Concanavalin A induces patching/capping of the platelet membrane glycoprotein IIb/IIIa complex.

Two recent reports have shown platelet patching/capping by concanavalin A (Con A). In these studies, Con A receptors were shown to mobilize from pseudopodia and lamellipodia to the central cell parts during platelet attachment and spreading. The molecular mechanism underlying Con A receptor capping was not examined in either study. Con a binds maximally to human platelet membrane glycoproteins IIb and IIIa. In order to test whether Con A-induced capping caused the capping of this membrane glycoprotein complex, we treated normal human platelets with unlabeled Con A. After fixation, platelets were further treated with mouse monoclonal antibodies against the membrane glycoprotein IIb/IIIa complex and stained with fluorescein isothiocyanate (FITC) tagged anti-mouse IgG. An average of 16% platelets manifested capping with one monoclonal antibody preparation (N = 2) and 12% with a second preparation (N = 2). Control studies showed that only 18% of normal human fresh platelets exhibit capping with FITC-Con A (N = 17). If platelets were first incubated with unlabeled Con A, followed by staining with FITC-labeled anti-Con A antibody, an average of 15% platelets manifested caps (N = 17). Capping was inhibited by methyl-alpha-D-mannopyranoside (a known inhibitor of Con A), at cold temperature and by pre-treatment of platelets with colchicine. Our studies confirm the earlier findings on Con A induced capping. Also, our findings suggest that the molecular mechanism for Con A receptor capping involves patching and capping of the platelet membrane glycoprotein IIb/IIIa complex. It is possible that glycoprotein IIb/IIIa redistribution might be intimately involved during platelet attachment and spreading.

Effect of methods of platelet resuspension on stored platelets.

Platelets are prepared from whole blood by differential centrifugation. Following their isolation as a platelet button, platelets are allowed to rest for a short period in the residual plasma before resuspension. In this study, the feasibility of resuspending platelets without this rest period is studied. A total of 35 platelet concentrates (PC) were prepared from blood collected in CPDA-1 and resuspended by one of the following four methods: (1) no resting period, PC placed on a rotator immediately after preparation, (2) a 1.5 hour rest period and gentle shaking prior to rotation, (3) no rest period and immediate gentle shaking prior to rotation, and (4) a 1.5 hour resting phase and no shaking prior to rotation. Following the previous processing, all platelet concentrates were stored for 72 hours on an elliptical platelet rotator at 20 to 24 degrees C to provide continuous agitation. A number of in vitro tests were used as indicators of platelet viability during storage. These included platelet morphology, pO2, pCO2, pH, osmotic recovery, number of platelets in the concentrate, and platelet volume distribution. Our findings demonstrate that platelets are of similar quality after storage in all of the four groups described. Our studies suggest that the resting phase is unnecessary for platelet preparation. The elimination of the resting phase would allow platelet concentrates to be available sooner and improve cost-effectiveness of platelet preparation.

Trends in hepatitis B surface antigen (HBsAg) prevalence in volunteer blood donors in Connecticut, 1973-1983.

A total of 1,573,769 blood donations from volunteer blood donors from Connecticut were tested for Hepatitis B Surface Antigen (HBsAg) from 1973 to 1983. The prevalence of HBsAg decreased over this 10 year period among first time as well as repeat donors. This decrease was much more pronounced in the first three years of the study (1973 to 1975) in repeat donors. Thereafter, prevalence was stable. In first-time donors, the largest decrease also occurred in the first three years. In contrast to repeat donors, however, marked fluctuations in the prevalence were noted in first time donors. As has been the case in previous studies, HBsAg prevalence in first-time donors was significantly higher than that observed in repeat donors. Our study encompasses the longest period of observation in comparison to previous reports on HBsAg prevalence among blood donors. While it has been previously established that a decrease in prevalence over time does occur among repeat donors, stabilization of prevalence after an initial reduction found in our study has not been previously documented. This may indicate an irreducible minimum prevalence rate in the repeat donor population. In addition, our study for the first time convincingly demonstrates a reduction in prevalence in first-time donors which may reflect better communications at the donor recruitment and nursing level.

Nature of platelet antibody in Evans syndrome: a case report.

The association of autoimmune thrombocytopenia and autoimmune hemolytic anemia (Evans Syndrome) has been recognized previously. Recently the present authors investigated a patient with this disorder. Both IgG and C3d were found on the patient's red cells and the serum contained a polyspecific IgG red cell antibody demonstrable only by the antiglobulin test. IgG antiplatelet antibody was demonstrated on circulating platelets as well as in the serum from the patient by using 125I-staphylococcal Protein A (ISPA) assay. Absorption of serum with normal platelets resulted in removal of platelet antibody, but not the red cell antibody. Furthermore, eluate prepared from platelets sensitized with patient serum contained an IgG antibody directed specifically against platelets, since it did not cross react with red cells. Our data suggest the presence of two distinct antibodies, one directed against platelets and the other against red cells in our patient with Evans Syndrome. Both these antibodies are IgG in nature and belong to subclass other than IgG3 since staphylococcal Protein A (SPA) binds to all subclasses of human IgG except IgG3.

