Identification and quantification of key phytochemicals in peas - Linking compounds with sensory attributes.

Pea protein isolates contain high-quality plant protein. However, they have sensory drawbacks, notably bitterness and astringency, that have limited their use in commercial foods. This study's aim was thus to identify the main phytochemicals in pea-based samples and to examine associations with sensory attributes. The phytochemical profiles of pea flour, pea protein isolates, and pea protein isolate fractions were characterized via UHPLC-DAD-MS. A total of 48 phytochemicals have been revealed: 6 phenolic acids, 5 flavonoids, and 1 saponin were identified and quantified, while another 9 phenolic acids, 10 flavonoids, and 6 saponins were tentatively identified. The impacts of protein extraction and fractionation were studied. These processes appear to have caused some compound degradation. It was found that 29 compounds were correlated with perceived bitterness and/or astringency. Therefore, these results show that certain phytochemicals can lead to negative sensory attributes in pea-protein-based products.

A B-cell mitogen from a pathogenic trypanosome is a eukaryotic proline racemase.

Lymphocyte polyclonal activation is a generalized mechanism of immune evasion among pathogens. In a mouse model of Trypanosoma cruzi infection (American trypanosomiasis), reduced levels of polyclonal lymphocyte responses correlate with resistance to infection and cardiopathy. We report here the characterization of a parasite protein with B-cell mitogenic properties in culture supernatants of infective forms, the cloning of the corresponding gene and the analysis of the biological properties of its product. We characterized the protein as a co-factor-independent proline racemase, and show that its expression as a cytoplasmic and/or membrane-associated protein is life-stage specific. Inhibition studies indicate that availability of the racemase active site is necessary for mitogenic activity. This is the first report to our knowledge of a eukaryotic amino acid racemase gene. Our findings have potential consequences for the development of new immune therapies and drug design against pathogens.

[Severe form of hereditary neuralgic amyotrophy without SEPT9 gene mutation]. / Névralgie amyotrophiante héréditaire à forme sévère sans mutation du gène SEPT9.

INTRODUCTION: Hereditary neuralgic amyotrophy (HNA) is a rare condition characterized by recurrent episodes of painful paralysis preferentially affecting the brachial plexus. It is often linked to a mutation in the SEPT9 gene. CASE REPORT: A 69-year-old female patient experienced a dozen episodes of severe neurological deficit mainly affecting the brachial plexus and the phrenic and recurrent nerves. The diagnosis of HNA without SEPT9 gene mutation was retained. DISCUSSION: HNA can have significant sequelae. A genetic heterogeneity exists and mutations in the SEPT9 gene may not be found. Immunomodulatory and corticosteroid treatments have sometimes proved to be effective.

Hemoglobin Djelfa beta98 (FG 5) Val leads to Ala: isolation and functional properties of the heme saturated form.

Hemoglobin Djelfa beta98 (FG 5) Val leads to Ala is a neutrally substituted unstable hemoglobin, exhibiting the same gross features as hemoglobin Köln beta98 (FG 5) Val leads to Met. In addition to the presence of a deheminized fraction, a heme saturated abnormal hemoglobin was visualized and isolated by high resolution electrofocusing. By functional studies of the fully heminized form, a slightly increased oxygen affinity, an impairment of heme-heme interaction and a decreased response to organic phosphates were demonstrated. These functional perturbations point out the importance of the beta98 invariant valyl residue, in the quaternary contacts. They can account for the poor oxygen delivery of erythrocytes.

Relationship between p53 gene mutations and multidrug resistance (mdr1) gene expression in myelodysplastic syndromes.

