Prediction of CD4 count from CD4 percentage: experience from three laboratories.

OBJECTIVE: CD4 counts have been used to monitor progression of disease in HIV infection as criteria for initiation of therapy, and to stratify and follow patients in clinical trials. Recently, the Centers for Disease Control and Prevention (CDC) has made CD4 counts part of the classification of HIV disease. Because a CD4 percentage may be the only laboratory information available, this study was initiated to determine whether the correlation between CD4 percentages and CD4 counts is sufficiently high to enable these measures to be substituted for each other. DESIGN, SETTING AND PATIENTS: One thousand consecutive CD4 measurements from the University of Washington (UW) were used to create a model that was tested using datasets of 1000 CD4 measurements each from Maryland Medical Laboratories (MML) and Rush-Presbyterian-St Luke's Medical Center (Rush). The patients were not selected for age, sex, risk group or treatment. All patients from MML and Rush were known to be HIV-positive, while the HIV status of all UW patients was unknown. RESULTS: The model predicted that a patient with a CD4 percentage > or = 14% would have a CD4 count > or = 200 x 10(6)/l(if CD4 percentage of 14% was used, 9% of patients would have a CD4 count > or = 200 x 10(6)/l), and a patient with a CD4 percentage > or = 27% would have a CD4 count > or = 500 x 10(6)/l(if CD4 percentage of 27% was used, 17% of patients would have a CD4 count > or = 500 x 10(6)/l). CONCLUSIONS: These CD4 percentage correlations may be useful when a white blood cell and lymphocyte count are not available to calculate the CD4 count.

CD7+ acute non-lymphocytic leukemia: evidence for an early multipotential progenitor.

Nineteen of 71 (26%) cases of acute myelogenous leukemia (AML) were found to express CD7, a cell surface marker found early during T lineage differentiation. These myeloid leukemias often expressed other lymphoid markers and frequently had rearranged T-cell receptor beta and immunoglobulin heavy chain genes. We propose that in CD7+ AML, the malignant transformation occurred in a CD7+ progenitor cell. CD7+ myeloid leukemic precursors may be capable of limited differentiation with loss of CD7 during the initial phase of the disease, but this capacity may diminish during the course of treatment such that CD7 expression persists.

Interference of recombinant soluble CD4 immunoglobulin G in flow cytometric measurement of CD4+ lymphocytes.

Recombinant soluble CD4 covalently linked to an immunoglobulin G heavy chain (rCD4-IgG) was evaluated clinically for the treatment of human immunodeficiency virus infection. The interference of rCD4-IgG with the measurement of peripheral blood CD4 lymphocytes by whole-blood lysis flow cytometric analysis was investigated using three commercial monoclonal antibody reagents. Addition of rCD4-IgG resulted in an artifactual decrease in measured CD4 number at rCD4-IgG levels of greater than or equal to 1 micrograms/mL; the threshold for this decrease was dependent on the concentration of monoclonal antibody in the commercial preparation used for the measurement of CD4. This artifactual decrease in CD4 cell count was observed in two patients who received rCD4-IgG intravenously. The apparent decrease in CD4 count was eliminated with the use of a single phosphate-buffered saline wash step before the addition of monoclonal antibody. rCD4-IgG can bind to anti-CD4 antibody and lower the measured CD4 cell count in vitro; this interference can be eliminated by a single or a double wash step and is necessary when using the whole-blood lysis flow cytometric technique of enumerating CD4 lymphocytes in patients receiving rCD4-immunoglobulin G.

Secondary T-cell lymphoproliferation after marrow transplantation.

Secondary lymphoproliferative syndromes in immunosuppressed patients have been characterized as polyclonal or monoclonal B-lineage disorders nearly always associated with Epstein-Barr virus (EBV) infection. The authors now report three patients with a distinctly different lymphoproliferative syndrome. Two patients with common acute lymphoblastic leukemia antigen (CALLA) (CD10)-positive acute lymphoblastic leukemia and one patient with acute myelogenous leukemia, respectively, received high-dose chemoradiotherapy followed by marrow transplantation from either an HLA-identical sibling or HLA-mismatched parent. All three patients developed severe graft-versus-host disease (GVHD), requiring immunosuppressive treatment with corticosteroids. A secondary malignant T-cell lymphoproliferation occurred 2, 21, and 43 months, respectively, after marrow transplantation. In all three cases the lymphoid cells expressed T-cell surface antigens and were morphologically and immunophenotypically distinct from the malignant cells present before transplantation. One tumor was of host cell origin, one was probably of donor origin, and the tumor origin in the third case could not be determined. The authors were unable to find any evidence for EBV, human T-cell lymphotropic virus type I or II, human immunodeficiency virus, or human herpesvirus 6.

