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Platelet membrane glycoprotein polymorphisms are candidate risk factors for thrombosis, but epidemiological data are conflicting. Thus, demonstration of a genotype-dependent alteration in function is desirable to resolve these inconsistencies. We investigated in vivo platelet activation in acute thrombosis and related this to platelet genotype. Frequencies of the 1b and 2b alleles of the HPA 1a/1b and HPA 2a/2b platelet glycoprotein polymorphisms were determined in 150 (52 men/98 women, mean age 58.3 years) patients with atherothrombotic stroke, and the influence of genotype on markers of platelet activation was assessed. Platelet P-selectin (CD62P) expression and fibrinogen binding was measured using whole blood flow cytometry within 24 h of stroke and 3 months later in 77 patients who provided a repeat blood sample. Results were compared with matched controls. Neither the 1b allele [allele frequency 0.11 vs. 0.13, odds ratio (OR) confidence interval (CI) 0.8 (0.5-1.3)] nor the 2b allele [0.09 vs. 0.07, OR (CI) 1.4 (0.8-2.4)] was significantly over-represented in patients. Increased numbers of activated platelets were found following stroke (acute mean P-selectin expression 0.64% vs. control 0.35%, P < 0.001; acute mean fibrinogen binding 1.6% vs. control 0.9%, P < 0.001). Activation persisted in the convalescent phase (P < 0.001 and P = 0.005 vs. controls for P-selectin and fibrinogen respectively). Expression of P-selectin and fibrinogen was not influenced by either the HPA 1a/1b genotype (P > 0.95 for each marker, Scheffe's test) or the 2a/2b genotype (P > 0.95 for each). Although persisting platelet activation is seen in atherothrombotic stroke, it is independent of HPA 1a/1b and 2a/2b genotypes. These data suggest an underlying prothrombotic state, but do not support the polymorphisms studied as risk factors for thrombotic stroke in this population.

Platelet dysfunction has a major contribution in bleeding after cardiopulmonary bypass (CPB) and transfusion of platelets is frequently used to secure haemostasis. Allogeneic platelets prepared for transfusion are functionally impaired. Autologous platelets harvested preoperatively require a shorter storage time before transfusion and their use also avoids the risks associated with transfusion of allogeneic blood products. For the first time, we have compared the functional quality of autologous platelets with allogeneic platelets prepared by two methods, immediately before infusion. Platelet activation was assessed by P-selectin expression and fibrinogen binding using flow cytometry. We also monitored the effects of CPB surgery and re-infusion of autologous platelets on platelet function. Autologous platelet-rich plasma (PRP) contained a significantly lower (P < 0.05) percentage of P-selectin-positive and fibrinogen-positive platelets compared with allogeneic platelet preparations, and also contained a significantly higher (P < 0.05) percentage of responsive platelets. Allogeneic platelets prepared by donor apheresis were more activated and less responsive than those produced by centrifugation of whole blood. In patients' blood, the percentage of platelets expressing P-selectin or binding fibrinogen increased significantly after CPB (P < 0.05), while the percentage of platelets responsive to in vitro agonists was decreased (P < 0.05 in autologous transfusion patients), consistent with platelet activation during the procedure. The percentage of activated platelets decreased (statistically not significant) after re-infusion of autologous PRP. P-selectin expression had returned to pre-CPB levels 24 h post-operatively. Autologous platelet preparations display minimal activation, but remain responsive. Conservation of platelet function may contribute to the potential clinical benefits of autologous transfusion in cardiopulmonary bypass.

In a retrospective review of transfusion errors in a large teaching hospital, we found the true incidence of errors to be at least four times the actual mistransfusion events detected. Seventy-five per cent of the errors were detected as near misses. The mistransfusions equated to 1/8610 compatibility procedures, and 1/27 007 units of blood issued, whereas the number of true transfusion errors equates to 1/2153 compatibility procedures and 1/6752 units of blood issued. The major error-prone activities included patient identification at phlebotomy and the final infusion of the blood product at the bedside. Of the cases, 95.2% were due to non-compliance with existing guidelines. Potential disasters were avoided only by the vigilance of the blood bank staff and the systems in place to detect errors.

Maternal administration of high-dose intravenous immunoglobulin (IVIgG) for treating fetal RhD haemolytic disease and alloimmune thrombocytopenias may be beneficial. Treatment failures, even when IVIgG is used optimally, may result from product differences. Using an in vitro placental perfusion model there was significant inhibition of placental anti-D IgG transfer with three commercial IVIgG preparations where circulating maternal IgG concentrations were > 20 g/l. One IVIgG product, which was not inhibitory, had lower circulating IgG levels (16.5 +/- 0.9 g/l) and significantly reduced placental transfer of total IgG, suggesting that the reduced functional activity of IgG from IVIgG preparations may correlate with poor clinical efficacy.

