Platelet-specific Antigens Research Articles

Plasma extracellular vesicle test sample to standardize flow cytometry measurements

BackgroundExtracellular vesicles (EVs) in body fluids are explored as disease biomarkers, but EV concentrations measured by flow cytometers (FCMs) are incomparable. ObjectivesTo improve data comparability, new reference materials with physical properties resembling EVs and reference procedures are being developed. The validation of new reference materials and procedures requires biological test samples. We developed a human plasma EV test sample (PEVTES) that i) resembles subcellular particles in plasma, ii) is ready-to-use, iii) is flow cytometry–compatible, and iv) is stable. MethodsThe PEVTES was prepared from human plasma of 3 fasting donors. EVs were immunofluorescently stained with antibodies against platelet-specific (CD61) and erythrocyte-specific (CD235a) antigens or lactadherin. To reduce the concentration of soluble proteins, lipoproteins, and unbound reagents, stained EVs were isolated from plasma by size-exclusion chromatography. After isolation, the PEVTES was filtered to remove remnant platelets. PEVTESs were diluted in cryopreservation agents, dimethyl sulfoxide, glycerol, or trehalose and stored at −80 °C for 12 months. After thawing, stained EV concentrations were measured with a calibrated FCM (Apogee A60-Micro). ResultsWe demonstrate that the developed PEVTES resembles subcellular particles in human plasma when measured using FCM and that the concentrations of prestained platelet-derived, erythrocyte-derived, and lactadherin+ EVs in the PEVTES are stable during storage at −80 °C for 12 months when stored in trehalose. ConclusionThe PEVTES i) resembles subcellular particles in plasma, ii) is ready-to-use, iii) is flow cytometry–compatible, and iv) is stable. Therefore, the developed PEVTES is an ideal candidate to validate newly developed reference materials and procedures.

Identification of a rare platelet-specific antigen HPA-10bw allele among ethnic Han Chinese population in Shandong

To study the polymorphism of human platelet antigen (HPA) system 10 among ethnic Han Chinese from Shandong, China so as to supplement the data of platelet donor bank in the region. Peripheral blood samples of platelet donors from the region were genotyped for HPA-10 alleles by PCR-sequence specific primer (PCR-SSP) and direct sequencing. Among 1401 donors, a rare heterozygote carrier of HPA-10w (a+b+) was identified, which gave an allelic frequency of approximately 0.035%. The detection of rare HPA-10bw antigen allele among ethnic Han Chinese from Shandong is useful for the diagnosis and prevention of neonatal alloimmune thrombocytopenia and post-transfusion purpura in the region.

Research progress on the immunological platelet transfusion refractoriness in patients with acute leukemia: a narrative review

: Acute leukemia (AL) is a malignant clonal disease of hematopoietic stem cells, many patients have achieved remission, long-term survival, and even cured. Thrombocytopenia is one of the main clinical manifestations of AL. With the development of blood transfusion medicine, platelet transfusion has become an important measure for the treatment of AL. However, platelet transfusion refractoriness (PTR) increases the risk of bleeding in patients and intensifies the demand for platelet transfusion, the occurrence of PTR is still a huge challenge for clinicians. This article reviews the research on the mechanism, prevention and treatment of immunological PTR in AL. PTR includes immune and non-immune factors, immune factors are an important reason for PTR. Some patients are prone to PTR, while others are not. The specific mechanism is currently unclear. Among the immune factors, humoral immunity is currently considered to be the main reason, including HLA antigen incompatibility, antibodies produced by platelet-specific antigens (HPA), and ABO blood group incompatibility, it has been discovered that anti-CD36-mediated alloimmune responses can produce platelet immune abnormalities. There is still a lack of effective methods to completely prevent and treat immunological PTR, we still need to conduct more in-depth research on the mechanism of immunological PTR in AL. Only by further clarifying the mechanism of immunological PTR can be possible to find more effective prevention and treatment methods.

