Data presentation.

The high data rates of flow cytometers make it imperative to use the best data presentation methods available. This unit discusses techniques designed to maximize user understanding of the data.

Complete replication of human sperm genome in egg extracts from Xenopus laevis.

To examine the ability of Xenopus egg extracts to support a complete replication cycle of human sperm genome, demembranated human spermatozoa were incubated with the extract from activated Xenopus laevis eggs. Most sperm heads were decondensed within 15 min. The heads became round within 30 min with diameters of 10-30 microns. The process of DNA replication in the pronuclei was monitored by two methods, bromodeoxyuridine incorporation and flow cytometry. The results indicate that DNA replication was initiated approximately 1.5 h after membrane structure formation and that it lasted up to 9 h. The amounts of DNA in most pronuclei were doubled by 4-9 h, depending on which donor toad was the source of the egg extract. Inclusion of the protein synthesis inhibitor, cycloheximide (100 micrograms/ml), had no obvious effect on human sperm DNA replication but appeared to prevent the pronuclei from degradation after a prolonged period (> 6 h) of incubation. After storage in liquid nitrogen for > 1.5 mo, the efficiency of the egg extracts in supporting sperm head decondensation and DNA replication was reduced for human sperm but not for Xenopus sperm. Possible applications of the use of Xenopus egg extract for human sperm activation and DNA replication are discussed.

A survey of current practices in clinical flow cytometry.

In mid-1995, a 40-question survey was distributed to determine the current practices of clinical flow cytometry laboratories. Forty-seven responses were received. Included in the survey were questions regarding the affiliation and size of the laboratory, the qualifications of the staff, the nature and number of assays performed, whether laboratory resources and specimen loads were changing, and how data were analyzed and interpreted. The results indicate considerable variability in many aspects of clinical cytometry, such as number of markers used for immunophenotyping leukemias and lymphomas, types of specimens analyzed for DNA-ploidy, and charges for specific tests.

Immunophenotypic analysis of the T cell receptor V beta repertoire in CD4+ and CD8+ lymphocytes from normal peripheral blood.

The antigenic specificity of the majority of T lymphocytes in the peripheral blood is determined by the combination of alpha and beta variable region chains present in the T cell receptor complex. Currently, the V beta chains are grouped into 25 families. Historically, determination of V beta usage has relied on detection of gene rearrangement on the nucleic acid level; however, with the increased availability of monoclonal antibodies to the product of these genomic rearrangements, immunophenotypic methods are rapidly becoming a reliable alternative method for studying the usage of V beta regions by T cells and T cell subsets. In the present study, multiparametric flow cytometry was used to determine the use of 10 V beta chains on CD4+ and CD8+ T cells in the peripheral blood of 28 normal donors. By obtaining absolute lymphocyte counts at the time blood was drawn, the absolute number of both CD4+ and CD8+ cells using particular V beta regions could be determined. Additionally, the intradonor consistency of V beta usage was examined by obtaining blood from 5 of the volunteers at an interval of approximately 1 year. The results of this study suggest a fairly homogeneous pattern of use for these V beta regions. The most striking longitudinal differences were observed in one individual who underwent a tonsillectomy midway between the T cell receptor V beta determinations.

Method to make paraffin-embedded breast and lymph tissue mimic fresh tissue in DNA analysis.

Determination of DNA ploidy has been found to be of diagnostic and prognostic value with regard to many solid tumors. Flow cytometric analysis of DNA ploidy is dependent on the binding of fluorescent dyes to DNA. Preserving cell morphology by fixing the tissue in formalin interferes with the binding of propidium iodide (PI) and other fluorescent dyes to DNA. This distortion of DNA content measurement can cause inaccuracies in DNA-ploidy determinations of formalin-fixed tissue specimens and precludes the use of appropriate DNA standards. Therefore, it has been impossible to determine accurately the DNA ploidy of formalin-fixed, paraffin-embedded (FFPE) tissues. Using formalin-fixed cells as a model for FFPE cells, we developed a thermal treatment method to reverse the effect of formalin on the binding of propidium iodide to DNA. Applying this approach to the preparation of FFPE lymph node and breast tissue for DNA analysis, we have developed a method that makes the binding of PI to the DNA of FFPE tissue mimic that of fresh tissue. Following dewaxing, rehydration, and trypsin treatment, the FFPE tissue, resuspended in PBS, was heated to 75 degrees C for 90 min to restore the PI binding to that of fresh cells. This method makes it possible to use fresh, DNA-diploid cells as an internal control and, thus, determine more accurately the DNA ploidy of tumors preserved in formalin and paraffin.

