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.

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.

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.

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.

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.

[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)

[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.

Distal renal tubular acidosis and ovalocytosis: a case report.

A 23-year-old man presented with osteoporosis, revealed by femoral fractures, and a history of nephrolithiasis, short stature, metabolic acidosis, hypokalemia and ovalocytosis, a red blood cell abnormality common in malaria endemic regions. Biological investigations led to the diagnosis of type 1 distal renal tubular acidosis (dRTA). Ovalocytosis and dRTA may co-exist in the same patient, since both can originate in mutations of the anion-exchanger 1 (AE1) gene, which codes for band 3, the bicarbonate/chloride exchanger, present in both the red cell membrane and the basolateral membrane of the collecting tubule alpha-intercalated cell.

Reticulocyte analysis using flow cytometry.

Automation of the reticulocyte count by means of flow cytometry has considerably improved the quality of this investigation. This article deals firstly with the reasons for the poor performance of the microscopic technique and with the physiological principles underlying identification and classification of reticulocytes using RNA labeling. It then outlines the automated methods currently on the market, which can be classified in three categories: a) "general-purpose" cytofluorometers, which in clinical laboratories usually deal with lymphocyte immunophenotyping; b) the only commercially available cytofluorometer dedicated to the reticulocyte count; this automat has the advantage of requiring no human intervention as it merely needs to be fed with samples; c) hematology analyzers with specific modules for automatic counting of reticulocytes previously incubated with a non-fluorescent dye. Of the various fluorescent markers available, thiazole orange, DEQTC iodide and auramine are most often used for this basic hematology test. The quality of the count, the availability of new reticulocyte indices (maturation index, percentage of young reticulocytes) and rapidity of the count give this test renewed value in the practical approach to the diagnosis of anemia, and also open new perspectives in the surveillance of aplastic anemia after chemotherapy or bone marrow grafting.

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.