Lab objective




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EXERCISE 8: Manual Leukocyte Count MLAB 1315 Hematology



MANUAL WBC
LAB OBJECTIVE

The student will be able to perform, within 20% accuracy as compared to the automated result, five manual white blood cell counts using the Unopette system.


PRINCIPLE

Free-flowing capillary or well-mixed anticoagulated (EDTA) venous blood is added to a diluent (ammonium oxalate) at a specific volume in the Unopette reservoir. The diluent lyses the erythrocytes but preserves leukocytes and platelets. The diluted blood is added to the hemacytometer chamber. Cells are allowed to settle for 10 minutes before leukocytes and platelets are counted.


SPECIMEN

EDTA-anticoagulated blood or capillary blood is preferred.


REAGENTS, SUPPLIES AND EQUIPMENT

Unopette reservoir no. 5854/5855 (1.98 ml of diluent + 0.02 ml blood = 1/100 dilution))

Ammonium oxalate 11.45 g

Sorensen’s phosphate buffer 1.0 g

Thimerosal 0.1 g

Purified water qs to 1000 mL

Unopette capillary pipet, 20-µL capacity

Hemacytometer and coverslip

Microscope

Lint-free wipe (Kim Wipes)

Alcohol pads

Hand counter

Petri dish with moist filter paper








Note on the Hemacytometer

The hemacytometer counting chamber is used for cell counting. It is constructed so that the distance between the bottom of the coverslip and the surface of the counting area of the chamber is 0.1 mm.




The surface of the chamber contains two square ruled areas separated by an H-shaped moat. These two squares are identical, allowing the technologist to duplicate the cell count. Each has a total area of 9 mm2 (1 mm on each side). These squares are divided into nine primary squares with an area of 1mm2. The four corner primary squares are used when counting leukocytes. (If the WBC is very low, you would count all 9 squares.) These 4 large corner squares contain 16 smaller secondary squares, each with an area of 0.04 mm2. All 25 secondary squares of the center primary square are used to count platelets, and each of these 25 squares is further divided into 16 smaller tertiary squares (see figure).


Cells which lie on LEFT and TOP boundary lines of the squares are counted. Cells which lie on the RIGHT and BOTTOM boundary lines of the squares are NOT counted. (This is to make cell counts statistically accurate.)


Hemacytometers and coverslips should meet the specifications of the National Bureau of Standards and are so marked by the manufacturer. A standardized coverslip should be used that has been ground to fit the specifics of the hemacytometer, ensuring a uniform depth and therefore a constant volume. A regular coverslip cannot be used.
QUALITY CONTROL

A normal control specimen should be counted. Perform estimated WBC from Wright-stained peripheral smear to confirm result.



NOTE: For this lab exercise, QC will be precision check with automated WBC.




PROCEDURE

Refer to figure for following instructions.

  1. Using the protective shield on the capillary pipette, puncture diaphragm of Unopette reservoir.

  2. Remove shield from pipette assembly by twisting.

  3. Holding pipette almost horizontally, touch tip of pipet to well-mixed blood. Pipet will fill by capillary action. Filling will cease automatically when the blood reaches the end of the capillary bore in the neck of the pipet.

  4. With a Kim Wipe, wipe the outside of the capillary pipet WELL (from top of capillary pipet to the bottom of the pipet) to remove excess blood that would interfere with the dilution factor. Avoid touching the tip of the pipet with a Kim Wipe to avoid removing any blood from the pipet.

  1. With one hand, squeeze reservoir slightly to force out some air while simultaneously maintaining pressure on reservoir.

  2. With the other hand, cover the opening of overflow back chamber of pipet with index finger. Place the pipet securely in neck of the reservoir. Twist the pipet firmly into the reservoir neck to form a tight seal.

  3. Release pressure from the first hand on reservoir. Then remove finger on the opposite hand from pipet opening. At this time negative pressure will draw blood into reservoir.

  4. Squeeze reservoir gently two or three times to rinse capillary bore forcing diluent up into, but not out of, overflow chamber, releasing pressure each time to return mixture to reservoir.

  5. Place index finger over upper opening and gently invert several times to thoroughly mix blood with diluent.

  6. Let mixture stand 10 minutes before charging the hemacytometer.to allow the red cells to lyse.

  7. While the Unopette is standing for 10 minutes, thoroughly clean the hemacytometer and its coverslip with an alcohol pad and then dry with a Kim Wipe.

