True risk of fetal/neonatal alloimmune thrombocytopenia in subsequent pregnancies: a prospective observational follow-up study.

OBJECTIVES: To assess neonatal platelet counts by comparing alloimmunised pregnancies from a Norwegian screening and intervention study with subsequent pregnancies from the same women. DESIGN: Prospective observational follow-up study. SETTING: A university hospital. POPULATION: HPA-1a immunised women from a large Norwegian screening study that gave birth to one or more children after the screening study ended (2004-2012). METHODS: Follow-up of maternal anti-HPA-1a antibody levels and neonatal platelet counts from the screening pregnancies were compared with subsequent pregnancies. None of the women received antenatal intravenous immunoglobulin (IVIG) treatment and neonatal platelet counts were therefore comparable. MAIN OUTCOME MEASURES: Change in neonatal platelet counts from one HPA-1a incompatible pregnancy to the next. Maternal anti-HPA-a1 antibody levels from one HPA-1a incompatible pregnancy to the next. RESULTS: Forty-five incompatible subsequent pregnancies were identified. Overall, the neonatal platelet count in the subsequent pregnancy was improved (18%), unchanged (52%), or worse (30%), compared with the corresponding screening pregnancy. There was one case of fetal intracranial haemorrhage (ICH) identified in the screening (intrauterine fetal death detected at 30 weeks of gestation) and no ICH cases recorded for the subsequent pregnancies. In cases where the platelet count was lower in the subsequent pregnancy, the maternal anti-HPA-1a antibody level was higher compared with the screening pregnancy. In comparison, the maternal antibody level was lower in subsequent pregnancies where the platelet count improved. CONCLUSIONS: In contrast to what is often stated, we found that the neonatal platelet count was increased or unchanged in the majority of subsequent pregnancies of HPA-1a-immunised women.

Foetal/neonatal alloimmune thrombocytopenia in Egypt; human platelet antigen genotype frequencies and antibody detection and follow-up in pregnancies.

BACKGROUND AND OBJECTIVES: Foetal and neonatal alloimmune thrombocytopenia (FNAIT) is studied mainly in Caucasian populations. Severe thrombocytopenia (<50×10(9)/L) gives risk of haemorrhage and the most feared complication is intracranial haemorrhage (ICH). In Caucasian populations anti-human platelet antigen (HPA)-1a antibodies are the cause of FNAIT in >80% of the cases. The aims of this project were to study the gene frequencies of HPA-1-5 and 15 alleles in an Egyptian population (Arabic), and to determine the frequency of HPA-1a and -5b immunisations in a cohort of Egyptian pregnant women. MATERIALS AND METHODS: Altogether 6974 pregnant women were included in the study. Genotyping was performed by polymerase chain reaction and antibodies were detected by flow cytometry and enzyme-linked immunosorbent assay. HPA-1-5 and 15 alleles were studied in 367 individuals. RESULTS: The HPA genotypes differed from genotypes published from different Caucasian and Chinese (Han) populations in HPA-1, -2, -3, and -5 systems with significant higher frequency of HPA-1b, -2b and -5b. The rate of HPA-1a alloimmunisation was found comparable to Caucasian populations. Severe thrombocytopenia was found in two newborns. No bleeding complication was reported. Anti-HPA-5b antibodies were detected in 4.4% of the pregnant women. Clinical consequences of these antibodies were not studied. CONCLUSION: The HPA-1bb and -5bb genotypes are more frequent in the Egyptian Arabic population studied compared to Caucasian populations. FNAIT due to anti-HPA-1a and -5b antibodies must be suspected in cases of neonatal thrombocytopenia. Further large prospective studies are needed to increase the knowledge of clinical complications related to HPA alloantibodies in populations with different genetic backgrounds.

Neonatal alloimmune thrombocytopenia is not what it was: a lesson learned from a large prospective screening and intervention program.

Controversies regarding the pathophysiology of neonatal alloimmune thrombocytopenia (NAIT) has hampered the development of consensus about how to identify, follow up and treat the women and children with this serious complication. One reason for this is that knowledge about the condition derived from previous retrospective studies do not necessarily conform with data derived from prospective investigations. The main obstacle to introduction of general screening programs to identify the pregnancies to treat, have been lack of reliable risk factors, and an effective treatment. Now, several recent prospective screening programs including up to 100,000 pregnant women has changed the understanding of the NAIT-pathology, and has shown that we are close to answering these critical questions.

Neonatal alloimmune thrombocytopenia in Norway: poor detection rate with nonscreening versus a general screening programme.

