAL
Intestines Intestinal macrophages o Stem cell pool BLOOD:
Peritoneum Peritoneal macrophages  Hematopoietic stem cells (CD34) o Circulating
Synovial tissue Type A cells o Mitotic (Proliferation) pool Neutrophil Pool
 CMP/Common Myeloid Progenitors (CNP): found in
Reproductive organ Reproductive organ macrophages
(CFU-GEMMs: Colony-forming the plasma
Bone Osteoclast
units-granulocyte, erythrocyte, o Marginal
Placenta Hofbauer/Hoffbauer cells monocyte, and megakaryocyte) Neutrophil Pool
 GMP (MNP): found
REFERENCE RANGES OF DIFFERENT WBC TYPES  Myeloblast adhering on the
WBC Type Relative Count Absolute Count  Promyelocyte blood vessel wall
Neutrophils 51-67% 1,600-7,260/uL  Myelocyte o Ratio: 50:50
Lymphocytes 25-33% 960- 4,400/uL o Storage (Maturation) pool (CNP:MNP)
Monocytes 2-6% 180-880/uL  Metamyelocyte
Eosinophils 1-4% 45-440/uL  Band
Basophils 0-1% 45-110/uL  Segmented neutrophils

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EOSINOPHILS BASOPHILS
NUCLEUS CYTOPLASM NUCLEUS CYTOPLASM
- Dark purple, usually - Filled with large, spherical granules of - Difficult to observe because of - Densely stained, dark violet to
has two lobes uniform size that stain bright orange overlying granules purple-black granules (variable
- IL-5 - Generally unsegmented of in size and unevenly
o promotes: terminal maturation, functional activation, and prevention bilobed distributed)
of apoptosis of eosinophils Toluidine Blue
o the most specific cytokine for eosinophil lineage
- a dye that can bind with acid mucopolysaccharides in blood cells to form
- Tissue destinations of eosinophils under normal conditions appear to
metachromatic complexes ("metachromasia" - histochemically defined
be the underlying columnar epithelial surfaces in the respiratory,
as a reaction product color that is considerably different from the color
gastrointestinal, and genitourinary tracts.
of the dye itself)
- Eosinophilia is a hallmark of allergic disorders.
- Before the application of the toluidine blue stain, the films are fixed first
- Eosinophil production is increased in helminthic infections, and in vitro
studies have shown that this leukocyte is capable of destroying tissue- in Mota's fixative.
invading helminths by secretion of MBP and eosinophil cationic protein - Basophil and mast cell granules are strongly metachromatic (granules
as well as the production reactive oxygen species are reddish-violet using toluidine blue)
- Eosinophils regulate mast cell function through the release of MBP - Toluidine blue stain is valuable in identifying basophils and mast cells,
(causes mast cell degranulation and cytokine production), and they also especially neoplastic forms in which the number of granules may be
produce nerve growth factor that promotes mast cell survival and considerably reduced.
activation
- Survival time of eosinophils in human tissues ranges from 2 to 5 days. - Some of the functions of the basophils are:
EOSINOPHIL GRANULES o Have IgE receptors on their surface membrane that, when cross-
linked by antigen, result in granule release
PRIMARY GRANULES SMALL LYSOSOMAL
o Induce B cells to synthesize IgE
- Formed during the promyelocyte GRANULES
o Involved in the control of helminth infections (promote
stage - Acid phosphatase eosinophilia & contribute to efficient worm expulsion)
- Contain: - Arylsulfatase B
- Least common WBC in normal peripheral blood
o Charcot-Leyden crystal protein - Catalase
 Disintegration products of Cytochrome b558
eosinophils
- Mast cell
- Elastase NOT leukocytes
 Hexagonal, bipyramidal -
- Eosinophil cationic Have several phenotypic and functional similarities w/ both basophils
crystals -
protein
 Colorless, but in: and eosinophil
 Hematoxylin: black Connective tissue cell that has large basophilic granules containing
LIPID BODIES -
 Eosin: red heparin, serotonin, bradykinin, and histamine (these substances are
 Trichrome stain: - Cyclooxygenase
released from the mast cell in response to IgE stimulation)
purplish red - 5-Lipoxygenase
BASOPHIL GRANULES
- 15-Lipoxygenase
Some of the contents of basophil granules are:
SECONDARY GRANULES - Leukotriene C4
- Formed throughout remaining synthase - Histamine
maturation - Eosinophil peroxidase - Interleukin-4
- Contain: - Esterase - Interleukin-13
o Major basic protein (core) - Chondroitin sulfates (e.g., heparin)
 Damages and kills SECRETORY VESICLES
parasites - Carry proteins from MONOCYTES
o Eosinophil cationic protein secondary granules to NUCLEUS CYTOPLASM
(matrix) be released into the - Round, horse-shoe shaped or - Abundant, blue-gray,
o Eosinophil-derived neurotoxin extracellular medium
(matrix) lobulated, usually with some containing fine, indistinct
o Eosinophil peroxidase (matrix) degree of folding or granules called azure dust
o Lysozyme (matrix) convolutions (ground-glass appearance)
o Catalase (core and matrix) - Chromatin: "lace-like" or - Small pseudopods or blebs
o β-Glucuronidase (core and "stringy" may be observed
matrix) - Nuclear vacuoles may be - Cytoplasmic vacuoles may be
o Cathepsin D (core and matrix) present present
o Interleukins 2, 4, and 5 (core) - Largest cell of normal blood (generally about two to three times the
o Interleukin-6 (matrix)
diameter of an RBC)
o Granulocyte-macrophage
colony-stimulating factor (core) - Monocytes are best identified by their strong positive reaction with
o Others NONSPECIFIC ESTERASE stain (by histochemical means)
- "The nonspecific esterase enzymes alpha-naphthyl acetate and
- Three methods of eosinophil degranulation butyrate esterase are used clinically to recognize cells of monocytic
1.) Classical exocytosis origin. If the enzyme is of monocytic origin, it is inhibited by sodium
o granules move to the plasma membrane, fuse with the fluoride; however, no sodium fluoride inhibition of enzyme occurs if the
plasma membrane, and empty their contents into the enzyme is of granulocytic or lymphocytic origin."- from p. 262 of Clinical
extracellular space Hematology: Theory and Procedures (4th ed.) by: Mary Louise Turgeon
2.) Compound exocytosis - Monocytes are said to be rich in muramidase.
o granules fuse together within the eosinophil prior to fusing - Normally, promonocytes undergo 2 mitotic divisions in 60 hours to
with the plasma membrane produce a total of 4 monocytes. However, under conditions of increased
3.) Piecemeal degranulation demand for monocyte, the promonocytes undergo 4 divisions to yield a
o Secretory vesicles remove specific proteins from the total of 16 monocytes in 60 hours
secondary granules. - Monocytes in the peripheral blood can be found in a marginal pool and
o These vesicles then travel to the plasma membrane and fuse a circulating pool (marginal pool of monocytes is 3.5 times the
to empty the specific proteins into the extracellular space. circulating pool).

LYMPHOCYTES
NUCLEUS CYTOPLASM
- Generally, deep purple, round, - Generally: Sky-blue or "Robin
oval, or indented egg" blue
- Nucleoli may be visible
- Lymphocytes NOT ONLY come from the bone marrow but also from the
thymus and the lymphatic system.
- T cells, B cells, Natural Killer cells (T & B cells: adaptive immunity) (NK
cells: innate immunity)

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- Three physiologic characteristics of lymphocytes:

1. Lymphocytes are predestined to migrate
2. Lymphocytes are a heterogeneous group of cells
3. Lymphocytes are not an obligate end cell (a mature cell committed
to perform a function and then die; ex. neutrophils)

VARIANT LYMPHOCYTE
- Nucleus: May range from extremely dense to pale and immature-
looking
- Cytoplasm: Deeply basophilic to pale blue and usually abundant
- Seen in nonmalignant reactive disorders
- Synonyms: reactive lymphocytes, atypical lymphocytes, virocytes,
stress lymphocytes, Downey cells, transformed lymphocytes,
transitional lymphocytes, and glandular fever cells.
- Not a separate subtype of lymphocyte, they are lymphocytes currently
reacting to a particular stimulus
NOTE:
TYPE I TYPE II TYPE III
- A.k.a. - Seen in: Infectious - Nucleus: finely - A thick coverglass accompanies the counting chamber.
Plasmacytoid mononucleosis reticulated - Ordinary coverglasses should NOT be used because they have uneven
lymphocyte, Turk’s - A.k.a. Infectious nuclear surfaces.
irritation cell mononucleosis cell chromatin - When the thick coverglass is in place on the platform of the counting
- Seen in German - Characteristic pattern chamber, there is a space exactly 0.1 mm. thick between it and the ruled
measles appearance: “flared platform; thus, each square millimeter of the ruling forms the base of a
skirt” or “fried egg” space holding exactly 0.1 cu.mm.

