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PubMed1997

The effect of cardiopulmonary bypass on circulating megakaryocytes.

Megakaryocytes (Mks) are found in the lungs and the blood stream as well as in the bone marrow. We modified a whole blood filtration method for Mks by immunostaining for CD61 using biotin streptavidin, and used this technique to study Mks and their morphology in the central venous and arterial circulations before, during and after cardiopulmonary bypass (CPB) in haematologically normal patients undergoing routine cardiac surgery. Blood samples were taken immediately after the insertion of central venous (V) and arterial (A) catheters and after thoracotomy, immediately before bypass. Further samples were taken after 60-90 min on-CPB and 180-240 min post-bypass. In comparison with the steady state before bypass, circulating Mk levels in blood on bypass increased dramatically, from (V) 10.93 +/- 3.94/ml (mean +/- SD) to 36.48 +/- 11.52/ml and from (A) 8.37 +/- 4.39/ml to 38.65 +/- 20.68/ml. This effect was still present, to a lesser extent, 180-240 min post-bypass. Circulating levels of Mks were consistently lower in the arterial circulation than in the venous circulation off bypass, but levels in the two circulations were comparable during CPB, confirming previous suggestions that the lungs are net removers of Mks from the circulation. Type 4 Mks, the largest and most normal morphologically, were rarely seen in arterial blood, but increased significantly during CPB, indicating that the lungs selectively remove large Mks. The lungs appear to play an active role in the regulation of Mk levels. This is lost during CPB and despite the extracorporeal 40 microm arterial line filter, large Mks enter the systemic circulation. More effective extracorporeal filtration of large Mks might reduce the neurological impairment seen in some patients who have undergone CPB.

Adult↗
PubMed1998

Feto-maternal alloimmune thrombocytopenia: antenatal therapy with IvIgG and steroids--more questions than answers. European Working Group on FMAIT.

The optimal antenatal therapy for fetal thrombocytopenia has not been determined. We analysed 37 cases managed by maternal therapy and observed a successful outcome of maternal treatment in 26% of IvIgG cases and in 10% of steroid-treated cases. The significance of a plateau of the fetal platelet counts during pregnancy, 41% of IvIgG cases and 20%, of cases treated with steroids, is uncertain. It may indicate a stabilization of thrombocytopenia, hence a beneficial effect of therapy, or the natural course of the platelet count in a low-risk pregnancy. Overall outcome was unpredictable, but amongst the therapy failures there were proportionally more severely affected siblings. Further multicentre studies are necessary to establish the optimal antenatal management of high-risk pregnancies.

Adrenal Cortex Hormones↗
PubMed1998

Linkage of four polymorphisms on the alphaIIb gene.

The subunits of the platelet integrin alphaIIb beta3 are encoded by two genes located on chromosome 17. Two pathologies are associated with structural modifications of this complex: Glanzmann's thrombasthenia and alloimmune thrombocytopenia. The former is a hereditary bleeding disorder, the latter is due to an immune response linked to the presence of specific epitopes defined by single amino acid substitutions called human platelet alloantigen (HPA) systems. Analysing the alphaIIb gene from 112 independent chromosomes, we have defined two new silent polymorphisms in complete linkage disequilibrium. They are reciprocally linked to HPA-3 and a previously reported 9 pb deletion in intron 21. Linkage of these four DNA markers spanning a 5 kb fragment of genomic DNA provides a new tool for analysing alphaIIb gene pathology and evolution.

Genetic Linkage↗
PubMed2000

A rapid one-stage whole-blood HPA-1a phenotyping assay using a recombinant monoclonal IgG1 anti-HPA-1a.

Severe neonatal alloimmune thrombocytopenia and patients with HPA-1a-specific antibodies require transfusion of HPA-1a-negative platelets. Identifying HPA-1a-negative donors requires simple and reliable typing methods. Most existing techniques use polyclonal antibodies, are time consuming and involve platelet isolation. We have used a horseradish peroxidase (HRP)-conjugated recombinant IgG1 anti-HPA-1a (CAMTRAN007) to develop a rapid and reliable enzyme-linked immunosorbent assay (ELISA), which eliminates sample preparation and reduces the incubation and wash steps associated with traditional sandwich ELISAs. The assay uses simultaneous incubation of the monoclonal antibody RFGP56 to capture GPIIbIIIa from whole blood and the recombinant IgG1 antibody to detect captured HPA-1a antigen. It allows 96 samples to be typed in less than 1 h and can be used on stored samples. Initial testing of 85 samples of known HPA-1a genotype demonstrated that HPA-1a-negative samples had OD values of < 0.266, whereas HPA-1a-positive samples had OD values of > 0.6. Testing of 1862 random donor samples in two blood centres confirmed these OD cut-off values and identified 45 HPA-1a-negative samples (2.4%), all except one giving OD values of < 0.2. The remaining HPA-1a-negative sample had an OD value of 0.303. The HPA-1a status on all the negative samples and an equivalent number of randomly selected positive samples was confirmed by flow cytometry and polymerase chain reaction with sequence-specific primers (PCR- SSP).