Analysis of T-cell receptor gene rearrangement in lymph nodes of patients with mycosis fungoides. Prognostic implications.

OBJECTIVE: To determine the prognostic value of analyzing lymph node (LN) DNA from patients with mycosis fungoides for the presence of a monoclonal T-cell population. DESIGN: Inception cohort study. SETTING: A tertiary care referral center in Seattle, Wash. PATIENTS: Fifty-five uniformly staged patients with the diagnosis of mycosis fungoides and who had a lymph node biopsy, 21 with clinically abnormal nodes and 34 with normal nodes. MAIN OUTCOME MEASURES: Lymph nodes were evaluated by Southern blot analysis for T-cell receptor beta-chain (TCRB) gene rearrangement and by histopathologic examination for the LN classification using the National Cancer Institute system. Patients were observed clinically for a mean (+/- SD) of 4.7 +/- 3.4 years. RESULTS: Patients with detectable TCRB gene rearrangement in lymph node DNA had an increased likelihood of a poor clinical outcome and a decreased probability of survival (P < .001 for both) compared with patients with the TCRB germline. Although patients with clinically enlarged nodes were more likely to have the TCRB gene rearranged, those with normal nodes and the TCRB gene rearranged also had a poor clinical outcome and a decreased probability of survival. Similar to those with the TCRB gene rearranged, most patients with advanced histopathologic changes (LN3 and LN4) had a poor prognosis. The presence of a rearranged TCRB gene, however, correctly predicted some patients with intermediate LN scores (LN2) who had a poor clinical outcome. CONCLUSIONS: Detection of a monoclonal T-cell population, as demonstrated by a rearranged TCRB gene on Southern blot analysis, in LNs of patients with mycosis fungoides is predictive of a poor clinical outcome and a reduced probability of survival. Lymph node TCRB gene analysis provides additional prognostic information for patients with mycosis fungoides with intermediate LN histopathology.

Treatment of large granular lymphocyte leukemia with oral low-dose methotrexate.

Morbidity and mortality in patients with T large granular lymphocyte (T-LGL) leukemia result from infections acquired during severe neutropenia. Optimum treatment for severe neutropenia remains undefined. We conducted an uncontrolled but prospective study of low-dose oral methotrexate, up to 10 mg/m2 weekly, in 10 patients with this disease. Therapeutic response was assessed by serial clinical evaluations and laboratory determinations including complete blood counts, lymphocyte phenotyping, and T-cell receptor gene rearrangement studies. A partial response was defined as a sustained increase in neutrophil count greater than 500/microL. A complete clinical remission was defined as achievement of a normal complete blood count and CD3+ LGL count. Previous prednisone treatment in eight of these patients had produced one clinical remission and four partial responses; tapering of prednisone in each of these patients resulted in recurrence of severe neutropenia. Five patients in this study received both methotrexate and tapering doses of prednisone. Complete clinical remissions on methotrexate were observed in five patients; an additional patient had a partial response. Molecular analyses of T-cell receptor gene rearrangement could not detect the abnormal clone in three of five patients achieving a complete clinical remission. Two weeks to 4 months of therapy were needed before attaining a neutrophil count greater than 500/microL. Complete and partial responses have been maintained on therapy, with a follow-up period ranging from 1.3 to 9.6 years. Low-dose oral methotrexate therapy is an effective treatment for some patients with LGL leukemia.

The pathophysiology of pure red cell aplasia: implications for therapy.

