Automation of a Factor XIII Activity Assay Utilizing a Plasma Blank Measurement.

Factor XIII (FXIII) is an essential coagulation factor that stabilizes fibrin clots and allows the clot to resist fibrinolysis. Inherited or acquired FXIII deficiency is a severe bleeding disorder with manifestations that can include fatal intracranial hemorrhage. Accurate FXIII laboratory testing is necessary for diagnosis, subtyping, and treatment monitoring. The recommended first-line test is FXIII activity, most commonly performed by commercial ammonia release assays. In these assays, it is important to perform a plasma blank measurement to correct for FXIII-independent ammonia production, which can lead to clinically significant overestimation of FXIII activity. Automated performance of a commercial FXIII activity assay (Technoclone, Vienna, Austria), including blank correction, on the BCS XP instrument is described.

Establishing reference intervals for von Willebrand factor multimers.

Background: von Willebrand factor (VWF) multimers (VWF:MM) methodologies are technically difficult, laborious, time consuming, non-standardized and results vary between laboratories. A new semi automated VWF:MM assay is available for routine use (Sebia). Due to lack of reference values for VWF:MM fractions, results interpretation can be challenging in some cases. The aim of this study was to determine reference intervals for low molecular weight (LMWM), intermediate molecular weight (IMWM) and high molecular weight (HMWM) multimers. Methods: By the international cooperation initiated between 4 countries (Estonia, Latvia, France, and USA) 131 samples of relatively healthy individuals were analyzed for VWF:MM (in total 51 males and 80 non-pregnant females aged 17-69 years). Reference intervals were calculated according to CLSI C28-A3 standard. Results: The proposed reference intervals for VWF:MM were calculated for LMWM 10.4-22.5%, IMWM 22.6-37.6%, HMWM 45.6-66.6%. Age related differences were seen in IMWM and HMWM (p<0.001 and 0.038). There was no gender related difference observed. Geographically LMWM results of France were different from the other regions (p<0.05). Conclusions: Quantification of VWF:MM fractions, in addition to qualitative assessment of VWF:MM patterns, has the potential to aid in differential diagnosis of von Willebrand disease (VWD) subtypes. The reference values calculated in this study can be used in future research to establish clinical decision limits.

Persistence of Ad26.COV2.S-associated vaccine-induced immune thrombotic thrombocytopenia (VITT) and specific detection of VITT antibodies.

Rare cases of COVID-19 vaccinated individuals develop anti-platelet factor 4 (PF4) antibodies that cause thrombocytopenia and thrombotic complications, a syndrome referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT). Currently, information on the characteristics and persistence of anti-PF4 antibodies that cause VITT after Ad26.COV2.S vaccination is limited, and available diagnostic assays fail to differentiate Ad26.COV2.S and ChAdOx1 nCoV-19-associated VITT from similar clinical disorders, namely heparin-induced thrombocytopenia (HIT) and spontaneous HIT. Here we demonstrate that while Ad26.COV2.S-associated VITT patients are uniformly strongly positive in PF4-polyanion enzyme-linked immunosorbent assays (ELISAs); they are frequently negative in the serotonin release assay (SRA). The PF4-dependent p-selectin expression assay (PEA) that uses platelets treated with PF4 rather than heparin consistently diagnosed Ad26.COV2.S-associated VITT. Most Ad26.COV2.S-associated VITT antibodies persisted for >5 months in PF4-polyanion ELISAs, while the PEA became negative earlier. Two patients had otherwise unexplained mild persistent thrombocytopenia (140-150 x 103 /µL) 6 months after acute presentation. From an epidemiological perspective, differentiating VITT from spontaneous HIT, another entity that develops in the absence of proximate heparin exposure, and HIT is important, but currently available PF4-polyanion ELISAs and functional assay are non-specific and detect all three conditions. Here, we report that a novel un-complexed PF4 ELISA specifically differentiates VITT, secondary to both Ad26.COV2.S and ChAdOx1 nCoV-19, from both spontaneous HIT, HIT and commonly-encountered HIT-suspected patients who are PF4/polyanion ELISA-positive but negative in functional assays. In summary, Ad26.COV2.S-associated VITT antibodies are persistent, and the un-complexed PF4 ELISA appears to be both sensitive and specific for VITT diagnosis.

