The natural history of maternal immunization against foetal platelet alloantigens

Foetomaternal alloimmune thrombocytopenia (FMAIT) occurs when maternal antibodies of an antigen-negative mother cause destruction of sensitized foetal platelets. In Caucasian populations, 6-12% of human platelet antigen (HPA)-1a-negative women develop anti-HPA-1a, and the incidence of clinically affected cases is estimated to be 10-20% of immunized women. This study was performed in order to elucidate the rate of maternal immunization, incidence of FMAIT and the likely outcome of the condition in Asians. Excluding two or more pregnancies during the period, serum samples from 24 630 pregnant women, mainly Japanese, were screened for antibodies against platelet alloantigens by means of mixed passive haemagglutination (MPHA) (Anti-HPA-MPHA, Olympus, Tokyo). Antibodies were detected in 0.91% (223/24 630) of the women's samples and the immunization rate was correlated with the number of pregnancies. Antibody specificity included anti-HPA-4b (49), anti-HPA-5a (three), anti-HPA-5b (168), anti-HPA-4b + 5b (one) and anti-Nak(a) (CD36) (two). No alloimmunization was observed within the HPA-1, HPA-2, HPA-3 or HPA-6 systems. Among HPA-4b- or HPA-5b-negative women, 24% or 14% estimated, respectively, had antibodies and 26% (10/38) or 10% (12/125) of neonates, respectively, born to these mothers developed thrombocytopenia. Two neonates born to mothers having anti-HPA-4b developed generalized purpura. No cases of intracranial bleeding or death due to FMAIT were recorded. Generalized purpura due to FMAIT occurs in one in 9359 (95% CI: 1 in 77 519-1 in 2591) pregnancies solely because of HPA-4b incompatibility.     

Platelet specific alloantigens.

The ability of platelets to aggregate and to form a platelet plug is central to the maintenance of normal hemostasis. When platelets have normal function, the severity of bleeding is related to the degree of thrombocytopenia. In patients with normal platelet production, the most common cause of thrombocytopenia is due to immune mechanisms that results in platelet injury and removal from the circulation. These mechanisms involve the binding of platelet-associated immunoglobulins and are classified as immune. Immune thrombocytopenias can be caused by autoantibodies (autoimmune thrombocytopenia), alloantibodies (isoimmune thrombocytopenia), or drug-induced immune complexes and conditions secondary to autoimmune disorders such as systemic lupus erythematosus. In this paper the focus is on alloimmune thrombocytopenias resulting from the formation of alloantibodies to platelet specific antigens. The clinical importance of the platelet alloantigens is due to their ability to elicit alloantibody production. Alloantigens, also referred to as isoantigens, are substances that induce the production of alloantibodies when they are infused into individuals of the same species who lack the specific alloantigen.     

Human platelet fraction arginine-vasopressin. Potential physiological role.

Arginine-vasopressin (AVP) immunoreactivity (Ir) has been found to be elevated in platelet-rich plasma. PlatAVP was defined as platelet-rich plasma Ir minus platelet-poor plasma Ir (Pavp). PlatAVP, Pavp, and synthetic AVP were found to have identical retention time on high performance liquid chromatography analysis and similar mobility on thin-layer chromatography. During a standard osmotic suppression-stimulation test, Pavp increased with plasma osmolality (Posm, mosmol/kg H2O); Pavp (pg/ml) = 0.98 (Posm -274.4), r = 0.57, P less than 0.001, n = 65; but PlatAVP was not significantly correlated with Posm and remained at 5 pg/ml. This PlatAVP concentration was estimated to represent a true intraplatelet AVP concentration of 0.4 to 3.7 X 10(-9) M. Binding studies on intact human platelets demonstrated specific binding sites for [3H]AVP (n = 16; BMax = 98 +/- 30 binding sites/platelet; Kd = 0.72 +/- 0.24 nM). This in vitro affinity association constant (Kd) was close to the estimated in vivo intraplatelet AVP concentration. Measurement of PlatAVP could estimate vasopressin bound to a specific platelet receptor.     

Human platelet stimulation by acetyl glyceryl ether phosphorylcholine.

