MODULE 1 – COMPLETE BLOOD COUNT

MODULE OUTCOMES
At the end of this module, the intern must be able to:
1. perform accurately complete blood count (CBC).
2. explain correctly the principle and the methodology of the tests consisting the CBC.
3. list completely the normal values of the CBC in conventional and SI units.
4. relate correctly results of the CBC to human diseases.

INTRODUCTION
The Complete Blood Count (CBC) is previously known as Full Blood Count (FBC). It consists of
Hemoglobin Concentration (Hemoglobin), Erythrocyte Volume Fraction (Hematocrit), Erythrocyte
Number Concentration (RBC Count), Leukocyte Number Concentration (WBC Count), Leukocyte Type
Number Fraction (Differential Count), Thrombocyte Number Concentration (Platelet Count), and RBC
indices. The World Health Organization (WHO) terminology may be used interchangeably with traditional
terms that is in brackets.

KEY CONCEPTS
I. HEMOGLOBIN
A. Clinical Significance and Reference Range
1. Reference range range vary with age and sex
Conventional units Factor SI units
Male 14 – 17.5 g/dL 10 140 – 175 g/L
Female 12.3 – 15.3 g/dL 10 123 – 153 g/L
2. Used to diagnose and follow the treatment of anemia
3. Increased inx polycythemia vera, in the morning, in smokers, strenuous exercise and in high
altitudes
4. Decreased in anemia, slightly lower values if the patient is lying down
B. Cyanmethemoglobin Method
1. Also known as Hemiglobincyanide method
2. Reference method approved by CLSI
3. Automated cell counters use some modification of this method
4. Measure all forms of hemoglobin except sulfhemoglobin (sulfhemoglobin is irreversible)
5. Uses Drabkin’s reagent to lyse RBCs and convert iron to the ferric state (Fe3+), forming
methemoglobin
Hb (Fe++) + Potassium ferricyanide Methemoglobin (Fe+++)
Methemoglobin + Potassium cyanide Cyanmethemoglobin
6. Drabkin’s Reagent
a. Contains:
i. Potassium ferricyanide [K3Fe(CN)6] – converts hemoglobin into methemoglobin
ii. Potassium cyanide (KCN) – converts methemoglobin to cyanomethemoglobin
iii. Dihydrogen potassium phosphate (KH2PO4) – shortens conversion time from 10-15
minutes to 3 minutes (0riginal Drabkin’s reagent uses NaHCO3)
iv. Nonionic detergent – enhances lysis of RBCs (e.g. Steroz and Triton X)
v. Distilled water
b. Stable for at least 6 months @ RT, protected from light by using an amber bottle.
c. Should be clear and pale yellow. If it becomes turbid, or loses its color, it should be
discarded.
d. Potassium cyanide is very poisonous. It must be kept in a locked cabinet at all times when
not in use.
7. Procedure
a. Pipette (Sahli) 20uL of blood to 5.0 mL of diluents. Specimen is EDTA whole blood.
b. Wipe outside of pipette. Rinse the pipette by drawing up and expelling the fluid in the tube
3×.
c. Mix well, and allowed to stand at room temperature for at least 3 minutes.
d. Read sample against reagent blank @ 540nm. If cloudiness appears in diluted blood
(abnormal plasma proteins or high WBC), centrifuge at 2000G for 5 minutes before taking
a reading.
e. Using table prepared from the calibration curve, record the concentration of hemoglobin.
8. Sources of Error
a. Lipemia – can be corrected using a patient blank

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 b. Increased WBCs and platelets – can be corrected by centrifuging test mixture and testing
hemoglobin on the supernatant fluid
c. HbS and HbC – dilute hemoglobin with distilled water
d. Increased globulins – use of dihydrogen potassium phosphate
e. Overanticoagulation – causes no effect on hemoglobin determinations
C. Other methods for hemoglobin concentration determination
1. Oxyhemoglobin procedure – Hb is converted to oxyHb by shaking with aqeous NH4OH;
imprecise
2. Acid Hematin Test uses HCl to convert Hb into acid hematin, followed by dilution with distilled
water drop by drop until the color matches that of the standard (comparator block)
3. Some automated instruments use sodium lauryl sulfate (SLS) to convert Hb to SLS-
methemoglobin. This method does not generate toxic wastes.

