An optimized procedure for Luminex-based human platelet antigen-specific antibody screening and identification (PakLx assay) with a cost-effective approach and improved sensitivity.

BACKGROUND AND OBJECTIVES: Detection of anti-platelet antibodies is required for the diagnosis of foetal/neonatal alloimmune thrombocytopaenia. The most commonly used methods for anti-platelet antibody detection are the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) and the Luminex bead assay (PakLx). However, for economic reasons, the use of the PakLx assay is limited. MATERIALS AND METHODS: In the present study, we evaluated the performance of an optimized protocol based on a half-volume of PakLx reagents. We compared two alternative procedures: one with a half-volume of all components including patient samples, and another based on a half-volume of reagents but a standard volume of patient sample. RESULTS: Our results obtained with a panel of 67 samples demonstrate improved sensitivity when using a standard sample volume. CONCLUSION: In the event of an inconclusive result with this optimized protocol (e.g., incomplete panel of positive Luminex beads), we recommend testing the sample with an alternative protocol (e.g., MAIPA or the original PakLx protocol).

Fetal and neonatal alloimmune thrombocytopenia: recommendations for evidence-based practice, an international approach.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) may result in severe bleeding, particularly fetal and neonatal intracranial haemorrhage (ICH). As a result, FNAIT requires prompt identification and treatment; subsequent pregnancies need close surveillance and management. An international panel convened to develop evidence-based recommendations for diagnosis and management of FNAIT. A rigorous approach was used to search, review and develop recommendations from published data for: antenatal management, postnatal management, diagnostic testing and universal screening. To confirm FNAIT, fetal human platelet antigen (HPA) typing, using non-invasive methods if quality-assured, should be performed during pregnancy when the father is unknown, unavailable for testing or heterozygous for the implicated antigen. Women with a previous child with an ICH related to FNAIT should be offered intravenous immunoglobulin (IVIG) infusions during subsequent affected pregnancies as early as 12 weeks gestation. Ideally, HPA-selected platelets should be available at delivery for potentially affected infants and used to increase the neonatal platelet count as needed. If HPA-selected platelets are not immediately available, unselected platelets should be transfused. FNAIT studies that optimize antenatal and postnatal management, develop risk stratification algorithms to guide management and standardize laboratory testing to identify high risk pregnancies are needed.

Antenatal management in fetal and neonatal alloimmune thrombocytopenia: a systematic review.

Several strategies can be used to manage fetal or neonatal alloimmune thrombocytopenia (FNAIT) in subsequent pregnancies. Serial fetal blood sampling (FBS) and intrauterine platelet transfusions (IUPT), as well as weekly maternal IV immunoglobulin infusion (IVIG), with or without additional corticosteroid therapy, are common options, but optimal management has not been determined. The aim of this systematic review was to assess antenatal treatment strategies for FNAIT. Four randomized controlled trials and 22 nonrandomized studies were included. Pooling of results was not possible due to considerable heterogeneity. Most studies found comparable outcomes regarding the occurrence of intracranial hemorrhage, regardless of the antenatal management strategy applied; FBS, IUPT, or IVIG with or without corticosteroids. There is no consistent evidence for the value of adding steroids to IVIG. FBS or IUPT resulted in a relatively high complication rate (consisting mainly of preterm emergency cesarean section) of 11% per treated pregnancy in all studies combined. Overall, noninvasive management in pregnant mothers who have had a previous neonate with FNAIT is effective without the relatively high rate of adverse outcomes seen with invasive strategies. This systematic review suggests that first-line antenatal management in FNAIT is weekly IVIG administration, with or without the addition of corticosteroids.

Maternal HPA-1a antibody level and its role in predicting the severity of Fetal/Neonatal Alloimmune Thrombocytopenia: a systematic review.

BACKGROUND AND OBJECTIVES: In Caucasians, fetal/neonatal alloimmune thrombocytopenia (FNAIT) is most commonly due to maternal HPA-1a antibodies. HPA-1a typing followed by screening for anti-HPA-1a antibodies in HPA-1bb women may identify first pregnancies at risk. Our goal was to review results from previous published studies to examine whether the maternal antibody level to HPA-1a could be used to identify high-risk pregnancies. MATERIALS AND METHODS: The studies included were categorized by recruitment strategies: screening of unselected pregnancies or samples analyzed from known or suspected FNAIT patients. RESULTS: Three prospective studies reported results from screening programmes, and 10 retrospective studies focused on suspected cases of FNAIT. In 8 studies samples for antibody measurement, performed by the monoclonal antibody immobilization of platelet antigen (MAIPA) assay, and samples for determining fetal/neonatal platelet count were collected simultaneously. In these 8 studies, the maternal antibody level correlated with the risk of severe thrombocytopenia. The prospective studies reported high negative predictive values (88-95%), which would allow for the use of maternal anti-HPA-1a antibody level as a predictive tool in a screening setting, in order to identify cases at low risk for FNAIT. However, due to low positive predictive values reported in prospective as well as retrospective studies (54-97%), the maternal antibody level is less suited for the final diagnosis and for guiding antenatal treatment. CONCLUSION: HPA-1a antibody level has the potential to predict the severity of FNAIT.

