A. Lopez Borrasca

Surface marker analysis in acute myeloid leukaemia and correlation with FAB classification.

Summary. The immunological phenotype of blast cells in 102 patients with acute myeloid leukaemia (AML) was analysed with a panel of 20 monoclonal antibodies and the enzyme terminal transferase, and correlated with the FAB classification. Although a partial correlation between these two approaches could be observed, almost every morphological group contained patients from more than one immunological phenotype. The M1 and M5a leukaemias showed the most undifferentiated phenotype, often lacking in specific myelomo‐nocytic antigens. The M3 formed a uniform group defined as My7+, Ia‐, FMC8+, a phenotype which was also observed in two cases of the microgranu‐lar variant. The granulocytic (CDwl5) and monocytic (CDwl4) antibodies crossreacted with some M5b and M2 leukaemias, respectively. Compared with M5a, the M5b leukaemias showed a large increase in the expression of CDw l4 antigen, confirming the validity of the morphological differentiation. Giyco‐phorin‐A was present in four out of five M6 leukaemias. TdT activity was demonstrated in 10% of AML cases, with a higher incidence among the monocytic variants: M4 and M5‐. Eleven AML were considered as unclassifi‐able according to the FAB criteria and in seven of them a megakaryoblastic cell population (GP IIb/IIIa+, GPIb+) was demonstrated; this confirms the need to include the subgroup of megakaryoblastic leukaemias within the AML. Finally, a possible immunological classification for AML is proposed.

Immunological phenotype of neoplasms involving the B cell in the last step of differentiation

Summary. The immunological phenotype of diseases involving the last step of B cell differentiation—multiple myeloma (MM, 38 patients) and Waldenströms macroglobulinaemia (WM, 12 patients)—was analysed with a panel of monoclonal antibodies (McAb) as well as conventional markers.

Development of acute leukaemia after idiopathic myelofibrosis.

AIMS: To determine the characteristics of blastic transformation of idiopathic myelofibrosis. METHODS: The clinical and haematological features, as well as the morphological characteristics of blast cells, were analysed in nine adults with blast transformation. RESULTS: Most of the patients were male and had enlarged spleens and livers. Five of the patients had normal platelet counts, while all had pronounced anaemia and a moderate degree of leucocytosis. The duration of the acute phase was usually short: 16 (SD 8) weeks. Most myeloid cell lineages--granulocytic, monocytic, and megakaryocytic--were similarly distributed. One patient also had a hybrid (lymphoid-myeloid) phenotype. The morphological assessment of blast cells agreed with immunophenotyping in five out of the nine cases. The onset of the blastic phase was not related to previous treatment. CONCLUSIONS: A pluripotential stem cell with preferential myeloid commitment would be the target cell of blast transformation in idiopathic myelofibrosis. Our immunophenotypic data do not support the concept of a preferential association between megakaryocytic lineage and the acute transformation of idiopathic myelofibrosis. The absence of previous treatment in some cases suggests that this kind of evolution is part of the natural history of idiopathic myelofibrosis.

CEREBROSPINAL FLUID LEVELS BETA2 MICROGLOBULIN AND FERRITIN IN LYMPHOPROLIVERATIVE DISORDERS

Several recent reports have emphasized the potential usefulness of determinations of plasma B2 microglobulin (B2m) and femtin in the evaluation and prognosis of some patients with lymphoproliferative disorders (LD) (1, 2, 3). Recently the level of B2m in cerebrospinal fluid (CSF) has been considered a useful marker of infiltration in the CNS during myeloproliferative and lymphoproliferative processes (4). To establish whether CSF B2m and ferritin concentrations reflect differences between patients with lymphoid neoplastic involvement in CNS and patients with bacterial meningitis, we have correlated their levels in both processes. Material and methods. CNS fluid was collected from 26 patients who had LD, with or without clinical evidence of CNS involvement. In 9 patients the diagnosis of CNS infiltration was based on the presence of tumor cells in CSF, along with compatible neurologic symptoms and signs. Specimens from 6 patients with bacterial meningitis (5 with Neisseria meningitis, 1 with Hemophilus influenza) were evaluated as well. Because CSF from normal donors was not available, our reference values of B2m and ferritin were determined on 10 patients with various diseases (6 cerebral trombosis, 2 headache and 2 head injury) with CSF normal. A sample of the fluid was centrifuged and stored at -20°C. B2m and ferritin levels in CSF were measured by radioimmunoassay using the “Phadebas B2 micro test” and “Phadebas ferritin test” (Pharmacia Diagnostic, Uppsala, Sweden). The statistical analyses used were Student’s t for B2m; for ferritin, the values do not have a normal distribution, which is why statistical analysis was camed out with nonparametric tests (Mann-Whitney’s U test) ( 5 ) . Results and comments. Table 1 and Figs. 1 and 2 show the results obtained. Comparing the different groups, there was statistic significance for B2m and ferritin between patients with lymphoid neoplastic involvement in CNS and patients with LD without CNS infiltration (p<O.OOl and p<O.Ol, for B2m and ferritin, respectively). Patients with neoplastic infiltration and patients with bacterial meningitis did not show any difference i-ese proteins. The levels of B2 m and fe&itin were serially determined in CSF from two patients with

