Plasma thrombopoietin levels as additional tool in clinical management of thrombocytopenic neonates.

Plasma thrombopoietin (Tpo) levels distinguish thrombocytopenia resulting from increased platelet destruction or decreased platelet production. We investigated whether measuring plasma Tpo levels in thrombocytopenic newborns is of diagnostic value to establish the underlying mechanism of thrombocytopenia.Tpo levels were measured with in-house developed ELISA in samples referred to our center because of thrombocytopenia noticed in the first 10 days of life. Clinical data were collected.Plasma Tpo levels <128 AU/ml were found in the majority (92%) of 121 newborns with immune-mediated thrombocytopenia (n = 104) and thrombocytopenia due to bacterial infections (n = 7); increased plasma Tpo levels (≥128 AU/ml) were found in thrombocytopenic newborns with severe asphyxia (n = 24). Highly increased plasma Tpo levels (>200 AU/ml) were found in thrombocytopenic neonates with congenital viral infections (n = 22) or amegakaryocytosis (n = 6). A plasma Tpo level <128 AU/ml excludes (negative predictive value 96%, 95% CI 90-99) severe asphyxia, congenital viral infections and amegakaryocytosis as the cause for thrombocytopenia in newborns.Increased plasma Tpo levels indicate that thrombocytopenia in newborns, as a result of various nonimmune disorders, is often caused by (temporary) bone marrow suppression/failure. Measurement of plasma Tpo levels provides the clinician with an additional tool to decide on the differential diagnosis, the necessity for subsequent diagnostics and treatment in neonates with thrombocytopenia.

Multicenter Study on Differential Human Neutrophil Antigen 2 Expression and Underlying Molecular Mechanisms.

BACKGROUND: The human neutrophil antigen 2 (HNA-2), which is expressed on CD177, is undetectable in 3-5% of the normal population. Exposure of these HNA-2null individuals to HNA-2-positive cells can cause immunization and pro-duction of HNA-2 antibodies, which can induce immune neutropenia and transfusion-related acute lung injury. In HNA-2-positive individuals, neutrophils are divided into a CD177pos. and a CD177neg. subpopulation. The molecular background of HNA-2 deficiency and the bimodal expression pattern, however, are not completely decoded. STUDY DESIGN: An international collaboration was conducted on the genetic analysis of HNA-2-phenotyped blood samples, including HNA-2-deficient individuals, mothers, and the respective children with neonatal immune neutropenia and regular blood donors. RESULTS: From a total of 54 HNA-2null individuals, 43 were homozygous for the CD177 *787A>T substitution. Six carried the CD177 *c.1291G>A single nucleotide polymorphism. All HNA-2-positive samples with >40% CD177pos. neutrophils carried the *787A wild-type allele, whereas a lower rate of CD177pos. neutrophils was preferentially associated with *c.787AT heterozygosity. Interestingly, only the *c.787A allele sequence was detected in complementary DNA (cDNA) sequence analysis carried out on all *c.787AT heterozygous individuals. However, cDNA analysis after sorting of CD177pos. and CD177neg. neutrophil subsets from HNA-2-positive individuals showed identical sequences, which makes regulatory elements within the promoter unlikely to affect CD177 gene transcription in different CD177 neutrophil subsets. CONCLUSION: This comprehensive study clearly demonstrates the impact of single nucleotide polymorphisms on the expression of HNA-2 on the neutrophil surface but challenges the hypothesis of regulatory epigenetic effects being implicated in the bimodal CD177 expression pattern.

