Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation.

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

Only Follow-Up of Memory B Cells Helps Monitor Rituximab Administration to Patients with Neuromyelitis Optica Spectrum Disorders.

INTRODUCTION: Neuromyelitis optica spectrum disorders (NMOSD) are identified as a spectrum of inflammatory demyelinating disorders involving the brain, spinal cord and optic nerves. These disorders require early diagnosis and highly active immunosuppressive treatment. Rituximab (RTX) has demonstrated efficacy in limiting relapse in NMOSD when using several administration schedules. We questioned if the CD19+ CD27+ memory B cell count was a more reliable marker to monitor RTX administration than the RTX plasma level and CD19+ B cell count. METHODS: We analyzed 125 blood samples from 17 NMOSD patients treated with RTX and also measured the level of anti-aquaporine-4 antibodies (anti-AQP-4 Abs), human anti-chimeric antibodies to the murine fragment of RTX (HACA-RTX Abs), and the RTX concentration. RESULTS: The mean follow-up time of the cohort was 7.4 (2-16) years. All patients improved with a mean EDSS going from 4 (1-8.5) to 2.7 (1-5.5). The mean interval between RTX infusions was 9.6 months with identification of prolonged responders. Total CD19+ B cell detection with the routine technique did not correlate to re-emergence of CD19+ CD27+ memory B cells. The RTX residual concentration did not correlate with the CD19+ CD27+ memory B cell count or with anti-RTX antibody production. CONCLUSION: In contrast to total CD19+ cell, detected with the routine technique, CD19+ CD27+ memory B cells are a reliable marker for biological relapse and allow a decrease in the frequency of infusions.

Therapeutic target of memory B cells depletion helps to tailor administration frequency of rituximab in myasthenia gravis.

Rituximab (RTX) has demonstrated efficacy in limiting relapses in myasthenia gravis (MG). We investigated the interest of CD27+ memory B cell monitoring in patients as a biological marker of clinical relapse. Twenty-four patients have been treated with RTX (375mg/m(2)/week-month as an induction treatment). Maintenance treatment consisted with either systematic treatment every 3months or only when CD27+ memory B cells were detectable. After the induction treatment, the mean infusions were 1.3/year compared with 4/year. We suggest that RTX administration frequency can be decreased safely by monitoring the re-emerging CD27+ memory B cells.

Intracytoplasmic detection of TCL1--but not ILT7-by flow cytometry is useful for blastic plasmacytoid dendritic cell leukemia diagnosis.

Diagnosis of blastic plasmacytoid dendritic cell neoplasm (BPDCN) or plasmacytoid dendritic cell leukemia (pDCL) is mainly based on immunophenotypical characterization of leukemic cells in blood or bone marrow samples. We tested by flow cytometry intracellular expression of the proto-oncogene T-cell leukemia 1 (TCL1), as well as membrane and intracellular expression of immunoglobulin-like transcript 7 (ILT7) in 21 pDCL samples and 61 non-pDC acute leukemia samples [i.e., 14 B-acute lymphoblastic leukemia (B-ALL), 9 T-ALL and 38 acute myeloid leukemia (AML)]. TCL1 is highly expressed in all pDCL samples while at a statistically lower level in all B-ALL and 34% of AML. Statistical analysis shows that intensity of TCL1 expression is a good marker for differential diagnosis of pDCL versus other acute leukemia (area under the receiver-operating characteristic curve, [AUC]: 0.96). By contrast, ILT7 positivity is limited to few pDCL samples and cannot be useful for diagnosis purpose. In conclusion, high intracellular intensity of TCL1 expression is currently the best marker for pDC lineage assignment by flow cytometry, which is particularly useful to distinguish pDCL from CD4(+) CD56(+/-) undifferentiated or monoblastic acute leukemia. Thus, intracellular TCL1 detection should be included in acute leukemia diagnosis panels used in hematology laboratories. © 2012 International Society for Advancement of Cytometry.

The osteopontin level in liver, adipose tissue and serum is correlated with fibrosis in patients with alcoholic liver disease.

