As clinicians, we rely heavily on reference ranges to define and interpret disease in daily practice. We operate under the assumption that these ranges are rigorously determined and inherently reliable, but we rarely question how they were established.
Before the 1960s, laboratories functioned independently, each developing its own “normal” ranges. These were based on vaguely defined groups of patients deemed “normal” and were specific to their own patient populations. However, as the philosopher, statistician and physician Edmond Murphy cleverly pointed out in A Scientific Viewpoint on Normalcy, “What is not ‘normal’ is ‘abnormal’ and therefore … to be condemned. Thus, it is difficult to control the consequences of identifying a group of subjects as abnormal.”
One striking example of a poorly defined “normal” range is the absolute neutrophil count (ANC). The ANC measures the number of neutrophils in the blood and is often used to assess risk of infection, medication or chemotherapy dosing, as well as eligibility for clinical trials. Most institutions define neutropenia as an ANC less than 1.5 cells/µl, but this reference range is not a one-size fits all. Previously, the term “benign ethnic neutropenia” described individuals with a clinically insignificant neutrophil count lower than 1.5 cells/µl due to a normal genetic variant known as the Duffy null phenotype.
The Duffy blood group system comprises two antigens, Fya and Fyb, which are expressed on erythrocytes. These antigens are encoded by Duffy alleles on the atypical chemokine receptor 1 (ACKR1) gene. A single nucleotide polymorphism in the ACKR1 promotor gene results in the loss of gene expression. Individuals who do not express either the Fya or Fyb antigens are referred to as Duffy null or Fy (a-,b-).
The Duffy null phenotype is most prevalent among individuals of African and some Middle Eastern ancestries, with more than 95 per cent prevalence in sub-Saharan Africa. These antigens serve as receptors for Plasmodium vivax, the most frequent and widely distributed parasite causing malaria. Therefore, it is believed that the Duffy null phenotype provides an evolutionary advantage in malaria-endemic areas.
Duffy antigens are also receptors for non-specific chemokines released into the bloodstream. Consequently, Duffy null individuals are not affected by these chemokines, which leads to the migration of neutrophils from the bloodstream to tissues. This means the Duffy null phenotype does not impact the production and functioning of neutrophils; rather, neutrophils are based in tissue and ready to mobilize when needed.
As a reminder, the ANC is the total number of mature neutrophils and band neutrophils per unit of blood. ANC only measures neutrophils circulating in the bloodstream, which explains the apparent lower ANC observed in Duffy null individuals. When accounting for Duffy-null status, one study showed that individuals with a Duffy-null phenotype had a median ANC of 2.8 cells/µl, ranging from 1.08 to 5.95 cells/µl. On the other hand, individuals with a non-Duffy-null phenotype had a median ANC of 5.0 cells/µl, ranging from 2.36 to 10.0 cells/µl.
Importantly, the lower median ANC and range seen in Duffy-null individuals do not confer an increased risk of infection or increased mortality risk. A multiethnic cohort study involving more than 26,000 individuals compared overall survival in neutropenic and non-neutropenic participants. When neutropenia was defined as an ANC less than 1.5 cells/µl, 0.6 per cent of Caucasian participants were considered neutropenic, and this was associated with significantly reduced overall survival. In contrast, 2.9 per cent of Black participants were considered neutropenic, but this did not confer an increased mortality risk. Using regression analysis, the study then determined that the ANC cutoff with optimal prognostic value in Black participants was 1.1 cells/µl. When it redefined neutropenia as an ANC less than 1.1 cells/µl, only 0.6 per cent of Black individuals were considered neutropenic, which was associated with a statistically significant reduction in overall survival.
Black patients are more likely to undergo unnecessary invasive testing.
Applying a Caucasian-based neutrophil reference range broadly has led to significant racial inequities in health care. Black patients, for example, have higher cancer morbidity and mortality at every stage of diagnosis. One likely contributing factor is their disproportionate exclusion from clinical trials. A review found that 41.4 per cent of prostate cancer clinical trials required an absolute neutrophil count (ANC) ≥ 1.5 cells/µl, while 89 per cent of excluded Black participants had a clinically insignificant ANC ≥ 1.0 cells/µl. Furthermore, Black patients are more likely to receive dose-reduced and delayed chemotherapy due to lower white blood cell counts.
Non-chemotherapeutic medications are also more likely to be underdosed in Black patients. For instance, azathioprine, a widely used immunosuppressant, was discontinued three times more often in patients with the Duffy null phenotype. Clozapine, which is contraindicated for patients with an ANC less than 1.5 cells/µl, is under-prescribed in Black patients.
Additionally, Black patients are more likely to undergo unnecessary invasive testing. One study showed that Black participants were more likely to have a bone marrow biopsy for isolated neutropenia without other hematologic concerns. Of those biopsied, 60 per cent of Black patients were diagnosed with clinically insignificant neutropenia, compared to 9 per cent of Caucasian patients. Only one Black patient was tested for Duffy antigens and was found to be Duffy null.
There has been a considerable delay in integrating this knowledge into medical practice, with many clinicians and educators remaining unaware of the implications of the Duffy null phenotype. More research is needed to redefine ANC reference ranges based on Duffy status, which then need to be implemented institutionally. The American Society of Hematology has initiated efforts to redefine these ranges to reflect Duffy status, a promising step toward addressing these disparities.
The term “benign ethnic neutropenia” suggests that individuals of European ancestry have the “normal” reference neutrophil count. Many institutions are now shifting away from this terminology and adopting “Duffy-null associated neutrophil count” (DANC) instead. Duffy antigen testing is accessible and available in transfusion medicine labs and many external labs.
Inappropriate reference ranges are a significant issue in various areas of medicine and perpetuate systemic racism. As medical experts constantly educated about the complexity of human beings, we must be cautious about labeling something as “abnormal” or “pathologic” simply because it falls two standard deviations from a certain population mean.
It is essential to remember that these values are based on a reference population that may be limited by demographic factors, including race and gender. These reference ranges should be interpreted in the context of the reference population, recognizing that normality in medicine is relative and context dependent.
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