Marianne Philippe

Follow-up of residual disease (MRD) in B lineage acute leukaemias using a simplified PCR strategy: evolution of MRD rather than its detection is correlated with clinical outcome

Summary Bone marrow samples of 16 patients (two adults and 14 children) with a B lineage acute lymphoblastic leukaemia (ALL), and in whom Ig heavy chain gene rearrangements were detectable at diagnosis using polymerase chain reaction (PCR), were studied during evolution using PCR. The VDJ junctional fragment of the Ig heavy chain rearranged gene was amplified at diagnosis. After length reduction by restriction digestion, the amplified fragment was recovered by chromatography, labelled using a specific hexamer as a primer and directly used as a clonospecific probe. The sensitivity of the PCR ranged from 1:104 to 1:105 cells, depending on the patients rearrangement. Residual disease (MRD) was detected in most of the patients achieving a complete remission after induction therapy, regardless of the long‐term outcome of treatment. However, in patients remaining in complete remission, the level of MRD showed a tendency to decrease and ultimately become undetectable for variable periods of time, while in patients eventually relapsing there was a trend for MRD to persist at stable levels and even to increase before relapse was clinically evident. We conclude that the use of a simplified methodology for obtaining a clonospecific probe from the Ig heavy chain gene, though less sensitive than the sequencing methodology, is a valuable and readily available tool to monitor MRD in a high proportion of B lineage ALL.

Application of the Six Sigma concept in clinical laboratories: a review.

Abstract Six Sigma is a global management strategy introduced to the industrial world in the 1980s. This methodology has been widely implemented in companies such as Motorola, General Electric, Allied Signal and many others, with tremendous success in terms of customer satisfaction and global profitability. To achieve similar benefits in the healthcare field, Six Sigma is currently being deployed in several laboratories around the world. Despite this situation, few articles have been published in the peer-reviewed literature on this subject. The aim of this article is to clarify the different aspects of Six Sigma and their potential applications in clinical laboratories, as well as to systematically review articles and books discussing Six Sigma strategy implementation in the laboratory field. Clin Chem Lab Med 2007;45:789–96.

Molecular characterization of femA from Staphylococcus hominis and Staphylococcus saprophyticus, and femA-based discrimination of staphylococcal species.

The femA gene encodes a protein precursor which plays a role in peptidoglycan biosynthesis in Staphylococcus aureus and is also considered as a factor influencing the level of methicillin resistance. A femA homologous gene was recently characterized in S. epidermidis, entailing the possibility of femA phylogenetic conservation in staphylococcal species. Accordingly, we assessed the presence of femA homologous genes in S. hominis and S. saprophyticus. Strategy for identification relied upon alignment of S. aureus and D. epidermidis femA sequences and upon identification of potentially conserved regions. Amplifications of portions of the femA genes were performed under permissive annealing conditions, by using several sets of primers designed to match the consensus regions. DNA sequencing of overlapping PCR fragments led to the characterization of the entire femA genes of S. hominis and S. saprophyticus, and provided more precise information on the femA start codon for all five species. The genomic organization of all these femA genes appeared highly conserved, with alternance of homologous and variable regions. On this basis, a consensus sequence of the femA gene was defined and interspecies variations were exploited to design strategies for staphylococci species-specific identification, including multiplex PCR amplification and a reverse hybridization assay.

Molecular expression of PSMA mRNA and protein in primary renal tumors.

Human prostate‐specific membrane antigen (PSMA), a 100‐kDa integral transmembrane glycoprotein, is considered to be a highly specific marker of the prostate gland, and has successfully been used as a marker of circulating prostatic epithelial cells. Extended PSMA homology has been demonstrated with a cDNA found in rat cerebral and renal tissues. In this study, we aimed to evaluate the expression of PSMA mRNA in a variety of human renal cancer tissues (n = 20) and cell lines (n = 12). Using reverse transcriptase‐polymerase chain reaction, DNA sequencing, blottings, and specific anti‐PSMA labelling with CYT 351 antibody, we identified PSMA mRNA and protein in normal and in neoplastic renal tissue. The sequence of the polymerase‐chain‐reaction products is identical to that of PSMA cDNA derived from prostate tissue. Immunological staining with the CYT 351 reveals that PSMA is expressed mainly in tubular cells. Since PSMA does not appear to be restricted to prostatic tissue, this novel biomarker may prove useful in the staging of renal cancer and in the search for the hematogenous spread of renal cells. Int. J. Cancer 80:799–803, 1999.

Evaluation of a new Sebia kit for analysis of hemoglobin fractions and variants on the Capillarys system.

Abstract We evaluated the analytical performances of the new Sebia kit for quantification of hemoglobin fractions (HbA, HbF and HbA2) and structural hemoglobin variants on the Capillarys® system. This automated capillary zone electrophoresis method uses an alkaline buffer with silica capillaries and spectrophotometric detection. Specimen stability was evaluated during 1 month. The reproducibility of migration and the imprecision of quantification were also investigated. Comparison with the Beckman P/ACE® system was performed on 202 samples. A total of 131 subjects without any hematological abnormality were analyzed to establish the HbA2 reference ranges based on our local population. Quantification of the Hb fractions and variants exhibited excellent stability for 4weeks of storage at 4°C, with CVs <0.3%. The imprecision of the migration normalized to that of HbA2 for all hemoglobins tested (fractions and variants) was low, with a CV of <2.5%. At physiological and pathological levels, total imprecision ranged from 1.9% to 4.6% for HbA2, from 0.6% to 9.7% for HbF, and from 0.6% to 1% for HbS. Statistical analysis revealed a small proportional negative bias for HbA2 (−8.6%). Small systematic bias (−0.2%) and proportional bias (−28%) were observed for HbF. No statistically significant difference was found for HbS. The reference range for HbA2 was 2.1–3.2%. The Capillarys® system is a fully automated and accurate system that gives high-resolution performance and displays appropriate characteristics for use as a routine method for the diagnosis of thalassemias and hemoglobinopathies.

