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Biomedical subjects

D Crisan

Publications and source records attributed to D Crisan.

At least 37 records · Page 2Linked to original sources
PubMed1995

Discordant morphologic features in bone marrow involvement by malignant lymphomas: use of gene rearrangement patterns for diagnosis.

Discordant morphology between lymph node or extra-nodal site and bone marrow (BM) involvement by non-Hodgkin's malignant lymphoma (NHL) is a common occurrence, causing diagnostic difficulties. Additional diagnostic problems are posed by lymphoid aggregates commonly found in the BM of elderly patients, the age group with the highest incidence of lymphoma. Morphologic features are used to distinguish between benign and malignant lesions but no feature is diagnostic and exceptions are numerous. Immunophenotyping is helpful for detecting B cell monoclonality, but it cannot detect T cell monoclonality. Unique B and T cell gene rearrangement patterns, the molecular "signature" of the lymphoma, can be used to detect monoclonal lymphoid populations. Finding the same rearrangement pattern in the BM as in the primary mass is proof of BM involvement by the same clone of malignant cells. We used B/T and Bcl-2 gene rearrangements to help diagnose cases with discordant morphology between primary site and BM. One hundred and seventy-five specimens, obtained from patients undergoing staging or restaging for NHL, were analyzed for B/T cell and Bcl-2 gene rearrangements by multiple restriction endonuclease digestion and Southern hybridization with 32P labeled JH, JK, CT beta, and Bcl-2 probes. Forty-two specimens (24%) from 24 patients showed discordant morphology: of 13 specimens with atypical lymphoid aggregates, only one had B cell gene rearrangement; of 15 specimens with morphologically benign lymphoid aggregates, one demonstrated B cell gene rearrangement; and of 14 specimens positive for NHL with different morphology than the lymph node, 13 were positive for B cell gene rearrangements. Molecular analysis can aid in the diagnosis of NHL, can establish a "baseline" for detection of recurrence, and is useful in monitoring therapy. These data suggest that it is also a tool for the pathologist in cases of discordant morphology between the primary tumor and BM, and should be strongly considered for each site.

B-Lymphocytes↗
PubMed1995

Separate clones in concomitant multiple myeloma and a second B-cell neoplasm demonstrated by molecular and immunophenotypic analysis.

The occurrence of multiple myeloma (MM) and a second B-cell neoplasm in the same patient is a rare event. We present 2 such patients, and provide evidence to support the presence of separate clones in these coexisting neoplasms. In the first case, MM became evident 14 months after the diagnosis of chronic lymphocytic leukemia (CLL). In past reports, most occurrences of this association, when investigated, have been regarded to be biclonal disease processes; however, with few exceptions, most were documented by immunologic studies alone. To establish the clonality in our case of CLL with MM, we examined both immunophenotypic data obtained by standard two-color flow cytometric analysis, and patterns of immunoglobulin gene rearrangement, using standard Southern analysis and hybridization with 32P-labelled JH and JK probes. This provided evidence for the presence in this patient of two separate monoclonal populations of B cells, manifested as light chain restrictions and gene rearrangements which differed in blood (CLL) and bone marrow (MM) samples. In the second case, MM presented simultaneously with bone marrow lymphocytosis and abnormal peripheral lymphocytes. Clonality studies on blood were not done. Bone marrow B-cell gene rearrangement studies, however, revealed the presence of three bands in the JK blot of significantly different intensities, suggestive of two monoclonal populations. A monoclonal population of small cells with surface B markers and surface IgM was demonstrated by flow cytometry, while a second population of larger cells with intracytoplasmic IgG matching the patient's serum monoclonal protein was detected by immunofluorescence microscopy. The results in these 2 cases expand previous findings of the rare association of MM with a second B-cell neoplasm, and demonstrate the usefulness of molecular diagnostic investigation.

B-Lymphocytes↗
PubMed1995

Myeloperoxidase mRNA detection for lineage determination of leukemic blasts: retrospective analysis.