Fetal and neonatal alloimmune thrombocytopenia: predictive factors of intracranial hemorrhage.

BACKGROUND: In Caucasians, fetal/neonatal alloimmune thrombocytopenia (FNAIT) is most frequently caused by maternal alloimmunization against the human platelet antigen HPA-1a. The most serious complication of severe FNAIT is intracranial hemorrhage (ICH). ICH mainly occurs in utero; therefore, there is a need to identify noninvasive predictive factors of ICH to facilitate early identification of this condition and to determine response to maternal therapy. STUDY DESIGN AND METHODS: We studied gynecologic and immunogenetic variables of severe cases of anti-HPA-1a FNAIT within three groups: Group I, FNAIT without ICH; Group II, FNAIT with ICH; and Group III, suspected FNAIT cases without detectable maternal anti-HPA-1a alloantibodies. RESULTS: ICH was associated with a poor outcome because it led to death in 59% of cases. Multigravida (two or more pregnancies) was overrepresented in Group II, consistent with the high concentrations of maternal HPA-1a alloantibody and the frequent detection of a strong newborn-specific HLA class I antibody response at delivery. The proportion of HLA-DRB4*01:01P (*01:01 or *01:03) women was similar in Groups I and II, but this allele was overrepresented in Group III, in which FNAIT was less severe than in the other two groups. Finally, antenatal intravenous immunoglobulin therapy tended to be more effective in HLA-DRB3*01:01(+)/HLA-DRB4*01:01P(+) women than for HLA-DRB3*01:01(+)/HLA-DRB4*01:01P(-) women. CONCLUSION: The number of gestations is a predictive factor of ICH in anti-HPA-1a-alloimmunized women. Maternal immunogenetic variables should be investigated in the context of maternal immunization and may predict response to maternal therapy in subsequent pregnancies.

HPA antibodies in Algerian multitransfused patients: Prevalence and involvement in platelet refractoriness.

BACKGROUND: Patients receiving cellular blood components may form HLA or HPA antibodies. The frequency and the specificity of HPA antibodies after a series of blood transfusions have never been reported in the Algerian population which is ethnically diverse and runs a higher risk of platelet alloimmunization due to high b allelic frequencies observed for the HPA systems. METHODS: 117 polytransfused patients were included in this study; the detection of HPA antibodies was performed by the Monoclonal Antibody-specific Immobilization of Platelet Antigens method (MAIPA). Post-transfusion platelet effectiveness was evaluated by the calculation of corrected count increment (CCI). RESULTS: The antibodies against platelets were detected in 10.26% of the patients. In this study, the platelet systems concerned by the alloimmunizations were specifically HPA-1, -3 and -5 with particular predominance of HPA-1. Twenty two patients were refractory to platelet transfusion, as assessed by a CCI; in which 64% have factors associated with increased platelet consumption. Platelet Immunization was found in 14% of platelet refractoriness (PTR) cases. 03 Anti-platelet antibodies were directed against GPIb-IX (n = 1), anti-HPA-1b (n = 1) and anti HPA-5b (n = 1) associated with anti-HLA antibodies in two cases. CONCLUSION: HLA and HPA alloimmunization is common among chronically transfused patients. PTR detection, identification of the underlying causes, and selection of the appropriate product for transfusion are fundamental to reduce the risk of major bleedings.

How do we treat fetal and neonatal alloimmune thrombocytopenia?

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a potentially devastating disease, seen in one in 800 to 1000 neonates. FNAIT is the most common cause of early-onset isolated severe neonatal thrombocytopenia in maternity wards. The complication of this disorder most to be feared is intracranial hemorrhage, leading to death or to neurologic sequels. As there is no systematic screening of at-risk pregnancies, FNAIT is often discovered when signs of bleeding are observed during pregnancy or at delivery. Platelet transfusion is required in case of bleeding or severe thrombocytopenia (<30 × 10(9) /L) during the 48-hour-postdelivery period. Diagnosis of alloimmunization is important for management of the index case and for subsequent pregnancies, due to the increasing severity of this syndrome as it recurs. Noninvasive antenatal therapy is based on maternal perfusion of intravenous immunoglobulins and risk stratification. In our experience, the addition of corticoids during the last trimester significantly improves the efficiency of treatment. Follow-up of antibody concentration during pregnancy may constitute a useful variable for therapy effectiveness.