HLA class II-associated genetic susceptibility in idiopathic progressive sensorineural hearing loss.

To investigate the association between genes in the major histocompatibility complex and inner ear disease susceptibility at the DNA level, high-resolution genotyping for HLA class II (HLA-DR, -DQ, -DP) was performed by polymerase chain reaction-sequence-specific oligonucleotide reverse dot blot and polymerase chain reaction-restriction fragment length polymorphism analysis in 34 patients with idiopathic progressive sensorineural hearing loss (PSHL) and in 214 controls. The frequencies of DRB1*0301, DRB3*0101, DQB1*0201, and DPB1*0401 were significantly increased in patients with idiopathic PSHL compared with controls. The DQB1*0301 allele was significantly decreased in the patients. A linkage disequilibrium was probably responsible for the concomitant increase of both DRB1*0301 and DRB3*0101 alleles in patients. The increase of DQB1*0201 in patients was associated with the DRB1*0301 allele. In addition, the telomeric DPB 1*0401 allele may act as an independent risk factor. The DQB1*0301 allele may have a protective role in the pathogenesis of idiopathic PSHL. These results suggest that the specific HLA class II gene products may confer susceptibility or resistance to idiopathic PSHL.

Prevalence of activated protein C resistance and analysis of clinical profile in thromboembolic patients. A Belgian prospective study

Objectives. To assess the prevalence of activated protein C resistance (APC‐R) among healthy subjects and thromboembolic patients and to determine the clinical characteristics associated with APC‐R.

20-25% lower concentrations of total and free prostate-specific antigen (PSA) after calibration of PSA assays to the WHO reference materials - analysis of 1098 patients in four centers

AIM To examine the potential clinical implications of the recalibration of total prostate-specific antigen (PSA) and free PSA (fPSA) assays to the World Health Organization (WHO) standard materials. MATERIAL AND METHODS Data from 1098 patients with or without clinically detected prostate cancer (PCa) from four independent cohort studies were compared using commercial assays calibrated to the traditional Hybritech PSA (PSA-Hyb) and fPSA (fPSA-Hyb) standards and to the WHO 96/670 (PSA-WHO) and 96/668 (fPSA-WHO) standards. The Access Immunoassay System (Beckman Coulter, Inc.) was used in all studies. RESULTS All studies showed 20% to 25% lower PSA and fPSA test results with the WHO-standardized assays. No significant change in %fPSA (fPSA/PSA x 100) was observed. Continuing to use the traditional clinical PSA cutoffs obtained with the Hybritech standard after changing to the PSA-WHO standard could result in up to one-third of prostate cancer cases being missed. CONCLUSIONS Manufacturers should fully inform laboratories about a calibration change and its clinical impact. Laboratory reports for PSA measurements should indicate the assays manufacturer and which calibration standard was used to avoid misleading information concerning PCa risk.

Prostate cancer screening: clinical impact of WHO calibration of Beckman Coulter Access prostate-specific antigen assays.

Abstract Background: The goal of this study was to evaluate the clinical impact of using the same total prostate-specific antigen (tPSA) and free PSA (fPSA) assays calibrated with World Health Organization (WHO) materials or with Hybritech Tandem-R calibrator. Methods: From the initial correlation study that included 150 patients, the clinical impact of the WHO calibration was simulated using a large cohort (n=4548) of referred patients. Interim reports of the European Study of Screening for Prostate Cancer (ERSPC) were used to evaluate the clinical outcomes of patients and the risk of prostate cancer (PCa). Results: WHO calibration of tPSA assays leads to a reduction of about 20% in measured results (tPSA WHO=0.81 tPSA Hybritech+0.04; fPSA WHO=0.78 fPSA Hybritech+0.00; %fPSA WHO=0.92 %fPSA Hybritech+0.00). The simulation showed that the WHO calibration is associated with a risk of missing 15% of PCa. Conclusions: The discrepancies between the two calibrations lead to significant clinical misinterpretation with decreased detection of PCa if tPSA cut-off thresholds are not adjusted. Clin Chem Lab Med 2010;48:285–8.

A panel of antibodies for the immunostaining of Bouin's fixed bone marrow trephine biopsies.

AIMS: To assess a panel of antibodies on Bouins fixed bone marrow trephine (BMT) biopsies. These biopsies are widely used in routine diagnosis of various haematological malignancies and may be the sole material available in many centres; however, information regarding the immunostaining of this material is lacking. METHODS: Biopsies were taken from 72 patients presenting with various haematological malignancies (leukaemia, 38; lymphoma, 14; multiple myeloma, 20). A panel of antibodies was assessed on Bouins fixed BMT biopsies by the alkaline phosphatase-antialkaline phosphatase method. RESULTS: Three B (MB2, LN-2, Ki-B5) and two T cell lineage antibodies (UCHL-1, CD3-r) reliably identified lymphoid cells, while MPO-r, Leu-M1/CD15, and KP-1/CD68 recognised cells from the myeloid or histiocytic/macrophage series. Reed-Sternberg cells were stained by LN-2, Leu-M1, and CD30. Antibodies specific for plasma cells (VS38) and hairy cells (DBA.44) gave a variable pattern of staining. Among the proliferation markers, proliferative cell nuclear antigen but not Ki-67 related antibodies were effective. CONCLUSION: This study presents a panel of antibodies with reactivity not restricted to common fixatives that are also suitable for Bouins fixed BMT biopsies.