BritishJourwl of Haematology, 1979, 43, 485-506.
Abstracts of Papers Presented at the Twentieth Annual General Meeting of the British Society for Haematology: Joint Meeting with Nederlandse Vereniging voor Hematologie, Aviemore, April 1979
SYMPOSIUM: NEW APPROACHES TO DIAGNOSIS AND THERAPY Plasmapheresis Techniques and their Clinical Application I. D. FRASER Regional Transfusion Centre, Southmead, Bristol Treatment of Aplastic Anaemia: ALG versus Bone Marrow Transplantation H. L. HAAK University Hospital, Leiden Treatment of Acute Leukaemia H. E. M. KAY Royal Marsden Hospital, London Bone Marrow Cultures for the Diagnosis of Haematological Diseases B. LOWENBERG University Hospital Dykrjigt, Rotterdam Stem Cell Cryopreservation in Leukaemia J. M. GOLDMAN Hammersmith Hospital, London
SCIENTIFIC SESSIONS Granulocyte-specific Alloantigen Loss in Chronic Myeloid Leukaemia H. J. V . D . REIJDEN, A. E. G. KR. V.D. BORNE,F. W. A. VERHEUGT, A. B. FLOOR-VANGENT,C . J. M. MELIEF and C. P. ENGELFRIET Department of Immunohaematology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, and University Laboratory of Experimental and Clinical Immunology, University of Amsterdam, Amsterdam The granulocytes of 23 patients with chronic myeloid leukaemia were examined with the indirect immuno-fluorescence test and some also with the micro-leucoagglutination test, with specific alloantisera against the NA1, NA2, N B l and N D l antigens and a specific granulocyte xeno-antiserum. In 10 cases a complete loss of alloantigens was detected. In one patient a partial loss was found. A significant correlation was established between this loss and an accelerated activity (impending blast crisis) of the disease.
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Correlation of Biochemical and Immunofluorescent Assays for Terminal Transferase in Acute Leukaemia K. F. BRADSTOCK, A . V. HOFFBRAND, G. JANOSSY, G. PRENTICE, K. GANESHAGURU, P. LLEWELLYN and F. J. BOLLUM* Departments ofImmunology and Haematology, Royal Free Hospita!, and *Uniformed Services University ofthe Health Sciences, Bethesda, Maryland, U.S.A. The level of terminal deoxyribonucleotidyl transferase (TdT) was determined in various forms of leukaemia and in normal regenerating bone marrow cells. T w o methods were compared: the biochemical assay and a single cell immunofluorescent analysis using purified anti-TdT antibody. The two assays gave mutually confirmatory results in almost all cases, and effectively distinguished between lymphoid (TdT+) and myeloid (TdT-) acute leukaemias. False negative results were seen rarely with either method: these were due to technical difficulties. The biochemical assay could not detect T d T + cells when these were present in proportions less than 5% ofa total population. The immunofluorescent assay was very sensitive and was able to detect T D T + cells in very small numbers. In addition, used in double and triple-marker combinations with other immunological markers (antisera to ALL antigen, Ia-like antigen, immunoglobulin and to T cell antigens) the assay was able to distinguish sub-classes of T d T cells a t a single cell level. Using this technique, T d T + cells were detected in regenerating marrows of treated patients with acute myeloid and lymphoid leukaemias, and also in aplastic anaemia patients treated with allogeneic bone marrow transplantation. This sensitive assay therefore could not distinguish between normal regenerating T d T + cells and residual leukaemic lymphoblasts. +