A prospective study of 7065 consecutive new pregnancies identified 230 with a positive screen, of which 27% (62/230) were 'enzyme-only' antibodies. 32 of these (52%) were potentially clinically important and were all of Rh specificity: 22 anti-E, seven anti-Cw, two anti-D and one anti-c. However, only three of these enzyme-only antibodies (one anti-D, one anti-c and one anti-E) became reactive by the indirect antiglobulin test (IAT) during the course of pregnancy, and all were detected in the routine 34-36-week maternal sample. No babies were affected, and we reaffirm that routine antibody screening by enzyme techniques is unnecessary.

The role of the platelet glycoprotein (GP) IIIa polymorphism HPA 1b (PlA2) in the risk of arterial thrombosis is controversial. We investigated the effect of the 1b allele on platelet fibrinogen binding by flow cytometry. Samples from 35 healthy platelet and plasma donors possessing the 1b allele were compared with 35 1a/1a donors. We found no allele-dependent difference in percentage of platelets binding fibrinogen (P = 0.6), nor in mean cell fluorescence (P = 0.3) following stimulation with ADP. These results render it unlikely that any relationship between the 1b polymorphism and arterial thrombosis is mediated by a significant effect on fibrinogen binding.

Using an in vitro perfusion model, therapeutic intravenous immunoglobulin (IVIgG) and IgG anti-D have been shown to cross the placenta from the maternal circuit to the fetal circuit. The transfer of all IgG species was linear with respect to time, and the amount of IgG transferred was proportional to the concentration of IgG in the maternal circuit ([IgG]m), but reached saturation at upper limits. With total [IgG]m at 6.5 g/l, 11.1 g/l or 26.2 g/l the increase in the fetal concentration of total IgG was 4.6 mg/l/h. 8.9 mg/l/h and 9.9 mg/l/h respectively. The rate of transfer of specific anti-D antibody to the fetal circuit was 0.026 IU/ml/h at a concentration of 38 IU/ml in the maternal circuit ([anti-D]m). High-dose therapeutic IVIgG added to the maternal circuit (total [IgG]m 29.2 g/l) significantly inhibited (P < 0.001) anti-D transfer to 0.004 IU/ml/n. Addition of the same IVIgG at a lower concentration (total [IgG]m 11.1 g/l) also reduced anti-D transfer, but only to 0.015 IU/l/h. The inhibitory effect of IVIgG does not appear to be mediated by anti-idiotypic or non-specific complexing with the anti-D, but may be the result of competition with IgG anti-D for placental Fc gamma receptors involved in the endocytotic uptake of IgG. The efficacy of IVIgG in this model suggests that it may be clinically useful in preventing HDN and other immune cytopenias, provided a sufficiently high dose is given.

The effects of intensive plasma exchange on the circulating levels of coagulation factors I, II, V, VII, VIII, IX, X and antithrombin III were determined. During courses of daily exchange marked cumulative reductions of coagulation factors may occur, particularly in the case of factors I, II and X, although usually remaining above the levels considered adequate for haemostasis. The extent of cumulative reduction and subsequent recovery differed for patients with different diseases. While antithrombin III levels were reduced during plasma exchange the results suggest that this may be partly due to consumption as well as physical removal. The very low incidence of haemorrhagic sequelae and absence of thrombotic events following plasma exchange at this Centre is explained by the maintenance of adequate levels of coagulation factors and of antithrombin III even during courses of daily plasma exchange.

An ADCC system has been developed using anti-D and papainized group O rhesus (D) positive red cells as the targets. Monocyte depleted mononuclear cell suspensions were effective in lysing appropriately sensitized red cells and papainization considerably enhanced the degree of specific lysis. Variation in culture volume and incubation in tubes or microplates were not critical to the degree of specific lysis obtained provided that the number of effector cells and target cells per culture was constant and the anti-D not diluted below the optimal concentration. Cytolytic activity was seen down to levels of 3 ng anti-D per culture. Specificity for lysis resided with the anti-D and not the effector cells. Several sources of anti-D were effective in inducing lysis of D positive red cells although individual variation was noted. Anti-c and anti-E were also shown to be effective in inducing specific lysis of red cells with the appropriate antigens.