Molecular HPA genotyping by microarray in Brazilian blood donors.

BACKGROUND: Human platelet antigens (HPA) polymorphisms may cause HPA alloimmunization, platelet (PLT) refractoriness, fetomaternal alloimmune thrombocytopenia, and posttransfusion purpura. Characterized by significant racial admixture, the Brazilian population might benefit from the knowledge about HPA frequency to guide decision-making concerning PLT transfusion. STUDY DESIGN AND METHODS: HPA frequencies were determined in 158 DNA samples from Brazilian blood donors by microarray for HPA-1 to -9, -11, and -15. A HPA-2 discrepancy was solved by polymerase chain reaction with sequence-specific primers (PCR-SSP) and sequencing. RESULTS: While a alleles were predominant for HPA-1 to -9 and -11, b alleles were absent for HPA-6, -7, -8, and -11. HPA-3 and HPA-15 had a higher prevalence of ab genotypes. One case of HPA-4ab and two cases of HPA-9abw were detected, the latter not previously described in Brazilian blood donors. One sample was not interpretable for HPA-2 due to a GPIb 468 C>G mutation; this donor was characterized as HPA-2ab by PCR-SSP and sequencing. CONCLUSION: Allele frequencies were comparable to those described in other Brazilian studies. Rare HPA-9 alleles were described in Brazilians for the first time. A mutation near the HPA-2 polymorphism suggests that complementary methods might be necessary in specific cases. PLT genotyping by microarray proved to be fast, accurate, and reliable.

Prediction of the fetal status in noninvasive management of alloimmune thrombocytopenia.

Fetal/neonatal alloimmune thrombocytopenia is the most common cause of severe thrombocytopenia in the fetus and in an otherwise healthy newborn. To counter the consequences of severe fetal thrombocytopenia, antenatal therapies have been implemented. Predictive parameters for fetal severe thrombocytopenia are important for the development of noninvasive strategy and tailored intervention. We report here data concerning 239 pregnancies in 75 HPA-1bb women. Analysis of the index cases (diagnosis of fetal/neonatal alloimmune thrombocytopenia) did not show any significant correlation between the severity of the disease and the maternal genetic background (ABO blood group and HLA-DRB3 allele). Subsequent pregnancies were managed, and therapy effectiveness was evaluated. The highest mean newborn platelet count was observed for a combination of intravenous immunoglobulin and steroids (135 × 109/L; 54 newborns) compared with intravenous immunoglobulin alone (89 × 109/L; 27 newborns). The maternal anti-HPA-1a antibody concentration measured before any treatment and before 28 weeks of gestation was predictive of the fetal status. The weighted areas under curves of the maternal alloantibody concentrations were predictive of therapy response. To conclude, this large retrospective survey gives new insights on maternal predictive parameters for fetal status and therapy effectiveness allowing noninvasive strategies.

The αIIb p.Leu841Met (Cab3(a+) ) polymorphism results in a new human platelet alloantigen involved in neonatal alloimmune thrombocytopenia.

BACKGROUND: Fetal-neonatal alloimmune thrombocytopenia (FNAIT) diagnosis relies on maternofetal incompatibility and alloantibody identification. Genotyping for rare platelet (PLT) polymorphisms allowed the identification of three families with suspected or confirmed maternofetal incompatibility for the αIIb-c.2614C>A mutation (Halle et al., Transfusion 2008;48:14-15). STUDY DESIGN AND METHODS: A polymerase chain reaction-sequence-specific primers amplification assay was designed to genotype the αIIb-c.2614C>A mutation. HEK293 cells expressing αIIb-Leu841 or αIIb-Met841 αIIbß3 forms were used to probe the reactivity of maternal sera from these families and to study the effects of the substitution on αIIbß3 expression and functions. RESULTS: Tested by flow cytometry (FCM), one serum sample specifically reacted with αIIb-Met841 but not with αIIb-Leu841 αIIbß3. This specificity revealed the αIIb-Leu841 polymorphism as a new alloantigen named Cab3(a+) . Cross-match testing using FCM also showed the Cab3(a+) antigen to be expressed at the PLT surface. As for anti-human PLT alloantigen (HPA)-3a (or -3b) and anti-HPA-9bw, detection of anti-Cab3(a+) alloantibodies appeared difficult and required whole PLT assays when classical monoclonal antibody-specific immobilization of PLT antigen test failed. In our FNAIT set, the immune response to Cab3(a+) maternofetal incompatibility could induce severe thrombocytopenias and life-threatening hemorrhages. The p.Leu841Met substitution has limited effects, if any, on local αIIb structure, preserving both αIIbß3 expression and functions. CONCLUSION: The Cab3(a+) polymorphism is a new rare alloantigen (allelic frequency <1%) carried by αIIb that might result in severe life-threatening thrombocytopenias. In Sub-Saharan African populations, higher Cab3(a+) gene frequencies (up to 8.2%; Halle et al., Transfusion 2008;48:14-15) and homozygous people are observed.