Analysis of Cell Kinetics in Human Myeloma by Double Label Flow Cytometry B. HOUWEN and J. H. SCARFFE* Division of Haematology, Department of Medicine, University of Groningen, and *Department of Medical Oncology, University of Manchester Growth kinetics of tumours and response to chemotherapy can be measured by 3H-thymidine labelling and by flow cytometry of cellular DNA content. Flow cytometry, extensively used for this purpose in acute leukaemia, is unable to distinguish between normal and tumour cells in a mixed population. However, ifa second parameter can be used for the detection of abnormal cells, data specific for the tumour cell population can be obtained. Multiple myeloma offers such an opportunity by labelling the cytoplasmic immunoglobulins of the plasma cells with fluorescent antisera. Therefore we investigated the possibilities of such a double label technique in flow cytometry and compared the results with a thymidine labelling index method. Nucleated bone marrow cells from 30 patients with multiple myeloma and from 14 patients with malignancies other than myeloma without marrow infiltration (controls) were fixed in an alcohol-glutaraldehyde mixture. The cells were then incubated with FITC conjugated antisera directed against the heavy and light chains of human immunoglobulins in order to stain the cytoplasmic immunoglobulins of the myeloma cells. After washing, the cells were incubated with propidium iodide to stain DNA. The samples were analysed in an Ortho@' 4800A Cytofluorograph and FITC (green) as well as propidium (red) fluorescence were recorded simultaneously. As a third parameter light scatter as an estimate of cell size was used. It appeared possible to separate fluorescent from non-fluorescent cells and to obtain a DNA-histogram from myeloma cells and the total population separately.
Abstracts of Papers
Correlations between the results for flow immunofluorescence and UV-microscopy were linear ( r = 0 . 8 9 ) , as were the correlations between DNA-histogram derived S-phase percentages and values obtained by 3H-thymidine labelling (r =0.92). This indicates that flow cytometric analysis of myeloma cells can be used for monitoring cell kinetics in these tumours.
Inhibition of Spontaneous Thymidine Incorporation in Non-T Lymphocytes by T-Cells in Hodgkin’s Disease B. DE PAUW,D. J. TH. WAGENER, J. M. C. WESSELS and C. HAANEN Division of Haematology, Department of Internal Medicine, St Radboud Hospital, University of Nijmegen If lymphocytes of patients with Hodgkin’s disease are cultured without adding any mitogen, and the DNA-synthesis is measured on the basis of thymidine uptake, increased values are found in comparison to normal controls. The increased incorporation is called spontaneous stimulation. Especially lymphocytes with a low specific gravity possessed this property. In order to study the spontaneously stimulated cells, the lymphocytes of 10 Hodgkin’s disease patients and 10 normal controls were divided into a fraction with a low and a fraction with a high specific gravity. Consecutively, both fractions were separated into a T (thymus dependant) and a non-T (thymus independant) subpopulation via sheep erythrocyte rosette sedimentation. Data, obtained after culturing the low density fractions, are not yet conclusive. The thymidine incorporation by high density T lymphocytes and by the unseparated fraction with a high specific gravity showed no significant difference between Hodgkin’s disease patients and normal controls. The high density non-T cells derived from the fraction which showed no spontaneous stimulation before the separation procedure, had a significantly increased thymidine incorporation (mean 5677 cpm; range 405-26 490 cprn) in Hodgkin’s disease compared to the corresponding cells from normal controls (mean 484 cpm; range 235-840 cpm). The apparent potency for D N A synthesis was unmasked after separation from T cells in the same density fraction. This indicates an inhibitory mechanism of the T cells on the proliferation of non-T cells in patients with Hodgkin’s disease. The mean inhibition, calculated on the basis of the following formula: cpmT x TOT, cpm non T x YOn o n T expected cpm = was 53%, ranging from 23% to 82%.
The inhibition i s possibly due to the presence of ‘suppressor’ T-cells; evaluation of the properties of the T cells is in progress.