After secondary immunizations of rhesus(D)-negative male volunteers with Rh(D)-positive red cells, changes were found in the in vitro transformation and antibody-dependent cell-mediated cytotoxicity (ADCC) capacities of the volunteers' lymphocytes. Responders, who produced anti-D, showed marked depressions of ADCC which were not found in non-responders. Responders and non-responders in general showed similar changes in lymphocyte transformation. The relationships between altered lymphocyte functions following immunization, immunoregulatory activity and responsiveness to the Rh(D) antigen are discussed.

Certain anti-D sera, selected on the basis of their agglutination characteristics in vitro, fail to induce lysis of Rh(D) positive red cells by lymphocyte mediated antibody dependent cell mediated cytotoxicity (ADCC). Further investigation revealed that the non-lytic anti-D blocked in an antigen specific manner the effect of other anti-D sera which were normally lytic in ADCC. Absorption selection studies and fractionation of a non-lytic anti-D serum showed that the blocking effect was associated with IgG anti-D. Antigen binding and lymphocyte Fc-receptor binding studies indicated that the non-lytic anti-D was bound to Rh(D) positive red cells and enabled them to be bound by lymphocytes, but failed to mediate ADCC.

Five patients with immunopathologic renal disease, 12 with malignant paraproteinaemia and one with myasthenia gravis underwent a total of 179 plasma exchanges on a continuous flow cell separator. Replacement fluids devoid of coagulation factors were used in 160 exchanges while 19 exchanges were replaced with Fresh Frozen Plasma. Coagulation screening was done immediately before and 30 min after each plasma exchange. Plasma fibrinogen concentrations fell to a mean of 25% of initial levels during individual exchanges. Sequential reduction to 10.7% was observed after five consecutive daily exchanges and in one patient fell to 1.2% after 10 daily exchanges. Low levels of fibrinogen could be maintained with daily or alternate daily exchanges. Platelet counts fell to a mean of 50% of pre-exchange levels during individual exchanges. Consecutive daily exchanges resulted in mean reductions to 20.7% after 5 d, but further reductions were not observed with longer periods of exchange. Platelet counts recovered to pre-exchange values during exchange intervals of 2 or more days. Mild clinical bleeding episodes, probably related to low platelet counts, occurred in one exchange in each of three patients. Haemostasis was rapidly achieved in these patients by infusions of platelet concentrates. Coagulation screening, including prothrombin ratio, thrombin time, reptilase time and partial thromboplastin time with kaolin showed progressively greater abnormalities as the intervals between exchanges shortened. The low incidence of clinical haemorrhagic episodes, three of 179 exchanges (2.2%), despite markedly abnormal coagulation parameters, emphasize the safety of the procedure even in moribund patients. We conclude that the use of FFP in intensive exchange programmes solely for the prevention of spontaneous haemorrhagic phenoma is unjustified.

The mechanism of lysis of anti-D coated human erythrocytes by human mononuclear K-cells was investigated. Red cell lysis was measurable after 30 min incubation and reached a maximum by 18--20 h. Cell-to-cell contact was necessary for lysis, phagocytosis was not a prerequisite, and intact microfilament function was required. The divalent cations Ca2+ and Mg2+ were both required for lysis to occur. Studies with metabolic inhibitors indicate that some RNA and protein synthesis is required for maximum expression of ADCC and intact microtubule function is essential. In the present system lysis was mediated by IgG1 anti-D antibodies and was significantly inhibited by IgG1 and IgG3 subclasses with some inhibition by IgG2 but not by IgG4, IgA or IgM. This suggests that the K-cell receptor is specific for IgG but that there is major cross-reactivity between IgG1 and IgG3. The inhibiting effect of hydrocortisone suggests that ADCC inhibition may be one mode of action of corticosteroids in ameliorating autoimmune haemolytic anaemia.

Conventional manual (enzyme and antiglobulin titres) and AutoAnalyser quantitation of anti-D sera were compared with the ability of the same sera to mediate lysis of Rh(D) positive red cells in an ADCC assay. AutoAnalyser (AA) quantitation correlated significantly with manual titration methods, although there were wide discrepancies between individual sera. The ADCC activity of the anti-D sera did not correlate with any of the conventional assays (AA, enzyme, antiglobulin). There were significant differences between anti-D sera obtained from females immunized during pregnancy and male volunteers immunized by deliberate injection. The female sera contained significantly less anti-D when assessed by AA, yet were significantly more active in ADCC activity at equivalent concentrations. These functional differences in anti-D activity could not be attributed to the absence of IgG subclasses (IgG1 and IgG3) known to induce ADCC. However, there was a relationship between ADCC and IgG1/IgG3 titres in that high ADCC activity was seen where the IgG3 titre was higher than the IgG1 titre. In the male anti-D sera IgG1 titres were greater than or equal to IgG3 titres and ADCC activity was very low.

