Smock, Kristi J.

ABSTRACT Background Platelet factor 4‐polyanion enzyme‐linked immunosorbent assays (ELISAs) are considered highly sensitive for diagnosing heparin‐induced thrombocytopenia (HIT), such that current practice guidelines recommend use of ELISA‐negative results to exclude HIT. Once HIT is ruled out, alternative, non‐heparin‐based anticoagulant treatments are ceased, and heparin reintroduction frequently occurs. Methods Antigen‐based and PF4‐dependent functional testing were used to study PF4/polyvinylsulfonate ELISA‐negative platelet‐activating antibodies in HIT‐suspected patients. Results Three patients with clinical presentations consistent with HIT tested negative in an ELISA using PF4‐polyvinylsulfonate (PF4/PVS), an antigenic target very commonly used for HIT antibody screening. All three patients demonstrated PF4‐dependent platelet activation in functional testing that was sensitive to blockade of platelet FcγRIIa receptors and inhibited by high concentrations of heparin, consistent with pathogenic HIT antibodies. Functional testing‐based screening of 500 ELISA‐negative patients identified three additional patients whose sera activated platelets in a PF4‐ and FcγRIIa‐dependent manner and had clinical histories consistent with HIT. Five of the six ELISA‐negative HIT patients were re‐exposed to heparin, which precipitated a decrease in platelet counts in all re‐exposed patients, and one patient developed a new thrombus. Conclusions Recognition of ELISA‐negative HIT is critical to avoid harm due to the cessation of alternative anticoagulation therapy and re‐exposure of these patients to heparin.

Abstract Measurement of D-dimer, a fibrin degradation product, is crucial for diagnosing thrombotic and fibrinolytic conditions. Despite its expanding utility, D-dimer testing faces challenges due to varying assay methodologies and nonstandardized reporting. To understand D-dimer reporting practices, the College of American Pathologists Hemostasis and Thrombosis Committee analyzed proficiency testing data from D-dimer surveys conducted between 2020 and 2023 across multiple laboratories. The data demonstrate that laboratories commonly report units that differ from the assay package insert when reporting their proficiency testing results. We sought to assess variability in reporting practices and opportunities for improvement in D-dimer reporting amongst participants. To accomplish this, we harmonized all D-dimer results to ng/mL fibrinogen equivalent units from one distributed sample and assessed distinct populations of entries per instrument/reagent combination. Notable trends emerged from this analysis. Data from laboratories using the same instrument/reagent combinations clustered around different means, suggesting issues with unit reporting and/or conversion. Entries reported in mg/L or ug/mL D-dimer units commonly exhibited significant deviations from the dominant population. For one instrument/reagent combination, most reported values differed by approximately twice the certificate of analysis value. This analysis highlights potential issues with D-dimer reporting in proficiency testing. Whether these issues translate into issues with clinical D-dimer reporting requires further study.

Context.— Clinical laboratories are increasingly implementing pharmacogenomic (PGx) testing. Although PGx is similar to genetic testing for other indications, there are unique aspects that laboratories should consider. Objective.— To aid clinical laboratories that are implementing clinical PGx testing by describing characteristics of PGx test design and validation, as well as approaches to reporting. Resources that are useful for clinical laboratories performing PGx testing will be highlighted. Design.— The College of American Pathologists formed a workgroup composed of laboratorians with expertise in clinical PGx testing. The workgroup included representatives from the Association for Molecular Pathology and the American College of Medical Genetics and Genomics. The workgroup reviewed pertinent literature, as well as experience from proficiency testing and from members’ laboratories. Results.— The workgroup recommends that laboratories implementing PGx consider the following concepts: testing platform, test design (ie, selection of pharmacogenes and variants/alleles), use of reference materials during test development and as controls during clinical runs, star allele and standard nomenclature systems, translations from genotype to predicted phenotype, and considerations for result reporting including making medication recommendations. The workgroup provides considerations when using report vendors, emphasizing the clinical laboratory’s role and responsibility when implementing such reporting tools from vendors. Conclusions.— Clinical laboratories should be familiar with the fundamentals of PGx, ensure that PGx testing meets the applicable regulatory requirements for all aspects of the clinical laboratory testing process, and follow recommendations for standardization of nomenclature and reporting.

