Alejandro Román

Flow cytometry and the study of central nervous disease in patients with acute leukaemia.

Central nervous system (CNS) leukaemia is still a matter of debate and new technologies are required to improve the classic morphological definition. One hundred and sixty‐eight cerebrospinal fluid (CSF) samples from 31 patients with acute leukaemia were analysed by flow cytometry and conventional cytology. Concordant positive and negative findings were found in 158 samples but 10 produced discrepant results. Cytology seemed to offer more precise information in one CSF sample and flow cytometric accuracy could be demonstrated in five samples. We conclude that flow cytometry is of great help in confirming CNS leukaemia and eliminating other conditions. Therefore, leukaemic patients can benefit from double cytological and flow cytometric CSF studies.

Flow Cytometric Analysis of Cerebrospinal Fluid Samples and Its Usefulness in Routine Clinical Practice

Low volume and few cells have hampered the use of flow cytometry for studying cerebrospinal fluid (CSF) in routine clinical practice, although information about the cellular phenotypes present in this type of sample is of great value in many diseases. We developed a novel flow cytometric strategy capable of identifying total CSF T lymphocytes and the CD4+ subset, even in CSF samples with as few as 1 leukocyte per 3 microL of sample. We also showed that identification of CD8+ T cells could be achieved in most samples, while B lymphocytes are detectable only in samples with more than 5 cells per microliter. These findings demonstrate the reliability of this method to improve the diagnostic accuracy of classic cytologic studies in many neurologic disorders.

Immunophenotype in chronic myelomonocytic leukemia: is it closer to myelodysplastic syndromes or to myeloproliferative disorders?

Chronic myelomonocytic leukemia (CMML) is a heterogeneous disease balanced between myelodysplastic syndromes (MDS) and myeloproliferative disorders (MPD). We used flow cytometry to describe and compare the immunophenotypic profile of 20 patients with CMML, 38 patients with MDS, and 20 patients with MPD. CMML and MDS only showed statistically significant differences (P<0.05) in CD56 monocyte expression. CMML and MPD showed significant differences in CD45 myeloid distribution, myeloid antigenic profile, CD56 and CD2 monocyte expression, and B-cell development. These data support the classic concept of CMML as part of MDS diseases and encourage including immunophenotyping among the studies to be performed in these diseases.

Evaluation of thrombophylic states in myeloma patients receiving thalidomide: a reasonable doubt

An increased incidence of deep venous thrombosis (DVT) has been described in multiple myeloma (MM). Its pathogenesis is multifactorial but the presence of other states of inherited or acquired thrombophilia can increase this hypercoagulability. Thalidomide has been used recently to treat refractory MM, and an increased risk of thrombosis has been reported when it is employed in combination with other chemotherapeutic agents (Zangari et al, 2001; Urbauer et al, 2002). We present a MM patient who was a homozygous carrier of the C677T mutation of the MTHFR gene who developed a DVT and a pulmonary embolism (PE) during thalidomide treatment. A 60-year-old man with stage IIIA Bence Jones MM was treated at diagnosis with conventional chemotherapy, and he achieved a complete response. During treatment, the patient suffered a DVT in the right subclavian vein associated with a central venous catheter. After disease relapse, thalidomide was initiated at 200 mg ⁄ d, in combination with 15-d cycles of dexamethasone. The thalidomide dose was increased by 200 mg ⁄ d every 2 weeks as tolerated. The patient reported constipation and drowsiness at the dose of 400 mg ⁄ d. At 800 mg ⁄ d, he had a slight polyneuropathy which disappeared when the dose was reduced to 600 mg ⁄ d. In the seventh month of treatment, the patient presented with progressive dyspnoea without other symptoms. On examination, the patient was obese, eupnoeic at rest and had normal cardiopulmonary examination. He had slight malleolar oedema without signs of DVT. Laboratory studies showed: haemoglobin 10Æ7 g ⁄ dl, and normal leucocyte count, platelet count, prothrombin time and activated partial thromboplastin time, and biochemically, fibrinogen 4Æ88 g ⁄ l, and proteinuria 700 mg ⁄24 h without evidence of Bence Jones protein. An arterial blood gas analysis showed hypoxaemia. D-dimer (by enzyme-linked immunosorbent assay) was 2428 ng ⁄ml (68–494 ng ⁄ml). The chest radiograph was normal and an electrocardiogram showed sinus tachycardia and repolarization disturbances. Pulmonary perfusion scintigraphy and helical computerized tomography were suggestive of PE. An echo-Doppler of the lower limbs detected a thrombosis in the right popliteal vein. Following the diagnosis of PE and DVT, anticoagulant treatment was initiated with good clinical results. A thrombophilia study indicated that he was homozygous for the C677T mutation of the MTHFR. High serum levels of homocysteine, 26 IU ⁄ml (normal < 15 IU ⁄ml), were also observed. Hyperhomocysteinaemia is associated with an increased risk of thrombosis. It is usually the result of acquired causes, although the MTHFR variant can contribute (Walker et al, 2001). Factor V Leiden and prothrombin G20201A, the most common genetic risk factors for thrombosis, were both negative. Other rare deficiencies of natural coagulation inhibitors, such as antithrombin III, protein S, protein C and plasminogen are detectable in less than 1% of the population. Our patient showed functional antithrombin III and plasminogen within the normal ranges. Acquired risk factors (antiphospholipid syndrome, oral contraceptives, major surgery) are also responsible for a large number of thrombotic events (Koster et al, 1993). The patient was negative for lupus anticoagulant and anticardiolipin antibodies. The patient had no family history of thrombosis. Thalidomide is an immunomodulator that inhibits the angiogenesis induced by vascular endothelial growth factor and fibroblast growth factor, although the exact mechanism of thalidomide in MM is unknown (Stirling, 2000). The percentage of DVT associated with treatment with thalidomide and chemotherapy including dexamethasone varies according to the series (4–28%) and a lower percentage of PE cases has been reported. This rate is greater than in MM patients not treated with thalidomide (Zangari et al, 2001; Zangari, 2002). A recent study indicates that a strong association exists between DVT and exposure to doxorubicin–thalidomide combination (Zangari et al, 2002). Nevertheless, no increase in the risk of thrombosis has been observed in MM treated with thalidomide as the only agent (2%). The thrombotic mechanism seems to be multifactorial in the DVT patients described. In our patient, the potential risk factors were MM, older age, obesity, relative immobility and genetic factors (MTHFR C677T homozygous with high levels of homocysteine), in addition to the treatment with thalidomide and dexamethasone. All cases of thrombosis described during the treatment with thalidomide have resolved favourably with anticoagulant treatment (Zangari et al, 2001; Urbauer et al, 2002), as in our patient. For this reason, the development of thrombosis is not an absolute contraindication for continuing treatment. Patients with a personal and family history of thrombosis must be investigated for genetic and acquired prothrombotic factors before they begin thalidomide treatment. Therefore, prophylactic anticoagulation should be considered in selected patients. British Journal of Haematology, 2003, 122, 159–167