PIPETS
RBC THOMA PIPET WBC THOMA PIPET
Markings 0.5, 1, 101 0.5, 1, 11
Color of the bead Red White/Colorless
Volume (bulb) 100 10
Image

WHITE BLOOD CELL COUNT

**Alternatively, one may also use a plain microhematocrit tube.

Background on Manual Hemocytometry:
DILUTING FLUIDS
- Counting Chambers (Hemacytometer/Hemocytometer) - used mainly to disperse blood cells to facilitate counting of cells
o According to TYPE: o According to RULINGS: CELLS DILUTING DILUTION OBJECTIVE AREA
COUNTED FLUID COUNTED
1.) Open type (Examples: 1.) Thoma WBCs 1% 1:20 10x 4 mm2
Spencer, Burker, Levy, 2.) Tuerk (leukocytes) ammonium 1:100 10x 9 mm2
Levy-Hausser) 3.) Neubauer oxalate, OR NRBCs in the sample are NOT lysed by
2.) Closed type (Ex. 4.) Improved Neubauer 3% acetic the WBC diluting fluid. Thus, NRBCs may
Thoma-Zeiss) 5.) Fuchs-Rosenthal acid, be counted with the WBCs and may cause
3.) Addis 6.) Bass-Jones OR 1% falsely ↑ WBC.
4.) Exton hydrochloric
5.) Petroff acid
Platelets 1% 1:100 40 x (phase) 1 mm2
o Most common hemacytometer: Levy chamber with improved
ammonium
Neubauer Ruling or Improved Neubauer Counting Chamber
oxalate
o Improved Neubauer Counting Chamber:
RBCs Isotonic 1:100 40 x 0.2 mm2
 This hemocytometer has two identical sides and both sides are
(erythrocytes) saline (5 small
counted.
squares of
 Chamber is 3 mm by 3 mm (divided into 9 square millimeters)
NOTE: central
 The 4 corner (large) squares - subdivided into 16 squares →
manual RBC square)
used for manual WBC count.
count = rarely
 The middle (central) square - subdivided into 25 squares → used
performed;
for manual RBC count
very
inaccurate

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WBC Count Procedure 2.) When a 1:20 dilution is used, the four large squares on one side of the
- The specimen may either be EDTA whole blood or blood from a skin chamber yield counts of 23, 26, 25, and 27. The four large squares on the
puncture. other side of the chamber yield counts of 28, 24, 29, and 27. What is the
- The diluting fluid lyses the non-nucleated RBCs to prevent their leukocyte count?
interference in the count.
One side: 23 + 26 + 25 + 27 = 101
- For the WBC count, the typical dilution of blood sample is 1:20.
Other side: 28 + 24 + 29 + 27 = 108
- If the count is below normal, a larger area may be counted (e.g., 9 mm2)
- For assessing the accuracy of the manual WBC count, perform a WBC Compute for the percentage difference:
estimate on a Wright stained PBS made from the same blood specimen.
- Clean the hemocytometer and coverslip with alcohol and dry carefully with ͳͲͺ െ ͳͲͳ
‫ ͲͲͳݔ‬ൌ ૟Ǥ ૠΨሺ൏ ͳͲΨǢ ܽܿܿ݁‫݈ܾ݁ܽݐ݌‬ሻ
a lint-free tissue. ͳͲͺ ൅ ͳͲͳ
ሺ ሻ
ʹ
1. Place the coverslip on the hemocytometer.
2. Make a 1:20 dilution (place 25 uL of well-mixed blood into 475 uL of WBC Compute for the WBC count:
diluting fluid) in a small test tube. ͳͲͶǤͷ‫Ͳʹݔ‬
3. Cover the tube and mix by inversion. ൌ ૞ǡ ૛૛૞Ȁ࢛ࡸ
4. Allow the dilution to sit for 10 minutes to ensure that, the RBC's have lysed: Ͷ‫Ͳݔ‬Ǥͳ
The solution will be clear once lysis has occurred. WBC counts should be
3.) When a 1:20 dilution is used, the four large squares on one side of the
performed within 3 hours of dilution.
chamber yield counts of 35, 14, 28, and 27. The four large squares on the
5. Mix again by inversion and fill a plain microhematocrit tube (blue ring).
other side of the chamber yield counts of 18, 24, 19, and 21. What is the
6. Charge both sides of the hemocytometer by holding the microhematocrit
leukocyte count?
tube at a 45-degree angle and touching the tip to the coverslip edge where
it meets the chamber floor.
One side 35-to14 + 28 + 27 = 104
7. After charging the hemocytometer, place it in a moist chamber for 10
Other side: 18 + 24 + 19 + 21 = 82
minutes before counting the cells to give them time to settle. Do NOT
disturb the coverslip.
Compute for the percentage difference:
(MOIST CHAMBER - may be prepared by placing a piece of damp filter
paper in the bottom of a Petri dish. An applicator stick broken in half can ͳͲͶ െ ͺʹ
serve as a support for the hemocytometer.) ‫ ͲͲͳݔ‬ൌ ૛૜Ǥ ૠΨሺ൐ ͳͲΨǢ ‫݈ܾ݁ܽݐ݌݁ܿܿܽ݊ݑ‬ሻ
ͳͲͶ ൅ ͺʹ
8. While keeping the hemocytometer in a horizontal position, place it on the ሺ ሻ
ʹ
microscope stage.
9. Lower the condenser on the microscope and focus by using the low-power CORRECTION OF WBC COUNT
(10x) objective lens (100x total magnification). The cells should be - Performed if there are >5 NRBCs (nucleated red blood cells) seen in 100
dispersed evenly in all of the squares. WBCs
10. For a 1:20 dilution, count all of the cells in the 4 corner squares, starting - Formula:
with the square in the upper left-hand comer.
o IMPORTANT:
 Cells touching the top and let lines = must be counted ("TLC")
 Cells touching the bottom and right lines = ignored ("BRI").
11. On the other side of the counting chamber, repeat the counting process. - Remember: result of the computation should be rounded off to the nearest
o IMPORTANT: hundreds.
 difference between the total cells counted on each side should
be < 10%. SAMPLE PROBLEM:
 greater difference could indicate uneven distribution (requires
that the procedure be repeated) - WBC count 15,000/mm3
 formula for percentage difference: - 12 NRBCs were seen (in 100 WBCs)
ܸଵ െ ܸଶ SOLUTION:
‫ ͲͲͳݔ‬ൌ ‫݁ܿ݊݁ݎ݂݂݁݅݀݁݃ܽݐ݊݁ܿݎ݁݌‬
ܸଵ ൅  ܸଶ
ሺ ሻ ͳͷǡͲͲͲ‫ͲͲͳݔ‬
ʹ ൌ ͳ͵ǡ͵ͻʹǤͺ͸Ȁ‫ݎ݋ܮݑ‬૚૜ǡ ૝૙૙Ȁ࢛ࡸ
ͳʹ ൅ ͳͲͲ
V1 = the larger number
V2 = the smaller number WBC DIFFERENTIAL COUNT
12. Average the number of leukocytes counted on the two sides. Using the A.) 100-cell differential
average, compute the WBC count using the following formula:
(Reminder. This is the general formula used for manual cell counts and can - Routinely performed
be used to compute any type of cell count)
B.) 200-cell differential
- Performed in instances when the WBC count is >40 x 109/L to increase
accuracy of results
- NOTE:
o 200-cell differential may also be performed in cases when:
 >10% eosinophils
 >2% basophils
 >11% monocytes
 More lymphocytes than neutrophils (except in children)
o Results are then divided by 2 (result reported in percentage).
SAMPLE PROBLEMS o Indicate in the report that 200 WBCs were counted.

