Analysis of cerebrospinal fluid cell populations with monoclonal antibodies.

Sixty-five samples of cerebrospinal fluid (CSF) were evaluated using an automated cytoflow method with the CD-Sapphire hematology analyzer in order to investigate possible relationships between cell population patterns and diagnostic groups and better understand the biology of neurological disease. A basic panel of CD markers, including CD3/4/8/19/138/HLA-DR, was used to analyze CSF samples from clinical and laboratory confirmed cases of multiple sclerosis, neuroborreliosis, viral and bacterial neuroinfective diseases, malignant infiltrations of meninges and scavenger macrophagic reactions of the central nervous system. The principles of immune response and the contribution of cytological 'disease-related patterns' for these nosological entities are described. The distinct patterns of lymphocyte subpopulations in neuroborreliosis appear to be characteristic and could possibly serve as diagnostic indicators. Further verification and research will be necessary to clarify the significance and nature of CD4+ CD8+ positive subset in cerebrospinal fluid.

Acute lymphoblastic leukaemia incidence in the UK by immunophenotype.

The incidence of acute lymphoblastic leukaemia (ALL) is described for both children and adults for the three major immunophenotypes: null, CD10-positive (CD+) which includes both common and pre-B types, and T-cell (including pre-T variants). The data are derived from a population-based specialist registry of leukaemias and lymphomas covering approximately one-half of England and Wales. Null ALL predominates in those under 1 year old and CD10+ ALL in the 1-7 year olds. There is a male excess at all ages for T-cell disease, which is particularly prominent in adolescents and young adults. The effect of socioeconomic levels is seen most clearly for CD10+ ALL in the childhood peak, where B-cell precursor disease occurs more frequently in areas of higher socioeconomic status.

Membrane transferrin receptor (TfR) and nuclear proliferation-associated Ki-67 expression in hemopoietic malignancies.

The expression of membrane transferrin receptors (TfRs), as defined by monoclonal antibody OKT9, and the nuclear proliferation-associated antigen Ki-67 were examined in 159 cases of hematological malignancy. Of the "chronic" B and T cell leukemias studied (n = 85), 61% showed less than 5% OKT9-positive cells and only 7% of cases were TfR+ (defined as greater than 20% positive cells). For comparison, the acute leukemias (n = 62) showed higher (p less than 0.001) TfR expression with 39% TfR+ cases, although there was considerable variation within diagnostic subgroups. Nuclear Ki-67 expression was generally insignificant (less than 1%) in chronic leukemias (78 of 88 cases), although two of eight B cell-type prolymphocytic leukemia and four of four cases of plasma cell leukemia showed greater than 10% Ki-67+ components. In contrast, 47% (31 of 66) acute leukemias had greater than 10% Ki-67+ cells, although there appeared to be no relationship between Ki-67 expression and leukemic type. Combined assessments of TfR and Ki-67 expression revealed a Ki-67- TfR- phenotype in 82% of chronic leukemias, compared with 28% of acute type, and a Ki-67+ TfR+ pattern was found in 27% of acute proliferations but not in any case of chronic leukemia. The determination of membrane TfR expression appears to have little value in the diagnostic differentiation of leukemias, whereas Ki-67 is considered to be a useful supplementary investigation in defining high grade tumors, in the recognition of prognostically poor cases of otherwise well defined low grade malignancy, and of potential value in resolving discrepancies between morphological and immunophenotypic features in leukemias of "intermediate" type.

Quantitative and qualitative enzyme studies of Hodgkin's disease-derived cell lines.

Any extensive analysis of Hodgkin (H) and Reed-Sternberg (RS) cells is limited by the scarcity of available material and by the common contamination with "by-stander" cells. The establishment of Hodgkin's disease-derived cell lines provides the opportunity to undertake studies in which large numbers of cells are required, as these cell lines are by definition monoclonal populations with unlimited cell growth. In this study, we analyzed the enzyme profiles of eight Hodgkin's disease cell lines (Co, Ho, Fox, HDLM-2, KM-H2, L428, L540, and L591) whereby cellular alpha-naphthyl acetate esterases, acid phosphatases, and dipeptidylpeptidase IV were examined quantitatively or qualitatively by IEF or chromatographic techniques. The results indicate that all of the H-RS cell lines examined had enzymatic features typical for lymphoid cells and, in particular, a monocyte/histiocyte origin of the cell lines could be excluded. Extrapolation of the available immunological, molecular biological, and enzymological evidence gained in vitro on cultured representatives of H-RS cells suggests a lymphoid origin for in vivo H-RS cells.

