Assay for “fetal hemoglobin levels” in erythrocytes: Impact of new In Vitro Diagnostics regulation and expanded applications in sickle cell disease management
Authors: Bruce Hewat Davis
Journal: American Journal of Hematology (2020)

The work of Hebert, Rakotoson et al. in this issue of the American Journal of Hematology1 points to evolving in vitro Diagnostics (IVD) testing of red blood cell (RBC) hemoglobin levels, now poised for a clear role in the personalized care and therapeutic management of sickle cell disease (SCD) patients, long known to be a genetically and clinically heterogenous patient population.2-4 The performance and validation studies detailed in this article and supplement material describe a laboratory developed flow cytometric test (LDT) capable of providing sufficient precision to measure threshold levels of hemoglobin F (HbF) in a clinically meaningful way. The study further provides clinical validation as to the utility of measuring HbF content in RBCs of SCD patients, not just as a prognostic test, but more likely a companion diagnostic in phase 3 clinical trials of new SCD therapies. The assay described by the French group is an “off label” adoption of a CE-IVD flow cytometric assay kit (Fetal Cell Count Kit, IQ Products, Groningen, NL), based upon anti-HbF monoclonal antibody detection and multiparameter flow cytometry. The original intended use is to measure fetomaternal hemorrhage (FMH) as a companion diagnostic for the use of Rh immune globulin therapy to prevent hemolytic disease of the newborn, where the target analyte is fetal RBCs, rather than adult RBCs containing lower HbF levels, so-called F cells. Why am I confident that this or a similar calibrated flow cytometric (or cell by cell measurement technology) assay satisfies an emerging clinical need? The evidence for continued development of therapeutics able of inducing the increased RBC levels of HbF in sickle cell disease is convincing both from economic and risk/benefit perspectives.5-10 These studies and others paint an increasing better understanding of the molecular regulation of hemoglobin switching and genetic heterogeneity in SCD and other hemoglobin variant disorders, such as thalassemia. The early studies with LDT versions of the HbF RBC assays make clear that patients may respond in either a homocellular or heterocellular HbF distribution fashion among RBCs, as reaffirmed by the current study by Hebert et al,1 the important parameter correlating with therapeutic response is the population size of RBCs having a threshold approximating >4 pg HbF per cell, seemingly where sufficient intercalating HbF prevents the polymerizing sickling phenomenon in SCD. It remains to be determined if the critical threshold for sickling prevention is a specific level of HbF in the cell or if merely achieving a certain proportion of HbF of the total cellular hemoglobin content is sufficient to predict sickling phenotype remains to be determined. The genetic heterogeneity of SCD and the proven inability of simple total blood or fractional HbF measurement levels to predict a therapeutic response to HbF-inducing SCD therapies makes it probable that rapid dose adjustments based upon RBC HbF levels are requisite to achieve the most desirous clinical outcomes, promising SCD patients near normal lifetimes with significantly lower healthcare costs. All the right reasons to adopt a new laboratory test are present, provided it is designed to meet both the clinical and laboratory demands - namely rapid 24/7 results obtained with technical ease. As best exemplified by the current COVID-19 testing crisis, the development of IVD assays and clinical acceptance has become more challenging as the regulatory climate changes, particularly in the EU with evolving CE marking and ISO guidelines for IVD testing after 2021. These regulations also impact diagnostic laboratories by requiring they utilize commercial CE marked IVD kits, unless they can demonstrate superiority through auditable and expensive validation testing, an exercise outside the scope of most diagnostic flow cytometry labs. Commercial IVD developers are already operating under the 2021 regulation due to the requisite development time for IVD devices. Additional EU IVD regulatory changes require that IVD assays provide measurement traceability for any analyte, found acceptable to the Joint Committee for Traceability in Laboratory Medicine (www.JCTLM.org); traceability in the case of fluorescence flow cytometry HbF RBC assays it would be the hemoglobin content (or fluorescent surrogate reagent fluorochrome) per RBC. Thus, pragmatically it is clear that any HbF RBC assay must be delivered to the clinical laboratory