Acetyl glyceryl ether phosphorylcholine (AGEPC) induced dose-dependent platelet aggregation and release of [3H]serotonin and platelet factor 4 in citrated human platelet-rich plasma. ADP scavengers or indomethacin prevented irreversible platelet aggregation responses induced by 0.2 microM AGEPC but had no effect upon platelet secretion; prostacyclin inhibited AGEPC-induced aggregation and secretion. EDTA or EGTA inhibited AGEPC-induced aggregation but had no effect on platelet secretion.     

Neutrophil alloantigens

Antibodies to neutrophil antigens can cause neonatal alloimmune neutropenia, autoimmune neutropenia, febrile transfusion reactions, and transfusion-related acute lung injury. Several neutrophil antigen systems have been described serologically, but only the human neutrophil antigen-1 (HNA-1) or NA and HNA-2 or NB systems have been well characterized biochemically and molecularly. HNA-1 antigens are located on FcgammaRIIIb, CD16. HNA-2 antigens are located on 58- to 64-Kd glycoprotein, CD177, and are encoded by a gene on chromosome 19 that belongs to the Ly-6 family. The function of the CD177 is not known, but the CD177 gene is highly homologous to a gene overexpressed in neutrophils from patients with polycythemia rubra vera called PRV-1. New polymorphisms in these antigen systems are still being described, but the complete understanding of these neutrophil antigen systems has been slow because of the complexity of these genes.     

Human megakaryocytes. II. Expression of platelet proteins in early marrow megakaryocytes

Analysis of various platelet proteins by immunofluorescence demonstrated that platelet glycoproteins Ib, IIb, and IIIa, as well as plasma factor VIII antigen (factor VIII:AGN), platelet factor 4, and fibronectin are present in the vast majority of morphologically recognizable megakaryocytes. In addition, a small number of lymphoid- like mononuclear marrow cells, representing approximately 1.4-- 2.9/10(4) marrow cells, was found to express the same platelet proteins. This population of early marrow megakaryocytes is analogous to small acetylcholinesterase-positive rat and mouse marrow cells. Fc receptors for IgG were expressed in all megakaryocytes and megakaryocyte precursors, whereas the Ia antigen was detected only on a proportion of mature megakaryocytes and not on only early or precursor megakaryocytes. Platelet glycoproteins Ib, IIb, and IIIa, as well as factor VIII:AGN, and platelet factor 4 were established as distinct markers for marrow megakaryocytes and may be helpful for identifying megakaryocytic cells as well as for monitoring events of megakaryocyte differentiation.     

B-lymphocyte alloantigens in Caucasians

Human B lymphocytes have been shown to have at least five polymorphic specificities defined by 32 antisera. The antisera were produced by absorption with pooled platelets to remove HLA activity and were selected out of over 400 tested sera. The sera that defined the five specificities had high correlation coefficients within a group (generally in the range of 0.6-0.9). As shown by the fit in the Hardy-Weinberg equilibrium, the five specificities appear to be determined by alleles at one genetic locus. No association between these specificities and HLA was noted.     

Human hexosaminidase isozymes. Assay of platelet activity for heterozygote identification during pregnancy

Identification of carriers of the Tay-Sachs gene during pregnancy is difficult because of the increase in serum of a heat stable hexosaminidase isozyme I (or P) as well as changes in the relative and absolute activities of the various molecular forms of the enzyme with advancing pregnancy. In contrast, isolation of blood platelets followed by ion exchange Chromatographic separation and assay of the hexosaminidase isozymes in platelet extracts by an automated method provides a sensitive and reliable method for heterozygote identification during pregnancy. This method appears superior to procedures involving thermal inactivation of extracts of peripheral blood leukocytes because of significant differences in the content of the hexosaminidase isozymes in granulocytes, lymphocyte and other cell types, as well as variations in the proportion of these cell types in samples of peripheral blood. It also alleviates the problem inherent in any method involving thermal inactivation of hexosaminidase A by avoiding possible interconversion of the various molecular forms of the enzyme associated with heating.     