Crystalline Inclusions in CLL
c.THIELEMANS, M. VANDER PLANCKE,* P. P O T V L I E G H E t and B. VANCAMP Departments ofHaematology and Anatomic Pathology, Academic Hospital V U B , and tDepartment ofHaematofogy, UIA, Belgium Crystalline inclusions in the cytoplasm of lymphocytes can be found in approximately 5% of all cases of CLL. In 21 reported cases these crystals were found to contain IgM-2, except in two cases where IgA-A was present. We studied six patients (five IgM-A, one IgA-A) with this characteristic at the cellular level using
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electronmicroscopy, immunofluorescence, enzymcytochemical morphology and surface receptor techniques. Morphologically two types of immunoglobulin (Ig) positive inclusions could be visualized: (1) rod-like crystals with ultrastructural periodicity of 4 nm, surrounded by the membrane of the endoplasmatic reticulum; (2) globular structures with vesicular appearance of the endoplasmatic reticulum. A monoclonal surface Ig of the same type and class was detected on the leukaemic lymphocytes. However, the percentage of SIg positive lymphocytes, EAC forming cells, and also E rosette counting was low, suggesting a non-expression of membrane receptors on these atypical lymphocytes. It seems plausible that these Ig crystals are a result of an intracellular Ig accumulation, related to an imbalance or arrest in the subsequent steps of the secretion process. Any explanation of this phenomenon should take into account the observation that cell inclusions were of the lambda light chain type.
Autologous Recovery following Allogeneic Bone Marrow Transplantation for Aplastic Anaemia
E. C. GORDON-SMITH, J. Hows and S. M. FAIRHEAD Royal Postgraduate Medical School, Hammersmith Hospital, London W12 OHS Twelve patients with severe acquired aplastic anaemia have been grafted with allogeneic marrow from HLA A and B identical MLR compatible siblings between 1975 and January 1979. Six of the patients have recovered normal haematological function but in two patients the reconstituted marrow is of recipient origin. O f the six patients who died, three failed to show evidence of engraftment. In the first case, a man of 45 who received immunosuppression with cyclophosphamide 50 mg/kg for 4 d and then marrow (2.6 x lo8 cells/kg) from his brother, there was prompt haematological recovery 3 weeks after the infusion of marrow. There was no GVH disease. He was discharged home 11 weeks after grafting and in September 1975 he returned to work as a printer. In December he developed a sore throat and extensive herpes simplex lesions of the face. Investigations revealed a return of pancytopenia and a severely hypoplastic marrow. He was discharged over the holiday period with a view to regrafting in the New Year. When he returned there were early signs of regeneration in the marrow and over the next 4 weeks haematological function recovered completely, including a return of CFU, from virtually zero at the time of relapse to normal levels. Blood group analysis and iso-enzyme patterns (Dr D. A. Hopkinson, Galton Laboratory, University College) showed several differences between the donor and recipient which by implication means autologous recovery. The patient remains entirely well. The second patient, a 19-year-old girl who had received gold injections for juvenile chronic polyarthritis, was transplanted with marrow from her sister (2.6 x lo8 cells/kg) following immunosuppression with cyclophosphamide as described above. The recipient was blood group B the donor 0 Group 0 blood products were used in her support post-grafting. In the fourth post-transplant week there was a rise in neutrophil count to a maximum of 1.0 x 109/1but she still required platelet and red cell support. The improvement in neutrophils was transient and she became refractory to platelet transfusions. She was reconditioned with horse anti-lymphocyte globulin and given a further infusion of marrow from the same donor (2.0 x lo8 cells/kg). During the 4 d of ALG administration there was a rise in granulocyte count but this was not maintained. Three weeks later the granulocyte count began to rise, followed shortly by the platelet and reticulocyte counts. The reticulocytes were group B + , and therefore of recipient origin. The return to normal haematological values was much slower in this patient, the platelets 6 months post grafting being 30 x lo9/]and currently (20 months after grafting) 150 X 1OY/l.In the past year she has had bilateral hip replacement without problems: At least 11 cases of autologous
Abstracts of Papers
bone marrow recovery have been reported out of 21 1 grafts for aplastic anaemia carried out in various centres in the U.S.A. and Europe. Whilst this represents only 5% ofall grafts, it represents nearly 20% of all patients who fail to show engraftment or who reject. The occurrence of autologous recovery indicates that maximum support should be continued even when an allograft has apparently been rejected.
