International standards for minimum potency of anti-A and anti-B blood grouping reagents: evaluation of candidate preparations in an international collaborative study.

BACKGROUND AND OBJECTIVES: The aim of the study was to evaluate lyophilized monoclonal IgM anti-A and anti-B preparations for use as international standards (IS) to specify recommended minimum potencies of anti-A and anti-B blood grouping reagents in tube tests. MATERIALS AND METHODS: The candidate IS for minimum potency of anti-A and anti-B blood grouping reagents, codes 03/188 and 03/164, respectively, were evaluated against a wide range of commercial anti-A and anti-B blood grouping reagents in an international collaborative study involving 16 laboratories in nine countries. Laboratories titrated 03/188 and 03/164 in parallel with as many commercial anti-A and anti-B blood grouping reagents, respectively, as were available to them, in tube tests according to specified haemagglutination methodology. Three of these laboratories and a further laboratory also titrated 03/188 and 03/164 in parallel with currently available reference preparations for anti-A and anti-B. The ratios of the mean endpoint titres of the anti-A and anti-B reagents to those of 03/188 and 03/164, respectively, within each laboratory were calculated. RESULTS: The ratios of the mean titres of the anti-A reagents to the mean titre of 03/188 within a laboratory fell within 0.062 and 4, i.e. the potencies of the anti-A reagents were between a sixteenth to four times as strong as 03/188. The ratios of the mean titres of the anti-B reagents to the mean titre of 03/164 within a laboratory also fell within 0.062 and 4, with one outlier. CONCLUSIONS: By international consensus, a 1 in 8 dilution of the candidate IS for anti-A, 03/188, and a 1 in 4 dilution of the candidate IS for anti-B, 03/164, were considered appropriate to define the recommended minimum potencies of anti-A and anti-B blood grouping reagents, respectively, in tube tests. On the basis of these results, preparations 03/188 and 03/164 were established by the World Health Organization as International Standards for Minimum Potency of Anti-A and Anti-B Blood Grouping Reagents respectively, and by the US Food and Drug Administration Center for Biologics Evaluation and Research as Minimum Potency Reference Reagents.

An International Standard for specifying the minimum potency of anti-D blood-grouping reagents: evaluation of a candidate preparation in an international collaborative study.

BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate a lyophilized monoclonal immunoglobulin M (IgM) anti-D preparation for use as an International Standard to specify a recommended minimum acceptable potency of anti-D blood-grouping reagents. MATERIALS AND METHODS: The candidate International Standard (99/836) for specifying the minimum potency of anti-D blood-grouping reagents was evaluated against a wide range of commercial anti-D blood-grouping reagents in an international collaborative study involving 20 laboratories in 13 countries. Laboratories titrated reconstituted 99/836, in parallel with as many commercial anti-D blood-grouping reagents as were available to them, in tube tests according to specified haemagglutination methodology for low-protein (e.g. monoclonal IgM) and high-protein (e.g. polyclonal) reagents. The ratios of the mean end-point titres of the reagents to that of 99/836 within each laboratory were calculated. RESULTS: The ratios of the mean titres of the low-protein reagents to the mean titre of 99/836 within a laboratory fell between 0.25 and 2 for 43 of the 45 low-protein anti-D reagents tested (i.e. the potencies of the low-protein reagents compared with 99/836 were between a 1:4 dilution of 99/836 to twice as potent as 99/836). The ratios of the mean titres of the high-protein reagents to the mean titre of 99/836 within a laboratory fell within 0.125 and 1 for eight out of the 10 high protein reagents tested. CONCLUSIONS: By international consensus, a 1:3 dilution of reconstituted 99/836 was deemed appropriate to define a recommended minimum acceptable potency of low-protein anti-D blood-grouping reagents. A 1:8 dilution of reconstituted 99/836 was deemed appropriate to define a recommended minimum acceptable potency of high-protein anti-D blood-grouping reagents. On the basis of the results presented here, 99/836 was established by the World Health Organization as the 1st International Standard for specifying the minimum potency of anti-D blood-grouping reagents, in tube tests.

International reference reagents: antihuman globulin. An ISBT/ICSH joint working party report. International Society of Blood Transfusion. International Committee for Standardization in Haematology.

An international working party has conducted a study designed to select a suitable reference reagent for antihuman globulin, to replace those first made available in 1987. The chosen preparation contains levels of anti-IgG and anti-C3 (anti-C3c and anti-C3d) potency that are considered suitable to serve for reference when evaluating either polyspecific antihuman globulin reagents or those containing their separate monospecific components. The reference material is available in 2-ml freeze-dried aliquots from seven assigned distribution centres.