Detection and monitoring of a concomitant atypical myeloproliferative disorder and chronic lymphocytic leukemia by flow-cytometric immunophenotyping.

OBJECTIVE: To illustrate the utility of a broad panel of monoclonal antibodies to detect secondary processes or unexpected characteristics of the primary blood dyscrasia. DESIGN: Case report and discussion. SETTING: Regional academic medical center. PATIENT: A 64-year-old man presenting with an apparent acute myeloid leukemia. INTERVENTIONS: Sequential immunophenotyping with a broad panel of monoclonal antibodies to monitor progression of disease and response to therapy. MAIN OUTCOME MEASURE: Identification and monitoring of the two atypical populations in this patient with correlation to the clinical status of the patient. RESULTS: Identification of an unsuspected mature lymphoid clone and characterization of the evolution of the myelomonocytic clone. CONCLUSION: The evolving mature lymphoid clone may have been overlooked in the context of a predominant atypical myeloproliferative process, particularly if a limited panel of monoclonal antibodies had been used for immunophenotyping. Sequential immunophenotyping was useful in monitoring the progression of each atypical process.

Immunophenotyping of congenital leukemia.

Congenital leukemia is a rare but well-documented disease in which a leukemic process is detected at birth or very shortly thereafter. An estimated 175-200 reports of congenital leukemia have appeared in the literature. The majority of the cases reported have not undergone thorough immunophenotyping, but rather have been assigned lineage based on cytochemical and morphological studies. Historically, a large proportion of congenital leukemias have been thought to be of the myeloid lineage, in contrast to pediatric leukemias in general, which are primarily lymphoid. The precise proportions of the lineage assignments may be distorted by the inclusion of cases of transient myeloproliferative disorders (TMD) as congenital leukemia. The immunophenotyping data available to date suggest that congenital leukemias are phenotypically heterogeneous, lacking any common distinguishing markers. The prognosis for congenital leukemias is usually poor if leukemoid reactive processes, such as TMD, are carefully excluded.

Congenital leukemia: report of two cases.

Congenital leukemia is a rare disease in which a leukemic process is present at birth or immediately thereafter. The majority of cases presented in the literature were reported prior to the availability of contemporary immunophenotyping methods, and lineage assignment was often made on the basis of morphology alone. Congenital leukemias may be of various lineages, although, historically, monocytic and myelomonocytic congenital leukemias appear to be the most prevalent. We present two cases of congenital leukemia with detailed immunophenotypic and cytochemical characterization. One case is of the lymphoid lineage, and the second is of myelomonocytic lineage. Neither patient displayed trisomy 21.

Alterations of T-cell receptor variable region expression in human immunodeficiency virus disease.

Since only a small percentage of CD4+ lymphocytes is infected at any one time during the course of human immunodeficiency virus (HIV) disease, a question central to the pathogenesis of HIV is whether or not the depletion of CD4+ lymphocytes is a random or selective event. The majority of peripheral blood T lymphocytes use alpha and beta variable chains as components of their T-cell receptor (TCR) complex. Depletion of CD4+ T lymphocytes from the peripheral blood may be dependent on the V beta chain expressed by the CD4+ cell, based on the hypothesis that HIV may encode a superantigen. Peripheral blood from normal controls and HIV+ patients was studied for alterations in the expression of various V beta chains of the TCR. Three-color flow cytometry was used to determine the expression of V beta 2, V beta 3, V beta 8, V beta 13, and V beta 19 on all lymphocytes and on both CD4+ and CD8+ lymphocytes independently. Alteration of the V beta chains in HIV+ disease was analyzed as a function of absolute CD4 count and Centers for Disease Control (CDC) stage of the patient. These data suggest that the loss of T helper (CD4) lymphocytes during the course of HIV disease may be a selective event. These data are consistent with the hypothesis that selective depletion of CD4+, V beta 19+ lymphocytes may be due to the encoding of a superantigen by HIV. Furthermore, using multicolor flow cytometry and stratifying patients by absolute CD4 counts (or stage of disease) may reveal immunologic changes that might otherwise be overlooked.

Quality control in flow cytometry for diagnostic pathology: II. A conspectus of reference ranges for lymphocyte immunophenotyping.

Immunophenotyping, as many other clinical assays, is interpreted only in the context of reference values obtained from healthy control individuals. While the use of these reference values, or ranges, has been commonplace in the clinical flow cytometry laboratory for well over a decade, there has been little consensus in standardizing how these values should be obtained, analyzed, or expressed. This report reviews the variables to be considered in establishing reference ranges and statistical methods which can be used. Additionally, examples are given of previously published reference ranges for a variety of specimens often submitted for immunophenotyping.

