A rapid isolation procedure of plasma membranes from human neutrophils using self-generating Percoll gradients. Importance of pH in avoiding contamination by intracellular membranes.

In this study we report an overall procedure for the isolation of both human polymorphonuclear neutrophils and their plasma membrane, by means of self-generating Percoll gradients. After efficient purification (40% yield), neutrophils were lysed by nitrogen cavitation and cellular structures quickly isolated in a one-step procedure. Plasma membrane recovery was monitored by [3H]concanavalin A and 5'-nucleotidase (EC 3.1.3.5) activity. We showed the latter activity is indeed present in human neutrophils. The procedure resulted in a good yield of plasma membrane, since 45% and 55% of total 5'-nucleotidase and [3H]concanavalin A activity, respectively, were recovered within two gradient fractions. Depending on the final pH of the Percoll gradient medium, endoplasmic reticulum markers contaminated either the plasma membrane or the granule fractions. At pH 9.05, NADH-ferricyanide reductase activity clearly separated from plasma membrane markers and displayed the same profile as CDPcholine:diacylglycerolcholine phosphotransferase (EC 2.7.8.2), a typical enzyme of endoplasmic reticulum. These results emphasize the need for strict monitoring of the pH of the gradient medium in subcellular fractionation of neutrophils.

Expression of basic fibroblast growth factor (bFGF) and FGF-receptors in human leukemic cells.

Recent reports have suggested that basic fibroblast growth factor (bFGF) could play a permissive role in hematopoiesis, in combination with specific colony-stimulating factors. We investigated the expression of bFGF and FGF-receptors (FGF-Rs) in leukemic cell lines of various hematopoietic lineages. Three protein isoforms of bFGF of approximately 18, 22 and 24 kDa were detected in the myeloid cell line K562, but not in myelomonocytic or lymphoid (T or B) cell lines. In vitro-induced differentiation of K562 cells did not change the pattern of expression of the different bFGF isoforms. Accordingly, the mRNA of bFGF was found expressed in K562, but not in other leukemic lines tested, as assayed by reverse transcript amplification (RT-PCR). Using the same technique, we searched for the presence of high affinity FGF-Rs on these cells: in eight out of ten cell lines tested, mRNA for at least one FGF-R among FGF-R1, FGF-R3 or FGF-R4 was expressed, whereas FGF-R2 was never detected. We found that two cell lines were responsive to bFGF in different biological assays: (i) in K562 myeloid cells induced to differentiate by hemin, preincubation with bFGF and heparin increased cell viability and decreased hemin-induced DNA fragmentation, without affecting erythroid differentiation; and (ii) in U937 monocytic cells, the production of plasminogen activator was increased by bFGF or aFGF in combination with heparin. Binding experiments with 125I-bFGF (up to 200 pM) in the presence of heparin revealed high affinity receptors on the K562 and U937 cell lines (1177 +/- 440 and 392 +/- 184 sites/cell, Kd = 61.7 +/- 8.6 and 43.1 +/- 13.5 pM, respectively). Thus our results strongly suggest that cells of hematopoietic origin could express functional FGF-receptors.

Predictive factors of postrenal transplant anemia.

OBJECTIVE: The aim of our study was to identify independent factors that might predict anemia at 6 months' (M6) and 12 (M12) months' posttransplantation. Postrenal transplant anemia was defined as a hemoglobin (Hb) level below 13 g/dL for men, and below 12 g/dL for women. We included 99 renal transplants performed in our department in 2001, for whom the graft was still functioning at 1 year. RESULTS: Anemia was observed in 78%, 35.5%, and 25% on day (D) 0, and at M6, and M12, respectively. Iron deficiency was observed in 14% of patients at D0, and 13% at M12. During the postoperative period, 59.8% of patients received at least one blood transfusion, whereas 37% of patients were prescribed recombinant erythropoietin (rEpo) therapy within the first few months posttransplantation. By multivariate analysis the independent predictive factors for anemia at M6 were rEpo therapy at D0, initial nephropathy, posttransplantation rEpo therapy, hematocrit at M3, platelets at D7 and sirolimus therapy. The independent predictive factors for anemia at M12 were rEpo therapy at D0 and platelets at D7, delayed graft function (DGF), serum creatinine, and creatinine clearance at M12, and Hb level at M6 were also checked. CONCLUSION: The prevalence of anemia is 25% at M12; DGF, renal function at M12, and anemia at M6 were independent risk factors of anemia at M12.

Neutrophils of healthy aged humans are normal.