DDAVP IN A NON-HAEMOPHILIC PATIENT WITH AN ACQUIRED FACTOR VIII INHIBITOR

Recently, de la Fuente et uZ(1985) have shown that the infusion of DDAVP (1 deamino-8-~ arginine vasopressin) in a patient with a low titre of acquired factor VIII inhibitor was able to induce a significant response of the activities related to the factor VIII/von Willebrand factor (FVIII/VWF) and was able to neutralize the circulating inhibitor. These authors wondered if DDAVP infusion might release sufficient factor VIII to obtain satisfactory levels in patients who have higher antibody titres. We have studied the response of FVIII/VWF activities to DDAVP infusion in a woman with a type I1 post-partum inhibitor, whose inhibitor titre at the time of study was 13 Bethesda units and whose factor VIII procoagulant activity was 3 u/dl. DDAVP (0.4 pg/kg) was added to 50 ml of physiological saline and infused over 30 min. Blood samples were collected immediately before and at 30, 120 and 300 min after infusion was ended.

Assessment of Multimeric Structure and Ristocetin-Induced Binding to Platelets of Von Willebrand Factor Present in Cryoprecipitate and Different Factor VIII Concentrates

Abstract. The multimeric structure of von Willebrand factor (vWF) and its ristocetin‐induced binding to platelets, using a simple and very sensitive radiomonoclonal antibody‐labeled vWF method, was compared in normal plasma, single‐donor cryoprecipitate (CP) and five different antihemophilic factor (AHF) concentrates. All the AHF showed a lack of larger vWF multimers, an abnormal ‘triplet’ pattern, and much lower vWF binding to platelets than that of plasma or CP, vWF being the lowest for those with a lesser proportion of larger vWF multimers. These results suggest that the combination of vWF multimeric analysis and the radiomonoclonal‐labeled vWF method may be very useful in the assessment of AHF preparations.

DDAVP in uremia.

Dr. V. Vicente, Departments of Hematology and Nephrology, University Hospital, Salamanca (Spain) Dear Sir, In recent years it has been shown that the intravenous injection of DDAVP (l-deamino-8-Darginine-vasopres-sin), a synthetic analogue of the antidiuretic hormone, provokes a rapid, marked, and transient increase of factor VΠI/von Willebrand factor (FVΠI/VWF) in normal subjects and patients with mild hemophilia A or von Wille-brand’s disease [1–3], Recently, Mannucci et al. [4] report that infusions of DDAVP shorten the prolonged bleeding time in patients with uremia. These authors suggest that this is dependent on the appearance in plasma of larger von Willebrand factor multimers than those present in the resting state. Uremia is commonly associated with wide variations in the levels of FVIΠ/VWF-related activities [5,6]. One of us has suggested that the response to DDAVP in subjects with elevated baseline concentrations of FVIΠ/VWF activities is accompanied by a decreased release from those storage pool(s) mobilized by DDAVP [7]. In the report of Mannucci et al. [4], levels of FVΠI/VWF activities were moderately elevated. We have evaluated the response of FVIΠ/VWF and bleeding time to DDAVP (0.3 μg/kg body weight) in 5 uremic patients (3 men and 2 women with an average age of 34 years, range 16–50) with chronic glomerulonephritis who were undergoing regular hemo-dialysis. These patients showed high levels of FVIΠ/VWF activities at the beginning of the study, which was begun 24 h after the end of dialysis (table I). The remaining clinical characteristics were similar to those described by Mannucci et al. [4]. Bleeding time and plasma collection were carried out immediately before DDAVP and again 1 h after the DDAVP infusion. Factor VIΠ/VWF activities were assayed as previously described [8]. Template bleeding time was assessed with the Simplate II device (General Diagnostic). Table I shows that the bleeding time was not Table I. Laboratory measurements before and after DDAVP in patients with uremia