Sezary cell-like leukemia: a distinct type of mature T cell malignancy.

We describe the clinical, ultrastructural, and immunophenotypical characteristics of four cases of an unusual type of T cell leukemia. Clinical features included high WBC, ranging from 26-148 x 10(9)/liter, bone marrow infiltration, splenomegaly, and lymphadenopathy. Skin involvement was not documented at presentation, but it was seen as a terminal event in one patient with a pattern of dermal lymphocytic infiltration different from that usually seen in Sezary syndrome. By ultrastructural analysis, the circulating lymphoid cells were indistinguishable from small Sezary cells in two cases, resembled large Sezary cells in one case, and consisted of a mixture of small Sezary cells and prolymphocytes in the remaining case. The cells from all cases had a mature T cell phenotype, TdT-, CD1a-, CD2+/-, CD3+, CD5+. In addition, the cells were either CD8+, CD4- or CD8+, CD4+ or CD4-, CD8-; and, in only one case, the findings were similar to those of Sezary syndrome cells: CD4+, CD8-, CD7-, BE-2+. In the latter case, serological and immunological assays were positive for HTLV-I while these were negative in two other patients investigated. The features of these patients suggest that Sezary cell leukemia is a distinct clinico-pathological entity although the alternative diagnosis of adult T cell leukemia/lymphoma could not be excluded in the HTLV-I+ case. Sezary cell leukemia appears to be resistant to current chemotherapy regimens and is associated with an aggressive clinical course and short survival.

Analysis of cost and effectiveness of pre-transfusion screening of donor blood and anti-malarial prophylaxis for recipients.

OBJECTIVES: To determine the prevalence of malaria in donor units in a low and a high endemic region in Kenya and evaluate the cost effectiveness of recipient anti-malarial prophylaxis and pre-transfusion screening (using an automated method) as options to prevent post transfusion malaria. DESIGN: A descriptive cross-sectional study. SETTING: Two regional blood banks, Nairobi and its environs (National Blood Transfusion Services, Nairobi) a low malaria endemic region and western region (National Blood Transfusion Services, Kisumu) high malaria endemic region. SUBJECTS: All the donated units were included in the study for analysis, during the duration of study, from the two study sites. MAIN OUTCOME MEASURES: Prevalence of malaria in donor units in low endemic area (Nairobi) and high endemic area (Kisumu). Cost per case prevented for the two options, Option I Prophylactic administration of anti-malarial (sulfadoxine pyrimethamine SP) drugs to recipients, and Option II pre-transfusion screening using an automated technique. RESULTS: A malaria prevalence of 0.67% was found in Nairobi and its environments (low endemic) and 8.63% for Kisumu and its environments (high endemic area). The cost analysis showed a cost per case prevented of Ksh.105 (US$1.4) adult, Ksh.52.5 (US$0. 69) and paediatric for the option of recipient prophylaxis using an SP based drug. The cost escalated to Ksh.592 (US$7.79) adult Ksh.444 (US$5.84) paediatric if the prophylaxis was upgraded to the recommended artemisinin derivative (ACT-artemisinin based combination) and for the option of pre-transfusion screening using an automated technique the cost was Ksh.2.08 (US$0.03). CONCLUSION: The prevalence of malaria in donors showed the expected regional variation in the low and high endemic areas and was comparable to data obtained elsewhere. If malaria positive donor units were to be excluded from the national blood supply, an estimated 5% (compared to 1.3% for human Immunodeficiency virus, 3.6% for hepatitis B virus and 1.3% for hepatitis C virus) would be wasted. The cost per case prevented of transfusion-associated malaria is considerably higher for recipient antimalarial prophylaxis than pre-transfusion screening using an automated technique. The cost escalates by five to seven times if the newer artemesinin based combination antimalarial drugs are adopted.

Blood donor haematology parameters in two regions of Kenya.