Polymorphonuclear functions are reported to be altered in aged humans. We have previously shown that chemotactic response, adherence, oxidative metabolism and Candida killing activity were abnormal in subjects over 70 years. These results lead us to investigate further the basic mechanisms of these alterations in a complementary series of elderly people over 75 years. However, we used in the present study the admission criteria of the SENIEUR protocol specially designed for immunogerontological investigations in humans. Neither the tests exploring the functions as a whole (migration and bacterial killing) nor those investigating the elementary components of these functions exhibited significant difference when compared to the sex-matched young controls. The discrepancy with our prior results is probably explained by the fact that the presently tested subjects had been selected according to more strict criteria. These data clearly demonstrate that neutrophils are intrinsically normal in the aged. Thus, it appears that the frequently observed neutrophil dysfunction in the elderly is due to the action of abnormal humoral components related to the aging process on otherwise normal neutrophils.

Isolation of bone marrow plasma cells by negative selection with immunomagnetic beads.

In order to isolate bone marrow plasma cells from patients presenting with multiple myeloma or monoclonal gammopathy of undetermined significance, we developed a method for purifying these cells by negative selection using monoclonal antibodies and immunomagnetic beads. The results presented here were obtained from 75 procedures. Purity was extremely variable (2-100%) and was dependent on the percentage of plasma cells in the original bone marrow sample with a 10% cut-off, beyond which purity was over 96% in all cases. The mean yield was about 20%. The cells collected were viable and suitable for immunophenotyping, semi-quantitative studies of oncoproteins, and PCR.

Atypical neutrophil alkaline phosphatase associated with impaired neutrophil functions.

We report the case of a healthy young man presenting with atypical neutrophil alkaline phosphatase (NAP) and reduced neutrophil chemotactic activity, but with no susceptibility to infection. NAP activity was low, kinetic parameters were modified and immunoreactive properties and subcellular distribution were abnormal. Neutrophil morphology was normal. A similar pattern was observed in the patient's healthy brother. The profile of the observed anomalies offers some similarity to that previously described in patients with chronic myelogenous leukaemia. However, in the present case, the NAP deficiency with impaired neutrophil function was present in two brothers with no haematological symptoms and is probably related to a non-acquired neutrophil abnormality. This observation of a primary NAP variant reinforces the hypothesis of a direct link between NAP activity and functional properties of neutrophils.

Simultaneous study of platelets and phagocytes in myeloproliferative disorders.

In order to determine the relationship between the anomalies affecting two types of blood cell in myeloproliferative disorders (MPD), a functional study was performed in individuals presenting with such diseases. Thus, platelet function was investigated by means of Ivy's method for bleeding time, platelet retention to glass beads, aggregation with epinephrine and density distribution on a discontinuous sucrose gradient. Simultaneously, three granulocyte functions, i.e. capillary tube random migration, particle ingestion activity and nitroblue tetrazolium dye reduction were studied. This investigation was carried out in 47 patients presenting with chronic myeloproliferative disorders (MPD): chronic granulocytic leukemia (18 cases), polycythemia vera (18 cases), myelofibrosis (6 cases) and essential thrombocythemia (5 cases). The results of the present study indicate that functional abnormalities are more frequent and more strongly marked in platelets than in phagocytes. The tests most affected were platelet density distribution and granulocytic random migration. Simultaneous assessment of platelet and phagocytic functions, though insufficient in itself to determine the type of MPD or to appraise the prognosis of the disease, could be useful in the diagnosis of some atypical cases of myeloproliferative disorders.

[Evaluation of an automatic analyzer of reticulocytes: the Sysmex R-1 000]. / Evaluation d'un analyseur automatique de réticulocytes: le Sysmex R-1 000.

The microscopic enumeration of reticulocytes is a tedious assay with poor reproducibility. Its results provide only fragmentory information on the kinetics of erythropoiesis. Thus, attempts have been made for its automation. This study presents results of an evaluation of the first machine exclusively devoted to reticulocyte analysis. The Sysmex R-1 000 uses flow cytofluorometry with argon laser, after precipitation of nucleic acids by auramine-O. The whole study was performed according to ICSH recommendations. At least 2,500 erythrocytes were examined in the microscopic reference technique, after 1 p. cent brilliant cresyl-blue staining. Within and between-batch precision studies gave CV not exceeding 5.46 and 8.72 p. cent respectively for reticulocyte numbers. In dilution study it was shown that no measured result was over 4 p. cent of the expected value, in the range 4-400 x 10(9)/l. There was no significant carry-over in the range tested (35-510 x 10(9)/l). Accuracy testing proved that the significant difference observed when microscopic enumeration was performed in routine conditions disappeared when at least 5,000 erythrocytes were examined. Keeping the samples at 4 degrees C limited the decrease of the reticulocyte number to less than 10 p. cent after 24 hours. Analysis of some particular clinical situations demonstrated the risk of spurious results in case of massive Plasmodium infestation, and the usefulness of obtaining the percentage of newly emerged reticulocytes, i.e. the most fluorescent, for a better estimate of the kinetics of erythrocyte production. This evaluation testifies the quality of the Sysmex R-1 000, and underlines the potential benefit of such analyzers in the equipment of the modern hematology laboratory.

