Chemiluminescent detection of gene rearrangements in hematologic malignancy.

A chemiluminescent based Southern blot assay is described for the detection of gene rearrangements in human hematologic malignancies. The DNA probes for regions of rearrangements within the immunoglobulin genes were labeled with digoxigenin-11-dUTP and detected by an antibody conjugated to alkaline phosphatase with Lumiphos 530 as a substrate. This assay has proven more sensitive than colorimetric assays and provides rapid turn-around times without the hazards of radioactive isotopes.

Methods in pathology. Identification of T-cell lymphomas in paraffin-embedded tissues using polyclonal anti-CD3 antibody: comparison with frozen section immunophenotyping and genotypic analysis.

While L26 (CD20) is now well established as a B-cell marker of high specificity for use in paraffin-embedded tissues, paraffin-reactive T-cell antibodies (UCHL1, MT1, Leu-22, DF-T1, and MT2) have not shown comparable lineage specificity. A new commercially available polyclonal antibody directed against a synthetic peptide sequence of the CD3 (T-cell) antigen has recently become available for use on paraffin sections. In order to evaluate the utility of this antibody, we studied CD3 expression in conjunction with L26 and leukocyte common antigen (LCA) in 15 T-cell and 20 B-cell non-Hodgkin's lymphomas (NHL), all genotypically confirmed by DNA hybridization and immunophenotyped by immunoperoxidase studies in frozen tissue. Ten of 15 T-cell NHLs (67%) showed unequivocal immunolabeling of neoplastic cells with anti-CD3 in paraffin-embedded tissue. Of the five negative cases, three were lymphoblastic lymphomas, and two were peripheral (postthymic) lymphomas (one anaplastic large cell, Ki-1 positive and one large cell, immunoblastic). CD3 expression was identical in paraffin and cryostat sections (100% concordance). Twenty of 20 B-cell NHLs were positive with L26 and LCA but were negative with anti-CD3. Other neoplasms examined, including three granulocytic sarcomas and 45 nonhematopoietic tumors, were similarly negative with anti-CD3. We conclude that polyclonal anti-CD3 is a sensitive and highly specific T-cell marker in paraffin-embedded tissue and, when used in conjunction with LCA and L26, that it can determine cell lineage in the majority of non-Hodgkin's lymphomas.

The role of cellular maturation in neutrophil heterogeneity.

Previous studies have shown that many neutrophil (PMN) characteristics are heterogeneous but the origin of PMN heterogeneity is unknown. It is unclear if PMN functional heterogeneity is secondary to maturational differences or due to distinct subpopulations of cells that possess different functional capacities. The PMN 31D8 antigen is a useful probe for evaluation of PMN subpopulations. The majority of PMNs (approximately 85%) exhibit a high intensity fluorescence after 31D8 monoclonal antibody (MoAb) labeling (31D8 enriched or "bright" PMNs) as determined by flow cytometric analysis. These cells are more functional than cells with low intensity fluorescence (31D8 diminished or "dull" PMNs). Various immunologic, clonogenic and functional techniques were used to study the expression of the 31D8 antigen in HL-60 cells and myeloid cells in order to evaluate antigenic and functional heterogeneity during morphologic maturation. The results of this study indicate that the percentage of 31D8 antigen positive (31D8 antigen enriched and diminished) bone marrow cells increases from 20 +/- 11% in myeloblast cells to 68 +/- 10% in promyelocytes, 93 +/- 2% in myelocytes and 99 +/- 1% in bands and PMNs. 31D8 antigen enriched cells first appear at the myelocyte stage (32 +/- 10%) and increase in bands (52 +/- 13%), marrow PMNs (62 +/- 13%) and peripheral blood PMNs (88 +/- 4%). These data indicate that the heterogeneous expression of 31D8 antigen in PMNs is due, at least in part, to maturational differences within the PMN population and raise the possibility that other heterogeneously expressed PMN characteristics are also maturationally derived. They also suggest that 31D8 antigenic expression may be a more precise indicator of myeloid functional maturation than maturation as identified by cellular morphology.

DNA ploidy in breast lesions. A comparative study using two commercial image analysis systems and flow cytometry.

DNA ploidy determinations on a series of 24 breast specimens were performed independently utilizing flow cytometry (FCM) and two separate commercially available computerized image analysis systems for image cytometry (ICM). The tissues analyzed were obtained from 20 carcinomas, 2 benign neoplasms and 2 benign reductive procedures. The results showed a close correlation between the DNA indices (DIs) obtained by all methods in 14 of the 24 cases. In four cases, all methods showed aneuploid peaks, but with differing DIs. In six cases (two benign and four malignant) FCM showed diploidy while ICM showed peridiploid cell populations. The results obtained with the two image analysis systems were in agreement for 20 of the 24 cases. ICM is an acceptable alternative to FCM for reproducible ploidy analysis. ICM-based measurements have the advantage of the visual discrimination of abnormal cells and therefore may have a greater sensitivity in identifying small aneuploid populations. Populations with DIs in the range of 1.0 to 1.3 need to be assessed carefully in ICM-based determinations due to the potential that these "aneuploid" peaks may represent shifted diploid populations.

Polymorphonuclear leukocyte heterogeneity in neonates and adults.

We have used a mouse monoclonal antibody (31D8) to determine whether differences in neutrophil (PMN) subpopulations might help explain decreased PMN chemotaxis in neonates compared with that of adults. 31D8 has been shown to bind heterogeneously to adult PMNs. Approximately 80% of the PMNs that strongly bind 31D8 (31D8 "bright") are the same cells that depolarize and migrate chemotactically when stimulated with the chemoattractant N-formyl-methionylleucylphenylalanine, while the 20% that weakly bind 31D8 fail to similarly respond. All neonatal PMNs bound 31D8 heterogeneously. There was a smaller population of 31D8 "bright" cells in neonates at birth (76% +/- 6%, n = 45) compared with that of neonates at three to 15 days of age (82% +/- 5%, n = 10, P less than 0.002) and both were smaller than that of adults (88% +/- 4%, n = 45, P less than 0.001 and P less than 0.001). Neonatal cord PMNs, which traversed a micropore filter in a modified Boyden chemotaxis chamber in the presence of a chemoattractant, had an increased percentage of 31D8 "bright" cells (89% +/- 7%) than did PMNs which remained above the filter (82% +/- 7%, n = 10, P = 0.034). PMN chemotaxis was less in neonates at birth (32.7 +/- 4.5 micron) than at three to six days of age (36.8 +/- 11.3 micron) and both were decreased compared with that of adults (69.1 +/- 12.4 micron, P less than 0.001 and P less than 0.001). These findings indicate that decreased PMN chemotaxis in neonates may be due in part to a smaller PMN subpopulation of highly motile cells.