Platelet HPA Typing of Platelet Donors in Zhangjiakou Area

To analyze the polymorphism of the HPA1-5,15 system of the donors in Zhangjiakou area. DNA was extracted from the blood samples of the donors, PCR- SSP method was used to divide HPA1-6, 15 genotype. The gene frequency and genotype frequency were calculated, compared with the difference and regiahal specificity of the populations in our country and foregiens was compared other populations. The gene expression in the HPA-1, HPA-2 and HPA-4 systems were all homozygous aa, and the donors who expressed homozygous bb was not exessed. Among them, one heterozygous ab expression was found in both HPA-1 and HPA-4 systems (1%), and 14 cases of heterozygous ab expression were found in HPA-2 system (14%). The gene expression in the HPA-5 system was mainly homozygous aa (98%), and a very few expressed homozygous bb (2%) was found. The degree of heterozygosity of gene expression in the HPA-3 and HPA-15 systems was relatively high. The proprotion of the expression of aa, ab and bb in the HPA-3 system was respectively 46%, 40% and 14%, the proprotion of the expression of aa, ab and bb in the HPA-15 system was respectively 21%, 64% and 15%. The gene frequency of platelet-specific antigen HPA1-5,15 system in zhangjiakou region shows local characteristics. The heterozygosity degree of gene expression in the HPA-3 and HPA-15 systems are both high, suggesting that they are more likely to result in alloimmunization and ineffective platelet transfusion, which should be pays attention to.

Analysis of Antigenic Determinants on Human Monocytes and Macrophages

Mo1, 2, 3, and 4, and Plt-1 are a series of five distinct antigens detected on the surface of human peripheral blood monocytes by mouse monoclonal antibodies. Mo2 and 3 are restricted to the monocyte-macrophage series, while Mo1, as previously reported, is also expressed by human granulocytes and null cells. Mo3, as distinguished from Mo1 and Mo2, is weakly expressed by virgin peripheral blood monocytes but becomes well expressed if monocytes are cultured overnight at 37 degrees C. Mo4 is coexpressed by monocytes and platelets, while Plt-1 appears to be a platelet-specific antigen whose detection on monocytes reflects adherence of platelets to monocyte membranes. That Mo2-4 are true monocyte antigens is demonstrated by their resynthesis following protease treatment of monocytes (Mol expression is resistant to proteolytic digestion). During myeloid-monocyte differentiation, the Mo antigens are infrequently expressed by immature myeloid cells but are found at higher frequency on leukemic monocytic forms. Macrophages from cultured peripheral blood monocytes and HL-60 cells exposed to lymphokines or phorbol diester express Mo1-4, but noncirculating peritoneal macrophages lack Mo3. The Mo antigens are differentiation markers whose expression reflects membrane heterogeneity during myeloid-monocyte-macrophage maturation.

Acute megakaryocytic leukemia: the Eastern Cooperative Oncology Group experience: Presented in part at the American Society of Hematology meeting, New Orleans, LA, December 1999.

AbstractAcute megakaryocytic leukemia (AMegL) is a rare subtype of acute myeloid leukemia (AML) evolving from primitive megakaryoblasts. Because of its rarity and the lack of precise diagnostic criteria in the past, few series of adults treated with contemporary therapy have been reported. Twenty among 1649 (1.2%) patients with newly diagnosed AML entered on Eastern Cooperative Oncology Group (ECOG) trials between 1984 and 1997 were found to have AMegL. The median age was 42.5 years (range 18-70). Marrow fibrosis, usually extensive, was present in the bone marrow. Of the 8 patients who had cytogenetic studies performed, abnormalities of chromosome 3 were the most frequent. The most consistent immunophenotypic finding was absence of myeloperoxidase in blast cells from 5 patients. In the most typical 3 cases, the leukemic cells were positive for one to 2 platelet-specific antigens in addition to lacking myeloperoxidase or an antigen consistent with a lymphoid leukemia. Myeloid antigens other than myeloperoxidase and selected T-cell antigens (CD7 and/or CD2) were frequently expressed. Induction therapy included an anthracycline and cytarabine in all cases. Complete remission (CR) was achieved in 10 of 20 patients (50%). Two patients remain alive, one in CR at 160+ months. Resistant disease was the cause of induction failure in all but 3 patients. The median CR duration was 10.6 months (range 1-160+ months). The median survival for all patients was 10.4 months (range 1-160+ months). Although half of the patients achieved CR, the long-term outcome is extremely poor, primarily attributable to resistant disease. New therapeutic strategies are needed.

Non-Alloimmune Mechanisms of Thrombocytopenia and Refractoriness to Platelet Transfusion.