[Computer-assisted validation system applied to hematology: Valab-hemato]. / Système de validation assistée par ordinateur appliqué à l'hématologie: Valab-hémato.

Validation of laboratory reports is the ultimate step before transmission of results to the clinician. The biologist checks the intrinsic consistency of the data as well as their possible medical value that is liable to lead to other investigations. Such a policy, when performed on all the data, is time-consuming, boring and uncertain. This step may be simplified by the use of a computerized expert system. The computer assisted validation system presented here concerns routine haematology data (Valab-haemato). Like its predecessor devoted to clinical chemistry (Valab-Biochem) it is based on the performance of a powerful inference engine which generates a decision-making tree for each report according to the data. This adaptability gives the system a capacity very close to human reasoning. In its haematology version the system deals with many variables including sex, age, origin of the patient (hospital ward), and the haematological data (blood cell count, differential, reticulocyte count, various information drawn from microscope examination of the blood smear as well as any report concerning the blood sample, erythrocyte sedimentation rate). Previous data are also taken into account, as well as the normal ranges, the values beyond which no result can be automatically validated and the delta-check. Some information definitely prevents validation of the results, others can be validated if they have been previously approved. Whereas the method of reasoning is fixed, all items are changeable in order to adapt the system to the type of activity of the laboratory.(ABSTRACT TRUNCATED AT 250 WORDS)

[CD30 positive pilotropic lymphoma]. / Lymphome pilotrope CD30 positif.

BACKGROUND: We report an unusual case of cutaneous CD30-positive lymphoma with pilar tropism and circulating Sezary cells which had a rapidly fatal course. CASE REPORT: A 78-year-old man presented erythematous infiltration of the face, a pruriginous eruption on the trunk and proximal portions of the limbs with small erythematopurpuric follicular papulae, and node enlargement in the inguinal and axillary areas. The rest of the clinical examination was normal. Circulating Sezary cells were found in significant numbers on two different blood smears. Histologic and immunohistochemistry examination of a skin biopsy evidenced medium to large sized lymphoid cell infiltration in a perifollicular localization. A few small cells penetrated the pilar apparatus. There was no follicular mucinosis. The tumoral cells expressed CD2, CD3, CD4 and 75 p. 100 were positive for CD30. Node aspiration showed lymphomatous cells and CD3+ and CD30+ lymphomatous infiltration was found on marrow smears. A T clone was evidenced both in blood and bone marrow leading to the diagnosis of pilotropic CD30-positive lymphoma. Chlorambucil and prednisone were given. The patient died 5 months later. DISCUSSION: The cytology findings suggest medium to large cell pleomorphic lymphoma. The circulating Sezary cells, the pilotropic eruption, and the rapidly fatal outcome suggest transformation of a Sezary syndrome into CD30-positive large cell lymphoma which has been described in fungoid mycosis.

[Value of of fine-needle aspiration cytology and MRI in parotid gland masses]. / Intérêt de la ponction cytologique et de l'IRM dans les tuméfactions parotidiennes.

INTRODUCTION: The objective of our study was to discuss the valve of fine-needle aspiration cytology (FNAC) and magnetic resonance imaging (MRI) in the diagnosis and treatment of parotid gland masses. MATERIALS AND METHODS: Forty patients were included in the prospective study. They had undergone clinical examination, FNAC and MRI before parotidectomy. The results of these examinations were compared with the corresponding histopathological diagnosis. RESULTS: When it is positive, FNAC is a good examination of malignant tumours (sensitivity 67%, specificity 79%, positive predictive value 86%, negative predictive value 100%). The MRI allows a good assessment of the tumoural mass and its anatomical relationships (sensitivity 55%, specificity 86%, positive predictive value 89%, negative predictive value 75%). If the T2 sequence shows reduced density (p < 0.05) or in case of bad limitation (p = 0.004), a malignant character is strongly suspected. CONCLUSION: In cases of parotid gland mass, where surgical intervention is necessary, there is no need of special investigations: however FNAC and MRI allow us to anticipate what operation will be required.

Linezolid-related pancytopenia in organ-transplant patients: report of two cases.