  8. To charge the hemacytometer, convert to dropper assembly by withdrawing pipet from reservoir and reseating securely in reverse position.

  9. Thoroughly mix the blood/diluent mix by swirling the assembly. Invert reservoir and discard the first 3 or 4 drops of mixture.

  10. Carefully charge cleaned hemacytometer with diluted blood by gently squeezing sides of reservoir to expel contents until chamber is properly filled. The chambers must be completely filled without bubbles (See A below), but the blood/diluent mixture must NOT overfill the chambers (See B below).






  1. Without disturbing the coverslip, place hemacytometer in moist Petri dish for 10 minutes to allow white cells to settle into the same plane. (Moistened filter paper retards evaporation of the plated specimen while standing.) Do not let the hemacytometer sit more than 15 minutes in the Petri dish because evaporation will begin to occur and cause erroneous counts.

  2. Without disturbing the coverslip, mount the hemacytometer on the microscope and lower the condenser.

  1. Procedure for counting WBC’s



  1. Under 10x magnification, scan to ensure even distribution. Leukocytes are counted in THE FOUR OUTSIDE large squares of counting chamber.

  2. Count cells starting in the upper the left top large corner square. Move to the upper right corner square, bottom right corner square, and end in the bottom left corner square.

  3. Count all cells that touch any of the upper and left lines, do not count any cell that touches a lower or right line.








  1. Calculation for counting 4 large squares:

1) Use the average total number of cells counted from both sides of the hemacytometer (One side includes the total number of cells counted in four large squares and the other side includes the total number of cells counted in its four large squares).

2) Final cell count is reported as the number of white blood cells per microliter (WBC/μL).


3) Formula:

Average of cells = the average of the total number of cells counted in the four large squares on BOTH sides of the hemacytometer
Correction for dilution = the dilution factor (which is the reciprocal of the blood

dilution) The dilution when using the WBC Unopettes is 1/100, so the dilution factor is 100.



Number of squares counted = four (4)
Volume of one large square = 0.1 μL
EXAMPLE:
WBC/µL = Avg # cells counted x 100

# squares counted x 0.1/µL


Number of cells counted in side 1 = 27

Number of cells counted in side 2 = 29

Average Number = 28
WBC/µL = 28 x 100 = 7.0 x 103/µL

4 x 0.1
RECORDING YOUR RESULTS

Use the form provided to report your results. Record WBC’s counted in each square before proceeding to the next square.
Precision: The ability to reproduce a test result on the same sample. To determine if the two counts are in close enough agreement, use the following procedure:


  1. Obtain the difference (C) between the Side A and Side B of the hemacytometer.

  2. Calculate 2 S.D. as follows:



  1. If C is less than 2 S.D., the results are acceptable. If greater, the counts must be repeated.

Example:

A = 430


B = 470

C = 40



C (40) is less than 2 S.D. (60) and therefore is acceptable. If C is greater than 2 S.D., the results are not precise (not close together enough) and the counts must be repeated. Re-clean the hemacytometer, re-mix the Unopette dilution, re-load the hemacytometer, etc.
Accuracy: The closeness of a test result to the true value. To determine if the two counts are accurate (within 20% of the automated count), use the following procedure:

Example:


Automated count = 7.0 x 103/μL.

7.0 x 103/L - 20% = 5.6 x 103/μL

7.0 x 103/L + 20% = 8.4 x 103/μL
Acceptable range is 5.6 - 8.4 x 103/μL
Manual count = 6.0 x 103/μL so manual count is acceptable.
REPORTING RESULTS

Normal Values

Newborn 9.0-30.0 x 103/μL

1 week 5.0-21.0 x 103/μL

1 month 5.0-19.5 x 103/μL

6-12 months 6.0-17.5 x 103/μL

2 years 6.2-17.0 x 103/μL

Child/adult 4.8-10.8 x 103/μL


NOTES

  1. Diluent and blood should be properly mixed before filling the hemactyometer.

  2. The hemacytometer must be properly filled to avoid erroneous results in manual cell counting. If the chamber is overfilled, as indicated by the presence of excess fluid in the moat of hemacytometer, clean hemacytometer and recharge chamber. As with all manual counts, the diluent sample must be thoroughly mixed before charging the hemacytometer, which must be properly filled.