The implementation of an antenatal screening programme for neonatal alloimmune thrombocytopenia (NAIT) is currently under debate. We evaluated the detection rate for NAIT in a nonscreened population of 661,200 births where NAIT was diagnosed on clinical indication. We did a cross-sectional comparison with a population of 100,448 human platelet antigen 1a (HPA1a)-screened pregnancies from three of the five health regions in Norway. In a nonscreening situation, 7.5 cases of NAIT were detected per year compared with 53 cases when screening was applied. The detection rate of NAIT in Norway was therefore 14% of the expected rate.

Cost-effectiveness of antenatal screening for neonatal alloimmune thrombocytopenia.

OBJECTIVES: To estimate the costs and health consequences of three different screening strategies for neonatal alloimmune thrombocytopenia (NAIT). DESIGN: Cost-utility analysis on the basis of a decision tree that incorporates the relevant strategies and outcomes. SETTING: Three health regions in Norway encompassing a 2.78 million population. POPULATION: Pregnant women (n = 100,448) screened for human platelet antigen (HPA) 1a and anti-HPA 1a antibodies, and their babies. METHOD: Decision tree analysis. In three branches of the decision tree, pregnant women entered a programme while in one no screening was performed. The three different screening strategies included all HPA 1a negative women, only HPA 1a negative, HLA DRB3*0101 positive women or only HPA 1a negative women with high level of anti-HPA 1a antibodies. Included women underwent ultrasound examination and elective caesarean section 2-4 weeks before term. Severely thrombocytopenic newborn were transfused immediately with compatible platelets. MAIN OUTCOME MEASUREMENTS: Quality-adjusted life years (QALYs) and costs. RESULTS: Compared with no screening, a programme of screening and subsequent treatment would generate between 210 and 230 additional QALYs among 100,000 pregnant women, and at the same time, reduce health care costs by approximately 1.7 million euros. The sensitivity analyses indicate that screening is cost effective or even cost saving within a wide range of probabilities and costs. CONCLUSION: Our calculations indicate that it is possible to establish an antenatal screening programme for NAIT that is cost effective.

Selecting and deselecting imatinib-resistant clones: observations made by longitudinal, quantitative monitoring of mutated BCR-ABL.

Resistance to imatinib during the treatment of chronic myeloid leukaemia (CML) is frequently associated with point mutations in the ABL gene encoding the ATP binding region likely to cause disease relapse. Early diagnosis and monitoring of these mutations may be important in order to prevent rapid expansion of resistant clones. We describe a quantitative mutation-specific PCR assay based on the readily available Taqman platform. Selectivity for the mutated target is conferred by mutation-specific primers destabilised by additional mismatches. The assay can be carried out in parallel to standard BCR-ABL quantification and is therefore more quickly compared to standard sequencing procedures. The sensitivity of the assay reaches 0.1%. It also allows for quantitative assessment of mutated clones. By analysing sequential samples of resistant subjects, we show how mutated clones were selected, maintained or deselected depending on the individual treatment setting. The high sensitivity and practical merits of this method makes it a good candidate for prospective molecular surveillance of patients at high risk for imatinib resistance.

Maternal anti-HLA class I antibodies are associated with reduced birth weight in thrombocytopenic neonates.

In this comparative cross-sectional study, possible associations between maternal anti-HLA class I antibodies and birth weight in neonatal thrombocytopenia are explored. Although commonly detected in pregnancies and generally regarded as harmless, it has been suggested that such antibodies might be associated with fetal and neonatal alloimmune thrombocytopenia (FNAIT). As a link between FNAIT due to human platelet antigen 1a-specific antibodies and reduced birth weight in boys has previously been demonstrated, we wanted to explore whether maternal anti-HLA class I antibodies might also affect birth weight. To examine this, suspected cases of FNAIT referred to the Norwegian National Unit for Platelet Immunology during the period 1998-2009 were identified. Pregnancies where the only finding was maternal anti-HLA class I antibodies were included. An unselected group of pregnant women participating in a prospective study investigating maternal-fetal hemodynamics at the University Hospital North Norway during the years 2006-2010 served as controls. Twenty-nine percent of controls had anti-HLA class I antibodies. The thrombocytopenic neonates had a significantly lower adjusted birth weight (linear regression, P=0.036) and significantly higher odds of being small for gestational age (OR=6.72, P<0.001) compared with controls. Increasing anti-HLA class I antibody levels in the mother were significantly associated with lower birth weight and placental weight among thrombocytopenic neonates, but not among controls. These results indicate that maternal anti-HLA class I antibodies in thrombocytopenic neonates are associated with reduced fetal growth. Further studies are needed to test if placental function is affected.

Monoclonal platelet antigen capture assays (MAIPA) and reagents: a statement.