The Clinical Significance of Histological Grading in Acquired Aplastic Anaemia in Adults H. L. HAAK, J. TE VELDE,* J. P. M. GERAEDTSt and L. J. M. S A B B E ~ Isolation W a r d , J . A . Cohen Institute, Leiden and Departments of *Pathology, t H u m a n Genetics, and Slrnrnunohaematology, University Hospital, Leiden In 30 patients suffering from acquired aplastic anaemia and observed since January 1974, the bone marrow biopsy specimens were scored according to a previous study (Haak te Velde (1977) British Journal ofHaematology, 35, 61). Fifteen cases conformed to grade I1 and an equal number to grade 111, before specific treatment. In these two groups differences were observed in clinical data, cytogenic and immunological findings. This suggests that histological grading may help to differentiate the clinical entities leading to the syndrome of aplastic anaemia. These differences were reflected in response to therapy, e.g. ALG, and development of leukaemia.
Immune Mediated Aplastic Anaemia: Normal Erythroid Burst Formation in T w o Patients after T-cell Depletion of Blood Mononuclear Cells C. A. SIEFF,B. TOROK-STORB,* R. SToRBt and J. W. ADAMSON* Haematology Department, The Hospital for Sick Children, London, *Haematology Research Laboratory, Veterans Administration Hospital, and ?Fred Hutchinson Cancer Research Center, Seattle, Wash., U .S. A . Recent reports that lymphocytes from patients with aplastic anaemia (AA) suppress haemopoiesis in vitro suggest that some of these cases have an autoimmune aetiology. Interpretation of these co-culture experiments is difficult, however, because of transfusion induced sensitization and HLA differences between co-cultured cells. To identify immune mediated AA we have assayed erythroid burst forming units in T-cell depleted AA patients’ blood cells. Ficoll-hypaque separated blood mononuclear cells were T-cell depleted by rosetting with sheep red blood cells. Remaining cells were designated ‘null+’. Erythroid bursts grew from both normal unfractionated and null+ cells. By contrast, unfractionated cells from 10 patients with AA formed no colonies. However, after T-cell depletion, normal appearing bursts grew from the null+ fraction of two patients. Immune mediated AA is suggested when (1) unfractionated AA cells do not form colonies while (2) AA null+ cells do; moreover, (3) the addition of AA T-cells abrogates that growth and (4) addition of T-cells from untransfused AA patients decreases colony growth from normal null+ cells.
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New Dimensions in Routine Haematology R. M. ROWAN,C. FRASERand J. H. GRAY Department oj'Haematology, Glasgow Royal Injirmary, Glasgow
The Coulter Counter Model S Plus generates four new parameters and can plot platelet size distribution. The red distribution width (RD W) is an expression ofpart of the size distribution spread of the red cells and is an attempt to express size distribution in a single numerical value. The platelet distribution width (PDW) is a similar expression for platelets. Increased values for either, indicate greater than normal anisocytosis and low values indicate tendency to monosize. The mean platelet volume (MPV) and the platelet crit (PCT) are self-explanatory. Normal values have been defined for these new parameters: R D W (N=88), mean 9.9 units, 2S1> range 8.5-11.3 units; P D W (N=113), mean 16.1 units, 2SD range 15.3-16.9 units; MPV (N=113), mean 8.0 fl, 2SD range 6.3-9.7 fl; P C T (N= 113), mean 0.224%, 2SD range 0.1464302%. O f the 1000 samples processed, red cell abnormality was detected in 64.8% of samples and platelet abnormality in 28.6%. Platelet data was rejected in 0 6 % . O f the 648 samples showing red cell abnormality, 94% would have been recognized by conventional parameters but only 6% on the basis of R D W abnormality alone. For clinical correlation, all samples showing abnormality in R D W were assessed. O f the 121 R D W abnormalities found, only 17 were reduced and included a wide range of unrelated clinical disorders; however, there were three patients with chronic renal failure. Increased R D W was found in 104 patients. Of these, patients with acute blood loss showed slight elevation of R D W in the range 11.5-12.0 units; patients with chronic blood loss, untreated iron deficiency