Cytogenetic and molecular analysis of an unusual case of acute promyelocytic leukemia with a t(15;17;17)(q22;q23;q21).

We present a 52-year-old female with a clinical history of acute myelocytic leukemia, probable acute promyelocytic leukemia (APL). Flow cytometry results were somewhat unusual. Specifically, the promyelocytic population showed partial positivity for antigens not usually expressed in APL (HLA-DR and CD117). The interpretation of these results was that the abnormal population contained a proportion of very early promyeolocytes that had not completely lost all their "precursor" antigens. Cytogenetic analysis of a bone marrow aspirate showed a t(15:17;17)(q22;q23;q21) in all cells analyzed. Fluorescence in situ hybridization (FISH) analysis using the PML-RARA DNA probe showed a positive signal pattern (fusion) in 100% of 200 total interphase and metaphase cells examined, confirming the presence of the PML-RARA rearrangement. Multicolor FISH, which produces 24 colors to differentiate all chromosomes in a single hybridization, was applied. This study confirmed the cytogenetic interpretation of the rearrangement. No material from any other chromosome was detected on the second smaller derivative chromosome 17. Additional studies using the RARA(17q21) break-apart DNA FISH probe showed that 17q21 (RARA) was not rearranged on the derivative chromosome 17 that received the q22-->qter segment from chromosome 15. The RARA locus on the smaller derivative 17 was the allele involved in the fusion in this three-way rearrangement. The signal pattern was consistent in 100% of interphase and metaphase cells scored. This unusual t(15;17;17) prompted us to investigate further using reverse-transcription polymerase chain reaction with primers from the 3' and 5' regions of both the RARA and PML loci. These studies showed that the PML-RARA fusion was present, but the complementary fusion RARA-PML, which is usually detectable, was absent. The patient is responding well to standard treatment protocols.

Thymopoiesis in HIV-infected adults after highly active antiretroviral therapy.

The thymus of HIV-seropositive patients can enlarge as CD4+ T cell counts increase on highly active anti-retroviral therapy (HAART). This may indicate development of new T cells or represent mature peripheral T cells recirculating to the thymus. To define the etiology of the enlargement, the thymuses of two HIV-infected individuals on HAART were biopsied. For more than 3 years before initiation of HAART, both patients (38 and 41 years of age) had documented CD4+ T lymphopenia. Peripheral blood samples were obtained to assess circulating CD4+ CD45RA+ CD62L+ T cells, which were thought to have recently developed in the thymus. Peripheral blood T cells from both patients and thymocytes from the second patient were also tested for levels of DNA episomes formed during T cell receptor gene rearrangement (T cell receptor rearrangement excision circles, TRECs). With HAART, peripheral blood CD4+ T cell counts increased from approximately 60/mm(3) to 552/mm(3) and 750/mm(3) for patients 1 and 2, respectively. Thymic biopsies from both patients showed normal thymus histology with active thymopoiesis. Percentages of peripheral blood CD4+ CD45RA+ CD62L+ T cells and quantitation of T cell TRECs also reflected active thymopoiesis in both patients. Thus, in these two HIV-seropositive adults examined after initiation of HAART, thymic enlargement represented active thymopoiesis. Thymopoiesis in adult AIDS patients may contribute to immune reconstitution even after prolonged CD4+ T lymphopenia.

Value of CD23 determination by flow cytometry in differentiating mantle cell lymphoma from chronic lymphocytic leukemia/small lymphocytic lymphoma.

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) share many morphologic and immunophenotypic features. In addition to histomorphologic examination, it is customary to use the absence of CD23 to differentiate MCL from CLL/SLL, based primarily on reported comparisons of immunohistochemical staining of tissue sections. These findings are widely extrapolated to flow cytometric analysis, although available data are contradictory and not sufficiently detailed. We compared expression of CD23 by flow cytometry in 22 cases of MCL and 25 cases of CLL/SLL. Lymphoma cells in 12 of 22 MCLs were negative for CD23, and 10 showed dim expression. In contrast, none of 25 CLL/SLLs were negative for CD23, 4 were dimly positive, and 21 were moderately or brightly positive. Thus, a significant proportion of MCL exhibited overlap of CD23 expression in the low-intensity range with CLL/SLL. Clinically, there was no correlation between the intensity of CD23 expression and clinical stage at diagnosis or survival. These findings emphasize that by flow cytometry, MCL can be differentiated reliably from CLL/SLL using CD23 if negative expression is observed. However, with dimly positive expression, interpretation should be cautious.

Establishing a pure lymphocyte gate for subset analysis by flow cytometry.

Development of a more cost-effective and efficient method of performing lymphocyte subset analysis is of continuing importance in clinical flow cytometry laboratories. Current two-color methods utilize forward and right angle light scatter and multiple tubes per sample and are thereby liable to gate contamination. Methods using three-color analysis with CD45 vs. right angle light scatter (RALS) gating cannot always exclude a contaminating nonlymphoid population. We have established a two tube approach to directly measure total T cells, T suppressor, and T helper subsets, total B cells and total natural killer cells. The technique involves staining of whole blood with a mixture of five monoclonal antibodies conjugated to three fluorochromes: CD4+CD19 fluorescein isothiocyanate (FITC), CD3+CD33 phycoerythrin (PE), CD45 peridin chlorophyll alpha protein (PerCP), CD8+CD16 FITC, CD3+CD33 PE, and CD45 PerCP. Analysis is performed using a single laser flow cytometer. This method has equivalent recovery to and improved purity of the lymphocyte gate when compared to well-established methods. These antibody combinations additionally allow clear separation of lymphocytes from other leukocytes and debris as well as separation of the T cell helper and suppressor subsets, natural killer cells and B lymphocytes. We additionally provide preliminary data that an accurate lymphocyte subset analysis can be performed on a single tube containing five antibodies (CD4+CD19 FITC, CD3+CD33 PE, and CD45 PerCP), although some measurements are performed deductively.

Detection of terminal deoxynucleotidyl transferase by flow cytometry: a three color method.

This short communication describes a three-color flow cytometric analysis procedure for terminal deoxynucleotidyl transferase (TdT). The method combines CD45-gating principles and a new permeabilization method based on a commercially prepared lysing reagent. While traditional permeabilizing fixatives are often cumbersome to use, distort cell light scatter, and fail to retain the fluorochrome peridin chlorophyll alpha protein (perCP), this simple method retains light scatter and does not affect perCP, making it ideal for three-color analysis. The procedure simply involves staining whole blood or bone marrow with CD45 perCP and an appropriate phycoerythrin (PE) conjugated monoclonal antibody (MoAb) and lysing with FACS lysing solution (Becton Dickinson, San Jose, CA). After washing the cells, fluorescein isothiocyanate (FITC) conjugated anti-TdT is added. The cells are washed, fixed with formaldehyde, and acquired on a flow cytometer compensated for three-color analysis. Display of CD45 perCP vs. right angle light scatter (RALS) allows identification of blasts. Dual color expression of anti-TdT FITC and a PE conjugated MoAb identifies TdT expression on blast populations defined by specific lineage associated antigens. This method is not only useful for TdT analysis but may also prove to be a valuable tool for looking at expression of other cytoplasmic antigens in combination with surface antigens on CD45-defined blast populations.