  3. The chamber must be well-cleaned and free of debris which could be mistaken for leukocytes.

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

  5. There are physiologic variations to consider when performing WBC counts. Higher WBC counts (leukocytosis) are seen following exercise, emotional stress, anxiety, and food intake. Increased leukocyte counts are also seen in certain disease conditions such as bacterial infections, inflammation and leukemias. Decreased leukocyte counts (leukopenia) are seen in viral disorders, radiation induced leukopenia, chemotherapy induced leukopenia, aplastic anemia and megaloblastic anemia.

  1. If more than 10 nucleated RBC’s are seen on the differential, the total leukocyte count should be corrected using the following calculation:

Corrected WBC = Total leukocyte count x 100

100 + # of NRBC’s/100 WBC’s on differential
REFERENCES

Harmening., Denise, Clinical Hematology and Fundamentals of Hemostasis, 3rd edition, pp. 593-599.

Turgeon, Mary Louise, Clinical Hematology - Theories and Procedures, 3rd edition, pp320-321.

Manual WBC Report Form for Lab Exercise
Student’s name:____________________________________________Date:________________
Unopette Lot #:_____________________________ Expiration Date:______________


Patient ID


Patient ID


Side 1

Side 2

Side 1

Side 2

1.

1.

1.

1.

2.

2.

2.

2.

3.

3.

3.

3.

4.

4.

4.

4.

Total:

Total:

Total:

Total:

Average:

Average:

Difference between results:____________

Calculate 2 S.D. for precision:

Is the difference less than 2 S.D.? ___________


Difference between results:____________

Calculate 2 S.D.for precision:

Is the difference less than 2 S.D.? ___________


Calculation of result:


Manual WBC result:______________________


Calculation of result:


Manual WBC result:______________________


Automated result:________________________

(Obtain from instructor for accuracy)

Automated result range 20%:______________


Automated result:________________________

(Obtain from instructor for accuracy)

Automated result range 20%:______________


Is manual result within 20% of automated result? _________________


Is manual result within 20% of automated result? _________________




STUDY QUESTIONS
Name _______________________________
Date_________________________________


2 pts

1.


2.Calculate the following white cell count if the dilution of the Unopette is 1/100. Show your calculations.

Side #1 Side #2

Square 1 = 30 white cells Square 1 = 27 white cells

Square 2 = 28 white cells Square 2 = 27 white cells

Square 3 = 25 white cells Square 3 = 24 white cells

Square 4 = 27 white cells Square 4 = 24 white cells





2 pts

2.


3.Calculate the following white cell count if the dilution of the Unopette is 1/100 and 9 squares were counted. Show your calculations.

 Total cells counted on side 1 = 36

Total cells counted on side 2 = 32


2 pts

3.


4.You are performing a manual white blood cell count. You have made the dilution and have filled the hemacytometer. A stat urinalysis is brought to the laboratory and you leave the hemacytometer sitting on the microscope stage while you perform the UA. Fifteen minutes later you return to complete the count. Should you count the dilution on the hemacytometer? Why or why not?




1 pt

4.


5.Why must the diluted blood sit in the Unopette for 10 minutes before it is plated on the hemacytometer?


1 pt

5.


6.Why must the loaded hemacytometer sit for 10 minutes before the manual WBC is read under the microscope?


2 pts

6.


7.You have performed a manual WBC and the counts from the two sides of the chamber are 232 and 268. Are the counts close enough to accept? Show your calculations.


2 pts

7.


8.State normal WBC reference ranges for:
Newborn ______________
Child/Adult_____________

8 pts

8.


9.State four sources of error and indicate if the cell count would be falsely increased or decreased.

A.
B.


C.
D.


1 pt

9.


10.State the diluent that is used for Unopette counts.



1 pt

10.


11.State the formula for correcting a WBC count if NRBCs are present.





2 pts

11.


12.Calculate the corrected WBC count for the following:

Show your calculation.


WBC count = 25,000/µL

# of NRBCs = 35/100 WBC




4 pts.


12.

13.Define leukopenia and leukocytosis.



  1. State 4 associated conditions for leukopenia.

A.
B.
C.
D.

2 pts.



  1. State 2 disease (pathologic) conditions for leukocytosis.

A.
B.



4 pts


  1. State 4 physiologic conditions that can cause an elevation in the WBC.

A.
B.
C.
D.


2 pt


  1. How can the two counts of a manual WBC be confirmed on a manual hemacytometer (precision)?



LAB EXERCISES MLAB 1315*




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