Traceability and Uncertainty of MINOCAL

DECLARATION of TRACEABILITY and UNCERTAINTY
HORIBA Medical calibrator

The purpose of this document is to describe the metrological traceability of values assigned to HORIBA
Medical calibrator: ABX Minocal Ref 2032002 and to estimate the calibrator assigned value uncertainty
component.
Assignment of Reference Values to Fresh Whole Blood
MKT-PMA-SASU-2012-0005 – Philippe Milian – Marketing dept Page. 1 of 3

Hematology Reference Methods
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 200 mL Class A volumetric flask filled with isotonic diluent. 2.4
mL of diluent is removed. Sample is added to the flask using a 400 L T.C. micropipet, followed by 2.0 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 L 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 100 mL Class A volumetric flask filled with the NCCLS
recommended reagent for the hemoglobincyanide (cyanmethemoglobin) method (1). Sample is added to
the flask using a 400 L T.C. micropipet. 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 NCCLS
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 NCCLS H7‐
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 L T.C. micropipet. 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).
BIBLIOGRAPHY
1. National Committee for Clinical Laboratory Standards. Reference and Selected Procedures for the
Quantitative Determination of Hemoglobin in Blood: Approved Standard‐Third Edition. NCCLS document
H15‐A3. Wayne, PA: NCCLS, 2000.
2. National Committee for Clinical Laboratory Standards. Procedure for Determining Packed Cell Volume by
the Microhematocrit Method: Approved Standard, NCCLS document H7‐A3. NCCLS, Wayne, PA: NCCLS,
2001.
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Determination of uncertainty (calibrator component)
The uncertainty associated with the calibration of the HORIBA Medical analyzer with the
ABX Minocal calibrator has been estimated by adding the following sources of uncertainty:
‐Uncertainty of the equipment used to determine the reference values: flask, pipette, single
aperture impedance counter (WBC, RBC), hemocytometer by phase‐contrast (PLT),
spectrophotometer (HGB) and hematocrit measurement (ruler).
Uncertainty as an absolute value:

Parameter Uncertainty
WBC (G/L 0.09
RBC (T/L) 0.03
HGB (g / dL) 0.06
HCT (%) 0.45
PLT (G/L) 5.4
Determination of total uncertainty
Total uncertainty is defined as the amount of error associated with reported patient results by the
HORIBA Medical hematology analyzers to reference methods when the analyzers are calibrated
using the ABX Minocal calibrator.
Three elements contribute to total uncertainty:
• the calibration system (working calibrators, primary and secondary calibrators, reference
measurement procedures…)
• the procedure (reagents, instruments, laboratory staff …)
• the sample
The overall expression of uncertainty is therefore:

Traceability and Uncertainty of MINOCAL

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Traceability and Uncertainty of MINOCAL

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DECLARATION of TRACEABILITY and UNCERTAINTY

HORIBA Medical calibrator

Assignment of Reference Values to Fresh Whole Blood

MKT-PMA-SASU-2012-0005 – Philippe Milian – Marketing dept Page. 1 of 3

MKT-PMA-SASU-2012-0005 – Philippe Milian – Marketing dept Page. 2 of 3

Uncertainty as an absolute value:

Determination of total uncertainty

The overall expression of uncertainty is therefore:

MKT-PMA-SASU-2012-0005 – Philippe Milian – Marketing dept Page. 3 of 3

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