II. HEMATOCRIT
A. Clinical Significance and Reference Range
1. Hct is the volume of RBC expressed over the percentage of the total whole blood volume
2. National committee for CLS recommends the term packed cell volume (PCV); hematocrit is the
term used to describe the method
3. Relationship of Hb to hct is 1:3, but may vary with the cause of anemia and the effect on the
RBC indices especially the MCV
4. Reference range range vary with age and sex
Conventional units Factor SI units
Male 41.5% – 50.4% 0.01 0.415 – 0.504 volume
fraction
Female 35.9% – 44.6% 0.01 0.359 – 0.446 volume
fraction

B. Microhematocrit Method
1. Spun microhematocrit is the reference manual method
2. Uses a Microhematocrit tube: 75 mm long, bore of about 1mm, holds 0.05 ml of blood
3. Procedure
a. Fill at least 2 capillary tubes approximately 2/3 to ¾ full or at least 5 cm. Fill tube at opposite
end of colored ring. Capillary tubes are color coded. Red is heparinized and used for non-
anticoagulated whole blood. Blue is plain and used when processing anticoagulated blood.
Anticoagulated blood should be centrifuged within 6 hours of collection if stored at RT
b. Seal unfilled end (end with colored ring) with nonadsorbent material (soft wax or plastic
modelling clay). Microhematocrit tube forms a 90 angle with the tray of clay. Clay plug
should be 4-6mm long.
c. Centrifuge for 5 minutes at 10,000 – 12,000 g (Henry) in microhematocrit centrifuge.
d. Read hct using the microhematocrit reading device. Results should be read within 10
minutes of centrifugation. Buffy coat should not be included in the reading.
e. Hematocrit may be expressed as a percentage (conventional) or, as a decimal fraction (S.I.)
Results must agree within ± 0.02 L/L. If they do not, repeat the procedure
4. Layers of blood after centrifugation
a. Top of the tube - Fatty layer
b. Second layer – Plasma
c. Third layer - Buffy coat
d. Bottom layer - Packed red cells
5. Sources of Error
a. Falsely low results
i. Incomplete sealing
ii. Short sample
iii. Over-anticoagulated blood (RBC shrinkage)
iv. Hemolysis
b. Falsely elevated results
i. Inadequate centrifugation (over centrifugation has no effect on hematocrit)
ii. Allowing the tubes to stand after centrifugation before reading
iii. Including buffy coat in the reading
iv. Hemoconcentration (prolonged application of tourniquet)
v. Abnormal RBC Morphology leading to trapped plasma. Trapped plasma is a small
amount of plasma that remains in the RBC portion of spun HCT. It is increased in
sickle cell anemia, hypochromic anemia, spherocytosis, macrocytosis, thalassemia
and may increase HCT by 0.02
c. Falsely elevated or low results
i. Insufficient mixing of blood prior to obtaining HCT sample

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 ii. Improper use of HCT reader
C. Macrohematocrit Method
1. Uses the Wintrobe tube; no longer used
2. Centrifuge WB at 2000-2300g for 30 minutes
3. Anticoagulant: Oxalate, Heparin, EDTA
D. Automated Techniques
1. Does not directly measures HCT
2. Calculated by automated counters by using the MCV and RBC count:
Hct = MCV (fL) × RBC (×1012/L)
10
3. MCV and RBC is directly measured by automated methods
4. Not affected by trapped plasma

III. CELL COUNT

A. Counting Chambers
1. Neubauer Counting Chamber
a. Total area = length × width = 3mm × 3mm = 9 mm2
b. Depth = 0.1 mm
c. Total volume = area × depth = 0.9mm2 × 0.1mm = 0.9 mm3
d. Counting Squares:
i. Area of the large square (WBC square) = 1mm × 1mm = 1mm2
ii. Volume of the large square = 1mm2 × 0.1mm = 0.1 mm3
iii. Area of the small central square (RBC square) = 0.2mm × 0.2mm = 0.04 mm2
iv. Volume of the small central square = 0.04 mm2 × 0.1mm = 0.004 mm3
2. Speirs–Levy Counting Chamber
a. Total area = 2mm × 5mm = 10 mm2
b. Depth = 0.2mm
c. Total volume = 10 mm2 × 0.2mm = 2.0 mm3
d. Used for eosinophil count because of its large volume
3. Fuchs–Rosenthal Counting Chamber
a. Total area = 4mm × 4mm = 16 mm2
b. Depth = 0.2mm
c. Total volume = 16 × 0.2mm = 3.2 mm3
d. Used for eosinophil count because of its large volume
B. Thoma Pipette
Thoma RBC Pipet Thoma WBC Pipet
Color of bead Red White
Outstanding marks 0.5, 1, 101 0.5, 1, 11
Volume of the bulb 100 10
Dilution range 1:100 – 1:1,000 1:10 – 1:100

