Development of a new staining protocol for the Kleihauer-Betke test to facilitate the reading of difficult cases.

BACKGROUND: The Kleihauer-Betke (KB) test allows the detection of fetal red blood cells (containing fetal hemoglobin, HbF) in the maternal blood to identify and quantify potential fetal-maternal hemorrhages. In certain cases, detecting fetal red blood cells with conventional staining is difficult. False-positive results or overestimation of the quantity of fetal red blood cells may occur in cases of maternal hemoglobinopathy. In this study, we developed a new staining protocol to facilitate the reading of difficult smears and improve the precision of the quantification of fetal red blood cells; we also analyzed the performance of this new method. This study assessed blood samples with and without hemoglobin abnormalities, which present difficulties when interpreting the KB test. METHODS: The new staining formula is based on an improved elution technique and the use of a different stain instead of hematoxylin. To test this staining method, 16 samples from patients with abnormal hemoglobin electrophoresis and 14 samples from patients with normal hemoglobin electrophoresis were analyzed using the KB test with the classical staining method and the new staining method. In addition, a second series was prepared using the same samples spiked with fetal red blood cells from newborn blood, to compare the accuracy of the two methods in identifying fetal red blood cells. RESULTS: In the 60 slides analyzed with both staining methods, we found that the new technique improved the accuracy from 78 to 85%; lowered the coefficient of variation between the operators, which decreased from 20.7% to 12.7%; increased the specificity in our population from 56 to 70%; and decreased the number of false-positive cases by 30%. CONCLUSIONS: We successfully developed a new staining technique that facilitates the reading of difficult slides and improves the specificity of the detection of fetal red blood cells. This technique is recommended as a secondary method to use before sending the sample for additional exploration.

The antigen specificity of the rheumatoid arthritis-associated ACPA directed to citrullinated fibrin is very closely restricted.

The major targets of the disease-specific autoantibodies to citrullinated proteins (ACPA) in synovium of rheumatoid arthritis (RA) patients are borne by the citrullinated α- and ß-chains of fibrin. We demonstrated that ACPA target a limited set of citrullinated fibrin peptides and particularly four multicitrullinated peptides which present the major epitopes. In this study, we established the clear immunodominance of the peptides α36-50Cit(38,42) and ß60-74Cit(60,72,74) which were recognised by 51/81 (63%) and 61/81 (75%) of ACPA-positive patients, respectively, more than 90% recognising one, the other or both peptides. We also identified the citrullyl residues αCit(42), ßCit(72) and ßCit(74) as essential for antigenicity, and at a lesser degree αCit(38). Then, we assayed on overlapping 7-mer peptides encompassing the sequences of the two peptides, 3 series of sera recognising either α36-50Cit(38,42) or ß60-74Cit(60,72,74) or both peptides. In each series, the reactivity profiles of the sera, largely superimposable, allowed identification of the two 4/5-mer overlapping epitopes (α: VECit(42)HQ and α': Cit(38)VVE), and the single 5-mer epitope (ß: GYCit(72)ACit(74)), all located to a flexible globular domain of fibrin on a topological 3D model. In conclusion, we demonstrated that only 3 immunodominant epitopes are targeted by ACPA on citrullinated fibrin stressing their actual oligoclonality. However, the reactivity to the 3 epitopes distinguishes three subgroups of patients. The closely restricted antigen specificity suggests that the autoimmune reaction to citrullinated fibrin is antigen-driven. The accessibility of the epitopes reinforces the hypothesis of a pathogenic role for ACPA via immune complexe formation in the synovial tissue.

Persistent oligoclonal CD4dimCD8+T cells in peripheral blood.

