Polymorphonuclear functions in Hodgkin's disease patients at diagnosis, in remission, and in relapse.

Five tests investigating different aspects of the nonspecific defense mechanisms including capillary tube random migration, particle ingestion activity, quantitative and histochemical nitroblue tetrazolium dye reduction by polymorphonuclear neutrophils, and serum lysozyme concentrations were performed in 46 patients with Hodgkin's disease. The anomalies observed in the active stage of the disease consisted of a decreased random migration, a high level of serum lysozyme, and an increased nitroblue tetrazolium reduction by resting phagocytes associated with a decrease in nitroblue tetrazolium reduction by stimulated phagocytes. The particle ingestion activity was normal. The serum lysozyme assay was the only test observed to normalize in the group of patients in remission. Its determination, therefore, offers an additional means of evaluating disease activity.

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.

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.

Necessity of the assessment of health status in human immunogerontological studies: evaluation of the SENIEUR protocol.

Disease is frequent in ageing, and the many conflicting results in studies of the ageing process can be due to the presence of factors such as underlying disease or the use of medication. For immunogerontology, a solution to this problem was initiated in 1984 by a working party of EURAGE, the European Community's Concerted Action Programme on Ageing and Diseases. A protocol defining strict admission criteria to studies of ageing, the SENIEUR protocol, was elaborated. This protocol intends to limit the influence of disease and/or medication and to standardize admission criteria to immunogerontological studies. In subjects fulfilling the SENIEUR criteria, we found less immunological defects with ageing than generally stated. This could mean that many studies performed in not-optimally healthy subjects describe defects that are not a consequence of the ageing process, but could be a result of underlying disease or of the influence of medication. For lymphocyte subsets, certain changes are only found in the comparison of SENIEUR groups of young and aged, while other changes are only found when non-healthy groups are compared. The occurrence of monoclonal gammopathies and autoantibodies was increased in ageing, but was also influenced by health status. Experience of other groups, and the objections against the protocol are discussed.

Monoclonal gammopathies in human aging: increased occurrence with age and correlation with health status.

To determine the incidence of monoclonal gammopathies (MG) in relation to the aging process as such, and to evaluate the influence of disease on the occurrence of MG, we studied 439 elderly subjects aged 75-84 years. These individuals were categorized into 4 groups on the basis of their health status. There was a group of "optimally healthy" elderly, a group of "apparently healthy" residents of homes for the aged, a group of geriatric outpatients and a group of randomly chosen inpatients from a general hospital. Whereas no MG were detected in a control group of healthy young subjects aged 25-34 years, the frequency of MG in the aged groups ranged from 11% in the "optimally healthy" aged group to 38% in the inpatients group. In a tentative classification according to possible cause, most of the MG belonged to the pathogenetic category of immunodeficiency. There was a clear association of the occurrence of monoclonal gammopathies of this category with the health status.

Admission criteria for immunogerontological studies in man: the SENIEUR protocol.

Immunogerontological studies in man have often led to conflicting results. One of the main reasons is the selection of the subjects to be studied. Admission criteria such as "apparently healthy" or "without overt disease" seem insufficient to exclude underlying disease which might influence the immune system and thereby the results. In an attempt to solve this problem, the SENIEUR protocol described in this paper was developed by a working party in the framework of the EURAGE Concerted Action Programme on Ageing of the European Community. This protocol establishes strict admission criteria for immunogerontological studies in man based on clinical information and laboratory data, and it sets limits to pharmacological interference. The use of this protocol will lead to standardization between centers and also to a closer study of the influence of age as such on the immune system. These findings in the immunologically "optimally aged" can also serve as reference values for immunogerontological studies in subjects who do not meet the SENIEUR criteria. In this way the use of this protocol can contribute to the dissection of the influence of disease versus ageing on the immune system.

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.

Polymorphonuclear functions and aging in humans.

To determine whether normal aging interferes with the functional capability of polymorphonuclear leukocytes (PMNs), 6 tests of PMN function were performed in 285 healthy subjects whose ages ranged from 20 to 97 years. A second selection based upon blood measurement and a review of medical histories 6 months later, eliminated 68 subjects. The 217 remaining persons were sub-classed by age into 7 groups including equal numbers of males and females. The functional properties of PMNs in the aged, when compared to those of younger adults, were characterized by: (a) a decrease in the chemotactic response in the 80+ age group: (b) increased adherence, with onset after age 70, maximal after age 80; (c) a progressive decrease of NBT dye reduction capability, up to age 70-79, followed by an unexplained increase of the mean value after age 80; (d) diminished Candida-killing activity, appearing in the 60+ group and becoming lowest in the oldest group; and (e) lack of changes in spontaneous migration and endocytosis. The mechanisms by which this impairment occurs are hypothetical. It is proposed that normal PMNs, after leaving the bone marrow, are influenced by various humoral components such as metabolic byproducts or immune processes altered by aging. Thus the defective PMN may represent only another victim of the aging process.

Phagocytosis in myeloproliferative disorders.