C. Cell Count Computation

1. Cell count/mm3 = # of cells counted × Factor
= # of cells counted × Dilution .

Total volume
= # of cells counted × Dilution .

(L × W × D) (# of squares counted)

IV. RED BLOOD CELL COUNT

A. Clinical Significance and Reference Range
1. Normal range vary with age and sex
Conventional units Factor SI units
Male 4.5 – 5.9 × 106/μL 106 4.5 – 5.9 × 1012/L
Female 4.5 – 5.1 × 106/μL 106 4.5 – 5.1 × 1012/L

2. Higher in newborns; slight decrease in childhood/adolescence and after 50 years of age

3. Highest in the morning and lowest in the evening
4. Increased in polycythemia, dehydration and high altitudes
5. Decreased in anemia and other disorders
B. Manual RBC Count
1. Counting of RBC by manual method is not recommended due to poor precision
2. Procedure
a. Using a thoma pipette draw blood up to 0.5 mark and dilute up to 101 mark with diluting
fluid (1:200). The dilutiuon in polycythemia 1:300 and in severe anemia 1:100.

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 b. Shake pipette for 3 minutes in mechanical shaker.
c. Discard first 4 drops of fluid from pipette and fill hemocytometer.
d. Allow hemocytometer to stand for 3-5 minutes.
e. Count RBCs in 5 small central squares.
f. Calculate the RBC count per mm3 by multiplying the # of RBCs counted in the 5 small
central squares by 10,000.
C. RBC Diluting Fluid
1. Should be isotonic to prevent lysis of RBCs
2. Hayem’s, normal saline solution (NSS), Dacie’s (Formol-Citrate), Toisson’s, Gower’s, Bethel’s,
and 3.8% Na Citrate
D. Rule of Three
1. Correlation of Hb, Hct and RBC count
2. Hb × 3 = Hct ± 3%
3. RBC (in millions) × 3 = Hb ± 0.5
4. RBC × 9 = Hct
5. Do not derive hemoglobin concentration by using the hematocrit value and vice versa (rule
apply only to RBCs of normal size and color)
6. If 'rule of 3" doesn't fit, consider clotted sample, cold agglutinin (warm sample and rerun), or
lipemic or icteric sample

V. WHITE BLOOD CELL COUNT

A. Clinical Significance and Reference Range
1. Normal range vary with age
Conventional units Factor SI units
4.4 – 11 × 103/μL 106 4.4 – 11.3 × 109/L

