Antiplatelet antibodies in cases of Glanzmann's thrombasthenia with and without a history of multiple platelet transfusion

Antiplatelet antibodies are known to be present in a wide spectrum of patients, which include chronic Idiopathic Thrombocytopenic Purpura (ITP), infections, etc., including Glanzmann''s thrombasthenia (GT) patients who receive multiple platelet transfusions. The presence of natural antibodies to platelet receptors is not studied in cases of GT. We studied the antiplatelet antibodies in 23 patients with GT, 15 of which had received multiple transfusions and eight that had not received transfusions, along with 50 cases of chronic ITP. The prevalence and specificity of platelet-bound antibodies were detected by inhibition assays using O-group platelets on flow cytometry. The mean antiplatelet antibodies in 15 patients of GT who had not received transfusions and eight patients with multiple transfusions was 8427 + 2131.88 and 9038 + 2856 antibodies/platelet, respectively, while in case of the 50 ITP patients studied, it was 22166 + 5616 antibodies/platelet (Normal Range 1500–3200 antibodies/platelet). We conclude that GT patients who have not received transfusions may develop antiplatelet antibodies to the missing/abnormal receptor. Whether this is due to a molecular mimicry or due to some other mechanism needs to be explored.     

Megakaryocytopoiesis in the mouse: Response to varying platelet demand

The response of murine megakaryocytopoiesis was studied under conditions of varying platelet demand. Twenty-four hours after mice were given a single injection of rabbit anti-platelet serum, megakaryocyte number and volume were increased, becoming maximal at 65 and 40 hr, respectively. Total body megakaryocytic colony-forming unit (CFU-M) numbers did not change until 90 hr, when a 35% increase in the experimental group was noted. The percentage of CFU-M in DNA synthesis in the experimental group was 38 ± 2% at 24 hr, 49 ± 1% at 40 hr, and returned to normal (11 ± 3%) at 90 hr. When mice were made thrombocytotic by platelet transfusions, both megakaryocyte number and volume were decreased compared to controls, while no difference was noted in the number and percentage of CFU-M in DNA synthesis. Finally, experiments were performed to examine the effect of platelet transfusions on regenerating marrow. Experimental mice were given platelet transfusions while control animals received platelet buffer solution. At sacrifice the number and volume of megakaryocytes in the experimental group (platelet count 2.568 × 10⁶/μl) were less than controls (platelet count 0.363 × 10⁶/μl), while the number and percentage of CFU-M in DNA synthesis were similar in both groups. These results demonstrate that CFU-M are not immediately responsive to acute changes in platelet demand. The data suggest that megakaryo-cytopoiesis is structured on at least two levels which are independently regulated.     

Use of platelet concentrate in eastern Ontario.

To better understand the reasons for the increasing use of platelet concentrate in Canada, we undertook a 4-month study of platelet concentrate transfusion in six eastern Ontario hospitals in 1985. A total of 4801 units of platelet concentrate were transfused on 687 occasions to 303 patients; the average number of transfusions per patient was 2.3, the average number of units per transfusion 7.0 and the average number of units per patient 15.8. The cardiovascular service used the largest proportion of units (28%), aortocoronary bypass grafting being the most common procedure. The mean pretransfusion platelet count for the medical and oncology services was about 30.0 X 10(9)/L, compared with 155.5 X 10(9)/L for the cardiovascular service. An increment in platelet count 1 hour after transfusion was noted with 238 (75%) of the transfusions for which the data were available; the average increment was 3.4 X 10(9)/L per unit of platelet concentrate transfused. When the data for patients who did not respond were excluded, the average increment was 6.9 X 10(9)/L. Single-donor platelet concentrate was requested for only half of the transfusions to which no response was detected. The current medical literature supports the appropriate use of platelet concentrate in patients with thrombocytopenia due to chemotherapy, but prophylactic platelet transfusion for patients undergoing cardiovascular bypass procedures is being questioned. We advise continued surveillance of the use of these products and re-evaluation of the aims of platelet transfusion therapy.     

