Blood Dilution Techniques in Hematology

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Questions and Answers

What does diluting the blood achieve in the counting process?

It facilitates the counting by suspending and dispersing red cells. (correct)

It alters the color of the red blood cells.

It makes the red cells stick together.

It preserves the cells for a longer duration.

What is the purpose of using EDTA in blood sample preparation?

It preserves the cells very well. (correct)

It acts as a diluent for the sample.

It increases the number of cells in suspension.

It enhances the color of the sample.

What dilution should be used for counting in a polycythemic patient?

1:10

1:50

1:400

1:200 (correct)

How should the RBC Thoma pipette be prepared for accurate counting?

By marking 0.5 with blood and 1 with diluent. Signup and view all the answers

Which color dot on a Thoma pipette indicates it is meant for RBC counting?

Red Signup and view all the answers

What should be avoided during the immersion of the pipette tip in the diluent?

Creating air bubbles. Signup and view all the answers

What is the maximum dilution range that can be prepared using the RBC Thoma pipette?

1:1000 Signup and view all the answers

What action is necessary after using the Neubauer chamber?

The chamber should be wiped with a tissue to clean. Signup and view all the answers

What is a significant drawback of Hayem’s diluting fluid?

It promotes yeast growth and cell clumping. Signup and view all the answers

Why must dilution fluids be isotonic?

To prevent shrinkage and bursting of cells. Signup and view all the answers

Which component is included in Gower’s solution for preventing rouleaux formation?

Sodium sulfate. Signup and view all the answers

What is the primary purpose of adding mercuric chloride in Hayem's diluting fluid?

To stabilize the solution. Signup and view all the answers

Which of the following is NOT a feature of Hayem’s diluting fluid?

It has corrosive effects on specimens. Signup and view all the answers

What is typically discarded during the charging of the counter chamber?

The first few drops from the pipette. Signup and view all the answers

What role does sodium chloride play in Gower’s solution?

It helps in preventing rouleaux formation. Signup and view all the answers

What must be avoided when shaking the pipette to mix the sample?

Shaking along the longitudinal axis. Signup and view all the answers

What is the dilution factor achieved by using the Thoma pipette method as described?

1:20 Signup and view all the answers

Why should the diluent used in counting white blood cells be hypotonic?

To eliminate red blood cells from the sample Signup and view all the answers

What is the purpose of letting the cells settle for 1 to 2 minutes after charging the counting chamber?

To ensure all cells are in the same plane for accurate counting Signup and view all the answers

Which of the following is an appropriate diluent for counting white blood cells?

2-3% acetic acid Signup and view all the answers

When using the low power objective to count cells, what should be done first?

Familiarize with the ruling of the chamber Signup and view all the answers

What is the significance of the mark at the short limb of the Thoma pipette?

Denotes the dilution factor Signup and view all the answers

In the counting procedure, how many corner squares should be counted?

4 squares Signup and view all the answers

What is a common mistake to avoid when counting cells across different chambers?

Counting only in one chamber Signup and view all the answers

What could cause a precipitated stain on a slide?

Drying during the staining period Signup and view all the answers

What is the significance of a 200-cell differential count?

It is required when leukocyte numbers exceed 35.0 x 10^9/L Signup and view all the answers

What is indicated by a platelet count of >25 platelets per OIO field?

Marked increase in the number of platelets Signup and view all the answers

Which of the following methods is NOT used for counting platelets?

Direct counting in sedimentation tubes Signup and view all the answers

What could indicate a 'marked increase' in neutrophil count?

Neutrophil count above 800,000/uL Signup and view all the answers

Which condition requires decreasing the number of platelets?

When WBC count is less than 1 platelet per OIO field Signup and view all the answers

Which reagent is NOT part of the procedure using Dameshek's method?

Ethanol Signup and view all the answers

What is the role of the phase contrast microscope in the platelet counting procedure?

To allow counting of platelets in small squares Signup and view all the answers

What is the primary reason that makes the Brecker-Cronkite method the most accurate for platelet counting?

It effectively swells platelets and hemolyzes red cells. Signup and view all the answers

What is a common source of error in platelet counting related to sample handling?

Inadequate mixing of the blood sample. Signup and view all the answers

Which factor contributes to the difficulty in counting platelets accurately?

Platelets can clump and adhere to surfaces. Signup and view all the answers

In the formula for platelet counting, what does the term 'dilution factor' refer to?

The ratio of sample to diluent before counting. Signup and view all the answers

What is one of the physiologic variations that can affect platelet counts?

Hormonal changes in the body. Signup and view all the answers

What error is specifically associated with the operator in platelet counting?