P glycoprotein, the product of multidrug resistance (mdr1) gene, is frequently expressed in advanced myelodysplastic syndromes (MDS) with an excess of bone marrow blasts and could explain their frequent resistance to chemotherapy. P53 gene mutations are also found in 10 to 15% of advanced MDS. Because it has recently been suggested that normal p53 suppressed, but that mutated p53 activated, the mdr1 gene promoter, we tried to correlate p53 mutations and P glycoprotein expression in 34 patients with MDS and an excess of bone marrow blasts (> 5%). P glycoprotein expression was assessed by immunocytochemistry using JSB1 monoclonal antibody and was found positive in 13 out of the 34 patients. p53 mutations were detected both by immunocytochemistry using three different monoclonal antibodies and by single stranded conformation polymorphism (SSCP) analysis of exons 5 to 8 of the P53 gene. Both methods detected a point mutation in 5 out of the 34 patients. Only one out of the 5 patients with a p53 mutation expressed P glycoprotein, as compared to 12 out of the 29 patients without p53 mutations. This suggested the mutant and normal p53 are not major determinants of the regulation of mdr1 expression in vivo, at least in MDS.

Detection of p53 mutations in hematological malignancies: comparison between immunocytochemistry and DNA analysis.

The wild type p53 protein has a short half-life and cannot be detected by immunohistochemistry on tissue sections. Mutated p53, on the other hand, has a prolonged half-life and becomes detectable by this method, so that its detection by immunohistochemistry in solid tumors is almost synonymous with mutation. We assessed the value of immunocytochemical analysis of p53 protein on blood or bone marrow slides in the detection of p53 mutation in hematological malignancies, by comparison with single-stranded conformation polymorphism (SSCP) analysis of exons 4 to 10 of the P53 gene. One hundred and twenty eight patients with acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), myelodysplastic syndromes (MDS), or chronic lymphocytic leukemia (CLL) were studied by both methods. Immunocytochemistry showed detectable levels of intracellular p53 in 19 cases (including 2/19 AML, 2/21 ALL, 11/48 MDS, 4/40 CLL). Staining by p53 antibodies was restricted to the nucleus of blasts in AML, ALL, and MDS, and of lymphocytes in CLL. In 16 of the 19 cases, SSCP analysis, followed by direct sequencing, showed a p53 missense mutation in exons 4 to 8 of the gene. In the remaining three cases, where the number of cells stained by p53 antibodies was small, no p53 mutation could be detected. On the other hand, SSCP and sequence analysis identified a p53 mutation in two patients who had negative immunocytochemical findings. Both cases had a nonsense mutation, presumably leading to reduced levels of truncated p53. Thus, overall, immunocytochemistry and SSCP gave concordant results in 123 of the 128 (96%) patients analyzed. Our findings show that immunocytochemistry on blood and bone marrow smears is a sensitive method of p53 mutation detection in hematological malignancies, except in the rare patients with chain-terminating mutations. Positive immunocytochemistry is found in some patients with normal SSCP findings, and could correspond to overexpression of a non-mutated p53, but also to p53 mutation in a minor proportion of the malignant cells, undetectable by SSCP.

Detection of BCR-ABL transcripts in chronic myeloid leukemia (CML) using an in situ RT-PCR assay.