Reversing the effect of formalin on the binding of propidium iodide to DNA.

Formalin, an excellent preservative of cellular morphology, is a commonly used fixative for tissue specimens in hospital pathology laboratories. This preserved material is a potential source of tissue for diagnostic and retrospective research studies on DNA using flow cytometry. Unfortunately, formalin interferes with the binding of propidium iodide (PI) and other fluorescent dyes to DNA, thus altering the measurement of DNA content by flow cytometry or image analysis. This interference has been attributed to the cross-linking of histones by formalin. Since formalin alters the measurement of DNA content in formalin-fixed and formalin-fixed, paraffin-embedded tissues, this study was designed to explore the use of various physicochemical methods to reverse the effect of the formalin on the binding of PI to DNA. This study demonstrates that resuspending formalin-fixed cells in PBS and heating them at 75 degrees C for at least 1 h prior to staining with PI restores the staining of the DNA to approximately the same fluorescence intensity as that of fresh tissue.

Treatment with recombinant IFN-gamma decreases cell surface CD4 levels on peripheral blood monocytes and on myelomonocyte cell lines.

The human cell surface protein CD4 is not only an important accessory molecule in the activation of MHC class-II-restricted T cells, but has also been implicated to be a receptor for the human immunodeficiency virus HIV-I on lymphoid and monocytic cells. We have found that a 24-h treatment of the promonocytic leukemia cell line U937 with rIFN-gamma decreases the expression of the CD4 Ag by 50% as measured by cytofluorographic analysis. The decrease in CD4 expression was dependent on the concentration of rIFN-gamma, with maximal effects occurring at 20 to 200 U/ml. The decrease appeared to be due to actual loss of the CD4 molecule from the cell surface rather than masking of a particular epitope, inasmuch as similar results were obtained with the OKT4 and OKT4A antibodies. The effect of rIFN-gamma to decrease CD4 expression was not due to a general loss of cell surface Ag, because the binding of OKM1 and anti-HLe-1 increased after rIFN-gamma treatment. Treatment of rIFN-gamma also decreased cell surface CD4 expression on the promyelocytic leukemia cell line HL-60, and on the monocytic cell line THP-1, although the extent of the decrease was less than on U937 cells. Freshly isolated normal peripheral blood monocytes treated for 48 h with rIFN-gamma bound much less OKT4 or OKT4A antibody than cells incubated in the absence of rIFN-gamma. Moreover, treatment with rIFN-gamma reduced the percentage of peripheral blood monocytes that were positive for the CD4 Ag. In contrast with the decrease in CD4 levels on rIFN-gamma-treated monocytes, treatment with rIFN-gamma had no effect on CD4 levels on peripheral blood T lymphocytes or T cell lines.

Modified histogram subtraction technique for analysis of flow cytometry data.

Analysis of flow cytometry histogram data by the subjective selection of an integration window can be a tedious and time-consuming task and is often inaccurate. A new method for automated calculation of the percent positive from immunofluorescence histograms is presented. This new method is a modification of the currently used method of channel-by-channel histogram subtraction. Its accuracy is compared to that of the channel-by-channel histogram subtraction method and to another currently used automated method, which selects an integration window by finding the channels that contain the most fluorescent 2% of a control histogram. The new histogram subtraction method is objective, easy to use, and is more accurate than other currently used automated analysis methods. PASCAL source code is given for each method of analysis.

The determination of an immunologically active dose of interferon-gamma in patients with melanoma.

This study was undertaken to determine an immunologically active regimen for the administration of recombinant gamma-interferon (rIFN-gamma). The patient population included patients with completely resected melanoma, stage I (Clark's level IV or V) or stage II. All patients exhibited no evidence of disease (NED) at the time of the study. Patients received rIFN-gamma by intramuscular (IM) injection daily for 15 days at 0.0001 mg/m2, 0.001 mg/m2, 0.01 mg/m2, 0.1 mg/m2 (ten patients/group), or 0.25 mg/m2 (five patients). Interferon (IFN) was well tolerated, with non-dose-limiting constitutional symptoms occurring in the majority of patients at 0.1 mg/m2 and 0.25 mg/m2. All five patients receiving 0.25 mg/m2 developed elevated transaminase levels, which led to a discontinuation of therapy in one patient. Immunological activity was assessed by serial measurements of natural killer (NK) cell activity, hydrogen peroxide production by monocytes, and changes in expression of Fc receptors and human leukocyte class II antigen (HLA-DR) on monocytes. These changes were determined at baseline (X2), six to seven time points during rIFN-gamma therapy, and two times after the last dose of rIFN-gamma. No changes were observed at the two lowest doses. At the 0.01 mg/m2 dose, all parameters were elevated but not as consistently nor to the same levels as seen following administration of 0.1 mg/m2. At 0.25 mg/m2, H2O2 production was enhanced, but unlike at 0.1 mg/m2, it declined during the last few days of IFN therapy. Subcutaneous (SC) administration was compared with IM administration using the 0.1 mg/m2 dose. SC administration resulted in enhanced H2O2 production and Fc receptor expression by monocytes. More consistent elevations in peroxide generation and higher levels of Fc receptor expression were seen following SC administration. No significant difference was found between the two routes of administration. A comparison of two schedules, daily and three times weekly, suggested that monocyte activation may return to normal 72 hours after IFN administration. Of the doses tested, 0.1 mg/m2 administered daily appeared to be the most effective biological response modifier (BRM) regimen, and because of ease of administration, we favor the SC route.

