ICSH Expert Panel on Cytometry Recommendations

Recommendations for Reference Method for the Packed Cell Volume

ICSH Standard 2001

Prepared by the Expert Panel on Cytometry of the International Council for Standardization in Haematology

Laboratory Hematology 7:148-170
© 2001 Carden Jennings Publishing Co., Ltd.

ABSTRACT

The manual, spun Packed Cell Volume (PCV, hematocrit) is a key measurement on which are based hematology instrument calibration, reference range determination and assignment of values to calibrators/controls. Until now, however, no true reference method for the PCV has existed. The International Council for Standardization in Haematology (ICSH) which met in Montpelier, France in May 2001 has now recommended a reference method for the PCV. This reference PCV method is fully traceable to the ICSH reference method for haemoglobinometry. Using carefully defined capillary tubes, the reference PCV can be calculated as the ratio between the hemoglobin of the whole blood and the hemoglobin of packed red blood cells with all plasma removed. The ICSH intends that this reference protocol be used primarily by manufacturers of capillary microhematocrit tubes, liquid calibrators and multichannel analyzers.

INTRODUCTION

The manual, spun Packed Cell Volume (PCV, hematocrit) is a key measurement, underpinning much of hematology. The calibration of virtually all hematology autoanalyzers can be traced in some way back to the PCV. Reference ranges for the hematocrit and red cell indices depend on the validity of this calibration, as do the assignment of expected values to calibrators and controls, and the assignment of target values¹ for statistical population-based quality control programs. Any errors in PCV assignment have far-reaching implications.

Although the manual, spun PCV is simple and inexpensive to perform, it is affected by several variables, including:

• Trapped plasma^{2, 3, 4}
• white blood cell and platelet contamination of the red cell layer⁵
• indistinct margin between red and white cell layers⁵
• non-flat tube seals⁵
• red cell dehydration⁶
• oxugenation state of the red cells⁷

Fortunately, these biases tend to counterbalance each other, so the net PCV error is typically small - less than one PCV unit.⁵ Although of little clinical consequence for individual patients, hematocrit errors such as these can have wide-ranging implications when applied globally. For example, biases as small as one PCV unit can result in the inappropriate deferral of up to 3.5% of potential blood donors.⁸ PCV errors of this order of magnitude are totally unacceptable in terms of autoanalyzer calibration/control and reference range determination. Even slight autoanalyzer miscalibration based on erroneous PCV values can lead to the incorrect categorization of patients being worked up for anemia.

Clearly, there is need for an unbiased "reference" method to validate working methods for the PCV. Although several such methods have been proposed, they are complex, time-consuming, and may require radiolabeled reagents^{9, 10} that are difficult to obtain and require that special licenses be obtained by the laboratory in which they are to be used.

The ICSH Expert Panel on Cytometry has prepared and approved the following recommended Reference PCV Method. This hemoglobin/MCHC-based reference method⁵ eliminates errors due to all of the above six factors. It requires only standard laboratory glassware, calibrated micropipettes, microhematocrit centrifuge and spectrophotometer. Either this reference method itself should be used when hematocrit values are needed for calibration or reference purposes, or if a secondary standard such as a capillary tube (see Appendix B) is certified for reference use, it should be traceable to this reference method.

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