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.

Reemergence of the International Normalized Ratio for the standardization of prothrombin time.

A survey of physicians demonstrated that half had knowledge of the International Normalized Ratio (INR) but none used the value for monitoring their patients because it was not available from the Coagulation Laboratory. The Laboratory then provided the INR value at a physician's request. A six month review of prothrombin time (PT) results showed that only the physicians from the Cardiology Clinic and the Hematology Clinic employed the INR for monitoring their patients. General Medical and Surgical, Vascular, and Orthopedic Clinics continued to use the PT in seconds. This dichotomy allowed the unique opportunity to compare variability of PT in patients followed by INR and those followed by PT in seconds. Inpatients on daily monitoring were used as the standard for close control. During a six month period, laboratory reports from all patients having regular PTs and/or INRs recorded were analyzed for mean level of PT maintained, variability between individual PTs in any given patient, and instances when the PT changed > or = 5 seconds (sec) or increased to > or = 30 sec. Physicians intended to keep the PTs between 16 and 19 sec (INR 2.0 to 3.0). Results showed statistically significantly lower values of PT, less variation in values of PT and a smaller fraction of patients with changes in PT of > or = 5 sec in the group followed by INR. This group was comparable to the inpatient group but significantly different from the outpatient group followed by PT in sec.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Immunological techniques to differentiate lymphoma from other malignancies.

Seven patients, who had lymph nodes or masses examined by both immunoperoxidase staining and flow cytometry, are presented to illustrate the value of each technique including a critical analysis of the current application of these techniques in the pathology laboratory. All seven patients had diagnoses established by immunoperoxidase staining using antibodies directed against: Leukocyte Common Antigen (LCA), Epithelial Membrane Antigen (EMA), Neuron Specific Enolase (NSE), Leu M1, B4 or chromagrafin and synaptosin. Flow cytometry, which could be more rapidly performed, when sufficient cells could be separated from the node or mass, was diagnostic in two of the seven cases. Flow cytometry failed to show abnormalities in Hodgkin's disease or solid tumors, but it was useful in rapid diagnosis of lymphoma, provided that the sample contained mostly involved tissue. Nodes in which there was a minor infiltration with lymphoma cells could only be detected by immunoperoxidase technique.

The role of flow cytometry in the diagnosis of lymphoma: a critical analysis.

Flow cytometry, now used routinely to aid in the classification of leukemias, is increasingly being evaluated as a rapid technique for determination of surface antigens on the cells teased from lymph nodes and other masses with suspected lymphoma. The present study reviews biopsy specimens from patients examined during a two year period which were sent for flow cytometry with a diagnosis of suspected lymphoma. Sixteen of 25 samples (64 percent) produced cell suspensions of sufficient quantity and quality to be diagnostically helpful. Results showed that in 9/16 (56 percent) the diagnosis of lymphoma or cancer could be suspected by flow cytometry alone, while 4/16 were consistent with the final tissue diagnosis of normal or reactive hyperplasia. Three samples that came from patients who had morphologic evidence of malignant disease on biopsy (two Hodgkin's disease and one large cell lymphoma) had flow cytometry results that were interpreted as normal. Flow cytometry is rapid and appears to be virtually diagnostic of non-Hodgkin's lymphoma when a majority of cells are B cells with an abnormal kappa/lambda ratio (> 4.0 or < 0.25). Nonhematologic malignancy can be suspected if less than 75 percent of the cells show CD45 (common leukocyte antigen). Hodgkin's disease cannot be detected by flow cytometry as it is currently used, and as many as 15 percent (1/6 in this study) of lymphomas may show normal results. It is extremely helpful when the biopsy sample actually contains the cells of interest in large proportion. Loss of architectural relationships in the course of processing specimens for flow cytometry is a major disadvantage when small foci of lymphoma or tumor cells exist together with large amounts of stroma or normal lymphocytes.

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.

Evaluation of apheresis platelet concentrates stored for 5 days in PL 732 bags.

To assess the functional viability of platelets collected by standard apheresis techniques using the Fenwal CS-3000 "closed system" and stored in Fenwal PL 732 plastic bags for 5 days at room temperature with agitation, a number of in vitro parameters (pH, morphology, platelet volume distribution, osmotic recovery, aggregation, and platelet-associated IgG) were examined as a function of storage time. During the first 24 hours of storage, minimal changes were observed in the test parameters with the exception of ADP-induced aggregation (75% decrease [10 uM], 84% decrease [5 microM]). Significant differences were observed between fresh (day 0) and 5-day-old platelet concentrates in all parameters except median platelet volume. These observed changes in in vitro test parameters with storage time are similar to those previously observed for comparably stored random single-donor platelet concentrates. Thus, the "closed-system" PL 732 apheresis platelet concentrates would be expected to be as effective in vivo as random single-donor platelet concentrates, while minimizing recipient exposure to transmissible agents of infectious disease.

5-day storage of platelet concentrates in CLX containers: effect of type of agitation.

To determine the degree of platelet damage produced by different modes of agitation during storage of concentrates for 5 days in CLX blood bags, we studied pH, platelet counts, release of LDH and beta thromboglobulin, morphology and osmotic recovery. Platelets were maintained at 20-24 degrees C on elliptical, 6-rpm circular, 2-rpm circular and flat bed agitators. At 72-120 h platelet concentrates stored on the flat bed shaker had significantly lower pH values than units stored on the elliptical or on either of the circular rotators (p less than 0.05). The percent LDH discharged was highest for the units stored on the elliptical rotator (p less than 0.05). Remaining tests of platelet function were not significantly different for concentrates stored on any of the four agitators. Flat bed shakers were unable to resuspend the platelet 'button' which formed after the final preparative centrifugation. Based on our in vitro studies, we conclude that due to problems with low pH values, flat bed shakers may not be optimal for storing platelet concentrates in CLX blood bags and that some other form of agitation should be used.

