1. Hemacytometer and Manual Cell Counts
Kimberly Murray
University of Phoenix
EDTC 560:
Applications of Multimedia and Web Page Design
June 4, 2007

2. Quote of the Day
“Education is a technique employed to open minds so that they may go from profound ignorance to thoughtful uncertainty” (Mitner, 1999).

3. Objectives Upon completion of this course, the student will:
Identify the dimensions of the Neubauer hemacytometer.
Identify sources of error in counting.
Calculate manual cell counts.

4. Hemacytometer Counting Chamber
A precision tool designed to standardize counting.
Used to count cells in peripheral blood or other body fluids.

5. Basic hemacytometer formula
Number of cells counted X dilution factor
total volume of the cells counted
total volume = # primary squares x 0.1 (depth)
This formula can be used with any counting chamber as long as one knows the dimensions for the chamber

6. Hemacytometer Components
Cover slip –NBS standardized
Moat
Fill trough
Chambers or
Counting grid
Diagrams by K. P. Murray, 2007

7. Sample is placed between cover slip and chamber
The design of the hemocytometer is to have the support for the cover slip at a specific height, resulting in a known depth of exactly 0.1mm.
Cover slip –NBS standardized
Moat
0.1 mm
Depth
National bureau of standards must be met for both the chamber and cover slip.
Sample
Diagrams by K. P. Murray, 2007

8. Do Not Use Disposable Cover Slip
Sample floats coverslip
Do not use a regular cover slip especially for viscous fluids. The sample will float disposable coverslips and the depth will be greater than 0.1mm.
Diagrams by K. P. Murray, 2007

9. Neubauer Hemacytometer Dimensions
Each side (chamber) has a ruled area
3 mm wide and 3 mm long
Divided into nine squares
The large squares are subdivided into 16
The middle square is subdivide into 25
Each of the 25 is again subdivided into 16
When the dimensions are known, one will always be able to calculate the area counted.

10. Dimensions of the Neubauer
Note: Center primary square has different divisions
Primary squares
1.0 mm
Secondary Squares
0.2mm
0.25mm
Tertiary squares mm
Diagrams by K. P. Murray, 2007

11. Hemacytometer Quality Control (QC)
Ensures accuracy
Performed by duplicating the counting area exactly.
For example:
If squares 1,3,5,7,9 are counted.
Then 10,12,14,16,18 are counted on the other side.
QC is duplicating the counts
if you count squares 1,2,3 then on the other side you count 10, 11, 12
Diagrams by K. P. Murray, 2007

12. Area Counted Depend on Cell Type
WBC
WBC . .
Platelet
RBC
RBC
RBC
RBC
RBC . .
WBC
WBC
Diagrams by K. P. Murray, 2007

13. RBC Count Method
Count the five inner tertiary squares on both sides of the chamber
on Peripheral Blood
Check difference in counts
Calculate RBC count using the basic hemacytometer formula . .
RBC
RBC
RBC
RBC
RBC . .
Diagrams by K. P. Murray, 2007

14. RBC Count Method If a Body Fluid count is less than 100 cells
count all 9 squares on both sides to provide greater accuracy.
Adjust the hemacytometer calculation accordingly . . . .
Diagrams by K. P. Murray, 2007

15. WBC Count Method
Count the four outer secondary squares on both sides of the chamber
Check difference in counts
Calculate WBC count using the basic hemacytometer formula
WBC
WBC
WBC
WBC
Diagrams by K. P. Murray, 2007

16. WBC Count Method If WBC count is less than 1000 cells
count all 9 squares on both sides to provide greater accuracy.
Adjust the hemacytometer calculation accordingly
Diagrams by K. P. Murray, 2007

17. Platelet (PLT) Area Counted
Diagrams by K. P. Murray, 2007

18. Neubauer Hemacytometer Calculation
Cells X dilution = cells/L
squares X 0.1
Note: 0.1 mm depth is constant in Neubauer Hemacytometer

19. Unopette – used today for manual counts
Components
Reservoir
Capillary pipette
Protective sleeve that also is used to open the reservoir.
Pre-measured diluents in vials.
Capillary pipette is specific for the diluent used.
The capillary tubes can not be interchanged.
Premeasured diluents in vials. And a specific capillary pipette for the diluent needed. The capillary tubes can not be interchanged.
Picture by K. P. Murray, 2007

20. Unopette System For RBC, WBC and PLT counts
RBC vial
Isotonic or physiologic saline
1/200 dilution
WBC only vials
Turk’s solution
1/20 dilution
WBC/PLT vials
Ammonium oxalate g
Sorenson’s buffer--1.0g
Thimerosal g
(preservative)
Distilled H2O qs to 1 liter
1/100 dilution
RBC can not resist osmotic pressures, as the WBC can, therefore RBC counts need isotonic solutions
Manual RBC counts in PB rarely done
Always performed on other body fluids

21. Unopettes Test name Sample diluent Type of diluent Final dilution WBC
Note: different final dilutions
Test name
Sample
diluent
Type of diluent
Final dilution
WBC
25 L
0.475 ml
TURKS- 3% acetic acid
1:20
RBC
10 L
1.99 ml
Isotonic saline
1:200
PLATELET
20 L
1.98 ml
1% ammonium oxalate
1:100
Eosinophils
0.775 ml
Phloxine B
1:32

