Reference Methods and Traceability
Hematology analyzers in R&D Systems' Quality Assurance Laboratory are whole blood calibrated to values obtained using these standard reference methods. Whole blood samples drawn from normal, healthy donors are collected in EDTA anticoagulant and analyzed within six hours of collection.
WBC: A 1:500 dilution is prepared using a 100 mL Class A volumetric flask filled with isotonic diluent. 1.2 mL of diluent is removed. Sample is added to the flask using a 200 μL calibrated positive displacement pipette, followed by 1.0 mL lysing agent. Counting is performed on a Coulter Counter Z series instrument. All counts are corrected for coincidence.
RBC: A 1:50,000 dilution is prepared using a 1000 mL Class A volumetric flask filled with isotonic diluent. Sample is added to the flask using a 20 μL T.C. micropipet. Counting is performed on a Coulter Counter Z series instrument. All counts are corrected for coincidence.
HGB: A 1:251 dilution is prepared using a 50 mL Class A volumetric flask filled with the CLSI recommended reagent for the hemoglobincyanide (cyanmethemoglobin) method (1). Sample is added to the flask using a 200 mL calibrated positive displacement pipette. The sample is filtered with a 0.2 µm filter immediately before reading. Readings are made at 540 nm in a colorimeter/spectrophotometer calibrated according to CLSI H15-A3 and ICSH recommendations (1).
HCT: Plain glass microhematocrit tubes (not coated with anticoagulant) are filled with sample, sealed with sealing putty and centrifuged for 5 minutes in a microhematocrit centrifuge according to the CLSI H07-A3 document (2). After centrifugation, the length of the whole column including the plasma, and the length of the red blood cell column, are viewed and measured using a microscope with graduated stage and an ocular micrometer. The hematocrit (packed cell volume) is calculated as the ratio of the two measurements. No correction is made for trapped plasma.
MCV: On some instruments MCV is the calibrated parameter instead of the HCT. The MCV is calculated from the HCT and RBC using the formula: MCV = HCT × 10/RBC
PLT: A 1:126 dilution is prepared using a 50 mL Class A volumetric flask filled with filtered 1% ammonium oxalate. Sample is added to the flask using a 400 μL calibrated positive displacement pipette. The dilution is plated onto a clean, dry Neubauer ruled phase type hemocytometer. The hemocytometer is left for 10 minutes in a humidified chamber. Using phase contrast optics, the platelets in the entire central square millimeter on both sides of the hemocytometer are counted. The two counts are averaged and multiplied by 1260 (dilution factor 126 × volume factor 10 = 1260).
- Clinical Laboratory Standards Institute. Reference and Selected Procedures for the Quantitative Determination of Hemoglobin in Blood: Approved Standard-Third Edition. CLSI document H15-A3.
- Clinical Laboratory Standards Institute. Procedure for Determining Packed Cell Volume by the Microhematocrit Method: Approved Standard-Third Edition. CLSI document H07-A3.
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