Multicenter evaluation of light transmission platelet aggregation reagents: communication from the ISTH SSC Subcommittee on Platelet Physiology.

BACKGROUND: Light transmission aggregation (LTA) is used widely by the clinical and research communities. Although it is a gold standard, there is a lack of interlaboratory harmonization. OBJECTIVES: The primary objective was to assess whether sources of activators (mainly adenosine diphosphate [ADP], collagen, arachidonic acid, epinephrine, and thrombin receptor activating peptide6) and ristocetin contribute to poor LTA reproducibility. The secondary objective was to evaluate interindividual variability of results to appreciate the distribution of normal values and consequently better interpret pathologic results. METHODS: An international multicenter study involving 28 laboratories in which we compared LTA results obtained with center-specific activators and a comparator that we supplied. RESULTS: We report variability in the potency (P) of activators in comparison with the comparator. Thrombin receptor activating peptide 6 (P, 1.32-2.68), arachidonic acid (P, 0.87-1.43), and epinephrine (P, 0.97-1.34) showed the greatest variability. ADP (P, 1.04-1.20) and ristocetin (P, 0.98-1.07) were the most consistent. The data highlighted clear interindividual variability, notably for ADP and epinephrine. Four profiles of responses were observed with ADP from high-responders, intermediate-responders, and low-responders. A fifth profile corresponding to nonresponders (5% of the individuals) was observed with epinephrine. CONCLUSION: Based on these data, the establishment and adoption of simple standardization principles should mitigate variability due to activator sources. The observation of huge interindividual variability for certain concentrations of activators should lead to a cautious interpretation before reporting a result as abnormal. Confidence can be taken from the fact that difference between sources is not exacerbated in patients treated with antiplatelet agents.

Serum from half of patients with immune thrombocytopenia trigger macrophage phagocytosis of platelets.

Humoral antiplatelet factors, such as autoantibodies, are thought to primarily clear platelets by triggering macrophage phagocytosis in immune thrombocytopenia (ITP). However, there are few studies characterizing the capacity and mechanisms of humoral factor-triggered macrophage phagocytosis of platelets using specimens from patients with ITP. Here, we assessed sera from a cohort of 24 patients with ITP for the capacity to trigger macrophage phagocytosis of normal donor platelets and characterized the contribution of humoral factors to phagocytosis. Sera that produced a phagocytosis magnitude greater than a normal human serum mean + 2 standard deviations were considered phagocytosis-positive. Overall, 42% (8/19) of MHC I alloantibody-negative ITP sera were phagocytosis-positive. The indirect monoclonal antibody immobilization of platelet antigens assay was used to detect immunoglobulin G (IgG) autoantibodies to glycoproteins (GP)IIb/IIIa, GPIb/IX, and GPIa/IIa. Autoantibody-positive sera triggered a higher mean magnitude of phagocytosis than autoantibody-negative sera. Phagocytosis correlated inversely with platelet counts among autoantibody-positive patients but not among autoantibody-negative patients. Select phagocytosis-positive sera were separated into IgG-purified and -depleted fractions via protein G and reassessed for phagocytosis. Phagocytosis was largely retained in the purified IgG fractions. In addition, we assessed serum concentrations of C-reactive protein, serum amyloid P, and pentraxin 3 as potential phagocytosis modulators. Pentraxin 3 concentrations correlated inversely with platelet counts among patients positive for autoantibodies. Taken together, sera from approximately half of the patients with ITP studied triggered macrophage phagocytosis of platelets beyond a normal level. An important role for antiplatelet autoantibodies in phagocytosis is supported; a role for pentraxins such as pentraxin 3 may be suggested.

Automated Light Transmission Aggregometry with and without Platelet Poor Plasma Reference: A Method Comparison.