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.

Evaluation of iron status in women identified by copper sulfate screening as ineligible to donate blood.

Assessment of venous hemoglobin (Hb), serum ferritin, and zinc protoporphyrin (ZPP) levels was carried out in women identified by CuSO4 screening as ineligible to donate blood. The correlation of log ferritin with ZPP was relatively poor (r = -0.580) but significant (p greater than 0.01). However, a ZPP level of 2.0 micrograms per g Hb or greater (upper limit of normal for first-time female donors) showed a predictive value of 0.85 for a ferritin level of 12 ng per ml or less in these donors. The correlation of hemoglobin concentration with ZPP level was significant (r = -0.667; p less than 0.001) in blood donors with ferritin levels at or below 12 ng per ml. ZPP level appears to be increased in iron-depleted (hypoferritinemic) blood donor in whom animals had developed or was developing (Hb less than 12.5 g/dl). Although direct measurements of venous hemoglobin and ferritin levels most accurately evaluate such blood donors, these tests are time-consuming and expensive and are currently not adaptable to bloodmobile operations. Copper sulfate screening has proved feasible in the bloodmobile setting, and the measurement of ZPP level has been used for mobile screening for lead poisoning. ZPP may be an inexpensive and useful screening test to determine a subset of donors who should receive supplemental iron or reduce their frequency of blood donation.

Iron supplementation in female blood donors deferred by copper sulfate screening.

Female blood donors with low hematocrit levels detected by copper sulfate screening were selected randomly to receive either 75 mg of iron per day, as ferrous gluconate, or a calcium phosphate placebo. Their ferritin, serum iron, total iron-binding capacity, zinc protoporphyrin, and hemoglobin values, as well as their suitability to donate blood, were determined initially (Visit 1) and at four follow-up visits (Visits 2-5). By the second visit, the serum ferritin and iron values of donors receiving iron supplementation differed significantly from those of donors receiving placebo. By the fifth visit, a less marked but significant increase in hemoglobin had occurred in the iron group, but not in the placebo group. At no time was there a significant difference between the groups' suitability to donate blood, with each group donating at almost half of their visits. The authors conclude that iron supplementation at this dose level in deferred female blood donors improves their iron status and hemoglobin levels, but does not significantly increase their suitability to donate blood as compared with the suitability of placebo-treated donors.

Effect of mode of agitation on storage of platelet concentrates in PL-732 containers for 5 days.

To determine the degree of damage induced by different modes of agitation during storage of platelets for 5 days in polyolefin (PL-732), we studied pH, platelet count, release of lactic dehydrogenase (LDH) and beta-thromboglobulin (beta-TG), morphology and osmotic recovery. Platelets were maintained at 20-24 degrees C on elliptical, 6 rpm circular, 2 rpm circular and flat bed agitators. Results showed that the most and least effective modes of agitation were the 2 rpm circular and the elliptical rotators, respectively. Elliptical rotators exhibited excessive release of LDH (46%) and beta-TG (51%) while the 2 rpm circular model produced significantly less discharge of these proteins (LDH 13%; beta-TG 30%; p less than 0.05). With elliptical units, by 120 h of storage, pH was often very alkaline (pH greater than 7.5) when platelet counts were under 1 x 10(9)/ml. Flat bed shakers and 6 rpm circular agitators were acceptable but flat bed units were unable to resuspend the platelet 'button' which forms after the final preparative centrifugation. The 2 rpm circular rotator showed significantly less LDH and beta-TG release than did the 6 rpm version (p less than 0.05) and permitted smooth resuspension of the platelet 'button'. Based on our in vitro studies, we conclude that elliptical rotators may not be suitable for storing PL-732 platelet concentrates and that some other form of agitation should be used.

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.

In vitro characteristics and in vivo viability of platelets contained in granulocyte-platelet apheresis concentrate.

A paired prospective study was performed to compare the in vitro storage characteristics and in vivo kinetics of platelets stored in granulocyte-platelet concentrates prepared by apheresis with platelets prepared from whole blood. Platelet and granulocyte-platelet concentrates were collected from five healthy volunteer autologous donors and stored for 16 to 18 hours at 20 to 24 degrees C with and without agitation, respectively. After storage, pH, platelet count, percent release of beta-thromboglobulin, morphologic score, and percent osmotic recovery were measured. In addition, the granulocyte-platelet concentrates were assayed for total leukocyte count, release of lysozyme, and by several in vitro tests of granulocyte function. The platelets in both products were labeled with 111In oxine and infused into the donors. The pH of both products was above 6.0 at the end of storage. The units stored as platelet concentrates compared with those stored as granulocyte-platelet concentrates showed a higher percent release of beta-thromboglobulin, 18.4 +/- 4.0 percent versus 5.9 +/- 3.2 percent (mean +/- SD), but significantly better morphologic scores, 676 +/- 21 versus 525 +/- 56, and better osmotic recovery scores, 72 +/- 10 percent versus 40 +/- 7 percent, respectively (all p less than 0.05). The platelet concentrates (compared with the granulocyte-platelet product) had significantly better in vivo recovery, 49.5 +/- 15.8 percent versus 38.9 +/- 11.5 percent, and survival, 6.1 +/- 1.3 days versus 2.4 +/- 0.4 days, respectively (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

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.