OBJECTIVES: To determine the status of blood donor haematology in two regional sites in Kenya and to assess the potential role of automated haematology in National blood bank process control. DESIGN: A cross sectional descriptive study. SETTING: Two regional blood banks--Nairobi and its environs (Blood Transfusion Services, Nairobi) and Western Region (National Blood Transfusion Services, Kisumu). MAIN OUTCOME MEASURES: Distribution, mean, median, and 95% percentile ranges of haemoglobin (Hb), red cell parameters (red cell count, haematocrit, MCV, MCH and MCHC), total and differential white blood cell (WBC) counts, and platelet counts in the two donor populations. RESULTS: A significant number of donations (16.5% in Kisumu and 3.4% in Nairobi) showed haemoglobin levels below the recommended National Blood Transfusion Service (NBTS) guideline of 42g/unit. Compared to Kisumu, Nairobi donors had significantly (p < 0.001) higher Hb, MCV and MCH values while the red blood cell counts and MCHC values were similar (p > 0.05). A low MCV (< 78 fl) was observed in 12.4% and 3.4% of Kisumu and Nairobi donors respectively. Both populations showed similar but significant frequencies (Kisumu, 21.3%; Nairobi, 18.7%) of mild neutropenia (< 1.5 x 10(9)/1), while eosinophilia (> 0.5 x 10(9)/1 in the tropics the cut off is > 0.6 x 109) was more prominent in Kisumu donors (18.8% versus 8.5%). Platelet counts were also significantly lower in Kisumu donors, with the prevalence of thrombocytopenia (< 150 x 10(9)/1) being considerably higher (15.9% versus 3.7%). CONCLUSIONS: A significant number of Kenyan donors showed abnormal haematology profiles that may indicate underlying pathology. Such abnormalities are not detected by current blood transfusion services screening practices and there may be a need to strengthen donor selection criteria to protect both donors and recipients.

Classification of large granular lymphocyte (LGL) and NK-associated (NKa) disorders.

Literature trends indicate an increasing awareness regarding the frequency, nature and clinical associations of abnormal and persistent expansions of lymphocytes with cytoplasmic granulation. These particular cells, which represent a minor normal lymphoid subpopulation and are widely referred to as large granular lymphocytes (LGL), generally (but not invariably) express monoclonal antibody-defined membrane NK-associated (NKa) determinants and appear to functionally correspond to those populations involved in cellular cytotoxicity. Increased proportions or absolute numbers of blood lymphocytes with LGL morphology and/or NKa+ phenotypes are associated with a diverse spectrum of clinical (haematological and non-haematological) disorders and may be broadly viewed as secondary (acute and chronic reactive) or primary in nature. Both primary and secondary LGL/NKa+ expansions may be persistent in type and the clinical distinction between the two may be difficult. A number of investigators have proposed schemes for the classification of these disorders but, because of their diversity, abnormal LGL/NKa+ expansions often defy rigid compartmentalisation. This communication examines the general basis of these classifications and illustrates their limitations by reviewing the data for 97 patients recorded in the largest (Yorkshire Leukaemia Group) survey to date of persistent LGL/NKa+ expansions.

Effect of folic acid and vitamin C supplementation on folate status and homocysteine level: a randomised controlled trial in Italian smoker-blood donors.

BACKGROUND: This trial sought to examine the effects of high dosage of folic acid and vitamin C supplementation on red blood cell folate (RCF), serum folate (SF) and homocysteine (Hcy) levels in subjects who smoke more than 15 cigarettes per day. METHODS: A prospective study of 100 Italian repeat blood donors was undertaken to measure RCF, SF and Hcy levels before and after 45 days of vitamin supplementation. All subjects were randomised into four groups: [A] folic acid (FA) 5 mg/day, [B] vitamin C 500 mg/day, [C] FA 5 mg/day plus vitamin C 500 mg/day [D] no supplementation. RESULTS: Before supplementation the median RCF, SF and Hcy levels were similar in the four groups; 32 (40%) subjects had an RCF level below 340 nmol/l, 15 (18.8%) had an SF level below 6.8 nmol/l and 21 (26.3%) had an Hcy level above 16 micromol/l. After 45 days the median RCF and SF levels were significantly (P<0.01) increased in all supplemented subjects. The median Hcy level was significantly (P=0.008) reduced in subjects supplemented with FA and significantly (P=0.01) increased in those supplemented with vitamin C alone. CONCLUSION: The supplementation with 5 FA mg/day is able to increase significantly both RCF and SF levels and reduce Hcy level in Italian smoker-blood donors.