Background Light transmission aggregometry (LTA) is considered the gold standard for the evaluation of platelet function but is labor-intensive and involves numerous manual steps. Automation may contribute to standardization. Here, we evaluate the performance characteristics of a new automated instrument, Thrombomate XRA (TXRA), and compare it against a manual instrument (PAP-8). Materials and Methods Leftover blood samples from blood donors or patients were tested in parallel with identical reagents and in identical concentrations both manually using PAP-8 and automated on the TXRA. In addition to precision and method comparison, an additional evaluation was performed on the TXRA against "virtual" platelet-poor plasma (VPPP) based on artificial intelligence. The main focus was on comparing the maximum aggregation (MA%) values. Results Precision for MA% ranged from 1.4 to 4.6% on TXRA for all reagents. Normal ranges for 100 healthy blood donors on both instruments were in a similar range for all reagents, with a tendency to slightly higher values with TXRA. Most agonists resulted in normally distributed MA%. Comparing 47 patient samples on both devices showed a good correlation for both slope and MA% with some differences in individual samples with epinephrine and TRAP. Correlation between the TXRA measurement against PPP and "virtual" PPP demonstrated excellent correlation. Reaction signatures of both devices were very similar. Conclusion TXRA provides reproducible LTA results that correlate with an established manual method when tested against PPP or VPPP. Its ability to perform LTA only from platelet-rich plasma without requiring autologous PPP simplifies LTA. TXRA is an important step not only for further standardizing LTA but also for a more widespread use of this important method.

Long-Term Outcomes after Vaccine-Induced Thrombotic Thrombocytopenia.

Vaccine-induced thrombotic thrombocytopenia (VITT), or thrombosis with thrombocytopenia syndrome (TTS), is a rare but serious complication of adenovirus-based vaccines against severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Observation of long-term outcomes is important to guide treatment of affected patients. This single-center consecutive cohort study included all patients diagnosed based on (1) vaccination 4 to 21 days before symptom onset, (2) signs or symptoms of venous or arterial thrombosis, (3) thrombocytopenia < 150/nL, (4) positive anti-platelet factor 4 (PF4) antibody, and (5) elevated D-Dimer > 4 times the upper limit of normal. Nine patients were enrolled. Acute management consisted of parenteral anticoagulants, corticosteroids, intravenous immunoglobulin (IVIG), and/or eculizumab. Eculizumab was successfully used in two patients with recurrent thromboembolic events after IVIG. Direct oral anticoagulants were given after hospital discharge. Median follow-up duration was 300 days (range 153 to 380). All patients survived the acute phase of the disease and were discharged from hospital. One patient died from long-term neurological sequelae of cerebral venous sinus thrombosis 335 days after diagnosis. Eight out of nine patients were alive at last follow-up, and seven had fully recovered. Anti-PF4 antibodies remained detectable for at least 12 weeks after diagnosis, and D-Dimer remained elevated in some patients despite oral anticoagulation. No recurrent thromboembolic events, other signs of VITT relapse, or bleeding complications occurred after discharge. In conclusion, VITT appears to be a highly prothrombotic condition. IVIG is not always successful, and eculizumab may be considered a rescue agent. Long-term management with direct oral anticoagulants appears to be safe and effective.

Diagnosing Immune Thrombocytopenia.

Although the detection of a characteristic autoantibody can prove immune thrombocytopenia (ITP), this diagnosis is often based on the exclusion of other causes of thrombocytopenia. Direct glycoprotein (GP)-specific tests have the property required to demonstrate such a characteristic autoantibody. In contrast, platelet-associated immunoglobulin G or antibody detection in plasma or serum is an insufficient diagnostic test. Moreover, data for commercial capture assays are sparse and their use is currently not recommended. A significant drawback of direct GP-specific tests is their low sensitivity, and a negative test result has no relevance. It is therefore also useful to establish a diagnosis of (primarily) hyperdestructive thrombocytopenia. A full blood count together with the immature platelet fraction has an excellent positive predictive value for ITP. Plasma glycocalicin has no apparent diagnostic value in identifying ITP patients, and conflicting data for TPO preclude its use for diagnostic purposes.

