Quality control of leucocyte-reduced blood components: overestimation of WBC content due to nucleated red blood cells.

Most methods for quality control of white blood cell (WBC) depletion in blood products are based on flow cytometric techniques. Nearly all commercial kits are based on propidium iodide staining of the DNA and subsequently counting those DNA based events as residual WBC. Here, we could show that a substantial proportion of those events are derived from nucleated red blood cells and therefore not specific for WBCs (e.g. in erythrocyte products 30%). We developed a flow cytometric method for residual WBC counting applying simultaneous DNA- and WBC-specific surface staining to enable this.

Repeated intrauterine IgG infusions in foetal alloimmune thrombocytopenia do not increase foetal platelet counts.

BACKGROUND AND OBJECTIVES: Foetal alloimmune thrombocytopenia (FNAIT) is often treated transplacentally with maternally administered i.v. immunoglobulins, but not all foetuses show a consistent platelet increase during such treatment. MATERIALS AND METHODS: We retrospectively analysed data from a cohort of ten foetuses with FNAIT treated by direct foetal immunoglobulin infusion. Foetal treatment was begun between 17 and 25 weeks and continued until 36 weeks with weekly cordocenteses and foetal immunoglobulin infusions. RESULTS: While foetal IgG levels increased steadily during weekly IgG infusions, foetal platelet counts remained unchanged. CONCLUSION: Our retrospective study presents a unique analysis of a historical cohort, contributing to the ongoing debate about the treatment of choice for foetal alloimmune thrombocytopenia.

Retrospective comparison of maternal vs. HPA-matched donor platelets for treatment of fetal alloimmune thrombocytopenia.

BACKGROUND AND OBJECTIVES: In fetal alloimmune thrombocytopenia (FAIT), transplacental maternal antibodies cause destruction of fetal platelets. FAIT is similar to fetal Rhesus haemolytic disease, but half of the affected fetuses are born to primiparous women. In 10-20% of cases, prenatal and perinatal intracranial haemorrhages are reported. Different therapeutic approaches have been described, including maternally administered high-dose intravenous immunoglobulin (high dose IVIG) without or with steroids or intrauterine transfusion (IUT) of compatible platelets. For the latter, the use of plasma-free maternal and donor platelets has been described, but a comparison of these two sources of platelets has not been reported. MATERIALS AND METHODS: We retrospectively analyzed the clinical courses of cases with FAIT treated with IUT of either HPA-matched donor platelets or maternal platelets, done by a single team between 1990 and 1997. In 57 pregnancies, FAIT was treated by repeated IUT with either maternal (15 fetuses) or donor platelets (42 fetuses). RESULTS: There was no procedure-related fetal or neonatal loss. Platelets from both sources reliably raised the fetal platelet counts. Donor platelet preparations contained more platelets and yielded higher fetal post-transfusion platelet counts, but maternal platelets were clinically equally effective. CONCLUSIONS: Donor and maternal platelet concentrates are effective sources for the treatment of FAIT.

Different behaviour of soluble CD40L concentrations can be reflected by variations of preanalytical conditions.

INTRODUCTION: Biomarkers reflecting an inflammatory or immunological response are increasingly offered to improve the risk stratification of patients. For example, current evidence suggests that soluble CD40 ligand (sCD40L) is elevated in patients with acute coronary syndrome. But only a few data are available to evaluate the influence of preanalytic conditions on sCD40L values. METHODS: Blood samples of seven healthy blood donors were collected in tubes without additives and in EDTA- or citrate-filled tubes at various storage conditions. Platelet count was modified by serum dilution, and sCD40L was measured in platelet-rich-plasma and in whole blood. sCD40L levels were determined by an commercially available ELISA-Kit. RESULTS: Immediately after blood sample assessment, sCD40L levels in serum samples were elevated (1258+/-820 pg/ml) compared to EDTA (64+/-32 pg/ml) and citrate (60+/-8.5 pg/ml) values. Additionally, sCD40L levels were dependent on storage duration. After platelet activation, sCD40L levels were significantly increased to 8278+/-2453 pg/ml and were significantly correlated to platelet count (r=0.96). CONCLUSIONS: Soluble CD40L levels were clearly influenced by preanalytical conditions and were dependent on storage duration, sample technique and platelet count. These influences should be considered by the determination and evaluation of sCD40L concentrations.

A hepatitis C virus (HCV) core protein derived peptide inhibits HCV specific lymphocyte proliferation.

