Biomedical subjects
Publications and source records attributed to J R Krause.
The der(1)t(1;19)(p12;p11) has not been previously reported in myelodysplastic syndrome (MDS). Fluorescence in situ hybridization (FISH) using chromosome 1- and chromosome 19-specific probes, performed on the bone marrow (BM) cells of this patient confirmed the initial karyotype, i.e., 47,XY,+der(1)t(1;19)(p12;p11).
The clinical application of B- and T-cell gene rearrangement analysis is discussed relative to diagnostic problems in specific lymphoproliferative disorders. This article reviews the use of the Southern blot hybridization technique for B- and T-cell gene rearrangement detection in the analysis of various hematopoietic lesions. Several case studies are presented and analyzed.
Karyotype, immunophenotype, and molecular studies are important in the evaluation of Acute Lymphocytic Leukemia as these data provide diagnostic as well as prognostic information. We present a case of acute lymphoblastic leukemia with unusual cytogenetics, 45,XY,i(7q),der(9)t(3;9)(q12;p22),del(12)(p12), :der(18)t(3;18)(p14;q22),-3. This karyotype is hypodiploid, showing loss of chromosome 3, a very rare occurrence. Hypodiploidy and translocations are suggestive of a poor clinical outcome. Cytogenetics also showed a chromosome 12p deletion which has been implicated in the oncogenesis of some acute leukemias. Immunophenotype by flow cytometry was positive for CD7 and CD10, T, and precursor B cell markers respectively. Given the specificity of CD7 for T cell processes, it was felt that the flow cytometry was more suggestive of a T cell process. Gene rearrangement studies showing a T cell receptor rearrangement helped confirm the T cell lineage of this malignancy. Hypodiploidy and T cell phenotype are indicators of poor prognosis. Interestingly this patient was refractory to two conventional chemotherapeutic protocols and finally responded to an unconventional protocol of high dose Ara C, etopside, and L asparaginase.
The white blood cell differential count represents a major portion of the workload in the hematology laboratory. The traditional procedure whereby a technologist examines a Romanowsky-stained blood film and classifies 100 cells is both labor intensive and imprecise. To significantly improve the precision, the technologist would have to count and classify at least 1000 cells, which is obviously impractical in terms of time and efficiency. Until the availability of the automated hematology instruments, it was not possible to obtain a differential count that could be regarded as statistically significant. Automation is therefore necessary and desirable for both economic and clinical reasons. Automation can also provide a number of instrument-derived parameters tha have attained diagnostic significance in their own right. Analytic and technologic advances have occurred with every generation of hematology instruments. As the means of analysis becomes more sophisticated, additional categories of cells may be expected to be classified, and current categories may be more accurately and precisely defined. It is hoped this will lead to even greater clinical utility, as well as provide quality assurance and assessment methods that need to be developed similar to other procedures in the clinical laboratory.
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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.
Readily identifiable intranuclear inclusions characterize parvovirus B19 cytopathic effect in formalin-fixed, paraffin-embedded material. Such inclusions are not apparent in air-dried smears of bone marrow aspirates. Brief formalin fixation of bone marrow smears, followed by either hematoxylin-eosin or Wright-Giemsa staining, permitted easy detection of parvovirus B19 inclusions in material from a renal transplant recipient with parvovirus B19 infection documented serologically and by electron microscopy. Formalin fixation of bone marrow and peripheral blood smears before hematoxylin-eosin or Wright-Giemsa staining may simplify the morphological diagnosis of parvovirus B19 infection.
DNA ploidy analysis was determined on 100 consecutive tumors from a wide variety of sites using both fresh and paraffin-embedded tissue on the same specimen. The correlation coefficient (r) value between the methods was 0.85. Aneuploidy was detected by both methods in 51/100 (51%) of the cases. Fresh tissue analysis yielded 10 additional cases (overall 61% aneuploidy) not detected on corresponding paraffin-embedded sections, whereas paraffin-embedded analysis detected 4 additional cases (overall 55% aneuploidy) not revealed by fresh tissue analysis. Fresh tissue analysis produced lower coefficients of variation and resulted in a cleaner preparation with less cellular debris. Fresh tissue analysis was also superior to paraffin for the detection of hypodiploid, near-diploid and multiple peaks. Analysis of paraffin-embedded material allows examination of archival tissue and provides a more rapid means of long-term follow-up and statistical correlations for prognostic studies. Although the overall correlation of both methodologies for DNA analysis showed a minimal variation in results, in our experience fresh tissue analysis has an advantage and is preferable, when available, for ploidy analysis.
