Hematology Quiz: Blood Diluting Fluids

Complete Blood Count and Other Routine Procedures

• A complete blood count (CBC) is the most common test in a hematology section.
• CBCs are used to assess patient conditions such as infections and malignancies.
• Learning outcomes related to CBC include discussing significance, enumerating parameters and function, understanding clinical conditions affecting results, explaining methods for determining hemoglobin and hematocrit, determining factors affecting macro and micromethod hematocrit determinations, and discussing the significance of reticulocyte count and erythrocyte sedimentation rate.

Topic Outline

• Complete Blood Count
• RBC Count
• WBC Count
• Differential Count
• Platelet Count
• Hemoglobin
• Hematocrit
• Reticulocyte Count
• Erythrocyte Sedimentation Rate

RBC Count

• Procedure: Dilute blood, charge the counting chamber, count cells, and calculate results.
• Dacie's fluid is the best diluent, maintains cell shape, and is suitable for long-term storage. It contains 40% formaldehyde.
• Hayem's fluid is not recommended due to yeast growth and cell clumping. It contains mercuric chloride, sodium sulfate, and sodium chloride.
• Gower's solution prevents rouleaux formation.
• Toisson's fluid has high specific gravity and stains WBCs, but may promote fungal growth. It contains sodium chloride, sodium sulfate, glycerin, and methyl violet.
• Bethell's fluid is used in emergency cases and contains sodium sulfate, sodium chloride, glycerin, sodium merthriolate, and distilled water.
• Normal saline solution (NSS) is stable and used as a preservative in emergency cases involving rouleaux formation or autoagglutination.

Charging the Counter Chamber

• A representative sample of the diluted mixture is transferred to the counting chamber.
• Pipettes should be mixed by shaking the pipette in any manner except along the longitudinal axis.
• The first few drops from the capillary system should be discarded.

Counting the Cell

• Use the high-power objective of the microscope to count red cells in five intermediate squares of the central ruled area.

Making the Calculations

• Count the RBCs in the specified area, considering the dilution factor and depth of the chamber.
• The formula for calculation is: RBC count = (avg. cell no. X dilution factor)/ (area counted X depth of counting chamber).

WBC Count

• The Thoma pipette method is similar to the red blood cell pipette but with modifications (smaller bulb, larger bore, short limb mark at 11 instead of 101).
• The dilution is achieved by drawing blood to the 0.5 mark and diluting fluid to the 11 mark.
• A hypotonic diluent is required for WBC counts, it colorizes/stains the nuclei of white blood cells. (e.g., 2-3% acetic acid, 1% hydrochloric acid with a drop of methyl/crystal violet).

Diluting the blood

• The dilution of the blood is vital for white cell counts; it hemolyzes mature red blood cells but not nucleated red cells.
• The WBC dilution range can be prepared from 1:10 to 1:100 using a Thoma pipette for WBCs.
• Blood should be drawn to the 0.5 mark on the pipette and the diluting fluid should reach to the 11 mark.

Charging the Counting Chamber

• A representative sample of the diluted mixture is transferred to the counting chamber.
• To avoid error, mix the pipette by shaking it but not along the longitudinal axis.
• Discard the first few drops of the diluted mixture.

Counting the Cells

• Scan the ruled area under low power to ensure good distribution of cells; then, count cells in the 4 corner squares of the central ruled area.
• Each corner square consists of 16 medium squares.

Making Calculations

• Use the correct area correction factor (1.25), depth correction factor (10), and dilution factor (e.g., 20 - for 1:20 dilution)
• Apply the formula: WBC count = [(number of white blood cells counted x 10 x 20) / 4]

Platelet Count

• Platelet count is the number of platelets in one milliliter of whole blood.
• Platelets are counted in 25 small squares of a larger square in the hemacytometer using a phase contrast microscope.

Methods in Platelet Counting

• Manual method (Indirect - Fonio's, Olef's, Dameshek; Direct - Guy and Leake, Rees and Ecker)
• Electronic method (Voltage-Pulse Counting, Electro-optical Counting), and unopette systems.
• Note: Platelets are stained light blue in the Rees and Ecker method.

Hemoglobin

• Hemoglobin (Hb) is the red, iron-bearing protein within erythrocytes.
• Hb carries oxygen and carbon dioxide.

Colorimetric Method

• Includes visual methods (Direct Matching - Talquist Scale; Direct Matching - Dare Hemoglobinometer), Acid Hematin (Sahli's Hellige, Haden-Hausser, Sahli-Adams, Haldane, Newcomer, Osgood), and Alkali Hematin.

Cyanmethemoglobin

• Blood is diluted in an alkaline Drabkin's solution (potassium cyanide, potassium ferricyanide, sodium bicarbonate) to measure hemoglobin.
• Readings are taken at 540 nm.
• Calculations involve a formula using the absorbance of the test sample and standard, typically, C test = (C standard × A test) / A standard

Hematocrit

• Hematocrit is the volume of packed red blood cells (RBCs) in a given volume of whole blood.
• It is reported as a percentage or volume/volume.

Methods of Hematocrit Determination

• Macromethods (Wintrobe, Haden's Modification, Van Allen, Sanford-Magath, Bray's)
• Micromethods (Adams')

Reticulocyte Count

• Reticulocytes are young red blood cells that lack a nucleus and have residual RNA.
• The reticulocyte count is used as an index of bone marrow activity and RBC production, and is used to monitor therapeutic measures for anemia.

Erythrocyte Sedimentation Rate (ESR)

• ESR is a non-specific test used as an index of inflammation, typically measured by the rate red blood cells sediment at the bottom of a tube.
• The rate of sedimentation is affected by factors such as plasma proteins, red blood cell characteristics, and the technique of measurement.

Automated Methods (ESR)

• Automated ESR systems (e.g., Ves-Matic 20/60 by Vega Biomedical) are automated instruments for ESR determination using liquid sodium citrate within evacuated tubes.

Sources of Error

• Various factors such as specimen handling, technical procedures, and the use of equipment can lead to errors in the measurement of the different blood work tests.
• For each type of test, different sources of errors are discussed in detail.