Fetal and Neonatal Alloimmune Thrombocytopenia-New Prospects for Fetal Risk Assessment of HPA-1a-Negative Pregnant Women.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare and potentially serious bleeding condition in the fetus/newborn. FNAIT is usually considered as the platelet counterpart of hemolytic disease of the fetus and newborn. In FNAIT, maternal alloantibodies against paternally inherited platelet antigens traverse the placenta and cause thrombocytopenia in the fetus/newborn. The most common and most serious cases of FNAIT among white people are caused by alloantibodies against the human platelet antigen 1a (HPA-1a), which is absent in 2.3% of women. Today, there is no screening for FNAIT, and for this reason, FNAIT is not suspected until an otherwise healthy child, born at term, presents with thrombocytopenia. Clinical management of subsequent pregnancies at risk of FNAIT is mostly based on the obstetric history. During the last 5 decades, hemolytic disease of the fetus and newborn caused by antibodies against RhD has successfully been prevented by administration of hyperimmune anti-D IgG drug products to RhD-negative women after delivery of an RhD-positive child. Similarly, a hyperimmune anti-HPA-1a IgG (NAITgam) is under development for the prevention of HPA-1a immunization and FNAIT. If NAITgam becomes licensed for FNAIT prophylaxis and national health authorities decide to include FNAIT screening in their antenatal health care programs, it will be necessary to improve today's tools for assessing the risk of FNAIT. Although the primary risk factor for HPA-1a immunization is platelet type HPA-1bb, not all HPA-1a-negative women develop anti-HPA-1a. The women who are HLA-DRB3:01:01 negative (72%) only rarely develop anti-HPA-1a, and for those few who become HPA-1a immunized, it is quite rare to have a child with severe thrombocytopenia. Determination of fetal HPA-1 type is important because 15% of HPA-1a-negative women will carry an HPA-1a-negative fetus and therefore not be at risk of FNAIT. The severity of FNAIT seems to be associated with the level of anti-HPA-1a. Hence, in Norway, for example, an Ab threshold of 3 IU/mL is used to distinguish between low- and high-risk pregnancies. The current review will discuss to what extent these analyses, as well as determination of subtypes of anti-HPA-1a (anti-ß3, anti-αIIbß3, and anti-αvß3) and Fc core fucosylation of anti-HPA-1a IgG, can be used as risk stratification tools.

How do I manage the platelet transfusion-refractory patient?

BACKGROUND: Platelet transfusion-refractoriness is a challenging and expensive clinical scenario seen most often in patients with hematologic malignancies. Although the majority of platelet transfusion-refractory cases are due to nonimmune causes, a significant minority are caused by alloimmunization against Class I human leukocyte antigens (HLAs) or human platelet antigens (HPAs). Such platelet transfusion-refractory patients can be effectively managed with appropriate antigen-negative products. STUDY DESIGN AND METHODS: Our institution has developed a diagnostic and management algorithm for the platelet transfusion-refractory patient with an early focus on identifying those cases caused by immune-mediated factors. Using physical platelet cross-matches to initially classify platelet transfusion-refractory patients as immune-mediated or not, cross-match-compatible inventory is then provided to immune-mediated patients, whereas subsequent HLA (with or without HPA) testing is performed. RESULTS: Our blood donor program performs Class I HLA typing of all repeat platelet donors to facilitate the identification of antigen-negative platelet units (virtual cross-matching) as well as the recruitment of HLA-matched donors. The platelet transfusion-refractoriness algorithm realizes an initial net cost savings once two apheresis platelets are saved from use for each newly identified, immune-mediated platelet transfusion-refractory patient. CONCLUSION: An algorithm utilizing physical platelet cross-matches, Class I HLA and HPA antibody testing, and upfront Class I HLA typing of platelet donors leads to overall resource savings and improved clinical management for platelet transfusion-refractory patients.

Non-invasive Prenatal Diagnosis of Feto-Maternal Platelet Incompatibility by Cold High Resolution Melting Analysis.

Fetal and Neonatal alloimmune thrombocytopenia (FNAIT) is a condition which could occur when pregnant women develop an alloimmunization against paternally inherited antigens of the fetal platelets. Approximately 80 % of FNAIT cases are caused by anti-HPA-1a, about 15 % by anti-HPA-5b and 5 % by other HPA antibodies. Only 2 % of the total population is HPA-1a negative (HPA-1b1b). The HPA-1a allele differs by one single nucleotide from HPA-1b allele, yet it represents around 27 % of total severe thrombocytopenias. HPA-1 was studied in serum cDNA from 12 volunteer pregnant women to determine their HPA-1 genotype by HRM (high resolution melting) PCR. When an homozygous HPA-1 gene was detected in a mother, a COLD HRM was performed to determine whether or not the fetal genotype differs from the mother's.The differences in the melting curve shapes allow us to accurately distinguish the three pregnants genotypes. The fetal heterozygous genotype of homozygous pregnant women was correctly detected by COLD PCR HRM in maternal serum. HPA-1 genotyping by HRM may be a useful aproach for genotyping all pregnant women in inexpensively. Moreover, when HPA-1 homozygosis was detected in a pregnant woman, fetal heterozygosis may be diagnosed by COLD HRM to select pregnancies for preventive monitoring.