1.) A manual leukocyte count was performed on an EDTA-anticoagulated

CASE:
specimen. The specimen was diluted 1:20, and a total of 150 leukocytes
were counted in the four corner squares of the hemocytometer. What is the - 29-year-old male patient
leukocyte count? - WBC count 10,000/uL
SOLUTION: - WBC differential count results (100-cell Differential):

Compute for the WBC count: N = 25

ͳͷͲ‫Ͳʹݔ‬ L = 68
ൌ ૠǡ ૞૙૙Ȁ࢛ࡸ What should the
Perform 200-cell
Ͷ‫Ͳݔ‬Ǥͳ M=2 differential on the
medtech do next?
E=4 same PBS
B=1

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- WBC differential count results (200-cell Differential): - Leukoerythroblastic Reaction

o Synonyms: Leukoerythroblastosis and Leukoerythroblastic anemia
N = 50 o Presence on a blood smear of:
L = 136  Immature neutrophils
What should the Divide each of  Nucleated RBCs
M=4
medtech do next? the values by 2
E=8  Teardrop RBCs
o Often, but NOT always, it is accompanied by neutrophilia
B=2 o A nonspecific reaction but provides important evidence of underlying
- The results are accurate disease or stress to the hematopoietic compartment
o This is strongly associated with PMF (Primary Myelofibrosis)
C.) 300 or 400-cell differential
- Striking and sustained leukoerythroblastic reactions are observed in
- Performed in instances when the WBC count is >100 x 109/L to increase
accuracy of results conditions involving the presence of a space-occupying lesion in the
marrow (myelophthisis) such as:
- If you will use the 300-differential count, divide the results by 3
o Metastatic tumor
- If you will use the 400-differential count, divide the results by 4 o Fibrosis
o Lymphoma
D.) 50-cell differential o Leukemia
- Performed in instances when the WBC count is <1.0 X 109/L
- Performed using a buffy coat smear - Mild and transitory leukoerythroblastic reactions may be observed in:
- Count 50 WBCs, then multiply the results by 2 to get a percentage o hemolytic anemia
o severe infections
REPORTED AS o cardiac failure
o uremia
- RELATIVE COUNT o megaloblastic anemia
o Gives the number of specific WBC type per 100 WBCs
o Not as informative (not as accurate) as the absolute count SOME CELLS THAT MAY BE FOUND DURING THE WBC
o Not completely useless; it gives an overview of/about the normality or DIFFERENTIAL COUNT
abnormality of the blood specimen
o Formula: NOTE: These cells are NOT counted as part of the 100 leukocytes but should be
mentioned in the comments when they are observed.
ܰ‫݁݌ݕݐܥܤܹ݂ܿ݅݅ܿ݁݌ݏ݂݋ݎܾ݁݉ݑ‬ CELLS DESCRIPTION ASSOCIATED CONDITIONS
‫ ͲͲͳݔ‬ൌ ܴ݈݁ܽ‫ݐ݊ݑ݋ܿ݁ݒ݅ݐ‬ሺΨሻ
ͳͲͲܹ‫݀݁ݐ݊ݑ݋ܿݏܥܤ‬ - nuclear remnants of - may be found normally in
lymphocytes few numbers
Smudge
cells

o Reference Ranges: - may be associated with

 Neutrophil = 51 – 67% - appearance similar to a chronic lymphocytic
 Lymphocyte = 25 – 33% thumbprint
leukemia (CLL)
 Monocyte = 2 – 6% - structureless chromatin
 Eosinophil = 1 – 4% - nuclear remnants of - may be found normally in
= 0 – 1%
Basket

 Basophil granulocytic cells few numbers

cells

- netlike chromatin - may be seen in some

- ABSOLUTE COUNT pattern leukemias
o Gives the number of specific WBC type per cubic millimeter of blood prolonged exposure to
- granulocytic cells with -
Necrotic

(mm3) EDTA
cells

pyknotic nuclei and an

o More informative (more accurate) than the relative count agranular cytoplasm - chemotherapy (rarely)
o Formula: R x W = A
o Reference Ranges:
 Neutrophil = 1,600 – 7,260/uL - neutrophils that have - overwhelming septicemia
Phagocytic

 Lymphocyte = 960 – 4,400/uL engulfed foreign bodies - bacterial and fungal

= 180 – 880/uL
cells

 Monocyte like bacteria and fungi infections

 Eosinophil = 45 – 440/uL - erythrophagocytosis (in
 Basophil = 45 – 110/uL which neutrophil engulfs
RBCs)
SAMPLE PROBLEM: - large cells (20 to 30 um) - considered a contaminant
- Patient's WBC Count = 10,000/mm3 which line the veins of venipuncture (found
WBC Differential count occasionally in blood
- - stretched and ovoid in obtained by venipuncture)
Relative count Absolute Count
Endothelial cells

appearance
o Neutrophil = 25% (0.25 x 10,000) = 2,500/uL - must not be confused with
- have a single nucleus clumps of malignant cells
o Lymphocyte = 68% (0.68 x 10,000) = 6,800/uL with dense chromatin
o Monocyte = 2% and no nucleoli
o Eosinophil = 4%
o Basophil = 1% - abundant cytoplasm
which appears
translucent
- Interpretation:
o Relative neutropenia: low neutrophil count in relation to 100 WBCs - usually found at feather
o Relative lymphocytosis: high lymphocyte count in relation to 100 edge
WBCs nude nuclei that stain found in newborns
Megakaryocyte

- -
dark purple may be found also in
fragments

o Absolute lymphocytosis -
aberrant platelet
RELATED TERMS production, myelofibrosis,
essential thrombocythemia
- Shift to the Left and Shift to the Right
o Shift to the Left - varies according to cell - found in newborns
Nucleated red blood

 increase in the number of young forms (usually neutrophils) maturity - may be found also in cases
cells (NRBCs)

 2 types: of hemolysis, leukemia,

myeloproliferative
DEGENERATIVE SHIFT TO THE REGENERATIVE SHIFT TO THE disorders, and others
LEFT LEFT
- Increase in the number of - Increase in the number of
young forms young forms
- Normal or ↓ WBC count - ↑ WBC count
*Reported to the physician but not included in the 100-cell differential count
- Ex. Tuberculosis - Ex. Appendicitis

o Shift to the Right

 Increase in the number of old forms (usually neutrophils)
 Ex. Pernicious anemia

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CASE STUDY

(NOTE: This case was based from "Quick Review Q and A's" by: VALERIE
DIETZ POLANSKY, M. Ed., MT(ASCP))

- A 40-year-old man was seen by his physician and he complained of

shortness of breath, coughing, and fever.
- Interpret the results of his hemogram:

RESULTS REFERENCE RANGES

WBC count 20.8 For both males & females:
4.5 to 11.5 x 109/L
RBC count 2.11 (↓) Males = 4.6 to 6.0 X 1012/L
Females = 4.0 to 5.4 X 1012/L
Hemoglobin 10.5 Males = 14-18 g/dL
Females = 12-15 g/dL
Hematocrit 24.5 Males: 40 to 54%
Females: 35 to 49%
RBC Indices
MCV 129.1 (↑) 80 to 100 fL
MCH 52.8 26 to 32 pg
MCHC 41.2 31 to 37 g/dL
RDW-CV 18.6 11.5 to 14.5%
PLT 232 150 to 450 x 109/L

- INTERPRETATION:
o Presence of cold agglutinins is suspected
 Due to low RBC count and high MCV
 Look for RBC clumping in the peripheral blood smear to confirm
(clumps of erythrocytes are counted as a single large RBC)

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WBC ANOMALIES
CONDITIONS/ ABNORMAL MORPHOLOGIC OR FUNCTIONAL DEFECT OTHER IMPORTANT REMARKS
WBCS or INCLUSIONS
Smudge Cells - nuclear remnants of lymphocytes - may be found normally in few numbers
- appearance similar to a thumbprint - may be associated with chronic lymphocytic leukemia
- structureless chromatin (CLL)

Basket Cells - nuclear remnants of granulocytic cells - may be found normally in few numbers
- netlike chromatin pattern - may be seen in some leukemias

Hand-mirror lymphocytes - a lymphocyte with a hand-mirror appearance - may be seen in certain types of ALL and AML
- may also be associated to infectious mononucleosis