by experienced IVD developers, meeting current regulatory requirements and contemporary clinical laboratory practice requirements. While the current proof of principle report describes a credible calibration method for the 4 pg HbF per RBC threshold, this LDT assay potentially has lot-to-lot variations and is susceptible to the known lot-to-lot variation in cell fixative (and permeability) reagents, particularly on a lab-to-lab basis. Manual software gating in flow cytometric assays is subjective and its accuracy is experientially based, which in the case of HbF RBC assays can be fully automated to improve reproducibility and precision.11 The second IVD device requirement is to address the actual clinical need in a format that allows both rapid testing to support SCD clinics and additionally address the Obstetrical demand for 24/7 FMH testing availability. Certainly, methods for rapid HbF RBC assays are known12-16 and existing CE IVD kits for FMH could easily be modified as done in the current study, additionally modern software algorithms are feasible and advocated to provide for fully automated high precision analysis of flow cytometric files and controls.11 These features would be requisite specifications for a commercial HbF RBC assay providing requisite rapid turnaround (< 60 minutes time to report), high precision, and moderate complexity ease of use within the laboratory workplace to allow for 24-hour availability, thus supportive of SCD outpatients clinics, patients in crisis and facilitate a more accurate adjustment of anti-D therapy in FMH patients. The format of any HbF content of RBC assay could take two pathways. The current IVD assays for FMH could be “improved” to both enhance the performance in FMH assessment and provide a new SCD solution. Alternatively, an assay for the SCD intent use could be developed separately from FMH assays. Currently in the absence of development of any new IVD solution with high precision and ease of use, the FMH testing has straddled heretofore two worlds of testing precision. Surprisingly most FMH testing remains performed with the manual microscopic method of Kleihauer Betke Braun with variable assay precision in the range of 25 - >100% (more of a qualitative assay in performance), clearly not acceptable for F cell HbF content assay, which requires the <5% CV precision afforded to rare event analysis by flow cytometry.2 What is the mostly likely course for an IVD company, two separate assays or a single improved assay? Years past the obvious choice would be to continue with the old test and just deal with the new application, but the 2021 EU regulatory rules again influence the decision. Another IVD company requirement with the new regulation is to re-register IVD products given CE IVD status under previous validation and documentation requirements under the new regulations. Thus, from a cost benefit perspective, one can make a cogent argument that since existing FMH IVD assays now require more validation (now including clinical studies) and the regents and platform of both assays are similar, that it is strategically wiser to re-engineer an improved assay format the meets the needs of both SCD and FMH testing. A fully calibrated CE IVD flow cytometric HbF RBC assay kit is sufficient and likely required as a companion diagnostic to support the initial new generation SCD treatments intended to increase RBC HbF levels, so likely Pharma will need a fully validated assay. As with reticulocyte analysis with improved parameters of immature reticulocyte fraction and reticulocyte hemoglobin content, it is conceivable that HbF RBC assays could migrate to automated blood cell counters,17 but more likely this platform migration would be a second-generation iteration and the anticipated development time would be a disservice to SCD patients needed improved therapeutic options. Thus, the testing and clinical communities concerned with SCD and FMH testing needs to prepare for both evolution of current FMH assays to more precise cytometric assays reporting both fetal RBC level and F Cell enumeration of subsets based upon HbF fractional or absolute content and plan for the changes outlined in the new IVD guidelines. The impact of a cytometric HbF content RBC assay on the prognosis of SCD patients is still being written, but for my two cents SCD patients are truly poised to benefit from an elegant bench to bedside story. The author receives royalty payments from several IVD companies related to sales of monoclonal antibodies and IVD assays for fetomaternal hemorrhage. None. The opinions expressed in this article are solely those of the authors for which no financial renumeration has been received.