Single amino acid charge switch defines clinically distinct proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1)-associated inflammatory diseases.

BACKGROUND: Hyperzincemia and hypercalprotectinemia (Hz/Hc) is a distinct autoinflammatory entity involving extremely high serum concentrations of the proinflammatory alarmin myeloid-related protein (MRP) 8/14 (S100A8/S100A9 and calprotectin). OBJECTIVE: We sought to characterize the genetic cause and clinical spectrum of Hz/Hc. METHODS: Proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) gene sequencing was performed in 14 patients with Hz/Hc, and their clinical phenotype was compared with that of 11 patients with pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome. PSTPIP1-pyrin interactions were analyzed by means of immunoprecipitation and Western blotting. A structural model of the PSTPIP1 dimer was generated. Cytokine profiles were analyzed by using the multiplex immunoassay, and MRP8/14 serum concentrations were analyzed by using an ELISA. RESULTS: Thirteen patients were heterozygous for a missense mutation in the PSTPIP1 gene, resulting in a p.E250K mutation, and 1 carried a mutation resulting in p.E257K. Both mutations substantially alter the electrostatic potential of the PSTPIP1 dimer model in a region critical for protein-protein interaction. Patients with Hz/Hc have extremely high MRP8/14 concentrations (2045 ± 1300 µg/mL) compared with those with PAPA syndrome (116 ± 74 µg/mL) and have a distinct clinical phenotype. A specific cytokine profile is associated with Hz/Hc. Hz/Hc mutations altered protein binding of PSTPIP1, increasing interaction with pyrin through phosphorylation of PSTPIP1. CONCLUSION: Mutations resulting in charge reversal in the y-domain of PSTPIP1 (E→K) and increased interaction with pyrin cause a distinct autoinflammatory disorder defined by clinical and biochemical features not found in patients with PAPA syndrome, indicating a unique genotype-phenotype correlation for mutations in the PSTPIP1 gene. This is the first inborn autoinflammatory syndrome in which inflammation is driven by uncontrolled release of members of the alarmin family.

Collection of peripheral blood progenitor cells on Day 4 is feasible and effective while reducing granulocyte-colony-stimulating factor exposure to healthy donors.

BACKGROUND: Peripheral blood progenitor cells (PBPCs) are the most common stem cell source for allogeneic transplantations. Analysis of our collection data obtained with a Spectra Optia device (Terumo) for apheresis demonstrated collection efficacies (CEs) exceeding our internal target levels of 5 × 10(6) CD34+ cells/kg body weight of the recipient when collected on Day 5. We thus aimed to investigate whether collection on Day 4 would lead to adequate amounts of PBPCs while minimizing granulocyte-colony-stimulating factor (G-CSF) exposure in healthy donors. STUDY DESIGN AND METHODS: We compared feasibility and effectiveness of Day 5 versus Day 4 collections with data obtained from 23 and 18 allogeneic procedures, respectively. RESULTS: Both groups were comparable with regard to donor and collection characteristics. Product characteristics as well as platelet loss, CE, throughput, and collection rate did not differ between both protocols. A higher contamination with white blood cells (WBCs; ×10(9) /L) was observed in products collected on Day 5 compared to Day 4 (231 [range, 181-299] vs. 203 [range, 165-239]; p = 0.004). A second apheresis procedure was required in three of 23 patients and three of 18 patients, respectively (p = 0.6) to obtain the required PBPC dose. CONCLUSIONS: PBPC apheresis on Day 4 seems as feasible and effective as collection on Day 5. Collection on Day 4 produces lower WBC content in the product and allows a reduction in G-CSF exposure to healthy donors.

Effectiveness of vitamin K in anticoagulation reversal for hip fracture surgery--a prospective observational study.