To determine the utility of marrow culture in defining the natural history and therapeutic response of pure red cell aplasia we have studied 37 patients. Patients were evaluated at the University of Washington before specific therapies (n = 21) or at the time of treatment failure in = 16). Evaluation included a medical and drug exposure history, a physical examination, a chest x-ray or computed tomography to rule out thymoma, lymphocyte immunophenotype studies, anti-nuclear antibody and rheumatoid factor determinations, marrow cytogenetics, and marrow progenitor cell cultures. Retrospective Southern analyses to detect human parvovirus B19 was performed in the 27 patients for whom sera was stored. Clinical follow-up was obtained to document therapeutic responses. Normal burst forming unit-erythroid (BFU-E) growth (>30 bursts/10(5) marrow mononuclear cells [MMNC]) in culture proved an outstanding predictor of clinical response, as 27 of 29 individuals with normal frequencies of erythroid bursts in culture responded to immunomodulating therapies (sensitivity 96%, specificity 78%, predictive value 93%, P = .0001 with two-tailed chi square analysis). Overall, 28 patients responded to either immunomodulating therapies or drug withdrawal. Twenty-four patients obtained a normal hematocrit (complete response [CR] and 4 additional patients became transfusion independent (partial response). Although responding patients often required several therapies, 20 of 24 (83%) patients who obtained a CR have sustained a normal hematocrit without maintenance therapy at the time of last follow-up (median 5 years). In contrast, of 8 patients with poor in vitro BFU-E growth (<6 bursts/10(5) MMNC), 7 failed to respond to any therapy and all died (median survival time 17 months). Our data suggest that in individuals, from whom BFU-E mature appropriately in culture, immunosuppressive drugs should be used sequentially until a CR is obtained and a durable remission is the expected outcome.

Polyclonal proliferation of activated suppressor/cytotoxic T cells with transient depression of natural killer cell function in acute infectious mononucleosis.

In acute infectious mononucleosis large numbers of atypical lymphocytes proliferate in response to B cells infected with Epstein-Barr virus, generally resulting in a self-limited illness. Although both T-cells and NK cells are known to be involved, the precise origin of the large granular lymphocytes in this disorder is incompletely understood. Using two-colour immunofluorescent flow cytometry, we sequentially examined the phenotype of selected T cell and NK cell subsets from nine patients with infectious mononucleosis. In parallel, we determined whether these lymphocytes utilized a restricted repertoire of the T cell receptor gene and also measured their NK activity. Our results show that in acute infectious mononucleosis there was a greater than three-fold increase in T lymphocytes with the phenotype CD2+, CD3+, CD8+ and DR+. A modest increase in Leu7(HNK1)+ and CD4+ T cells was also seen. In addition, there was a three-fold increase in cells coexpressing CD3- and CD16+, the phenotype reported to represent most NK cells. In spite of this latter finding, however, a marked decrease in NK function was found at the time of diagnosis, gradually returning to normal by day 28. Finally, Southern blot analysis of DNA from patient lymphocytes showed polyclonal rearrangements of the T cell receptor beta chain gene. These studies indicate that the proliferation of activated suppressor/cytotoxic T lymphocytes in acute infectious mononucleosis is polyclonal and is associated with transient depression of NK function.

Comparison of the cellularity and presence of residual leukemia in bone marrow aspirate and biopsy specimens in pediatric patients with acute lymphoblastic leukemia (ALL) at day 7-14 of chemotherapy.

BACKGROUND: Many pediatric chemotherapy protocols for treatment of ALL require a bone marrow examination at day 7 or day 14 after initiation of therapy. The usefulness of a bone marrow biopsy, in addition to an aspirate, has been a frequently asked question. PROCEDURE: This study addresses the evaluation of bone marrow cellularity and presence of residual leukemia in both aspirate and biopsy specimens in 45 consecutive pediatric patients (ages 1-19 years, 19 females, and 26 males) with ALL 7-14 days after initiation of therapy. DISCUSSION: 20/45 patients showed evidence of residual leukemia by bone marrow biopsy; 16/20 (80%) of these had evidence of residual leukemia in the aspirate specimen. Of the 4 aspirate specimens that did not demonstrate residual leukemia, 2 had <5% blasts and 2 had too few cells in the aspirate for evaluation. Of the 25/45 bone marrow biopsy specimens with no detectable residual leukemia, 14 of the aspirates had <5% blasts, and 11 had too few cells in the aspirate for evaluation. 13/45 (29%) of the aspirates had too few cells for a differential count. The bone marrow cellularity judged from the aspirate specimen was considered to be low (0-1+) in 34/45 patients. Of these 34 patients, the bone marrow biopsy showed hypocellularity (<20% cellularity) in 12/34, moderate cellularity (20-79% cellularity) in 14/34, and hypercellularity (>79% cellularity) in 8/34. CONCLUSIONS: We conclude that both the bone marrow aspirate and biopsy specimens provide important information in evaluating the response to chemotherapy in pediatric patients with ALL at day 7-14 of induction chemotherapy. The aspirate alone may be misleading in terms of cellularity in many patients and may not provide evidence of residual leukemia.