T helper lymphocytes are important regulatory cells for the immune response in chronic hepatitis C. They recognize peptides, which are generated from the viral proteins by antigen processing and are bound to MHC (major histocompatibility complex) class II molecules. However, antigen processing might also result in non-immunogenic peptide fragments that can modify T cell activation. - To identify such peptide fragments in hepatitis C, we studied binding of 15 synthetic HCV core derived peptides to MHC class II molecules of 9 human homozygous typing B cell lines (HT-BCLs) as well as T cell proliferation in 41 HLA-typed patients with chronic hepatitis C. - We identified a peptide (HCV core aa 59-83) which bound to 7 HT-BCLs, whereas PBMC of only 2 out of 36 patients with the corresponding HLA-DR alleles proliferated in response to this peptide. Competition experiments indicated that small amounts of peptide aa 59-83 specifically inhibited the proliferative response to the recombinant core protein but not to core derived immunogenic peptides. Our data show that a peptide fragment from the HCV core region aa 59-83 can interfere in vitro with immune recognition of the HCV core protein.

Increased frequency of the HLA-DR15 (B1*15011) allele in German patients with self-limited hepatitis C virus infection.

BACKGROUND: A genetically determined resistance or susceptibility to chronic hepatitis C virus infection (HCV) may make an important contribution to the course of liver disease and may be linked to the human major histocompatibility complex. DESIGN: Twenty-one subjects with self-limited HCV infection as assessed by the presence of HCV antibodies, absence of HCV-RNA and normal levels of aminotransferases for 2 years were identified from a large pool of blood donors. The frequency of HLA serotypes of these individuals was compared with 49 consecutive patients with chronic hepatitis C. RESULTS: We detected a significantly higher prevalence of HLA-DR15 in patients with self-limited HCV infection than in patients with chronic hepatitis C (10/21 vs. 6/49; relative risk 6.5; P = 0.02; corrected for multiple comparisons). To confirm HLA assignments by serotyping we also performed sequencing of HLA-DR types in the 27 patients (9 with self-limited infection, 18 with chronic hepatitis C) who had been enrolled during 1995-96. This analysis confirmed the predominance of HLA-DR15 (HLA-DRB1*15011) in self-limited HCV infection (4/9 vs. 1/18; relative risk 13.6; P = 0.03). CONCLUSIONS: Our data suggest that HLA-DR15 (B1*15011) might constitute an important genetic factor for the elimination of the hepatitis C virus in Germany.

[Molecular mechanisms and indicators of thrombogenesis after heart valve replacement]. / Molekulare Mechanismen und Indikatoren der Thrombogenese nach Herzklappenersatz.

Complex interactions among constituents of blood, components of the subendothelial extracellular matrix, and artificial surfaces of cardiovascular devices are involved in the thrombogenesis following heart valve replacement. Recently, the molecular basis of some of these interactions has been studied in detail. These insights have extended our understanding of interactive processes between platelet receptors, adhesive macromolecules, and abnormal flow conditions during platelet adhesion and aggregation. On the basis of new experimental data, it is concluded that circulating nonactivated platelets are capable of recognizing surface-bound fibrinogen via their glycoprotein receptor GPIIb-IIIa and become activated as a consequence of this specific interaction. In addition, a molecular mechanism has been proposed indicating that high-shear stress created by prosthetic heart valves can induce platelet aggregation. For this platelet reaction, multimeric von Willebrand factor is essential; its structure function relationship and its interaction with the platelet membrane GPIb receptor have been elucidated. The progress made provides specific targets for selective antithrombotic strategies. Moreover, new molecular activation markers have become available which permit appropriate control of established and experimental antithrombotic regimens. This paper reviews some aspects of recent advances in this area.

[Treatment with blood components and coagulation factor concentrates--modern concepts of hemotherapy]. / Behandlung mit Blutkomponenten und Gerinnungsfaktorenkonzentraten--Moderne Konzepte zur Hämotherapie.

Transfusion of whole blood is the most common and successful organ transplantation world wide. It is in use longer than any other transplantation procedure. With the exception of uses in situations of crisis, treatment with whole blood is nowadays obsolete. This is due to enormous progress in preparation technologies. As a result blood from volunteer donors is separated before therapeutic use into its cellular and plasmatic components. The latter may be further processed into subfractions or secondary products. The advantages of this strategy are evident: a) the crude pharmaceutic material blood is used optimally, b) the isolated components may be stored over periods appropriate for their stability be it in a cryopreserved state or after biochemical, immunologic or biological modification, c) the patient receives only the blood components he needs, d) the transfusion of unnecessary fractions of blood is avoided, e) the rate of transfusion-associated adverse events is reduced, f) the physician has a basis for customized hematotherapy. Important implications concern logistics, economy, differentiated therapy and drug safety consideration. This contribution gives an overview on progress made in therapy of hematologic and oncologic patients with blood products and concentrates of coagulation factors. It demonstrates selected aspects of actual interest for optimal hemotherapy.