INTRODUCTION: Vitamin K antagonists are often used for anticoagulant treatment in hip fracture patients. The optimal handling with such anticoagulants is unclear. We aimed to determine when anticoagulation reversal occurred after vitamin K administration and how often prothrombin complex concentrates (PCCs) were administered. We compared patients' treatments and outcomes with those of a control group not receiving treatment for anticoagulation. PATIENTS AND METHODS: A total of 402 geriatric hip fracture patients were included in this observational study. We collected data on treatment for anticoagulation, time to surgery, and reasons for delay of surgery. In patients taking vitamin K antagonists, we measured the INR (international normalized ratio) on admission and prior to surgery, along with the frequency of PCC administration. Finally, we compared in-hospital mortality and complications between patient groups. RESULTS: A total of 62 (15%) patients received phenprocoumon prior to their fractures. Surgery was delayed in these patients compared to controls (27h; 95%CI 23-31 vs. 16h; 95%CI 19-19; p=0.001), but surgery delay >48h (n=5; 8%) was not due to a failure of INR reversal. The main reason for these delays was a lack of capacity for surgery. The average INR on admission was 2.1 (±0.7; range 1.0-3.5) in patients taking phenprocoumon, which decreased to 1.3 (±0.3; range 1.0-1.6) until surgery. PCCs were administered to 19% of patients. We found no differences in the in-hospital mortality (6.2% vs. 8.1%, p=0.575) or complication rates (12.9% vs. 9.4%, p=0.364). CONCLUSION: The use of vitamin K seemed to be sufficient for anticoagulation reversal in geriatric hip fracture patients, and it generally led to timely surgery; despite this success, PCCs were sometimes administered for logistical reasons.

Non-transient "self-sustaining" heparin-induced thrombocytopenia: 4-year persistence of a platelet-activating PF4/heparin-antibody status without heparin exposure.

The more we come to understand the pathophysiology of heparin-induced thrombocytopenia (HIT) syndrome, the more we realize that HIT is a rather unusual immune response. One peculiar feature of HIT is the transient character of the antibodies. After cessation of exposure to heparins, the antibodies tend to disappear after 40-100 days. If re-immunization occurs, it generally takes at least 4 days to redevelop antibodies (if they are formed at all). We report about a patient who most likely developed platelet-activating IgG-specific platelet factor 4 (PF4)/heparin antibodies after knee surgery, experienced a transient ischemic attack years later [when HIT was diagnosed by using PF4/heparin ELISA] and presented a high number of these antibodies even 4 years after this first diagnosis of HIT without further re-exposure to heparin.

Anti-human neutrophil antigen-3a induced transfusion-related acute lung injury in mice by direct disturbance of lung endothelial cells.

OBJECTIVE: Antibodies against human neutrophil antigen-3a (HNA-3a) located on choline transporter-like protein 2 induce severe transfusion-related acute lung injury (TRALI). This study aims to identify the mechanism implicated in anti-HNA-3a-mediated TRALI. APPROACH AND RESULTS: Our analysis shows that anti-HNA-3a recognizes 2 choline transporter-like protein 2 isoforms (P1 and P2) on human microvascular endothelial cells from lung blood vessels but reacts only with the P1 isoform on neutrophils. Direct treatment of HNA-3a-positive endothelial cells with anti-HNA-3a, but not with anti-HNA-3b, leads to reactive oxygen species production, increased albumin influx, and decreased endothelial resistance associated with the formation of actin stress filaments and loosening of junctional vascular endothelium-cadherin. In a novel in vivo mouse model, TRALI was documented by significant increase in lung water content, albumin concentration, and neutrophil numbers in the bronchoalveolar lavage on injection of human anti-HNA-3a in lipopolysaccharides-treated, as well as nontreated mice. Interestingly, although neutrophil depletion alleviated severity of lung injury, it failed to prevent TRALI in this model. Infusion of anti-HNA-3a F(ab')2 fragments caused moderate TRALI. Finally, mice lacking nicotinamide adenine dinucleotide phosphate oxidase (NOX2(y/-)) were protected from anti-HNA-3a-mediated TRALI. CONCLUSIONS: These data demonstrate the initiation of endothelial barrier dysfunction in vitro and in vivo by direct binding of anti-HNA-3a on endothelial cells. It seems, however, that the presence of neutrophils aggravates barrier dysfunction. This novel mechanism of TRALI primarily mediated by endothelial cell dysfunction via choline transporter-like protein 2 may help to define new treatment strategies to decrease TRALI-related mortality.