and untreated megaloblastic anaemia showed moderate elevation in the range 12.0-14.0 units; treatment of either iron deficiency or megaloblastic states produced a marked elevation as did blood transfusion in these patients of the order 144-20.0 units. Certain conclusions may be drawn from these observations. Firstly, although both the MCV and R D W are size measurements, they vary independently of each other. The R D W does not give any indication of direction of shift in cell size. Secondly, it appears that the increase in R D W predominantly reflects treatment rather than primary disease. Thirdly, an increased R D W does not parallel the reticulocyte count closely, it follows it. Finally, decrease in the R D W is of no proven value. It seems that the R D W simply indicates that increased anisocytosis exists. The same sequence of study was followed for the new platelet parameters. 52.1% of the total abnormality was flagged by the platelet count. 47.9% of the total was flagged by abnormality in the new parameters only. The MPV was abnormal on 106 occasions. I t was reduced in 13 patients, all of whom were myelo-suppressed for whatever reason. It was increased in 93 patients, the predominant clinical events being blood loss, vasculopathy, treated vitamin Blzlfolate deficiency and autoimmune thrombocytopenia in relapse. The P D W was abnormal on 139 occasions. It was reduced in 28, comprising an extremely miscellaneous collection of disease entities. It was increased in 121, and in 63% of these the increase was associated with an increase in the MPV. The PCT does not appear to give any useful information. Certain conclusions can be made from this limited study. If the MPV and P D W are meaningful, there is a vast amount of hitherto unsuspected platelet abnormality. The MPV gives more specific information than the PDW. H o w do the new parameters measure up in their ability to define clinical entities? O n the red cell side, much more information may be obtained from a full plot of the red cell size distribution rather than the part expression given by RDW. Such plotting facility is not available on the Model S Plus. O n the platelet side, more information may be obtained by manipulation of the platelet size distribution graph. This facility is available on the Model S Plus.
Abstracts of P a p e r s
Platelet Membrane Glycoproteins: a Comparative Study of Various Polyacrylamide Gel Electrophoretic Techniques G. T. E. ZONNEVELD, C. S. P. JENKINS and J. W. TEN CATE Department of Haematology, University Hospital Wilhelmina Gasthuis’, Amsterdam There is increasing evidence to support the fact that platelet membrane glycoproteins have a major role in platelet function. Abnormalities in glycoprotein content have been observed in the platelet membranes of patients with certain bleeding disorders using polyacrylamide gel electrophoresis (PAGE) in SDS-phosphate buffer. However, the glycoprotein defects associated with these disorders represent gross abnormalities of the platelet membrane; more subtle defects of platelet components and in particular of the surface glycoproteins may exist. Therefore other PAGE techniques together with radioactive labelling of the outer platelet surface could be of value in order to define more clearly these abnormalities. As platelet samples from patients are only available in limited quantities it is desirable to employ a PAGE technique where maximum resolution of the glycoproteins is obtained while maintaining sensitivity. Several PAGE techniques were compared. The system of Weber & Osborne (1969, Journal of Biological Chemistry, 244, 4406) gives the best separation of glycoprotein I, and Ib, and is suitable for the study of patients with the Bernard-Soutier syndrome (BSs). An adaptation of the system ofFairbanks et a1 (1971, Biochemistry, 10,2606) gives the best separation of glycoprotein 111, and IIIb, and this system is suitable for studying patients with Glanzmann’s disease (GT disease). The mobilities of the major and minor glycoproteins were correlated in these systems according to Clemetson et al (1977, Biochimica et Biophysica A d a , 464, 493) using platelet membranes of healthy volunteers and patients with known glycoprotein defects (GT disease and BSs). It was found that the mobilities were highly reproducible.