BACKGROUND: Osteopontin (OPN) plays an important role in the progression of chronic liver diseases. We aimed to quantify the liver, adipose tissue and serum levels of OPN in heavy alcohol drinkers and to compare them with the histological severity of hepatic inflammation and fibrosis. METHODOLOGY/PRINCIPAL FINDINGS: OPN was evaluated in the serum of a retrospective and prospective group of 109 and 95 heavy alcohol drinkers, respectively, in the liver of 34 patients from the retrospective group, and in the liver and adipose tissue from an additional group of 38 heavy alcohol drinkers. Serum levels of OPN increased slightly with hepatic inflammation and progressively with the severity of hepatic fibrosis. Hepatic OPN expression correlated with hepatic inflammation, fibrosis, TGFß expression, neutrophils accumulation and with the serum OPN level. Interestingly, adipose tissue OPN expression also correlated with hepatic fibrosis even after 7 days of alcohol abstinence. The elevated serum OPN level was an independent risk factor in estimating significant (F ≥ 2) fibrosis in a model combining alkaline phosphatase, albumin, hemoglobin, OPN and FibroMeter® levels. OPN had an area under the receiving operator curve that estimated significant fibrosis of 0.89 and 0.88 in the retrospective and prospective groups, respectively. OPN, Hyaluronate (AUROC: 0.88), total Cytokeratin 18 (AUROC: 0.83) and FibroMeter® (AUROC: 0.90) estimated significance to the same extent in the retrospective group. Finally, the serum OPN levels also correlated with hepatic fibrosis and estimated significant (F ≥ 2) fibrosis in 86 patients with chronic hepatitis C, which suggested that its elevated level could be a general response to chronic liver injury. CONCLUSION/SIGNIFICANCE: OPN increased in the liver, adipose tissue and serum with liver fibrosis in alcoholic patients. Further, OPN is a new relevant biomarker for significant liver fibrosis. OPN could thus be an important actor in the pathogenesis of this chronic liver disease.

Comparison of nine blood tests and transient elastography for liver fibrosis in chronic hepatitis C: the ANRS HCEP-23 study.

BACKGROUND & AIMS: Blood tests and transient elastography (Fibroscan™) have been developed as alternatives to liver biopsy. This ANRS HCEP-23 study compared the diagnostic accuracy of nine blood tests and transient elastography (Fibroscan™) to assess liver fibrosis, vs. liver biopsy, in untreated patients with chronic hepatitis C (CHC). METHODS: This was a multicentre prospective independent study in 19 French University hospitals of consecutive adult patients having simultaneous liver biopsy, biochemical blood tests (performed in a centralized laboratory) and Fibroscan™. Two experienced pathologists independently reviewed the liver biopsies (mean length=25±8.4 mm). Performance was assessed using ROC curves corrected by Obuchowski's method. RESULTS: Fibroscan™ was not interpretable in 113 (22%) patients. In the 382 patients having both blood tests and interpretable Fibroscan™, Fibroscan™ performed similarly to the best blood tests for the diagnosis of significant fibrosis and cirrhosis. Obuchowski's measure showed Fibrometer® (0.86), Fibrotest® (0.84), Hepascore® (0.84), and interpretable Fibroscan™ (0.84) to be the most accurate tests. The combination of Fibrotest®, Fibrometer®, or Hepascore® with Fibroscan™ or Apri increases the percentage of well classified patients from 70-73% to 80-83% for significant fibrosis, but for cirrhosis a combination offers no improvement. For the 436 patients having all the blood tests, AUROC's ranged from 0.82 (Fibrometer®) to 0.75 (Hyaluronate) for significant fibrosis, and from 0.89 (Fibrometer® and Hepascore®) to 0.83 (FIB-4) for cirrhosis. CONCLUSIONS: Contrarily to blood tests, performance of Fibroscan™ was reduced due to uninterpretable results. Fibrotest®, interpretable Fibroscan™, Fibrometer®, and Hepascore® perform best and similarly for diagnosis of significant fibrosis and cirrhosis.

Noninvasive procedures to evaluate liver involvement in HIV-1 vertically infected children.

OBJECTIVES: : Progressive liver injury is a concern in HIV-infected children exposed to long-term antiretroviral drugs and to the cytopathic effect of HIV. Yet liver biopsy is usually considered too invasive to be repeated in these patients. The aims of this study are to evaluate the feasibility of noninvasive hepatic investigations in HIV-1-infected children, assess the prevalence of signs of liver affection, and analyse the influence of the HIV disease severity and the exposure to antiretroviral therapy. MATERIALS AND METHODS: : A cross-sectional study conducted in 26 HIV-1 vertically infected children ages 8 to 18 years old. Liver function was assessed with standard serum biochemical markers, FibroTest, ActiTest, SteatoTest, Forns index, aspartate aminotransferase to platelet ratio index, ultrasound, and Fibroscan. RESULTS: : Nineteen (>60%) children had signs of liver affection on at least 1 of the test results: 13 (50%) had elevated liver enzymes, 15 (63%), 8 (33%), 5 (21%), and 5 (21%) had abnormal FibroTest, ActiTest, Forns index, and aspartate aminotransferase to platelet ratio index results, respectively. Four children (17%) had mild liver steatosis on ultrasound. Fibroscan measures were significantly higher in patients than in age-matched healthy children. Patients with elevated Fibroscan measures also had significantly higher FibroTest results. Age, HIV stage N in the Centers for Disease Control and Prevention classification and exposure duration to nucleoside reverse transcriptase inhibitor and non-nucleoside reverse transcriptase inhibitor drugs were the main risk factors for hepatotoxicity. CONCLUSIONS: : More than half of our population of HIV-infected children had biological and/or radiological signs of liver affection. Regular follow-up of liver function is necessary in these patients, which is now possible with noninvasive procedures.