[A prospective assessment of the diagnostic value of MAIPA test in idiopathic thrombocytopenic purpura].

OBJECTIVE: To evaluate the clinical significance of MAIPA test in diagnosis of idiopathic thrombocytopenic purpura (ITP) and in the differential diagnosis of antoimmune thrombocytopenias from nonimmune thrombocytopenias. METHODS: A total of 321 thrombocytopenic patients (118 males, 203 females) from 14 centers were studied. A modified monoclonal antibody immobilization of platelet antigen (MAIPA) method was used to detect the platelet glycoprotein-specific autoantibodies (anti-GP IIb/IIIa, anti-GPIb/IX) to double-blindly evaluate its sensitivity and specificity for the diagnosis of ITP and to investigate the impact of the antibodies on platelet count. RESULTS: The results showed that for the diagnosis of ITP, anti-GPIIb/IIIa, anti-GPIb/IX and both of them had the sensitivity of 39.75%, 32.64% and 55.23%; the specificity of 97.56%, 93.94% and 92.68%; the positive predictive value of 97.94%, 93.98% and 95.65%; the negative predictive value of 35.71%, 32.35% and 41.53%; and the total efficiency of 54.51%, 48.29% and 64.80%, respectively. The positivity of the autoantibodies in immune thrombocytopenias was incredibly higher than that in nonimmune thrombocytopenias. The platelet counts in the immune thrombocytopenias with autoantibody positivities were significantly lower than those without the autoantibodies. The platelet counts were negatively correlated with the concentration of the autoantibodies. The levels of anti-GPIIb/IIIa or anti-GPIb/IX or both of them dropped or disappeared in patients being responsive to steroid therapy. CONCLUSION: MAIPA assay is proved to be of great value for the diagnosis of ITP and for differential diagnosis of immune thrombocytopenias from nonimmune thrombocytopenias.

[Treatment of chronic immune thrombocytopenic purpura. Looking for something better. Review]. / Tratamiento de la púrpura trombocitopénica inmune crónica. Buscando algo mejor. Revisión.

Chronic Immune Thrombocytopenic Purpura (cITP) has become a field of multiple therapeutic assays. More than 20 types of treatment have been developed to obtain a favorable and prolonged platelet response. The treatment of cITP is oriented to inhibit the antiplatelet antibodies production by interference with the macrophage of the reticulum endothelial system and a blockade of the antigenic response with a decrease in the amplification of the immunological response. Steroids of the glucocorticoids type and splenectomy constitute the first line of treatment. Failure of these treatments leads to the use of second line drugs such as non steroid immuno-supressors and the immunoglobulins type IgG and anti-D. Therapeutic assays with others immunomodulators have been reported. The introduction of new drugs destined to increase the megakaryocytic bone marrow platelet production, has opened a new way to treat the cITP. However, the splenectomy remains as the simplest, safest and most effective treatment in cITP. The principal criteria does not have to be focused on obtaining a normal platelet count, but to reach safe hemostatic levels in absence of hemorrhage, for a prolonged time. On the other hand, despite the persistence of thrombocytopenia, the hematologist can choose to maintain the patient with no treatment and with only a strict clinical observation. It is obvious that the cost-benefit from the different treatments is inclined towards those of lower cost and minimal secondary effects.

External quality assessment of platelet serology and human platelet antigen genotyping: a 10-year review.

BACKGROUND: In this review, the results of an external quality assessment (EQA) over 10 years of platelet (PLT) serology and of human platelet antigen (HPA) polymorphisms genotyping are shown. The detection and identification of PLT antibodies and the distinction between PLT-specific antibodies and HLA Class I antibodies are evaluated. STUDY DESIGN AND METHODS: Each year, serum samples from five patients and four donor blood samples for DNA typing were distributed. Laboratories could participate as a screening laboratory (SL; n = 7) or as an identification laboratory (IL; n = 8). RESULTS: SLs scored 57 to 100 percent correct positive and 91 to 100 percent correct negative results in the detection of PLT-specific antibodies. SLs only using a PLT immunofluorescence test (PIFT) scored less well than those using a PLT glycoprotein-based antibody detection method. ILs scored 70 to 100 percent correct positive and 87 to 100 percent correct negative results for, respectively, the detection and identification of PLT-specific antibodies. Both the specificity and the sensitivity for the detection and identification of PLT-specific antibodies were not as good in ILs using solid-phase enzyme-linked immunosorbent assay methods as in those using the monoclonal antibody immobilization of PLT antigens (MAIPA) assay. For HPA-1, -2, -3, and -5 genotyping, incorrect results were observed only twice in 280 genotyping assays. CONCLUSION: The data underscore the necessity of using the most accurate methods with a high level of knowledge, experience, and technical training. For screening purposes, it is not sufficient to use only the PIFT, whereas for identification of PLT-specific antibodies, the MAIPA assay is the superior assay.