Antigens, Human Platelet↗
PubMed2000

Relationship between bleeding time, aspirin and the PlA1/A2 polymorphism of platelet glycoprotein IIIa.

A single nucleotide T to C transition of the gene encoding glycoprotein IIIa leads to a common diallelic polymorphism Leu-33-->Pro (PLA1/A2). We studied the relationship between the PlA1/A2 polymorphism and platelet function in 80 healthy men, aged 20-25 years. Before aspirin ingestion, bleeding time (BT) was shorter in carriers of the PlA2 than in carriers of the PlA1/A1 allele. At 4 h after ingestion of 300 mg of aspirin, BT became prolonged, and the intergroup difference was enhanced. In seven out of 26 PLA2 allele carriers, aspirin shortened BT on average by 30 s, compared with only one among 54 subjects with the PlA1/A1 genotype. Thus, BT both at baseline and after aspirin depends on the PlA1/A2 polymorphism of glycoprotein IIIa. Carriers of the PlA2 allele appear to be more resistant to the antithrombotic action of aspirin.

Adult↗
PubMed2001

Automated CD61 immunoplatelet analysis of thrombocytopenic samples.

Revision of the current decision point for prophylactic platelet transfusion in thrombocytopenic patients requires the availability of a method that is able to provide accurate platelet counts to as low as 1 x 109/l. This study is the first to evaluate the immunoplatelet method (CD61-Imm) of the haematological analyser Cell-Dyn 4000 in direct comparison with the flow cytometric procedure. Additionally CD61-Imm results were compared with CD4000 optical (Plto) counts in the ranges 20-547 x 109/l (n = 127) and 1-35 x 109/l (n = 107). The immunoplatelet and Plto results were in good agreement between 20 x 109/l and 547 x 109/l, but for samples of < 25 x 109/l the Plto tended to overestimate the counts. We determined the limits of detection (LD) and quantification (LLQ) for all three methods using standard statistical procedures. The LD for the flow cytometric CD41a method was 0.02 x 109/l compared with 0.009 x 109/l and 1.73 x 109/l for the CD61-Imm and Plto methods respectively. The LLQCV = 15% for the CD41a method was 1.8 x 109/l compared with 1.6 x 109/l and 18.0 x 109/l for the CD61-Imm and Plto procedures. In conclusion, (i) the CD61-Imm method performance is at least equivalent to the reference flow cytometric method, and (ii) in severe thrombocytopenia the CD61-Imm count is superior to the Plto count.

Antigens, CD↗
PubMed2001

Provision of platelet support for fetuses and neonates affected by severe fetomaternal alloimmune thrombocytopenia.

Severe fetomaternal alloimmune thrombocytopenia requires urgent treatment with compatible platelet concentrates. As prompt treatment is sometimes delayed owing to the unavailability of compatible platelets, we established an accredited platelet donor panel to provide effective and timely transfusion support for fetal and neonatal therapy. After a mass screening programme of over 60,000 blood donations, 45 HPA-1a-negative donors with no antibodies to HPA, HLA, red cell antigens and granulocytes/lymphocytes, and with low titre anti-A and/or -B were accredited. All accredited donors were fully genotyped for HPA-1, -2, -3 and -5 by PCR-SSP. Ninety-one per cent of the accredited donors were also negative for HPA-5b.

Antigens, Human Platelet↗
PubMed2001

Apoptosis and megakaryocytic differentiation during ex vivo expansion of human cord blood CD34+ cells using thrombopoietin.