This statement concerning the monoclonal-specific immobilization of platelet antigens (MAIPA) has been written on behalf of the International Society of Blood Transfusion--Working Party on Platelet Immunology. The MAIPA technique is considered as the gold standard reference technique in platelet immunology. The assay performed with reagents labelled for 'research only' is acceptable as long as it is regularly evaluated by participation of laboratories in national or international workshops held with reference laboratories.

Coeliac disease in subjects with secondary hyperparathyroidism.

OBJECTIVE: Coeliac disease (CD) may present in its classical form with diarrhoea and weight loss, but also with atypical symptoms that are both related and unrelated to malabsorption. Osteomalacia or osteopenia following malabsorption of calcium and vitamin D is known to occur in patients with CD, and in such cases secondary hyperparathyroidism (SHP) caused by low serum calcium levels is frequently found. However, the prevalence of CD in subjects with SHP has not been reported. MATERIAL AND METHODS: In the Tromsø study 2001, serum parathyroid hormone (PTH) and calcium were measured in 7954 subjects of whom 6061 were eligible for follow-up. From this group, 97 subjects with SHP (serum PTH> or =6.5 pmol/l and serum calcium <2.40 mmol/l) and 104 matched control subjects were re-examined with serological tests for CD (anti-tissue transglutaminase, anti-gluten IgA and IgG). RESULTS: CD was diagnosed in 4 subjects, all from the original SHP group. At the re-examination, only 29 of the 97 subjects with SHP still had elevated serum PTH levels. Among these were 3 of the subjects with CD. When grouping the serological test results as negative, borderline or positive, there was a significant difference between the SHP group and the controls for anti-tissue transglutaminase and anti-gluten IgA (p<0.05). CONCLUSIONS: Subjects with SHP, at least when SHP is persistent, should be tested for CD.

Degradation of amyloid proteins with protease I from Aspergillus oryzae. In vivo increase in SAA clearance rate after enzyme infusion.

The effect of the thrombolytic enzyme protease I from Aspergillus oryzae (brinase) on the amyloid protein AA was investigated. The effect of the enzyme on purified, low molecular weight protein AA was very high. Protein AA in intact amyloid fibrils suspended in neutral buffer was also degraded by the enzyme, although at a much lower rate. Amyloid fibrils in tissue sections could also be attacked and removed, leaving the tissue structure fairly intact. The in vivo effect of brinase on protein SAA was demonstrated in rabbits by an increase in the clearance rate of SAA after enzyme infusion.

[Significance of maternal alloantibodies for neonatal thrombocytopenia]. / Maternelle alloantistoffers betydning ved trombocytopeni hos nyfødte.

BACKGROUND: Thrombocytopenia has been shown to be present in about 1% of unselected newborns. Alloantibodies to platelets induce the most severe thrombocytopenia. MATERIAL AND METHODS: Samples from 195 mothers who had just given birth to thrombocytopenic children, were analysed by platelet antigen genotyping and detection of platelet specific antibodies. RESULTS: 75 mothers were typed human platelet antigen (HPA) 1bb, and in 65 mother, anti-HPA 1a antibodies could be detected. Anti-HPA 5b antibodies were detected in three samples and anti-HLA antibodies in 73 samples. INTERPRETATION: Alloantibodies were shown to be an important cause of thrombocytopenia in the new-born children and anti-HPA 1a antibodies were, as expected, the most common platelet-specific antibody involved. Anti-HLA class 1 antibodies were detected as the only antibody in 51 cases of thrombocytopenia. Though it is not yet formally shown, this may indicate that anti-HLA class 1 antibodies may cause thrombocytopenia in the fetus and new-born. Based on the assumption that neonatal alloimmune thrombocytopenia is present in 1:1,000 new-born, 25% of the neonatal alloimmune thrombocytopenia cases in Norway are verified by laboratory analysis. Alloantibodies to thrombocytes are of clinical importance in future pregnancies and transfusions. The cost and benefit of a national screening program for anti-HPA 1a antibodies in all pregnant women should be carefully considered.

High-density lipoprotein has different binding capacity for different apoproteins. The amyloidogenic apoproteins are easier to displace from high-density lipoprotein.

Purified human amyloid protein A (AA) or serum amyloid protein A (SAA) was incubated with normal human high-density lipoprotein (HDL). After ultracentrifugation the amount of AA or SAA associated with HDL was measured. It was found that the binding capacity of HDL for SAA was higher than that for AA. Incubation of these in vitro associated HDL-AA and HDL-SAA complexes with purified apo AI or apo AII resulted in varying degrees of displacement of the associated AA or SAA from HDL. Under the experimental conditions used, apo AI was able to displace AA from HDL, while apo AII was able to displace both SAA and AA. This indicates that the binding capacity of HDL is different for SAA and AA. Mouse acute-phase HDL was isolated and the native complexes were incubated with human apo AII. SAA2, the amyloidogenic SAA variant in mice, was displaced from HDL to a greater extent than SAA1, indicating a lower binding capacity for the amyloidogenic SAA variant for the HDL complexes.