Quantitation of cell-surface beta 2-microglobulin (beta 2 m) using a comparative antibody inhibition assay.

An improved immunoenzyme assay for measuring cell-surface beta 2-microglobulin (beta 2 m) is presented in which quantitation is achieved by reference to soluble beta 2 m-induced inhibition of horseradish peroxidase-labelled rabbit antibody. Some consideration is given to kinetics of binding and dissociation, and their implications for interpretation are discussed.

Fractionation and further characterization of granulocytic and monocytic alpha-naphthyl acetate (ANAE) esterases.

Following characterization of myeloid nonspecific esterases by isoelectric focusing (IEF), two main groups of alpha-naphthyl acetate (ANAE) esterase isoenzymes were defined and fractionated from cytoplasmic extracts by chromato focusing techniques according to differences in their isoelectric points (pI). The first of these ANAE enzyme groups was common to leukocytes of both granulocytic and monocytic lineage, while the other, which characteristically comprised a group of isoenzymes within the pI range 5.5-6.1, was specifically associated with monocytic differentiation. The properties of the two purified ANAE enzyme fractions were compared by inhibition (heat and sodium fluoride) and further electrophoretic studies, and the results discussed in relation to the cytochemical characterization of these enzymes as markers of specific myeloid cell differentiation.

Inhibitor studies of purified haemopoietic (myeloid) cell esterases. Evidence for the existence of distinct enzyme species.

Human myeloid cells synthesize and express two major species of esterase, defined by isoelectric focusing (IEF). The first of these (MonEst) is specifically associated with haemopoietic cells of monocytic lineage, whereas the other species (ComEst) is common to all myeloid cells (granulocytes and monocytes) irrespective of lineage affiliation. Having recently purified these two species of human myeloid cell esterase, this present study extensively investigated the effects of 17 different inhibitors on their ability to hydrolyse the synthetic substrate alpha-naphthyl acetate (alpha NA). Significant inhibition of both ComEst and MonEst was exerted by 1% sodium dodecyl sulphate (SDS) and 1.0 mM diethyl pyrocarbonate (DEPC), but the patterns of inhibition for the two esterase species with the remaining compounds studied differed considerably; for example, 0.2 mM phenylmethylsulphonyl fluoride (PMSF), 5.0 x 10(-3) M dichloroisocoumarin (DCIC) and 0.1 mM N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) all inhibited MonEst but not ComEst. Mechanisms of inhibition were also examined and these studies established that SDS, PMSF, DCIC and TPCK irreversibly inactivated MonEst whilst the inhibition of ComEst by SDS was reversible. Analysis of inhibition kinetics further showed that (a) the reversible inhibition of both ComEst and MonEst by sodium fluoride (NaF) was noncompetitive (with Ki values of 1.28 and 0.01 mM, respectively, indicating a marked difference in sensitivity); (b) the inhibition of MonEst by PMSF was of 'mixed' noncompetitive-competitive type; and (c) that DEPC exerted noncompetitive inhibition with similar Ki values (0.05 mM) for both esterase species. These observations unequivocably demonstrate that ComEst and MonEst are unrelated enzyme species, with a common ability to hydrolyse alpha NA, and that these esterase show marked differences with respect to their active sites as adjudged by inhibitor sensitivities. These observations are particularly relevant to the histochemical analysis of these enzymes and to the elucidation of their in vivo functions.

Trichloroethylene and cancer: epidemiologic evidence.