Thrombopoietin (TPO), the primary regulator of megakaryocytopoiesis, plays important roles in early haematopoiesis. Previously, we have demonstrated that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of cord blood (CB) CD34+ cells. In this study, we have demonstrated that the TPO-induced apoptotic cells belong to the megakaryocytic (MK) lineage and that initially expanding MK progenitors declined along with the appearance of TPO-induced apoptosis. Human CB CD34+ cells were expanded in serum-free conditions with TPO. Multidimensional flow cytometry using simultaneous measurement of apoptosis and immunophenotyping showed that the TPO-induced apoptotic cells appeared in CD61+ fractions. Immunocytochemical analysis of the fluorescent activated cell-sorted fractions showed that the apoptosis-associated CD44low fraction expressed CD61. Clonogenic assay revealed 7.4 +/- 0.50-fold increase of total megakaryocyte colony-forming units (CFU-MKs) during the initial 9 d. Thereafter, the number of CFU-MKs decreased in parallel with the increase of apoptosis. When the MK colonies were subdivided according to size, the proportion of large colonies progressively decreased, while that of medium and small colonies increased. In particular, from d 6 small colonies became predominant. These results suggested that the MK progenitors matured as they expanded during ex vivo expansion with TPO and then proceeded to apoptosis.

Antigens, CD↗
PubMed2001

Cord blood megakaryocytes do not complete maturation, as indicated by impaired establishment of endomitosis and low expression of G1/S cyclins upon thrombopoietin-induced differentiation.

Cord blood (CB) has successfully been used as a stem cell source for haemopoietic reconstitution. However, a significant delay in platelet engraftment is consistently found in CB versus adult peripheral blood (PB) or bone marrow transplants. We sought to determine whether or not CB megakaryocytes have reached terminal maturation and, hence, full thrombopoietic potential. A comparative analysis of megakaryocytes cultured from either CB or PB progenitors in the presence of thrombopoietin (TPO) showed a similar differentiation response, although proliferation was 2.4 times higher in CB than in PB cells. Importantly, the TPO-induced ploidy level was notably different: whereas 82.7% of CB megakaryocytes remained diploid (2N) at the end of the culture, more than 50% of PB megakaryocytes had reached a DNA content equal to or higher than 4N. Western blot and flow cytometry analyses revealed that only polyploid PB megakaryocytes expressed cyclins E, A and B, whereas cyclin D3 was detected in both fetal and adult megakaryocytic nuclei. These data suggest that establishment of endomitotic cycles is impaired in CB megakaryocytes, associated with a differential regulation of G1/S cell cycle factors. We believe that the relative immaturity of fetal megakaryocytes could be a contributing factor to the delayed platelet engraftment in cord blood transplantation.

Antigens, CD↗
PubMed2001

Comparison of megakaryopoiesis in vitro of paired peripheral blood progenitor cells and bone marrow harvested during remission in patients with acute myeloid leukaemia.

We have studied paired peripheral blood progenitor cells (PBPC) and bone marrow (BM) samples from 12 acute myeloid leukaemia (AML) patients following intensive chemotherapy, and assessed direct granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), megakaryocyte CFU (CFU-Mk) numbers and the production of CD61+ (platelet glycoprotein IIIa) cells in suspension culture in response to various haemopoietic growth factor combinations. We found that CFU-GM and BFU-E numbers per 105 mononuclear cells were similar in both AML PBPC and BM harvests; CFU-Mk numbers, however, were significantly higher in PBPC than BM. In addition, the higher total white cell count of the PBPC harvests meant that PBPC have much higher numbers of total progenitors per collection. CD61+ cell numbers in suspension cultures of AML PBPC and BM were lower than those of harvested normal marrow. However, response to pegylated recombinant human megakaryocyte growth and development factor (PEGrHuMGDF) both alone and in combination with other growth factors was qualitatively similar to that of normal BM. As with normal BM, response to PEGrHuMGDF alone did not increase further with addition of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin 6 (IL-6) or erythropoietin (EPO) in the AML PBPC and BM. Further responses over PEGrHuMGDF alone were seen when added with stem cell factor (SCF) or with a combination of SCF + IL-3 + EPO in both AML PBPC and BM cultures; however, the magnitude of the response was greater in the PBPC cultures. Response to PEGrHuMGDF + IL-3 was seen in the PBPC cultures but not in the AML BM. These data suggest that, in AML patients, there are proportionally more megakaryocyte progenitor cells in the mobilized PBPC than in the BM harvests, which would explain the more rapid platelet recovery following PBPC autografts.

Acute Disease↗
PubMed2002

Human platelet antigen 1 (HPA 1) genotyping with 5' nuclease assay and sequence-specific primers reveals a single nucleotide deletion in intron 2 of the HPA 1a allele of platelet glycoprotein IIIa.