Preparing platelet concentrates from banked blood stored for 1-5 days by using tetracycline antibiotics

It was shown that storage of banked blood up to five days did not change number of platelets and their functional integrity was retained rather well. That made possible the development of a method for preparation of viable platelet concentrates (PC) from stored blood. This method is based on the reversibility of the tetracycline antibiotics inhibitory effect on platelet activation process. According to the results of our in vitro studies PC from stored blood seem to be suitable for clinical usage. This tetracycline method of PC preparation from stored blood may provide a more efficient utilization of available donor blood to meet the ever-expanding needs for PC transfusions.     

Autologous cryopreserved platelets and prophylaxis of bleeding in autologous bone marrow transplantation

Autologous platelets were harvested and cryopreserved in eight consecutive patients elected for ablative chemotherapy and autologous bone marrow transplantation (ABMT) for solid malignancy. There was a 19% loss in platelet count after the freeze thaw and wash procedure; with an in vitro functional loss of 40-60%. No correlation could be found for individual platelet transfusions between in vitro functional tests and in vivo recovery. Six consecutive patients received a total of 16 autologous platelet transfusions in the aplastic phase of ABMT. No bleeding was observed during the study period and there was no CMV infection in the recipients. While improvement in freezing and subsequent handling is desirable, autologous cryopreserved platelets can safely be used for the prophylaxis of bleeding during aplasia in patients treated with ABMT.     

Transfusion strategy for patients in therapeutic aplasia. Experience of the Henri Mondor Hospital apropos of 2 successive protocols

Retrospective analysis of two transfusion protocols applied in our institution to the bone marrow transplanted patients was conducted. Granulocyte transfusions should be only proposed as a therapeutic treatment to patients with severe well documented bacterial infection resistant to an adapted antibiotherapy. Leukocyte-depleted blood products reduce the incidence of HLA-immunization but do not influence the frequency of CMV infections. Random single donor platelet concentrates (obtained by cytapheresis) could decrease the incidence of polyspecific HLA-antibodies in comparison with the use of random standard platelet concentrates. The best transfusion protocol should associate leukocyte-depleted blood products with transfusion of prophylactic single donor platelet concentrates. In our institution, this protocol is less expensive than the protocol with prophylactic white blood cell transfusions and has the same cost than other protocols using standard blood products.     

Effect of six weekly transfusions on canine marrow grafts: tests for sensitization and abrogation of sensitization by procarbazine and antithymocyte serum.

We have previously shown that blood transfusions can immunize a dog and lead to rejection of a subsequent marrow graft despite lethal total body irradiation (TBI). Sensitization to histocompatibility antigens induced by two prior transfusions of whole blood could be overcome by a regimen of procarbazine and anti-thymocyte serum (ATS) preceding TBI. The current study investigated a) whether this regimen could abrogate sensitization induced by six weekly transfusions given from days --50 to --15 preceding a marrow graft, and b) whether platelet survival studies and two in vitro tests of immunity could predict marrow graft rejection. All donor-recipient pairs were histoincompatible, unrelated, and of different breed. Twenty-two recipients received platelet concentrate transfusions and eight received whole blood transfusions. Recipients were given 1200 R TBI and a graft of marrow and peripheral blood leukocytes from the transfusion donor on day 0. Three of 15 recipients (20%) given procarbazine, 12.5 mg/kg i.v. on days --8, --6, and --4, and ATS, 0.6 ml/kg subcutaneously on days --7, --5 and --3, Rejected their grafts, whereas 11 of 15 dogs (73%) not given procarbazine and ATS rejected their grafts (p less than 0.01). Serum lymphocytotoxic antibodies, peripheral leukocyte migration inhibition, and in vivo donor platelet recovery and survival were studied in those recipients receiving six weekly transfusions and in 18 other recipients receiving a single donor transfusion 3 months before marrow grafting. No significant correlation was found among these in vitro and in vivo tests of sensitization. Sensitization to marrow grafts was not reliably detected by the presence of cytotoxic antibodies or leukocyte migration inhibition. Platelet survival, however, was positively correlated with the results of marrow grafting in 12 of 15 (80%) evaluable recipients (p approximately 0.15).     