Prospective epidemiologic study of the outcome and cost-effectiveness of antenatal screening to detect neonatal alloimmune thrombocytopenia due to anti-HPA-1a.

BACKGROUND: To assess the value of antenatal screening to detect neonatal alloimmune thrombocytopenia (NAIT) due to anti-HPA-1a, a prospective study was carried out to quantify the potential clinical benefits and determine whether screening would be cost-effective. STUDY DESIGN AND METHODS: An observational prospective controlled study was carried out on 26,506 pregnant women over 2 years. HPA-1a phenotyping was performed in the first trimester and women confirmed HPA-1a-negative were tested for anti-HPA-1a during pregnancy, at delivery, and 10 to 14 days after birth. Babies of HPA-1a-negative women were tested at delivery for thrombocytopenia and examined for signs of bleeding. Economic evaluation was undertaken on the basis of the data collected during the study. RESULTS: Twenty-five of 318 women (7.9%) had anti-HPA-1a detected for the first time. Eight women (43 per 100,000) gave birth to babies with NAIT, and 5 (27 per 100,000) had severe thrombocytopenia. Three babies had mild signs of bleeding, and no cases of intracranial hemorrhage (ICH) or fetal loss were detected. It is estimated that it would cost 60,596 pounds (98,771 US dollars) to detect a case of severe NAIT, where anti-HPA-1a has been identified for the first time, and 1,151,323 pounds (1,876,656 US dollars) to prevent a case of ICH, assuming that detection allowed successful intervention. CONCLUSIONS: Our data suggest that severe HPA-1a NAIT is underdiagnosed in the absence of routine antenatal screening. Serious bleeding complications and ICH, however, occur less frequently in first cases of NAIT than suspected from the literature, and the costs of screening and possible intervention must be balanced against the procedural risks.

Survey of the use and clinical effectiveness of HPA-1a/5b-negative platelet concentrates in proven or suspected platelet alloimmunization.

The optimal treatment of neonatal alloimmune thrombocytopenia (NAIT) is the transfusion of compatible donor platelets. The National Blood Service in England has established panels of "accredited" donors negative for human platelet antigens HPA-1a and HPA-5b, the most commonly implicated alloantigens. We have retrospectively surveyed the frequency of use and clinical effectiveness of donations collected over a 13-month period from the Oxford accredited panel. Ninety-five per cent of hyperconcentrated platelets (HPCs) collected were issued, all for intrauterine transfusion to fetuses at risk of NAIT due to the presence of maternal platelet alloantibodies and previously affected siblings. Thirty-one per cent of paediatric platelet concentrates (PPCs) collected were issued, of which 57% were used for cases of suspected NAIT. Fifty-four per cent of adult therapeutic doses collected were issued; 5% of these were used in cases of suspected NAIT or proven post-transfusion purpura (PTP). Good increments were seen in most NAIT cases transfused with HPCs or PPCs, and a moderate increment in the one PTP case. We conclude that the establishment of accredited panels is justified and enables delivery of a clinically effective treatment for NAIT. Increased use and cost-effectiveness could be achieved by the delivery of an educational programme to neonatal unit clinical staff to increase the awareness and appropriate treatment of NAIT.

Platelet transfusion trigger in difficult patients.