Trichloroethylene is an organic chemical that has been used in dry cleaning, for metal degreasing, and as a solvent for oils and resins. It has been shown to cause liver and kidney cancer in experimental animals. This article reviews over 80 published papers and letters on the cancer epidemiology of people exposed to trichloroethylene. Evidence of excess cancer incidence among occupational cohorts with the most rigorous exposure assessment is found for kidney cancer (relative risk [RR] = 1.7, 95% confidence interval [CI] 1.1-2.7), liver cancer (RR = 1.9, 95% CI(1.0-3.4), and non-Hodgkin's lymphoma (RR = 1.5, 95% CI 0.9-2.3) as well as for cervical cancer, Hodgkin's disease, and multiple myeloma. However, since few studies isolate trichloroethylene exposure, results are likely confounded by exposure to other solvents and other risk factors. Although we believe that solvent exposure causes cancer in humans and that trichloroethylene likely is one of the active agents, we recommend further study to better specify the specific agents that confer this risk and to estimate the magnitude of that risk.

Modes of action of trichloroethylene for kidney tumorigenesis.

This article focuses on the various models for kidney toxicity due to trichloroethylene (TCE) and its glutathione-dependent metabolites, in particular S-(1,2-dichlorovinyl)-l-cysteine. Areas of controversy regarding the relative importance of metabolic pathways, species differences in toxic responses, rates of generation of reactive metabolites, and dose-dependent phenomena are highlighted. The first section briefly reviews information on the incidence and risk factors of kidney cancer in the general U.S. population. Epidemiological data on incidence of kidney cancer in male workers exposed occupationally to TCE are also summarized. This is contrasted with cancer bioassay data from laboratory animals, that highlights sex and species differences and, consequently, the difficulties in making risk assessments for humans based on animal data. The major section of the article considers proposed modes of action for TCE or its metabolites in kidney, including peroxisome proliferation, alpha(2u)-globulin nephropathy, genotoxicity, and acute and chronic toxicity mechanisms. The latter comprise oxidative stress, alterations in calcium ion homeostasis, mitochondrial dysfunction, protein alkylation, cellular repair processes, and alterations in gene expression and cell proliferation. Finally, the status of risk assessment for TCE based on the kidneys as a target organ and remaining questions and research needs are discussed.

Expression of MHC class I and class I-like gene products on the cell membrane of mature and immature T cells.

Beta 2-microglobulin (beta 2m) constitutes the 12 kD light chain of the MHC-encoded 43-kD glycoprotein HLA-ABC molecules, which are expressed on most nucleated cells. The T6 (CD1a) antigen is expressed on thymocytes in reciprocal manner to HLA-ABC, having a similar structure to the HLA-ABC molecule. We have determined the expression of beta 2m, HLA-ABC and the T6 antigen on the cell-surface of CD1a+ and CD1a- thymocytes (defined by NA1/34 expression), and have shown that despite immunophenotypic differences between these two stages of thymic maturation, the quantity of beta 2m-associated molecules expressed was not significantly different. The nature of the heavy (alpha) chain associated with beta 2m, however, differed since HLA-ABC had largely replaced T6 on the surface of CD1a- thymocytes. We also determined the expression of beta 2m and HLA-ABC on the surface of peripheral blood CD4+ and CD8+ T-cells, and showed that the CD8+ population expressed higher levels of these antigens than CD4+ T-cells, with no detectable excess beta 2m over HLA-ABC for either subpopulation. In addition, thymocytes expressed fewer beta 2m-associated determinants than peripheral blood T-cells. These results indicate an increase in the expression of beta 2m-associated molecules with differentiation from thymic to peripheral T-cell, with a further increase in such molecules expressed on the CD8+ compared to the CD4+ peripheral T-cell subpopulation.

Quantitative and qualitative studies of leukaemic cell dipeptidylpeptidases II and IV.

Cellular concentrations of dipeptidylpeptidases (DAP) II and IV were determined by fluorimetric assay in 152 cases of leukaemia and 24 permanent T-cell lines. Whilst its estimation appears to have few diagnostic applications, it was found that DAPII levels were generally higher in prolymphocytoid CLL variants (CLL-Pro) compared with "typical" CLL cases. Qualitatively, DAPII extracted from leukaemic B- and T-cells did not differ significantly with respect to molecular weight (Mr 82 kD: FPLC gel filtration) or substrate affinity (Km). Similarly, a single molecular weight species (Mr 189 kD) of DAPIV was also detected in all leukaemic sonicates examined although, in quantitative terms, increased DAPIV levels were primarily associated with a subgroup of CD4-positive postthymic malignancies. Studies of immature T-cell proliferations however indicated that DAPIV expression was unrelated to either stage of immunological differentiation or CD4 expression. No evidence of lineage restriction was found for either enzyme and it is concluded that variations in DAP cytochemical staining patterns reflect quantitative rather than qualitative differences.