A single center comparison between three different apheresis systems for autologous and allogeneic stem cell collections.

Three different apheresis systems were used in our center for the collection of peripheral blood progenitor cells (PBPCs): COM.TEC (Fresenius Healthcare), COBE Spectra, and Spectra Optia (both from Caridian BCT). We compared 131 autologous and 56 allogeneic apheresis procedures to elucidate feasibility and effectiveness of the different systems. Collection efficiacy varied significantly with lowest results obtained with COBE Spectra. COM.TEC and Spectra Optia produced lower WBC contamination than COBE Spectra, but at the expense of higher product volume and longer apheresis time. High collection efficacy and a low product volume may be favorable characteristics of the Spectra Optia.

Anti-protamine-heparin antibodies: incidence, clinical relevance, and pathogenesis.

Protamine, which is routinely used after cardiac surgery to reverse the anticoagulant effects of heparin, is known to be immunogenic. Observing patients with an otherwise unexplained rapid decrease in platelet count directly after protamine administration, we determined the incidence and clinical relevance of protamine-reactive antibodies in patients undergoing cardiac-surgery. In vitro, these antibodies activated washed platelets in a FcγRIIa-dependent fashion. Using a nonobese diabetic/severe combined immunodeficiency mouse model, those antibodies induced thrombocytopenia only when protamine and heparin were present but not with protamine alone. Of 591 patients undergoing cardiopulmonary bypass surgery, 57 (9.6%) tested positive for anti-protamine-heparin antibodies at baseline and 154 (26.6%) tested positive at day 10. Diabetes was identified as a risk factor for the development of anti-protamine-heparin antibodies. In the majority of the patients, these antibodies were transient and titers decreased substantially after 4 months (P < .001). Seven patients had platelet-activating, anti-protamine-heparin antibodies at baseline and showed a greater and more prolonged decline in platelet counts compared with antibody-negative patients (P = .003). In addition, 2 of those patients experienced early arterial thromboembolic complications vs 9 of 584 control patients (multivariate analysis: odds ratio, 21.58; 95% confidence interval, 2.90-160.89; P = .003). Platelet-activating anti-protamine-heparin antibodies show several similarities with anti-platelet factor 4-heparin antibodies and are a potential risk factor for early postoperative thrombosis.

Implication of transfected cell lines for the detection of alloantibodies against human neutrophil antigen-3.

BACKGROUND: Alloantibodies against human neutrophil antigen-3 (HNA-3) are responsible for the fatalities reported in transfusion-related acute lung injury. Consequently, reliable detection of these alloantibodies is mandatory to improve blood transfusion safety. In this study, we developed stable cell lines for the detection of HNA-3 antibodies. STUDY DESIGN AND METHODS: HEK293T were transfected with HNA-3a or HNA-3b constructs and sorted by flow cytometry according to high surface expression. Transfected cells were tested with sera containing HNA-3 antibodies in flow cytometry and antibody capture assay (ACA). The results were compared with granulocyte agglutination test and granulocyte immunofluorescence test. RESULTS: In flow cytometry, 12 of 14 HNA-3a sera reacted specifically with HNA-3aa cells. One serum sample showed positive reaction with HNA-3bb cells. All HNA-3b sera recognized HNA-3bb cells. No reaction was observed with broad reactive antibodies against HLA Class I. In ACA, all HNA-3a sera (12/12) showed positive reactivity with HNA-3aa cells with no cross-reactivity with HNA-3bb cells. Again, all HNA-3b sera reacted with HNA-3bb cells only. Furthermore, genotyping of 249 individuals detected a new HNA-3 allele caused by a nucleotide substitution C>T at Position 457 leading to L(153)F mutation in choline transporter-like protein-2. This mutation impairs polymerase chain reaction with sequence-specific primers based HNA-3a typing. However, analysis with cells expressing F(153) isoform showed that this mutation did not alter the binding of HNA-3 antibodies. CONCLUSIONS: This study demonstrated that HEK293T cells expressing stable recombinant HNA-3 are suitable for the detection of HNA-3 alloantibodies allowing reliable screening of blood products.