The phagocytic function was investigated by means of four tests (capillary tube random migration, phagocytosis of yeast particles, quantitative nitroblue tetrazolium dye reduction and whole-blood bactericidal activity for Staphylococcus aureus) in 57 patients who had myeloproliferative disorders: 24 had chronic granulocytic leukemia, 22 had polycythemia vera, six had myelofibrosis, and five had essential thrombocythemia. This study confirms the previously reported functional anomalies of phagocytosis in all the myeloproliferative disorders and shows that, despite these anomalies, the increased number of phagocytes allows an efficient whole-blood bactericidal activity, essential for the nonspecific host defence mechanisms.

Chronic granulomatous disease with leukocytic glucose-6-phosphate dehydrogenase deficiency in a 28-month-old girl.

A 28-month-old girl, whose parents are first cousins, was hospitalized following a series of severe infections. Results of functional granulocytic tests permitted the diagnosis of chronic granulomatous disease (lack of nitroblue tetrazolium dye reduction, impaired bactericidal activity for Staphylococcus aureus but normal activity for Streptococcus foecalis). Random migration was also impaired, and leukocytic glucose-6-phosphate dehydrogenase (G6PD) activity was decreased (37% of the normal mean). In contrast, erythrocytic G6PD activity was normal. Similar leukocytic studies of both parents revealed a moderate decrease of the mother's leukocytic G6PD activity (62% of the normal mean). This case represents an additional argument in favor of the recessive autosomal transmission of chronic granulomatous disease in females.

Heterogeneity of erythrocyte pyruvate kinase deficiency and related metabolic disorders in patients with hematological diseases.

In several patients suffering from congenital non-spherocytic hemolytic anemia or from malignant hemotological disorder associated with erythrocyte pyruvate kinase (PK) deficiency, a metabolic study has been carried out involving the following biochemical determinations: assay of red cell enzyme activities; estimation of glucose consumption; measurement of the rate of glycolytic intermediates; and, in some cases, enzyme purification and characterization of the PK variant. Metabolic equilibrium most probably does not depend on kinetic characteristics of PK molecules. Furthermore, the data obtained allow separation of cases with congenital non-spherocytic hemolytic anemia (hereditary defect) and acquired PK deficiencies.

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.

Inflammatory/haematopoietic cytokine production by human bone marrow adipocytes.

Adipocytes are now considered as secretory and endocrine cells. White and brown adipocytes synthesize and secrete a variety of cytokines, among a number of peptide and non-peptide products. Some of these cytokines, particularly IL-6 and TNF-alpha, appear multifunctional since they may be involved in the control of adipose mass, inflammatory response and haematopoiesis. Bone marrow adipocytes are another abundant type of adipocytes, but their precise role in humans is poorly understood. In the present study, we demonstrate that, in contrast to non-medullary adipocytes, human bone marrow adipocytes in primary culture secrete only trace amounts of IL-1 beta and TNF-alpha, but, they secrete significant and regulated levels of IL-6. These results reinforce the concept of functional heterogeneity of adipose tissues according to their anatomical localization, and indicate that bone marrow adipocytes may contribute to the complex network of cytokines involved in the control of haematopoiesis.

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)

[Recommended algorithms of prescription in the diagnosis of iron deficiency and overload]. / Algorithmes de prescription recommandés pour le diagnostic d'un déficit et d'une surcharge en fer.

In view of the recent development of new tests of biochemistry and molecular biology the assessment of iron status should be reconsidered and updated. The French Society of Clinical Biology (SFBC) and the French Society of Hematology (Cellular Hematology Group) recommend algorithms in the diagnosis of iron deficiency and iron overload bearing in mind the best efficiency and health economy. These recommendations are based on the known sensibility and specificity of each test. The analytical requirements for the determination of the tests as well as the clinical and biological signs evoking an iron deficiency or overload are recalled.

[Etiological aspects of reactive hemophagocytoses: retrospective study in 99 patients]. / Aspects étiologiques des hémophagocytoses réactionnelles: étude rétrospective chez 99 patients.

We describe the causes of reactive hemophagocytic process in a retrospective study including 99 patients. The main diagnosis were: lymphomas (18 cases), pyogenic bacteria infections (15 cases), herpes virus infections (12 cases), other infections (multiple, parasitic, fungal, mycobacterial, unidentified) (11 cases), acute hepatitis (five cases), systemic lupus erythematosus (three cases). We also found numerous other diseases involving the reticuloendothelial system. The cause remained undetermined in 16 cases. Lymphoma accounted for 64% of the cases in previously healthy patients who had been febrile for more than 10 days at the time of the diagnosis of reactive hemophagocytic process, and for 31% in HIV-positive patients. Lymphomas were rare (5%) in non HIV-positive, immunosuppressed patients. In this setting and in previously healthy patients who had been febrile for less than 10 days, infectious diseases were widely dominant (respectively 60% and 86% of the cases). Those were mainly due to pyogenic bacteria and to herpes virus. A rapidly fatal evolution occurred in some cases of lymphomas-related hemophagocytic process. These data support the choice of aggressive investigations in order to diagnose lymphoma in previously healthy patients presenting with reactive hemophagocytic process who have been febrile for more than 10 days, and in selected HIV-patients. Such a procedure is not recommended in the other cases.

[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.