Page 687 - Clinical Hematology_ Theory _ Procedures ( PDFDrive )

CHAPTER 32 ■ Laboratory Manual: Manual Procedures in Hematology 671

HEMATOLOGY PROCEDURES (continued)

coverslip or the hemocytometer is place over the chambers 5. Begin counting in the upper le -han square an count

or cell counting. T e our large outer squares are use or the leukocytes, which appear as roun ark particles.

leukocyte counting. Continue to count in the remaining three squares.

T e variation in the number o cells counte in the our
Quality Control squares shoul not i er by more than 5 to 10 cells.

A normal control specimen shoul be counte . 6. Count the leukocytes on the other si e o the hemocytom-

eter; this total shoul be within 10 cells o the other si e.
Procedure Average the count rom both si es.

1. Follow the manu acturer’s irections or specimen prepa- 7. T e total leukocytes counte on each si e are a e

ration an f lling o the counting chamber. together an ivi e by 2 to obtain the average. It is
2. o each si e o the chamber o a clean hemocytometer important to note that the istribution o cells shoul be

with a coverslip, loa a small amount o the ilute bloo . roughly equivalent through the count an that no clumps
Allow the counting chamber to sit covere , with a moist- o cells shoul be seen. I clumping is note , another ilu-

ene f lter paper in hal o a Petri plate, or a ew minutes tion must be ma e.
to allow the cells to settle an the erythrocytes to com- 8. Soak the hemocytometer in a 10% bleach solution to is-

pletely lyse. in ect. Discar the capillary pipette an contaminate
3. Place the hemocytometer un er the 10× microscopic supplies in a biohazar bag.

objective (low power) to ocus using either a phase-con-
trast optics or bright f el (lowere con enser) at 10× CALCULATIONS

magnif cation or the total count. Erythrocytes shoul be Shortcut Formulas
lyse . T e nucleus o polymorphonuclear segmente neu-

trophils will be bright, but the lymphocyte nucleus will be otal leukocyte count rom 4 large corner squares × 0.05 =
9
roun . WBCs × 10 /L

4. T e leukocytes in all large our corner squares that are or
urther rule into 16 smaller squares in each large square, otal leukocyte count rom 4 large corner squares × 50 =

o each si e o the chamber, shoul be counte (see WBCs/µL
Fig. 32.1). I cells touch the inner or mi le lines o two

a jacent lines, or example, upper an le -han si e, they Classic Formula
can be counte . Cells touching the outer lines or the oppo- Dilutional Factor = 20. T is represents the reciprocal o the

site a jacent lines shoul not be counte . 1:20 ilution o bloo .
Number o squares counte = 4

Area o each square = 1 mm 2

Depth o solution = 0.1 mm

otal number o cells counte × Reciprocal
W W o the ilution = cell mm 33

/
Number o sqquares counte × Area o each

×
square Depth o the solution

R R Example: I the average total o leukocytes counte was 180,

the total leukocyte count woul be:

R 180 20 3 9 )
×
90 10 /µ
.
×
41mm 2 × 0 1mm = . × Lor 9 0 10 L. × / (SI units
R R
Reporting Results

Re erence value: 4.5 to 11.0 × 10 /L
9
W W Procedure Notes

Sources of Error

Erroneous results can be ue to contaminate iluting ui ,

incorrect ilution or loa ing o the hemocytometer, or an uneven

FIGURE 32.1 Neubauer counting chamber. (R, re cell area; istribution o leukocytes in the counting chamber. Prompt

W, white cell area.) counting o cells is important to the accuracy o the count.

(continued)