Refractoriness to platelet transfusion is a common clinical problem encountered by the transfusion medicine specialist. It is well recognized that most causes of refractoriness to platelet transfusion are not a consequence of alloimmunization to human leukocyte, platelet-specific, or ABO antigens, but are a consequence of platelet sequestration and consumption. This review summarizes the clinical factors that result in platelet refractoriness and highlights recent data describing novel biological mechanisms that contribute to this clinical problem.

Glycophorin A-based exclusion of red blood cells for flow cytometric analysis of platelet glycoprotein expression in citrated whole blood.

Objectives Analysis of platelet glycoprotein (GP) expression by flow cytometry is applied for diagnostic confirmation of GP-associated thrombocytopathies. While platelet-rich plasma may be used for distinct identification of target events, this strategy is not feasible for small sample volumes or for patients showing low platelet counts and/or giant platelets. However, also the use of whole blood (WB) is hampered by the difficulty to discriminate platelets from red blood cells (RBC) in such patients. To circumvent these limitations, we evaluated the feasibility of a RBC gating-out strategy. Methods In addition to platelet GPIb, GPIIa/IIIa, as well as P-selectin (CD62P), citrated whole blood (CWB) samples were stained for RBC-specific glycophorin A (CD235a). CD235a-negative platelet events were further discriminated by forward-/side-scatter characteristics and platelet GP expressions analyzed relative to that of a healthy control sample processed in parallel. Results Established reference intervals allowed for clear identification of decreased GPIIb/IIIa- or GPIb expression pattern in samples of patients with confirmed Glanzmann thrombasthenia or Bernard-Soulier syndrome, respectively. It could be shown that the analysis of 2,500 platelet events is sufficient for reliable GP expression analysis, rendering the proposed method applicable to samples with low platelet counts. Conclusions This study demonstrates the feasibility of CD235a-based exclusion of RBC for platelet GP expression analysis in CWB. In contrast to direct staining of platelet-specific antigens for target identification, this indirect gating out approach is generally applicable independent of any underlying platelet GP expression deficiency.

Acquired Glanzmann thrombasthenia associated with platelet desialylation

BackgroundThe notable discrepancy between platelet count and bleeding manifestations in immune thrombocytopenia (ITP) patients with acquired Glanzmann thrombasthenia (GT) has been described. ObjectivesWe aimed to examine the mechanisms responsible for thrombocytopenia and the bleeding phenotype in a patient with acquired GT. Patient, methods, and resultsA patient with primary ITP underwent splenectomy due to steroid intolerance. Despite platelet count normalization, bleeding continued. Platelet aggregometry was abnormal with all agonists except for ristocetin. Flow cytometry demonstrated the presence of antiplatelet antibody, which caused dose‐dependent inhibition of fibrinogen and PAC‐1 binding, induction of neuraminidase‐1 expression as well as platelet desialylation in donor platelets. Indirect monoclonal antibody immobilization of platelet specific antigen assay (MAIPA) confirmed specificity to αIIbβ3 only, corroborated by binding on Chinese hamster ovary (CHO) cells expressing human glycoprotein αIIbβ3 but not GP Ib/IX. Both desialylation and neuraminidase expression were observed with plasma adsorbed on Ib/IX CHO cells and with the immunoglobulin G (IgG) fraction. Desialylation was inhibited in the presence of anti‐Fc‐gamma receptor IIa (FcγRIIa) antibody. A nonobese diabetic/severe combined immunodeficient ITP murine model was established, which showed rapid hepatic donor platelet clearance in the presence of patient IgG. Treatment of mice with the neuraminidase inhibitor oseltamivir significantly reduced antibody‐induced platelet destruction. ConclusionsWe report the first case of a patient with acquired GT due to ITP with FcγRIIa mediated platelet desialylation, independent of platelet activation. Treatment with neuraminidase inhibitor may prevent platelet clearance by anti‐αIIbβ3 antibodies.