Methods of minimal residual disease (MRD) detection in chronic myelogenous leukemia (CML) include chromosome analysis, reverse transcriptase polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) techniques. We report a novel method to detect intracellular BCR-ABL messenger on single cells using in situ RT-PCR, which can be performed on blood and marrow slides, without extraction of the nucleic acid. After cellular permeabilization and fixation, the mRNA BCR-ABL was reverse transcribed and amplified by PCR using digoxigenin-labelled dUTP. The reaction was revealed with the anti-digoxigenin FITC antibody. On the fluorescent microscope, a strong positive green fluorescence signal was observed in 98-99% cells in Ph1-positive cell lines. A faint signal was detected in 1.5% and 2% of negative cell lines. Likewise, a faint signal was found in 1.6-2.8% of the cells in five normal controls (mean 2.2 +/- 1.1%). The positive threshold for in situ RT-PCR was therefore determined as mean + 2 s.d. = 4.4% cells. We used in situ RT-PCR by comparison to cytogenetics (at least 30 mitoses examined), and two-step RT-PCR (10(-6) sensitivity in our hands) in bone marrow samples from 13 CML patients: two patients at diagnosis and 11 patients in hematological remission after alpha interferon (three patients), hydroxyurea (one patient) autologous bone marrow transplantation (BMT) (one patient) and allogeneic BMT (six patients). In the two diagnostic patients, 90 and 95% cells were respectively strongly positive by in situ RT-PCR. In the six patients treated by allogeneic BMT, the median percentage of positive cells was 2.4% (range 1.8-3.2). All six patients had normal karyotype and negative two-step RT-PCR results. In the five other patients, two were treated by hydroxyurea alone or autologous BMT, and 11 and 13% of the cells were strongly positive; three were treated with interferon and 14-62% of the cells were positive, generally weakly. All five patients had persistence of Ph1 (in 9-56% mitoses), and positive RT-PCR results after one round. In conclusion, in situ RT-PCR can specifically identify cells with BCR-ABL transcript and its results are concordant with those of karyotype and RT-PCR. Because of its limited sensitivity and specificity, however, it appears to have limited value in the analysis of MRD. On the other hand, it can evaluate the presence and intensity of BCR-ABL fusion transcript at the single cell level, and this could be useful in treatment monitoring.

bcl-2 expression in myelodysplastic syndromes and its correlation with hematological features, p53 mutations and prognosis.

We looked for bcl-2 protein expression by immunocytochemistry on bone marrow slides from 51 cases of myelodysplastic syndrome (MDS), of whom 25 received some form of chemotherapy. Forty-six of them had at least 20% bcl-2 positive blasts and the median percentage of positive blasts was 80%, whereas myeloid cells beyond blasts were always negative. No correlation was found between bcl-2 expression and the FAB type of MDS, CD34 expression and P-glycoprotein expression. A strong correlation between weak bcl-2 expression and the presence of a p53 mutation detected by SSCP analysis and direct sequencing was found. Response to chemotherapy (intensive chemotherapy or low-dose Ara-C) and survival were not significantly influenced by the intensity of bcl-2 expression in blasts, although there was a trend for better response to chemotherapy and longer survival in patients with strong bcl-2 expression. This trend was no longer found, however, if patients with a p53 mutation were excluded. Our findings show that blasts from a majority of MDS cases have bcl-2 expression and that strong bcl-2 expression is not associated with a poor prognosis. The correlation between weak bcl-2 expression and p53 mutation suggests a possible downregulation of bcl-2 gene expression by mutated p53, the mechanism of which remains to be established.

Expression of the multidrug resistance P-glycoprotein and its relationship to hematological characteristics and response to treatment in myelodysplastic syndromes.

Expression of P-glycoprotein (PGP), the product of the multi-drug resistance mdr1 gene was studied by immunocytochemistry on bone marrow slides using JSB1 monoclonal antibody and the alkaline phosphatase-antialkaline phosphatase (APAAP) and avidin-biotin-peroxidase (ABC) techniques in 82 cases of untreated myelodysplastic syndromes (MDS), of whom ten had evolved to AML (MDS-AML). The relationship between PGP expression, myeloperoxidase activity and immunophenotype of blast cells, karyotype and outcome was also analyzed. PGP expression was found in the blasts of 34 of the 82 patients (41%), the majority of blasts being stained in positive cases. PGP positivity was rare in 'low risk' MDS (RA and RARS: 2/12 cases) as opposed to 'high risk' MDS (RAEB, RAEB-T, CMML: 25/60 cases) and MDS-AML (7/10 cases) (p = 0.04). PGP expression was positively correlated to the presence of myeloperoxidase activity in less than 3% of blasts (p = 0.025), and CD34 antigen expression (p = 0.04), whereas CD33 antigen expression had borderline significance (p = 0.07), demonstrating that PGP expression predominated in blasts with an immature phenotype. An abnormal karyotype, and especially the presence of monosomy 7, was not correlated to a higher incidence of PGP expression, however. There was a trend for more frequent progression to AML and for shorter survival in PGP-positive cases, but differences with PGP-negative cases were not significant. Twenty patients received intensive anthracycline-Ara-C chemotherapy and ten (50%) achieved complete response, including 9/13 (69%) PGP-negative cases and 1/7 (14%) PGP-positive cases (p = 0.03). Twenty other patients were treated with low-dose Ara-C and ten (50%) responded (complete or partial response). PGP-positivity did not negatively affect response to low-dose Ara-C: 4/11 responses in PGP-negative, and 6/9 responses in PGP-positive patients (p = 0.18). Because the treatment choice in advanced MDS (especially between anthracycline-Ara-C or low-dose Ara-C, chemotherapy) is difficult, our preliminary therapeutic results suggest that the analysis of PGP expression could have practical importance in MDS. These findings however, will have to be confirmed on larger numbers of patients. Clinical trials using drugs potentially reverting mdr, activity could also be warranted in MDS.