Comparison of benzo[a]pyrene DNA adduct formation in the skin of two strains of mice selected for resistance (DBA/2) or susceptibility (C3H/HeN) to contact dermatitis.

Two strains of mice were selected for resistance (DBA/2) or susceptibility (C3H/HeN) to contact dermatitis. Benzo[a]pyrene-DNA adduct formations was compared in the two mouse strains by a postlabeling procedure to determine if there was a significant effect. Results showed that adduct profiles in DBA/2 and C3H/HeN dermis were qualitatively similar. The total binding levels were higher in DBA/2 mice on the d 2 and the d 10. DNA adduct formation has been shown to inversely correlate with skin allergy induction. Data suggest that the expression of the genes responsible for the differences in responsiveness to chemical induced contact dermatitis in mouse may play an important role in benzo[a]pyrene-DNA adduct formation.

Antitumour immune response and cancer vaccination: the critical role of dendritic cells.

Increasing the capacity of the immune system to mediate tumour regression has been a major goal in tumour immunology. Progress towards this goal has been recently aided by the identification of immunogenic cancer antigens and by a better understanding of the mechanisms of T-cell immune response and tumour escape. A rare antigen-presenting cell--the dendritic cell (DC)--appears to be the key to these mechanisms. The possibility of generating these cells in vitro from blood precursors has initiated a new era in cancer immunotherapy. Using DC as a cancer vaccine adjuvant has led to reports of measurable immune responses, and, in a few cases, to complete disease responses in patients with B-cell lymphoma and melanoma.

[Allergy to the bites of blood-sucking insects: clinical manifestations]. / Allergie aux piqûres d'insectes hématophages: manifestations cliniques.

Blood-sucking insects and other arthropods may occur allergic reactions when stinging. Usually, inflammatory symptoms are mild with a locoregional extension. Pruritus may be painful and sometimes patients experienced a functional impotency. Systemic reactions are possible. Anaphylaxis is rare and rather observed with ticks (Ixodes and Argas) and kissing-bugs. The circumstances and mechanisms of the sting related to the knowledge of the insect biology elicit the clinical symptoms. Skin prick-tests are usually not practicable. Indeed, the diagnosis remains based upon anamnestic and entomological data because the poor sensibility of in vitro tests. However, some immunological investigations gave signs of interest for prospective studies. Symptomatic drugs and prevention advices must be purchased for exposed patients.

Prevalence of respiratory symptoms and increased specific IgE levels in West-African workers exposed to isocyanates.

Respiratory symptoms and immunological effects from chronic exposure to isocyanates (toluene diisocyanate) were studied in a cross survey of workers from West African factories producing paints and polyurethane foam. A questionnaire, a pulmonary function test, immunoglobulin E (IgE) levels, radioallergosorbent test (RAST) and an atmospheric sample to quantify isocyanate exposures were carried out in the workplace for each worker. Ninety-six workers, of whom 44 had occupational isocyanate-induced asthma, were included in the study. Twenty-four viral-infected subjects were excluded from the immunological study. Specific antibodies to isocyanates were detected in two of the symptomatic individuals. This low proportion appeared to be a common feature of this disease. The prevalence of isocyanate-induced asthma in a West African working population appears to be significant in the context of chronic human exposure, as current data are based on excessive acute exposure due to an accident as seen in India.

Mechanisms of occupational asthma induced by isocyanates.

Isocyanates are some of the most important low molecular weight chemicals associated with occupational asthma. These compounds are often volatile and they are highly reactive on mucous membranes, especially the conjunctivae and respiratory tract. Despite numerous data derived from experimental and clinical investigations, there is no agreement concerning the real mechanisms involved in isocyanate-induced occupational asthma. In fact, the cause of occupational asthma is multifactorial. The aim of this paper is to review the involved physiological causes of isocyanate-induced asthma; the main mechanisms are immunological, pharmacological and/or irritative.

Adherent-free generation of functional dendritic cells from purified blood monocytes in view of potential clinical use.