A clinical, histopathological, and molecular study of two cases of VEXAS syndrome without a definitive myeloid neoplasm.

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is caused by somatic mutations in UBA1 and is identified by a genotype-driven method. This condition affects unrelated men with adultonset inflammatory syndromes in association with hematologic manifestations of peripheral cytopenia and bone marrow myeloid dysplasia. Although bone marrow vacuolization restricted to myeloid and erythroid precursors has been identified in patients with VEXAS, the detailed clinical and histopathological features of peripheral blood and bone marrows remain unclear. The current case report describes the characteristic hematologic findings in patients with VEXAS, including macrocytic anemia, thrombocytopenia, marked hypercellular bone marrow with granulocytic hyperplasia, megaloblastic changes in erythroid precursors, and the absence of hematogones in addition to prominent vacuoles in myeloid and erythroid precursor cells. Characterizing the clinical and hematologic features helps to raise awareness and improve diagnosis of this novel, rare, but potentially underrecognized disease. Prompt diagnosis expands the general knowledgeable and understanding of this disease, and optimal management may prevent patients from developing complications related to this refractory inflammatory syndrome and improve the overall clinical outcome.

Cryptogenic oozers and bruisers.

Bleeding disorders with normal, borderline, or nondiagnostic coagulation tests represent a diagnostic challenge. Disorders of primary hemostasis can be further evaluated by additional platelet function testing modalities, platelet electron microscopy, repeat von Willebrand disease testing, and specialized von Willebrand factor testing beyond the usual initial panel. Secondary hemostasis is further evaluated by coagulation factor assays, and factor XIII assays are used to diagnose disorders of fibrin clot stabilization. Fibrinolytic disorders are particularly difficult to diagnose with current testing options. A significant number of patients remain unclassified after thorough testing; most unclassified patients have a clinically mild bleeding phenotype, and many may have undiagnosed platelet function disorders. High-throughput genetic testing using large gene panels for bleeding disorders may allow diagnosis of a larger number of these patients in the future, but more study is needed. A logical laboratory workup in the context of the clinical setting and with a high level of expertise regarding test interpretation and limitations facilitates a diagnosis for as many patients as possible.

Direct oral anticoagulant (DOAC) interference in hemostasis assays.

Direct oral anticoagulants (DOACs) are a group of direct coagulation factor inhibitors including both direct thrombin inhibitors and direct factor Xa inhibitors. These medications may cause hemostasis assay interference by falsely increasing or decreasing measured values, depending on the analyte. Considering the potential for DOAC interference in a variety of hemostasis assays is essential to avoid erroneous interpretation of results. Preanalytic strategies to avoid DOAC interference include selecting alternatives to clot-based hemostasis assays in patients taking DOACs when possible and sample collection timed when the patient is off anticoagulant therapy or at the expected drug trough. Clinical laboratories may also provide educational materials that clearly describe possible interferences from DOAC, develop testing algorithms to aid in detection of DOAC in submitted samples, use DOAC-neutralizing agents to remove DOACs before continuing with testing, and write interpretive comments that explain the effects of DOAC interference in hemostasis tests. Using a combination of the described strategies will aid physicians and laboratorians in correctly interpreting hemostasis and thrombosis laboratory tests in the presence of DOACs.

Clinical, immunophenotypic and genomic findings of NK lymphoblastic leukemia: a study from the Bone Marrow Pathology Group.

