Manual Total Leucocyte Count Sop

MANUAL LEUKOCYTE COUNT

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Medical Unit Laboratory


UNOPETTE 5856 TECHNIQUE

PRINCIPLE

Changes in the QBC count are seen with many infectious, hematologic, inflammatory, and neoplastic
diseases. The variety of diseases makes the WBC a nonspecific test. Its degree of elevation or
depression from reference values often correlates with the severity of the disease process. Following
changes in the WBC count over time can also provide information about the course of an illness.

Performing the WBC count involves diluting a volume of whole blood in a specified volume of a
solution that lyses RBCs. The WBCs are not lysed and can therefore be counted by adding the diluted
specimen to a hemacytometer. Under 100X magnification using bright-light microscopy, leukocytes are
counted. At this magnification their nuclear morphology is clearly visible.

Leukocyte counts obtained with UNOPETTE Test 5856 compare favorably with those obtained using
the Thomas White Cell Pipette with 3% acetic acid. Each sample is run in duplicate.

REAGENTS AND EQUIPMENT

1. Equipment

Microscope equipped for 100X magnification.

Hemacytometer w/cover slip (Neubauer hemacytometer)

2. Materials/Reagents

http://74.125.153.132/search?q=cache:DycTbZhgFz8...+USING+NEUBAUER+CHAMBER&cd=8&hl=en&ct=clnk&gl=in (1 of 8)6/9/2009 12:13:17 PM


UNOPETTE Reservoir No. 5856 containing 0.475 ml of diluent mixture :

Glacial acetic acid . . . 28.6 ml

Distilled water. . . . . qs to 1 liter

UNOPETTE Capillary Pipette. . . 25 ul capacity

Petri plate

Caution: If reservoir is squeezed too hard, the specimen may be expelled through the top of the
overflow chamber, resulting in contamination of the fingers.

Dilution Ratio: Sample to total volume 1:20

3. Preparation–If room temperature EDTA blood is being used the blood should be placed on the
blood rocker for 10-15 minutes to allow adequate mixing of the blood cells. If the blood was
refrigerated, mix the tube for at least 20 minutes. Clean the microscope lens cleaner and paper.

4. Performance Parameters–NA

5. Storage Requirements–Store at room temperature.

STANDARD PRECAUTION: Patient specimens and all materials coming into contact with them should be
handled as if capable of transmitting infections and disposed of with proper precautions. Gloves should be worn
when handling all specimens.

SPECIMEN

1. Patient Preparation–N/A



2. Type–Free-flowing capillary or thoroughly mixed anticoagulated venous blood. EDTA is the
anticoagulant of choice.

3. Handling Conditions–If leukocyte count cannot be performed immediately after blood is
diluted, store reservoir at room temperature. Perform count within three (3) hours of making
dilution.

QUALITY CONTROL

Test is performed in duplicate and results must agree within 10%. If not, repeat test.

At least one cell count control specimen must be analyzed, or a procedural control employed for each 8
hours of patient testing. If infrequently performed, perform prior to all patient testing. This can be
performed with a previously assayed patient sample or by comparison with a visual blood film
concentration estimate.

INTERFERENCES

Nucleated erythrocytes (nuRBC) are preserved in 3% acetic acid and are indistinguishable from
leukocytes at 100X magnification. To distinguish between the two, a differential count is done on a
stained blood smear, noting the number of nucleated red cells per 100 leukocytes. If the number of
nucleated red cells is high, the procedure should be repeated and an average taken. A corrected
leukocyte count must then be made using the following formulas:

No. of nuRBC ÷ 100 + no. of nuRBC x leukocytes/cu mm

= absolute number of nuRBC/cu mm

Leukocytes/cu mm – absolute number of nuRBC/cu mm = corrected leukocyte count



Example: For a blood smear showing 25 nucleated red cells per 100 leukocytes and a total leukocyte
count of 10,000, the following calculations would be done:

25 x 10,000 = 2,000

125

10,000 – 2,000 = 8,000 leukocytes/cu mm

PROCEDURE

PRECAUTION: Patient specimens and all materials coming into contact with them should be handled as if
capable of transmitting infections and disposed of with proper precautions. Gloves should be worn when
handling all specimens.

1. PUNCTURE DIAPHRAGM. Using the protective shield on the capillary pipette, puncture the
diaphragm of the reservoir as follows:

a. Place reservoir on a flat surface. Grasping reservoir in one hand, take pipette assembly in other
hand and push tip of pipette shield firmly through diaphragm in neck of reservoir, then remove.

b. Remove shield from pipette assembly with a twist.

2. ADD SAMPLE. Fill capillary with whole blood and transfer to reservoir as follows:

a. Holding pipette almost horizontally, touch tip of pipette to blood. Pipette will fill by capillary
action. Filling is complete and will stop automatically when blood reaches end of capillary bore
in neck of pipette.



b. Wipe excess blood from outside of capillary pipette, making certain that no sample is removed
from capillary bore.
c. Squeeze reservoir slightly to force out some air, but do not expel any liquid. Maintain pressure
on reservoir.
d. Cover opening of overflow chamber of pipette with index finger and seat pipette securely in
reservoir neck.
e. Release pressure on reservoir. Then remove finger from pipette opening. Negative pressure will
draw blood into diluent.
f. Squeeze reservoir gently two or three times to rinse capillary bore, forcing diluent into, but not
out of, overflow chamber releasing pressure each time to return mixture to reservoir.
g. Place index finger over upper opening and gently invert several times to thoroughly mix blood
with diluent.

h. Let stand for ten (10) minutes to allow red cells to hemolyze. Leukocyte count should then be
performed within three (3) hours.