ELECTROBLOTTING OF FACTOR VIII/VON WILLEBRAND FACTOR MULTIMERS AFTER ELECTROPHORESIS IN SDS–AGAROSE GEL, DISCONTINUOUS BUFFER SYSTEM

The paper by Furlong & Peake (1983) provides an interesting system to analyse factor VIII/von Willebrand factor (FVIII/vWF) multimeric structure by transferring the FVIII/vWF multimers to a nitrocellulose paper from SDS-agarose gel. This method shows that normal plasma multimers appear as single bands, whereas FVIII concentrates besides having more abundant lower molecular weight multimers they appear with a ‘doublet’ pattern. Type IIA von Willebrand’s disease (vWd) have wider bands for the lower molecular weight multimers than those seen in normal plasma. We have been working on FVIIIIvWF multimeric structure using a variation of the discontinuous buffer system of Laemmli with SDS-2% agarose gels (Zimmerman et al, 1983) that allows better resolution of FVIII/vWF structure. Recently we have been able to transfer the FVIII/vWF multimers from SDS-2% agarose gels to nitrocellulose paper. The method was similar to that described by Furlong & Peake (1983) with some modifications. The transfer

The phenotype of L-CFU and its correlation with the immunological characteristics of the blast cell population in AML.

SummaryThe membrane phenotype of AML clonogenic cells (L-CFU) was analyzed in 19 AML patients using an in vitro culture technique after a complement-mediated lysis assay employing a panel of six monoclonal antibodies (McAb) -HLA-DR, FMC56 (CD9), FMC27 (CD9), CD14, CD15, CD41a-. Our results show that L-CFU has a heterogeneous but immature phenotype lacking on the expression of differentiation antigens (CD14, CD15, CD41a). In addition, we observed that the L-CFU phenotype is different from that of the whole blast cell population. Interestingly, L-CFU showed a higher expression of HLA-DR antigens with respect to their progeny. Upon analyzing whether the L-CFU phenotype was related to both the morphological and immunological features of AML blast cells, it was observed that, while there is no correlation with the FAB classification, there was a partial relationship between the immunological phenotype of AML blast cells and that of L-CFU. Accordingly, the more immature AML cases showed a more differentiated L-CFU phenotype (HLA-DR+, CD9+, FMC27+) when compared with cases with a more mature blast cell phenotype. These results suggest that those AML cases with a relatively immature myeloblastic phenotype may arise from a progenitor cell that has undergone partial differentiation and that is unable to acquire myeloid differentiation antigens, while those AML cases with mature blast cells might emerge from a very early L-CFU that has the capacity to undergo a greater degree of differentiation.

Ulcer necrotic legs as first manifestation of protein S deficiency

Protein S is a vitamin K-dependent plasma protein which increases the rate of inactivation of factor Va by activated protein C. Recently, several families with members affected by a hereditary deficiency of this protein asociated with thrombosis have been reported [1-3]. Generally, the first thrombotic event in these patients appears towards the end of the second decade of life. In this report we describe two siblings (FD and MD) aged 29 and 27, who at the age of 10 and 12, respectively, presented with leg ulcers, especially in maleolar areas, with no apparent cause; both of them failed to respond to the treatment prescribed. At the age of 19, FD had clinical symptoms of deep vein thrombosis in his left leg complicated by pulmonary emboli. He received anticoagulant orally for 6 months, after which his leg ulcers improved. At the age of 23 years, abdominal pains appeared, accompanied by splenomegaly, melena and hematemesis. An exploratory laparotomy was performed and a diagnosis of mesenteric and portal vein thrombosis was established. The other brother MD presented at 24 years with abdominal episodes similar to that of his brother. After laparotomy extensive mesenteric vein thrombosis was found. In both cases, immediately after surgery oral anticoagulant therapy was started. This led to the disappearance of leg ulcers and no other thrombotic manifestations reappeared. Three years later, the two siblings were admired to our hospital in order to study their hypercoagulability state. Coumarin therapy was discontinued in both cases and replaced by heparin. Fifteen days after stopping oral anticoagulant therapy and of receiving intravenous administration of vitamin K, total and free protein S levels were measured by electroimmunoassay technique using rabbit antiserum purchased from Diagnostica Stago (Asni6res, France). Free protein S antigen was measured in the supernatant from plasma precipitated with PEG 8000 [4]. Total protein S antigen from the propositi were 41% and 31% (normal range 63-147), whereas free protein S antigen was undetectable in both cases (normal range 52-131). At the time of study, the propositi had normal levels of biological and immunological activities of factors II,