Exploring the relationship of drug-induced neutrophil immaturity & haematological toxicity to drug chemistry using quantitative structure-activity models.

An investigation of the relationships between physicochemical features of ten antipsychotic drugs and previously reported influence of these drugs on neutrophil maturity was made. A quantitative structure-activity relations (QSAR) approach was adopted, in which several numerical parameters describing physicochemical characteristics of the antipsychotics were estimated. Possible connections between these parameters and neutrophil maturity were explored. Influence of drug physicochemistry on the incidence of agranulocytosis and neutropenia reported in the literature was documented. Overall it was found that drugs with the greatest tendency to induce neutrophil immaturity (chlorpromazine, clozapine and olanzapine) also showed the greatest tendency to cause agranulocytosis and neutropenia. Moreover marked induction of neutrophil immaturity occurred with compounds of moderately amphipathic character, whose amphipathic indices (AI) fell in the range 3-5; higher or lower AI values correlated with less immaturity. Consideration of the QSAR findings suggest that toxicity could be associated with selective uptake into the most fluid intracellular membranes, those of the endoplasmic reticulum and the outer mitochondrial membrane. The AI hazard zone (AI = 3-5) does constitute a predictive tool to assess risk of agranulocytosis and neutropenia arising from antipsychotic and other psychoactive drugs - and not only risk arising from medication but also from experimental or even proposed compounds.

Formation of immature neutrophil leucocytes in schizophrenic patients treated with various antipsychotic drugs: comparisons and predictions.

The neutrophils of schizophrenic patients taking antipsychotic drugs were evaluated. Neutrophil immaturity was assessed by determining mean nuclear lobe number in peripheral blood smears of patients and controls. Subjects were patients medicated with typical (upenthixol (n 6), uphenazine (n 7), haloperidol (n 23), thioridazine (n 15), and triuoperazine (n 6)) or atypical (olanzapine (n 15), risperidone (n 10), and sulpiride (n 7)) antipsychotic drugs. Controls (n 58) were healthy, non-medicated clinical and academic staff. Mean lobe number was determined using light microscopy and examining 300 neutrophils per individual. For subject and control groups, means and medians of mean lobe numbers, and also mean white cell and neutrophil counts, were determined. Means for each group were compared using the Mann-Whitney U test; variances using F ratios. Mean lobe numbers of all patients were decreased compared to controls. The left shift occurring in patients medicated with haloperidol, olanzapine, risperidone, thioridazine, and triuoperazine was signícantat P 0.0001; for upenthixol P 0.001, and for sulpiride P 0.05. The left shift for uphenazine was not statistically signíant. For one patient, mean lobe numbers were obtained before and after medication with olanzapine commenced, and a lowering of mean lobe number was seen. Although the coefficient of variation in the patient groups was large compared to the controls, nevertheless more than half of the patients had mean lobe numbers outside the observed range of values seen in the control population. White blood cell and neutrophil counts in patients and controls were not signiécantly different. This study demonstrated that patients taking antipsychotic drugs have immature neutrophils, but normal total white cell and neutrophil numbers. The effect was seen with both typical and atypical antipsychotic drugs, and is probably drug-induced. It is possible that mean lobe number may predict patients at risk from neutropenia or agranulocytosis, as is also suggested by an analysis of the relative numbers of literature reports of neutrophil pathology for these drugs. It is of interest that olanzapine, which has been considered a haematologically non-hazardous drug, was shown to be associated with a significant decrease in mean lobe number.

Red cell antigens and renal transplantation.

Donor-specific transfusion was performed with and without cyclosporine between haplomismatched relatives prior to living-donor renal transplantation. Red cell antigen mismatching was not taken as a contraindication to DST. Of 80 patients included in the trial; eleven were ABO-mismatched, 15 were Rh(D)-mismatched, and a further 11 were transfused in the presence of atypical red cell antibodies (anti-D, -C, -Fya, -Kell -N, -H/I -I, -P1, -Wra). Patients were randomized to receive cyclosporine (10 mg/kg) daily during DST or not (control group). The presence of atypical red cell antibodies, with the exception of Rh anti-D, did not appear to influence DST or renal transplantation. DST did not act as a primary stimulus to Rh anti-D production but stimulated preexisting anti D levels. ABO mismatching did not appear to influence DST or subsequent renal transplantation except in one group A [corrected] patient who received group O [corrected] blood and cyclosporine. This patient developed a severe, but self-limiting, autoimmune hemolytic anemia due to auto-anti A antibodies. A similar group A patient in the control group developed an auto-antibody with no clinical sequelae. The influence of cyclosporine on the development of this auto-antibody is uncertain. We conclude that, with the exception of preexisting anti-D antibodies, minor red cell antigen disparities should not preclude pretransplant conditioning with donor-specific transfusions.

Autologous bone marrow transplantation for myeloma patients using PNA- and CD19-purged marrow rescue.