Evaluation and monitoring of response to platelet transfusion therapy: experience from a tertiary care center

ABSTRACT Objective: Refractoriness to platelet transfusion is defined as Corrected Count Increment (CCI) <5000 after two sequential ABO identical transfusions. Immune causes include alloimmunization to HLA and/or platelet-specific antigens. Analysis of various factors leading to platelet refractoriness would help in the management of the patient in a timely manner. Materials and Methods: A prospective observational study on platelet transfusions from November 2013 to June 2015 was conducted. Demographic information, pre, and post-transfusion platelet counts and relevant clinical details were noted. Among patients who were considered refractory to platelet transfusions, platelet factor 3 assay was performed to detect antiplatelet antibodies. Data were analyzed using SPSS software. A P value of <0.05 was considered to be statistically significant. Results: A total of 1190 patients received platelet transfusions during the study period. Among these only 339 (28.5%) patients received transfusions on two or more occasions, of which 237 (69.91%) were considered non-refractory. Among the 102 (30.1%) refractory cases non-immune causes for refractoriness were present in 97 (95.1%) patients and antiplatelet antibody was positive in 18 (17.64%) patients. Bleeding and medication have shown to have significantly contributed to refractoriness (p = 0.025 and 0.003) respectively. Use of medication was independently associated with refractoriness. Conclusion: Non-immune causes are more profound in leading to a poor response to platelet transfusions. Recognizing the cause of poor response and treating the underlying cause would help in getting a better response.

Establishment of Flow Cytometric Immunobead Array Assay for Quantitation of Platelet-Specific Antibodies and Its Application

To establish a flow cytometric immunobead array assay (FCIA) to quantify platelet antibodies and to explore its application in the diagnosis and treatment of ITP. The guantitative standard curve was established by binding the human IgG of known concentration on antibody-coated microbeads; at the same time, the platelet-specific antigen and antibody complex was captured and levels of platelet antibodies were detected using the microbeads coated by 5 kinds of antibodies against platelets suca as GPIX (SZ1), GPⅠb (SZ2), GpⅢa (SZ21), GPⅡb (SZ22) and p-selection (SZ51). The fluorescence signal detected by flow cytometry were transformed into the conentration of platelet antibodies in samples through the quantitative standard curve, thereby establishing the method for quantititive detection of platelet-specific antibodies in plasm samples (FCIA), moreover the property, efficiency and clinical application of establishod FCIA method were evaluated. The FCIA could detect 5 kinds of antibodies against GPIX, GPⅠb, GpⅢa, GPⅡb and β-selection within a broad range of 33.29-1280 ng/ml, 45.17-1280 ng/ml, 42.07-1280 ng/ml, 46.40-1280 ng/ml, 42.48-1280 ng/ml and 42.48-1280 ng/ml respectively, and their recovery rates were 115.23%, 112.58%, 117.47%, 107.64% and 112.67% respectively. The intra-assay coefficient of variation (CV) for anti- GPIX, -GPⅠb, -GpⅢa, -GPⅡb and p-selection antibodies was 3.54%, 3.63%, 4.66%, 6.43% and 6.67% respectively, and the inter-assay CV for above mentioned antibodies were 10.89%, 7.57%, 10.34%, 6.95% and 10.72% respectively. The detection showed that the levels of 5 kinds of platelet-specific antibodies in ITP group all were higher than those in non-ITP and healthy control groups (P＜0.01). The sensitivity, specificity and accuracy of quantitatively detecting 5 kinds of antibodies for diagnosis of ITP by FCIA were 68.29%, 84.98% and 78.95% respectively, while the sensitivity, specificity and accuracy of detecting 5 kinds of antibodies by modified indirect MAIPA were 41.46%, 90.41% and 72.81% respectively. The established quantitative FCIA for detection of antibodies provides a powerful tool for diaghosis and evaluation of therapeutic efficacy and prognosis of ITP patients.

Establishment of Flow Cytometric Immunobead Array Assay for Quantitation of Platelet-Specific Antibodies and Its Application