Myeloperoxidase (MPO) mRNA is an early myeloid marker; its detection in the morphologically and immunophenotypically primitive blasts of acute undifferentiated leukemia (AUL) establishes myeloid lineage and allows reclassification as acute myelogenous leukemia with minimal differentiation (AML-MO). We have previously reported a procedure for MPO mRNA detection by RT-PCR (reverse transcription-polymerase chain reaction) and an adaptation for use of routine hematology smears. This variant procedure allows retrospective analysis of mRNA and is used in the present study to evaluate the lineage of leukemic blasts in seven cases with morphology and cytochemistry consistent with AUL. All hematology smears used in this study were air-dried, unstained or Wright-stained and stored at room temperature for periods varying between 3 days and 2 years. MPO mRNA was detected in six cases, establishing the myeloid lineage of the blasts and the diagnosis of AML-MO. In the remaining case, the blasts were MPO mRNA negative, confirming the diagnosis of AUL. The RT-PCR procedure for retrospective mRNA analysis is useful in the clinical setting, due to its high specificity and sensitivity, speed (less than 24 h), safety (no radioactivity) and convenient use of routine hematology smears; it is particularly attractive in clinical situations when fresh or frozen specimens are no longer available at the time when the need for molecular diagnostics becomes apparent.

Acute Disease↗
PubMed1994

Polymerase chain reaction in the diagnosis of chromosomal breakpoints.

Polymerase chain reaction-based methods for the detection of translocation-induced gene rearrangements are now widely used for diagnostics and patient monitoring. This article concentrates on two of the best studied chromosome translocations resulting in specific gene rearrangements and oncogene activation: the Philadelphia translocation of chronic myelogenous leukemia and acute leukemias, and the t(14;18) translocation of follicular lymphomas.

Chromosome Aberrations↗
PubMed1993

Retrospective DNA analysis using fixed tissue specimens.

The recent explosion of scientific and technical knowledge in the field of molecular biology has allowed us to make important advances in our understanding of the molecular basis of many human diseases. This technology has now entered the clinical laboratory where identification of specific genetic sequences can aid in the precise diagnosis of hematologic and other malignancies, inherited diseases, specific infectious agents, and inherited predisposition to disease. In addition, it can be applied to prenatal diagnosis, paternity testing, identification of minimal residual disease following treatment, and assessment of drug sensitivity or resistance. In many cases in diagnostic pathology, the need for molecular analysis often is not realized until after a critical tissue specimen has been fixed, embedded, and examined microscopically. Thus, there is a clear need for development of techniques that would allow the retrospective study of archival tissues that have been fixed and embedded in paraffin. This review examines in depth those factors which influence the quality of the DNA available from fixed embedded tissues and discusses the usefulness of polymerase chain reaction amplification in obtaining sufficient diagnostically useful DNA from archival specimens. It is hoped that this review will aid the diagnostic pathologist interested in the application of molecular techniques in the retrospective study of fixed embedded tissues.

DNA↗
PubMed1993

A new procedure for cell lineage determination in acute leukemias. Myeloperoxidase mRNA detection.

Determination of cell lineage in acute leukemias is essential for diagnosis and treatment. Detection of myeloperoxidase (MPO) mRNA establishes myeloid lineage of leukemic blasts that may be too primitive to be identified as myeloblasts based on morphology, cytochemistry, or immunophenotype. A highly specific and sensitive new procedure for MPO mRNA detection has been developed using HL-60 cells. It involves a microprocedure for total cellular RNA extraction, reverse transcription, and specific amplification of target sequences in the resulting MPO cDNA, by the polymerase chain reaction. Specific primers are designed to amplify an 89-base pair (bp) sequence from the signal peptide, 179 and 318-bp sequences from the start and end, respectively, of the heavy-chain sequence, and a 255-bp sequence overlapping the proregion and light chain. The correct-size amplification products, detected electrophoretically, demonstrate MPO mRNA expression in the leukemic cells analyzed. The sensitivity of this new procedure was evaluated on serial concentrations of HL-60 cells and was found to be 10-10(4) cells depending on the MPO cDNA amplified sequence. No amplification products were obtained using peripheral blood lymphocytes as a negative cellular control. The specificity of the procedure is demonstrated by Southern blotting and hybridization with 32P-labeled oligonucleotide probes specific for each of the amplified sequences. An additional advantage of this procedure is availability of results in 8-24 h, compared with 1-2 weeks for conventional RNA methods.