Myeloperoxidase Deficiency - Low or absent myeloperoxidase enzyme - Commonly benign (other bactericidal systems prevent most
(Alius-Grignaschi Anomaly) - Normal cell morphology infections)
Chronic granulomatous - An inherited disorder characterized by defects in the - Frequent infections especially in children
disease (CGD) respiratory burst oxidase system (cells engulf but are - Tests:
unable to kill microorganisms) o Chemiluminescence
- Normal neutrophil morphology o Nitroblue Tetrazolium Test (NBT)
- Formation of granulomas can obstruct hollow organs
Niemann-Pick Disease - Deficiency of sphingomyelinase (an enzyme needed to - Rare autosomal recessive disease
(NPD) break down lipids) - More commonly seen in Ashkenazi Jews
- Foam cell (a.k.a. Pick cell): a macrophage whose - Signs of the disease begin in infancy
cytoplasm is swollen by many small lipid droplets - With poor physical development
- Spleen and liver are greatly enlarged
- Disease is often fatal by three years of age

Gaucher Disease - Defect or deficiency in the catabolic enzyme: - Most common of the lipidoses
β-glucocerebrosidase - Tests:
- Gaucher Cell o Chitotriosidase
o found in the bone marrow o Periodic acid-schiff stain
o large macrophage with small, eccentric nucleus
o cytoplasm is distended by Glucocerebrosides
o “crumpled tissue paper” or onion skin

Hypersegmented - Nucleus has > 6 lobes - Megaloblastic anemias, myelodysplastic syndromes,

Neutrophils hereditary neutrophil hypersegmentation, myelokathexis
- NOTE: Myelokathexis pertains to a rare hereditary condition
characterized by normal granulocyte production;
nevertheless, there is impaired release into the blood (leads
to neutropenia). Here, neutrophils appear hypermature.
There may be hypersegmentation, hypercondensed
chromatin, and pyknotic changes in this condition.
Pelger-Huet Anomaly - Failure of the neutrophil nucleus to segment - Most common genetic disorder of WBCs
(a.k.a. True/Congenital PHA) - Pelger-Huet Cell - Autosomal dominant disorder
o “pince nez" or "spectacle" form of neutrophil nucleus - Decreased nuclear segmentation (bilobed, unilobed), coarse
o Hyposegmented neutrophil (with 1 to 2 nuclear lobes) chromatin clumping pattern potentially affecting all
o Unilobed = round, ovoid, or peanut shaped leukocytes, although morphologic changes are MOST
o Bilobed = spectacle-like ("pince nez") morphology obvious in mature neutrophils.
with nuclei attached by a thin filament - A result of a mutation in the lamin β-receptor gene
- Lamin β-receptor = an inner nuclear membrane protein;
Acquired PHA plays a major role in leukocyte nuclear shape changes that
aka: Pseudo-PHA occur during normal maturation
- Psuedo-Pelger Huet Cell - Homozygous PHA: all neutrophils are affected and
o neutrophils that resemble Pelger-Huet cells demonstrate round nuclei
(hyposegmented and hypogranular)
- Heterozygous PHA: 55% to 93% of the neutrophil population
o may be seen in acute myeloid leukemia, chronic
are affected (there is generally a mixture of all of the
myeloproliferative neoplasms, and myelodysplastic
aforementioned nuclear shapes)
syndromes (MDS), HIV infection, tuberculosis,
Mycoplasma pneumoniae, and severe bacterial - Neutrophils show normal granulation in true PHA.
infections - Neutrophils in PHA appear to function normally.

Lupus Erythematosus (LE) - Usually a neutrophil that has ingested the antibody- - Usually an in vitro phenomenon
Cell coated nucleus of another neutrophil or has engulfed the - Found in systemic lupus erythematosus (SLE) but may
homogenous, globular nuclear mass of a destroyed cell also be found in comparable connective tissue disorders

Rieder Cell - Similar to normal lymphocytes BUT the nucleus is - Found in chronic lymphocytic leukemia or can then be
notched, lobulated, and cloverleaf-like artificially formed through blood film preparation

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Flame Cell - An abnormal plasma cell with intensely eosinophilic or - Found in IgA Myeloma
A.k.a. Thesaurocyte "flamingo cytoplasm”

Grape Cell - An abnormal plasma cell with a cytoplasm that is - Found in Multiple Myeloma
A.k.a. Morula cell, Mott cell completely filled with Russell Bodies (round, discrete
globules containing antibodies)

Alder-Reilly Anomaly - Characterized by dense azurophilic granulation in all - Granulation results from an abnormal deposition and storage
types of leukocytes of mucopolysaccharides
- Found in mucopolysaccharidoses (ex. Sanfilippo
syndrome, Hurler syndrome, Hunter syndrome)

Chediak-Higashi Syndrome - Basic defect: golgi complex (responsible for granule - Partial albinism is observed due to abnormal packaging of
assembly) melanosomes (patient has silvery hair, pale skin and suffers
- Characterized by the presence of large/giant, abnormal from photophobia)
cytoplasmic granules in phagocytes (granulocytes and
monocytes), and occasionally in lymphocytes
- Abnormal granules in phagocytes are: Peroxidase (+)
- Abnormal granules in lymphocytes are: Peroxidase (-)
Hairy Cells - Small lymphocytes with little cytoplasmic projections - Found in Hairy Cell Leukemia
- TRAP (+) – Tartrate Resistant Acid Phosphatase; a - Nearly all blood cells contain 7 non-erythroid isoenzymes of
cytochemical stain acid phosphatase (0, 1, 2, 3, 3b, 4, and 5)
- Isoenzyme 5 (tartrate resistant & is produced in abundance
by Hairy Cells)

Tart Cells - A monocyte that has ingested a whole lymphocyte or - May be seen in drug sensitivity
a nucleus (with an identifiable nuclear chromatin)
Toxic Granulations - Altered primary granules (present because of rapid cell - Described as dark-blue to black granules found in the
maturation) cytoplasm of neutrophils
- Look similar with Alder-Reilly granules - Seen in severe infections and chemical poisoning (ex.
- Exclusive for neutrophils lead poisoning)

Auer Rods - Linear projections of primary granules - Seen in certain types of acute myelogenous leukemia
- Faggot cell: an abnormal WBC with bundles of Auer rods (AML)
in its cytoplasm

Reed-Sternberg Cell - Has a characteristic “owl’s eyes” appearance - Presence of these cells is the definitive histologic
- A large lymphoid cell which may demonstrate two nuclei characteristic of Hodgkin's Disease
(with eosinophilic nucleoli) and an abundant cytoplasm

Lazy Leukocyte Syndrome - Poor neutrophil response to chemotactic agents - Also characterized by neutropenia, recurrent infections
Job Syndrome - Neutrophils have poor directional motility - Also characterized by recurrent severe bacterial infections,
A.k.a. skeletal abnormalities, and elevated levels of IgE
Hyperimmunoglobulinemia E
syndrome
Jordan's Anomaly - Characterized by the presence of fat-containing vacuoles - May be seen in muscular dystrophy and ichthyosis
in granulocytes and monocytes
Döhle Bodies - Round or oval blue-staining cytoplasmic inclusions found - Found in: pregnancy, severe burns, aplastic anemia, scarlet
in neutrophils (arranged in parallel rows and consisting of fever, and other infectious diseases, and following
ribosomal RNA) administration of toxic agents
- Döhle bodies are typically found in band and segmented
neutrophils and often in cells containing toxic granulation

May-Hegglin Anomaly - Characterized by the presence of gray-blue spindle- - Also characterized by leukopenia, variable
shaped inclusions in the cytoplasm of granulocytes and thrombocytopenia, giant platelets, and large Döhle body-like
monocytes inclusions in neutrophils, eosinophils, basophils, and
monocytes

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DÖHLE BODIES VS. MHA INCLUSIONS - REMINDERS