The routine use of panels of monoclonal antibodies has been complementary to the French-American-British (FAB) leukemia classification, and has unmasked the occurrence of mixed acute leukemia (myeloid-lymphoid). It is widely accepted that children with Down's syndrome (DS) have a high incidence of acute leukemia. There is an extensive body of literature emphasizing the cytogenetic findings in these children. However, information as to the immunophenotype is often limited to the lymphoid surface determinants. The authors report two children with DS whose leukemic blasts were studied with a panel of 17 monoclonal antibodies (myeloid, lymphoid, and megakaryocytic) by flow cytometric examination and were classified as biphenotypic acute leukemia. The blast population coexpressed myeloid and T-cell surface markers. The lymphoid origin was ruled out on the basis of negative terminal deoxynucleotidyl transferase and molecular analysis demonstrating germline configuration for the JH and beta TCR genes.
Rearrangement of the immunoglobulin heavy chain and of the T-cell receptor beta subunit was analyzed by using restricted polymerase chain reaction (PCR). To differentiate between the germline configuration and the rearranged genome in a DNA sample extracted from lymphocytes, we compared the ratio of the amplified products. The intensity of amplification of the intron region (JHF) upstream of the first joining region was compared to the intensity of joining region 6 of the immunoglobulin heavy chain. The number of the amplification cycles in the PCR was designated in such a way that the ratio of JHF/JH6 was less than one in the rearranged configuration. As the concentration of clonal B-lymphocytes with the rearranged genome in the sample increased the amplification of the JHF intron proportionally decreased. We used the same approach for the two constant regions of the T-cell receptor beta chain. As one of the intron regions of the constant sequence became depleted by rearrangement so the amplification of the particular region decreased. Therefore, the absence or decreased concentration of a particular product of amplification indicated deletion and thus rearrangement of the genome in the leukemic B- or T-lymphocytes. The threshold of detection of cells with the rearranged genome on a photograph of agarose gel loaded with the particular amplified regions and staining with the ethidium bromide is less than 10% by densitometric tracing and 25-50% by visual evaluation. This novel approach allows the detection of the rearranged DNA sequences in a 2 day span. Hence, it can serve as a diagnostic tool for the identification of clonal expansion of lymphocytes in acute leukemias and lymphomas in particular and for the detection of deleted genomic regions in general.
Chromosome studies were carried out after a 24-hour harvest of unstimulated bone marrow aspirate cell cultures from a 75-year-old male with a clinical diagnosis of acute myelomonocytic leukemia (FAB M4). Analysis of nine cells after trypsin-Giemsa banding (GTG) revealed two cell lines with a mosaic chromosome pattern, 46,XY/46,XY,t(7;19)(q22;p13.3). A review of the recent literature reveals one case of childhood ALL with a 46,XY/46,XY,t(7;19)(q11;q13) chromosome pattern [1] and a 46,XY,t(3q;11q),t(7q;19p),t(15;17)(q26;q22) in one patient with ANLL (FAB M3) [2]. The t(7;19)(q22;p13.3) seen in our case has not been reported as the sole specific clonal chromosome rearrangement in myeloid neoplasia. Interestingly, the plasminogen activator inhibitor type I, multi-drug resistance, and erythropoietin genes are located at band 7q22 and the insulin receptor gene is located at band 19p13.3. Both sites contain fragile site loci. The possible role of these fragile sites, genes, or other genes in the rearrangement can only be surmised.
One hundred twenty-six effusion samples from 102 patients were examined by cytology and flow cytometry (FCM). Overall, there was an 84% correlation between cytologic and FCM results. Of the 36 malignant cases determined by cytologic examination, FCM revealed an aneuploid peak in 20 (56%). Image analysis (IA) performed on the malignant cytologic cases with a diploid flow pattern detected two additional aneuploid peaks. In addition, FCM indicated three aneuploid cases in which cytologic characteristics were initially interpreted as benign (false negative). Aneuploidy was therefore detected in 64% of the malignant effusion specimens by FCM and IA. Twenty-three of the total of 24 aneuploid cases detected by FCM were associated with malignancy (predictive value = 96%). The one nonmalignant case was that of hemorrhagic pancreatitis with infected pseudocyst. FCM is an excellent tool when moderate to large numbers of tumor cells are present, whereas use of IA is advantageous for specimens containing smaller numbers of malignant cells because these can be directly analyzed. When an aneuploid peak is present, a diagnosis of malignancy must be suspected, and, if the initial cytologic screen is negative, a critical review of the cytology slides is justified. In those cases with an equivocal atypical cytology report and an abnormal cytometric histogram, additional investigation is warranted. In some malignancies the tumor cells will be diploid (in this study 36%) and neither FCM nor IA will add to tumor detection, leaving cytologic examination as the definitive technique.