Polymorphism of platelet membrane glycoprotein IIIa: human platelet antigen 1b (HPA-1b/PlA2) is an inherited risk factor for premature myocardial infarction in coronary artery disease.

Conflicting results of an association between the human platelet antigen 1b (HPA-1b or PlA2) allele and the risk of myocardial infarction and coronary artery disease have been reported. To assess the reason for this discrepancy, we determined the HPA-1 genotype in 298 men who had undergone coronary angiography, including 124 individuals with myocardial infarction, 83 individuals with coronary artery disease but no history of myocardial infarction, and 91 control patients. Among patients with acute or recent onset myocardial infarction (< 1 year), the prevalence of HPA-1b was higher than among patients with coronary artery disease but without myocardial infarction (33 percent vs. 14 percent, p = 0.016). In patients under 60 years of age this difference was even more pronounced (45 percent vs. 15 percent, p = 0.003). Unlike conventional risk factors HPA-1b does not represent a risk factor for coronary artery disease itself but appears to be associated with increased platelet thrombogenicity.

Genetic typing of human platelet antigen 1 (HPA-1) by oligonucleotide ligation assay in a specific and reliable semi-automated system.

Genotyping of platelet alloantigens with the possibility of using any type of cellular material as a source of DNA has become a preferred procedure, particularly in thrombocytopenic patients when platelet counts are too low for phenotyping. Recently human platelet antigen 1 (HPA-1) has been identified as an inherited risk factor for coronary thrombosis. The different detection methods currently used have disadvantages for large-scale DNA diagnosis, including the need for electrophoresis (allele-specific restriction enzyme analysis, amplification with sequence-specific primers) or the potential risk of reduced specificity (allele-specific oligonucleotide hybridization). In this report we describe the adaptation of an automated oligonucleotide ligation assay to genotype HPA-1 in polymerase chain reaction (PCR)-amplified DNA samples. HPA-1a and HPA-1b phenotypes corresponded to the results of the different genotyping assays. The genotypes determined with the ELISA-based PCR-oligonucleotide ligation assay were in 100% concordance with the results obtained by conventional allele-specific restriction enzyme site analysis and PCR amplification with sequence-specific primers. The automated oligonucleotide ligation assay provides a rapid, reliable, nonisotopic method to genotype human platelet antigens that can rapidly be applied to large population screening.

[Intrauterine transfusion in fetal alloimmunothrombocytopenia: comparison of maternal and fetal weight-adjusted IgG therapy with exclusive fetal thrombocyte transfusion]. / Intrauterine Hämotherapie bei fetaler Alloimmunthrombozytopenie: Vergleich der maternalen und fetalgewichtadaptierten IgG-Therapie mit ausschliesslicher Thrombozytentransfusion des Feten.

Fetal alloimmune thrombocytopenia is caused by maternal immunization against a fetal platelet antigen and transplacental transfer of the antibody into the fetal circulation. Since 10-20% of the fetuses or newborns are threatened by intracranial hemorrhages, early management is required. Fetal blood sampling should be started between the 20th and 22nd week of gestation to assess fetal phenotype and platelet count. Different concepts to elevate the fetal platelet count have been discussed: maternal intravenous immunoglobulins, fetal intravenous immunoglobulins, or only repeated fetal platelet transfusions. Our investigations suggested that platelet transfusions in short intervals appear to be the only effective regimen to increase platelet counts in thrombocytopenic fetuses at risk.

T- and B-cell responses to different hepatitis C virus antigens in patients with chronic hepatitis C infection and in healthy anti-hepatitis C virus--positive blood donors without viremia.

As the host's immune response may determine the course of hepatitis C virus (HCV) infection, we studied the humoral and cellular immune responses to HCV-related antigens in subjects with different outcomes of HCV infection. Lymphoproliferative responses and circulating antibodies to a panel of HCV core- and E1-related 25-mer peptides were examined in 10 healthy anti-HCV-seropositive blood donors (group A) and in 29 patients with chronic hepatitis C (group B). In addition, cellular recognition of recombinant HCV proteins (core, NS3, NS4A, NS5A, NS5B) were investigated. In group A, stronger T-cell responses were detected against both HCV proteins (core, P = .03; NS4, P = .005; NS5B, P = .03) and peptides. Proliferation was induced by the same peptides in each group, defining at least five distinctive epitopes within core (amino acids [aa] of 20-44, aa 39-63, aa 79-103, aa 118-152 and aa 148-172) and three regions within E1(aa 198-252, aa 308-372, and aa 368-392). Subjects with strong T-cell responses had low or no detectable levels of peptide-specific antibodies, and vice versa. In particular, T-cell responses were more common in group A; B-cell responses were more common in group B. From our data, we conclude that a benign course of HCV infection may be the consequence of the effective activation of T-helper lymphocytes.

