Jean-Luc Gala

Marrow residual disease (RD) in patients with B-cell chronic lymphocytic leukemia (B-CLL) who are in complete remission (CR) after treatment with purine analogues is reported to have a prognostic value, but sample dilution, factors interfering with marrow aspiration, or undetectable immunoglobulin rearrangement can affect the assessment of RD by molecular or immunologic methods. As demonstrated for hairy cell leukemia and follicular lymphoma, bone marrow trephine biopsy specimen immunostaining (BMT/IS) can successfully detect residual malignant cells. The aim of this study was to use BMT/IS and computerized image analysis (CIMA) of bcl-2-positive cells to quantify RD in B-CLL patients in CR, after achievement of CR and more than 1 year later. This methodology was compared with other conventional techniques, i.e., cytologic, flow cytometric, cytogenetic, and molecular analysis. BMT/IS readily detected RD in every trephine biopsy specimen examined, either after CR or at distant follow-up. CIMA allowed an objective quantification of residual B-CLL cells, as evidenced by the correlation with semiquantitative polymerase chain reaction results. Both analyses indicated a progression of RD. This finding was also supported (but inconsistently) by the other techniques. CIMA with an interstitial labeling index, therefore, seems to be a reproducible and sensitive method to detect persistence and progression of RD in patients with B-CLL. This method could apply to other hematologic malignancies infiltrating the bone marrow.

Detection of intracellular antigens by flow cytometry requires effective fixation and permeabilization of the cell membrane. This study compares three fixation/permeabilization techniques: two commercial chemical reagents, the ORTHOPermeaFix^TM (OPF) and the FIX&PERM Cell Permeabilization Kit^® (F&P), and a novel method based on microwave heating (MWH). They have been applied to the detection of two nuclear (p53 and rb/p105) and two cytoplasmic (bcl-2 and mdr-1/gp-170) antigens, using positive- and negative-control cell lines and peripheral blood mononuclear cells. Western blotting was performed as a control of protein expression. For the four antigens assessed, cellular morphology, discrimination between intact cells and debris, percentage of positive cells, and mean fluorescence intensity were examined. For this last parameter, the assessment of the MWH technique was performed using SD and a graphical approach inspired by the concepts described by Bland and Altman (Lancet 1986;346:1085–7) as well as Petersen et al. (Clin Chem 1997;43:2039–46). The statistical analysis shows that MWH is comparable to the commercial methods and that its reproducibility is also equivalent to OPF and F&P. As assessed for some of the most clinically relevant intracytoplasmic and intranuclear antigens, the MWH method appears to be a valuable and inexpensive alternative. It is worth noting that, unlike commercial reagents, MWH altered surface antigens. Interestingly, this feature, which would prevent cell selection on the basis of combined membrane and intracellular epitopes, is associated with a decrease of nonspecific background fluorescence.

Multiplex amplification of femA and mecA genetic determinants allowed an early and rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) in endotracheal aspirates of mechanically ventilated patients. femA and/or mecA amplification and bacteriological results were concordant in 57 of 60 samples. In all three discrepant cases, complementary bacteriological tests confirmed the presence of MRSA first identified by molecular analysis. These results underline the value and rapidity of this molecular diagnosis for MRSA infection and control surveillance in intensive care units. Rapid MRSA detection is expected to have a significant clinical impact not only on patient outcome but also on the costs for isolation and treatment.

Circulating prostate cells can be detected in cancer patients by using reverse transcriptase–PCR (RT-PCR) assay for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) mRNA. A quality-control study involving a conventional RT-PCR assay was performed and, surprisingly, detected both transcripts in many negative control cell lines and in normal blood samples. The existence of an illegitimate transcription of the PSA and PSM genes was evidenced by sequence analysis of several PSM and PSA-PCR products. Sequencing indeed demonstrated the presence of a PSA or PSM polymorphism in some but not all the cell lines and patient samples, as well as a heterozygous mutation (G to A; Asp to Asn) in the Jurkat cell line. Moreover, the amount of PSA transcript in MCF-7, a PSA-negative breast line, increased after incubation with cycloheximide. Interestingly, the frequency of positivity was as high as 12% in male samples if only tested once, but dropped to 3% upon multiple testing of the same cDNA. This highlights the stochastic effects in RT-PCR results at high sensitivity, hence the importance of repetitive testing in clinical samples. Decreasing the number of cycles avoided the amplification of illegitimate transcripts but also affected the limit of detection, as evidenced with PSA and PSM cDNA containing plasmids, mixing of LNCap with normal blood samples, and the PSA-PSM-negative K562 cell line. The current data raise the need for a multicentric standardization of the RT-PCR methodology used to amplify PSA and PSM transcripts.

The presence of the 20210A allele of the prothrombin gene has recently been shown to be a risk factor of venous thromboembolism, probably mediated through increased prothrombin levels. The aim of the study was to determine the frequency of the prothrombin 20210A allele in 193 consecutive unselected patients with venous thromboembolism and 100 healthy controls and to analyze the clinical profile associated with this new inherited thrombophilic factor. In agreement with previous reports, we found a frequency of 7.3% of heterozygous carriers of the 20210A allele among patients and 1% among controls. We confirm that plasma prothrombin levels are more elevated in the individuals bearing the prothrombin 20210A allele compared with those who do not. We did not find any relationship between the presence of the prothrombin 20210A allele and either a family history of thromboembolism, the rate of recurrences or the age at disease onset. However, the co-inheritance in the same individual of both prothrombin 20210A allele and factor V Leiden was associated with a significantly lower age at disease onset suggesting a synergistic contribution of both abnormalities.

AIMS: To assess a panel of antibodies on Bouin's 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 Bouin's 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 Bouin's fixed BMT biopsies.

Beta-thalassaemia, a widespread autosomal recessive disorder, occurs sporadically in Northern and Western European countries. Molecular analysis of the beta-globin gene has been carried out in 30 members of 15 unrelated indigenous Belgian families which presented with non sideropenic hypochromic and microcytic anaemia. For all of them, extensive search failed to find an ancestor at risk for the disease. The beta-globin genes were first screened for frequent beta-thalassemic mutations by dot-blot hybridization with specific radiolabeled oligonucleotide probes. Direct automated fluorescence-based DNA sequencing and, in one case, Southern blotting were also used. All the 30 patients were found to be heterozygous for a beta-thalassemic mutation. Eight different mutations were identified. Among these, four are commonly found in the Mediterraneans: codon 8 (-AA), IVS-I-1 (G-->A), IVS-1-6 (T-->C) and codon 39 (C-->T); three have occasionally been reported: initiation codon (T-->C) and codon 35 (C-->A) and a rare deletion of 12.6 kb which removes all the beta-globin gene and its flanking regions. A new mutation, a -CC deletion at codon 38/39 was found in one family. These results both at the biological and molecular level show that beta-thalassaemia exist in indigenous Belgian families with no known ancestor a risk for the disease. They also show that clinicians and biologists should keep in mind the existence of beta-thalassaemia in indigenous Belgian families when investigating hypochromic and microcytic anaemia in patients whom the past familial history does not evocate a risk for the disease.