3. CHARGE HEMACYTOMER. Mix diluted blood thoroughly by inverting reservoir to
resuspend cells.

a. Convert to dropper assembly by withdrawing pipette from reservoir and reseat securely in reverse
position.

b. To clean capillary bore, invert reservoir, gently squeeze sides and discard first three or four drops.
c. Carefully charge hemacytometer with diluted blood by gently squeezing sides of reservoir to
expel contents until chamber is properly filled. If fluid flows into the grooves at the edges of the
chamber or if air bubbles are seen in the field, the chamber is flooded and must be cleaned with
distilled water, dried with lens tissue, and reloaded. If the chamber is underloaded, carefully add
additional fluid until properly loaded.
d. Place the loaded hemacytometer into a petri dish with a piece of dampened tissue to keep the
hemacytometer from drying out. Allow 5 minutes for the cells to settle.



4. COUNT AND CALCULATE. A leukocyte count is performed with a Neubauer
hemacytometer as follows:

a. Once the cells have settled, place the hemacytometer on the microscope. Use the low-power lens
to locate the 4 corner squares (A, B, C, D)

b. Switch to the high power lens. Count all of the leukocytes in the four (4) corner squares,
including cells touching the lines at the top and on the left. Do not count any cells that touch the
lines on the right or at the bottom.
c. Multiply the total number of cells counted by 50 to get total leukocyte count.

EXAMPLE: If 120 cells are counted, total count is: 120 x 50 = 6000 leukocytes/cu mm.

LIMITATIONS OF PROCEDURE

A highly elevated leukocyte count may make accurate counting difficult. In this instance, a secondary
dilution should be made. When calculating the total count, adjust the formula to allow for secondary
dilution.

In order to avoid increasing the imprecision of particle counting, an increased number of hemocytometer
squares must be enumerated when there is leukopenia or thrombocytopenia.

NORMAL VALUES

Adult . . 4,500 – 11,000 WBC/cu mm

One-year old . 6,000 – 17,500 WBC/cu mm

Newborn. . 9,000 – 30,000 WBC/cu mm



INTERPRETATION

Because the main white cell groups, e.g., neutrophils, eosinophils, basophils, moncytes, and
lymphocytes, represent physiologically independent systems under varying controls, it is difficult to
define a normal white blood cell count. The lower limits of the leukocyte count may be depressed to
3,000 cell/ cu mm in white individuals and to 1,500 cells/cu mm in blacks and still be within physiologic
limits. The count is not only influenced by race but also by age and somewhat by sex. As neutrophils
make up a major sector of the total white cell count, their fluctuations materially affect the result of the
total count. The neutrophilic peripheral blood pool is visualized as having two compartments, the
circulating and the marginal pools. The first is evaluated by the peripheral blood count and represents
about 50% of the available peripheral white cells. It is in constant flux as there is a rapid exchange from
one pool to the other. Demargination of neutrophils is encouraged by epinephrine, stress, exercise, time
of day, exposure to light, the postprandial state, lithium, and inflammation, while margination is favored
by anesthesia, barbiturates, and tranquilizers. Repeat white cell count should be performed at the same
time with the patient in basal condition.

PROCEDURE NOTES

Sources of error

1. Apparatus – non-optically plane cover glasses, dirty glassware, inaccurate rulings on chamber,
chipped pipette tips

2. Personal technique

q Poor specimen collection – excessively squeezing a specimen from a finger stick
q Not thoroughly mixing blood
q Inadequate shaking
q Failure to discard first 4 drops
q Not loading chamber properly (overfilling/underfilling, trapped air bubbles)
q Counting cells inaccurately (skipping cells, counting cells twice, counting on wrong borders)
q Calculation error
q Clerical error



3. Inherent errors in hemocytometery

q Field errors – related to the random distribution of cells on the counting chamber
q Statistical error – occurs when total number of cells is too low to give statistical
confidence in result (this error is reduced when larger numbers of cells are counted)

REFERENCES

1. Wintrobe MM: Clinical Hematology, ed 6, Philadelphia, Lea & Febiger, 1967, p. 425.

2. Data on file at Becton Dickinson and Company, Rutherford, NJ. 07070. Available upon request.

3. Lampasso JA: Changes in hematologic values induced by storage of ethylenediaminetetracetate
human blood for varying periods of time. Amer. J. Clin. Path. 49: 443 (Mar) 1968.

4. Davidsohn I, Henry JB (eds): Todd-Sanford Clinical Diagnosis by Laboratory Methods, Ed 14,
Philadelphia, W.B. Saunders Company. 1969, p. 156.

5. Altman PL, Dittmer DS (eds): Biology Data Book, Washington, D.C. Fed. Amer. Soc. Exp. Bio.
1964, pp 272, 273.

4/29/07 Section - Hematology Procedures Page of 5


Manual Total Leucocyte Count Sop

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