We have established a 5' nuclease assay (5' NA) for human platelet antigen (HPA) 1a/b allelic discrimination. The assay is based on the simultaneous amplification and detection of the two targets in a one-tube system. The results are read optically, immediately after termination of the polymerase chain reaction (PCR), and no post-PCR processing is necessary. This genotyping procedure is less time-consuming and cheaper than our conventional sequence-specific primer PCR (SSP-PCR), which is run as a two-tube test, with verification of the results after electrophoresis in agarose gel. The reduction of analytical steps, simplification of the procedure and potential for automation were important advantages for our choice of system. This test system is more suitable for large-scale testing and fits better for our screening programme for HPA 1bb determination. DNA from 1093 individuals were tested in parallel with the SSP-PCR and the 5' NA. One thousand and ninety-one samples gave identical results in SSP-PCR and 5' NA. Upon repeated testing, two samples consistently came out as HPA 1bb in SSP-PCR and HPA 1ab in 5' NA. DNA sequencing revealed a defect located in an intronic area that corresponds to the consensus primer used for the SSP-PCR HPA 1a typing.

Alleles↗
PubMed2002

Human platelet alloantigen polymorphism in Glanzmann's thrombasthenia and its impact on the severity of the disease.

Glanzmann's thrombasthenia (GT) is an autosomal recessive disorder of platelets caused by the deficiency or abnormality of platelet receptors. Several platelet alloantigen systems reside on glycoprotein (GP) IIb and GPIIIa, of which the human platelet antigen 1 (HPA-1) system is important. Studies have shown that, in the normal population, the HPA-1b phenotype results in increased platelet aggregation and increased fibrinogen binding, increasing the risk of myocardial infarction. GT produces severe bleeding, but in a subset of patients has a relatively milder course. Forty-one GT patients and 100 healthy control subjects were genotyped for platelet alloantigens HPA-1 to HPA-6, using PCR-ASA (polymerase chain reaction-allele-specific amplification), and for GPIIb-IIIa expression and fibrinogen binding using flow cytometric techniques. Platelet alloantigen distributions were similar in the patient and control groups. With the exception of the two HPA-1b/1b homozygous patients (> 10%), 25 GT patients had less than 5% aggregation to 6 micro mol/l ADP, and 16 patients showed between 5% and 10% aggregation to 6 micro mol/l ADP. Seven out of 37 patients with HPA-1a/1a phenotype showed 1-5% fibrinogen binding and GPIIb-IIIa receptors. The two HPA-1b/1b patients showed 34.6% and 32% fibrinogen binding and > 10% GPIIb-IIIa receptors. This study determined the platelet alloantigen distribution in a large cohort of unrelated GT patients from western India. GT patients homozygous for HPA-1b/1b had higher levels of platelet aggregation and fibrinogen binding as well as a milder course, as evidenced by infrequent epistaxis and no transfusion requirement to date.

Adolescent↗
PubMed2003

The role of P-selectin in the immune destruction of platelets.

Antibody-mediated platelet destruction is a poorly understood process, although several lines of evidence suggest that Fcgamma receptor (FcgammaR)-expressing splenic macrophages may be involved. In this study, chemiluminescence (CL) was used to measure the in vitro metabolic response of human monocytes to platelets sensitized with a human immunoglobulin (Ig)G1 recombinant antihuman platelet antigen-1a (anti-HPA-1a) antibody (B2G1; P-hrIgG1). CL responses were inhibited, but not abrogated, in the presence of 10 micro g/ml human IgG or murine IgG2a, suggesting that FcgammaRI was principally involved. Experiments to determine the effect of Fab fragments to FcgammaRII found that CL responses to P-hrIgG1 were significantly enhanced, indicating that crosslinking of monocyte FcgammaRII by platelet-bound hIgG may modulate concomitant activation by FcgammaRI. Several observations suggested that the CL responses to P-IgG were dependent on the activation of resting platelets during their co-culture with monocytes and their subsequent P-selectin-mediated adhesion. First, the magnitude of the CL response was related to the level of P-selectin expression following platelet activation with alpha-thrombin. Second, CL responses were inhibited in the presence of antibodies that block the binding of P-selectin to P-selectin glycoprotein ligand-1 but not when platelets were pretreated and then washed. Third, the addition of anti-HPA-1a to monocytes from HPA-1a-negative donors preincubated with HPA-1a-positive platelets resulted in rapid CL responses. Finally, PGI2 inhibited the CL response to resting P-hrIgG1. Thus, evidence is presented that the interaction of human monocytes with P-hrIgG1 is mediated by FcgammaRI, modulated via FcgammaRII, and enhanced by the presence of P-selectin on the platelet membrane.