Involvement of peripheral blood cells in multiple myeloma: chromosome changes are the rule within circulating plasma cells but not within B lymphocytes.

The mononuclear cells in the peripheral blood are implicated in the myeloma process especially with the presence of peripheral blood plasma cells (PBPC) and clonal B lymphocytes found using phenotypic or gene rearrangement techniques. The purpose of this study was to look for aneuploidy in the two main B cell components of the peripheral blood: PBPC and CD20-positive B lymphocytes. Conventional cytogenetics (CC) or DNA content analysis and fluorescence in situ hybridization (FISH) with centromeric probes were performed on bone marrow plasma cells (BMPC) of 21 patients with multiple myeloma and peripheral blood cells were studied as follows: immunostaining to look for PBPC and to assess their number, image analysis cytometry for the determination of their DNA content, and FISH chromosomes analysis. FISH was performed using probes against the chromsomes that were lost or gained in BMPC and was coupled with immunostaining of the relevant light chain or CD20 antigen to study PBPC or B lymphocytes, respectively. Monotypic PBPC were found in 16 patients. Their DNA content was the same or nearly the same as for BMPC and they exhibited the same monosomies or trisomies as those found within their BM counterpart. By contrast, DNA content of mononuclear cells other than PBPC was within normal ranges, and in 13 of 15 patients CD20-positive B lymphocytes failed to show chromosomal changes by FISH analysis. In two patients however, a few CD20+ cells with lymphoid morphology exhibited chromosome changes, hypothesizing that a few cytogenetically abnormal B cells without plasmocytic morphology may circulate. From these data, we conclude that PBPC share the same genetic abnormalities as BMPC and thus belong to the malignant clone, whereas most peripheral blood B lymphocytes are unrelated to the tumor clone.

Detection of BCR-ABL transcripts in chronic myeloid leukemia (CML) using a 'real time' quantitative RT-PCR assay.