BACKGROUND: Routine CD4/CD8 T-cell phenotyping may shows a small fraction of CD4dimCD8+ T cells with a homogeneous appearance as described for lymphoproliferative syndromes or chronic infections. The aim of this study was to elucidate the significance of CD4dimCD8+ T cells and their degree of diversity. METHODS: Phenotyping was performed in 272 samples from healthy donors, elderly patients, and immunocompromised (human immunodeficiency virus or renal transplantation) patients. RESULTS: The CD4dimCD8+ T cells had decreased fluorescence intensity for CD4 but not for CD8. The frequency of patients with CD4dimCD8+ T cells (>20 cells/microl; 10.3% of patients with human immunodeficiency virus and 7.7% with renal transplantation) was not significantly different when compared with healthy donors (9.7%). The CD4dimCD8+ T cells did not express the activation marker CD69. The CD8 of CD4dimCD8+ T cells expressed the heterodimeric (beta) isoform. In 13 of 26 samples, the apparently highly homogeneous CD4dimCD8+ T cells expressed one predominant T-cell receptor Vbeta clonotype. These predominant clonotypes were widely distributed among patients: Vbeta 5.2, 17, 2, 3, 5.1, 13.1, 14, and 20. CONCLUSIONS: Whether these findings demonstrate an oligoclonal reaction to chronic inflammation or an emerging lymphoproliferative disorder must be elucidated in a long-term longitudinal study. By analogy to monoclonal gammopathy, we propose to name this phenomenon "oligoclonal clonopathy of undetermined significance."

Loss of heterozygosity in tumor tissue in hormonal receptor genes is associated with poor prognostic criteria in breast cancer.

The estrogen receptors (ESRα and ß) and the androgen receptor (AR) mediate genomic and non-genomic effects on breast tumor growth and proliferation. We analyzed 101 breast cancer patients for allelic loss in microsatellites located in regulatory regions of the ESRs and AR genes in breast cancer tumors. The loss of heterozygosity (LOH) at these loci was found in 36.2% of tumor tissues (ductal carcinoma cases), for 19% of cases at the ESRα locus, for 16% at the ESRß locus, and for 10% at the AR locus. The LOH in at least one of the two ESR loci was correlated to poor prognosis criteria: ESR-negative status (P = 0.007), PR-negative status (P = 0.003), high Scarff-Bloom-Richardson (SBR) grade (P = 0.0007), high MIB-1 proliferation index (P = 0.02), and diminished apoptosis potential (TP53-positive status, P = 0.018). When AR was also considered, the LOH in at least one of the three loci was associated with ESR-negative status (P = 0.036), PR-negative status (P = 0.027), high SBR grade (P = 0.005), high mitotic index (P = 0.0002), TP53-positive status (P = 0.029), and proliferating index (high MIB-1, P = 0.03). Allelic loss was observed in 26% of normal tissue adjacent to tumor with LOH at the ESRα locus and in 7.1% of tumors with LOH at the ESRß locus. The LOH in tumor tissue in the regulatory regions of ESRα, ESRß, and AR genes has potentially synergistic effects on tumor proliferation, histological aggressiveness, down-regulation of ESRα and progesterone receptor (PR) genes, and is an early genetic alteration in cancer that is possibly involved in passage to estrogen independence.

[Toward pertinent analytical objectives for haematological parameters]. / Vers les objectifs analytiques pertinents pour les paramètres de l'hémogramme.

Usually, the blood cell counting activity in haematology laboratory uses the comparison of IQC values to the target values proposed by the manufacturer. We intended to improve the monitoring of the proper functioning of our analytical measure system for 17 main haematologic parameters. To set the allowable critical limits of IQC, we propose our reflection based on several elements: benchmark and expert recommendation, clinical requirements, statistical indicators of the laboratory calculated using IQC values (3 levels, 2 different lots, 2 haematology analysers and 2 passage modes) and the EEQ values, during four months. We exploited the reports obtained from the middleware (our own IQC values), and the interlaboratory comparison reports (obtained from SNCS and EuroCell websites) and we compared our performances to the Ricos objectives, to set clearly argued allowable limits for IQC values. Finally, the allowable limits correspond to the imprecision limits stated by Ricos for 14 parameters (desirable for 11 parametres and minimal for 3 parameters) and personalized limits (more exigent than desirable Ricos limits) for 3 parameters of blood cell counting.

Variability of CD3 membrane expression and T cell activation capacity.

BACKGROUND: AlphabetaT cells have a wide distribution of CD3 membrane density. The aim of this article was to evaluate the significance of the CD3 differential expression on T cell subsets. Analysis was performed on healthy donors and renal transplant patients by flow cytometry. The results obtained are: (1) CD3 expression was widely distributed (CV = 38.3 +/- 3.1 to 43 +/- 2.3%). (2) The CD4, CD8, CD45 and forward scatter were similarly distributed. (3) The diversity of CD3 expression was directly related to the clonotypes: gamma9, non gamma9 from gammadeltaT cells and Vbeta clonotype from alphabetaT cells (e.g., Vbeta3FITC 7,980 +/- 1,628 Vbeta8PE: Vbeta20-FITC 11,768 +/- 1,510). (4) Using a computer simulation, we could confirm differential kinetics of T cell activation according to the initial parameters. Finally, in vitro activation was significantly higher on Vbeta8 and Vbeta9 (high CD3) compared with Vbeta2 and Vbeta3 (low CD3, P = 0.040-0.0003). In conclusion, T cells have highly heterogeneous CD3 expression, possibly predetermined and with clear functional significance.