Fast and low-cost direct ELISA for high-throughput serological HPA-1a typing.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal alloantibodies against fetal human platelet antigens (HPAs), mostly caused by anti-HPA-1a. Population-based screening for FNAIT is still a topic of debate. Logistically and financially, the major challenge for implementation is the typing of pregnant women to recognize the 2% HPA-1a-negative women. Therefore, there is need for a high-throughput and low-cost HPA-1a-typing assay. STUDY DESIGN AND METHODS: A sandwich ELISA was developed, using a monoclonal anti-GPIIIa as coating antibody and horseradish-peroxidase-conjugated recombinant anti-HPA-1a, as detecting antibody. The ELISA results were compared to an allelic discrimination PCR-assay. In phase I, samples from unselected consecutive pregnant women were tested with both assays. Phase II was part of a prospective screening study in pregnancy and genotyping was restricted to samples with an arbitrary set, OD < 0.500. RESULTS: The ELISA was optimized to require no additional handling (swirling or spinning) of stored tubes. During phase I, 506 samples were tested. In phase II, another 62,171 consecutive samples were phenotyped, with supportive genotyping in 1,902. In total 1,585 HPA-1a negative and 823 HPA-1a positive women were genotyped. The assay reached 100% sensitivity with a cut-off OD from 0.160, corresponding with a 99.9% specificity and a false-HPA-1a negative rate of 0.03. CONCLUSION: A high-throughput, low-cost, and reliable HPA-1a phenotyping assay was developed which can be used in population-based screening to select samples for testing of presence of anti-HPA-1a. Because plasma from tubes of 3- to 6-days-old samples can be used, this assay is applicable to settings with suboptimal conditions.

Detection of platelet autoantibodies to identify immune thrombocytopenia: state of the art.

Immune Thrombocytopenia (ITP) is diagnosed by exclusion of other causes for thrombocytopenia. Reliable detection of platelet autoantibodies would support the clinical diagnosis of ITP and prevent misdiagnosis. We optimized our diagnostic algorithm for suspected ITP using the direct monoclonal antibody immobilization of platelet antigens assay (MAIPA), which evaluates the presence of platelet autoantibodies on the glycoproteins (GP) IIb/IIIa, Ib/IX and V bound on the patient platelets. The direct MAIPA was shown to be a valuable technique for the detection of platelet autoantibodies and could possibly become a guide for optimizing therapy towards a more personalized treatment of ITP.

A multicenter validation of recombinant ß3 integrin-coupled beads to detect human platelet antigen-1 alloantibodies in 498 cases of fetomaternal alloimmune thrombocytopenia.

BACKGROUND: Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by human platelet (PLT) antigen (HPA) incompatibility. Beads coupled with recombinant ß3 integrins, displaying the biallelic HPA-1 epitopes (rHPA-1), have been shown to detect HPA-1a alloantibodies implicated in FMAIT. This report describes a multicenter validation of the beads using the results of well-characterized samples to define the optimum parameters for analysis of a large cohort of 498 clinical samples. STUDY DESIGN AND METHODS: Fifty-one blinded quality assurance (QA) samples were tested by six laboratories to standardize the rHPA-1 bead assay and to develop an algorithm for sample classification. Five laboratories retrieved samples from 498 independent FMAIT cases, previously tested by the monoclonal antibody-specific immobilization of PLT antigens (MAIPA) assay, from their local archives for testing with the rHPA-1 beads. The results were evaluated using a mathematical algorithm developed to classify the samples. RESULTS: The QA samples gave a mean concordance of 94% between the bead and MAIPA assays, while 97% concordance was observed with the FMAIT samples. Of the 15 discrepant samples, seven were positive by the beads but negative by MAIPA, while the contrary was observed for eight samples. Overall, the bead assay achieved 98% sensitivity for HPA-1a antibody detection in FMAIT and 98.7% specificity compared to the local MAIPA. CONCLUSION: The rHPA-1 bead assay is a rapid 3-hour assay for the sensitive detection of HPA-1 antibodies. Its ease of use would enable prompt detection of maternal HPA-1a antibodies in suspected FMAIT cases, which is important supportive evidence for treatment by transfusion with HPA-1b1b PLTs.

A new bead-based human platelet antigen antibodies detection assay versus the monoclonal antibody immobilization of platelet antigens assay.