The implementation of surface plasmon resonance technique in monitoring pregnancies with expected fetal and neonatal alloimmune thrombocytopenia.

BACKGROUND: Maternal anti-HPA-1a alloantibodies are responsible for most cases of severe fetal and neonatal alloimmune thrombocytopenia (FNAIT). The presence of HPA-1a alloantibodies in maternal blood alone does not predict the fetal platelet (PLT) count, and the predictivity of antibody titers determined by enzyme immunoassays (EIAs) is debated. In contrast to EIA, surface plasmon resonance (SPR) provides information on antibody-binding properties. STUDY DESIGN AND METHODS: Sequential sera from pregnant women with expected FNAIT were assessed for HPA-1a alloantibodies using SPR. Group I (n = 6) was treated with intravenous immunoglobulin (IVIG) and steroids beginning at 19 weeks of gestation (w.g.), and Group II (n = 4) received intrauterine PLT transfusions (IUT) beginning at 22 w.g. Maternal alloantibodies were quantified using an HPA-1a monoclonal antibody (MoAb) as a standard. Antibody avidity was determined as the ratio of B700 (end of the dissociation phase) to B350 (end of the association phase); the area under the curve (AUC) was calculated to determine overall antibody binding. RESULTS: After 22 w.g., alloantibody characteristics remained stable in both groups, while there was a steep decrease in B700 and B350 values between 16 and 22 w.g. (assessed only in Group I), indicating a decrease in anti-HPA-1a alloantibody concentrations. Interestingly, the AUCs of the last maternal sample before elective delivery appeared to be correlated with fetal and neonatal PLT counts (p = 0.014 and 0.017, respectively). CONCLUSION: SPR provides quantitative information on HPA-1a alloantibody characteristics in addition to monoclonal antibody-specific immobilization of platelet antigens. SPR results can be calibrated using a MoAb standard and should be further assessed for a potential correlation with fetal PLT count.

New K103 ß3 allele identified in a context of severe neonatal thrombocytopenia.

BACKGROUND: A new ß3 allele was identified in a severe case of neonatal alloimmune thrombocytopenia (<7 × 10(9) /L). STUDY DESIGN AND METHODS: Diagnosis was done by use of monoclonal antibody-specific immobilization of platelet (PLT) antigen for serologic analyses and polymerase chain reaction (PCR)-sequence-specific primers (SSP) and PCR-restriction fragment length polymorphism (RFLP) for genotyping. Direct sequencing of PCR product was done and mutant αIIbß3 expressed in HEK-293 cells. RESULTS: Serologic analysis revealed in the maternal serum an anti-human PLT alloantigen (HPA)-1a alloantibody associated to an anti-α2ß1. Anti-HPA-1a alloimmunization diagnosis was confirmed by genotyping showing maternofetal incompatibility. However, investigation of rare HPA polymorphisms revealed discrepant HPA-16b assignation between PCR-RFLP and PCR-SSP. Sequencing revealed a new c.385C>A mutation in the ß3 coding sequence resulting in a false assignation of the HPA-16b allele by PCR-RFLP. This mutation leads to a Q103K substitution in mature ß3. The K103-ß3 form of the complex was expressed in HEK-293 cells but did not react with the maternal serum. CONCLUSION: We have characterized a new rare allele (frequency < 1%) of ß3 that yields false HPA-16b genotyping in PCR-RFLP. This new case of false typing assignation emphasizes the necessity to use two genotyping techniques in diagnosis. This particularly applies for rare HPA polymorphisms when PLT phenotyping cannot be used.

Characterization of the novel HLA-B*51:296 allele by next-generation sequencing.

B*51:296 differs from B*51:01:01:01 by one nucleotide substitution at position 1030 in exon 6.

Characterization of the novel HLA-B*40:450 allele by next-generation sequencing.

B*40:450 differs from B*40:01:02:01 by one nucleotide substitution at position 5 in exon 1.

Characterization of the novel HLA-DRB1*11:260 allele by next-generation sequencing.

DRB1*11:260 differs from DRB1*11:03:01 by one nucleotide substitution at position 484 in exon 3.