A novel enzyme-linked immunosorbent assay method for the detection of human neutrophil antigen-2a antibodies.

BACKGROUND: Antibodies to human neutrophil antigen (HNA)-2a are responsible for a number of immune-mediated neutropenia disorders. Although several methods exist for the identification of anti-HNA-2a, all these methods have several limitations. In this study, a solid-phase enzyme-linked immunosorbent assay (ELISA) using recombinant HNA-2a antigen (rHNA-2a) allowing rapid detection of HNA-2a antibodies was developed. STUDY DESIGN AND METHODS: Soluble rHNA-2 was generated by transfection of insect cells with CD177 vector. Purified rHNA-2a was immobilized on microtiter wells coated with anti-CD177 and was applied to analyze 10 sera containing HNA-2a antibodies. For the evaluation of the ELISA method, results were compared with the standard assay, MAIGA (monoclonal antibody antigen capture assay) for detection of neutrophil antibodies. RESULTS: The specificity of HNA-2a antibodies in all sera was confirmed by immunoblotting. Sera were then tested simultaneously in ELISA and MAIGA assays. Nine of 10 sera showed positive reactions in ELISA, whereas only 9 of 10 sera reacted in the standard MAIGA assay. All HNA-2a antibodies were detectable in MAIGA when diluted sera were applied. No reaction was observed with different sera containing neutrophil-reactive antibodies (6 anti-HNA-1a, 4 anti-HNA-1b, and 20 anti-HLA Class I and II) in ELISA. All HNA-2a antibodies were detectable in MAIGA when diluted sera were applied. Notably, sera containing anti-proteinase 3 (PR3) from patients with Wegener's granulomatosis reacted in MAIGA. In contrast, this antibody showed no reaction in ELISA with purified rHNA-2a. CONCLUSIONS: These results demonstrated that ELISA with rHNA-2a provides a good method for detecting HNA-2a antibodies in human serum. This assay enables to exclude the presence of autoantibody against PR3 in patient's sera, which cannot be differentiated from anti HNA-2a with current serologic methods.

Transfusion-associated graft-versus-host disease.

Transfusion-associated graft-versus-host disease (TA-GvHD) is a rare complication of blood transfusion that has a fatal outcome in most patients. It is caused by the transfusion of viable T cells present in blood products that are not rejected by the transfusion recipient, either because of recipient immunodeficiency or because of a common HLA haplotype between the blood donor and recipient. Because effective treatment is not available, risk identification and prevention are of central importance. Among the potential risk factors that have been discussed to date, a definite hazard for developing TA-GvHD has been recognized for HLA-matched transfusions or transfusions from blood relatives, intrauterine and exchange transfusions, patients with congenital immunodeficiency syndromes, bone marrow transplantation, stem cell transplantation, or lymphomas. Patients at possible TA-GvHD risk who will require further evaluation include patients with hematologic malignancies, solid tumors, or solid organ transplantation. Although postulated, an increased risk for term or preterm newborns and patients with HIV/AIDS has not thus far been demonstrated.