Quantitative competitive RT-PCR techniques have been developed to detect BCR-ABL fusion transcripts in CML but they are hardly reproducible. In this work, we have developed BCR-ABL quantification by real time RT-PCR using the ABI PRISM 7700 (Perkin Elmer), a new technique which allows simple and rapid quantification of a target sequence during the extension phase of PCR amplifications. A fluorogenic probe labeled with both a reporter dye at the 5' end and a quencher-dye at the 3' end hybridizes to the target sequence on the third exon of the ABL gene. The exonuclease activity of the Taq DNA polymerase cleaves the probe and releases the reporter dye, resulting in an increase in the fluorescence signal. The absolute copy number of the target sequence (BCR-ABL) or a control gene (ABL) in an unknown sample can then be calculated using a calibration curve prepared from a set of BCR-ABL RNA standards, and results are expressed as a BCR-ABL/ABL ratio. In our hands, the sensitivity of a serial dilution of total RNA from a positive cell line (K562) in a negative cell line (HL60) was 10(-4). Fifteen CML patients in cytogenetic CR, including 11 allografted patients, two autografted patients and two patients treated by IFN were studied sequentially by this new real time quantitative RT-PCR technique in parallel with conventional qualitative two round nested RT-PCR. The two autografted patients showed high BCR-ABL/ABL ratio in all samples. The two patients treated by IFN showed a progressive decrease in the ratio. In the 11 allografted patients, four were sequentially studied 2 years or more after allo-BMT, and all ratios were below 10(-4). The four patients remained in clinical and cytogenetic CR. The seven other allografted patients were studied immediately after the procedure. Three of them showed a progressive decrease in the BCR-ABL/ABL ratio which reached 10(-4) 7 months after allo-BMT. The three patients remained in hematologic and cytogenetic CR. The remaining four allografted patients had progressive increase of BCR-ABL ratio; three developed cytogenetic relapse 9, 11, 28 months after allo-BMT, and the last patient remained in cytogenetic CR in the bone marrow but developed granulocytic sarcoma. Results of real-time quantitative RT-PCR were in agreement with those of qualitative two round nested PCR. However, evolution changes in the results of real-time quantitative RT-PCR often preceded those of the conventional technique: a decrease of the BCR-ABL/ABL ratio preceded progression from first round to second round positivity and then negativity with the classical technique; conversely, an increase in the ratio preceded evolution with the classical technique. Thus, real-time quantitative RT-PCR may show better correlation with clinical and cytogenetic evolution than conventional qualitative techniques and may help in making early therapeutic decisions in CML, especially after molecular relapse.

Philadelphia negative, BCR-ABL positive adult acute lymphoblastic leukemia (ALL) in 2 of 39 patients with combined cytogenetic and molecular analysis.

We performed cytogenetic and molecular analysis of the BCR-ABL rearrangement by polymerase chain reaction (PCR) in 39 consecutive cases of adult acute lymphoblastic leukemia (ALL). Eleven patients had a Philadelphia (Ph) chromosome. Thirteen patients had a BCR-ABL rearrangement, involving minor breakpoint cluster region (m-bcr, situated in intron 1 of the BCR gene) in 11 cases, and major breakpoint cluster region (M-bcr, 'specific' of chronic myeloid leukemia) in the remaining two cases. All of the 12 BCR-ABL cases studied immunologically were of early B, CALLA-positive immunophenotype. The 13 BCR-ABL positive cases included the 11 Ph-positive cases, and two patients with normal karyotype at diagnosis. In the two Ph-negative BCR-positive cases, seven (patient 1) and 18 (patient 2) mitoses had been examined at diagnosis. In patient 1, Ph negativity at diagnosis could certainly be explained by the small number of mitoses analyzed, as a Ph chromosome was found at relapse. This was less probable in patient 2, who raised the issue of whether authentic Ph-negative BCR-ABL-positive ALL exists (as in the chronic myeloid leukemia model) or not. Whatever the explanation, our results suggest that molecular detection of BCR-ABL should be more widely used in B-lineage ALL.

Good correlation between RT-PCR analysis and relapse in Philadelphia (Ph1)-positive acute lymphoblastic leukemia (ALL).

We sequentially performed cytogenetic analysis and RT-PCR analysis of BCR-ABL transcripts in 17 cases of Ph1-positive ALL who had achieved hematological complete remission (CR) with intensive chemotherapy (CT). Sixteen cases were studied prospectively. All but one of the patients had reached cytogenetic CR, but cytogenetic has low sensitivity in predicting relapse. Twelve patients relapsed, three died in first CR and two were alive in first CR. Two of five, two of four, and five of nine patients who were allografted (in first or second CR), autografted and received consolidation CT, respectively, achieved negative two-round PCR in the bone marrow (BM): three died in CR, three remained in CR with negative two-step PCR in the BM and three relapsed after 22 to 28 months. In all cases, relapse was preceded by switch to PCR positivity in the BM by 4 to 6 months. The remaining nine patients remained PCR-positive in the BM and relapsed after 2 to 16 months. In the four autografted cases, PCR was positive at the time of bone marrow harvest. The two patients who received a purged transplant achieved negative PCR and prolonged CR, whereas the two patients who received an unpurged transplant remained PCR positive and relapsed. In 34% of the samples where analysis was concomitant, sensitivity of PCR proved lower in the blood than in the BM. These findings show that RT-PCR is a useful tool in the monitoring of MRD in Ph1 positive ALL.