Human platelet lysate current standards and future developments

A state‐of‐the‐art workshop focused on the use of human platelet lysate (HPL) for cell therapy. The meeting established that HPL is used mainly as an adjunct material for ex vivo expansion of mesenchymal stem/progenitor cells (MSCs), where it is successfully used as a substitute for fetal bovine serum. HPL manufacturing as a cell expansion supplement is currently not yet uniformly standardized with regard to platelet source and production methodology. There are very few reports of HPL preparations manufactured specifically for direct clinical use. There exists an urgent need for controlled clinical studies for HPL and for standardization of product definition. Workshop participants also stated a need for consensus minimum release criteria to allow for better product definition and to limit variability in performance. The increasing use of cell‐based therapies including MSCs has led to an increasing demand for HPL, either produced in blood establishments or large‐scale manufacture by biopharmaceutical companies. The use of pooled donor platelets for HPL production may require the implementation of pathogen inactivation procedures and/or removal steps to improve the safety of advanced cell therapy products. There should also be a requirement for thorough risk assessments and risk mitigation steps, including the qualification of suppliers and identification of ingredients as well as meticulous monitoring of product quality and safety profiles. State‐of‐the‐art regulatory approaches for HPL used for human cell propagation and PRP in direct clinical applications were reviewed.     

Surface alloantigens of plasma cells

A serological study of immunoglobulin-forming cells of the mouse, normal and malignant, shows that they lack all known surface differentiation antigens of the thymocyte-lymphocyte axis: TL, θ, Ly-A, Ly-B, and MSLA. Two systems of normal alloantigens are expressed on these cells, H-2 and a new system named PC. The gene Pca (Plasma cell antigen) which specifies PC.1 alloantigen segregates as a mendelian dominant not closely linked with H-2. This cell surface antigen is absent from thymocytes, leukemias, and very probably from thymus-derived lymphocytes also; it is present on cells of the liver, kidney, brain, and lymph nodes as well as on hemolytic plaque-forming cells of the spleen, and on myelomas. So PC.1 is properly classified as a differentiation alloantigen. The strain distribution of PC.1 does not conform to that of any known immunoglobulin allotype or cell surface alloantigen previously described. Thus the cell surface antigens of immunoglobulin-producing cells are clearly different from those of cells belonging to the thymocyte-lymphocyte axis. Each family of cells has distinctive alloantigens, and the two families share alloantigens of only one known system, H-2. This implies that either immunoglobulin-producing cells are not derived from thymic lymphocytes, or if they are, the program responsible for the transition must include extensive revision of cell surface structure.     

Status of platelet collection and platelet transfusion.

Platelet product derived from single donor plateletpheresis is required to reduce the risks of adverse reactions by blood transfusion. The objectives of this study are to evaluate the status of platelet collection and its efficacy by various kinds of plateletpheresis equipment and to assess the achievement of platelet transfusion by platelet product derived from a single donor. Since the blood centers have introduced some kinds of efficient plateletpheresis equipment, large units of platelet products have been supplied mainly for the patients. Amicus and CCS might be preferable plateletpheresis machines because of their collection efficiencies and wider indication for donors. The average number of donors of platelet product per patient has recently reached nearly 1.0, and around 90% of patients have received platelet product derived from a single donor in the recent several years. However, platelet transfusion derived from a single donor has not yet been completely achieved. Each regional blood center should seriously consider the efficacy of each plateletpheresis equipment and arrange the equipment to collect platelets more effectively to achieve platelet transfusion from a single donor.     

Human platelet antigen-2 and -3 genotyping by PCR-SSP

SUMMARY. Allele-specific PCR using sequence specific primers (PCR-SSP) is a simple and reliable technique to detect point mutations in genes. We have developed a PCR-SSP to enable the detection of a C-T mutation at position 482 of the GPIb gene and a T-G mutation at position 13962 in exon 26 of the GPIIb gene. These point mutations are at the basis of the HPA alloantigens 2a, 2b and 3a, 3b respectively. One primer of each primer set has a 3' nucleotide complementary to the DNA sequence coding for one allele. PCR product is only produced when the corresponding DNA is present and thus the genotype is determined by the presence or absence of a band in agarose electrophoresis of PCR products. A second set of primers in the same reaction yields a product regardless of the HPA genotype to control the efficiency of the PCR amplification. The HPA-2 and -3 genotypes determined in this way were in strict concordance with those established by conventional genotyping using PCR followed by restriction enzyme digestion (PCR-ASRA). PCR-SSP is a rapid and reliable technique that can be used for the determination of alleles which code for platelet alloantigens.