BACKGROUND: The performance of a newly developed Luminex bead-based platelet (PLT) antibody detection method (PAKLx) was compared with the monoclonal antibody immobilization of PLT antigens (MAIPA) assay and the LifeScreen Deluxe Luminex bead-based HLA Class I antibody detection method (LMX). STUDY DESIGN AND METHODS: Six sera containing anti-human PLT antigen (HPA)-1a (n=2), HPA-1b, HPA-2b, HPA-3a, or HPA-5b were tested in titration. A total of 194 sera, including HPA-1a, -1b, -2a, -2b, -3a, -5a, and -5b antibodies with or without HLA antibodies (n=63); glycoprotein (GP) IV antibodies (n=1); PLT autoantibodies (n=3); HLA antibodies (n=45); and samples with no PLT-reactive antibodies (n=82), were tested in both assays. RESULTS: Comparable levels of sensitivity were obtained for the MAIPA and PAKLx. The PAKLx showed four (6%) false-negative results in 67 sera with HPA or GP-reactive antibodies: anti-HPA-3a (n=1) or anti-HPA-5b (n=3). The PAKLx showed in 10 of the total 194 samples (5%) the presence of antibodies not detected by the MAIPA. This concerned broadly GP-reactive antibodies (n=7), anti-GPIIb/IIIa combined with anti-HPA-3a (n=1), anti-HPA-1a (borderline, n=1), and anti-GPIV (n=1). Testing 175 sera for anti-HLA Class I antibodies in the PAKLx and LMX showed four discrepant results: PAKLx negative and LMX positive, n=3 and n=1, respectively. CONCLUSION: For the vast majority of the specimens tested (93%) the results of the PAKLx were in concordance with the MAIPA. The PAKLx is a fast, easy to perform, and sensitive PLT antibody screening method.

Management and outcome of 35 cases with foetal/neonatal alloimmune neutropenia.

AIM: The aim of this study was to provide an overview of foetal/neonatal alloimmune neutropenia (FNAIN), together with advice on the clinical management. METHODS: Neutrophil serology in the Netherlands is centralised at Sanquin Diagnostic Services. We examined FNAIN cases between January 1, 1991, and July 1, 2013, to determine the number of cases diagnosed, the relationship with human neutrophil antigen (HNA) antibody, the clinical presentation and therapeutic interventions. RESULTS: We identified 35 FNAIN cases. The detected HNA antibodies were as follows: anti-HNA-1a (n = 7), anti-HNA-1b (n = 12), anti-HNA-1c (n = 2), anti-HNA-2 (n = 8), anti-HNA-3a (n = 1), anti-HNA-5a (n = 1) and anti-FcγRIIIb (n = 4). No infections were diagnosed in 14 neonates, and the other 21 neonates suffered from omphalitis (n = 6), urinary tract infection (n = 1), candida mucositis (n = 1), fever of unknown origin (n = 6) and sepsis (n = 7, 20%). Parity, gestational age, birthweight, neutrophil counts and antibody specificity were not significantly different for cases with, and without, infections. All the infected children were treated with antibiotics. No children died. CONCLUSION: More than half (21) of the 35 cases of FNAIN presented with infections and most implicated were HNA-1a, HNA-1b and HNA-2. Treatment with antibiotics seemed adequate. A neonatal neutropenia workflow model for use in neonatal intensive care units is presented.

Neonatal alloimmune neutropenia due to immunoglobulin G antibodies against human neutrophil antigen-5a.

BACKGROUND: Neonatal alloimmune-mediated neutropenia (NAIN) due to maternal alloantibodies directed against one of the human neutrophil antigens (HNAs) can cause severe infections. NAIN has been described as caused by antibodies against HNA-1a, -b, -c, -2a, -3a, or -4a, but not by antibodies against HNA-5a. RESULTS: Blood from a 3-week-old newborn and from his parents was sent to our laboratory because of suspicion of NAIN. Granulocyte-specific antibodies were present in the maternal antiserum and reactive with the paternal granulocytes. The specificity of the maternal alloantibodies was shown to be anti-HNA-5a by the monoclonal antibody immobilization of granulocyte antigens assay. The mother was genotyped HNA-5a negative, and the father was genotyped homozygous HNA-5a positive. CONCLUSION: We identified a first case of NAIN due to maternal alloantibodies against HNA-5a.

The association and functional relevance of genetic variation in low-to-medium-affinity Fc-gamma receptors with clinical platelet transfusion refractoriness.