An MRI comparison of anatomical changes related to surgical treatment of prolapse by vaginal or abdominal route.

PURPOSE: The aim of this study was to determine whether or not abdominal or vaginal access is the best choice for treating genital prolapse in term of anatomical postoperative results, using an MRI pre and postoperative assessment. MATERIALS AND METHODS: Prospective study was conducted on 43 consecutive patients planned for surgery for genital prolapse from October 2001 to February 2002. The patients were studied with dynamic and static MRI sequence 4 months after surgery to indicate surgical effects on organ position. The position was evaluated with respect to the pubo coccygeal line in a dynamic sequence. RESULTS: Fifteen patients had their prolapse corrected by laparotomy always associated with sub-total hysterectomy, anterior and posterior prosthetic mesh with promontory fixation to the prevertebral ligament. Sixteen others were subjected to vaginal route with vaginal hysterectomy, paravaginal suspension followed in all cases by suspension of the vaginal cuff using Richter's technique and myorraphy of the levators. Finally, 12 patients benefited from a suspension of a sacrospinous suspension and myorraphy of the levators, without paravaginal suspension. Measure of the MRI organ location showed no significant difference except for bladder position in vaginally operated women (P = 0.02). Vaginal length and axis were comparable in all groups. CONCLUSION: Our study confirmed the objective effectiveness of the anatomical prolapse corrections conducted by abdominal route using sacropexy or by vaginal route using sacrospinous fixation. The correction provided by vaginal route always results in a return of the bladder and the vaginal fundus to their normal positions, which clearly demonstrates the short-term effectiveness of these surgical suspensions. Prolapse corrections by vaginal route did not result in any shortening of the vagina or postoperative change in vaginal orientation. Further evaluations will be needed to assess the long-term results.

Interest of immunomodulation as a mean to improve the preparation of polyclonal and monoclonal antibody reagents.

Immunomodulation by monoclonal antibodies (mAbs) was investigated in mice in order to improve the preparation of antibody reagents. Three different types of representative immunogens were chosen: a human soluble protein (secretory immunoglobulin A, SIgA), a bacterial polysaccharide from E. coli K1 and an envelope protein from the hepatitis B virus. These Ag are all of importance for diagnosis and exhibit different levels of immunogenicity. Antibody-mediated enhancement was observed against restricted and defined regions of each immunogen i.e.: the Fab epitopes of SIgA, the preS1 domain of the HBV envelope and associated cell wall components of the capsular PS. The epitopes which were enhanced appeared to be different from those recognized by the modulating mAb. Negative modulations were also observed. Moreover, new epitopes seemed to be generated. In both cases the level and direction of the modulation were irrespective of isotypy and affinity of the mAbs. Interestingly the positive modulatory effect was found to be correlated with an in vitro assay based on the binding of immune complex to antigen-presenting cells.

Determination of DNA antibodies in normal and pathological sera by a new counterimmunoelectrophoresis method.

A quantitative counter-immunoelectrophoresis technique has been applied to the evaluation of antibodies against native and single-stranded DNA. Anti-DNA antibodies have been found at high dilutions in patients with systematic lupus erythematosus, without correlation with the existence of renal lesions or with the degree of DNA binding assessed by Farr assay. Significant precipitates were also observed at significantly lower dilutions in other pathological situations and in normal subjects, posing the problem of the nature of the precipitates in these cases.

Immunoglobulin and T-cell receptor delta gene rearrangements are rarely found in myelodysplastic syndromes in chronic phase.