Regulation of human large granular lymphocyte and T cell growth and function by recombinant interleukin 2: induction of interleukin 2 receptor and promotion of growth of cells with enhanced cytotoxicity.

Human large granular lymphocytes (LGL), which account for virtually all natural killer activity, and T cells were separated from low and high density fractions of Percoll gradients, respectively. We were unable to detect interleukin 2 receptors (IL 2R) on fresh LGL and T cells using flow cytometric analysis with anti-Tac monoclonal antibody or radiolabeled probes with [125I]anti-Tac. IL 2R messenger ribonucleic acid was not observed in fresh LGL or T cells. Although phytohemagglutinin induced the expression of IL 2R on purified LGL and T cells, recombinant IL 2 (rIL 2) alone induced IL 2R messenger ribonucleic acid, IL 2R, and proliferation of LGL but not of T cells. The high level of cytotoxicity of cultured LGL against K562 cells was directly dependent on rIL 2. When T cells were costimulated by phytohemagglutinin and rIL 2 for 3 days, only very low levels of cytotoxicity were generated. Proliferation and cytotoxicity against K562 cells inhibited the culture of LGL in the presence of anti-Tac antibody for 3 days. LGL began to grow more rapidly after 5 days in culture with rIL 2 alone. When rIL 2 were removed from growing LGL for 1 day, proliferation completely stopped, and cytotoxicity was no longer detected. These data indicate that rIL 2 induces IL 2R expression in fresh LGL at the transcriptional level, promoting growth and enhancing cytotoxicity. More importantly, the presence of rIL 2 is necessary and sufficient to induce proliferation of resting LGL and to maintain the growth of LGL with potent lytic activity.

Natural killer cell activity in the rat. VI. Characterization of rat large granular lymphocytes as effector cells in natural killer and antibody-dependent cellular cytotoxic activities.

The present study examined rat natural killer (NK) cells, which mediate not only NK activity but also antibody-dependent cellular cytotoxicity (ADCC). NK and ADCC activities were compared with regard to organ distribution, strain distribution, Percoll fractionation of the effector cells, effects of aging, and potential to be augmented by biological response modifiers (BRM). Like NK activity, appreciable ADCC activity was observed in peripheral blood leukocytes (PBL), splenic leukocytes (SPL), and peritoneal exudate cells (PEC), but not in cell preparations from the peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), bone marrow (BM), and thymus (THY). ADCC activity, when compared with NK activity, was significantly higher in PBL but the same or lower in SPL and PEC. In terms of strain distribution, a high NK/ADCC strain (rnu/rnu), four intermediate NK and high ADCC strains (PVG/RTLRL, Lewis, PVG/OLA, and F344), an intermediate NK/ADCC strain (WF/N), and a low NK/ADCC strain (Buffalo) were observed. Fractionation of effector cells on discontinuous Percoll gradients revealed that both NK and ADCC activities were associated with relatively high-density large granular lymphocytes (LGL). In contrast, ADCC but little or no NK activity was associated with lower density LGL. However, the NK activity of this lower-density LGL population could be elicited following the in vitro incubation with a number of BRM, including rat interferon (IFN) and OK-432, but not rat interleukin-2 (IL-2). In general, the ADCC activity of both higher and lower density LGL-enriched cell populations correlated with both the frequency of FC gamma R+ LGL and the percentage of LGL binders to antibody-coated P815 target cells. The present study also has shown that in contrast to NK activity, which remained relatively stable with age, ADCC activity from F344 but not WF/N rats increased until 30-50 wk of age. This increase of ADCC activity in older F344 rats was accompanied by an increase in the percentage and absolute number of lower density FC gamma R+ LGL. This study demonstrates a number of similarities and differences between NK and ADCC activities in the rat. These findings should be useful for further examining and comparing the in vivo development and biological role of these two effector arms of the immune system.