To establish a flow cytometric immunobead array assay (FCIA) to quantify platelet antibodies and to explore its application in the diagnosis and treatment of ITP. The guantitative standard curve was established by binding the human IgG of known concentration on antibody-coated microbeads; at the same time, the platelet-specific antigen and antibody complex was captured and levels of platelet antibodies were detected using the microbeads coated by 5 kinds of antibodies against platelets suca as GPIX (SZ1), GPⅠb (SZ2), GpⅢa (SZ21), GPⅡb (SZ22) and p-selection (SZ51). The fluorescence signal detected by flow cytometry were transformed into the conentration of platelet antibodies in samples through the quantitative standard curve, thereby establishing the method for quantititive detection of platelet-specific antibodies in plasm samples (FCIA), moreover the property, efficiency and clinical application of establishod FCIA method were evaluated. The FCIA could detect 5 kinds of antibodies against GPIX, GPⅠb, GpⅢa, GPⅡb and β-selection within a broad range of 33.29-1280 ng/ml, 45.17-1280 ng/ml, 42.07-1280 ng/ml, 46.40-1280 ng/ml, 42.48-1280 ng/ml and 42.48-1280 ng/ml respectively, and their recovery rates were 115.23%, 112.58%, 117.47%, 107.64% and 112.67% respectively. The intra-assay coefficient of variation (CV) for anti- GPIX, -GPⅠb, -GpⅢa, -GPⅡb and p-selection antibodies was 3.54%, 3.63%, 4.66%, 6.43% and 6.67% respectively, and the inter-assay CV for above mentioned antibodies were 10.89%, 7.57%, 10.34%, 6.95% and 10.72% respectively. The detection showed that the levels of 5 kinds of platelet-specific antibodies in ITP group all were higher than those in non-ITP and healthy control groups (P＜0.01). The sensitivity, specificity and accuracy of quantitatively detecting 5 kinds of antibodies for diagnosis of ITP by FCIA were 68.29%, 84.98% and 78.95% respectively, while the sensitivity, specificity and accuracy of detecting 5 kinds of antibodies by modified indirect MAIPA were 41.46%, 90.41% and 72.81% respectively. The established quantitative FCIA for detection of antibodies provides a powerful tool for diaghosis and evaluation of therapeutic efficacy and prognosis of ITP patients.

Abnormal Platelet Phenotypes in Patients with Myelodysplastic Syndromes

Background: Hemorrhage and infection are two main causes of death in patients with myelodysplastic syndromes (MDS), mainly due to thrombocytopenia and neutropenia respectively. However, it is becoming increasingly clear that platelet dysfunction can also affect the process of hemostasis and anti-infection. Several studies have evaluated platelet activation in MDS and found platelet dysfunction, but immune-related function of platelets in patients with MDS has not been investigated yet. The aim of this study was to evaluate activation function and immune-related function of platelets in MDS by testing platelet activation-associated phenotypes and immune-associated phenotypes. Methods: We included 29 MDS patients and 22 healthy subjects, and MDS patients were divided into different subgroups (low-risk group and high-risk group; untreated group and treated group; pre-transfusion group and post-transfusion group) according to IPSS-R score, hypomethylating agents (HMAs) therapy and platelet transfusion history to observe phenotype changes under different factors. Platelet light scatter properties, expression of CD41a, expression of activation-associated phenotypes (CD62p and CD63) and expression of immune-associated phenotypes (CD154 and TLR4) were systematically detected by multi-parameter flow cytometry (MFC). Then correlations between phenotypes and clinical indexes were analyzed. Results: Compared to the healthy controls, decreased expression of platelet-specific antigen CD41a was shown in MDS patients (P&lt;0.05). No difference was found in platelet light scatter properties between MDS patients and healthy subjects (P&gt;0.05). Significantly decreased expression frequency and intensity of activation phenotype CD63 were found in patients with MDS (P&lt;0.05). Low-risk MDS showed lower expression frequency while high-risk MDS showed reduced mean fluorescence intensity (MFI) of CD63. Decreased expression of CD154 and TLR4 was found in MDS patients (P&lt;0.05) which was significantly elevated after HMAs therapy (P&lt;0.05). Particularly, high-risk MDS patients again exhibited reduced MFI of CD154 and TLR4 (P&lt;0.05). Correlation analysis showed that the expression of activation-associated phenotypes was not significantly correlated with the severity of bleeding events, but expression of CD62p was negatively correlated with white blood cell count (WBC) (r=-0.496, P=0.006; r=-0.498, P=0.006), absolute neutrophil count (ANC) (r=-0.512, P=-0.005; r=-0.442, P=0.016), fibrinogen (FIB) (r=-0.415, P=0.025; r=-0.510, P=0.005), and D-dimer (DD) (r=-0.547, P=0.002; r=-0.601, P=0.001), and positively correlated with prothrombin time (PT) (r=0.409, P =0.028; r=0.489, P=0.007). Expression of immune-associated phenotypes was not significantly correlated with the severity of infection events (P &gt; 0.05). Conclusion: MDS patients displayed defective expression of both activation- and immune-associated platelet phenotypes, with differential mechanisms between low-risk and high-risk group regarding phenotype alterations. CD62p showed clinical relevance in regard to WBC count and coagulation indexes, and HMAs treatment significantly improved CD154 and TLR4 expression on platelets. Taken together, the findings confirmed impaired platelet phenotypes in MDS which may assist in the diagnosis and identification of MDS patients. Disclosures No relevant conflicts of interest to declare.

