Answering your questions

Editor's note: Anthony Kurec, MS, H(ASCP)DLM, answers questions from two readers. Anthony is Clinical Associate Professor, Emeritus, at SUNY Upstate Medical University in Syracuse, NY.

Is it possible to report the absolute calibrated value with its reference range when a physician asks for a Total Eosinophil Count? It is our understanding that the calibrated value of the instruments with five different settings is more exact than any other manual method used to count eosinophils. Many physicians keep ordering this. What is your opinion or recommendation?

Eosinophils are produced by the bone marrow, circulate in the peripheral blood for a short time, and eventually localize in various tissues and organs such as the skin, lungs, and GI tract.¹ Eosinophilia is generally considered when the total eos count is greater than 450-500 eos/µL of blood and is most often associated with parasitic infections, some bacterial infections, drug or other allergies, respiratory disorders, GI disorders, certain immunodeficiences, various malignancies and leukemias, and connective tissue diseases. Eosinopenia has been associated with stress, some acute bacterial infections, and steroid use.²

Total or absolute eosinophil counts can be useful in managing these various disorders and would be ordered when a CBC and differential present with elevated eosinophil numbers. Today's automated cell counting and differentials are highly sensitive and reliable in making this determination. Alternatively, manual chamber counting methods, which have been around for many years, can be used but come with a number of potential inherent sources of error (dilutional errors, dirty or broken pipettes, counting chamber imperfections, and chamber filling errors). Depending on the technologist's skill and type of counting chamber used, the coefficient of variation (CV) ranges from 6.5% to 15%, whereas automated methods tend to be less than 3%.³ In the evaluation of other body fluids where cell numbers are often low, CVs have been observed as high as 45%.⁴ Evaluation for total eosinophils using a manual method would most likely be prone to a greater margin of error than use of the automated method.

1. Kremyanskaya M, Ackerman SJ, Butterfield JH, et al. Eosinophilia, eosinophil-associated diseases, chronic eosinophil leukemia, and hypereosinophilic syndromes. In: Hoffman's Hematology: Basic Principles and Practice. 6th ed. Elsevier-Saunder: Philadelphia, PA; 2012.
2. Holland SM, Gallin J. Disorders of granulocytes and monocytes. In: Longo DL, Fauci AS, Kasper DL, et al, eds. Harrison's Principles of Internal Medicine. 18th ed. New York, NY: McGraw-Hill; 2012.
3. Vajpayee N, Graham S, Bem S. Basic examination of blood and bone marrow. In: McPherson RA, Pincus MR, eds. Henry's Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Elsevier-Saunders: Philadelphia, PA; 2012.
4. Karcher DS, McPherson RA. Cerebrospinal, synovial, serous body fluids, and alternative specimens. In: McPherson RA, Pincus MR, eds. Henry's Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Elsevier-Saunders: Philadelphia, PA; 2012.

RE: CAP Question CHM.13720 Maximum Dilution

I know that I have to have in my procedures the maximum allowable dilution. Do I have to prove that a 1:10 or a 1:20 provides analytically precise results? And if so, do I have to repeat that documentation on a biennial basis?

When a test result exceeds the analytic measurement range (AMR), the sample requires dilution to bring it within acceptable range. While CAP does not mandate what the maximum dilution should be for any one test, it does state that each laboratory must establish the maximum dilution rate for each applicable analyte.¹ The maximum allowable number of dilutions must be clinically appropriate, validated for accuracy, approved by the laboratory director, and reviewed on an annual basis. How often revalidation of the procedure is needed is also up to the laboratory director. It would definitely be required with changes in instrumentation, methodology, or other significant alteration of the testing protocol, and clearly outlined in your procedure manual. Specimen results that exceed the maximum dilution level for some tests might be appropriately reported as greater than whatever the maximum dilutional level is, if this is acceptable to your laboratory director.

This is from CAP's Laboratory Accreditation Manual: Maximum Dilution/Concentration: For analytes that may have results falling outside the limits of the AMR, check to see that the laboratory procedure specifies the maximum concentration or dilution that may be performed to obtain a reportable numeric result. Establishment of allowable dilutions and concentrations is performed when a method is first placed into service and is reviewed annually thereafter as part of the procedure manual review by the laboratory director or designee.²

References

1. Kiechle FL. CAP Today. Q&A. September 2012.
2. Sharkey FE. CAP Laboratory Accreditation Manual. 2011;62.