Flow cytometry in the transplant laboratory.
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Antibodies↗
Biomedical subjects
R A Bray
Publications and source records attributed to R A Bray.
CD34+ progenitors and colony-forming units-granulocyte macrophage are recruited during large-volume leukapheresis and concentrated by counterflow centrifugal elutriation.
The recruitment of mononuclear cells (MNCs), colony-forming units-granulocyte macrophage (CFU-GM), lymphocyte subpopulations, and CD34+ progenitor cells was studied during large-volume (15-25 L blood processed) peripheral blood stem cell (PBSC) harvests. Normal donors (n = 13) underwent a 4-hour leukapheresis designed to maximize PBSC yield (blood flow rate, 85 mL/min). Mean (+/- SD) volume processed was 17.7 +/- 0.4 L, and yield was 2.4 +/- 0.7 x 10(10) white cells containing 99 percent MNCs and 1.3 mL red cells per L of blood processed. Postapheresis hematocrit, platelets, and MNCs were reduced from preapheresis values by 7, 35, and 23 percent, respectively (p < 0.05). In nine donors, the component was collected as four 1-hour samples, and culturing of CFU-GM and flow cytometric analysis of lymphocyte subpopulations and CD34+/HLA-DR+ cells were done in individual samples. Total CFU-GM were 2.4 +/- 1.4 x 10(6) (3.0 +/- 1.8 x 10(4) CFU-GM/kg) and lymphocytes were 20.8 x 10(9), with 75 percent CD3+ T cells, 10 percent CD19/CD20+ B cells, and 17 percent natural killer cells. A more than twofold increase in CFU-GM and CD34+ cells was noted over the course of the 4-hour procedure (p < 0.05). In four donors, the leukapheresis component underwent counterflow centrifugal elutriation (CCE), which separated it into four fractions in an attempt to concentrate CD34+ and CFU-GM progenitors and to deplete T-lymphocytes on a large scale. There was a 1.8-, 4.6-, 3.9-, and 0.32-fold increase in CFU-GM in the four fractions relative to the unseparated component.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Improved staining method for the simultaneous flow cytofluorometric analysis of DNA content, S-phase fraction, and surface phenotype using single laser instrumentation.
We have developed an improved technique for triple staining that permits the simultaneous flow cytofluorometric analysis of cell surface antigens, bromodeoxyuridine incorporation into DNA, and DNA quantification using 7-amino-actinomycin D. PHA-activated human peripheral blood lymphocytes were incubated with bromodeoxyuridine and stained for cell surface phenotype with phycoerythrin-labeled monoclonal antibodies. Stained cells were fixed serially with 1% paraformaldehyde and 45% ethanol. Fixed cells were sequentially stained with an anti-BrdUrd monoclonal antibody followed by a FITC-conjugated goat anti-mouse antibody and incubated with 7-amino-actinomycin D. Hypotonic buffer was employed for all procedures after fixation. Stained-fixed cells were analyzed by flow cytofluorometry for simultaneous green (525 nm), orange (570 nm), and red (greater than 650 nm) fluorescence. Utilizing this staining technique, we were able to analyze simultaneously cell phenotype, DNA synthesis, and total cellular DNA content with single laser excitation.
Antibodies, Monoclonal↗
Stephen Prudhoe 1911-1992.
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History, 20th Century↗
Conflicting phylogenetic hypotheses for the parasitic platyhelminths tested by partial sequencing of 18S ribosomal RNA.
Partial sequencing of the 18S ribosomal RNA in nine parasitic and one free-living species of platyhelminth was used to test hypotheses on the phylogenetic relationships among the major groups. The eucestodes, amphilinideans, gyrocotylideans and monopisthocotylideans appeared as a monophyletic assemblage in a cladistic analysis of the data, with a very close association between the gyrocotylideans and monopisthocotylideans. The polyopisthocotylidean monogeneans were paraphyletic to the monopisthocotylideans. The digeneans appeared to be a sister group to the monogeneans and eucestodes, while the temnocephalidean was closely related to the free-living polyclad.