2. Higher in newborns, 10 – 30 × 109/L ; 1 year old, 6 – 17 × 109/L; adult, 4 – 11 × 109/L

3. Diurnal variation: highest in the afternoon, lowest in the morning
4. Physiologic leukocytosis (without tissue damage): smoking, exercise, emotional stress, anxiety,
release of epinephrine, pregnancy
5. Pathologic leukocytosis: bacterial infection, appendicitis, leukemia, HDN.
6. Leukopenia: viral disease, typhoid fever, measles, brucellosis, Ra, LE, liver cirrhosis, infectious
hepatitis, radiation, drug therapy
B. Manual WBC Count
1. EDTA should be used; heparin is unsatisfactory as an anticoagulant.
2. Performed as a check on the validity of electronic methods for calibration; back up method
3. WBC Diluting Fluid
a. Lyses the erythrocytes so that they will not obscure the leukocytes.
b. Must be refrigerated and filtered frequently to remove yeasts and molds.
c. 1% ammonium oxalate, 2-3% glacial acetic acid with gentian violet, 1% hydrochloric acid,
and Turk's solution (glacial acetic acid and methyl violet)
4. Procedure
a. Place WB on rotator for at least 1 or 2 minutes or hand invert at least 60 times.
b. Attach mechanical pipette suction to WBC pipette. Draw blood exactly to 0.5 mark and
dilute up to 11 mark with diluting fluid (1:20). Use RBC pipette or Unopette system if WBC
is ↑.
i. 1:100 dilution for WBC >30 × 109/L
ii. 1:200 dilution for WBC 100-300 × 109/L
iii. 1:10 dilution for WBC <3.0 × 109/L
c. Mix pipette for approximately 3-5 minutes in a mechanical pipette shaker. Or place thumb
over open tip and vigorously rotate pipette back and forth, moving only the wrist for 30-45
seconds.
d. Discard 4 drops or ¼ of mixture in bulb onto a piece of gauze.
e. Fill/charge hemotocytometer (tip of pipette at 45°) and stand for approximately 1-10 minutes
to allow cells to settle in moist chamber.
f. Count on 4 corner squares using LPO (10× objective) on 2 ruled area and get the average.
No more than 10 variations between the 4 squares.
g. Include in the count the WBC seen on the lines of 2 sides of each square counted, using
the letter ⎣ “or” ⎤ as guide.
h. Calculate the WBC count by multiplying the # of WBCs counted in the 4 squares by 50
(report result as number per mm3) or 0.05 (report result as number × 109/L)
i. Clean chamber with 95% alcohol.
5. Sources of Error
a. False decrease
i. Failure to mix specimen before sucking

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 ii. Failure to mix pipette with specimen
iii. Bubbles in pipette (while sucking blood)
b. False increase
i. Diluting fluid contaminated with blood
ii. Bubbles in pipette (while sucking diluting fluid)
iii. Drawing blood to far past appropriate mark
C. Corrected WBC Count
1. The diluting fluid used for WBC count destroy/hemolyze all non-nucleated RBCs. Nucleated
RBCs are not lyzed by WBC diluting fluid and will be counted as WBC (causing a falsely
increased WBC count)
2. Target cells and sickle cells also don’t lyse in diluting fluids.
3. Correct WBC count if ≥ 5 nRBC/100 WBC
4. Corrected/True WBC count = (Total count × 100) / (100 + No. of NRBCs)

VI. BLOOD SMEAR

A. Blood Smear Preparation
1. Faster film drying (thin) has better cell detail.
2. Slow film drying (thick) will show contraction of cells
3. Blood smears should be made within 5 hours of collection from blood anticoagulated with EDTA
B. Wedge Blood Smear
1. Aka spreader-slide, push smear, 2 slide method
2. Most widely used, easy to perform
3. Smearer or spreader slide with chamfered or beveled corners is recommended
4. Size of glass slide = 3-inch × 1-inch or 75-mm × 25-mm
5. Procedure:
a. Place a drop of blood: 2-3 mm in diameter, 0.25 inch (or 1 cm) from the end or frosted area
of a glass slide.
b. Position spreader slide 30-45° to the stationary slide and bring it back into the drop of blood
c. Allow blood drop to spread along the back edge of the spreader slide and push forward with
a “smooth and rapid” stroke. Smear should terminate 0.5 inch near the end of the slide.
d. Air dry rapidly (manually or with an electric fan/cool air blower).
e. Label on frosted end of slide or in the thicker end after it has dried.
6. Factors affecting thickness of blood films
a. Angle predicts thinness or thickness of smear
b. Increasing the angle leads to thick smears (for specimens with low Hct)
c. Decreasing the angle leads to thin smears (for specimens with extremely high Hct)

Extremely thick smears are caused by: Extremely thin smears are caused by:
i. decreased pressure i. increased pressure
ii. increased angle of spreader slide ii. decreased angle of spreader slide
iii. increased size of drop of blood iii. decreased size of drop of blood
iv. increased speed of spreader (too iv. decreased speed of spreader (too slow a
fast a spread) spread)

