Manual techniques remain important for reference and investigating discrepant automated results. Hemoglobin is measured using cyanmethemoglobin or direct spectrometry methods. Packed cell volume is assessed using microhematocrit centrifugation. Cell counts use a hemocytometer, with red cells counted at 1:200 dilution and white cells at 1:20 dilution with acetic acid and stain. Platelets are counted at 1:20 dilution in ammonium oxalate. Proper microscope use includes adjusting eyepieces, using coarse and fine focus, examining at different magnifications, and cleaning lenses with tissues.

1. Manual CBC
Aliaa Adel EL-Feshawy, MSc
Clinical Pathology Department
Faculty of Medicine-Menoufia University

2. MANUAL TECHNIQUES
Despite the widespread use of automated instruments, the manual
techniques remain important both as reference methods and in the
investigation of blood samples that appear to give discrepant
results with automated analyzers.

3. Hemoglobin concentration
(1) Cyanmet-hemoglobin method is the Reference Method for
determining the Hb concentration of blood.
 Principle:
 Dilution of blood in a solution containing potassium cyanide and
potassium ferricyanide.
 Hb, Met-Hb & carboxy-Hb but not S-Hb are converted to
Cyanmet-Hb .
 The absorbance of the solution is measured in a
spectrophotometer at 540 nm (546 nm Filter).

4.  Diluent
The modified solution, Drabkin-type reagent pH 7.0–7.4.
 The diluent should be clear and pale yellow in color.
 If stored at room temperature, in a brown borosilicate glass
bottle, the solution keeps for several months.
 If the ambient temperature >30°C, the solution should be stored
in the refrigerator but brought to room temperature before use.
 Reagent must be discarded if:
1. Turbid.
2. pH outside 7.0–7.4 range.
3. an absorbance other than zero at 540 nm against a water
blank.

5.  Method:
1. Make 1 in 201 dilution of blood by adding 20 μl of blood to 4 ml
of diluent.
2. Stopper the tube containing the solution and invert it several
times.
3. Let the test sample stand at room temperature for at least 5 min.
4. Pour into a cuvette and read the absorbance in a spectrometer
at 540 nm against a reagent blank.
5. The absorbance of the test sample must be measured within 6
hours of its initial dilution.

6.  Standard
 The absorbance of a commercially available cyanmet-
hemoglobin standard should also be compared to a reagent blank
in the same spectrometer as the patient sample.
 The standard should be kept in the dark and, to ensure that
contamination is avoided, any unused solution should be discarded
at the end of the day on which the ampoule is opened.
 Calculation of Hb Concentration
Hb (g/l) = A* of test sample / A of standard × Conc. of standard ×
Dilution factor (201)/1000
A* = absorbance

7. (2) Direct Spectrometry
 Hb of a diluted blood sample can be determined by
spectrometry without the need for a standard, provided that the
spectrometer has been correctly calibrated.
 The blood is diluted 1:251 with cyanide–ferricyanide and the
absorbance is measured at 540 nm.
 Hb is calculated as follows:
Hb ( g/l) = (At × 16114 × dilution factor) / ( 11 × d × 1000)
Hb ( g/l) = At × 36.77
At = absorbance of test sample
16114 = monomeric molecular weight of hemoglobin
dilution = 251 when 10 µl of blood is diluted in 2.5 ml of reagent
11.0 = milli molar coefficient extinction
d = layer thickness in cm
1000 = conversion of mg to g.

8. Packed cell volume (Hematocrit)
The packed cell volume (PCV) can be used as a simple
screening test for anemia, as a reference method for calibrating
automated blood count systems and as a rough guide to the
accuracy of Hb measurements.
The PCV × 100 is about three times the Hb expressed in g/dl
e.g. 0.36 × 100 is approximately 12 (Hb)× 3.

9.  Microhematocrit Method
The microhematocrit method is carried out on blood contained in
capillary tubes 75 mm in length and having an internal diameter of
about 1 mm.
The tubes may be plain for use with anti-coagulated blood
samples or coated inside with heparin for the direct collection of
capillary blood.
The centrifuge used for the capillary tubes provides a centrifugal
force of c 12000 g and 5 min centrifugation results in separation of
the column of blood into red cells, buffy coat and plasma.

10.  Method
1. Allow blood from a well-mixed specimen or from
a free flow of blood by skin puncture, to enter
the tube by capillarity, leaving at least 15 mm
unfilled.
2. Then seal the tube by a plastic seal.
3. After centrifugation for 5 min, measure the
proportion of red cells to the whole column.

11.  Factors affecting accuracy of micro-hematocrit method:
 Anticoagulant:
 K2-EDTA is recommended because K3-EDTA causes shrinking
of the red cells reducing the PCV by about 2%.
 Anticoagulant concentration in excess of 2.2 mg/ml may also
cause a falsely low PCV as a result of cell shrinkage.
 Blood Sample:
The test should be performed within 6 hours of collecting the
blood sample but a delay of up to 24 hours is acceptable if the
blood is kept at 4°C.
 Failure to mix the blood sample adequately will produce an
inaccurate result.

12. Manual cell counts using counting chambers
 Counting Chamber (hemocytometer)
 Improved Neubauer ruling: This has nine 1 mm × 1 mm ruled
areas which when covered correctly with the special thick cover
glass each contain a volume of 0.1 μl of diluted blood .
 The sample is introduced between the chamber & the cover
glass using a pipette and the preparation is viewed using ×40
objective and ×10 eyepieces.
 With Improved Neubauer chambers, the cells in squares are
counted, including the cells that touch the bottom and left-hand
margins of the small squares.

13. Improved Neubauer ruling

14. (1) The red cell count
 Diluent: saline
 Dilution 1: 200
 Calculation: R1+R2+R3+R4+R5×10,000 = count/µl
 Red cell indices
MCV(fl) = Hct/ RBC
MCH(pg) = Hb/ RBC
MCHC(g/dl) = Hb / Hct

15. (2) The white cell count
 Diluent: 2 % Glacial acetic Acid colored pale
violet with drop of Leishman stain.
 Dilution: 1: 20 (0.1 ml of well-mixed blood
“lack of adequate mixing is a major source of
error” to 1.9 ml of diluent).
 Calculation: W1+W2+W3+W4 × 50 = count/µl

16. (3) The platelet count
 Diluent: 1% aqueous ammonium oxalate (To lyse
RBCs)
 Brilliant cresyl blue can be added to the diluent.
This stains platelets light blue and facilitates their
identification.
Dilution 1: 20
 Calculation: P1+P2+P3+P4+P5 × 1000 = count/µl

17. Setting up and using a microscope

18.  If you need to move or lift the microscope do so using only the
arm .
 Adjust the position of the eye pieces so that they match your inter-
pupillary distance and look at the slide through the eye pieces,
making sure that the light is at a comfortable level.
 Use the coarse and then the fine focus.
 Examine the slide with the × 10 objective, then rotate in a × 40
objective.

19.  When you have finished working, rotate back in the lowest
power objective and lower the stage.
 Do not leave the microscope turned on when you are away from
your workstation.
 Keep the microscope clean. Dust can be removed with a small
brush. Lenses should be cleaned only with lens tissues. These
can be moistened with methanol.
 Cover the microscope with a dust cover when not in use.