Intrauterine transfusion with red cells and platelets.

Having direct access to the fetoplacental circulation by ultrasound-directed needle puncture has led to therapeutic interventions for fetal anemia and thrombocytopenia. Most cases of red cell alloimmunization associated with fetal anemia are caused by the antibody to the D red cell antigen. The intravascular transfusion of red cells to a hydropic fetus in such cases has notably improved survival. Nonimmune hydrops fetalis due to maternal parvovirus infection has also been treated successfully with the intravascular transfusion of red cells, whereas fetomaternal hemorrhage has not proved amenable to such therapy. Sensitization to the PLA-1 platelet antigen is the most common cause of fetal thrombocytopenia in maternal platelet alloimmunization. Fetal platelet transfusions have not proved to be a practical therapeutic modality for this disorder owing to the short half-life of the platelets. Platelets transfusions to the fetus just before delivery may avert the need for cesarean section in cases of severe thrombocytopenia.     

Supportive haemotherapy in bone marrow transplantation

The intensive supportive haemotherapy which has to be used in bone marrow transplantation is discussed, taking mainly into account platelet transfusions. Ways to avoid alloimmunizations against platelet antigens, especially HLA-ABC antigens, are shown (use of HLA-AB homozygous donors or of cross-reacting groups).     

Contribution of perfusion techniques to the evaluation of the hemostatic effectiveness of platelet concentrates.

Perfusion systems allowing the morphometric analysis of platelet interactions with vessel subendothelium under flow conditions have been applied to evaluate the quality and function of stored platelets. Studies performed in vitro indicate that despite the existence of storage lesions, platelets in concentrates stored for up to 5 days retain their ability to interact with the subendothelium. Perfusion studies ex vivo with nonanticoagulated blood from anemic-thrombocytopenic patients have shown the critical hemorrheological role of red blood cells facilitating platelet interactions with subendothelium. Similar studies performed on severely thrombocytopenic patients who received transfusions of platelets stored at 4 degrees C indicate that incompletely viable platelets can contribute to primary hemostasis through procoagulant mechanisms. The latter results suggest that storage lesions which contribute to impairment of platelet function may result in enhancement of platelet procoagulant activities. Perfusion techniques have contributed to the evaluation of the hemostatic effectiveness of platelet concentrates. These techniques will provide a useful model to test the impact of new storage technologies on platelet hemostatic function.     

Prophylactic platelets in dengue: survey responses highlight lack of an evidence base

Dengue is the most important arboviral infection of humans. Thrombocytopenia is frequently observed in the course of infection and haemorrhage may occur in severe disease. The degree of thrombocytopenia correlates with the severity of infection, and may contribute to the risk of haemorrhage. As a result of this prophylactic platelet transfusions are sometimes advocated for the prevention of haemorrhage. There is currently no evidence to support this practice, and platelet transfusions are costly and sometimes harmful. We conducted a global survey to assess the different approaches to the use of platelets in dengue. Respondents were all physicians involved with the treatment of patients with dengue. Respondents were asked that their answers reflected what they would do if they were the treating physician. We received responses from 306 physicians from 20 different countries. The heterogeneity of the responses highlights the variation in clinical practice and lack of an evidence base in this area and underscores the importance of prospective clinical trials to address this key question in the clinical management of patients with dengue.     

Steady Increment of Immature Platelet Fraction Is Suppressed by Irradiation in Single-Donor Platelet Components during Storage

Circulating immature platelet fraction (IPF) reflects real-time thrombopoiesis and correlates with platelet recovery from thrombocytopenic presentations. To understand the dynamics of IPF in platelet transfusions, we quantified the %-IPF in single-donor platelet components (SDP) during prolonged storage. %-IPF significantly increased from baseline by day 5 post-donation. Absolute IPF counts (A-IPC) had similar significant increments. However, gamma-irradiation suppressed the increments of %-IPF and A-IPC by >50%. Ultrastructural analysis of SDP units at day 10 showed well preserved morphology of immature platelets. Our findings suggest that IPF might actively expand ex-vivo and may have a longer shelf life than their mature counterparts. Closer study of IPF may be of critical clinical importance for transfusion practices.     