Animals↗
Identification of a population of large granular lymphocytes obtained from the rheumatoid joint coexpressing the CD3 and CD16 antigens.
In this study, we phenotypically characterized large granular lymphocytes (LGL) among the synovial fluid mononuclear cells obtained from rheumatoid arthritis (RA) patients. Cytochemical and flow cytometric studies revealed an increased percentage of LGL in the synovial fluid mononuclear cells obtained from patients with RA compared to those without RA. Flow cytometric analysis revealed an expanded population of cells expressing a rare phenotype: CD3 +/CD16+. While these cells are seen in very low percentages in normal individuals (less than 2%) and have been reported in T cell lymphoproliferative disorders. In 20/30 RA patients studied, these cells constituted from 20 to 80% of the total synovial fluid mononuclear cells. Furthermore, studies of synovial fluids with greater than 20% CD3 +/CD16+ cells failed to show significant cytolytic activity even after incubation with recombinant interleukin-2, while fluids with less than 20% CD3 +/CD16+ cells possessed normal cytolytic activity. Studies of matched blood and fluid in eight patients with RA demonstrated a significantly increased percentage of CD3 +/CD16+ cells in synovial fluid compared to peripheral blood. Modulation and adsorption studies did not provide evidence that the CD16 antigen present on these CD3 cells was due to passive adsorption of soluble CD16 antigens. Thus, while the relevance of these cells in the pathogenesis of RA is not clear, this report identifies CD3 +/CD16+ cells in a disease state other than T cell lymphoproliferative disorders.
Antigens, Differentiation↗
Two populations of CD56 (Leu-19)+/CD16+ cells in bone marrow transplant recipients.
Bone marrow transplant (BMT) recipients are severely immunocompromised during the early post-transplant period as their immune systems recapitulate. Among the first cells to repopulate the peripheral blood are natural killer (NK) cells. Studies in normal subjects have revealed that the majority of NK cells express CD16 and CD56 (Leu-19). Such NK cells express low density, dim, Leu-19 (Leu-19D+). A small percentage of high-density, bright, Leu-19 cells (Leu-19B+) which are CD16- have also been reported in normal subjects. In this study we observed that peripheral blood mononuclear cells (PBMC) from BMT recipients contained two distinct populations of Leu-19+ cells that co-expressed CD16 and these populations could be separated on the basis of differential Leu-19 and CD16 fluorescence intensities. Leu-19B/CD16D and Leu-19D/CD-16B cells were present in four of nine BMT patients studied. Cells from one BMT recipient with a very large expansion of the Leu-19B+ population (46% of PBMC by flow cytometry) were studied in detail. While this patient is not characteristic of all BMT recipients, the large number of Leu-19+ cells that could be isolated from him allowed extensive analysis of a previously unreported population of cells. Our data suggest that BMT recipients can be an important group of subjects to evaluate NK cell subsets as these cells mature and, presumably, differentiate in a regenerating immune system.
Adolescent↗
T cells from patients successfully treated with OKT3 do not react with the T-cell receptor antibody.
The mechanism of OKT3 therapy is complex and may include depletion of circulating CD3 cells, modulation of the CD3 molecule, and/or functional inactivation of T cells. Although the absolute number of circulating CD3 cells in OKT3-treated patients is used to monitor therapy, many laboratories assign CD3 numbers based on reactivity with OKT3. These CD3 numbers could be artificially low since the epitope recognized by OKT3 may already be occupied. Using a monoclonal antibody against a different CD3 epitope, we detected CD3 expression on T lymphocytes from 18/18 OKT3-treated patients. Nonetheless, OKT3 therapy in these patients was clinically successful, suggesting that monitoring patients solely for CD3 is uninformative. Since CD3 is associated with the T-cell receptor (TcR), we also evaluated alpha-TcR-1, a monoclonal antibody which detects a conformational determinant of the CD3/TcR alpha/beta complex, and found that less than 1% of the CD3 cells from OKT3-treated patients reacted. Furthermore, these cells were unresponsive to allogeneic stimulation. However, when patient cells were cultured overnight in the absence of OKT3, both alpha-TcR 1 binding and responsiveness to allogeneic stimulation became detectable. Thus, the monitoring of patients treated with OKT3 can be more informative if lymphocytes are tested for reactivity with alpha-TcR-1 and an alpha-CD3 antibody other than OKT3.