Linezolid is a recent oral antibiotic used in drug-resistant Gram-positive cocci infection. We herein report on the first two cases of linezolid-related pancytopenia in organ-transplant patients. Both patients had methicillineresistant Staphylococcus aureus infections. Pancytopenia, i.e. aregenerative anemia, neutropenia and thrombopenia, developed 3 weeks and 5 weeks after initiating linezolid therapy at a conventional dosage (600 mg bid). There were no other confounding causes of pancytopenia, which resolved promptly after withdrawing linezolid. Because of the potential hazards of pancytopenia in immunosuppressed organ-transplant patients, we advocate the cautious use of linezolid for transplant patients.

Blood cell parameters do not change during physiological human ageing.

Despite the considerable amount of data published during the last 30 years concerning the hematological status of the aged, practitioners are still confronted with the problem of 'normal' or 'physiological' values in these subjects. Indeed, significantly different values are presented in the literature as normal for the aged, apparently depending on the various types of populations studied. Criteria used to define the subjects as 'normal' could be the main factor responsible for this discrepancy. Based on this hypothesis, we determined the values of usual blood cell parameters in two groups of apparently healthy subjects of over 75 years of age. Subjects of group A (16 men and 13 women) were selected according to the stringent criteria of the Senieur protocol and subjects of group B (15 men and 20 women) were recruited using 'conventional' admission criteria. For both groups, sex-matched young controls aged 20-29 years were tested in parallel. No clinically significant alteration was observed in group A, whereas more pronounced differences were found in group B. Our results confirm the necessity for stringent selection criteria in biological investigations in gerontology. Moreover, they demonstrate that blood cell parameters are not altered in the aged, provided that the latter are really 'healthy'.

Evaluation of a new haematology analyser for whole blood count and full differential (NE-8000).

We evaluated the fully automated haematology analyser TOA Sysmex NE-8000 over a three-month period according to the ICSH protocol using as reference techniques a Coulter STKR counter and microscope examination for WBC differential and cell morphology. The NE-8000 employs aperture impedance to perform cell counts, with a sheath fluid to focus cells hydrodynamically prior to counting and sizing. WBC are differentiated into five populations. A combination of aperture impedance, radio frequency measurement and differential cell shrinkage is used, eosinophil and basophil percentages being established in two separate channels and subtracted from the total granulocyte count in order to give the value for neutrophils. Analysis of 1060 samples processed by the closed sampling automode demonstrated satisfactory counting performance. Among the WBC differentials obtained from 100 samples, neutrophil, eosinophil and lymphocyte results correlated well with those from microscopic examination of blood smears performed according to the NCCLS standard H20 T protocol. Differences observed in the percentages of basophils were of no biomedical significance. A comparative study for monocytes showed poor correlation for values below 5% and above 10%, best results being obtained in the intermediate range 5-10%. The NE-8000 also demonstrated good reliability for detection of abnormal cells.

Bone marrow enrichment technique for detection and characterization of scarce abnormal cells.

A frequent problem encountered in analysis of bone marrow aspirates is the small number of suspect cells in the material. We present a method for concentration of the cells in bone marrow aspirates which yields smears suitable for immunocytochemical techniques. Bone marrow sampling is performed in two steps: a first aspirate is used to prepare conventional smears and a second aspirate is submitted to two-step centrifugation to separate and collect the nucleated cells without use of a separation medium. Sufficient material is obtained to prepare a large number of films, thus allowing immunocytochemical investigation with a wide panel of monoclonal antibodies. The proportions of the different cell types are similar to those observed in conventional smears and cell morphology is unaltered. This enrichment procedure improves the accuracy of routine cytological bone marrow examination, may be easily applied in laboratories performing bone marrow studies and in our hands has proved of value for the detection and characterization of both malignant blood diseases and bone marrow dissemination of carcinoma.

Human neutrophils are not severely injured in conditions mimicking social drinking.

In an attempt to assess the effect of alcohol per se on human polymorphonuclear neutrophils (PMN), irrespective of other physiopathological parameters, we examined neutrophil function in healthy volunteers who had taken a single large dose of whisky. Before and at different times after ingestion, several PMN properties were simultaneously tested including random migration, in vitro chemotaxis, adherence, aggregation, cytochrome C reduction, phagocytosis, bacterial killing, intracellular cAMP and cGMP contents, myeloperoxidase, and neutrophil alkaline phosphatase scores. Only phagocytosis of Staphylococcus aureus was significantly depressed after alcohol ingestion. Adherence was inhibited only in some individuals when their respective blood alcohol levels were the highest. Both alterations were moderate and reversible. These data point out the limited effect of occasional alcohol consumption on the different facets of neutrophil behavior. These findings suggest that factors other than alcohol itself could be concerned in the marked PMN dysfunction well established in chronic alcoholism.