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Q&A column, 9/17

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Editor: Frederick L. Kiechle, MD, PhD

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Q. I received a sample with very high hemoglobin grossly. When I ran the sample on the Cell-Dyn Ruby, it was unable to calculate the parameters related to Hgb. I diluted the EDTA blood and ran the test again. In this scenario, should I multiply all the indices and Hgb-related parameters with the dilution factor? Which parameters should I multiply with the dilution factor?

A. The vast majority of automated hematology analyzers, including the Abbott Cell-Dyn Ruby system, determine Hgb (hemoglobin concentration) by first lysing the red cells of a known volume of input sample, diluting the lysate in an oxidant-containing solution, and inferring the hemoglobin concentration by absorptiometry relative to known standards. In contrast, the RBC parameter (or red blood count) is a directly computed count of the number of red cells in a known dilution of input sample; likewise, the MCV (or mean corpuscular volume) is calculated from the histogram of red cell sizes, which are determined by passing an aliquot of the input sample through a flow cytometer. The remaining red cell parameters including Hct (hematocrit), MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration), and RDW (red cell distribution width) are typically calculated from these empirically determined results (see Wintrobe’s Clinical Hematology for the equations defining these values).

Thus, if an input blood sample must first be diluted prior to testing (in cases, for example, of erythrocytosis in which neat-tested samples result in analyzer errors or unexpected results), only some of the complete blood count parameters need be adjusted by the dilution factor. Notably, Hgb and RBC will need to be adjusted as both of these quantities will have been reduced by the process of dilution. Since the distribution of red cell size, which is used to determine MCV and RDW, is not altered by dilution of the sample prior to analysis, these parameters are not subject to adjustment. However, since Hct is calculated using the RBC (diluted) and MCV (not diluted) values, this parameter must also be adjusted for dilution. Finally, the astute reader will also note that for MCH and MCHC, the dilution factors to adjust both Hgb and RBC will cancel out in their calculations, so these parameters do not need to be adjusted.

It should be noted that laboratories are advised to fully validate their protocols, including those that may necessitate manual sample dilution prior to automated analyses. One easy way to do this in the above context would be to perform a serial dilution process. Linearity of results in such a dilution experiment serves as a means of reassurance that the analysis workflow in the context of sample predilution is valid.

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