Antibodies, Monoclonal↗
Identification and functional characterization of mononuclear cells by flow cytometry.
Flow cytometric technology has evolved tremendously over the past 5 years. Among the major advancements have been the development of monoclonal antibodies, fluorescent labels, and computer software that makes it possible to perform routine multiparameter analyses. The clinical application of such analyses requires that standards and quality control procedures be established. Areas that need to be specifically addressed include sample preparation, staining, instrument calibration, and sample and data analysis. This study describes the basic elements involved in establishing such procedures and explores the use of multiparameter flow cytometric analysis in the characterization of peripheral blood mononuclear cells. Two-color, flow cytometric analysis of T, B, and natural killer cells can provide important insights into the biologic features of these cells as well as significant diagnostic information.
Antibodies, Monoclonal↗
The detection and interpretation of urinary eosinophils.
We determined the clinical diagnosis for 183 patients who had urine samples examined for the presence of eosinophils. Urine samples were examined with both Hansel's stain and Wright's stain. A total of 11% of these patients had eosinophils detected in the urine. A variety of clinical conditions were associated with eosinophiluria. Urinary tract infection and acute interstitial nephritis were most common, each accounting for 25% of the total patients with eosinophiluria. Eosinophiluria proved to be a good predictor of acute interstitial nephritis. Hansel's stain was superior to Wright's stain in detecting eosinophils in urine. In particular, Hansel's stain increased the sensitivity of eosinophiluria for acute interstitial nephritis (63% vs 25%) as well as its positive predictive value (50% vs 25%).
Acute Disease↗
Discordant expression of CD3 and T-cell receptor antigens on lymphocytes from patients treated with OKT3.
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Antibodies, Monoclonal↗
The flow cytometric crossmatch. Dual-color analysis of T cell and B cell reactivities.
The flow cytometric crossmatch (FCXM) has become an increasingly utilized method to detect low levels of anti-donor antibodies (e.g., anti-HLA) in potential renal allograft recipients. Anti-donor antibodies not apparent in the standard complement-dependent crossmatch, but detectable by the FCXM, are often associated with increased episodes of graft rejection and early graft failure. In this study we examined several parameters of the FCXM in order to establish a standardized methodology. First, we observed that optimal staining results were obtained when the secondary antibody was an Fc-specific F(ab'), anti-human IgG. In contrast to an anti-whole immunoglobulin antibody, the anti-Fc specific reagent did not react with surface immunoglobulin on B cells but was reactive with cytophilic immunoglobulin present on CD16+ cells. Next we determined that dualcolor analysis was superior to single-color analysis both for the evaluation of T cell reactivities and for the discrimination of T cell from B cell reactivities. Additionally, dual-color analysis revealed that the density of class I histocompatibility antigens on B cells is greater than on T cells, indicating that B cells may be a more sensitive target for detecting low levels of anti-class I antibodies. Finally, we determined that a shift in the mean fluorescence intensity of greater than 10 channels on a 256-channel, 3-decade log scale was indicative of a positive FCXM. The data presented in these studies provide the basis for performing standardized dual-color FCXM with increased sensitivity and specificity.
B-Lymphocytes↗
Preferential expression of neo-CRP epitopes on the surface of human peripheral blood lymphocytes.