Recombinant leukocyte A interferon therapy for advanced hairy cell leukemia. Therapeutic and immunologic results.

Hairy cell leukemia is a lymphoproliferative disorder characterized clinically by cytopenias. Standard therapy following variable periods of disease stability consists of splenectomy that often restores normal hematologic parameters for periods ranging from weeks to years. Fifteen patients (five without prior splenectomy or chemotherapy) were treated with 3 X 10(6) units per day of recombinant leukocyte A interferon and 14 of 15 patients completed eight weeks of therapy and were evaluated for response. There was one complete and 12 partial responses for an overall response rate of 93 percent. All of these patients' conditions have remained in complete or partial remissions and they continue to receive interferon with a median follow-up of six months. Coincident with the normalization of peripheral blood counts was a return of natural killer activity and normalization of immunologic surface markers as determined by monoclonal antibodies. This study confirms and extends earlier observations with natural alpha-interferon and indicates that recombinant leukocyte A interferon in low daily doses is also very effective treatment for hairy cell leukemia. In fact, it may be the best single modality of therapy for inducing both hematologic and immunologic recovery of these patients and deserves consideration as initial therapy.

Immunomodulatory effects of poly(I,C)-LC in cancer patients.

Poly(I,C)-LC was administered in low (1 mg/m2) and intermediate (4 mg/m2) doses to cancer patients by intramuscular injection or intravenous infusion to evaluate the immunomodulatory effects. Natural killer cell (NK) activity was elevated slightly at the low dose and remained unchanged overall, but some depression was observed at the 4 mg/m2 intravenous dose. Monocyte function was elevated in all groups of patients, as was the interferon-induced enzyme 2'5'-oligo-A synthetase. These increases were observed at the 1 mg/m2 intramuscular dose, despite a lack of detectable circulating serum interferon (IFN). In regard to cell surface markers, poly(I,C)-LC induced an increase in OKT10-positive cells and a small but consistent trend toward increases in the ratio of Leu-3/Leu-2-positive cells. Lymphocyte proliferation in response to concanavalin A was depressed by poly(I,C)-LC administration. Although an optimum immunomodulatory dose and schedule was not determined, the data indicate that low doses produce significant changes in immune function and that induction of detectable levels of circulating interferon is not required for poly(I,C)-LC to have biological effects.

Reactivity of anti-asialo GM1 serum with tumoricidal and non-tumoricidal mouse macrophages.

All peritoneal macrophage (pM phi) populations studied exhibited some binding of the anti-asGM1 serum as assessed by flow cytometry. The levels of reactivity varied quantitatively among populations, depending on the combination of eliciting and activating agents employed prior to the harvest of pM phi. Resident pM phi contained a very small percentage (4%) of cells that were strongly asGM1+. Any treatment of these cells that induced them to become stimulated or activated increased the percentage of highly asGM1+ cells. Treatments that enhanced anti-asGM1 binding including eliciting pM phi with proteose peptone (16% asGM1+) or Brewer's thioglycollate medium (66% asGM1+), treatment with the activating biological response modifiers (BRMs) MVE-2 (12% asGM1+) and P acnes (18% asGM1+), or treatment with both peptone + MVE-2 (37% asGM1+) or peptone + poly IC/LC (33%). Increased expression of anti-asGM1 was accompanied by some increase in the reactivity of the various pM phi populations to treatment with anti-asGM1 serum. This conclusion was based on the reduced viabilities of cells treated with both an eliciting agent and an activating agent prior to in vitro treatment with anti-asGM1 + C, as well as by reductions in cytolytic activity of pM phi elicited with peptone and activated by MVE-2, following anti-asGM1 treatment in vitro or administration in vivo. Conversely, the cytolytic activity of resident pM phi activated in vivo by MVE-2 or heat-killed P acnes, agents that induced relatively small increases in the percentage of asGM1+ cells, was resistant to the effects of in vivo and/or in vitro treatment with doses of anti-asGM1 serum that inhibit NK activity. These results indicate that stimulation of pM phi by eliciting or activating agents can increase the level of expression of asGM1. This increased expression of asGM1 may be a useful marker for some aspects of macrophage heterogeneity, but increased expression is not necessarily directly related to expression of tumoricidal activity. In fact, the results of this study demonstrate that anti-asGM1 serum can be used for specific depletion of NK activity in vivo in normal mice and in mice treated with at least some BRMs. However, the results also demonstrate that the use of eliciting agents, particularly thioglycollate, or eliciting agents in conjunction with activating agents can cause pM phi to become reactive with anti-asGM1 serum.