Fast protein liquid chromatography (FPLC) of leukaemic cell N-acetyl beta-D hexosaminidases.

Leukaemic myeloid and lymphoid cell N-acetyl beta-D glucosaminidase (hexosaminidase) enzymes were fractionated by Fast Protein Liquid Chromatography (FPLC) using high-resolution ion exchange (Mono-S and Mono-Q), gel filtration (Superose-6) and chromatofocusing (Mono-P) columns. Although only one molecular weight species was detected in haemopoietic cells, with an apparent mass of 129 kD, "isoenzyme" variants defined by differences in molecular charge were considerably more diverse than previously thought. Separation of the major Hex forms (A and B) by chromatofocusing indicated that intermediate (I) components were present in most acute leukaemias irrespective of lineage supporting the concept that the I form is a non-specific marker of haemopoietic immaturity. Substrate and inhibitor studies further revealed that leukaemic cell hexosaminidases hydrolyse galactopyranosides at significantly lower rates than glucopyranosides and that the hydrolysis of N-acetyl beta-D glucosamine is inhibited by both glucosamine and galactosamine products.

Prolymphocytoid transformation of CLL: a clinical and immunological study of 22 cases.

The clinical, morphological and immunological features of 22 cases of chronic lymphocytic leukaemia in 'prolymphocytoid' transformation (CLL-Pro) are reported. Immunophenotypic patterns in CLL-Pro differ from CLL by the appearance of significant (greater than 15%) FMC7-positive components and/or increased SIg densities in most cases. Membrane TU1 and MRBC receptor expression was similar to that found in typical CLL. Morphologically, all cases showed a mixture of small lymphocytes and larger nucleolated 'prolymphocytes' although the degree of prolymphocytoid change was unrelated to immunological patterns. Clinically, the cases behaved in a very heterogeneous fashion, with some patients dying rapidly following transformation despite treatment, while others even if untreated had a long, stable and relatively benign course. It was not possible to predict which patients would do badly from immunological or morphological features but the presence of more than one involved lymph node site and the occurrence of B-symptoms appeared to identify a group that did badly. Immunological assessments were however important in therapeutic terms, drawing distinction between CLL-Pro variants and prolymphocytic leukaemia (PLL).

Prolymphocytoid variants of chronic lymphocytic leukaemia: an immunological and morphological survey.

Of 417 cases of chronic lymphocytic leukaemia (CLL) examined morphologically, 346 (83%) showed low (less than 10%) numbers of cells with prolymphocytoid (PLC) morphology. Immunological studies in 267 of these cases, with particular reference to membrane surface immunoglobulin (SIg) densities and FMC7 expression, revealed that of 223 cases with insignificant prolymphocytoid components, 185 were phenotypically consistent with typical CLL whilst the other 38 cases showed significant (greater than 20% positive cells) FMC7 expression and/or increased SIg density. In contrast, 40 of the 44 cases with greater than 10% PLC showed atypical immunophenotypic features even though the expression of individual membrane components associated with B-cell differentiation appeared to be unrelated. MRBC receptor expression showed little correlation with the degree of prolymphocytoid change although all TU1- cases showed greater than 10% PLC. The results suggest that abnormal phenotypic features, similar to those observed in "prolymphocytoid" transformation, may be found in cases of CLL in the absence of morphological change.

Serum enzyme and ferritin concentrations in acute leukaemia.

Serum ferritin concentrations were determined in 142 untreated cases of acute leukaemia. No correlation between type of leukaemia as defined by morphology and immunology and the level of serum ferritin was found. Samples were also tested for lactate dehydrogenase (LDH), phosphohexose isomerase (PHI), B-glucuronidase (B-gluc), leucine aminopeptidase (LAP), and C-reactive protein (CRP) levels. Serum ferritin was significantly correlated with serum PHI, LAP, and LDH concentrations but not with leukaemic mass as assessed by total white blood cell count (WBC). Ferritin and CRP levels were also significantly correlated suggesting that ferritin may behave to some extent like an acute phase reactant in acute leukaemia.