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CALPAHI, JUSTINE JADE A. MLS 312 0962C

EXPLORE_EO8: THE BLOOD CELL COUNT: HEMOCYTOMETER METHOD

QUESTIONS FOR RESEARCH:

1. In case of conditions associated with abnormally low or high red blood cell count such as anemia (low cell
count) or polycythemia vera (high cell count), how was the procedure and computation for manual RBC count
adjusted to compensate for these conditions?
In polycythemia or erythremia, when the number of RBC is markedly increased, the blood is drawn to
0.3 mark of the RBC pipette and the diluent to 101. This gives a ratio of dilution of 1:333.33 and in the formula
the dilution factor varies accordingly. In case the number of cells is markedly decrease as in anemia, the
procedure may be modified by :
a. Sucking blood to mark 1 and the diluent to 101; the dilution is 1:100 and in the formula the dilution
varies accordingly.
b. Sucking the blood in the usual standard manner but counting more squares other than the standard
5 medium squares and in the formula, the area factor varies accordingly.
If 10 medium squares are used = area factor is 2.5
If 15 medium squares are used = area factor is 1.67
If 20 medium squares are used = area factor is 1.25
If 25 medium squares are used = area factor is 1.0

2. From the listed diluting fluids for RBC and WBC count, construct a table showing the composition,
advantage and disadvantage of each.
DILUTING COMPOSITION ADVANTAGES DISADVANTAGES
FLUIDS
RBC count
Gower’s -12.5g sodium sulfate -precipitates protein in cases of
solution -33.3g glacial acetic acid hemoglobinemia and
-200mL water hyperglobulinemia
-Rouleaux formation is inhibited in
this fluid (perhaps more than
others.)
Hayem’s -0.25g mercuric chloride -Gives better results, -More expensive
solution -2.5g sodium sulfate -Contains sodium sulfate that -The fluid has to be
-0.5g sodium chloride prevents bacterial or fungal growth renewed frequently to
-100mL distilled water -mercuric chloride serves as avoid RBC clumping.
antiseptic
Toison’s 1g sodium chloride -stains WBC -Because of Glycerine,
solution -8g sodium sulfate -high specific gravity RBC tend to settle on the
-30g glycerin surface of the counting
0.025g methyl violet chamber very slowly
-180mL distilled water -supports the growth of
fungi
 Dacie’s -3.13g Tri sodium citrate -cheaper and commonly used
solution -1mL commercial -cell shape is preserved
formaldehyde(37% -can be kept for a long time (best
formalin) diluent)
-100mL distilled water -Formaldehyde prevents growth of
bacteria and fungus.
Strong’s
solution
Bethel’s -5g sodium sulfate
solution -1g sodium chloride
-20mL glycerin
2mL sodium merthiolate
-200mL distilled water
Eagle’s
solution
WBC count
2% Acetic -2mL glacial acetic acid Gentian violet has an antifungal
acid -1mL Gentian violet function-
-100mL distilled water -Thymol – (pinch) – prevent the
growth of fungus.
-Glacial acetic acid lyses the red
cells.
Turk’s -2mL acetic acid -The diluting fluid is not isotonic
solution -1gtt-methyl violet and the very dilute acid solution
-100mL distilled water lyses RBC but not WBC.

3. What are the sources of error in blood cell counting?

Sources of error during blood cell counting may be due to equipment or technical errors. Sources of error due
to equipment include chipped tips, obscure markings, poor calibration, non-optically planed cover glass, and
unclean or wet pipettes. Sources of error due to technical errors include too much pressure on skin puncture,
prolonged tourniquet application, improper filling of pipette, failure to wipe off pipette tip, inadequate mixing,
and erroneous counting of cells.

4. What is the Poison’s Law of Distribution? How is it applied in blood cell counting using hemocytometry?
Poisson’s Law of Distribution is a discrete probability distribution that expresses the probability of a given
number of events occurring in a fixed interval of time or space if these events occur with a known constant
mean rate and independently of the time since the last event. In hemocytometry, the said law states that cells
settle in a random manner. This implies that even though there are differences in cell counts in each square,
they must not go beyond the acceptable difference which is greater than or equal to 15 for white blood cell
count and 20 for red blood cell count. Otherwise, the count is considered as invalid and it is recommended to
charge the hemocytometer again.
5. What is the “inverted L rule?” How is it applied in hemocytometry?
The “inverted L rule” means not counting the cells touching the middle line at the bottom and right sides of the
counting areas. The point of this "rule" is to avoid double counting. If you count all 4 lines on a given square,
then when you move to the adjacent square and do the same you will have counted the cells on the line that
forms the border between those 2 squares twice. This may be hard to visualize with a fairly low cell density,
but at higher densities when you will see cells on every line it's quite clear. This rule ensures that as you
progress through the squares you will have the true total.
6. In the computation for dilution factor, what is the rationale for subtracting 1 unit from the total volume (i.e.,
TV – 1)?
1 unit must be deducted from the total volume when computing for the dilution factor because in the procedure
before charging the hemocytometer, a few drops of blood are discarded from the Thoma pipette. These first
few drops are mainly diluting fluid, hence the need to subtract 1 unit from the total volume.

References:
Batra, S. (2018). Total red blood cell (RBC) count using hemocytometer / neubauer’s chamber (micro
dilution & macro dilution method). Retrieved from: https://paramedicsworld.com/hematology-
practicals/total-red-blood-cell-rbc-count-using-hemocytometer-neubauer-chamber-microdilution-
macrodilution/medical-paramedical-studynotes

Dhage, H. (n.d.). How is RBC Count Done?. Biology Discussion. Retrieved from:
https://www.biologydiscussion.com/hematology-2/blood-cells/how-is-rbc-count-done-types-blood-
cells-biology/80446

Haight, Frank A. (1967), Handbook of the Poisson Distribution, New York, NY, USA: John Wiley & Sons,
ISBN 978-0-471-33932-8

MT Notes.(2017). Enumeration of Blood Cells. Retrieved from: http://mt-

lectures.blogspot.com/2017/08/lecture-12a-enumeration-of-blood-cells.html

Sobolewski, P. (2014). Re: During cell counting in a hemocytometer, why should we not have the cells
touching the middle line at bottom and right?. Retrieved from:
https://www.researchgate.net/post/During_cell_counting_in_a_hemocytometer_why_should_we_not_
have_the_cells_touching_the_middle_line_at_bottom_and_right/540ebe2fd4c118af0e8b45cc/citation/
download.

Aliko, Valbona. (2015). Re: Why does Turk's reagent just eliminate RBC without affecting WBC?.
Retrieved from:
https://www.researchgate.net/post/Why_does_Turks_reagent_just_eliminate_RBC_without_affecting_
WBC/54fed0b1d2fd6429648b45c2/citation/download.