Mechanism of Activation of Prekallikrein B. N. BOUMA and J. H. GRIFFIN Department $ Haematology, University Hospital, Utrecht, and Scripps Clinic and Research Foundation, La Jolla, U.S.A.
Kallikrein, a proteolytic enzyme, effects the release of vasodepressor peptides or kinins (including bradykinin) from kininogens. These kinins can increase vascular permeability, contract smooth muscles, produce pain and influence the migration of leucocytes. Kallikrein can be derived from tissue or from plasma and the plasma enzyme circulates as an inactive zymogen prekallikrein. Plasma kallikrein is capable of generating fibrinolytic activity by activation of plasminogen, and it participates in the intrinsic pathway of blood coagulation, because kallikrein is capable of activating factor XII. Purified prekallikrein appeared to consist of two forms with very similar molecular weights. Both forms have a single polypeptide chain near 85 000 molecular weight. Factor XII, converts prekallikrein into kallikrein by limited proteolysis. The generation of kallikrein activity correlates directly with the extent of proteolytic cleavage. Kallikrein still has a molecular weight of 80 000. It consists of fragments held together by disulphide bonds. Three fragments were observed with apparent molecular weights of 52 000, 42 000 and 37 000. T w o forms of factor XII, were tested for capability of activating prekallikrein. The two chain form of factor XII, with a molecular weight of 80 000 (U-XII,) and the 28 000 single chain fragment (P-XIIJ were both capable of activating prekallikrein. The activation of
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prekallikrein by u-XII, but not by fl-XII, was potentiated by the presence of high molecular weight kininogen and by the presence of a negatively charged surface (dextran sulphate). High molecular weight kininogen formed a complex with prekallikrein as was shown by cross-immunoelectrophoresis. It is this high molecular weight kininogen which binds prekallikrein to a negatively charged surface where it can be activated by factor XII,. Once activated, the kallikrein molecules dissociate into the surrounding space.
Fibrinogen Manchester: an Abnormal Fibrinogen with Delayed Fibrinopeptide A Release D. A. LANE,M. VANROSS, V. V. KAKKAR, J. BOTTOMLEY,* K . DHIR,* L. P. J. HoLTt and J. E. MACIVER* Thrombosis Research Unit, King’s College Hospital Medical School, London, and *Department of Haematology* and tDepartment of Rheumatology, Manchester Royal Infirmary, Manchester An investigation has been carried out of a coagulation defect occurring in three generations of a family as an autosomal dominant trait. Coagulation tests revealed slightly elevated fibrinogen concentration, normal FDP level yet prolonged thrombin clotting time, suggesting a congenital dysfibrinogenaemia. Further plasma clotting studies demonstrated no pH dependence of the defect, little correction by calcium ions, but a total correction when normal and patient plasma was mixed in equal amounts. A dysfibrinogenaemia was confirmed when fibrinogen purified from plasma of one of the patients exhibited delayed clotting with thrombin. A small delay in the rate of polymerization of monomers was evident when purified patient fibrin monomers were compared with those of normals. Electrophoretic, immunoelectrophoretic and immunodiffusion experiments were able to detect neither structural nor immunological abnormalities associated with the intact molecule or with the Aa, Bfl and y chains of fibrinogen. Factor XIII, induced crosslinking of fibrin appeared normal as judged by SDS polyacrylamide gel electrophoresis. However, using a specific radioimmunoassay to fibrinopeptide A the major defect has been localized to a delay in the rate of release of this peptide by thrombin when the patient fibrinogen is converted to fibrin.