Antigens, Human Platelet↗
PubMed2003

European collaborative study of the antenatal management of feto-maternal alloimmune thrombocytopenia.

The aims of this study were to determine whether the severity of fetomaternal alloimmune thrombocytopenia (FMAIT) in the current pregnancy could be predicted from the history of FMAIT in previous pregnancies, and to assess the effects of different types of antenatal intervention. Fifty-six fetuses were studied that all had a sibling affected by FMAIT due to human platelet antigen 1a (HPA-1a) alloimmunization. Cases with a sibling history of antenatal intracranial haemorrhage (ICH) or severe thrombocytopenia (platelet counts of < 20 x 109/l) had significantly lower pretreatment platelet counts than cases whose siblings had less severe thrombocytopenia or postnatal ICH. Maternal therapy resulted in a platelet count exceeding 50 x 109/l in 67% of cases. None of the fetuses managed by serial platelet intrauterine transfusions (IUT) suffered ICH following treatment. However, several serious complications arose with fetal blood sampling (FBS). Overall, intervention improved outcome, as three study cases suffered from antenatal ICH and three others died whereas 15 study cases had a sibling with an ICH, eight of whom died. The results of this study suggest that the start of therapy can be stratified on the basis of the sibling history of FMAIT, and support the use of maternal therapy as first-line treatment.

Antigens, Human Platelet↗
PubMed2004

A rapid and specific whole blood HPA-1 phenotyping by flow cytometry using two commercialized monoclonal antibodies directed against GP IIIa and GP IIb-IIIa complexes.

The human platelet antigen 1 (HPA-1) system has been implicated in rare but severe diseases, such as neonatal alloimmune thrombocytopenic purpura, post-transfusion purpura and immune platelet refractoriness. We developed a flow cytometry assay for HPA-1 phenotyping using two commercial monoclonal antibodies, P2 and SZ21, directed against glycoprotein (GP) IIb-IIIa and GP IIIa respectively. One hundred and twenty-seven healthy controls were studied and ratios of mean fluorescence intensity for P2 and SZ21 discriminated between HPA-1a homozygotes and heterozygotes. These two monoclonal antibodies, coupled with flow cytometry represent a rapid and reliable tool for platelet HPA-1 typing to aid diagnosis.

Antibodies, Monoclonal↗
PubMed1996

Cross-reactive antibodies between HIV-gp120 and platelet gpIIIa (CD61) in HIV-related immune thrombocytopenic purpura.

We have previously demonstrated that immune platelet destruction observed in an AIDS-free HIV-infected patient was associated with the presence of a cross-reactive antibody recognizing both HIV-glycoprotein (gp)120 and platelet gpIIIa (CD61). We have now investigated the presence of such antibodies in other HIV-infected patients, together with the molecular structure of the cross-reactive epitope. Platelet gpIIb/IIIa antibodies were characterized in sera from HIV-infected patients with immune thrombocytopenic purpura by means of an ELISA and a radio-immunoprecipitation procedure (RIP). The platelet antibodies were purified and tested for their ability to recognize HIV-gp. We also tried to characterize the antibody target epitope on HIV-gp120 using recombinant gp and synthetic peptides. IgG with anti-gpIIb/IIIa activity were detected, by means of an ELISA with purified gpIIb/IIIa, in 101/138 (73%) sera from HIV-infected patients with immune thrombocytopenic purpura. The platelet antibodies were purified from 23 sera by absorption/elution on purified immobilized platelet gpIIb/IIIa, and recognition of gpIIIa was confirmed in eight cases with a RIP. Furthermore, the presence of a cross-reactive antibody between HIV-gp120 and platelet gpIIIa was demonstrated in 18/18 patients (including the eight with a confirmed gpIIIa antibody) by the ability of the serum HIV-gp160/120 antibodies to bind to purified gpIIb/IIIa. The cross-reactive epitope was shown to be independent of the carbohydrate moieties of gp120, since deglycosylation of two recombinant (r)-gp120s did not abolish antibody binding. However, the antibody did not recognize synthetic gp120 peptides spanning 355 of the 516 amino acids of gp120, particularly the four regions exhibiting sequences of four or five consecutive amino acids that are identical between r-gp120 and gpIIIa. Our results thus support the hypothesis that the cross-reactive antibody recognizes the conformational structure of gp120. These results strongly suggest that molecular mimicry between HIV-gp120 and platelet gpIIIa may be important in the pathogenesis of immune thrombocytopenia in AIDS-free HIV-infected patients.

Antigens, CD↗