We have examined the effect of recombinant human tumor necrosis factor (TNF) upon granulocyte kinetics in cancer patients in a phase I clinical trial. TNF was given to each patient intravenously over 2 h at varying doses. A marked drop in the total white blood cell count, absolute polymorphonuclear leukocyte (PMN) count, and absolute monocyte count occurred reproducibly at 30 min after TNF initiation. Also noted was a drop in the absolute lymphocyte and eosinophil counts, both of which reached their nadir at approximately 4 h. A marked increase in immature PMN leukocytes (bands) was noted beginning at 1 h. These changes were statistically significant. Statistically significant increases in hemoglobin and hematocrit occurred at the 30 min time point but subsequently decreased to approximately 90% of pretreatment baseline. Additionally, the platelet count decreased, reaching its nadir approximately 6 h after TNF initiation. In four serial studies in patients on the highest dose of TNF, the granulocyte adhesion protein CD11b was shown to increase on the surface of the PMN leukocytes by as early as 7-15 min after initiation of TNF infusion. In each of these, expression of CD11b antigen increased prior to the disappearance of PMN leukocytes from the peripheral circulation. A similar finding was obtained for monocytes. This work indicates that within 30 min of intravenous infusion of TNF, mature granulocytes and monocytes have left the peripheral circulation. This is followed by an apparent bone marrow response indicated by an outpouring of bands. The initial granulocyte and monocyte emigration from the peripheral circulation is preceded at highest-dose TNF by increased cell surface expression of CD11b for both cell types, suggesting a causal relationship between these temporally linked events.
In North America, the dose of warfarin has been unintentionally increased during the past two decades because of failure to recognize the effect on the anticoagulation test of less sensitive tissue thromboplastins. Although there still is some controversy, the suggested dose of warfarin has now been adjusted downward to reduce the risk of bleeding. These revised dosing recommendations incorporate the international normalized ratio (INR), which takes into account the source of thromboplastin. However, there is considerable variability in the sensitivities of thromboplastin from manufacturer to manufacturer and lot to lot. Therefore, prothrombin times (PTs) are not comparable from laboratory to laboratory without knowing the sensitivity of the thromboplastin. Unless laboratories adopt a standardized tissue thromboplastin, the PT should be reported as an INR.
The leukemia cells from 63 children with acute leukemia were evaluated by flow cytometry with a panel of monoclonal antibodies that included lymphoid and myeloid lineage-specific antigens. Surface markers from patients with acute lyphocytic leukemia (ALL) did not correlate with their FAB classification except for L3 leukemia. Myeloid leukemias of FAB class M1-M4 were positive for CD13 and CD33, whereas CD14 and MY8 were only detected in FAB class M4 leukemia. Mixed leukemias were subclassified as intralineage (B+T+) or interlineage (B+ or T+/M+). Interlineage leukemias represented 5.6% of ALLs, and all patients are alive after treatment with ALL protocols. Interlineage mixed leukemias represent 7.9% of all leukemias occurring in 3.7% of ALLs and 33% of acute myeloid leukemias (AMLs). All children with mixed interlineage leukemias are alive after treatment with the protocol for the dominant leukemia; however, follow-up periods are too short to predict final outcome. The high proportion of mixed interlineage leukemias in AMLs supports Greaves' theory of lineage promiscuity, that is, there is a normal period of hematopoietic development when individual cells co-express multiple lineage antigens on the cell surface.
Flow cytometric analyses have become commonplace in the clinical laboratory for determining cell lineage and for quantitation of cells bearing a given phenotype. Because these assays are being conducted to support diagnoses or assist in determining therapy, it is crucial to ensure that these tests are highly accurate and reproducible within a laboratory and among laboratories involved in similar endeavors. This quality assurance has been slow evolving in clinical flow cytometry for a variety of reasons: the exquisite sensitivity and delicacy of the instrumentation that recognize previously undetectable variations in staining; the constant improvement of the hardware and software; the rapid development of new techniques and reagents of clinical interest; and the failure of any existing specialty or subspecialty to encompass all aspects of flow cytometry. This article provides an overview of quality assurance necessary for the flow cytometric analysis of cell surface markers. Practical experience, published studies, and suggested guidelines from accreditation agencies have been combined to develop the text.
The white blood cell differential continues to be one of the most widely performed clinical laboratory procedures. However, its clinical usefulness is affected by sampling error and, to some extent, by classification criteria. It is also labor intensive and expensive to perform. Automated leukocyte differential instrumentation addresses many of the sources of error that occur with the manual differential. Current state-of-the-art instrumentation will give results that equal or exceed the routine manual differential. Because these instruments also examine red blood cell and platelet parameters, as well as providing white blood cell information, they can better screen for significant abnormalities as well as greatly reduce the expensive and time-consuming manual differential procedures.