Human umbilical vein endothelial cells display high-affinity c-kit receptors and produce a soluble form of the c-kit receptor.

Stem cell factor (SCF) is a hematopoietic growth factor produced by fibroblasts and endothelial cells that stimulates the growth of primitive hematopoietic cells. SCF triggers cell growth by binding to the c-kit receptor. Because endothelial cells can respond to certain hematopoietic growth factors, we tested human umbilical vein endothelial cells for display of the c-kit receptor and examined the effect of SCF on endothelial cell proliferation, adhesion molecule expression, and production of tissue factor. Quantitative binding experiments with 125I-SCF showed both high-affinity (Kd = 42 pmol/L) and low-affinity (Kd = 1.7 nmol/L) c-kit receptors. There were approximately 1,100 high-affinity c-kit receptors, and 5,400 low-affinity c-kit receptors per endothelial cell. Enzyme immunoassays showed that endothelial cells released soluble c-kit receptor and SCF. The transmembrane form of SCF was detected by indirect immunofluorescence analysis using monoclonal or polyclonal anti-SCF receptor antibodies. The addition of SCF (100 ng/mL) did not alter endothelial cell proliferation over a 7-day period. Similarly, there was no change in the release of tissue factor or expression of inducible endothelial adhesion molecules (intercellular adhesion molecule-1, endothelial-leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1) measured by enzyme-linked immunosorbant assay at 4 and 24 hours after SCF addition. The neutralizing anti-c-kit receptor monoclonal antibody SR-1 blocked binding of 125I-SCF to the c-kit receptor by 98% but did not alter endothelial cell proliferation or adhesion-molecule expression. c-kit receptors were also detected on adult endothelial cells lining small blood vessels in normal human lymph nodes. These data indicate that normal human endothelial cells produce SCF and show high-affinity c-kit receptors that have the capacity to dimerize. The lack of response to exogenous SCF may be because of intracellular activation of the c-kit receptor via autocrine production of SCF. Alternatively, SCF and c-kit may play a role other than stimulation of proliferation, adhesion-molecule display, or tissue factor production by endothelial cells. The production of soluble c-kit receptors by normal human endothelial cells may serve to regulate the bioactivity of SCF within the bone marrow microenvironment.

Chimeric BCR-abl messenger RNA as a marker for minimal residual disease in patients transplanted for Philadelphia chromosome-positive acute lymphoblastic leukemia.

We correlated polymerase chain reaction (PCR)-detectable BCR-abl fusion transcripts with cytogenetic status in 24 patients with acute lymphocytic leukemia (ALL). Of 10 Philadelphia chromosome negative (Ph-) patients, only one was found to exhibit a BCR-abl fusion transcript. Fourteen patients with Ph+ ALL, including eight in clinical remission, exhibited PCR-detectable BCR-abl rearrangements. A detectable Ph chromosome was present in only five of the eight patients in clinical remission. Of the three cytogenetically negative, BCR-abl-positive patients, two eventually succumbed to post-bone marrow transplantation (BMT) relapse. The third died of early transplant complications. Serial PCR analyses were performed on four Ph+ ALL patients in clinical remission who underwent allogeneic BMT. One patient who was PCR negative on post-BMT days 21 and 75 became PCR-positive on day 116 and died in relapse on day 154. One patient was weakly positive for BCR-abl on day 23, negative on day 56, but died of transplant complications on day 124. Two patients exhibited no post-BMT BCR-abl rearrangements and remain well on days 279 and 371. Our findings suggest that PCR analysis may be useful in the early identification of relapse in patients transplanted for Ph+ ALL.

Serum reactivity to human T-cell leukaemia/lymphoma virus type I proteins in patients with large granular lymphocytic leukaemia.

To investigate the possibility that the recently recognised syndrome, leukaemia of large granular lymphocytes, could be associated with human T-cell leukaemia/lymphoma virus type I (HTLV-I), sera from 12 patients with this type of leukaemia were tested by the use of western-blot techniques for IgG antibodies to proteins related to human T-cell leukaemia/lymphoma virus type I (HTLV-I). Sera from 6 patients, including 2 patients with rheumatoid arthritis, reacted with p19 or p24 retroviral proteins or both. In contrast, no sample from 32 patients with uncomplicated rheumatoid arthritis, 27 with Felty's syndrome, 11 with other connective tissue disorders, or 21 normal individuals reacted with HTLV-I. The results suggest that leukaemia of large granular lymphocytes may be associated with a retrovirus related to HTLV-I.