Flow cytometric analysis in platelet crossmatching using a platelet suspension immunofluorescence test.

BACKGROUND: The sensitivity of flow cytometric measurement of platelet antibodies in a crossmatch technique was investigated. STUDY DESIGN AND METHODS: The corrected count increment after platelet transfusion was compared with the fluorescence ratio determined by flow cytometric measurement. RESULTS: When crossmatching was performed before transfusion(s) in alloimmunized patients, a fluorescence ratio < or = 1.7 was associated with satisfactory responses (corrected count increment > or = 7.5), and the predictive values for negative and positive crossmatch results were 94 and 87 percent, respectively. Analysis of antigen preservation during platelet storage with antibodies to HLA alpha-chain, HLA-B27, HPA-1a, and HPA-3a showed that platelets can be stored, refrigerated, for up to 4 weeks without significant loss of HLA class I and HPA-1a. There was a slight but continuous loss of HPA-3a upon storage. CONCLUSION: Flow cytometric measurement of fluorescence in the platelet suspension immunofluorescence test can be used for platelet crossmatching, with sensitivity and predictive values comparable to those of previously described techniques and with the advantage of automation.

Characterization of amyloid proteins AA and SAA as apolipoproteins of high density lipoprotein (HDL). Displacement of SAA from the HDL-SAA complex by apo AI and apo AII.

An AA-like protein with a molecular weight of 8600 complexed to high-density lipoprotein (HDL) was demonstrated in several acute-phase sera with high levels of SAA. The protein 'apo AA' (to distinguish it from tissue AA) was isolated by elution from sodium dodecyl sulphate (SDS)-polyacrylamide gel, and showed antigenic identity with purified tissue protein AA in double immunodiffusion. Normal HDL was shown to bind purified tissue AA in vitro. When the in vitro-associated HDL-AA complexes were given intravenously to mice during induction of amyloidosis, human AA was incorporated in the amyloid fibrils. Both apo AI and apo AII were shown to displace SAA from acute phase HDL when added to HDL-SAA complexes in vitro. This might be of importance in amyloidogenesis, as the liver and the small intestine, which are the main sites for AI and AII synthesis, are also sites of early amyloid deposition.

Degradation of amyloid proteins by different serine proteases.

A protein extract was obtained from normal human serum by adsorption to unsubstituted Sepharose 4B. This extract contained one or several enzymes with SAA and AA degrading capacity. The optimal pH for degradation of SAA was about 7.3. On fractionation of the enzyme extract on Sephadex G-160, the active component was eluted in the V0 peak. The V0 fraction, which on double immunodiffusion analysis was found to contain alpha 2-macroglobulin, was also active against synthetic substrates used to determine the activity of thrombin and plasma kallikrein. Gel filtration under dissociating conditions and molecular weight estimation further indicated the presence of those enzymes in the preparation. Several serine proteases which are known to be inhibited by alpha 2-macroglobulin possessed AA and SAA degrading activity. On degradation of SAA, an intermediate split product with molecular weight similar to AA was formed. Kallikrein, plasmin and elastase were also able to degrade intact amyloid fibrils suspended in phosphate-buffered saline.

In vitro enhancement of AA-degrading activity in human plasma with the plasminogen activator streptokinase.

Radiolabelled protein AA was coupled to cyanogen-bromide-activated Sepharose 6 MB and used as a substrate to determine the AA-degrading activity of enzymes in solution. The applicability of the substrate was tested with an elastase preparation known to have AA-degrading activity. The substrate was used to determine the AA-degrading activity in fractions of normal human serum in the presence and absence of the plasminogen activator streptokinase. The AA-degrading activity was increased in fractions in which streptokinase-induced plasminogen activation had occurred. The increase in activity could be inhibited with antibodies to plasminogen. AA-degrading activity could also be increased in whole human plasma by the addition of streptokinase.

A re-examination of patients with previously detected autoantibodies to smooth muscle.

A group of patients which previously had been found to possess smooth muscle antibody (SMA) activity was re-examined for persistence of the autoantibody. The mean observation time was four years (range 0.5-9 years). Thirty out of 140 patients, who initially had SMA associated with rheumatic or other symptoms, had autoantibody activity at re-examination. Most were low in titer, and none of the 30 had developed chronic liver disease. Twenty-four patients with SMA at the first testing were analyzed because of liver pathology. Five had CAH at that time. SMA in low titer associated with liver pathology was a transient phenomenon. SMA in high titer combined with liver pathology seems to be a persistent condition with respect to both parameters, although no additional patients developed CAH between the first testing and re-examination.