Severe thrombocytopenia in Epstein-Barr virus-induced mononucleosis.

Severe thrombocytopenia is a rare complication of Epstein-Barr virus-induced infectious mononucleosis. We evaluated the clinical and laboratory data from seven patients seen between 1976 and 1985 whose lowest platelet counts varied from 3 to 25 x 10(9) per liter. Five of the seven patients were initially thought to have either acute leukemia or idiopathic thrombocytopenic purpura; eventually, however, primary Epstein-Barr virus infections were confirmed in all patients. Two of six patients tested had antiplatelet antibodies during the acute phase of their illnesses. Eight additional patients with acute disease who had only mild thrombocytopenia (94 to 144 x 10(9) per liter) were also tested for platelet antibodies with negative results. Steroid therapy was administered to three patients and platelet transfusions to one. All seven patients recovered with no serious hemorrhagic sequelae.     

A uniform‐shear rate microfluidic bioreactor for real‐time study of proplatelet formation and rapidly‐released platelets

Platelet transfusions, with profound clinical importance in blood clotting and wound healing, are entirely derived from human volunteer donors. Hospitals rely on a steady supply of donations, but these methods are limited by a 5‐day shelf life, the potential risk of contamination, and differences in donor/recipient histocompatibility. These challenges invite the opportunity to generate platelets ex vivo. Although much progress has been made in generating large numbers of culture‐derived megakaryocytes (Mks, the precursor cells to platelets), stimulating a high percentage of Mks to undergo platelet release remains a major challenge. Recent studies have demonstrated the utility of shear forces to enhance platelet release from cultured Mks. In this study, we performed a computational fluid dynamics (CFD) analysis of several published platelet microbioreactor systems, and used the results to develop a new 7‐µm slit bioreactor—with well‐defined flow patterns and uniform shear profiles. This uniform‐shear‐rate bioreactor (USRB‐7µm) permits real‐time visualization of the proplatelet (proPLT) formation process and the rapid‐release of individual platelet‐like‐particles (PLPs), which has been observed in vivo, but not previously reported for platelet bioreactors. We showed that modulating shear forces and flow patterns had an immediate and significant impact on PLP generation. Surprisingly, using a single flow instead of dual flows led to an unexpected six‐fold increase in PLP production. By identifying particularly effective operating conditions within a physiologically relevant environment, this USRB‐7µm will be a useful tool for the study and analysis of proPLT/PLP formation that will further understanding of how to increase ex vivo platelet release. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1614–1629, 2017     

Influence of storage on signal transduction pathways and platelet function.

The use of platelet concentrates in prophylactic and therapeutic transfusions has increased considerably. The design of suitable storage bags and improvements in procurement, processing, and storage practices have contributed significantly to the quality of stored platelets and their increased shelf life. There continues to be activation of platelets during procurement of blood, shipment, processing, and storage. By using appropriate preventive measures, inhibitors of platelet activation and protectants, platelet activation can be minimized. Although platelets seem to recover from initial activation and function normally, their in vitro response varies depending on the degree of activation occurring during preparative procedures. By and large, response to weak agonists diminishes rapidly during aging in storage. Further studies are essential to determine the reason for the development of this acquired defect. The normal response of stored platelets to a potent agonist such as thrombin suggests that signal generation and transduction mechanisms are not significantly compromised during storage. Epinephrine-mediated membrane modulation may contribute significantly to their improved in vivo performance during transfusions.     