SIZE SHAPE PAS REACTION CONTENT o Depending on the method, peroxidase positive granules may produce
(PERIODIC ACID-SCHIFF) a red-brown, dark brown or black color.
Döhle Smaller Round Positive Ribosomal o In one method, the red blood cells develop diffusely brown color
bodies RNA because of pseudoperoxidase activity in hemoglobin
MHA Larger Spindle- Negative Messenger o 3,3-diaminobenzidine (DAB) tetrahydrochloride, which is the
inclusions shaped RNA substrate being used in one of the methods, is a potential carcinogen.
The following precautions should be taken when handling this reagent
GENERAL OVERVIEW OF LEUKEMIA & LYMPHOMA or its solutions:
 Wear protective clothing (including gloves, lab coat, and mask)
LEUKEMIAS LYMPHOMAS when weighing out powders.
- Overproduction of various - Solid malignant tumors of the lymph  For all pipetting, use mechanical aids.
types of immature or nodes and related WBCs in the  Clean up spills instantly.
mature cells in the bone bone tissue  Wash hands properly after completion
marrow and/or peripheral - The distinctive cell type is the  Weigh benzidine in hood.
blood (in most types of lymphocyte - Peroxidase enzyme is sensitive to lights.
leukemia) - Malignant cells are initially confined o Smears should be stained immediately or kept in the dark.
- Frequently involves to the organs containing o Smears that are older than 2 weeks or that have been exposed to
WBCs of the mononuclear phagocytic cells such excessive light should NOT be reported as peroxidase negative.
myelogenous or as lymph nodes, spleen, liver, and - Cyanide-resistant Peroxidase stain
lymphocytic cell types bone marrow. Lymphomas can spill o May be used to detect eosinophilic leukemia
- Malignant cells easily over into the circulating blood and o Eosinophilic Leukemia is poorly understood.
trespass the blood-brain demonstrate a leukemic-appearing o The peroxidase of eosinophils is different from that in other
barrier picture on a peripheral blood film. granulocytes because of the activity of the enzyme in the presence of
sodium cyanide.

LEUKEMIA
- Malignant neoplasm of the blood-forming tissues of the bone marrow, SUDAN BLACK B (SBB)
spleen, and lymph system - Reactions parallel those of the MPO's except in that peroxidase enzyme is
- Generalizations (true most of the time but not all the time): only found in primary granules
o More blasts: shorter, more fatal course of disease o MPO (-), SBB (+) = using old smears (>2 weeks) or smear has been
o ↑ WBC count with shift to the left: increased numbers of young forms exposed to excessive light
of WBCs - Stains sterols, neutral fats, phospholipids (found in the primary and
o M:E (myeloid to erythroid) ratio of 10:1
secondary granules of neutrophils and lysosomal granules of monocytes)
 Normal: 2:1 to 4:1
o Type of anemia usually present in cases of acute leukemia: - Sudan Black B stain - most sensitive stain for granulocytic precursors.
normocytic, normochromic anemia
- POSITIVE:
ACUTE LEUKEMIAS CHRONIC LEUKEMIAS o Promyelocyte, myelocyte
- Described by symptoms of - Described by symptoms of long o Metamyelocytes, bands, and segmented neutrophils: STRONGLY
short duration duration POSITIVE
- Numerous immature cell - Mostly mature cell forms in the o Leukemic blasts
forms in the bone marrow bone marrow and/or peripheral blood o Auer rods
and/or peripheral blood - Total WBC counts range from o Eosinophils
- Increased total WBC extremely elevated to lower than
count normal
- WEAKLY POSITIVE or NEGATIVE:
FAB CLASSIFICATION OF LEUKEMIAS o Myeloblasts
o Monocytic cells
- FAB: French-American-British Classification
- An old way of classifying leukemia - NEGATIVE:
o New: WHO Classification o Lymphocytes and its precursors
- Based on morphology of cells in Romanowsky-stained smear o Megakaryocytes and platelets
o Romanowsky-stained smear o Erythrocytes
 Group of stains that contain methylene blue and a halogenated
fluorescein dye (e.g. eosin Y and eosin B)
REMINDERS
Examples: Wright’s. Giemsa, May-Grunwald stain

-
o Brownish-black cytoplasmic granules are seen in myelocytic
- Based on cytologic and histochemical characteristics of cells involved
precursors.
- Cytochemical stains: MPO & SBB o Monocytes demonstrate few small brownish-black granules.
MYELOPEROXIDASE (MPO) o Eosinophilic granules are brown and commonly demonstrate central
pallor (granules are positive at their periphery with negative centers)
- Enzyme found in primary granules of:
o Neutrophils and Eosinophils
o Lymphoid cells rarely stain.
o Monocytes (to a certain extent) o Sudan Black B: stain may be performed on a specimen that is several
months old.
- Used in differentiating blasts of AML from those of ALL (+)
o Reagents in SBB staining are NOT considered to be carcinogenic.
- (+) Peroxidase stain rules out ALL
o Disadvantages of the SBB reaction:
POSITIVE:
 Time necessary to perform the stain (1 to 2 hours)
 False-positive reactions are possible in disorders characterized
-
o Neutrophilic granulocytes (except normal blasts)
o Auer rods by cytoplasmic lipid vacuoles (such as Burkitt's lymphoma and
o Leukemic blasts in FAB M1, M2, and M3 occasionally, acute lymphocytic leukemia)
o Eosinophils  Increased background staining on bone marrow specimens
(attributable to the fatty nature of the bone marrow itself)
- WEAKLY POSITIVE or NEGATIVE:
o Monocytes

- NEGATIVE:
o Myeloblasts
o Basophils
o Lymphocytic cell series
o Erythrocytic cell series

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LYMPHOCYTIC LEUKEMIAS NON-LYMPHOCYTIC LEUKEMIAS/MYELOGENOUS LEUKEMIAS

- Generally: - Generally:
o Myeloperoxidase: negative (-) o Myeloperoxidase: Positive (+)
o Sudan Black B: negative (-) o Sudan Black B: Positive (+)

ACUTE LYMPHOCYTIC LEUKEMIA ACUTE MYELOGENOUS LEUKEMIA (AML)

- Most common form of childhood leukemia
Classification by French, American, British Cooperative Group (FAB):

Subgroup
-
Origin Remarks
ALL L1 ALL L2 ALL L3
Patients 70% of adult 70% of adult Rare in children
ALL ALL and adults M0 -AML, with minimal differentiation
Also known as
Burkitt-type of -MPO: Neg (-)
leukemia -SBB: Neg (-)
Immunologic CALLA (CD10) TdT sIg M1 -AML, without maturation
markers TdT CD19 -May demonstrate Auer rods (linear
CD19 CD20 projections of primary granules)
CD20 CD22 M2 - AML, with maturation
CD24
Cell size Homogenous Heterogeneous Homogeneous - Most common subtype of AML
population of population of population of large - May demonstrate Auer rods
small blasts large blasts blasts (with nuclear M3 Myelocytic - Acute Promyelocytic Leukemia (APL)
and cytoplasmic - DIC (Disseminated Intravascular
vacuoles) Coagulation)
Nucleus Uniformly Irregular Round to oval o A.k.a Defibrination syndrome,
round; Small Consumption coagulopathy
Nucleolus Single Single to Two to five o A consequence of disease
several
- Faggot cells
Chromatin Slightly Fine Coarse with clear
o Abnormal WBC with bundles of Auer rods
reticulated parachromatin
with o Faggot: means bundles of sticks
perinucleolar - M3V: “APL, microgranular variant”
clumping o Cells have the characteristic “butterfly",
Cytoplasm Scant, blue Moderate, pale Moderate, blue, “bowtie”, or “apple core” nuclei
prominently M4 Myelocytic - Acute Myelomonocytic Leukemia (AMML)
vacuolated Monocytic - Naegeli Monocytic Leukemia
CYTOCHEMISTRY - 2nd most common subtype of AML
ALL L1 ALL L2 ALL L3
- May demonstrate Auer rods
Periodic acid Schiff (PAS) + + -
Methyl Green Pyronine (MGP) - - + - M4E
Oil Red O (ORO) + + + o AMML, with increased marrow
eosinophils
- L1 & L2 are sometimes positive in ORO while L3 is always positive
M5 Monocytic - Acute Monocytic Leukemia
By use of immunologic markers: - A.k.a Schilling leukemia
- M5a: AMoL, poorly differentiated
E rosettes Surface Ig Serum anti-ALL o Seen in children
T-ALL + - - o >80% monoblasts in BM
B-ALL - + - - M5b: AMoL, well differentiated
Common ALL - - + o Seen in middle aged adults
Null ALL - - - o <80% monoblasts in BM
M6 Erythrocytic - Acute Erythroleukemia
CHRONIC LYMPHOCYTIC LEUKEMIA Myelocytic - A.k.a DiGuglielmo’s Syndrome
- May demonstrate Auer rods
- Most common type of leukemia in the elderly
- Type of anemia: macrocytic, normochromic
- Persistent lymphocytosis
- Erythroid cells in AML M6: PAS = Strongly (+)
- Presence of increased number of Smudge cells and Rieder cells in the o Normal erythroid cells should be PAS (-)
peripheral blood smear M7 Megakaryocytic - Acute Megakaryocytic Leukemia
o Smudge cells
- Requires immunocytochemical staining for
 nuclear remnants of lymphocytes
accurate diagnosis (Factor VII stain: +)
 thumbprint appearance
o Rieder cells - AML subtypes that demonstrate Auer rods: M1, M2, M3, M4, M6
 lymphocytes with notched, lobulated, or cloverleaf-like nucleus
AS-D

acetate
α-naphthyl butyrate

- Examples of clinical variations of CLL:

Chloroacetate (SE)

o Hairy-cell leukemia
Factor VIII stain

 Lymphocytes with little cytoplasmic projections

esterase (NSE)

esterase (NSE)

 Cytochemical stain: Tartrate Resistant Acid Phosphatase - Conditions

α-naphthyl

TRAP (+)
Naphthol

o Lymphosarcoma cell leukemia

MPO

SSB

o Prolymphocytic leukemia

THE SOLID TUMOR COUNTERPARTS OF ALL, CLL, and OTHER M1, M2, M3 + + + - - -
TYPES OF LEUKEMIA M4 (AMML) + + + + + -
LEUKEMIA TYPE SOLID TUMOR COUNTERPART M5 (AMoL) - +/- - + + -
Acute Lymphoblastic Leukemia Lymphoma, poorly differentiated; M6 (Erythroleukemia) +/- +/- +/- - - -
(ALL) lymphocytic M7 (Megakaryocytic - - - - Localized +
Chronic Lymphocytic Leukemia Lymphoma, well-differentiated; leukemia) positivity
(CLL) lymphocytic - SE: Specific Esterase
Monocytic Leukemia Reticulum cell sarcoma - NSE: Non-Specific Esterase
Acute Myelogenous Granulocytic Chloroma
- Myelocytic origin: Positive in MPO, SBB, SE
Leukemia
- Monocytic origin: Positive in NSE (Butyrate), NSE (Acetate)
Plasma cell leukemia Myeloma
Stem cell leukemia Lymphoma, undifferentiated

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WHO (World Health Organization) Classification of AML (updated: 2016) CHRONIC MYELOGENOUS LEUKEMIA (CML)

1.) AML with certain genetic abnormalities - Also known as: Chronic Granulocytic Leukemia
o AML with a translocation between chromosomes 8 and 21 [t(8;21)] - Characterized by the presence of Philadelphia Chromosome (Ph1)
o AML with a translocation or inversion in chromosome 16 [t(16;16) or o First described in 1960 by Peter C. Nowell, a faculty member of the
inv(16)] University of Pennsylvania, Philadelphia, as an unfamiliar small
o APL with the PML-RARA fusion gene chromosome present in the WBCs of patients with CML
o AML with a translocation between chromosomes 9 and 11 [t(9;11)] o Due to reciprocal translocation involving the long arms of
o AML with a translocation between chromosomes 6 and 9 [t(6:9)] chromosome 9 and 22 (results in the formation of the BCR-ABL1
o AML with a translocation or inversion in chromosome 3 [t(3;3) or fusion gene)
inv(3)] o 90% of patients with CML have Ph1; not all patients have Ph1
o AML (megakaryoblastic) with a translocation between chromosomes chromosome
1 and 22 [t(1:22) o Presence of Ph1 = good prognosis
o AML with the BCR-ABL1 (BCR-ABL) fusion gene
o AML with mutated NPM1 gene
o AML with biallelic mutations of the CEBPA gene (that is, mutations in
both copies of the gene)
o AML with mutated RUNX1 gene*
*Still a "provisional entity" meaning it's NOT yet clear if there is sufficient
evidence that it is a unique group.
2.) AML with myelodysplasia-related changes
3.) AML related to previous chemotherapy or radiation
4.) AML, NOS (not otherwise specified)
o includes cases of AML that DO NOT fall into one of the above
groups; similar to the FAB classification
o AML with minimal differentiation (FAB M0)
o AML without maturation (FAB M1)
o AML with maturation (FAB M2)
o Acute myelomonocytic leukemia (FAB M4) - Three clinical phases:
o Acute monoblastic/monocytic leukemia (FAB M5) o Chronic phase
o Pure erythroid leukemia (FAB M6) o Accelerated phase
o Acute megakaryoblastic leukemia (FAB M7) o Blast crisis
o Acute basophilic leukemia - Must be differentiated from LEUKEMOID REACTION (LR)
o Acute panmyelosis with fibrosis o Not a disease, but a description only
5.) Myeloid sarcoma (aka: Granulocytic sarcoma or Chloroma) o Excessive leukocytic response in the peripheral blood
6.) Myeloid proliferations related to Down syndrome o Confused with CML
7.) Undifferentiated and biphenotypic acute leukemias o WBC count: greater than 50 X 109/L (with neutrophilia and a marked
o Are NOT strictly AML left shift [presence of immature neutrophilic forms])
o Leukemias that have both lymphocytic and myeloid features. o LR most frequently refers to neutrophils, but the increased count may
o They are sometimes called MPALs (mixed phenotype acute be due to an increase in other types of WBCs
leukemias) - LEUKOCYTE (NEUTROPHIL) ALKALINE PHOSPHATASE (LAP/NAP)
TEST
CASE STUDY o Generally used to distinguish LR from CML
- A 45-year-old man was admitted to the hospital with complaints of fatigue,  Cannot be differentiated using Wright’s stain
malaise, and severe pain on his left side. Based on the results of the o Principle: ↑ LAP activity can be observed in neutrophils that have
hemogram, what is the most likely diagnosis? undergone normal growth.
 Neutrophils are the only type of WBCs that has LAP activity
RESULTS REFERENCE RANGES o KAPLOW’S METHOD
WBC count 247.5 (↑) For both males & females:  Principle: Hydrolysis of sodium alpha naphthyl phosphate by
4.5 to 11.5 x 109/L alkaline phosphatase produces a colored precipitate with a
RBC count 1.40 (↓) Males = 4.6 to 6.0 X 1012/L diazotised amine.
Females = 4.0 to 5.4 X 1012/L  Reagents:
Hemoglobin 4.9 (↓) Males = 14-18 g/dL
Females = 12-15 g/dL Fixative Methanol and Formalin
Hematocrit 13.5 (↓) Males: 40 to 54% Buffer Propanediole
Females: 35 to 49% Substrate Sodium alpha naphthyl phosphate
RBC INDICES Initial stain Brentamine-fast gamet salt
MCV 96.4 80 to 100 fL
Counterstain Aqueous Mayer’s Hematoxylin
MCH 35.0 26 to 32 pg
MCHC 36.3 31 to 37 g/dL
RDW-CV 19.3 11.5 to 14.5%
PLT 22 150 to 450 x 109/L PROCEDURE
WBC DIFF COUNT 1. Immerse dry blood smear in fixative for 30 seconds.
NEUTRO 2 51 to 67% 2. Pour onto smear the working substrate (made up of buffer, substrate and
BANDS 1 0 to 6% initial stain) and allow to stand for at least 10 minutes.
LYMPH 6 25 to 33% 3. Rinse with distilled water and dry.
MONO 0 2 to 6% 4. Counterstain for 10 to 15 minutes.
EO 0 1 to 4% 5. Rinse with distilled water and mount in mounting solution like glycerol.
BASO 0 0 to 1% 6. Examine immediately under the microscope and look for the presence of
BLASTS 86 (↑) reddish-brown to black precipitate of alkaline phosphatase activity in the
PROMYELOCYTES 5 cytoplasm of neutrophils.
RBC - Moderate anisocytosis and poikilocytosis 7. Count 100 segmented neutrophils and bands on a stained smear (use the
MORPHOLOGY particular kind of stain needed for this procedure) and score each of these
- 4 nucleated RBCs
cell as:
OTHER FINDINGS - Auer rods
SCORE DESCRIPTION
0 no reddish brown to black precipitate
ANSWER: ACUTE MYELOGENOUS LEUKEMIA 1+ slightly diffused reddish brown to black precipitate
2+ moderately diffused reddish brown to black precipitate
- Very high WBC count
3+ heavily diffused reddish brown to black precipitate
- Normocytic, normochromic anemia 4+ very heavily diffused reddish brown to black precipitate
- Presence of immature cells - After which total the individual scores of the 100 neutrophils.
- Presence of Auer rods - Normal Kaplow’s Score = 20-100
- GENERALLY:
o ↑ LAP score – LR
o ↓ LAP score – CML