7. Characteristics of an ideal blood film:

a. Smear should cover 2/3 to 3/4 (at least half the length) of the glass slide but should not
occupy the whole length of the slide,
b. Narrower than the slide; should not occupy the lateral edge of the slide
c. Gradual transition from thick to feathery edge
d. No waves, streaks, holes (oily areas) or bubbles
e. Should terminate in a straight feathered end; finger shaped (not bullet shaped)
f. When slide is held up to light, thin portion of the film has a “rainbow” appearance
g. Whole drop of blood is picked up and spread
8. Disadvantages:
a. Cell distribution in slide is size dependent
i. Large cells (monocytes, eosinophils, blasts)
ii. Smaller cells (RBC, lymphocytes, segmenters) in the middle
b. Leads to greater trauma of cells (“basket” or “smudge” cells)
C. Other Types of Blood Smears
1. Cover Glass Smear
a. 2 cover glass are used (size of coverslip = 22 × 22 mm)
b. Harder to master; uniform overlapping of cells
c. Utilized extensively for BM exams
d. Procedure:

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 i. Place small drop of blood (2mm in diameter) on surface of a coverslip and place another
coverslip on top (8 pointed star overlapping).
ii. When spread is almost complete, pull 2 coverslips apart.
iii. Air dry rapidly, stain and mount (blood-film down) on glass slide.
2. Automated Blood Films
a. Wedge (Push) Type – stimulates the manual spreader-type technique
b. Centrifugal (Spinner) Type/ Spun Smear
i. Drops of blood placed on center of slide and spun for a period of time
ii. Random and monolayer distribution of cells
3. Buffy Coat Smear
a. Centrifuge blood in Wintrobe tubes for 15 minutes at 1500g or 6 minutes at 1000g and
aspirate buffy coat
b. Indications:
i. If WBC count is <1.0 × 109/L and a 100-cell differential is desired
ii. Finding reactive, immature or abnormal cells (e.g. blasts) that are present in small
numbers
iii. Finding megaloblastic nucleated cells, hypersegmented neutrophils, plasma cells and
tumor cells
iv. Easier to locate bacteria or parasites. Example: mature RBCs containing malarial
parasites are concentrated on top of the RBC layer (more fruitful than the “thick drop”
preparation).
4. Thick and Thin Blood Film
a. Used when looking for blood parasites such as malaria
i. Thick smear: Detection
ii. Thin smear: Identification
b. Use the corner of a slide to spread blood the size of a dime
c. Thickness: newspaper print is visible through the blood
d. Air dry @ least 2-4 hours @ RT, or preferably overnight
D. Blood Staining
1. For best results blood should be stained 2-3 hours after collection
2. Types of stain:
a. Nonvital polychrome stain - stains present produce multiple colors when applied to cellular
elements (example: Romanowsky stains)
b. Nonvital monochrome stain - stains specific cellular components (example: Prussian blue,
visualize iron granules in RBC)
c. Supravital monochrome stain - used to stain specific cellular components in the living state;
no fixatives are used in the staining process (examples: New methylene blue, precipitate
RNA in reticulocytes and neutral red with brilliant cresyl green as counterstain, for Heinz
bodies)
3. Romanowsky Stain
a. Routinely used to stain peripheral blood and BM smears
b. Considered to be polychromatic stain
c. Basically contains methylene blue and eosin
i. Methylene blue – a basic dye, (+) charged, stains the acidic components of the cell
(nucleus) blue/purple
ii. Eosin – an acidic dye, (-) charged, stains the basic component of the cells (cytoplasm)
orange/pink
iii. Buffer - monobasic K phosphate, dibasic Na phosphate; pH 6.8 (6.4-6.8) ideal for blood
and BM staining; pH 7.2 used when looking for malarial parasites (Schuffner granules)
d. Examples of Romanowsky stains:
i. Wright’s
• Most commonly used routine peripheral blood smear
• Oxidized methylene blue and eosin
• Preparation: 1g Wright powder + 500mL chemical pure, acetone-free methanol.
Incubate for 1 week at 37°C and then at RT or ref temp. (Incubation can be
shortened by adding 0.1g Giemsa powder per 500mL stain).
• Methanol is used to fix the cells to the slide (fixative)
ii. Wright-Giemsa
• Combines eosin and methylene blue in methanol with glycerin
• Giemsa contains azures (oxidative products of methylene blue)
• Produce more delicate staining characteristics
• Alone, Giemsa is inadequate for staining RBCs, platelets, and WBC cytoplasm
iii. Others: Giemsa, Leishman, May-Grunwald, Jenner, MacNeal’s
4. Staining Methods