Neonatal Intracranial Hemorrhage with a Dramatic Outcome Due to Maternal Anti CD36 Antibodies

Fetal/neonatal allo-immune thrombocytopenia (FNAIT) results from maternal immunization against fetal platelet-specific antigens (HPA) inherited from the father. Most cases involve HPA located on glycoproteins (GP) IbIX, IaIIa and IIbIIIa. Iso-immunizations can also occur in the absence of expression of membrane proteins, such as GPIIb or GPIIIa in Glanzmann patients. CD36 (also called glycoprotein GPIV) deficiency is observed in 3 to 5% of Asian and African populations. We report here the case of a 41-year-old Canadian woman originated from Africa, who delivered a male dead new-born at 39 weeks of gestation. A massive intracranial haemorrhage was identified as being the obvious cause of death. No platelet antibody against GPIbIX, IaIIa, and IIbIIIa was identified by the gold-standard Monoclonal Antibody-specific Immobilization of Platelet Antigens (MAIPA) assay. Surprisingly, anti CD36 iso-antibodies were identified in the maternal serum with a new bead-based multiplex assay. The CD36 gene was sequenced for both parents, and a mutation was identified on Exon 10 of the mother’s CD36 gene, which was absent for the father: NM_000072.3:c.975T&gt;G inducing a STOP codon at position 325 of the mature protein. The absence of CD36 expression on the mother’s platelets was confirmed by flow cytometry.

Atomic-Level Dissection of the Polyclonal Immune Response to the Human Platelet Alloantigen, HPA-1a (PlA1)

Alloantibodies to platelet-specific antigens are responsible for two clinically-important bleeding disorders: Post-transfusion purpura and fetal/neonatal alloimmune thrombocytopenia (FNAIT). The HPA-1a/1b (also known as PlA1/A2) alloantigen system of human platelet membrane glycoprotein (GP)IIIa is controlled by a Leu33Pro polymorphism, and is responsible for ~80% of the cases of FNAIT. Local residues surrounding polymorphic residue 33 are suspected to have a profound effect on alloantibody binding and subsequent downstream effector events. To define the molecular requirements for HPA-1a alloantibody binding, we generated transgenic mice that expressed murine GPIIIa isoforms harboring select humanized residues within the PSI and EGF1 domain, and examined their ability to support the binding of a series of monoclonal and polyclonal HPA-1a-specific antibodies. Humanizing the PSI domain of murine GPIIIa was sufficient to recreate the HPA-1a epitope recognized by some HPA-1a-specific antibodies, however humanizing distinct amino acids within the linearly distant, but conformationally close, EGF1 domain was required to enable binding of others. Using a series of transgenic mice and recombinant constructs, we reveal previously unsuspected, complex heterogeneity of the polyclonal alloimmune response to this clinically important human platelet alloantigen system. High-resolution mapping of this alloimmune response may improve diagnosis of FNAIT by providing cells that distinguish specific alloantibody subpopulations, and should facilitate the rational design and selection of contemplated prophylactic and therapeutic anti-HPA-1a reagents. DisclosuresCurtis:Ionis Pharmaceuticals: Consultancy.

High-resolution mapping of the polyclonal immune response to the human platelet alloantigen HPA-1a (PlA1)

AbstractAntibodies to platelet-specific antigens are responsible for 2 clinically important bleeding disorders: posttransfusion purpura and fetal/neonatal alloimmune thrombocytopenia (FNAIT). The human platelet-specific alloantigen 1a/1b (HPA-1a/1b; also known as PlA1/A2) alloantigen system of human platelet membrane glycoprotein (GP) IIIa is controlled by a Leu33Pro polymorphism and is responsible for ∼80% of the cases of FNAIT. Local residues surrounding polymorphic residue 33 are suspected to have a profound effect on alloantibody binding and subsequent downstream effector events. To define the molecular requirements for HPA-1a alloantibody binding, we generated transgenic mice that expressed murine GPIIIa (muGPIIIa) isoforms harboring select humanized residues within the plexin-semaphorin-integrin (PSI) and epidermal growth factor 1 (EGF1) domains and examined their ability to support the binding of a series of monoclonal and polyclonal HPA-1a–specific antibodies. Humanizing the PSI domain of muGPIIIa was sufficient to recreate the HPA-1a epitope recognized by some HPA-1a–specific antibodies; however, humanizing distinct amino acids within the linearly distant but conformationally close EGF1 domain was required to enable binding of others. These results reveal the previously unsuspected complex heterogeneity of the polyclonal alloimmune response to this clinically important human platelet alloantigen system. High-resolution mapping of this alloimmune response may improve diagnosis of FNAIT and should facilitate the rational design and selection of contemplated prophylactic and therapeutic anti–HPA-1a reagents.