Therapy with intravenous immunoglobulin G (ivIgG) during pregnancy for fetal alloimmune (HPA-1a(Zwa)) thrombocytopenic purpura.

We have evaluated the effect of maternal intravenous immunoglobulin G (ivIgG) treatment on platelet counts in fetal alloimmune thrombocytopenia. Seven patients were studied. All of them were multiparous women who had been immunized against the HPA-1a antigen during previous pregnancies and had given birth to at least one severely thrombocytopenic infant. In this study, umbilical blood collection was performed first at the 20th week of gestation and repeated 2-13 times (mean 6 times), depending on the degree of fetal thrombocytopenia. Fetal platelet counting was combined with intrauterine transfusion of 20-30 ml of HPA-1a-negative platelet concentrates to prevent bleeding following umbilical cord puncture. Initial fetal platelet counts ranged from 10,000 to 91,000 per microliters. Maternal treatment with ivIgG (1 g per kg body weight; mean dose 70 g) was given once a week over 7 weeks. In five of seven cases, the basal platelet count did not rise and in two of these cases, it decreased during maternal ivIgG treatment. In one fetus, the baseline platelet count increased from 10,000 to 35,000 per microliters during ivIgG, and in another fetus from 23,000 to 64,000 per microliters. Our observations suggest that ivIgG has no definite benefit for fetal alloimmune thrombocytopenia. Since platelet counts can be very low, careful fetal monitoring by umbilical blood sampling is required. Frequent platelet transfusions in short intervals may be necessary to increase platelet counts in extremely thrombocytopenic fetuses.

[Prenatal management of fetuses with alloimmune thrombocytopenia]. / Pränatale Betreuung von Feten mit Alloimmunthrombozytopenie.

Fetal alloimmune thrombocytopenia is caused by maternal immunization against a fetal platelet alloantigen and transplacental transfer of the antibody into the fetal circulation. Since 10-20% of the fetuses or newborns are threatened by intracranial hemorrhages (ICH) early management is required. Intensive prenatal monitoring should be performed if a maternal HPA-1a antibody is known and a previous infant suffered from thrombocytopenia and/or ICH. Fetal blood sampling (FBS) should be started at 20th to 22nd weeks of gestation to assess fetal phenotype and platelet count. Different concepts to elevate the fetal platelet count have been discussed: corticosteroids, maternal intravenous immunoglobulins (ivIgG), fetal ivIgG and repeated fetal platelet transfusions. In a European survey with data from five centres maternal corticoid treatment and ivIgG infusion were accompanied by increasing fetal platelet counts in only 20 and 24% of the cases, respectively. In fetuses with very low platelet counts only transfusions of compatible platelets in short intervals are able to sustain a safe platelet count. Fetuses with mild thrombocytopenia should be monitored by subsequent FBS since it could be shown that platelet counts tend to decline during gestation. To avoid bleeding complications during and after FBS which was observed in about 5% of the cases every cord vessel puncture should be covered by a platelet transfusion. As no safe and non-invasive therapy exists for fetal alloimmune thrombocytopenia the value of prenatal screening programs in unaffected pregnancies is questionable.

[Fetal weight-adjusted intrauterine IgG therapy in neonatal alloimmune thrombocytopenia]. / Fetalgewichtadaptierte intrauterine IgG-Therapie bei neonataler Alloimmunthrombozytopenie.

Fetal alloimmune thrombocytopenia is caused by materno-fetal transfer of platelet antibodies. Since the thrombocytopenic fetus is threatened by intracranial hemorrhage, prenatal observation and, if necessary, treatment is required. However, the benefit of therapeutic options, including intravenous IgG (ivIgG), platelet transfusions or fetal IgG transfusions is still controversial. In this study we have evaluated the effect of intrauterine IgG and intraumbilical platelet transfusions on fetal platelet counts. All patients were multiparous women who were immunized against the Zwa antigen during previous pregnancies and had given birth to at least one severely thrombocytopenic infant. First umbilical blood was sampled at the 20th week of gestation. Fetal treatment of IgG was given, on av erage, over 9 weeks. In all cases, fetal IgG levels rose significantly whereas platelet counts did not increase following fetal IgG treatment. We conclude that fetal IgG infusions have no detectable effect on fetal allo-immune thrombocytopenia. Since platelet counts can be very low as early as 20 weeks of gestation, careful fetal monitoring by umbilical blood sampling is essential. Platelet transfusions in short intervals appear to be the only effective regimen to increase platelet counts in thrombocytopenic fetuses at risk.