BACKGROUND: Inadequate responses to platelet transfusions (i.e., platelet transfusion refractoriness [PLT refractoriness]) are a serious problem. Multiple factors contribute to low yields upon platelet transfusion, among which are platelet-reactive allo-antibodies. Platelet-reactive allo-antibodies occur in up to 30% of patients receiving multiple transfusions, and presumably lead to rapid destruction of the transfused platelets via receptors for IgG, the Fc-gamma receptors (FcγRs). Genetic variation in FcγRs is associated with susceptibility to immune thrombocytopenia, in which autoantibodies against platelets cause thrombocytopenia. OBJECTIVES: We hypothesized that genetic variation in FcγRs may also influence PLT refractoriness in allo-immunized patients and could help in identifying the patients at risk. PATIENTS/METHODS: Patients with severe PLT refractoriness for whom diagnostic testing for allo-immunization was requested in the period of 2005 to 2013 were retrospectively included. A case-control study was performed comparing patients in whom platelet-reactive antibodies were detected (n = 181) with ethnically matched healthy controls (n = 180) to determine differences in all known functional copy number variations and single nucleotide polymorphisms in FcγRs. RESULTS AND CONCLUSIONS: None of the tested FcγR genetic variations seemed associated with the development of severe PLT refractoriness. In contrast to observations in immune thrombocytopenia, genetic variation in FcγRs does not seem to influence the chance to develop PLT refractoriness. Our results do not support determination of FcγR genetic background as a means to identify patients most at risk for PLT refractoriness.

Alloantibodies against low-frequency human platelet antigens do not account for a significant proportion of cases of fetomaternal alloimmune thrombocytopenia: evidence from 1054 cases.

BACKGROUND: Maternal alloantibodies against the five common human platelet antigen (HPA) systems (HPA-1 to -3, -5, and -15) are found in only 20% of cases referred for fetal and neonatal thrombocytopenia (FMAIT) investigations. The question asked was whether mismatches for the remaining 11 low-frequency HPAs (HPA-4 and -6bw to -17bw) might in part explain the remaining 80% of cases. STUDY DESIGN AND METHODS: A total of 1054 paternal DNA samples from referred FMAIT cases (among which 223 cases where antibodies against a common HPA were found) were genotyped for 11 low-frequency HPAs as well as a recently discovered polymorphism (ITGA2B-C2320T). The initial genotyping was carried out by TaqMan and potential heterozygotes were confirmed by DNA sequencing. Clinical and serologic data were collected for each case with a heterozygote father. RESULTS: In total, eight heterozygous fathers were identified: four for HPA-6w, one each for HPA-10w and -11w, and two for HPA-12w. Maternal antibodies against the corresponding antigen were identified in four of the eight cases. In two of these cases, antibodies against HPA-1a and HPA-1b were also found. CONCLUSION: It was concluded that the minor alleles of HPA-4 and -6bw to -17bw are exceptionally rare in the Caucasian population and therefore do not explain the large number of FMAIT referrals which test negative for the common HPA antibodies.

Glycoprotein V: the predominant target antigen in gold-induced autoimmune thrombocytopenia.

Autoimmune thrombocytopenia is generally caused by autoantibodies against glycoprotein (GP) IIb-IIIa or GPIb-IX and occasionally against GPIa-IIa or GPV. By investigating 38 rheumatoid arthritis (RA) patients on gold therapy, 10 with profound thrombocytopenia and 28 nonthrombocytopenic controls, we showed that in all 10 patients with thrombocytopenia, the platelet autoantibodies preferentially targeted GPV but the presence of gold was not required for their reactivity. Elevated levels of platelet-associated IgG (PAIgG) were observed in 8 of the 10 patients in whom the tests were performed. In 5 patients with sufficient autologous platelets, the GPV specificity of PAIgG was confirmed. Tests with GPV transfectants revealed that the antibodies reacted with GPV independent of GPIb alpha, GPIb beta, or GPIX. Autoantibodies recognizing GPV were not seen in the 28 nonthrombocytopenic control RA patients. Thus, GPV seems to be targeted in gold-induced autoimmune thrombocytopenia.