Loss of Kindlin-3 in LAD-III eliminates LFA-1 but not VLA-4 adhesiveness developed under shear flow conditions.

Leukocyte adhesion deficiency (LAD)-III is associated with homozygous stop codon mutations in Kindlin-3, the hematopoietic member of the Kindlin family of integrin coactivators. In addition, a subgroup of LAD-III patients has a homozygous splice junction mutation in and reduced expression of the Rap-1 guanine nucleotide exchange factor, CalDAG-GEFI (CDGI). In this study, we compared the adhesive properties of the leukocyte function-associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4) integrins in both primary and activated leukocytes derived from these 2 LAD-III subgroups. Primary lymphocytes lacking both Kindlin-3 and CDGI lost all firm T-cell receptor-stimulated LFA-1 adhesiveness, in contrast to LAD-III lymphocytes deficient in Kindlin-3 alone. Effector T cells expanded from all tested LAD-III variants expressed normal CDGI, but lacked Kindlin-3. These Kindlin-3-null effector T cells exhibited total loss of inside-out LFA-1 activation by chemokine signals as well as abrogated intrinsic LFA-1 adhesiveness. Surprisingly, VLA-4 in Kindlin-3-null resting or effector lymphocytes retained intrinsic rolling adhesions to vascular cell adhesion molecule-1 and exhibited only partial defects in chemokine-stimulated adhesiveness to vascular cell adhesion molecule-1. Deletion of the putative beta(1) Kindlin-3 binding site also retained VLA-4 adhesiveness. Thus, our study provides the first evidence that Kindlin-3 is more critical to LFA-1 than to VLA-4-adhesive functions in human lymphocytes.

Optimal correlation between different instruments for Fibrotest-Actitest protein measurement in patients with chronic hepatitis C.

OBJECTIVES: Combination of alpha 2-macroglobulin, haptoglobin, apolipoprotein-A1, gamma-glutamyl transpeptidase, total bilirubin and alanine aminotransferase measurements allows to determine the Fibrotest-Actitest score, an alternative to liver biopsy in hepatitis C virus infection. The aims of this study were to evaluate the analytical variability of the Fibrotest-Actitest proteins alpha 2-macroglobulin, haptoglobin and apolipoprotein-A1, and to assess their impact on the Fibrotest-Actitest scores. METHODS: We compared 129 sera from hepatitis C virus infected patients for alpha 2-macroglobulin, haptoglobin and apolipoprotein-A1 levels obtained with the Immage (Beckman-Coulter) and the BNProspec (Dade-Berhing) automates. We evaluated Fibrotest-Actitest results obtained with the two nephelemeters. RESULTS: Optimal correlation was found for alpha 2-macroglobulin (Y=1.05X + 0.01, correlation coefficient: 0.98) and haptoglobin (Y=1.05X - 0.07, correlation coefficient: 0.98). Apolipoprotein-A1 levels, as determined by Immage, were slightly lower than those obtained by BNProspec (Y=0.86X - 0.02, CC=0.95). When Fibrotest-Actitest scores obtained with the two protein measurements were compared adjusting for apolipoprotein-A1 from Immage, the concordance rate was 0.903+/-0.096, with only 2/107 patients showing minimal discordance>0.10 for Fibrotest, and 1.00+/-0.06 for Actitest, with no discordance>0.10. CONCLUSIONS: Measurement of apolipoprotein-A1, included in the Fibrotest-Actitest score, depends on the equipment used. Such discordance is of little clinical consequence for liver fibrosis evaluation in hepatitis C virus patients.

Serum C-reactive protein: a non-invasive marker of alcoholic hepatitis.