Base Sequence↗
PubMed1992

Amplification of intermediate-size DNA sequences from formalin and B-5 fixed tissue by polymerase chain reaction.

Retrospective analysis of DNA from paraffin-embedded fixed bone marrow biopsy specimens is possible if preceded by amplification of the DNA sequences of interest by the polymerase chain reaction (PCR). These fixed specimens yield degraded DNA that may not be suitable for direct analysis by conventional digestion and hybridization methods. This limitation is circumvented by PCR amplification and subsequent analysis of the amplified products. The model used in this study is the amplification of a 725 base-pair (bp) beta-globin gene sequence encompassing the sickle-cell anemia point mutation, followed by Cvn I digestion. The beta A beta A, beta A beta S, and beta S beta S genotypes are derived from analysis of the allele-specific digestion patterns. Two fixatives were compared: neutral-buffered formalin and a mercury-based fixative (B-5) routinely used for bone marrow biopsies. DNA extracted from B-5-fixed bone marrow specimens was found to be more degraded than DNA from neutral-buffered, formalin-fixed bone marrow aliquots from the same specimens. PCR amplification of the 725 bp beta-globin gene sequence was successful with DNA from formalin-fixed bone marrow specimens, but not with DNA from B-5-fixed identical specimens. Analysis of the amplified product by Cvn I digestion resulted in correct genotype derivation for all patients, normal controls and positive controls (patients diagnosed with sickle-cell anemia or trait). These results indicate that intermediate-size DNA sequences can be amplified and analyzed when DNA is extracted from formalin-fixed bone marrow specimens.(ABSTRACT TRUNCATED AT 250 WORDS)

Anemia, Sickle Cell↗
PubMed1991

Chronic granulocytic leukemia: reassessment of morphologic and cytogenetic characteristics in Ph1-positive and Ph1-negative cases.

33 cases of chronic granulocytic leukemia (CGL) were reassessed to determine if, by strict morphologic criteria. Philadelphia chromosome (Ph1)-negative CGL exists as a diagnostic entity and if Ph1-positive CGL could be distinguished from Ph1-negative CGL. Cases were reassessed using published criteria and, of 11 Ph1-negative cases, only 4 could be reclassified as myelodysplastic syndromes or undifferentiated chronic myeloproliferative disorder. Of the morphologic parameters evaluated, peripheral blood basophilia and bicytopenia proved to be good discriminators between Ph1-positive and Ph1-negative cases. As a group, Ph1-negative cases were more heterogeneous and tended to have lower hemoglobin, WBC, platelet count and absolute eosinophilia. Chromosomal abnormalities other than Ph1 were seen only in the Ph1-positive cases. Based on these findings, we conclude that Ph1-negative CGL constitutes a heterogeneous group, a subgroup of which is morphologically identical with the Ph1-positive CGL. The parameters that best discriminate between Ph1-positive and Ph1-negative cases are peripheral blood absolute basophilia and bicytopenia.

Adolescent↗
PubMed1991

Digoxin-like immunoreactivity in serum from neonates and infants reduced by centrifugal ultrafiltration and fluorescence polarization immunoassay.

We evaluated the TDx Digoxin II (Abbott) modified procedure for interference from digoxin-like immunoreactive factors (DLIF) in pediatric patients. The effectiveness of centrifugal ultrafiltration as a means of removing DLIF interference from the serum of such patients was assessed. We used sera from 40 patients who had not received digoxin, whom we divided into two age groups: 30 neonates (less than 34 days postpartum) and 10 infants (younger than six months). Digoxin-like immunoreactivity was detected in 34 of 41 (83%) neonatal specimens (range 0.2-1.0 micrograms/L) and 16 of 25 (60%) infants' specimens (range 0.2-1.3 micrograms/L). Centrifugal ultrafiltration of serum specimens from these patients reduced but did not eliminate the DLIF interference in some specimens. A comparison of concentrations of DLIF in serum with various other patients' characteristics demonstrated a strong correlation (r = 0.915; P = 0.0001) between DLIF and serum bilirubin in the infants. Apparent digoxin concentrations from 19 serum and serum ultrafiltrate samples collected from 13 patients (four neonates and nine infants) who were treated with digoxin showed a good correlation (r = 0.97); however, the serum samples showed a positive bias of 0.39 microgram/L. We conclude that the TDx Digoxin II modified procedure is still subject to considerable DLIF interference in these two pediatric populations. This interference can be reduced in some serum specimens, but cannot be eliminated completely as others reported.