A new method of in vitro bone marrow purging using a lectin and monoclonal antibody in combination has been used for the first time in vivo. Two patients with advanced myeloma were treated with high-dose melphalan and total body irradiation and then rescued with autologous bone marrow which had been purged in vitro to remove malignant cells by using a combination of a plasma cell-binding lectin (peanut agglutinin, PNA) and the anti-B lymphocyte monoclonal antibody anti-CD19, bound to magnetised microspheres. Both patients showed rapid engraftment of the purged bone marrow and remain well 36 and 46 months later with normal bone marrow morphology, although one patient still has a low level of circulating paraprotein. This is a promising form of therapy for what has been an invariably fatal condition.

A method for clinical purging of myeloma bone marrow using peanut agglutinin as an anti-plasma cell agent, in combination with CD19 monoclonal antibody.

Previous studies have shown that the lectin peanut agglutinin (PNA) binds bone marrow plasma cells in the majority of patients with myeloma and does not bind to normal haemopoietic progenitors. This lectin has been used in combination with anti-CD19 monoclonal antibody (moAb) in a system for purging myeloma bone marrow. This has now been scaled up for application to ex vivo treatment of large volumes of bone marrow suitable for autologous bone marrow transplantation. Four bone marrow harvests from patients with myeloma containing 9.5 +/- 4.9% plasma cells were depleted of erythrocytes and mature granulocytes by Ficoll separation using the Haemonetics V50 cell separator. The mononuclear fraction was then purged with magnetic beads coated with PNA and anti-CD19 moAb. The system proved highly efficient with removal of all detectable plasma cells and CD19+ cells. Average mononuclear cell recovery following purging was 71% of the concentrated marrow with 78% yield of CFU-GM. Normal progenitor recovery related to patients' weight is predicted to be adequate for haemopoietic reconstitution following ablative chemoradiotherapy. This system is therefore feasible for large-scale clinical purging.

Autologous Bone Marrow Transplantation in Hodgkin's Disease-A Single Centre U.K. Experience.

Twenty-five patients with relapsed or primary resistant Hodgkin's Disease were treated with high dose combination chemotherapy comprising cyclophosphamide, carmustine (BCNU) and etoposide followed by autologous bone marrow transplantation (ABMT). Sixteen (67%) of the twenty-four patients who survived the treatment schedule attained complete response (CR) and eleven of these remain disease free at a median time post ABMT of 16 months (range 6 to 27 months). Two of the patients who relapsed died at 7 and 17 months and the remaining three patients are alive with persistent disease at 12, 14 and 18 months. Four patients showed partial response. Three of these developed disease progression soon after the procedure and all died within ten months. The remaining patient achieved CR following further chemotherapy. There was a tendency for patients with bulky mediastinal disease, extra-nodal disease and heavy pre-treatment to fail to achieve CR.

Antipsychotic drugs result in the formation of immature neutrophil leucocytes in schizophrenic patients.

Subclinical abnormality of neutrophil populations of patients suffering from schizophrenia and medicated with antipsychotic drugs was evaluated using cellular immaturity as a criterion. Neutrophil maturity of patients and controls was compared by determining mean nuclear lobularity in peripheral blood smears. White blood cell and neutrophil counts were made. Subjects were patients medicated with chlorpromazine (n = 17) or clozapine (n = 48). Controls (n = 58) were healthy, non-medicated clinical and academic staff. Determination of mean lobe number involved assessment of 300 neutrophils per individual. For subject and control groups, means and medians of mean lobe numbers and mean white cell and neutrophil counts were determined. Means for each group were compared using the Mann-Whitney U-test; variances using F ratios. Means of lobe numbers of both patient populations were significantly different (p < 0.0001) compared to controls. Two-thirds of patients had mean lobe numbers outside the control range. Dose-response (mean lobe number) plots were significant for patients medicated with both chlorpromazine and clozapine. White cell and neutrophil counts in patients and controls did not differ significantly. For six patients, mean lobe numbers were obtained before and after medication commenced and all showed lowering of mean lobe number. The mean lobe number of the one patient who subsequently suffered from agranulocytosis was at the low end of the patient range. Thus, patients medicated with antipsychotic drugs typically have immature neutrophils, but normal white cell and neutrophil numbers. This effect is probably drug-induced. Mean lobe number may predict patients at risk from agranulocytosis.

Immunisation of staff of a regional blood transfusion centre with a recombinant hepatitis B vaccine.

Medical, nursing, laboratory and other staff of the Mersey Regional Blood Transfusion Centre at risk of acquiring hepatitis B were vaccinated against the virus with a recombinant vaccine. Altogether, 86% staff developed greater than 10 mIU/ml antibody to hepatitis B virus surface antigen after immunisation but the proportion was lower in older staff. After a further injection of vaccine was given to poor and non-responders the overall proportion responding rose to 93%. The difference in response between the sexes was significant. Women had a higher rate of response and, among those persons responding, women developed a higher concentration of antibody.

Prothrombin fragment F1 + 2: correlations with cardiovascular risk factors.