Clinical trial of young red blood cells prepared by apheresis.

Transfusion of young red blood cells (YRBC) with prolonged survival should result in increased intervals between transfusions and, therefore, decreased transfusion-associated iron loading. A prospective clinical trial comparing YRBC transfusions prepared by apheresis versus washed or frozen red cell transfusions was performed in five children with transfusion-dependent thalassemia. A total of 152 YRBC units, evaluated by reticulocyte enrichment and pyruvate kinase activity, were transfused. While a slightly longer interval between transfusions was observed during the time period of YRBC versus the time period after (30.0 +/- 1.5 days versus 27.9 +/- 1.1 days, respectively, p less than 0.02), there was no associated decrease in mg of iron transfused per kg. The effectiveness of transfused YRBC units was less than predicted by in vitro and in vivo studies.

HLA alloimmunization with leukocyte concentrates from HLA-matched and HLA-non-matched donors in patients with Hunter's syndrome.

The incidence and characteristics of HLA alloimmunization following transfusions of leukocyte concentrates as a source of enzyme replacement were determined in male patients with Hunter's syndrome. Five patients were given leukocyte concentrates from HLA matched donors (Group I) and another five patients received leukocyte concentrates from non-HLA matched donors (Group II). No other blood products were transfused in either group. Immune response pattern of HLA alloimmunization measured as the proportion of screening cells manifesting cytotoxicity with patient's sera obtained during the follow-up period was similar in both groups. HLA alloimmunization is seen with transfused leukocyte concentrates from either HLA-matched or non-HLA-matched donors in patients with Hunter's syndrome. There appears to be a trend for an earlier onset of HLA alloimmunization with fewer transfusions when leukocyte concentrates from non-HLA-matched donors are transfused as compared to leukocyte concentrates from HLA-matched donors. Once HLA alloimmunization occurs, immune response patterns appear similar with either leukocyte product.

Absence of hemolytic disease of fetus and newborn despite maternal high-titer IgG anti-Ku.

Anti-Ku seen in K(o) (Kell-null) individuals has previously been shown to cause severe hemolytic transfusion reactions. Maternal anti-Ku can cause none or moderate to severe hemolytic disease of the fetus and newborn (HDFN). In two of four previously described HDFN cases, intrauterine transfusions were required because of severe anemia. We report a case in which maternal anti-Ku did not cause HDFN. Standard serologic methods were used for RBC antibody screening and identification, adsorption and elution of RBC antibodies, and antigen typing. A gravida 3, para 3 (G3P3) woman was first evaluated in 2006 and was found to have an IgG RBC antibody that reacted against all panel RBCs in the anti-human globulin phase. A panel of RBCs treated with DTT did not react with the antibody. The antibody failed to react with one example of K(o) RBCs. The patient's RBCs typed negative for the following Kell blood group antigens: KEL1, KEL2, KEL3, KEL4, KEL6, KEL7, KEL11, KEL13, and KEL18. These results established the presence of anti-Ku in maternal serum. The newborn was group A, D+ and required phototherapy for hyperbilirubinemia, but did not require transfusion. The woman was seen again in January 2010 during the third trimester (G4P3). At this time, anti-Ku titer was 256. She delivered a healthy group O, D+ baby boy at 37 weeks' gestation. Cord RBCs were 4+ for IgG by DAT. An eluate reacted with all RBCs tested, but did not react when tested against a panel of DTT-treated RBCs. K(o) phenotype is rare to begin with, and the maternal anti-Ku formation may require more than one pregnancy. Therefore, cases that can be evaluated for anti-Ku­related HDFN are rare. Our case contributes to serologic and clinical aspects of such rare cases.

Platelet preservation in large containers.

The influence of the size of the container on platelet concentrate storage at 20-24 degrees C was examined. Baseline studies were obtained with 50 platelet concentrates in 300-ml PL-146 plastic containers. Experimental studies included 6 platelet concentrates each in 300-, 600- and 2,000-ml containers made of PL-146 plastic. Platelet count, total platelets, pO2, pCO2, pH, glucose consumption, lactate production, platelet morphology and recovery by platelets to osmotic stress were monitored during storage. During storage, the best pH and morphology values were observed with 2,000-ml containers. The lowest glucose consumption and lactate production were also associated with the 2,000-ml containers. Intermediate improvement in these parameters was noted in 600-ml containers. Recovery from osmotic stress was better in 2,000- and 600-ml containers as compared to 300-ml containers. In addition, characteristic changes in pO2 and pCO2 during storage suggest that improved platelet preservation in larger containers is due to improved gas exchange conditions obtained with increased surface area available for gas exchange.