An Immunoenzymehistochemical Method for the Demonstration of Fibrin and Fibrin Degradation Products Separately J. J. EMEIS, W. NIEUWENHUIZEN and J. LINDEMAN* (introduced by P. Brakman) Gaubius Institute TNO, Leiden, and Department of Pathology, University Medicaf Centre, Leiden Recently, we described an indirect immunoenzymehistochemical method for the demonstration of fibrin microthrombi in tissue sections (Craane et a1 (1978) Histochemistry, 57,97). Using the same animal model (rats with induced disseminated intravascular coagulation), we subsequently developed a method for differentiating between fibrinogen, fibrin and fibrin degradation products (FDP). This differentiation is based on the following observations: (a) Only fibrin is retained in paraformaldehyde-fixed, paraplast-embedded tissue; while (b) FDP and fibrinogen are retained in ethanol-fixed, paraplastembedded tissue; (c) The FDP D and E do not react with antisera against the constituent Au-, BP- and y-chains of fibrinogen, in contrast to fibrinogen, fibrin and early FDP. (d) FDP do react with antisera against fibrinogen and FDP. Thus, it could be shown that in rats with DIC the early FDP, derived from the Au- and Bfl-chains of fibrin, are cleared through the kidneys, while the terminal FDP D and E are taken up by liver macrophages.
Abstracts o j P a p e r s
As our anti-rat antisera cross-react with human fibrin-related material, the method proved applicable to human autopsy or biopsy material. In cases of DIC, the same distribution of FDP was found.
Dissociate Factor VIII and Fibrinolytic Response to DDAVP C. A. LUDLAM, I. R. PEAKE and A. L. BLOOM Department of Haematology , University Hospital of Wales, Heath Park, Card13 The intravenous administration of the vasopressin analogue l-deamino-8-~-arginine vasopressin (DDAVP) increases both the circulating factor VIII concentration and fibrinolytic activity. Previous studies have demonstrated that procoagulant factor VIII (FVIIIC) and factor VIII related antigen (FVIIIRAG) rise in parallel in normal subjects and most patients with von Willebrand’s disease (vWd). In this study we have followed the factor VIII and fibrinolytic response to intravenous DDAVP in several patients with various types of vWd and haemophilia. One patient with classical vWd (FVIIIC 9 u/dl, FVIIIRAG 3 u/dl, ristocetin cofactor activity 12.0 fl, 17.0% had a disorder associated with platelet hyperdestruction, 13.5% had granulocytic leukaemia or sideroblastic anaemia, 10.6% miscellaneous thrombocytopenias and 46.2% Mediterranean MT, either isolated (36.1%) or accompanied by a haematological disorder (10.1 %). Platelet kinetic studies in 10 such cases demonstrated normal platelet life span together with moderate hypersplenism. No relevant aetiology was found in the remaining 12.7%, approximating the 10.5% predicted from the distribution of mean volumes in controls. The frequency of M T was three cases out offour in SLE, 6/9 in ITP, 4/10 in sideroblastic anaemia, 11/45 in AML, 6/32 in CML and 1/17 in megaloblastic anaemia. I n a sample of 1000 consecutive volume distributions there was a high correlation between the mean and Sr) of volumes (a corollary of the log normality of platelet volumes), but the mean and SD of log volumes behaved like nearly independent parameters. In addition to showing that M T is not pathognomonic of platelet hyperdestruction, these data are compatible with the notion that platelet volume parameters essentially reflect the combined rates of growth and demarcation of the platelet territories in megakaryocytes.