[Determination of intestinal strontium absorption--assessment and validation of routinely manageable test procedures]. / Die Bestimmung der intestinalen Strontiumabsorption--Etablierung und Validierung eines routinemässig anwendbaren Testverfahrens.

Intestinal strontium absorption has been discussed recently as an indirect measure for calcium uptake. Prerequisite for the clinical use of an oral strontium test is the availability of a reliable procedure including controlled strontium supply, sample pretreatment and analysis as well as the assessment of normal values. In the present study, a group of young females (n = 33; 24.0 +/- 2.7 y; BMI 21.5 +/- 1.9) received an oral dose of 2.27 mmol strontium in a standardized breakfast that contained 0.625 mmol calcium. Before and 220 min after the bolus serum strontium concentrations were determined by means of atomic absorption spectrophotometry (coefficient of variation: within day 4.8%, n = 10; day-to-day 9.5%, n = 8). The error of the method was 2.7%. Calculation of the fractional strontium absorption rate considered the respective distribution volume (extracellular fluid; either estimated using body weight or determined by means of bioimpedance analysis [BIA]). Average absorption rates were 13.3 +/- 3.1% and, considering BIA measurement 13.6 +/- 2.6%, respectively. Smoking, exercise and, use of oral contraceptives showed no effects. Our oral strontium test is characterized by excellent reliability, easy handling and low costs and, thus, is suitable for routine use.

Maternal intravenous immunoglobulin treatment does not prevent intracranial haemorrhage in fetal alloimmune thrombocytopenia.

In fetal alloimmune thrombocytopenia (FAIT) the fetus is threatened by intracranial haemorrhage (ICH); therefore early diagnostic and therapeutic intervention is required. We followed the clinical course of a 30-year-old woman during her fifth pregnancy after she had given birth to a child with alloimmune thrombocytopenia due to anti-Zwa. The fetus was monitored by 13 fetal blood samplings (FBS) always followed by transfusion of either maternal or compatible donor platelets. Intravenous immunoglobulin (ivIg) treatment of the mother was begun at 20 weeks of gestation when the fetal platelet count was 36 x 10(9)/l. The fetal platelets were typed Zwa positive by DNA analysis. Despite 11 weeks of maternal ivIg treatment fetal platelet counts progressively declined to 6 x 10(9)/l and ICH occurred. Subsequently, the fetus was successfully managed by intrauterine platelet transfusions at shorter intervals (3-5 days) and elective Cesarean section was carried out at 35 weeks of gestation. We conclude that maternal ivIg treatment does not prevent ICH in FAIT. The treatment of choice for severely affected cases is serial FBS combined with transfusion of compatible platelets.

[Prenatal substitution therapy in fetal alloimmune thrombocytopenia]. / Pränatale Substitutionstherapie bei fetaler Alloimmunthrombozytopenie.

Fetal alloimmune thrombocytopenia is caused by maternofetal transfer of platelet antibodies. Since the thrombocytopenic fetus is threatened by intracranial hemorrhage, prenatal observation and, if necessary, treatment is required. However, the benefit of therapeutic options, including intravenous IgG (ivIgG) or platelet transfusions, is still controversial. In this study we have evaluated the effect of maternal ivIgG and intraumbilical platelet transfusions on fetal platelet counts in 7 cases. All patients were multiparous women who were immunized against the Zwa antigen during previous pregnancies and had given birth to at least one severely thrombocytopenic infant. First umbilical blood was sampled at the 26th week of gestation. Maternal treatment of ivIgG was given over 7 weeks, the mean number of intrauterine platelet transfusions was 5.9. In 6 of 7 cases the basal platelet count before transfusion did not rise or even further decreased during maternal ivIgG treatment. In one case the baseline platelet count increased from 18,000/microliter to 60,000/microliter during ivIgG. We conclude that, in general, ivIgG alone has no detectable effect on fetal alloimmune thrombocytopenia. Since platelet counts can be very low as early as 20 weeks of gestation, careful fetal monitoring by umbilical blood sampling is required. Platelet transfusions in short intervals appear to be the only effective regimen to increase platelet counts in extremely thrombocytopenic fetuses.