22. Loading Chamber using a Unopette
Picture by K. P. Murray, 2007

23. “Wet Box” prevents solution from drying out and altering count
Settle cells to the same optical plane
WBC
2-3 minutes
PLT
10 minutes (take the longest to settle)
RBC
Picture by K. P. Murray, 2007

24. Acceptable differences between chambers of the hemacytometer
No more than 10 cell variation between the 4 squares for the WBC count.
The difference between the highest and the lowest number should not be greater than 25 cell for the 10 tertiary squares (5 squares per side) the RBC count.
Diagrams by K. P. Murray, 2007

25. How to Tell If Sample has Uneven Distribution
Difference between individual squares exceeds 10%
Difference between each chamber count exceeds 20 %
REPEAT COUNT with a freshly loaded hemacytometer, if the count does not match the above criteria.

26. WBC Count Reporting Report WBC Number (period) one decimal X 103/ L
Number. decimal X 103/ L
Example: 10.9 X 103/ L
WBC has one decimal place, if sample is peripheral blood.
For other body fluids (BF), report whole number, no scientific notation or abbreviations.

27. RBC Count Reporting Report RBC Number (period) two decimals X 106/ L
Number. two decimals X 106/ L
Example: 4.13 X 103/ L
RBC has two decimal places, if peripheral blood.
For other body fluids (BF), report whole number, no scientific notation or abbreviations.

28. Platelet Count Reporting
Report PLT
Whole number (no decimal) X 103/ L
Example: 428 X 103/ L
Platelet count does not have a decimal point.
Other body fluids do not report platelet counts.
Verbal reporting always give the full number i.e. “Dr. the platelet count on Ms Smith is 428, 000”

29. Platelet Count Reporting
Verbal reporting always give the full number.
For example, “Doctor Jones, the platelet count on Ms. Smith is 428,000”.
DO NOT just say the count is 428.
The doctors or the nurse may think only four hundred (transfusion time!!!), rather than a normal count of 400 thousand.
Verbal reporting always give the full number i.e. “Dr. the platelet count on Ms Smith is 428, 000”

30. Reference Units Notice the similarities and differences between units
Conventional units
Example
SI units
WBC
X 103/ L
4.5 X 103 / L
X 109 / L
4.5 X 109 / L
RBC
X 106/ L
5.21 X 106/ L
X 1012 / L
5.21 X 1012 / L
HGB
g/dL
15.4 g/dL
g/L
154 g/L
MCV fl
88 fl
MCH pg
29 pg
MCHC %
33 %
330 g/L
PLT
182 X 103/ L
182 X 109 / L

31. Sources of Error Failure to mix sample prior to dilution
Pipeting technique
Uneven distribution in chamber
Failure to remove excess blood from outside of pipette
Squeezing out some fluid from reservoir [Unopette]
Nature of sample:
Clotted
not mixed thoroughly
Errors caused by apparatus:
1. chipped pipette tips
2. obscure markings on pipettes
3. non-optically plane cover glasses (wrong cover slips)
4. dirty glassware
5. inaccurate rulings on chamber( use certified by the Bureau of Standards)
3) Errors caused by personal technique:
Pipetting- not wiping the outside of tip
1. not thoroughly mixing blood
2. inadequate shaking (thoma pipettes)
3. failure to discard first 4 drops
4. not loading chamber properly (under or overfilling, trapped air bubbles)
Moving cover slip
5. counting cells inaccurately (skipping cells, counting cells twice, counting on wrong borders)
6. calculation error
7. clerical error
(3) Inherent errors in hemocytometry include:
1. "field errors" - relates to the random distribution of cells on the counting chamber
2. 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)

32. Sources of Error Cell aggregates (clots)
Bacteria ( may be mistaken for plt)
Dirt, talc, debris
Drying of the chamber
taking too long to count
Falsely increase counts

33. Fill problems Correct fill Coverslip Air bubbles Overfill Underfill
Diagrams by K. P. Murray, 2007

34. Problem 1 A blood sample was diluted1:20. On a Neubauer hemacytometer
140 WBC counted in the four outer squares on side A.
147 WBC counted on the same four squares on side B.
What is the WBC count?

35. Problem 1 answer WBC 140 and 147 ‘match’ and average 143.5
Depth = 0.1 (always with a Neubauer)
143.5 cells x 20 (dilution factor)
4 (squares) x 0.1(depth)
WBC = 7,175
WBC =7.2 x 10 3 /dL

36. Problem #2
A tech counted both side of the hemacytometer on an undiluted spinal fluid sample.
22 WBC were counted and no RBC.
What is the WBC count?
How is it reported?

37. Problem #2 solution Both side area = 18 mm2 {(1x1) (9+9)} Depth = 0.1
Both side count = 22
Dilution = 1
Answer: WBC = 12 cells / dL
NOTE: no decimal or scientific notation because it is a body fluid and one does not report a fraction of a cell.

38. References
Barry T. Mitzner, (1999), D.V.M, Southeast Vetlab, Inc., Retrieved on July 12, 2003 from
Murray, K.P.,(2007) Hematology Lab Photographs (personal collection)
Murray, K.P.,(2007) Hematology Diagrams and Animations (personal collection)
END