There is general consensus that a prophylactic pre-transfusion trigger at 10.000 platelets/microL in stable oncohematological patients is as safe as the traditional trigger of 20.000/microL, and that perioperative triggers at 50.000 and 100.000/microL are adequate in most surgical and neurosurgical conditions respectively. Guidelines on the trigger and other issues related to platelet transfusion can be found in nine documents published during 1987-2001 by the National Institutes of Health (NIH), the British Committee on Standardization in Hematology, the Royal College of Physicians of Edinburgh, the College of American Pathologists, the American Society of Anesthesiology and the American Society of Clinical Oncology (ASCO). Although consensus may be less evident on specific triggers for 'difficult' patients, the following triggers, listed by progressively increasing levels, have been proposed in the literature and have found general agreement: a stable oncohematological recipient: 10.000; lumbar puncture in a stable pediatric leukemic patient: 10.000; thrombocytopenia secondary to gpIIb/IIIa receptor inhibitors [corrected]:10.000; bone marrow aspiration and biopsy: 20.000; gastrointestinal endoscopy in cancer: 20.000-40.000; disseminated intravascular coagulation: 20.000-50.000; fiber-optic bronchoscopy in a bone marrow transplant recipient: 20.000-50.000; neonatal alloimmune thrombocytopenia: 30.000; major surgery in leukemia: 50.000; thrombocytopenia secondary to massive transfusion: 50.000; invasive procedures in cirrhosis: 50.000; cardiopulmonary bypass: 50.000-60.000; liver biopsy: 50.000-100.000; a nonbleeding premature infant: 60.000; neurosurgery: 100.000. The proposed values must be considered within the context of careful clinical evaluation of each individual patient, and attention should be given to the power of discrimination of platelet counters at low counts and to the prompt availability of good quality platelet products in the case of emergency.

Screening primiparous women and newborns for fetal/neonatal alloimmune thrombocytopenia: a prospective comparison of effectiveness and costs. Immune Thrombocytopenia Working Group.

A prospective study was conducted in three maternity wards to compare the medical outcomes and the costs of two screening strategies for the detection of fetal/neonatal alloimmune thrombocytopenia (FMAIT). A total of 2066 primiparas and 6081 newborns were included. Fifty-two primiparous women with HPA-1b phenotype were found, and 45 were followed during pregnancy. Four women developed antibodies, and two fetuses exhibited FMAIT; therefore, the prevalence of anti-HPA-1a was 2 per 1000, and the prevalence of FMAIT 1 per 1000. Forty-eight thrombocytopenic newborns were found out of a total of 5632 blood samples. Five were HPA-1a children whose mothers were HPA-1b. The cost-effectiveness of screening all primiparous women was $45,000 and of screening all newborns is $18,000-per anti-HPA-1a alloimmunization diagnosed. Costs per fetal death or disability averted were $500,000 for the primiparous strategy and $225,000 for the newborn strategy. In conclusion, screening newborns for neonatal alloimmune thrombocytopenia is more cost-effective than screening primiparous women.

Platelet phenotyping in carriers for Glanzmann's thrombasthenia: a simple screening test for assessment of the molecular defect.

Glanzmann's thrombasthenia (GT) is a recessive autosomal bleeding disorder characterized by the abnormality of aggregation due to a platelet glycoprotein (GP) IIb-IIIa deficiency or a dysfunctional complex. Molecular abnormalities have been localized on the gene coding for GP IIb or IIIa. The aim of our work was an attempt to obtain indirectly information on the putative localization of the molecular defect in patients with GT type I or II by the determination of the HPA-1 (GP IIIa) and HPA-3 (GP IIb) alloantigenic systems' expression in GT carriers. If GT results from a defective GP IIb gene, a GT carrier would appear homozygous for HPA-3 by serology, because the normal gene product will be expressed while the abnormal GP IIb gene product will not be present. Conversely, if the abnormality is in the GP IIIa gene, such an individual would appear homozygous for HPA-1. Therefore, the heterozygous status for HPA would result from the normal expression of the two genes for the considered alloantigenic system. Among the four families studied with informative members, our presumptions were strengthened by the preliminary genetic results in one family showing a mutation in the GP IIb gene. Thus, serology could be a simple screening test for the possible defective gene responsible for GT allowing molecular investigation focusing only on GP IIb or IIIa gene.

The therapeutic value of HPA-1a-negative platelet transfusion in post-transfusion purpura complicated by life-threatening haemorrhage.

Post-transfusion purpura (PTP) was diagnosed in a female patient 1 week after transfusion for hysterectomy. Despite treatment with oral steroids and intravenous immunoglobulin, platelet counts remained low, and the patient developed profuse rectal bleeding. Random platelet transfusion is reported to be ineffective in PTP. We report a case in which the transfusion of HPA-la negative platelets provided good increment and clinical haemostasis before intravenous immunoglobulin could have had an effect.

Difficulties in the antenatal assessment of neonatal alloimmune thrombocytopenia.

In this paper we present a patient with an initially questionable history of neonatal alloimmune thrombocytopenia (NAT) due to materno-fetal HPA-1a (PLA1) incompatibility. No circulating antibodies were detectable in untreated maternal serum, but an adsorption/elution technique enabled the demonstration of the platelet-specific anti-HPA-1a (anti-PLA1) in maternal serum. Cordocentesis at 35 weeks of gestation revealed a fetal platelet count of 18 x 10(9)/l. Intrauterine platelet transfusion with HPA-1a (PLA1)-negative donor platelets was performed prior to cesarean section.