Comparison of the effects of Ficoll-Hypaque separation and whole blood lysis on results of immunophenotypic analysis of blood and bone marrow samples from patients with hematologic malignancies.

We compared flow cytometric immunophenotyping results obtained by using the lysed whole blood method of sample preparation with those obtained by using Ficoll-Hypaque-separated cells on 44 consecutive specimens from patients with various hematologic malignancies. When the samples were analyzed as a group, seven antigens (CD2, CD3, CD5, CD11c, CD20, CD22, and CD34) demonstrated significantly different percentages of positively staining cells. When the samples were grouped by disease, results for patients with acute lymphocytic leukemia were discordant for CD22 and HLA-DR and results for patients with hairy cell leukemia were discordant for CD34. Most of the differences, however, were not with antigens critical to the evaluation of the malignancy. Additionally, the most frequent reason for differences in the percentage of positive cells was due to isotype control-based placement of the quadrant markers and not an actual discrepancy in staining. However, analysis of the CD34 antigen yielded eight instances in which staining of Ficoll-Hypaque-separated cells was essentially negative, but a clearly positive population was evident with the lysed preparation. This finding has important implications because of the prognostic significance of this antigen. Further studies are needed to determine the cause of this phenomenon.

Stress-associated reductions of cytotoxic T lymphocytes and natural killer cells in asymptomatic HIV infection.

OBJECTIVE: Previous research has documented a possible relation of stress and depression to cell-mediated immunity. The authors examined how stressful events and depression may affect key parameters of cellular immunity in subjects with and without HIV infection. METHOD: Data were collected on 99 asymptomatic HIV-positive and 65 HIV-negative homosexual men as part of an ongoing, longitudinal study. Criticisms of previous studies of psychoimmunity were addressed by 1) using a comprehensive, semistructured interview to measure the objective context of stressful events, 2) double labeling of lymphocytes with monoclonal antibodies to measure subsets of cytotoxic/suppressor T lymphocytes and natural killer (NK) cells, and 3) controlling for circadian effects and methodological factors. RESULTS: In the HIV-positive men, severe stress was significantly associated with reductions in NK cell populations and a subset of T cells thought to represent cytotoxic T effector cells, particularly the CD8+ T cells expressing the CD57 antigen. In the HIV-negative men, no clear and consistent relation between stress and immune system measures was found. Depression was not correlated with any variables in either of the groups, perhaps due to the low levels of depressive symptoms. CONCLUSIONS: The findings suggest that stress is associated with reductions in killer lymphocytes (decreased NK cell and cytotoxic T lymphocyte phenotypes). The data provide evidence that stress may alter cell populations that provide cytotoxic defense against infection in HIV-positive men and indicate that the clinical significance of stress-related changes in cytotoxic T lymphocytes and NK cells in HIV infection warrants further study.

Two color flow cytometric analysis of concomitant acute myeloid leukemia and chronic lymphocytic leukemia.

During evaluation for macrocytic anemia and thrombocytopenia, a 74-year-old female was found to have a leukocytosis with two apparent populations of malignant cells identified in her peripheral blood smear and bone marrow aspirate. Morphologic characteristics of the two cell types were unusual, and cytochemical assays yielded ambiguous results. Two-color flow cytometric analysis demonstrated two distinct cell populations with immunophenotyping patterns consistent with chronic lymphocytic leukemia (CD5+/CD20+) and acute myelocytic leukemia (CD33+/CD34+), detected concurrently. The concomitant appearance of CLL and AML has been reported only rarely. The use of two-color flow cytometry to differentiate the populations demonstrates the utility of this technology in resolving unusual hematological malignancies.

Comparison of a lysed whole blood method to purified cell preparations for lymphocyte immunophenotyping: differences between healthy controls and HIV-positive specimens.

Although the majority of clinical laboratories now use a lysed whole blood (LWB) method for routine immunophenotyping, researchers wishing to perform other types of studies with lymphocytes from HIV+ patients may still need to use purified cell preparations, such as peripheral blood mononuclear cells (PBMC). A comparison study of the two methods was performed, using peripheral blood specimens from normal donors and from patients infected with the human immunodeficiency virus (HIV+). Reproducibility studies and several types of holding studies (both before and after specimen processing) were also performed. The results suggest that the two different methods of sample preparation have different effects upon abnormal patient specimens than those observed in healthy controls. Immunophenotyping results derived from the two different methods cannot be considered equivalent for the purposes of quantitating the presence of a particular type of cell.