OBJECTIVE: To determine the diagnostic accuracy of C-reactive protein (CRP) for alcoholic hepatitis in heavy drinkers. MATERIAL AND METHODS: A total of 101 heavy drinkers (67 M, 34 F) with elevated transaminase activity and negative HBsAg, anti-HCV and anti-HIV antibodies were included in the study. All patients underwent standard liver function tests, CRP determination and liver biopsies. None of the patients had signs of infection or inflammatory disease and none of them were taking antibiotics. The severity of alcoholic hepatitis was assessed semi-quantitatively using a Metavir-derived scoring system. The receiver operating curve (ROC) for CRP was constructed to assess different areas under the curve (AUCs) and the best threshold value for predicting alcoholic hepatitis (an AUC of 1.0 for an ideal test and of 0.5 for a less indicative test). RESULTS: Pathological signs of alcoholic hepatitis were found in 29 patients (30%) and significant fibrosis (F > 1) in 46 (45.1%). CRP increased significantly with the severity of acute alcoholic hepatitis (p<0.001). Total bilirubin (OR = 1.03 CI 95% (1.01-1.06), p=0.04) and CRP (OR = 1.1 CI 95% (1.02-1.19), p=0.01) were independent factors for predicting alcoholic hepatitis. The area under the ROC curve of CRP was 0.78. Using optimized cut-off values (CRP > 19 mg/L), the sensitivity, specificity, positive, negative predictive value and diagnostic accuracy were 41%, 99%, 92%, 81% and 82%, respectively. CONCLUSION: CRP is an accurate marker of alcoholic hepatitis.

Influence of beta 1 integrin intracytoplasmic domains in the regulation of VLA-4-mediated adhesion of human T cells to VCAM-1 under flow conditions.

The VLA-4 integrin supports static cell-cell, cell-matrix adhesion, and dynamic interactions with VCAM-1. Although functions for well-conserved beta(1) integrin cytoplasmic domains in regulating static cell adhesion has been established, the molecular basis for beta(1) integrin-dependent arrest on VCAM-1 under flow conditions remains poorly understood. We have transfected the beta(1) integrin-deficient A1 Jurkat T cell line with beta(1) cDNA constructs with deletions of the NPXY motifs and specific mutations of tyrosine residues. Deletion of either NPXY motif impaired static adhesion induced by CD2 or CD47 triggering or direct beta(1) integrin stimulation. In contrast, PMA-induced adhesion to VCAM-1 was unaffected by deletion of the NPIY motif and only slightly impaired by deletion of NPKY. Moreover, deletion of the NPIY motif resulted in enhanced rolling and reduced arrest on VCAM-1 under shear flow conditions. In contrast, deletion of the NPKY motif did not alter arrest under flow. Although tyrosine to phenylalanine substitutions within two NPXY motifs did not alter static adhesion to VCAM-1, these mutations enhanced arrest on VCAM-1 under flow conditions. Furthermore, although deletion of the C'-terminal 5 AA of the beta(1) cytoplasmic domain dramatically impaired activation-dependent static adhesion, it did not impair arrest on VCAM-1 under flow conditions. Thus, our results demonstrate distinct structural requirements for VLA-4 function under static and shear flow conditions. This may be relevant for VLA-4 activity regulation in different anatomic compartments, such as when circulating cells arrest on inflamed endothelium under shear flow and when resident cells in bone marrow interact with VCAM-1- positive stromal cells.

Analytical variability of the Fibrotest proteins.

OBJECTIVES: The analytical variability of the Fibrotest (FT) parameters raises the issue of the test's reliability for routine use. Whereas standardization has been proposed by the International Federation of Clinical Chemistry (IFCC) for specific proteins, few data are available concerning the actual transferability of the FT proteins, i.e. haptoglobin, apolipoprotein A1 and alpha2 macroglobulin. The aim of this study was to evaluate the analytical variability of the FT proteins. DESIGN AND METHODS: During the FIBROPACA study, we evaluated 112 sera from patients with hepatitis C infection who underwent liver biopsy. We compared measurements of haptoglobin, apolipoprotein A1 and alpha2 macroglobulin by the autoanalyzers Immage (Beckman-Coulter) and the FT reference BNProspec (Dade-Behring). RESULTS: Optimal concordance was found for haptoglobin (correlation: y = 1.05x -0.09; correlation coefficient = 0.98). However, apolipoprotein A1 as determined with Immage was globally 12% lower than with BNProspec (correlation: y = 0.88x -0.05; correlation coefficient = 0.91) and alpha2 macroglobulin values were 40% greater with Immage than with BNProspec (correlation: y = 1.40x -0.46; correlation coefficient = 0.96). CONCLUSIONS: Inter-technique analytical variability of the Fibrotest parameters remains a major issue. After IFCC standardization of specific proteins, some discrepancies remain for alpha2 macroglobulin and, to a lesser extent, for apolipoprotein A1. National and international quality control programs would be useful to monitor analytical performance of protein assays.