Regional programs for surgical bone banking.

The findings of a survey of 12 regional programs engaged in collection, storage, and distribution of surgical bone grafts are described in this report. In approximately one year, 1944 grafts (mostly femoral heads) were collected. The overall discard rate for the grafts was 30%. An unacceptable medical history, and laboratory evidence of positive screening tests for hepatitis B surface antigen and human immunodeficiency virus (HIV) antibodies accounted for 34.0%, 3.4%, and 1.2% of the discard rate, respectively. Eighty-seven percent of the grafts were used for three surgical procedures, i.e., revision hip surgery, spinal fusions, and treatment of nonunited fractures. The practices and experiences of the regional program described in this report of surgical bone collection and transplantation appear to be similar to those previously described for the community-hospital-based institutional programs. Regional programs represent an alternative approach to institutional programs in surgical bone banking.

Labile coagulation factors in thawed fresh frozen plasma prepared by two methods.

Two preparations of fresh frozen plasma were studied. The plasma from CPDA-1 whole blood units was either frozen within 6 h (FFP-I) or 18-20 h (FFP-II) of phlebotomy. Average activity for factor VIII for FFP-I (n = 6) and FFP-II (n = 19) was 102% and 55%, respectively (p less than 0.01). Average activity for factor V and VII in both products was approximately 100%. Factor XI activity (n = 13) in FFP-II averaged 84%. We conclude that FFP-I and FFP-II are equivalent in terms of coagulation factor activity except for factor VIII.

Removal by centrifugation of leukocytes and red cells from apheresis platelets.

Platelets prepared by discontinuous flow centrifugation contain significant numbers of leukocytes and red cells. Platelet products often are centrifuged further to remove this cellular "contamination." A method is described by which platelet products obtained by cytapheresis were prepared using an additional slow centrifugation step (121 X g for 7.5 minutes). In a production environment, we removed an average of 97 percent of leukocytes and most of the contaminating red cells with this method, while retaining 89 percent of the platelets in the final product. This method is an improvement over methods described previously and was consistently effective in routine use.

Simulation analysis of platelet production and inventory management.

To maximize the availability of platelet concentrates (PC) and minimize their outdate, competing criteria, a computer simulation model of platelet production and distribution was developed. Based on 2 years of actual platelet orders placed with a regional blood center, the simulation program generated daily platelet orders, and also calculated mean demand and standard deviation of demand for each day of the week. The number of PC to be produced on a given day was calculated from: PC to be produced = (mean demand for that day of the week) + Tx (standard deviation of demand for that day of the week)-(PC in inventory on the given day), where T is a selected multiple of the standard deviation. As T increases, availability is maximized, but outdating is expected to increase. Conversely, lower T is associated with less availability, but lower outdate. At the simulated platelet demand level (about 735 PC per week), with 3-day platelet storage life, 99% availability is associated with 1% outdate and distribution of PC of less than 1 day average age. 100% availability, with no outdate is predicted for a 5-day storage life, with no further improvement in logistics with 7-day storage life, at this level of demand. Although logistics of platelet production and distribution vary from center to center, the simulation analysis is generally applicable, and a formal plan has the great advantage of predictive, rather than reactive, platelet production.

Preparation of young red cells for transfusion using the Fenwal CS 3000 cell separator.

A pheresis procedure was devised to isolate young red cells by centrifugation using the Fenwal CS 3000 continuous flow cell separator. Young red cell enriched products were collected in a 2.5-3-hour procedure. Large numbers of white cells and platelets were collected with the red cells, but cryopreservation and subsequent washing removed 99% of the contaminating cells. At the completion of all processing a product yielding 70% of the total hemoglobin content of a standard frozen/deglycerolized red cell unit was produced. Autologous radiochromium survival of young red cells, measured in 12 normal donors, showed an average 24-hour recovery of 89.9% with a T50Cr of 40.8 days. In paired autologous studies (N = 4) there was a mean increase of 35% in the observed T50Cr of young red cells as compared to standard frozen red cells.

Rifampin-induced immune thrombocytopenia. A case report.