Clinical diagnosis of adult patients with acute megakaryocytic leukemia.

Acute megakaryocytic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML), which is challenging to diagnose due to frequent myelofibrosis (MF) and a low percentage of blast cells. In the present study, clinical characteristics and experimental observations in 9 adult patients diagnosed with AMKL, who were recruited by the Sino-U.S. Shanghai Leukemia Co-operative Group, were analyzed in order to summarize the diagnostic experience and provide recommendations on diagnosing AMKL. All the patients were diagnosed according to the 2008 World Health Organization diagnostic criteria. The mean age of the patients with AMKL was 59 years (range, 53–68 years). A total of 8 patients had different degrees of anemia, and 2 patients had <5% marrow blasts present in the bone marrow; however, the percentage of positive cells with cluster of differentiation (CD)41 and CD61 expression was >20%, as demonstrated by flow cytometry. A total of 6 patients were positive for platelet-specific antigens, as indicated by immunocytochemistry. Furthermore, 7 patients presented with moderate or marked MF, as demonstrated by a bone marrow biopsy. Karyotypic analysis indicated that 6 patients had abnormal karyotypes. Only 1 patient exhibited the Janus kinase 2V617F mutation. Treatment efficiency was notably poor, with a median survival time of 6.0 months (range, 1.1–24.0 months). In conclusion, the diagnosis of AMKL requires a combination of the results of bone marrow smears and bone marrow biopsy, immunophenotype or immunohistochemistry. We recommend that routine immunophenotypic analysis should include the CD41 and CD61 markers for diagnosing acute leukemia when bone marrow morphology does not indicate the diagnosis.

Extensive human platelet specific antigens typing of blood donors of different geographical origin to manage platelet transfusion in alloimmunized patients: Experience from a transfusion center in Northeastern Italy

Aims: As a consequence of global migration flows the risk of HPA alloimmunization may be increased for recipients of platelets from ethnically diverse donors. In this study, the frequencies of HPA in donors of different geographical origin were determined in a Northeastern Italy Transfusion Department. Methods: One hundred and ninety-nine apheresis platelet donors, including Europeans (n = 154) and North Africans (n = 29), were enrolled. DNA was extracted with a commercial kit (Bee-Robotic). Extensive HPA genotyping was performed with BloodChip ID HPA (Progenika-Grifols). Chi-squared test for Hardy-Weinberg equilibrium was used to compare frequencies. Results: The allele a was clearly predominant for HPA 1-11 in both European and North African donors. The allele b was absent for HPA 6-7-8-10-11 in Europeans and for HPA 4-6-7-8-9-10-11 in North Africans. For HPA-15, allele b was more frequent than allele a in North Africans. One case of HPA-4ab and one case of HPA-9abw were detected in Europeans. All HPA genotypes were consistent with Hardy-Weinberg equilibrium. Gene frequencies between both ethnic groups were similar excepting HPA-2 (p = 0.0342). Conclusion: In our population, there should not be clinically relevant increased risk of post-transfusion purpura or platelet transfusion refractoriness secondary to antibodies to HPAs for recipients of platelet concentrates from blood donors of European and North African ethnicity.