There is increasing interest in dendritic cells (DC) that are capable of initiating antitumor immune responses. An in vitro cell differentiation method has recently been developed that uses GM-CSF and IL-4 to generate human DC from adherent blood mononuclear cells cultured on tissue culture plastic. These cells are competent for antigen uptake but express relatively low levels of co-stimulatory molecules and thus correspond to immature resident tissue DC. We have adapted this method to consider some variables that are pertinent to clinical use, including a large scale differentiation of functional DC in a culture system suitable for clinical use. We report here that sizable numbers of monocytes purified by elutriation from blood leukocytes and cultured in Teflon bags develop with high efficiency into typical DC, as defined by morphology and membrane phenotype. When compared with usual adherent DC, cells generated under our adherent-free conditions exhibited lower CD1a expression and antigen capture capacity, but maintained the ability to present soluble antigens to T cells. They neoexpressed a high level of the co-stimulator molecule B7-2 (CD86) and was potent accessory cells for T cell proliferation, but they lacked the CD83 marker of DC full maturation. This study may constitute a prerequisite step for clinical investigations in tumor immunotherapy.

Allergy to latex, avocado pear, and banana: evidence for a 30 kd antigen in immunoblotting.

Allergens of natural latex, latex gloves, avocado pear, and banana extracts were investigated by an immunoblotting technique in sera of patients experiencing associated latex and fruit allergies. Extracts were separated by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. After incubation with patients' sera, IgE antibodies were revealed by a goat anti-human IgE alkaline-phosphatase conjugate. Seventeen serum samples from patients with well-documented latex allergy were studied. Among these patients, 10 demonstrated an allergy to avocado pear sometimes associated with banana. In sera from patients with latex and fruit allergy, prominent IgE binding was revealed at about 30 kd with latex and fruit extracts. Serum controls remained negative. Cross-inhibition of immunoblotting confirmed that this main allergen is linked to a common epitope present in latex and fruits. This must be related to clinical findings and previous observations of cross-reactivity.

Review of the problems involved in using enzymes in blood group serology--provision of freeze-dried ICSH/ISBT protease enzyme and anti-D reference standards. International Council for Standardization in Haematology. International Society of Blood Transfusion.

Proteolytic enzyme preparations and techniques used routinely in blood group serology for the detection of atypical patient antibodies prior to transfusion vary widely and are often poorly standardised. Recent advances have been made in the use of biochemical methods to standardise and stabilise the potency of the enzyme preparations used. A joint working party of the International Council for Standardization in Haematology (ICSH) and the International Society of Blood Transfusion (ISBT) has investigated possibilities for the provision of standards for the protease preparations and techniques. The specification for these standards was that the performance of enzyme reference preparation in the reference technique should be of equivalent sensitivity to the ICSH/ISBT LISS spin indirect antiglobulin test using a titration series of a reference weak anti-D, and be free from false-positive reactions. The working party circulated materials for evaluation in inter-laboratory trials, followed by a laboratory workshop meeting to achieve agreement on the specification for reference materials and methods. Reference freeze-dried papain at 0.6 azoalbumin units and weak anti-D preparations (91/562) have been prepared and validated to meet these specifications. The performance of a test enzyme preparation in the technique for which it is recommended for use should be at least equal to that of the reference papain preparation, by the reference two-stage technique in terms of sensitivity, using a titration series of the reference anti-D, and freedom from false-positive reactions, using six fresh inert sera. The reference papain and weak anti-D can also be used to calibrate the level of proteolytic activity required in other procedures in blood group serology, such as new technology methods for antibody detection, and automated and microplate cell grouping procedures. These preparations and an agreed method for their use are now available from listed centres as ICSH/ISBT and Food and Drug Administration reference materials.

Toluene diisocyanate-induced conformational changes of serum albumin: a study on repeated inhalations in guinea-pigs.

High responder lines of Hartley guinea-pigs were sensitized by repeated inhalations of toluene diisocyanate (TDI). After 3 weeks, we demonstrated a degree of TDI substitution of the serum-albumin-enriched fraction (AEF) and we ascertained the sensitization of the most exposed animals using PCA methodology. Fourier transform infrared spectroscopy (FT-IR), used to investigate conformational changes in AEF, highlighted the structural modifications of the native protein conformation. Such crucial changes may support, at least in part, the relationship between TDI exposure and triggering of hypersensitivity reactions.

[T lymphocytes in the circulating blood and pleural fluid of patients with tuberculosis or cancer]. / T lymphocytes du sang circulant et du liquide pleural chez des patients atteints de tuberculose évolutive ou de cancer.