Comparison of posttransfusion recoveries achieved with either fresh or stored platelet concentrates

The effectiveness of platelet concentrate transfusion depends on such variables as blood bag material, donor--recipient compatibility, and time elapsed between donation and transfusion. To study the latter a corrected thrombocyte increment for recovery in the recipients was evaluated with 108 platelet transfusions in 31 patients. In 83 treatment programs, the mean recovery at the one-hour post-transfusion time point was 8.6 X 10(9) platelets/l with fresh platelets and 5.9 X 10(9) platelets/l with stored platelets. Significantly better recovery was achieved with freshly prepared platelet over the total of platelet concentrates stored for up to 96 hours; however, if the recoveries in different patient groups given stored platelets were considered separately in terms of storage times of up to 48 h or 48-96 h, the good recovery with fresh platelets was significantly better only when compared to the older (p = 0.034) but not to the younger group of stored platelets. In patients with signs indicating enhanced platelet destruction (fever, splenomegaly, disseminated intravascular coagulation) the transfusion with fresh platelet concentrates gave a significantly better recovery compared to stored platelet concentrates (p = 0.028), whereas in the absence of such signs the recovery produced by fresh concentrates was not significantly higher than with stored concentrates. These findings may be relevant for the logistics in blood banking.     

Neonatal alloimmune thrombopenia

88 families in which 84 cases of neonatal alloimmune thrombocytopenia (NAT) occurred, were studied. In 84 families, the NAT was the consequence of an incompatibility in the PLA system. Furthermore, the phenotype HLA-DR3 increases greatly the risk of immunisation (RR: 76,5). The importance of the risk of neurological sequellae was shown by the clinical study (about 25% of the surviving neonates). The occurrence of the accident at the first birth of a PLA1 positive child in a sibship was frequent (59%). In addition, the NAT recurred at each birth of a PLA1 positive child with only five exceptions. All of them concern a female neonate and this might be meaningful. Therapeutical data are heterogeneous and difficult to interpret. However, it appears that the prevention of obstetrical traumatism by caesarean section and compatible platelet transfusions are useful. It is too early to evaluate the efficacy of prenatal transfusions of mother's washed platelets. However, in the two cases in which we use them, they gave a good and sustained platelet count increment. The prenatal diagnosis of NAT and the PLA grouping of the foetus has been proposed in three cases and are feasible at 20 weeks of pregnancy.     

Intravenous immune globulin use in patients with human immunodeficiency virus-related thrombocytopenia who require dental extraction.

Five patients with human immunodeficiency virus (HIV)-related immune thrombocytopenia who were undergoing dental extraction were treated with intravenous immune globulin (IVIG). All patients received IVIG, 1 gram per kg, the day before the dental extraction and again the day of the dental extraction. Four patients had a previous history of minor clinical bleeding. The median baseline platelet count before extraction was 20 X 10(9) per liter (range 13 to 44). The median peak platelet count was 100 X 10(9) per liter (range 56 to 528) following infusion. This peak response was achieved by day 2 in 3 patients and by days 5 and 7 in 1 patient each. No patients had complications or toxicity from the infusions or perioperative bleeding. No patients required blood product transfusions for the surgical procedure. In conclusion, IVIG infusion should be considered in patients with HIV-related immune thrombocytopenia requiring surgical procedures when a prompt rise in platelet count is desired.     

Transformation of human mesenchymal cells and skin fibroblasts into hematopoietic cells

Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs) and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza) and the growth factors (GF) granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy.     

Thrombotic thrombocytopenic purpura treated with plasma exchange or exchange transfusions.

Of 40 patients with thrombotic thrombocytopenic purpura, 17 were treated with plasma exchange, 15 with exchange transfusions, and 6 with both types of therapy. One patient died before being treated and another patient was seen but not treated. Plasma exchange was performed daily for a mean of seven exchanges per patient. The replacement fluid during plasma exchange was fresh frozen plasma in all cases. The complete response rates for each type of treatment were as follows: 88% for plasma exchange (15 patients), 47% for exchange transfusions (7 patients), and 67% for exchange transfusions and plasma exchange (4 patients). Clinical and laboratory factors were examined for any statistically significant association with therapy response. Treatment with plasma exchange was statistically the initial factor most strongly associated with prognosis. Paresis, paresthesias, seizures, mental status change, and coma showed no association with response to treatment. Some of the laboratory factors that did not show significant association with treatment response were the initial creatinine, hemoglobin, platelet count, lactate dehydrogenase, and total bilirubin. This study supports the hypothesis that plasma exchange has significantly improved the prognosis of patients with thrombotic thrombocytopenic purpura. These patients should be treated aggressively regardless of the severity of their symptoms.