Inconsistent handling of samples. Signup and view all the answers

Which of the following best describes the electronic method of platelet counting?

It removes red cells from the blood before measurement. Signup and view all the answers

What is the typical error percentage in the Brecker-Cronkite method?

10 – 15 % Signup and view all the answers

Study Notes

Diluting the Blood

The blood is diluted to facilitate counting and disperse red blood cells.
A 1:200 dilution is commonly used for red blood cell counts.
The dilution range for red blood cell counts using the Thoma pipette is 1:100 up to 1:1000.
EDTA is used as an anticoagulant to preserve blood cells.
A pipette should be fully immersed in the diluent to avoid air bubbles.
The tip of the pipette should be wiped clean with a tissue.
For anemic samples, a lower dilution (1:20, 1:100, or 1:10) may be required.

Diluting Fluids

Hayem's solution can lead to rouleaux formation and autoagglutination of cells.
- Composition:
  - Sodium chloride: 0.85 grams
  - Distilled water: 100.0 mL
Gower's solution prevents rouleaux formation and precipitates protein in cases of hemoglobinemia and hyperglobulinemia.
- Composition:
  - Sodium sulfate anhydrous: 12.5 grams
  - Sodium chloride: 2.0 grams
  - Distilled water: 100 mL
3.8% sodium citrate is isotonic and prevents cell shrinkage or bursting.
- Composition:
  - Sodium chloride: 3.8 grams
All diluting fluids should be isotonic to prevent cell shrinkage and bursting.

Charging the Counting Chamber

RBC Thoma pipette: Used for red blood cell counts.
WBC Thoma pipette: Used for white blood cell counts.
- The color of the dot on the pipette distinguishes them - red for RBC and white for WBC.
Charging procedure:
- A representative sample of the diluted mixture is transferred to a counting chamber.
- The pipette should be mixed by shaking, but not along the longitudinal axis.
- Discard the first few drops (3-4) from the capillary system.
WBC Thoma pipette dilution range: 1:10 - 1:100.

Counting Cells

White blood cells:
- Dilution: 1:20 for white blood cell counts.
- Diluting fluid: Should be hypotonic to eliminate red blood cells and stain white blood cell nuclei.
  - Example diluents:
    - 2-3% acetic acid
    - 1% HCl with 1 drop of methyl violet or crystal violet
- Counting procedure:
  - Familiarize yourself with the ruling of the chamber.
  - Let the cells settle for 1-2 minutes.
  - Use the low-power objective to locate and scan the ruled area.
  - Count the cells within the 4 corner squares (4 sq.mm.) which each have 16 medium squares.
Differential count:
- A 200-cell differential should be performed when the leukocyte count is in excess of 600,000-800,000/uL.
Causes of precipitated stain:
- Unclean slides
- Drying during staining
- Inadequate washing
- Failure to hold the slide horizontally during initial washing
- Inadequate filtration of the stain

Platelet Count

Platelet count is the number of platelets per liter or milliliter of whole blood.
Counting procedure:
- Count platelets in the 25 small squares in the large center square (1 mm^2) of the hemacytometer using a phase contrast microscope.
Platelet count formula:
- Platelet count = Average platelet count x Depth factor x Dilution factor x Area correction factor
Brecher-Cronkite method:
- Most accurate method for platelet counting.
- Formula:
  - Platelet/uL = Platelet x 5 x 10 x 100
Dameshek's method (indirect):
- Uses brilliant cresyl blue, sodium citrate, sucrose, and formalin.
- Counterstained with Wright's stain.
Reasons why platelets are hard to count:
- Easily disintegrate.
- Small size can lead to mistaking them for debris.
- Easily clump.
- Uneven distribution of blood.
Sources of error in platelet counting:
- Handling error.
- Operator error.
- Equipment and reagent error.
- Inherent/field error.
Causes of platelet clumping:
- Initiation of platelet aggregation.
- Clotting before blood reaches the anticoagulant.
- Imperfect venipuncture.
- Delay in sampling.
Physiologic variation in platelet counts:
- < 5 platelets/OIO field: Decrease in platelet number.
- 5-20 platelets/OIO field: Adequate platelet supply.
- 25 platelets/OIO field: Increase in platelet number.

Electronic Method for Platelet Counting

Red blood cells must be removed from whole blood by sedimentation or centrifugation.

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Description

This quiz covers the critical techniques used in diluting blood samples for red blood cell counts, including the appropriate dilutions and anticoagulants. It also explores different diluting fluids and their compositions, such as Hayem's and Gower's solutions, highlighting their effects on cell behavior. Test your knowledge of these essential hematological practices.