ABSTRACT BACKGROUND Platelet factor 4-polyanion enzyme-linked immunosorbent assays (ELISAs) are considered highly sensitive for diagnosing heparin-induced thrombocytopenia (HIT), such that current practice guidelines recommend use of ELISA-negative results to exclude HIT. Once HIT is ruled out, alternative, non-heparin-based anticoagulant treatments are ceased, and heparin reintroduction frequently occurs. METHODS Antigen-based and PF4-dependent functional testing were used to study PF4/polyvinyl sulfonate ELISA-negative platelet-activating antibodies in HIT-suspected patients and mice immunized with PF4/heparin. RESULTS Three patients with clinical presentations consistent with HIT tested negative in an ELISA using PF4-polyvinylsulfonate (PF4/PVS), an antigenic target very commonly used for HIT antibody detection. All three patients demonstrated PF4-dependent platelet activation in functional testing that was sensitive to blockade of platelet FcγRIIa receptors and inhibited by high concentrations of heparin, consistent with pathogenic HIT antibodies. Functional testing-based screening of 500 ELISA-negative patients identified three patients whose sera activated platelets in a PF4- and FcγRIIa-dependent manner, and had clinical histories consistent with HIT. Five of the six ELISA-negative HIT patients were re-exposed to heparin, which precipitated a decrease in platelet counts in all re-exposed patients, and one patient developed a new thrombus. To advance the study of ELISA-negative HIT antibodies, mice were immunized with PF4/heparin, and functional and antigenic assays were simultaneously used to successfully identify an ELISA-negative, PF4-dependent platelet-activating murine monoclonal antibody that recapitulated the serological characteristics of ELISA-negative HIT patients. CONCLUSIONS Recognition of ELISA-negative HIT is critical to avoid harm due to the cessation of alternative anticoagulation therapy and re-exposure of these patients to heparin.

Abstract Background There have been conflicting messages about the influence of female sex hormones on women's health, with historical messaging indicating that use of estrogen and/or progesterone in peri- or postmenopause poses a significant clinical risk to cisgender women. More recent guidance indicates that the benefit of hormone therapy (HT) outweighs the risks for symptomatic women. Exogenous estrogen use is also indicated for contraception and gender-affirming care. Despite the potential for broad applications, robust reference intervals for estradiol, progesterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are lacking, and guidelines indicate that measurement of 17-β-estradiol (E2), progesterone, LH, or FSH does not facilitate care in women who may be experiencing menopausal symptoms or women taking exogenous HT. Content Here we review the physiological roles of estrogen, progesterone, LH, and FSH. We examine the modes of administration for estrogen and progesterone, clarify the nomenclature related to exogenous hormone use, and comprehensively review the literature for studies evaluating normal concentrations of these female gonadal axis hormones during the menstrual cycle. The content primarily focuses on cisgender women, but some aspects of these hormones in transgender women will also be discussed. Summary Currently, E2, LH, FSH, and progesterone reference intervals for women remain incomplete. Although there are a variety of clinical indications that benefit women using HT, symptoms and shared decision-making should guide care. Collaborative efforts between clinicians and laboratory professionals to better define therapeutic or reference intervals for these hormones can advance women's health globally.

Background: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe thrombotic thrombocytopenia that predominantly occurs after adenoviral vector-based vaccines. VITT patients produce anti-platelet factor 4 (PF4) antibodies with restricted clonality (i.e., mono/oligoclonal) with lambda light chains. Long-term follow-up studies in VITT show a disconnect in the persistence of anti-PF4 antibodies, whereby ELISA-positive antibodies persist significantly longer than platelet-activating antibodies. The mechanistic basis of this is unclear and could be explained by a couple of hypotheses: (1) Epitope spreading where, with time, the specificity of platelet-activating anti-PF4 antibodies may “spread” to recognize only PF4 complexed to heparin vs. (2) Persistence of clonally restricted platelet-activating anti-PF4 antibodies which decrease in titers with time such that their levels are insufficient to crosslink platelet FcGRIIa and induce platelet activation. Methods: Acute, convalescent patient sera and affinity-purified anti-PF4 antibodies were evaluated in antigen-based, functional (platelet-activation-based), and mass spectrometry studies as previously described (Blood, 2022 Jul 7;140(1):73-77). The median time to last follow-up was 243 days after the acute episode (range: 114-664 days). Results: Follow-up samples were obtained from 6 VITT patients after Ad26.COV2.S vaccination. At last follow-up, anti-PF4 IgG antibodies reactive in PF4/polyanion ELISA were detected in the sera of 5/6 VITT patients. However, only one of these samples stimulated platelet activation in the PF4-dependent P-selectin expression assay (PEA; 199 days after acute presentation). Affinity-purified anti-PF4 antibodies were detected by ELISA in all follow-up samples and three of the six affinity-purified anti-PF4 antibody samples activated platelets in the PEA. Four of the six VITT patients demonstrated a monoclonal anti-PF4 antibody, while one each had a biclonal and triclonal profile. Five VITT patients produced IgG lambda light chain antibodies, while one produced an IgG kappa. All six VITT patients demonstrated synonymous anti-PF4 antibody profiles when comparing acute and follow-up antibody repertoires, without evidence of VITT patients producing new anti-PF4 antibodies after the acute episode. In all cases, levels of anti-PF4 antibodies were significantly lower in convalescent samples relative to acute samples. Given this data on the persistence of clonally-restricted VITT antibodies, samples from another VITT patient with thrombocytopenia (100-150,000/uL) persisting for more than three years were similarly evaluated. This patient, who experienced severe thrombosis after ChAdOx1 nCoV19 vaccination, demonstrated persistent ELISA and PEA positivity even three years after symptom onset but had not experienced breakthrough thrombosis while on anticoagulation therapy. Serum protein electrophoresis (SPEP) did not detect a monoclonal gammopathy. Anti-PF4 antibodies immuno-enriched from a sample obtained >2 years after acute presentation demonstrated a monoclonal profile. While non-availability of an acute sample for anti-PF4 antibody characterization precluded a firm determination of whether this “Long VITT” case represented a subset of monoclonal gammopathy of thrombotic significance (MGTS; Blood 2023 Apr 6;141(14):1772-1776; New Eng J Med 2024 Aug 8), the chronicity of thrombocytopenia, presence of a monoclonal anti-PF4 antibody and persistently positive anti-PF4 serology supports this diagnosis. Conclusions. Clonally-restricted VITT antibodies persist for months-years with no evidence for the production of novel anti-PF4 antibodies after the acute event. Decreased abundance of anti-PF4 antibodies leads to negative functional results with follow up samples. Some platelet-activating antibodies can persist for very long periods (>3 years) and mediate persistent thrombocytopenia, representing an SPEP-negative MGTS-like state. This study also demonstrates the first example of a VITT antibody with a kappa light chain. The requirement for anticoagulation in patients demonstrating ELISA-positive/functional test-negative results is not clear, but the persistence of mono/oligoclonal anti-PF4 antibodies raises the possibility for the upregulation of pathogenic antibody production in proinflammatory settings.