Circulating microparticles in pancreatic tumor patients and their roles in mediating leukocyte adhesion in intact microvessels

Pancreatic cancer is one of the nation's deadliest cancers with a dismal 6 percent five‐year survival rate. Pancreatic cancer characteristically creates an abnormal inflammatory or desmoplastic response, which may play a role in its resistance to systemic therapy and aggressive metastasis. Recent studies in circulating microparticles (MPs), the small plasma membrane‐derived vesicles (0.1–1um) released during cell activation, indicated that elevated MPs occur in many cardiovascular diseases, as well as in cancer patients. Since the released MPs carry a range of antigens depending on the type and activation state of their parental cells, they have been considered as biomarker to indicate the disease status and predict the prognosis. The objectives of our study are to investigate the relationship between pancreatic cancer and vascular inflammation and evaluate whether circulating MPs can be a potential biomarker for pancreatic cancer prognosis and treatment outcomes. In this study we characterized the profile of circulating MPs in patients with malignant and non‐malignant pancreatic diseases, investigated the interaction of cultured pancreatic cancer cell‐derived MPs (PACMPs) with blood and endothelial cell‐derived MPs, and explored the roles of PACMPs in mediating vascular inflammation. Flow cytometry was used to quantify plasma MPs, and identify MP cell origins using antibodies directly against cell specific antigens. Compared with healthy controls (n=8), patients with pancreatic cancer (n=11) and pancreatic cyst (nonmalignant, n=7) all had elevated levels of MPs. About 90% of total plasma MPs were from platelets, leukocytes, and endothelial cells. Over 50% of the MPs existed as aggregated units expressing multiple parental cell surface antigens. Electron microscopy and atomic force microscopy delineated the aggregation profiles of plasma MPs. When PACMPs were introduced into cancer patients’ plasma, more than 50% of the plasma MPs bound with PACMPs, and 32% of the plasma MPs that bound with PACMPs express platelet specific antigen. The interaction between PACMPs and vascular endothelial cells were investigated in individually perfused rat venules. After perfusing vessels with PACMPs followed by annexin V staining, confocal images show a significant number of adhered PATMPs in the microvessel walls. After resuming blood flow in PACMP perfused vessels, leukocyte adhesion was significantly increased from 3 ± 1 (control) to 13 ± 3 per 100 um of vessel length. Our results showed that both patients with malignant and non‐malignant pancreatic diseases had elevated circulating MPs, indicating an association between pancreatic diseases and inflammation. The increased plasma MPs, mainly from blood cells and ECs origins, and PACMPS are highly adhesive. They not only interact with each other, but also interact with normal ECs and mediate leukocyte adhesion, which may play important roles in propagation of the inflammation and contribute to pancreatic cancer progression and metastasis.Support or Funding InformationSupported by HL056237, HL084338 and DK097391.

Polymorphism of Platelet Specific Antigens (HPA-1-5, 15) in Han Donors in the North Area of Henan Province

To study the gene polymorphism distribution characteristics of human platelet HPA-1-5 and 15 blood group antigens and construct a certain scale of platelet HPA database in the north area of Henan Province so as to provide platelet apheresis for clinical departments. Using polymerase chain reaction with sequence-specific primers (PCR-SSP), the genotyping of HPA-1-5 and 15 system was carried out; the periperal blood of 500 healthy Han donors in north area of Henan Province was collected randomly, the gene and genotype frequencies were detected by direct counting method, and the population distribution frequncy of HPA genes was analyzed by Hardy-Weinberg balance test, and compared with other regions and ethnics by using χ(2) test. There was statistically significant (P < 0.05) of increase HPA-3b and HPA-5a in North area of Henan Province, compared with Chinese Han population; the HPA-3b and 5a increase and HPA-2a decrease were statistically significant (P < 0.05), compared with Ethnic minority of China. There was partly increase of HPA-1a, 2a, 3a and 5a, compared with different regions and ethnic in abroad. HPA allele genes of 500 Han donors in the North area of Henan Province were as follows: 0.985 and 0.015 for 1a and 1b; 0.924 and 0.076 for 2a and 2b; 0.469 and 0.531 for 3a and 3b; 1.000 and 1.000 for 4a and 5a; 0.532 and 0.468 for 15a and 15b, respectively. HPA allele gene frequencies were 1aa0.970, 1ab0.030; 2aa0.848, 2ab0.152; 3aa0.222, 3ab0.494, 3bb0.284; 4aa1.000; 5aa1.000; 15aa0.282, 15ab0.500, 15bb0.218. Compared with other regions and ethnic, HPA gene frequencies partly had statistical significance. Distribution of HPA allele frequencies in the North area of Henan province is in accordence with the Hardy-Weinberg law. There are race and regional differences in HPA allele gene frequencies, compared with other regions and countries. And the HPA systems HPA-3 and 15 display the genetic polymorphisms, which provides a theoretical basis for the relevant research of the same type platelet infusion and alloimmune thrombocytopenia.