Clonality, in MDS, can only be assessed in patients with chromosomal rearrangements or in females heterozygote for X chromosome restricted polymorphisms. "Illegitimate" rearrangements of the immunoglobulin heavy chain (IgH) gene and incomplete rearrangements involving V delta 2 and D delta 3 segments of the T-cell receptor delta (TcR delta) gene are seen in some cases of AML, and AML post-SMD, and can be detected by a sensitive PCR method. In order to analyse clonality in additional cases in MDS, we looked for Ig H and TcR delta gene rearrangement by PCR in 95 cases of MDS. A rearrangement of the Ig H gene was seen in 2 of the 95 patients: in the circulating blood of 2 of the 36 cases of chronic myelomonocytic leukaemia (CMML) and in none of the marrow samples of the other 59 MDS. A rearrangement of the TcR delta gene (involving V delta 2 and D delta 3 segments) was seen in three cases (in the circulating blood of two other CMLL patients, and in the bone marrow of another MDS patient). Twenty-five of the 90 cases of MDS with negative PCR findings, in addition to the five cases with positive PCR findings underwent Southern blot analysis of Ig H and TcR delta genes, and PCR analysis of V delta 1 and J delta 1 segments of the TcR delta gene. Those examinations were normal in all the cases tested. In patients with positive PCR findings for Ig H or V delta 2 D delta 3 rearrangements, the proportion of rearranged cells was evaluated at 1-5% in four cases, and 5-10% in the remaining patient. Because the analysis was performed on total circulating leukocytes or total nucleated marrow cells, the nature of the clonal population in positive cases (lymphoid cells? myeloid cells? blasts?) could not be determined. From a practical point of view, Ig H and TcR delta gene rearrangements seem to very rare in MDS, and cannot be used as clonality markers in most cases.

FISH analysis with a YAC probe improves detection of LAZ3/BCL6 rearrangement in non-Hodgkin's lymphoma.

INTRODUCTION: Chromosomal translocations involving the chromosome 3q27 region are common in B-cell non-Hodgkin's lymphoma (NHL), mainly diffuse large cell lymphoma (DLCL) and less often in follicular lymphoma. Most of these rearrangements involve the same major translocation cluster (MTC) on the 3q27 region, disrupting the LAZ3/BCL6 gene. Some of those translocations are difficult to detect by cytogenetic analysis and/or Southern-blot analysis. In the present report we used a FISH assay to improve the detection of LAZ3/BCL6 rearrangements. METHODS: We isolated a YAC clone (803g3), containing the BCL6 gene, in order to analyze by FISH 19 cases of B-cell non-Hodgkin's lymphoma with cytogenetically detectable 3q27 rearrangement, including reciprocal translocation in 11 cases, deletion in two cases, and addition of undefined chromosomal material on 3q27 in six cases. RESULTS: In the 11 cases with reciprocal translocation, FISH results confirmed cytogenetic data and showed disruption of the LAZ3 region: four t(3;4)(q27;p13), two t(3;11)(q27;q23.1), four t(3;14)(q27;q32) and one t(2;3)(p12;q27). In two of the cases, reciprocal t(3;14) was associated with other cytogenetically detectable abnormalities of 3q27, but FISH showed that they did not affect the LAZ3 gene region. FISH demonstrated a reciprocal translocation with LAZ3 gene rearrangement in two of the six patients with add 3q27: one t(3;11) and one t(3;14). In the two patients with del(3q27), one had two 3q27 FISH signals and one had only one 3q27 FISH signal, but no LAZ3 gene rearrangement was observed. CONCLUSION: We have identified a YAC containing the LAZ3/BCL6 gene. This YAC probe could be useful in clinical practice to demonstrate LAZ3 rearrangements by FISH analysis on tumor samples in NHL.

Laboratory evaluation of the Abbott Cell DYN 3500 5-part differential.