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Disorders with increased (↑) Disorders with decreased (↓) FLOW CYTOMETRY
Kaplow’s (LAP) score Kaplow’s (LAP) score
- Characterized by Leukemoid - CML - Most common clinical application of flow cytometry diagnosis of leukemias
reaction - Paroxysmal nocturnal and lymphomas
o Third trimester of hemoglobinuria - Originally designed to evaluate physical properties of cells based on their
pregnancy - Sideroblastic anemia ability to deflect light
o Polycythemia vera Myelodysplastic syndrome
o Infections
- - Most significant discovery that led to the improvement of low cytometry and
o Intoxication its subsequent widespread application in clinical practice: development of
monoclonal antibodies.
- Although the term low cytometry suggests the measurement of a cell, this
SAMPLE PROBLEM procedure is successfully applied to study other particles, including
chromosomes, microorganisms, and proteins.
Score No. of neutrophils LAP score - Flow cytometry measures physical antigenic and functional properties of
0 32 0 particles suspended in a fluid.
1+ 24 24 - A flow cytometer is composed of fluidics, a light source (laser), multiple
2+ 21 42 detectors, and a computer.
3+ 15 45
- Main advantage of flow cytometry over other techniques: its ability to
4+ 8 32
quickly and simultaneously analyze multiple parameters in a large number
Total 143 = LR
of cells
Other factors that differentiate CML from LR:
- Specimens most commonly analyzed: bone marrow, peripheral blood,
CML Leukemoid lymphoid tissues, body cavity fluids and solid tissues.
reaction - For peripheral blood and bone marrow specimens
Leukocyte in the peripheral Blasts/promyelocytes Usually o Must be processed within 24 to 48 hours from time of collection
blood myelocytes o Collected into a tube or container with an anticoagulant (preferably
Toxic granulation Absent Present heparin) and are transported to a flow cytometry laboratory at room
Eosinophils/Basophils Increased (↑) Decreased (↓) temperature
LAP Decreased (↓) Increased (↑) - Flow cytometry can describe antigen expression on many viable cells.
Philadelphia chromosome Usually present Absent Currently, 17 antigens can be detected at the same time on an individual
(Ph1) cell. This is accomplished by the conjugation of monoclonal antibodies to a
Splenomegaly Usually prominent Mild (if present) variety of fluorochromes that can be detected directly by a flow cytometer.
Platelet count >600 or <50 x 109/L Normal - Cells must pass separately (one by one) through the illumination and
detection system of a low cytometer in order for them to be analyzed
individually. This is accomplished by injecting a cell suspension into a
LYMPHOMA stream of sheath fluid. This method, called hydrodynamic focusing, creates
a central core of individually aligned cells surrounded by a sheath fluid.
As particles are illuminated, they emit fluorescent signals registered by
Hodgkin’s Disease/Hodgkin’s Lymphoma
-
A. detectors. These results are later converted to digital output and analyzed
using low cytometry software.
- In addition to fluorescence, scatter signals are recorded. The detector
CLASSIFICATIONS OF HODGKIN'S LYMPHOMA
located directly in line with the illuminating laser beam measures forward
Rye Classification WHO (World Health Organization)
scatter (FS or FSC), which is proportional to particle volume or size. A
(1966) Classification
photo detector positioned to the side measures side scatter (SS or SSC),
Nodular Sclerosis Nodular lymphocyte predominant Hodgkin
which reflects surface complexity and internal structures such as granules
Lymphoma (5% of Hodgkin lymphomas)
and vacuoles.
- Characterized by: Popcorn cells (a.k.a. L
- FS, SS, and fluorescence are displayed at the same time on the instrument
and H cells; Lymphocytic & Histiocytic)
screen and registered by the computer system.
Lymphocyte Classical Hodgkin Lymphoma (95% of
predominant Hodgkin lymphomas) LINEAGE-ASSOCIATED MARKERS COMMONLY ANALYZED IN ROUTINE
Mixed cellularity FLOW CYTOMETRY
Lymphocyte Subtypes: Lineage Markers Lineage Markers
depleted - Nodular sclerosis (70% of cases) CD34 CD19
- Mixed cellularity (20% of cases) CD117 CD20
B-lymphocytes

Immature
- Lymphocyte rich (5% of cases) Terminal deoxynucleotidyl CD22
- Lymphocyte depleted (5% of cases) transferase κ Light chain
CD33 λ Light chain
Granulocytic/ CD13
SUBTYPE OF CLASSICAL NEOPLASTIC CELLS Monocytic CD15
HODGKIN LYMPHOMA CD14
Nodular Sclerosis RS cells; Hodgkin cells, lacunar cells CD71 CD2
Erythroid
T-lymphocytes

Mixed Cellularity RS cells; Hodgkin cells Glycophorin A CD3

Lymphocyte-Rich RS cells; Hodgkin cells CD41 CD4
Lymphocyte-Depleted RS cells; Hodgkin cells CD42 CD5
*RS: Reed-Sternberg Cell Megakaryocytic CD61 CD7
CD8
B. Non-Hodgkin's Lymphoma
- involve mostly the B cells
- some of the subtypes include:
o Burkitt's Lymphoma
 Associated with EBV
o CTCL (Cutaneous T Cell Lymphoma)
 Most common type of cutaneous T-cell lymphoma (CTCL)
 Characteristic cell: Sezary cell

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CYTOCHEMISTRY AUTOMATION IN HEMATOLOGY

- Study of chemical constituents of cells
- Useful for acute leukemias BASIC COMPONENTS OF MOST HEMA ANALYZERS
- Specimens: bone marrow, lymph nodes, peripheral blood
- For enzymatic techniques: fresh smears - Hydraulics - (aspirating unit, dispensers, dilutors, mixing chambers,
- For non-enzymatic techniques (like PAS/SBB): may remain stable for aperture baths and/or flow cells, and hemoglobinometer)
months if stored at room temperature - Pneumatics - (vacuums and pressures for operating valves and moving the
sample through the system)
- Enzyme found in primary granules of. - Electrical systems (electronic analyzers and computing circuitry for
o Neutrophils and Eosinophils processing data)
o Mono (to a certain extent)
Used in differentiating blasts of AML from those of ALL
MYELOPEROXIDASE (MPO)

- ELECTRICAL IMPEDANCE
- (+) peroxidase stain rules out ALL
- POSITIVE: - a cell counting principle
o Neutrophilic granulocytes (except normal blasts) **
o Auer rods** - a.k.a: Electronic Resistance or low-voltage direct current (DC) resistance
o Leukemic blasts in FAB M1, M2, and M3 or "Coulter Principle"
o Eosinophils - developed by Coulter in the 1950s
- WEAKLY POSITIVE or NEGATIVE: - MOST COMMON methodology used
o Monocytes - Radiofrequency (RF) a.k.a, alternating current (AC) resistance is a
- NEGATIVE: modification sometimes used in conjunction with DC electronic impedance
o Myeloblasts
o Basophils - Cell counting and sizing is based on the detection and measurement of
o Lymphocytic cell series changes in electrical impedance (resistance) produced by a particle as it
o Erythrocytic cell series passes through a small aperture.
- Reactions parallel those of the MPO's - Particles such as blood cells are non-conductive but are suspended in an
- Stains sterols, neutral fats, phospholipids (found in the primary electrically conductive diluent
and secondary granules of neutrophils and lysosomal granules - As a dilute AS & dilute suspension of cells is drawn through the aperture,
of monocytes) the passage of each individual cell momentarily increases the impedance
POSITIVE (resistance) of the electrical path between two submerged electrodes that
SUDAN BLACK B (SBB)