Aging↗
PubMed1990

Polymerase chain reaction: amplification of DNA from fixed tissue.

The polymerase chain reaction (PCR) allows the analysis of DNA from biologic samples containing only nanogram quantities of DNA. We used DNA purified from fresh or frozen peripheral blood (PB) leukocytes and formalin, or B-5 fixed bone marrow aspirate clots (BM). A sequence of the beta-globin gene was amplified via the PCR then hybridized with allele specific oligonucleotide probes for hemoglobin A, S, and C. All DNA preparations, including formalin and B-5 fixed BMs, were successfully amplified; the hybridization of the amplified products resulted in patterns consistent with the hemoglobin phenotype for all patients. PCR can be used on DNA from many sources; retrospective studies using paraffin embedded fixed tissue are possible because extremely small amounts of DNA present in fixed tissue can be successfully amplified.

Adult↗
PubMedDate not supplied

The influence of novel platelet aggregation inhibitors on human blood platelet clustering and retention effected by some polymer materials, in vitro.

Human blood platelet clustering and retention in vitro induced by medical grade Cuprophane and Silastic and by non-medical grade polyethylene and Mylar, are discussed. The influence of synthetic compounds on polymer-induced platelet clustering and retention has been evaluated, interpreted in terms of physicochemical parameters, and correlated with ADP- and human alpha-trombin-stimulated aggregation; the Pearson product-moment correlation coefficient for a series of bis (alkylnipecotoylamino) alkanes was computed at r = 0.98 between (i) mylar-induced cluster formation and (ii) ADP-effected aggregation, and at r = 0.94 in corresponding correlations with thrombin as an inducing agent. All experiments were carried out in vitro. Due to the paucity of polymer specimens meeting the stipulated specifications, several evaluations had to be limited to triplicate and duplicate determinations.

Blood Platelets↗
PubMedDate not supplied

Molecular diagnostic testing for determination of myeloid lineage in acute leukemias.

Determination of myeloid vs. lymphoid cell lineage in acute leukemias is essential for diagnosis, prognosis, and treatment. However, some leukemic cells are too primitive to be identified based on conventional morphology, cytochemistry, and immunophenotype criteria. The gene for myeloperoxidase (MPO) is central to the lineage designation of myeloid cells and their function. Expression of MPO messenger ribonucleic acid (mRNA) may serve to determine the myeloid lineage of primitive leukemic cells that do not express the final product, MPO. Recently, a procedure has been developed for detection of MPO mRNA in leukemic cells, based on message amplification by reverse transcription-polymerase chain reaction (RT-PCR). Three variant polymerase chain reaction (PCR) methods, developed in view of future diagnostic applications of the RT-PCR procedure are reported: (A) nested PCR, using a second pair of internal primers for reamplification of the first PCR product; (B) multiplex PCR for concomitant amplification of up to seven sequences encompassing all regions of MPO mRNA; (C) adaptation of the standard PCR procedure and the two variant methods for use of cellular material from air-dried unstained or Wright stained smears. These variant procedures developed on the HL-60 cell line make the RT-PCR detection of MPO mRNA easily applicable as a new diagnostic test in acute leukemias and confer the versatility needed in the clinical setting. The advantages of our new procedure for MPO mRNA detection are: high sensitivity and specificity, speed, simplicity, and safety. Myeloid lineage of leukemic blasts can be established, confirmed, or ruled out in 6 to 10 hours, making results available at the time of bone marrow sign-out.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