AbstractVon Willebrand factor (VWF) level and/or function is altered in von Willebrand disease (VWD), the most common heritable bleeding disorder worldwide. Laboratory assessment of VWF is continually evolving. Historically, the primary method for the assessment of VWF platelet-binding activity was the ristocetin cofactor assay (VWF:RCo). Contemporary alternative measures of VWF platelet-binding activity include VWF:GPIbR (recombinant; using ristocetin), VWF:GPIbM (recombinant; gain-of-function mutant), and monoclonal antibody. Recently, the American Society of Hematology, International Society on Thrombosis and Haemostasis, National Hemophilia Foundation, and World Federation of Hemophilia collaboration issued guidelines recommending the use of newer assays of VWF platelet-binding activity (VWF: GPIbM, VWF: GPIbR) over VWF:RCo, given known limitations of the VWF:RCo assay. Despite this recommendation, the newer VWF:GPIbM and VWF:GPIbR assays are not United States Food and Drug Administration cleared, limiting their availability in the United States. We sought to assess assay utilization trends, agreement of VWF testing methods, and imprecision of VWF testing (based on assigned sample type) from the College of American Pathologists Proficiency Testing Surveys. The analysis confirms that, while VWF antigen testing has low imprecision, the various VWF activity assays have significant interassay variability, with VWF:RCo showing greater imprecision than the newer GPIb-binding assays. The overall trends in assay utilization reflect the barriers to complete compliance with modern VWD diagnostic guidelines in North America.

AbstractVon Willebrand factor (VWF) level and/or function is altered in von Willebrand disease (VWD), the most common heritable bleeding disorder worldwide. Laboratory assessment of VWF is continually evolving. Historically, the primary method for the assessment of VWF platelet-binding activity was the ristocetin cofactor assay (VWF:RCo). Contemporary alternative measures of VWF platelet-binding activity include VWF:GPIbR (recombinant; using ristocetin), VWF:GPIbM (recombinant; gain-of-function mutant), and monoclonal antibody. Recently, the American Society of Hematology, International Society on Thrombosis and Haemostasis, National Hemophilia Foundation, and World Federation of Hemophilia collaboration issued guidelines recommending the use of newer assays of VWF platelet-binding activity (VWF: GPIbM, VWF: GPIbR) over VWF:RCo, given known limitations of the VWF:RCo assay. Despite this recommendation, the newer VWF:GPIbM and VWF:GPIbR assays are not United States Food and Drug Administration cleared, limiting their availability in the United States. We sought to assess assay utilization trends, agreement of VWF testing methods, and imprecision of VWF testing (based on assigned sample type) from the College of American Pathologists Proficiency Testing Surveys. The analysis confirms that, while VWF antigen testing has low imprecision, the various VWF activity assays have significant interassay variability, with VWF:RCo showing greater imprecision than the newer GPIb-binding assays. The overall trends in assay utilization reflect the barriers to complete compliance with modern VWD diagnostic guidelines in North America.