Inflammatory pleurisies in the adult are particularly rich in CD4 lymphocytes, capable of inducing and facilitating the immune reaction. The aim of this study was to determine the percentage of CD4 and CD8 lymphocytes in pleural fluid and blood in 13 patients with tuberculous pleurisy and 12 patients with pleural neoplasms, to establish the diagnostic value of this analysis. Independent of the etiology of the effusion there is a concentration of CD4 T lymphocytes in the pleural fluid compared to the blood (49.52 +/- 12.36% and 39.28 +/- 6.74%, respectively, p value less than 0.001). However, there was a similar concentration for the two types of disorders studied with 59.92 +/- 7.26% for tuberculous pleurisies and 45.83 +/- 6.26% for neoplastic effusions, thus this technique does not make the diagnostic orientation any clearer.

Double phenotyping of immunoregulatory T cell subsets in patients with allergic asthma.

In order to determine whether the dissection of helper/inducer (CD4+) and suppressor/cytotoxic (CD8+) lymphocyte subsets with Leu 8 reagent would reveal any differences between allergic asthma patients and non-atopic controls, we compared in both groups the 'true helper' T cell subset (Leu 8- CD4+), responsible for the major helper effect, and one of the suppressor T cell subpopulations (Leu 8- CD8+). Peripheral blood mononuclear cells from sixty-nine individuals, including nineteen extrinsic asthmatics, fifteen intrinsic asthmatics, seventeen patients with chronic obstructive lung disease and eighteen healthy controls, were comparatively analysed. Although total CD4+ cells and total CD8+ cells were similar for all groups, we found in the extrinsic asthma patients group a significant increase in the number of 'true helper' T cell sublineage (Leu 8- CD4+) and of suppressor cells expressing Leu 8- CD8+ phenotype. Such imbalances may be implicated in the pathogenesis of atopic asthma.

T-lymphocyte subsets in pleural fluids: discrimination according to traditional and monoclonal antibody-defined markers.

T-lymphocyte subpopulations in pleural fluid and in peripheral blood from 17 patients admitted for pleural effusion were identified by E-rosette formation and delineated by monoclonal antibodies OKT3 (peripheral T-cells), OKT4 (helper/inducer cells), and OKT8 (suppressor/cytotoxic cells). We studied 13 patients with specified pleural diseases (tuberculosis, malignancies, connective tissue diseases, congestive heart failure) and 4 patients with non-specified pleural diseases. Our findings showed that the percentage of T-cells increases in pleural fluid versus peripheral blood whatever the diagnosis is, and that these T-cells are predominantly helper/inducer cells. Moreover, a recently described T-lymphocyte subpopulation, which expresses neither T3 nor other monoclonal antibody-defined markers, seems to be concentrated in the pleural fluid, especially in tuberculosis and malignant effusions. Although T-lymphocyte delineation seems to fail to aid in etiological diagnosis of pleurisy, such determinations could provide informations about local pathogenic mechanisms.

Failure to distinguish ultrastructurally between T4+ (helper) and T8+ (suppressor/cytotoxic) T-cell subsets.

Human peripheral T-cell subpopulations revealed by monoclonal antibodies by means of a rosetting method were isolated by micromanipulation and submitted to electron microscopic analysis. The T3+ subset (total T cells) displayed a high degree of heterogeneity, including multiple transitional forms, from cells with a high nuclear to cytoplasmic ratio and rare organelles to cells with a low nuclear to cytoplasmic ratio and a complex system of cytoplasmic organelles. T4+ (inducer/helper) and T8+ (suppressor/cytotoxic) cell subpopulations were shown to have no evident distinguishing characteristics. They both displayed the same morphological variation mentioned for T3+ lymphocytes. On morphometric analysis, these two cell subsets were very similar, with only slight differences for cell surface roughness, volume of mitochondria, extent of nuclear indentation, and surface area of the rough endoplasmic reticulum. The significance of these minor morphological differences is discussed.

[Phenotype of T lymphocytes and macrophages obtained by broncho-alveolar lavage of human lung]. / Etude du phénotype des lymphocytes T et des macrophages recueillis par lavage broncho-alvéolaire du poumon humain.

T lymphocytes and macrophages, isolated and purified from broncho-alveolar lavages performed in normal controls and in patients with various alveolar structure diseases were identified using monoclonal antibodies against different membrane markers. For T lymphocyte subsets, our results are consistent with previous observations showing a large number of lung helper T cells in patients with sarcoidosis with high-intensity alveolitis. For macrophage subsets, we pointed out, for all cases, a weak expression of monocyte markers.