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Editor: Frederick L. Kiechle, MD, PhD

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Q. Automated differentials are broken down into absolute counts for neutrophils, lymphocytes, monocytes, eosinophils, and basophils. The absolute neutrophil count is a useful index for physicians in clinical decision-making. When performing a manual differential that contains immature cells such as metamyelocytes and myelocytes, do you report an absolute count on all of the individual cells in the myelocytic line, or do you group them together and calculate one ANC? What about lymphocytes and reactive lymphocytes?

A. White blood cell differential counts, traditionally performed manually, were reported as percentages of total WBCs. Given the potential issues in interpreting percentages, including errors in manual calculations of absolute counts, together with readily available absolute counts on automated CBC analyzers, reporting absolute differential counts is recommended.1 Automated CBC analyzers typically provide a five-part or six-part differential count including absolute neutrophil count, absolute monocyte count, absolute lymphocyte count, absolute eosinophil count, absolute basophil count, and, in some platforms, absolute immature granulocyte count.

An absolute neutrophil count, or ANC, historically calculated from manual differentials, is: total WBC count × (% segmented neutrophils + % bands)/100. This is the calculation used to define thresholds for neutropenia and its significance in early clinical studies.2,3 When providing manual ANCs, the calculations should therefore include neutrophils and bands but not other immature granulocytes (i.e. metamyelocytes, myelocytes, promyelocytes). Immature granulocytes, when present, may be reported as their individual absolute counts (i.e. absolute metamyelocyte count, absolute myelocyte count, absolute promyelocyte count), or combined into one absolute immature granulocyte count. On the other hand, total granulocyte count includes immature granulocytes and is calculated as: total WBC count × (% segmented neutrophils + % bands + % myelocytes + % metamyelocytes + % promyelocytes)/100.

An automated ANC is calculated as: total WBC count × (% neutrophils)/100. Since automated analyzers do not distinguish bands from segmented neutrophils, the percent neutrophils enumerated by automated analyzers includes both stages. Metamyelocytes, myelocytes, and promyelocytes are generally not part of the automated ANC in analyzers that generate a six-part differential, as these are separately enumerated as immature granulocytes. ANCs from analyzers reporting a five-part differential are technically total granulocyte counts, as percent neutrophils in such analyzers may include metamyelocytes, myelocytes, and promyelocytes, if present. When immature granulocytes are extremely low to absent, the automated total granulocyte count approaches an ANC.

Manual absolute lymphocyte counts, or ALCs, should ideally include lymphocytes and reactive lymphocytes. Thus a manual ALC calculation would be: total WBC count × (% lymphocytes + % reactive lymphocytes)/100. This combined ALC provides a more consistent measure of total lymphocytes and trends over time. In our experience, splitting the ALC into two categories (lymphocytes and reactive lymphocytes) is not important in clinical decision-making and leads to fluctuations in reported counts due to interobserver variability. Moreover, thresholds for determining presence of an absolute lymphocytosis are based on an increase in total lymphocytes, and splitting the ALC into two numbers may lead to missed recognition of an absolute lymphocytosis. Automated ALCs may also include reactive lymphocytes. In some analyzers, reactive lymphocytes may fall under a separate “large unstained cells” category. If these LUCs are deemed to be reactive lymphocytes on review of the corresponding peripheral smear, and the automated differential is being released, then the reported ALC calculation would be as follows: total WBC count × (% lymphocytes + % LUCs)/100.

In our laboratory, we report absolute differential counts only, with a single reported ALC for all lymphocytes. If reactive lymphocytes are noted, a descriptive comment regarding the presence of reactive lymphocytes is appended to the ALC. Malignant lymphoma cells, if present, are also included within the reported ALC category, with an interpretive comment appended. A notation regarding proportion of the total ALC that is malignant appearing by morphology may be included in the interpretive comment as needed. In the case of chronic lymphocytic leukemia, distinction of malignant versus nonmalignant lymphocytes is not reliable on morphologic grounds, and thus a single ALC is advisable; this also allows clinicians easier monitoring of lymphocyte doubling time.

  1. Etzell JE. For WBC differentials, report in absolute numbers. CAP TODAY. 2010;24(3):12.
  2. Hijiya N, Onciu M, Howard SC, et al. Utility of automated counting to determine absolute neutrophil counts and absolute phagocyte counts for pediatric cancer treatment protocols. Cancer. 2004;101(11):2681–2686.
  3. Pizzo PA. Management of fever in patients with cancer and treatment-induced neutropenia. N Engl J Med. 1993;328(18):1323–1332.

Parul Bhargava, MD
Professor of Clinical Laboratory Medicine
Director of Clinical Laboratories
Moffitt-Long Hospital
University of California San Francisco
Member, CAP Hematology/Clinical
Microscopy Resource Committee

Q. I read about the new approaches to TB testing (CAP TODAY, April 2018). Why and in what employment screening settings is the two-step skin test recommended?

A. When a person is infected with Mycobacterium tuberculosis, a delayed-type hypersensitivity reaction, detected by the tuberculin skin test (TST) administered using the Mantoux technique, generally develops two to eight weeks after the infection. However, if an infected person is tested several years after the infection occurred, the TST may be negative in some individuals. If such individuals are tested again, this second TST may be positive because the first test stimulated or “boosted” their ability to react, hence the term booster phenomenon. Because this change in the TST result (i.e. negative to positive) may be interpreted mistakenly as a conversion, experts at the Centers for Disease Control and Prevention recommend the two-step method for baseline or initial testing when using the TST to screen for latent M. tuberculosis infection in those who will be tested periodically, such as health care workers who are screened annually. In this situation, the first TST is placed on initial hire. If this TST is positive, the person has latent M. tuberculosis infection and should be evaluated accordingly. However, if the first TST is negative, a second is placed one to three weeks after the first. If the second test is positive, the person is considered to have latent infection; if it is negative, the person is not infected. Thereafter, annual testing is performed with a single TST, and a positive result indicates conversion, most likely due to a recent infection with M. tuberculosis.

  1. Latent tuberculosis infection: a guide for primary health care providers. Centers for Disease Control and Prevention website. www.cdc.gov/tb/publications/ltbi/default.htm. Accessed Jan. 16, 2019.
  2. Testing health care workers. Centers for Disease Control and Prevention website. www.cdc.gov/tb/topic/testing/healthcareworkers.htm. Accessed Jan. 16, 2019.

Gail L. Woods, MD
Member, CAP Microbiology
Resource Committee

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