In this report, a case of rifampin-induced immune thrombocytopenia with the following characteristics is described: (a) thrombocytopenia follows intermittent drug administration; (b) onset occurs within hours of drug ingestion; (c) IgG antirifampin antibody binds in vitro to normal platelets only in the presence of rifampin; (d) thrombocytopenia resolves quickly in the absence of rifampin; (e) using immunofluorescence microscopy, IgG binding to normal platelets was seen with the patient's serum only in the presence of rifampin, and (f) using fluorescence spectrofluorometry, an absence of rifampin binding to normal platelets was demonstrated. Although the serological studies are not definitive, the mechanism of thrombocytopenia in the patient can best be explained by the formation of immune complexes composed of rifampin-antirifampin antibody binding to platelets causing their rapid clearance from the circulation.

Problems with a walking donor transfusion program.

A walking donor transfusion program is outlined in detail. A total of 205 transfusions from 72 walking donors were given to 57 newborns in a Newborn Intensive Care Unit over a nine-month period. The average recipient weighed 1,762 g and the average transfusion was 15 ml of blood. Because a suitable walking donor was not always available when a transfusion was needed, 19 units of regular adult blood were also used to support the program. No immediate or delayed transfusion reactions were noted, but one fatal incident of serum hepatitis transmission occurred. Our experience suggests that a walking donor program carries an inherent significant risk of transmission of hepatitis and alternative methods with strict blood bank control are needed to assure maximum safety in neonatal transfusion.

Pedi-pack transfusion in a newborn intensive care unit.

Two hundred and ninety-one transfusions using 221 pediatric frozen red blood cell packs (Pedi-Packs) were given to 141 newborn babies and infants in the newborn intensive care unit. In 18 patients, 47 transfusions were studied for transfusion and clinical characteristics. Two possible hemolytic episodes are described in detail and remain unexplained. Blood loss for laboratory tests was found to average 3.1 ml/kg per day spent in the newborn intensive care unit. The rise in hematocrit was found to be excellent. Overall, the transfusion of thawed pediatric red blood cell packs was found to be convenient, safe and effective. Because of pretesting possibilities with the use of this source of red blood cells, one of the problems associated with a walking donor program is eliminated.

Platelet viability following storage for 5 days. Influence of holding whole blood for 8 hours at 20 to 24 degrees C before concentrate preparation.

Regional blood centers frequently need to hold units of whole blood at 20 to 24 degrees C for several hours after phlebotomy so that sufficient platelet concentrates can be prepared to meet the increasing need. We have evaluated the in vivo viability and in vitro properties of platelets that were prepared from whole blood drawn into citrate-phosphate-dextrose-adenine (CPDA-1) either immediately after phlebotomy or after an 8-hour hold at 20 to 24 degrees C. Platelet concentrates were stored for 5 days at 20 to 24 degrees C in polyolefin containers (PL 732, Fenwal) with end-over-end tumbler agitation. The autologous in vivo recovery (mean +/- SD) and one-half disappearance of 51Cr-labeled platelets prepared immediately after phlebotomy were 44.4 +/- 9.4 percent and 4.0 +/- 0.5 days, respectively. Platelets prepared after the delay of 8 hours showed a recovery of 44.5 +/- 8.4 percent and a one-half disappearance of 4.1 +/- 0.4 days. After 5 days of storage, platelet concentrates showed a mean pH of 7.21 +/- 0.20 when prepared immediately after phlebotomy, and of 7.22 +/- 0.15 when prepared after an 8-hour delay. Mean morphology scores were 280 +/- 33 and 302 +/- 27 for platelets from units prepared immediately after phlebotomy or after a holding period of 8 hours, respectively. Platelets underwent synergistic aggregation after 5 days of storage, independent of the length of time that the units of whole blood were held prior to centrifugation. These studies indicate that platelet concentrates prepared from units of whole blood held initially for 8 hours can be stored for 5 days at 20 to 24 degrees C and survive satisfactorily in vivo and retain in vitro characteristics.

Four crossmatch methods to select platelet donors.

We employed four crossmatch techniques to select platelet donors for refractory patients. Forty-four donor-recipient pairs were studied in 32 patients. Analysis of effectiveness of platelet transfusions revealed that only 18 percent of transfusions gave a borderline response; the remainder were either effective or not effective at all. The corrected predictive values of three crossmatch tests were as follows: enzyme-linked immuno-specific assay, 81 percent; platelet immunofluorescence test, 73 percent; and lymphocytotoxicity, 70 percent (p greater than 0.05). The predictive value of these tests did not differ in HLA-matched versus unmatched platelet transfusions. Donor selection by lymphocytotoxicity compatibility did not appear to be useful if donors were selected by either of the other two methods. The fourth test, antiglobulin-modified lymphocytotoxicity, offered no advantage over lymphocytotoxicity. Our data suggest that platelet crossmatching assays are a useful adjunct to the selection process for the platelet donor in addition to ABO, Rh, and HLA matching.