The study reports the performance of the Abbott CD3500 automated hematology analyzer for the enumeration and delineation of leukocyte populations for both adult and pediatric samples, and the ability of this instrument to detect the presence of abnormal cells. Samples from 542 individual patients either attending medical practitioners or during hospitalization were examined and then subdivided for the purposes of this study into 106 samples from newborn infants (< days), 145 samples from older children (15 days to 14 years) with non-oncologic disorders, 100 samples from normal adults, and 191 samples from oncology patient (97 adults and 94 children). The leukocyte differentials provided by both the Abbott CD3500 and the Coulter STKS were compared with those obtained from conventional morphology (two observers, total of 400 leukocytes). The sensitivities and specificities of the blast, immature granulocyte (IG) and NRBC "flags" were also determined. For the non-oncology adult (n=100) and pediatric (n=145) cohorts, automated differentials were given in all samples with the CD3500, whereas the STKS did not provide a differential analysis for 20 of the 145 (14%) pediatric samples, 11 of these were absent for no obvious reason. However, for the evaluable cases, the performances of the CD3500 and the STKS were broadly similar and generally correlated well with the manual reference procedure. The results for the newborn samples were less consistent with wider 95% confidence intervals (CI) noted. For example, the CD3500 (which reported a differential for all 106 samples studied) gave CI values of +/-14.4% for neutrophils, +/-14.6% for lymphocytes and +/-8.1% for monocytes. For comparison, the STKS (which did not provide a differential in 15% of 79 samples analyzed; insufficient material being available from the remaining 27 of 106 newborn samples) gave CI values of +/-21.9% for neutrophils, +/-23.5% for lymphocytes and +/-8.2% for monocytes. For all samples, the sensitivity of the blast flag on the CD3500 was 85% with a specificity of 91% (STKS: sensitivity, 75%; specificity, 85%); the sensitivity of the CD3500 IG flag was 72% with a specificity of 76% (STKS: sensitivity, 75%: specificity, 73%); and the sensitivity of the NRBC flag was 43% with a specificity of 94%(STKS sensitivity, 37%; specificity, 88%). This study confirms competitive performance levels for the CD3500 in the analysis of normal adult samples and suggests positive performance advantages in the study of neonatal, pediatric, and leukopenic samples.

CD19 and immunophenotype of bone marrow plasma cells in monoclonal gammopathy of undetermined significance.

AIMS: To determine whether a particular phenotype or antigen is preferentially related to monoclonal gammopathies of undetermined significance (MGUS). METHODS: Bone marrow specimens from 56 patients with MGUS were stained immunocytochemically (ABC peroxidase) for CD38, CD56, CD9, CD10, CD19, CD20, CD22, and MB2. Specimens from patients recently diagnosed with multiple myeloma and reactive bone marrow samples were studied in parallel. RESULTS: CD38 was expressed on all plasma cells from all MGUS samples tested, while 36% were positive for CD56, CD9 and MB2 were both expressed strongly; CD20 was moderately expressed, and staining for CD10 and CD22 was uncommon. For these five B cell antigens there was no clear difference between their expression in MGUS and in multiple myeloma. A great difference was found for CD19: in MGUS this antigen was expressed on 2-91% of plasma cells (mean 35%) and 77% patients had > 10% positive plasma cells; in multiple myeloma its expression was low and only 12% patients had > 10% positive plasma cells. When these results were converted to numbers of CD19 positive plasma cells per 100 nucleated bone marrow cells, reactive bone marrow and MGUS specimens had a similar number of positive plasma cells. There was no correlation between expression of any of the antigens tested. CONCLUSIONS: Many of the so-called pre-B, B or activation antigens are present on plasma cells from MGUS specimens, and expression of CD9, CD10, CD20, CD22, MB2, and CD38 in MGUS was very similar to that in multiple myeloma. CD56 was frequently expressed in MGUS. In this series CD19 was highly expressed in MGUS but not in multiple myeloma. Plasma cells bearing this antigen could represent the non-neoplastic process and determination of its expression could be useful for the diagnosis of MGUS.