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 a. Manual: Rack method, Dip (incubation) method
b. Automated: Platen type, Carousel type, Dip-type instruments
E. Criteria for a Good Stain
1. Smear should be reddish brown
2. Microscopically:
a. RBC – salmon pink
b. WBC nuclei – purple-blue
c. Platelets – purple-blue to lilac with red-purple granules
3. Additional criteria:
a. Eosinophils – orange granules
b. Neutrophils – pinkish tan cytoplasm
c. Monocytes – gray ground glass cytoplasm with many tiny red-purple granules
d. Bacteria – blue
e. Malarial parasite – sky blue cytoplasm and red purple chromatin
4. Staining Problems
Staining
Causes Remedies
Problems
Too blue Thick film Shorten staining time
Overstained Less stain/more diluent
Alkaline reagent New buffer/decrease pH
Too red Understained Lengthen time
Overwashed Shorten wash time
Acid reagent (metOH → formic acid) New buffer/fresh methanol
Precipitates Dirty slides (dusts) Clean slides
Underwashed Wash properly
Failure to keep slides in horizontal Keep slides in horizontal
position position
Inadequate stain filtration Filter stains
5. Other causes:
a. Excessively Blue Stain: excessive staining time, inadequate washing, old smear, protein
abnormality, use of heparinized blood sample, very high WBC count with many blasts, low
Hct
b. Excessively Red Stain: insufficient staining time, very thin smears, contaminants like
chlorine

VII. DIFFERENTIAL COUNT

A. Clinical Significance and Reference Range
1. Performed to determine the relative number of each type of WBC and at the same time a study
of RBC, WBC and platelet morphology is performed.
2. 3-part differential: granulocytes, lymphocytes, monocytes
3. 5-part differential: neutrophils, eosinophils, basophils, lymphocytes, monocytes
Relative Value Absolute Value
Neutrophil, Segmented 50 – 70% 1.8 – 7.8 × 109/L
Neutrophil, Band 0 – 5% 0 – 0.70 × 109/L
Eosinophil 1 – 3% 0 – 0.45 × 109/L
Basophil 0 – 2% 0 – 0.20 × 109/L
Lymphocyte 18 – 42% 1.0 – 4.8 × 109/L
Monocyte 2 – 11% 0 – 0.80 × 109/L
4. Relative Count
a. Percentage of cells counted
b. Conventional reporting
c. Relative count = (Absolute count/WBC count) × 100
5. Absolute Count
a. Actual # of cells per liter of blood
b. Preferred and more accurate method of reporting
c. Absolute Count = WBC count × Relative count
B. Approach to Evaluation
1. Check slide identification.
2. Perform patient specimen orientation. Review automated count report
3. Low Power Examination
a. Scan first the counting areas of the slide including the lateral and feather edge. Locate rare
abnormal cells which may be examined more closely under higher magnification.
b. Check feather edge for fibrin clots.

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 c. Examine RBC distribution patterns, size and shape. Avoid the feather edge, RBC appear
flattened from pressure of slide making.
d. Examine film edges for excessive leukocytes
e. Verify stain quality
4. High Power Examination
a. Examination of smear may be used to double check the WBC count. Do not report a WBC
count based on an estimate using the blood smear.
b. To estimate the WBC count:
# of cells / HPF WBC count
2–5 4 – 7 × 109/L
4–6 7 – 10 × 109/L
6 – 10 10 – 13 × 109/L
10 – 20 13 – 18 × 109/L