Eight monoclonal antibodies (mabs) to the Rh antigen D produced by Epstein-Barr virus transformed B-lymphoblastoid cell lines from two individuals have been compared for their behaviour in in vitro cell-mediated assays. Three IgG1 Glm(1,17) and two IgG3 G3m(21) mabs from one donor and three IgG1 Glm(3) mabs from another were used. IgG3 anti-D mabs induced greater adherence and phagocytosis of sensitized red cells by U937 monocytes than IgG1 anti-D mabs or the polyclonal anti-D. Minimum sensitization levels for rosetting and phagocytosis by U937 monocytes were 2,000 molecules IgG/cell for IgG3 and 5,000 molecules/cell for IgG1 mabs; maximum rosetting mediated by both IgG1 and IgG3 mabs was obtained at 15,000-20,000 molecules/cell. The IgG3 anti-D mabs were comparable to polyclonal anti-D in mediating binding of sensitized red cells to gamma-interferon stimulated monocyte-derived cultured macrophages and were markedly more effective than the IgG1 anti-D mabs. However, in lymphocyte ADCC assays, only anti-D mabs which were IgG1 Glm(3) were effective in mediating high levels of lysis of sensitized red cells, unlike the IgG1 Glm (1,17) or IgG3 G3m(21) mabs. Minimum sensitization levels required for this lymphocyte-mediated red cell lysis were found to be approximately 5,000 molecules/cell with one IgG1 Glm(3) mab; maximum lysis with this mab was obtained at 10,000 molecules/cell. Polyclonal anti-D containing both IgG1 and IgG3 was effective in all three assays. These observations suggest that different isotypes and allotypes of anti-D antibodies mediate red cell removed or destruction by monocyte or lymphocyte effector cell through functionally dissimilar Fc receptor interactions.

Alzheimer's disease may arise from an interaction between a conventional infective agent and a particular disease susceptibility (related to the HLA-A or B locus). HLA antigens and antibodies to conventional infective agents were examined in 14 patients with pre-senile dementia. Most of the sample probably suffered Alzheimer's disease, though one subject may have had Pick's disease. No particular HLA type or antibody was associated with the sample.

An in vitro homologous system using human Rhesus alloantibodies and target erythrocytes labelled with 51Cr has been used to demonstrate the ability of normal peripheral blood lymphoid cells to lyse antibody-coated human red cells. The results obtained suggest that this type of mechanism may be relevant to certain haemolytic diseases in man.

Chronic mucocutaneous candidiasis with hypoparathyroidism in a 6-year-old-boy is described. In addition to defects of in vivo and in vitro correlates of delayed-type hypersensitivity to Candida albicans the child also had abnormalities of neutrophil function in terms of their capacity to respond by chemotaxis to a known attractant and to kill suspensions of C. albicans. Dialysable transfer factor was given on six occasions at intervals of between 26 and 45 days. Neutrophil chemotaxis (optimal conditions) was restored following each of the six injections, neutrophil chemotaxis (sub-optimal conditions) following five of the six injections and candidicidal capacity following four of the six injections. The effects of transfer factor were transient requiring repeated injections. The Candida delayed-type hypersensitivity skin test was restored to normal but lymphocyte transformation to Candida extract was not consistently positive following treatment. There was a slight clinical improvement following therapy. These abnormalities of neutrophil and lymphocyte function point to the complexity of chronic mucocutaneous candidiasis. The improvement in neutrophil chemotaxis and candidicidal capacity following treatment suggests that transfer factor may be a heterogeneous group of molecules, some of which affect granulocytes and restore defects in their function.

Three monoclonal IgG1 anti-Rh(D), UCH D4, ARC 7D5 and UKTS FC3, produced by Epstein-Barr virus transformed cells from Rh(D)-sensitized individuals, were compared with polyclonal single donor anti-D sera and therapeutic immunoglobulin preparations in antibody dependent cellular cytotoxicity (ADCC) and macrophage binding tests. When assayed at equal anti-D concentrations monoclonal antibodies varied considerably in their ADCC and macrophage binding activities: only UKTS FC3 showed significant activity in both assays, but these were substantially lower than those of the polyclonal anti-D sera and immunoglobulins. When examined in different combinations the monoclonal antibodies showed little synergism in mediating red cell destruction by the effector cells. Factors which might contribute to the diverse ADCC and macrophage binding activities of the monoclonal anti-Ds of the same IgG subclass are discussed.