Practical Physiology: Manual WBC Count

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Questions and Answers

What is a common cause of low cell counts during manual counting?

Insufficient mixing of the cell suspension (correct)

Inclusion of contaminants in the sample

Overfilling the counting chamber

Long delay in performing the count after filling the chamber (correct)

What condition is indicated by an increase in white blood cell count?

Anemia

Thrombocytopenia

Leukocytosis (correct)

Leukocytopenia

Which factor may contribute to inaccurate high white blood cell counts?

Mixing the blood with diluent thoroughly

Working swiftly after preparing the sample

Using a properly calibrated pipette

Drawing excessive blood into the pipette (correct)

Which of the following reference ranges is applicable to adult males for white blood cell count?

4-11 109 /L Signup and view all the answers

What is a likely reason for leukocytopenia in patients?

Long-term use of broad spectrum antibiotics Signup and view all the answers

How many squares are there in the central large square?

25 Signup and view all the answers

What is the depth of the chamber used for counting white blood cells?

0.1 mm Signup and view all the answers

Which squares should not be counted according to the counting rules?

Cells touching the lower and right limits Signup and view all the answers

What dilution should be used when the white cell count is very high?

1:100 Signup and view all the answers

What should be avoided to ensure accurate counts in the haemocytometer?

Contaminating the diluent fluid Signup and view all the answers

What happens if the chamber is left in direct light before counting?

It leads to evaporation Signup and view all the answers

What is the recommended procedure when the white cell count is very low?

Repeat with a smaller dilution Signup and view all the answers

How many small squares are formed when each of the 25 squares is subdivided into 16 squares?

256 Signup and view all the answers

What is the main purpose of the manual white blood cell counting test?

To count the number of white blood cells in a blood sample. Signup and view all the answers

Which of the following solutions is used to dilute the blood sample for WBC counting?

Buffered ammonium oxalate or glacial acetic acid. Signup and view all the answers

When performing the WBC count using a Neubauer counting chamber, what is the dilution ratio of the blood specimen?

1:20. Signup and view all the answers

What type of microscope is typically used to count the white blood cells in this practical?

Compound microscope. Signup and view all the answers

Identify a common source of error in the manual WBC counting procedure.

Failing to completely lyse the red blood cells. Signup and view all the answers

What is the normal reference range for white blood cells in adult males?

5,000 to 10,000 cells per cubic mm. Signup and view all the answers

Which type of fluid is primarily used for diluting white blood cells in this procedure?

1% Acetic acid. Signup and view all the answers

What is the objective magnification used during the counting of white blood cells?

10x objective. Signup and view all the answers

What is the first step in the procedure for counting white blood cells?

Transfer 380µL of 1% acetic acid into a small Kahn tube Signup and view all the answers

What is the dilution achieved by mixing 20µL of blood with 380µL of acetic acid?

1:20 Signup and view all the answers

How long should the solution be left to ensure complete lysis of the cells?

2-3 minutes Signup and view all the answers

What is the recommended magnification for counting the leukocytes in the haemocytometer?

x10 Signup and view all the answers

What should be done if there is more than a 10% difference in cell counts between the two sides of the chamber?

Repeat the test with a new sample Signup and view all the answers

Which part of the haemocytometer should be cleaned before use?

The central area Signup and view all the answers

What is the size of the Neubauer chamber's counting grid?

3 mm x 3 mm Signup and view all the answers

When filling the haemocytometer, which side should be disregarded when counting overlapping cells?

None of the sides Signup and view all the answers

Study Notes

Practical Physiology: Manual White Blood Cell Count

Lab: Practical physiology lab (3), 2nd year, 2023-2024
Lecturer: Karrar Abduljabar Alqershi, M.Lab.Med
Topic: Manual White blood cells count

Learning Objectives

Understand the purpose, idea and principle of manual WBC counting
Count and interpret results
Understand calculation procedures
Know sources of errors
Know normal reference ranges (adult males and females)
Recognize when WBCs increase or decrease and why

Purpose of the Test

The student will perform and count white blood cells using Neubauer's counting chamber (hemocytometer)

Idea of the Test

Blood is diluted in buffered ammonium oxalate or glacial acetic acid to lyse red blood cells (RBCs).
This preserves platelets, leukocytes, and reticulocytes.
After complete lysis, the solution is clear, allowing for accurate counting.

Principle of the Test

Blood is diluted 1:20 with WBC diluting fluid (1% acetic acid).
Cells are counted using a counting chamber (low power 10x or high power 40x objective).
Undiluted blood cell counts are calculated and reported as white blood cells per cubic millimeter of whole blood

Materials and Equipment

1% Acetic acid (WBC diluting fluid)
Pipettes and tips
20µL Sahli pipette
Kahn tubes
EDTA anticoagulated or capillary blood
Microscope
Improved Neubauer haemocytometer
Ethanol 70%
Lancets

Method

Transfer 380µL of 1% acetic acid to a tube.
Wipe finger with alcohol, prick, and collect 20µL blood into the pipette and tube.
Mix the blood and acetic acid.
Clean and dry the haemocytometer and coverslip.
Place the coverslip on the haemocytometer.
Fill the chamber with the diluted blood sample.
Allow cells to settle.
Count WBCs in the four corner squares of the haemocytometer's grid.
Exclude cells touching the edge.
Count WBCs in all 4 corner squares, maintain consistency for each count.
The test may be rerun if there's a significant difference between opposing corner squares.

Counting Area

Neubauer chamber grid: 3mm x 3mm; has 9 squares.
Each square has 1mm x 1mm, which is further subdivided into 16 squares.
Each small square has an area of 0.0025 mm².
The depth of the chamber = 0.1 mm
Each grid has depth 0.1 mm.

Calculation

Count WBCs from the 4 corner squares.
Add the WBC counts (from all squares) from each side.
Calculate the average WBCs per chamber.
Calculate the number of WBCs per cubic mm; use WBCs per chamber multiplied by the dilution (20) and divide by depth of chamber(0.4)

Clinical Significance

Increased (Leukocytosis): Infection, stress, inflammatory disorders, leukemia.
Decreased (Leukocytopenia): Chemotherapy, radiation, long-term broad-spectrum antibiotic use.

Notes

Perform repeated count at higher dilution (1:100 or 1:200) when WBC count is extremely high.
Use lower dilutions (1:10 or 1:5) for exceptionally low counts.
Do not contaminate the diluent with dust or cells.
Perform counts promptly after filling the chamber. Avoid direct light.
Possible error sources: insufficient blood, poor mixing, improper filling, incorrect counts, etc.

References

Hematology practical manual, RMIT University, 2014.
https://emedicine.medscape.com/article/956278-overview.
Hoffbrand's essential hematology, 7th edition, 2016

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Description

This quiz focuses on manual white blood cell counting techniques as part of a practical physiology lab for second-year students. Students will learn about the methods for counting and interpreting WBC results, sources of errors, and the significance of normal reference ranges. Key practical skills such as using a hemocytometer and understanding dilution principles are emphasized.