5. Oil Immersion Examination

a. Do WBC differential counting just behind the feather edge where the RBC are not
overlapping (moderately thin area)
b. Classify each cell until 100 cells are counted
c. Approximation of platelet is also made
6. Notes to Remember!
a. 200 cell differential required if >10% eosinophils, >2% basophils, >11% monocytes and/or
more lymphocytes than neutrophil (except in children)
b. 50 cell differential allowed if WBC < 1.0 × 109/L. Remarks must be made that only 50 cells
were counted
c. “Shift to the left” means ↑ immature WBC (immature granulocytic cells), blast to stab
d. “Shift to the right” means ↑ of mature neutrophils (segmenters)
C. Counting Methods
1. Cross sectional or crenellation
a. WBCs are counted in consecutive fields as the blood film is moved from side to side
2. Longitudinal
a. Ideal method if the smear is thin enough
b. WBCs are counted in consecutive fields from tail toward the head of the smear
3. Battlement
a. Uses a pattern of consecutive fields beginning near the tail on a horizontal edge: count 3
consecutive horizontal edge fields, count 2 fields toward the center of the smear, count 2
fields horizontally, count 2 fields vertically to the edge
D. Consensus
1. The following shall be indicated in the comments/remarks part of the report, as applicable
(PCQACL):
a. If the manual method was used for part or all of the tests required
b. Any abnormalities noted such as WBC, RBC, and PLT abnormalities
c. Incidental findings, e.g. inclusion bodies, malaria and other organisms
d. Toxic changes (e.g., toxic granules, Dohle bodies, toxic vacuoles)

VIII. RBC INDICES

A. Clinical Significance and Reference Range
1. Useful in the morphologic characterization of anemia
2. Calculations introduced by Wintrobe
3. Reference range
Conventional units Factor SI units
MCV 80 – 96 um3 1 80 – 96 fL
MCH 27.5 – 33.2 pg 1 27.5 – 33.2 pg
MCHC 33.4 – 35.5 % 0.01 0.334 – 0.355

B. Mean Corpuscular Volume (MCV)

1. MCV = Hct/RBC × 10
2. Indicates average volume of RBC
3. 1 femtoliter (fL) = 10−15 L = 1 cubic micrometer (μm 3)
4. Interpretation:
a. Macrocytic RBC (>96 fL) seen in megaloblastic anemia, hemolytic anemia with
reticulocytosis, liver disease and normal newborn
b. Normocytic RBC (80-96 fL)
c. Microcytic RBC (<80fL) seen in IDA, thalassemia, sideroblastic anemia and lead poisoning

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 C. Mean Corpuscular Hemoglobin (MCH)
1. MCH = Hb/RBC × 10
2. Average content (weight) of Hb in the RBC
3. 1 picogram (pg) = 10−12 g
4. Less valuable than the MCV and MCHC
5. Interpretation
a. Increased in macrocytic anemia
b. Decreased in microcytic anemia and hypochromic anemia
D. Mean Corpuscular Hemoglobin Concentration (MCHC)
1. MCHC = Hb/Hct × 100
2. Average concentration of Hb in each individual RBC in g/dL
3. Interpretation:
a. Hyperchromia (>35.5%) seen in the presence of spherocytes
b. Normochromic (33.4-35.5 %)
c. Hypochromia (<33.4%) seen in IDA and thalassemia
d. >38% MCHC should not occur.
e. <22% MCHC should not occur; lipemic plasma or abnormal Hb (S or C)
E. Red Blood Cell Distribution Width (RDW)
1. RDW = SD of MCV/Mean MCV × 100
2. Indicates degree of anisocytosis (variation in cell size)
3. Determined from the RBC histogram
4. Normal value: 12-17%
5. ↑ RDW seen in IDA, post-transfusion, post-treatment (e.g. iron, B12, or folic acid therapy),
idiopathic sideroblastic anemia, in the presence of two concurrent deficiencies (iron and folic
acid deficiency)

FURTHER READING
1. Brown, B. A. (1993). Hematology: Principles and procedures (6th ed.). Estados Unidos: Lea & Febiger.
2. Keohane, E. M., Smith, L. J., Walenga, J. M., Rodak, B. F. (2016). Rodak's hematology: Clinical
principles and applications (5th ed.). St. Louis, MO: Elsevier.
3. Lo, R. W., Liu, E. B., Orillaza, M. A., Faundo, A. C., & J., D. C. (2012). PCQACL'S Standardization and
harmonization of complete blood count in the Philippines (1st ed.). Quezon City: C & E Publishing.
4. McPherson, R. A., & Pincus, M. R. (2011).
5. Henry's Clinical Diagnosis and Management by Laboratory Methods E-Book. Saunders.
6. Stiene-Martin, E. A., Lotspeich-Steininger, C. A., & Koepke, J. A. (1998). Clinical Hematology:
Principles, procedures, correlations. Philadelphia: Lippincott-Raven.
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