Analysis of human platelet glycoproteins IIb-IIIa and Glanzmann's thrombasthenia in whole blood by flow cytometry

Antibodies that bind to human platelet membrane glycoproteins IIb and IIIa were used to develop methods for analyzing platelet membrane components by flow cytometry. Platelets were tentatively identified by their low-intensity light scatter profiles in whole blood or platelet- rich plasma preparations. Identification of this cell population as platelets was verified by using platelet-specific antibodies and fluorescein-conjugated antiimmunoglobulin. Two-parameter analysis of light scatter versus fluorescence intensity identified greater than 98% of the cells in the "platelet" light scatter profile as platelets due to their acquired fluorescence. Both platelet-rich plasma and whole blood were used to study platelet membrane glycoproteins IIb and IIIa on a single cell basis in an unwashed system. Prostacycline was included in these preparations as a precautionary step to inhibit platelet aggregation during analysis. Flow cytometry is a successful technique for rapid detection of platelet membrane defects such as Glanzmann's thrombasthenia. Platelets from Glanzmann's thrombasthenic individuals were readily distinguished from platelets with normal levels of glycoprotein IIb and IIIa and from platelets with glycoprotein levels characteristic of heterozygote carriers of this disorder. This technique provides a sensitive tool for investigating platelet functional defects due to altered expression or deficiency of platelet surface proteins.     

A training model for external cephalic version.

The Royal College of Obstetricians and Gynaecologists (RCOG) has recommended that all women with an uncomplicated pregnancy and a breech presentation at term should be offered external cephalic version (ECV) but only 43% of consultants in England practice ECV. There is a need to train clinicians to perform ECV safely and effectively. A model abdomen is described which fulfils these requirements. The model is adjustable to represent a continuum from an easy procedure to an impossible procedure. The model was used to train and assess two midwives in the technique of ECV. This model is a valuable method of both training a clinician to perform ECV and also providing an objective method of assessing their technique. It has the potential to maximise the success rate of the newly trained clinician prior to performing ECV on women.     

Anogenital HIV RNA in Thai men who have sex with men in Bangkok during acute HIV infection and after randomization to standard vs. intensified antiretroviral regimens

Introduction

HIV transmission risk is highest during acute HIV infection (AHI). We evaluated HIV RNA in the anogenital compartment in men who have sex with men (MSM) during AHI and compared time to undetectable HIV RNA after three-drug versus five-drug antiretroviral therapy (ART) to understand risk for onward HIV transmission.

Methods

MSM with AHI (n=54) had blood, seminal plasma and anal lavage collected for HIV RNA at baseline, days 3 and 7, and weeks 2, 4, 12 and 24. Data were compared between AHI stages: 1 (fourth-generation antigen-antibody combo immunoassay [IA]–, third-generation IA–, n=15), 2 (fourth-generation IA+, third-generation IA–, n=9) and 3 (fourth-generation IA+, third-generation IA+, western blot–/indeterminate, n=30) by randomization to five-drug (tenofovir+emtricitabine+efavirenz+raltegravir+maraviroc, n=18) versus three-drug (tenofovir+emtricitabine+efavirenz, n=18) regimens.

Results

Mean age was 29 years and mean duration since HIV exposure was 15.4 days. Mean baseline HIV RNA was 5.5 in blood, 3.9 in seminal plasma and 2.6 log₁₀ copies/ml in anal lavage (p<0.001). Blood and seminal plasma HIV RNA were higher in AHI Stage 3 compared to Stage 1 (p<0.01). Median time from ART initiation to HIV RNA <50 copies/ml was 60 days in blood, 15 days in seminal plasma and three days in anal lavage. Compared with the three-drug ART, the five-drug ART had a shorter time to HIV RNA <1500 copies/ml in blood (15 vs. 29 days, p=0.005) and <50 copies/ml in seminal plasma (13 vs. 24 days, p=0.048).

Conclusions

Among MSM with AHI, HIV RNA was highest in blood, followed by seminal plasma and anal lavage. ART rapidly reduced HIV RNA in all compartments, with regimen intensified by raltegravir and maraviroc showing faster HIV RNA reductions in blood and seminal plasma.     

Blood donor-, apheresis-, and transfusion-related activities: results of the 1991 American Association of Blood Banks Institutional Membership Questionnaire

Approximately 2154 regional blood centers and hospital-based blood banks and transfusion services responded to the 1991 American Association of Blood Banks Institutional Membership Questionnaire that elicited data from 1990. Information from 2144 institutions was considered valid. Questionnaire topics were donor blood collections, hemapheresis, perioperative cell salvage, component usage, and transfusion-associated diseases. Institutional members reported collecting 9.3 million units, of which 90.9 percent were for allogeneic use in the community, 6.0 percent were for autologous use, and 3.1 percent were directed donations. The percentage of directed-donor units that were crossed over for allogeneic use (51%) was greater than the percentage of units transfused to the designated patient (49%). Only 12.5 percent of institutions reported obtaining specific consent for transfusion. Of the 15.4 million transfused blood components, 8.5 million were red cells, 4.1 million were platelets, 1.8 million were fresh-frozen plasma, and 0.9 million were cryoprecipitate. There were 1263 reported cases of transfusion-associated hepatitis. Approximately 44 percent of the patients who were tested proved positive for hepatitis B surface antigen, and 80 percent of the patients who were tested proved positive for antibody to hepatitis C. The questionnaire's aggregate results can be used to assess current patterns of blood donation and transfusion activities.     

Monoclonal antibodies to human platelet glycoprotein IIb beta that initiate distinct platelet responses

Hybridomas secreting monoclonal antibodies (MoAbs) to human platelet membrane glycoprotein IIb (GPIIb) were prepared by fusing cells of a mouse myeloma line to spleen cells from a BALB/c mouse immunized with purified GPIIb. Six of the hybridomas secreted MoAbs that recognized epitopes on the 23,000-dalton, disulfide-linked subunit of GPIIb, GPIIb beta. All six of these MoAbs agglutinated platelets in the absence of calcium. The agglutination titers of three of the MoAbs, however, were enhanced between 2 and 6 log2 dilutions when titrated in the presence of mmol/L of calcium. The enhancement in titer was the result of MoAb- induced platelet activation followed by platelet aggregation, a reaction that could also be initiated by the monovalent Fab fragments prepared from one of the MoAbs. The MoAbs did not significantly agglutinate platelets from patients with Glanzmann's thrombasthenia, confirming biochemical evidence that there is a paucity of GPIIb beta in the membranes of these cells. Our results show that MoAbs to epitopes on GPIIb beta initiate distinct platelet responses; therefore, they should be useful for studying the ways in which regions of surface glycoproteins are involved in platelet-platelet interactions. In addition, these reagents may prove of value in diagnosing and typing patients with Glanzmann's thrombasthenia.     

The new consultation: developing doctor-patient communication. David Pendleton, Theo Schofield, Peter Tate, Peter Havelock (eds). (143 pages, {pound}19.95.) Oxford University Press, 2003. ISBN 0-19-263288

The consultation is the core of medicine. Consultations makeup more than two-thirds of the workload of a family doctor,but the number of books on the topic is surprisingly small.Those who have read these authors' first book