Plasma prothrombin fragment F 1 + 2 (F1 + 2), thrombin-antithrombin complexes (TAT), fibrinogen and factor VII were related to variables associated with increased cardiovascular risk in 86 plasma donors (49 male and 37 female). F1 + 2 had a log-normal distribution and increased significantly with age and body mass index (BMI). Significantly, higher F1 + 2 levels were found in smoking compared with non-smoking males and in indolent males compared with males taking regular exercise. Higher levels were found in subjects with a parental history of ischaemic heart disease than in those lacking such a history. F1 + 2 correlated strongly with increasing cholesterol in males. Fibrinogen was significantly higher in male smokers than male non-smokers but did not vary with age or BMI. Factor VII correlated strongly with cholesterol and to a lesser extent with fibrinogen, F1 + 2 and BMI, but not with smoking. F1 + 2 correlated more closely with risk factors for cardiovascular disease than fibrinogen and factor VII, and consistently reflected the difference in cardiovascular risk when correlated with risk factors which have markedly different effects between the sexes. It promises to be a useful predictive marker of ischaemic heart disease.

Assessment of severity of haemolytic disease of newborn by monocyte monolayer assay.

The present study was undertaken to estimate the predictive value of antibody titration, antibody quantitation and monocyte monolayer assay (MMA) in assessment of severity of haemolytic disease of the newborn (HDN). Serum samples from 45 alloimmunized mothers, with anti-D(23), anti-c(10), anti-K(6), anti-E(5) and anti-e(1) were taken for the study. The results obtained were compared and the efficiency of each technique in predicting the severity of HDN was assessed. Antibody quantitation and MMA (phagocytic index) correlated well with severity of HDN in mothers with anti-D antibodies. Antibody quantitation (anti-D) had a positive predictive value of 54.5 per cent and negative predictive value of 85.7 per cent while MMA had a positive predictive value of 75 per cent and a negative predictive value of 100 per cent. These findings suggest MMA to be a good negative predictor of HDN but not a good positive predictor of haemolytic disease of the newborn.

Red cell antibody screening and identification: a comparison of two column technology methods.

Two commercial column techniques for use in antibody screening and identification procedures were tested in parallel with 1000 random samples sent for ante-natal serological investigation. The DiaMed ID microtyping system uses a sephadex gel contained in microtubes, either neutral or impregnated with anti-human globulin (AHG), for use in two-stage enzyme methods and LISS indirect antiglobulin testing (IAT) respectively. The Ortho Biovue technique consists of a slurry of micro glass spheres which act as the filter to retain haemagglutination reactions within the matrix. Columns containing AHG also possess a macromolecular density barrier to prevent test serum from passing into the column and neutralising the AHG. Both systems offer the advantage of 'no-wash' IAT, which minimises the potential for problems and errors associated with conventional spin-tube techniques. In this comparison of the two column methods, antibody detection rates were found to be similar and the sensitivity of both methods was comparable, although the Biovue technique was prone to exhibit equivocal results, particularly in the IAT.

Developing techniques in blood transfusion.

Expansion of transfusion medicine has led to an increasing awareness of the importance of its practice. Specialists in this branch of haematology whose main aim is to provide adequate and safe supplies of blood (and blood products) and to ensure these are used appropriately, are increasingly aware that to do this efficiently requires the development and utilization of new laboratory and technical procedures. Review of cross-matching techniques has led to the introduction of more rapid methods using low ionic strength saline. Use of monoclonal antibodies for blood grouping has made use of new technology, whilst allowing scarce human plasma to be used more appropriately for therapeutic purposes. Similarly, the implementation of a more rational approach to blood ordering, as in a maximum surgical blood-order schedule, allows for the more efficient use of donor blood. The use of microtitre plates for grouping and cross-matching techniques allows for speed and economy in the transfusion laboratory. Their use is also associated with increased automation and computer use. The possibility of using solid-phase techniques, monocyte-macrophage assays and antibody-dependent cellular cytotoxicity assays introduces new techniques differing markedly from time-honoured liquid-phase serology methods. The application of flow cytometry, which has already been shown to be useful in many aspects of haematology, is also of benefit in the field of blood transfusion science. Safety of blood transfusion is an important aspect of its practice and has led to the introduction and development of screening tests for donor blood to exclude infection risks from such organisms as HIV-1, hepatitis B and non-A, non-B hepatitis. Another approach to ensure the safety of transfused blood has been increased usage of autologous transfusion by means of both predeposit donation and intraoperative cell salvage.

Uses of Column Technology in Blood Transfusion.

Microtubes in which columns of gel or microspheres are contained have gained widespread acceptance for routine serological testing. The use of columns for blood grouping, antibody screening and identification, and compatibility testing have all been well described. Columns have also been shown to perform well in the identification of antibody immunoglobulin subclasses and in the investigation of drug induced hemolytic anemia. The development of multi skilled laboratories has been facilitated by the introduction of these techniques and anxieties about loss of traditional skills have proved to be unfounded. The development of automation able to identify, handle and interpret large numbers of column tests has also led to their introduction into busy serological laboratories. New developments to increase the range of tests for which gel columns can be used include the use of columns containing specific monoclonal antibodies for the diagnosis of paroxysmal hemoglobinuria. The ability to easily and accurately identify irregular antibodies in patients serum associated with similarly accurate grouping and compatibility testing has led to improved patient care and management and a more efficient and effective delivery of transfusion practice.