-
o Promyelocyte, myelocyte are located on each side of the aperture.
o Metamyelocytes, bands, and segmented neutrophils →
STRONGLY POSITIVE - The number of pulses generated during a specific period is proportional to
o Leukemic blasts the number of particles or cells.
o Auer rods - The amplitude (magnitude) of the electrical pulse produced indicates the
o Eosinophils cell's volume.
- WEAKLY POSITIVE or NEGATIVE: - Radiofrequency (RF) - otherwise known as RF resistance or high-voltage
o Myeloblasts electromagnetic current measures conductivity
o Monocytic cells
- Total volume of cell is proportional to the change in DC.
- NEGATIVE:
o Lymphocytes and its precursors - Cell interior density (e.g., nuclear volume) is proportional to pulse
o Megakaryocytes and platelets size/change in the RF signal.
o Erythrocytes - DC and RF voltage changes may be detected simultaneously.
- Differentiate acute granulocytic leukemias from monocytic - Two-dimensional distribution cytogram or scatterplot → plots conductivity
leukemias (RF) and impedance (DC) of the cells
- SUBSTRATES: Scatterplot - displays dusters of cells number of dots represents the
o α-naphthyl acetate and α-naphthyl butyrate (nonspecific)
-
concentration of a specific cell types
o Naphthol AS-D Chloroacetate (specific)
- α-naphthyl acetate esterase (NSE) TYPES OF ERRORS WHICH MAY BE ENCOUNTERED:
o POSITIVE:
 Monocytes (strong positive reaction) A.) Instrumental Errors
 Positive for other certain cell types
o Aperture plugs - most common problem in cell counting; produce
o WEAKLY POSITIVE OR NEGATIVE:
 Granulocytes POSITIVE (+) ERROR
 Lymphoid cells (Lymphocytic cell lines) o Bubbles in the sample (caused by to vigorous mixing) - produce
o NEGATIVE: POSITIVE (+) ERROR
 Monocytes (with NaF inhibition) o Extraneous electrical pulses - produce POSITIVE (+) ERROR
α-naphthyl butyrate esterase (NSE)
ESTERASES

- o Excessive lysing of RBCs - produces NEGATIVE (-) ERROR

o POSITIVE: o Improper settings of aperture current/threshold will cause EITHER
 Monocytes POSITIVE (+) or NEGATIVE (-) ERROR
 Positive for other certain cell types B.) Nature of the Specimen
o WEAKLY POSITIVE OR NEGATIVE: o Giant platelets - may be counted as RBCs or WBCs
 Granulocytes o Fragments of WBC cytoplasm (may be seen in leukemia therapy) may
 Lymphoid cells (Lymphocytic cell lines)
o NEGATIVE: be counted as platelets or RBCs
 Monocytes (with NaF inhibition). o Some abnormal RBCs (for example: Sickle Cells, extremely
Naphthol AS-D Chloroacetate esterase hypochromic cells, Target cells) resist lysis – may cause falsely
elevated (↑) WBC counts
-
o POSITIVE.
 Promyelocyte, myelocyte, metamyelocyte, bands,
segmented neutrophils, Leukemic myeloblasts
 Auer rods
o WEAKLY POSITIVĚ OR NEGATIVE:
 Monocytic cells
o NEGATIVE
 Myeloblasts (variable)
 Monoblasts, promonocytes and monocytes
- Stain for GLYCOGEN
ACID-SCHIFF

REACTION
PERIODIC

- Useful in identifying FAB M6 leukemia

(PAS)

- RBCs in erythroleukemia (FAB M6) are STRONGLY POSITIVE

- Normal erythroid precursors are PAS negative

- Enzyme marker for primitive lymphoid cells

DEOXYNUCLEOTIDYL
TRANSFERASE (TdT)

- Strong TDT activity - observed in approximately 90% of patients

with ALL
TERMINAL

- Helpful in the recognition of the "lymphoblastic transformation"

of CML

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BLOOD CELL HISTOGRAMS OTHER RELATED NOTES

- Are provided by many high-volume-instruments to provide size distribution REFERENCE RANGES FOR SOME HEMATOLOGIC TESTS
of the different cell populations. TESTS REFERENCE RANGES**
- The volume, given in cu.mm. or fL, is plotted against the relative frequency RBC count Males = 4.6 to 6.0 x 1012/L
for platelets, WBCs, and RBCs. Females = 4.0 to 5.4 x 1012/L
- These types of histograms will provide an approximate number of cells on Red Cell Distribution Width- 11.5% to 14.5%
the y-axis and the cell size on the x-axis. Coefficient of Variation (RDW-CV)
Reticulocyte Count 0.5 to 1.5%
RBC histograms Hemoglobin Males = 14-18 g/dL
Females = 12-15 g/dL
- The instrument being used counts those cells with volume sizes between Hematocrit Males: 40 to 54%
36 L and 360 L as erythrocytes. Females: 35 to 49%
- If the RBC's are larger than normal, the curve will shift toward the right Erythrocyte Sedimentation Rate Modified Westergren:
- If the RBC's are smaller than normal the curve will shift to the left Males: 0 to 10 mm/hour
- If the histogram curve is bimodal, then there is two population of red blood Females: 0 to 15 mm/hour
cells as might be seen when a patient received a blood transfusion. Wintrobe:
- Other conditions that will cause a bimodal distribution curve are cold Males: 0 to 9 mm/hour
agglutinin disease, hemolytic anemia with schistocytes present, or anemias Females: 0 to 20 mm/hour
with different size cell populations. Mean Corpuscular Volume (MCV) 80 to 100 L
- The RBC histogram can measure cells as small as 24 fL. Mean Corpuscular Hemoglobin 26 to 32 pg
- Those cells that are counted in the 24 to 36 L range are rejected as RBC's (MCH)
and not included in the RBC count. Mean Corpuscular Hemoglobin 31 to 37 g/dL or %
Concentration (MCHC)
- Leukocytes are present in the diluted fluid containing RBCs, but their
numbers are statistically insignificant in the count WBC count For both males & females:
4.5 to 11.5 x 109/L
- The instrument computer can be calibrated to compensate for the presence
WBC Differential Count (Relative Count)
of leukocytes.
Neutrophils 51 to 67%
- If the leukocyte count is significantly elevated, the erythrocyte histogram
Lymphocytes 25 to 33%
will be affected.
Eosinophils 1 to 4%
WBC histograms Monocytes 2 to 6%
Basophils 0 to 1%
- Provide a count and plot of cells in the WBC aperture bath larger than 45 Platelet count 150 to 450 x 109/L
fL. **Reference ranges are for adults using frequently-used units.
- Normal WBC histograms have three (3) distribution peaks:
**The difference between 1mm and 1 uL is INSIGNIFICANT.
1. First peak (45-90 L)
o small mononuclear population of cells (i.e., lymphocytes)
2. Second peak (90-160 L)
o minor population of large mononuclear cells (i.e. monocytes)
o an increase in the number of cells in this size range can also
represent abnormal cell types (such as the immature precursor
of cell types found in patients with leukemia).
3. Third peak (160-450 fL)
o normal mature types of granulocytes.

ABNORMAL WBC HISTOGRAM PATTERNS

1.) Region code (R) fags

o signal irregularities in the WBC distribution and will appear next
to the differential parameters that are in error.
o The "R" stands for the region.
o R1 - Warns of increased interference in the area left of the
lymphocyte peak (approx. 35 L)
o typically caused by sickled RBCs, nucleated RBCs, or
clumped and giant platelets being counted in the WBC
aperture bath.
o R2 - Warns of excessive overlap of cell populations at the
lymphocyte/mononuclear cell boundary (approximately 90 fL)
caused by the presence of abnormal cell types, such as atypical
lymphocytes, blast, or plasma cells.
o R3 - warning is caused by excessive overlap of cell populations
at the mononuclear/granulocyte boundary (approximately 160
1L), which is due to the increased presence of immature
granulocytes (i.e., bands, metamyelocytes).
o R4 - Warning is caused by the extension of the cell distribution
past the upper end of the WBC threshold (approximately 450 fL).
This most commonly occurs when the granulocyte population is
very high.
o RM - is the error code for multiple region overlap.

2.) Other tags:

o H- when a parameter value is higher than the set normal limit.
o L- when a parameter value is lower than the set normal limit.

PLATELET Histograms

- Platelet derived histograms (via the electrical impedance method) are

obtained from volume sizes of 2 to 20 L.
- The actual counting takes place in the RBC aperture.

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