Natural killer (NK) cells are lymphocytes of the native immune system that play a pivotal role in host defense and immune surveillance. While the conceptual view of NK-neoplasms is evolving, little is known about the rare NK lymphoblastic leukemia (NK-LL), which remains as a provisional entity in the 2016 WHO Classification. The goal of this study is to characterize NK-LL cases and compare with other CD56 co-expressing acute leukemias. We identified 105 cases, diagnosed as NK-LL (6), CD56+ acute undifferentiated leukemia (AUL) (6), CD56+ T-lymphoblastic leukemia (T-LL) (51), and CD56+ acute myeloid leukemia (AML) (42). Compared to AUL patients, NK-LL patients were significantly younger (p = 0.021) and presented with higher white blood cell (WBC) (p = 0.037) and platelet counts (p = 0.041). Flow cytometry showed more frequent expression of cytoplasmic CD3 (cCD3, p = 0.064) and CD33, (p = 0.065), while HLA-DR was significantly absent from NK-LL (p = 0.035) compared to AUL. Compared to T-ALL, NK-LL cases showed less frequent cCD3 (p = 0.002), CD4 (p = 0.051), and CD10 expression (p = 0.06). The frequency of abnormal karyotypes was similar between NK-LL, AUL, and T-ALL. The mutational profile differed in four leukemia groups, with a significance enrichment of NOTCH1 (p = 0.002), ETV6 (p = 0.002) and JAK3 (p = 0.02) mutations in NK-LL as compared to AML. As compared to T-ALL, NK-LL cases showed a higher number of total mutations (p = 0.04) and significantly more frequent ETV6 mutations (p = 0.004). Clinical outcome data showed differences in overall survival between all four groups (p = 0.0175), but no difference in event free survival (p = 0.246). In this largest study to date, we find that that NK-LL shows clinical presentation, immunophenotypic and molecular characteristics distinct from AUL, T-ALL, and AML. Our findings suggest NK-LL is a distinct acute leukemia entity and should be considered in the clinical diagnosis of acute leukemias of ambiguous lineage.

D-Dimer Varies Widely Across Instrument Platforms and is Not a Reliable Indicator of Periprosthetic Joint Infections.

The D-dimer test is a component of the modified scoring criteria for periprosthetic joint infection (PJI). The performance of the D-dimer test varies greatly among laboratories because of the lack of standardization. Laboratories may use different assays and will produce widely varying results for the same sample. This study used published proficiency testing data from 3903 laboratories to demonstrate the variability in D-dimer results and estimate the misclassification rate of patients using the proposed cutoff for the test as a component of PJI criteria. Given the variability in D-dimer results, a clinically significant percentage of patients are likely to be misclassified. The data illustrate that a universal cutoff for this marker in the context of assessment for PJI is not appropriate. Each site must conduct a study to determine an appropriate cutoff for their unique testing platform.

What have we learned from coagulation laboratory participation in external quality programs?

Coagulation laboratory external quality assurance (EQA) programs provide information that satisfies regulatory requirements and improves laboratory quality, patient care, and safety. In addition to the value to individual laboratories, data from EQA programs benefits the laboratory community in multiple ways by providing a snapshot of laboratory practice and summarizing the performance of various methods in identifying normal and abnormal specimens and the effects of therapies or interfering substances. This review article aims to summarize and provide examples of some of the important lessons learned from coagulation EQA data, including issues related to non-standardization, imprecision, and the effects of interfering substances.

Protein C Deficiency.

Protein C (PC) deficiency is a heritable or acquired risk factor for thrombophilia, with presentations varying from asymptomatic to venous thromboembolism to neonatal purpura fulminans, a life-threatening disorder. Hereditary PC deficiency is caused by mutation in the PC (PROC) gene located on chromosome 2q14.3. Heterozygous and acquired PC deficiencies are more common than homozygous deficiency. The recommended initial laboratory test measures PC activity using either clot-based or chromogenic methods. There are numerous potential interferences in PC activity testing that may result in either false-positive (falsely low activity) or false-negative (falsely normal or elevated activity) results. In the present review, we discuss common clinical presentations; laboratory testing, with a focus on potential assay interferences; treatment options; and prognosis in patients with PC deficiency.

Chromogenic factor VIII activity assay.

Factor VIII (FVIII) may be measured by three different methodologies in the clinical laboratory: one-stage clotting assay, two-stage clotting assay, and chromogenic assay. These assays differ in ease of use, variety of reagents available, sensitivity to mild hemophilia A, and interference from lupus anticoagulants. This review will outline the methodology for each of the FVIII activity assays, with a discussion of assay interferences and variability. In some cases, chromogenic FVIII activity assays may be preferable to clot-based assays, and these clinical situations will be reviewed as well.