Automated Readout of Test Results of a BG 15 Autoanalyser Blood Grouping Machine in a Hospital Transfusion Service V. A . J. M. KUNST,J. H. BLOOand P. GEERDINK Transjiusion Service, University Hospital, Nijmegen A BG 15 Blood Grouping machine with an automated electronic readout device was used for AUO
Abstracts of Papers
(A1-Ar included) and rhesus D ( C and E included) testing (Rechsteiner & Benjamin (1976) Vox Sanguinis, 30, 445). Samples of 33 692 persons were investigated. About 70% were donor samples and 30% patient samples. The overall rejection rate was 6.7%; in 5.7% the ABO grouping was rejected and in 1.0% the rhesus D typing was not possible. When the rejected samples were run a second time, about half of them were still correctly determined. The rejection rate in the patient samples was somewhat higher (7.4%) than in the donor samples (5.1%). This was connected with the fact that 23% of the rejected patient samples contained irregular (mostly non-specific cold) antibodies in contrast to only 8% of the rejected donor samples. False results were given nine times (0.03%). Three time5 the ABO group was incorrect and six times the rhesus D group was determined negative instead of D” (4 x ) or positive (2 x ). In one of these cases a rhesus D positive patient was determined as D negative after transfusion of 3 units rhesus D negative donor blood. In vitro mixing experiments revealed that addition of 15-25% rhesus D negative blood to a rhesus D positive sample gives a rhesus D negative result in the Blood Grouping machine.
The Analysis of Leukaemic Cell Populations by Automated Cytochernistry using the Technicon Haemalog D
K. G. PATTERSON, J. D. M. RICHARDS, F. R. FELLINCHAM and A. H. GOLDSTONE Department of Haematology, U C H , London The Haemalog D performs three cytochemical staining reactions on blood or marrow suspensions, Peroxidase, Esterase and Alcian Blue. Each of 10 000 cells is assessed in respect of light absorption (cell staining intensity) and light scatter (cell size). By means of electronic thresholding, the different cell populations are separated on the basis of their size and staining intensity. Thus in the peroxidase channel eosinophils stain intensely, distinguishing them from neutrophils having moderate peroxidase activity and lymphocytes having little activity. Monocytes are counted on their own esterase channel, and basophils in their Alcian Blue channel. In addition to numerical differential counts, the instrument also generates an oscilloscopic XY display of 10 000 cells positioned according to their size and staining. In the peroxidase channel, large unstained cells (LUC) and cells of higher peroxidase activity than the neutrophil population (HPX) are counted. In chronic myeloid leukaemia the neutrophil population is characteristically large and poorly delineated at its edges and there is a high ‘HPX’ percentage. Acceleration of the disease is characterized by fragmentation and dispersion of this myeloid load with increasing HPX and increasing LUC. In acute myeloid leukaemia, the majority of blasts lie in the large unstained cell and ‘neutrophil’ areas. Acute lymphoblastic leukaemia is also associated with a high LUC percentage, but the major population of cells lies in the ‘lymphocytic’ range of size and peroxidase activity. Myelomonocytic leukaemia is characterized by a disperse pattern of peroxidase activity representing monocytoid cells a t all stages of differentiation, in addition to fluoride sensitive esterase positivity.
Serum Anti T Levels in Metastatic Melanoma K . HASHMI, J. G. CHANC* and N. T H A T C H E R t Haematology Department, Hope Hospital, Salford, *Department of Haematology and t University Department of Medical Oncology, Christie Hospital, Manchester
The T antigen, precursor to the M and N blood group antigens, has been demonstrated on the surface
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of cells of human breast and gastrointestinal cancers. The T antigen is not expressed on the surface of normal human cells. However, human serum contains anti T agglutinins, which are thought to arise due to continual antigenic stimulation from bacteria in the gastrointestinal tract. Anti T levels are now known to be depressed in the serum of patients with breast, lung and gastrointestinal cancers, and may act as a non-specific tumour marker. Anti T levels were estimated by the ability of diluted test serum to agglutinate neuraminidase treated pooled group 0 red cells under standard conditions. The greatest dilution which produced an agglutination was read as the titre of the serum and the anti T levels were expressed as titres. It was found that the titres remained unchanged after storage at -20°C for periods longer than 6 months. A normal range was established from 50 healthy blood donors, selected at random. Fifty-six patients with metastatic melanoma were studied. Their pretreatment anti T levels were significantly lower than the normal range (P