Induction of macrophage secretion of tumor necrosis factor alpha through Fcgamma receptor IIa engagement by rheumatoid arthritis-specific autoantibodies to citrullinated proteins complexed with fibrinogen.

OBJECTIVE: Macrophage-derived tumor necrosis factor alpha (TNFalpha) is a dominant mediator of synovitis in rheumatoid arthritis (RA). This study was undertaken to assess whether and how immune complexes (ICs) formed by the interaction of disease-specific autoantibodies to citrullinated proteins (ACPAs) with their main synovial target antigen, citrullinated fibrin, contribute to TNFalpha production by macrophages. METHODS: An in vitro human model was developed in which monocyte-derived macrophages were stimulated with ACPA-containing ICs that were generated by capturing ACPAs from RA sera on immobilized citrullinated fibrinogen. Cellular activation was evaluated by TNFalpha assay in culture supernatants. Selective blockade of IC interactions with the 3 classes of Fcgamma receptors (FcgammaR) was used to assess the contribution of each receptor to macrophage activation. In addition, 2 citrullinated fibrin-derived peptides bearing major ACPA epitopes were tested for their capacity to inhibit formation of macrophage-activating ACPA-containing ICs. RESULTS: ACPA-containing ICs induced a dose-dependent TNFalpha secretion by macrophages from 14 of 20 healthy donors. The macrophage response was systematically higher than that of the paired monocyte precursors. TNFalpha secretion was not reduced by blockade of FcgammaRI or FcgammaRIII, but was strongly repressed when interaction of ICs with FcgammaRII was prevented. The 2 citrullinated peptides significantly inhibited ACPA reactivity to citrullinated fibrinogen and, when tested together, almost completely abolished formation of macrophage-activating ICs, thereby diminishing the secreted TNFalpha levels. CONCLUSION: Our model demonstrates the inflammatory potential of ACPA-containing ICs via engagement of FcgammaRIIa at the surface of macrophages, strongly supporting their pathophysiologic involvement. Continuing dissection of these molecular pathways could open the way to new therapeutic approaches in patients with RA.

Significance of unconventional peripheral CD4+CD8dim T cell subsets.

Routine T cells phenotyping occasionally reveals a CD4+CD8dim T cell subset with an apparently homogeneous dot plot. The aim of this study was to elucidate their immunological significance from analysis of 31 healthy donors, 21 elderly and 220 immune deficient patients. CD4+CD8dim T cells expressed reduced levels of CD8 (11-17,000 compared to 96-128,000 mol/cell on CD8+ T Cells). CD4 was expressed at the same level as on CD4+ T cells. The occurrence of raised CD4+CD8dim T cells (> 20 cells/muL) was similar in kidney transplant recipients (28.4%) and healthy donors (26%). It was somewhat lower in HIV+ patients (19.7%) possibly due to virally induced CD4+ T lymphopenia. However, an age effect is possible because the occurrence was raised (33.3%) in 70 volunteers (chi2 test NS). On the other hand, the size of the CD4+CD8dim subset was not correlated with age. CD4+CD8dim T cells did not express the activation markers CD69 (n = 220) or CD25 (n = 10) and expressed the homodimeric (alphaalpha) isoform of CD8, suggesting they are related to mucosal immunity (MALT). We selected 29 patients with unambiguous dot plots. In 26 of them one predominant TCR Vbeta clonotype was expressed on 18 to 94% of CD4+CD8dim T cells and never on more than 10% of conventional T cells. The predominant clonotypes were Vbeta8 (n = 5), Vbeta2 (n = 4), Vbeta13.1 and Vbeta 21 (n = 3 each). Whether this reveals a chronic stimulation or an emerging lymphoproliferative disorder must be elucidated. We propose to name this entity: "Oligoclonal Clonopathy of Undetermined Significance (OCUS)."