Answering your questions

May 1, 2012


We've recently changed our policy on body fluid cell counts.

1) All fluids are to get 5-part diffs rather than just mono / poly / other.

2) Rather than having a defined set of CSF tube #s that we do specified tests on, we are to do precisely as the order reads (e.g., cell count on 1 and IF > 10 RBCs; do count on 4; or culture and gram stain on different tubes, etc.) Many of us feel that this is not giving best value in either results or cost. What is your view on these matters?


While the accepted standard for CSF cell counts is to use tube 3 or 4, some clinicians find it helpful to perform additional testing to determine if the RBCs present are the result of a traumatic tap or from an actual central nervous system bleed. Because visual examination does not aid in this assessment, this is usually accomplished by counting the RBCs in the first tube. If the count of tube 1 is dramatically higher than tubes 3 or 4, it can be assumed the tap was traumatic.

—Donna L. Canterbury, MT (ASCP)SH
Hematology Supervisor—Core Laboratory
University of Virginia Health Systems
Charlottesville, VA


How long can a blood specimen collected in a serum separator tube to test chemistries “sit” before it is centrifuged?


Per BD SST product literature, a freshly collected specimen is to be inverted five times to adequately mix the silica particles to help activate the clot. Once sufficiently inverted, the specimen should sit for a minimum of 30 minutes to complete clot activation before centrifugation. The specimen can be centrifuged up to two hours post collection. Specimens should not be re-centrifuged. Consult the BD website for technical information at

—Barbara Strain, MA, SM(ASCP)
Director Supply Chain Analytics
University of Virginia Health System
Charlottesville, VA

We have been using a formula to correct a hemoglobin from a lipemic sample for 10 to 15 years now. The formula is hgb=MCV x RBC divided by 29.8. We then use the calculated hemoglobin to recalculate the MCH & MCHC. We remember finding the calculation in Medical Laboratory Observer, but have lost the reference. Can you confirm the corrected hemoglobin formula?


The equation (Hb g/dL = MCV x RBC/2.98 x 10 you ask about was published by Kalache et al in 1991.1 Kalache et al reported having tested 200 samples of varying MCV and established a constant ratio of MCV/MCH of 2.98 (s.d. 0.085). Using the standard MCH calculation of MCH = Hb g/dL x 10/ RBC without the power of 10 and rearranging the ratio:

MCV/MCH = 2.98

MCV/(Hb g/dL x 10/RBC) = 2.98

MCV x RBC/Hb g/dL x 10 = 2.98

MCV x RBC/2.98 x 10 = Hb g/dL

Kalache et al went on to test 60 samples for hemoglobin and then spiked the samples with lipids to simulate lipemia. They used the plasma hemoglobin method2 to correct the hemoglobin. They compared the estimated hemoglobin to the measured and corrected hemoglobins. The linear regression of the measured hemoglobin and estimated hemoglobin was 0.98. They felt this was superior to the corrected hemoglobin assay that had a larger SD and CV than the estimated method.

Two points are important. First, they were using a Coulter Model S+I, which used the cyanmethemoglobin method for hemoglobin determination and pulse height determinations for MCV. Second, they did not publish the data for the 200 samples for readers to inspect. For these two reasons alone, any modern users should validate the equation using their instruments.

Kalache's report is essentially a technical validation of the Rule of Three. The standard rule of thumb is that 3 x Hb g/dL = Hct %. The Coulter instrument that Kalache used measured MCV and calculated the Hct, the opposite of the manual determinations of Hct with calculation of MCV. But if you take the Rule of Three and rearrange the equations, you get:

3 x Hb = Hct

3 x Hb = MCV x RBC/10

Hb = MCV x RBC/3 x 10, which is essentially the equation that Kalache et al reported.

The Rule of Three is notoriously unreliable when the cell morphology is not normal, particularly when there is hypochromia. Kalache's samples included those with MCVs as low as 46 fL, which would suggest likely hypochromia. However, as there is no subsequent published report that we could find that reproduces Kalache's findings and he did not present the portion of the data establishing the ratio, the equation should be validated using samples with a wide variety of morphology, particularly for use with laser analyzers and those using methods other than cyanmethemoglobin for Hb determination.

—Kathy Doig, PhD
Professor, Biomedical Laboratory Diagnostics
Associate Dean, College of Natural Science
Michigan State University
East Lansing, MI


  1. Kalache GR, Sartor MM, Hughes WG. The indirect estimation of hemoglobin concentration in whole blood. Pathology. 1991 Apr;23(2):115-117.


  2. Nicholls PD. An improved method for obtaining reliable blood counts with hyperlipaemic samples on an automated particle counter. Med Lab Sci. 1983;40:397-400.


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