Antibodies specific for C-reactive protein (CRP) have been reported to react with certain human peripheral blood lymphocytes (PBL); however, the nature of the antigen has not been clearly defined. In the present study we identified the CRP antigenicity on PBL as a CRP neoepitope not seen on the native-CRP molecule. Neo-CRP epitopes are expressed when the native pentameric form of CRP is dissociated into free subunits. Commercial anti-CRP antisera were found to possess a significant proportion of specificities (up to 16% of the total reactivity) directed against neo-CRP antigenicity. Since similar reagents had been used in previous studies on the reactivity of anti-CRP antisera with PBL, we set out to determine if either native- or neo-CRP epitopes were preferentially expressed on PBL. We prepared antisera monospecific for native-CRP and neo-CRP, respectively, and characterized these reactivities in both direct and indirect enzyme immunoassays. When analyzed by flow cytometry, anti-neo-CRP but not anti-native-CRP antiserum was found to react with normal PBL. F(ab')2 fragments of affinity-purified anti-neo-CRP had identical activity, and the reactivity against CRP was absorbed by reagents expressing neo-CRP but not native-CRP epitopes. Flow cytometric analyses of monocyte-depleted PBL from 25 normal donors detected a mean of 23.8 +/- 5.8% anti-neo-CRP-positive cells, a higher proportion of PBL expressing the CRP antigen than previously reported. Our findings indicate that a molecule identical to, or cross-reactive with, a neo-antigenic form of CRP is present on the surface of a significant proportion of normal human PBL.
Antigens, Surface↗
C-reactive protein antigenicity on the surface of human peripheral blood lymphocytes. Characterization of lymphocytes reactive with anti-neo-CRP.
Previously, we have shown that antibodies specific for C-reactive protein determinants, not present on the native molecule, termed neo-CRP, also react with a significant percentage of PBL. In the present study, cells were evaluated by flow cytometry using alpha-neo-CRP antisera and mAb specific for lymphocyte subsets. With use of either monocyte-depleted PBL or Percoll-enriched large granular lymphocytes, we observed an overlap between cells reactive with alpha-neo-CRP and cells bearing the surface markers CD16, CD11b, Leu-7, and/or Leu-19, which are expressed on NK cells. In addition, we showed co-expression of the neo-CRP antigen with CD19, CD20, and HLA-DR, cell surface markers which are expressed on B lymphocytes. The major proportion of CD3+ cells failed to exhibit co-expression of neo-CRP. Single parameter flow cytometric analyses demonstrated that cells reactive with alpha-neo-CRP exhibited a bimodal staining pattern based on fluorescence intensity: high intensity neo-CRPbright and low intensity neo-CRPdim. Two-color analysis revealed that neo-CRPbright cells co-expressed CD19, CD20, and HLA-DR, whereas neo-CRPdim cells co-expressed CD16, CD11b, Leu-7, and Leu-19. Anti-neo-CRP also reacted with PBL obtained from patients with CD16+ lymphoproliferative disorders and from patients with chronic lymphocytic leukemia of B cell origin, but not with cells from patients with T cell or myeloid leukemias. The alpha-neo-CRP cells from patients with NK cell expansions showed dim fluorescence, whereas patients with B cell expansions showed bright fluorescence, consistent with the staining patterns observed with normal PBL. In addition, cell lines of T cell, B cell, NK cell, myeloid, and erythroid origin were evaluated for reactivity with alpha-neo-CRP. The cloned NK cell line NK 3.3 reacted as neo-CRPdim, but the B cell lines BL41, BL41/95, T1, T2, and CESS all reacted as neo-CRPbright. The cell lines K562, Molt-4, Hut-78, HL-60, U-937, and THP-1-0, which lack characteristic NK and B cell markers, did not react with alpha-neo-CRP. Additional study of the two-color histograms revealed a distinct diagonal staining pattern that was observed only when cells were co-stained with alpha-neo-CRP and either alpha-CD16 (alpha-Fc gamma RIII) or antibody IV3 (alpha-CDw32; alpha-Fc gamma RII). This finding suggests a 1:1 relationship between Fc gamma R on both NK and B cells and determinants recognized by alpha-neo-CRP.(ABSTRACT TRUNCATED AT 400 WORDS)
Antigen-Antibody Reactions↗
T-cell receptor gene rearrangement and expression in human natural killer cells: natural killer activity is not dependent on the rearrangement and expression of T-cell receptor alpha, beta, or gamma genes.
To test the hypothesis that the T-cell receptor (Tcr) gamma gene encodes a natural killer (NK) cell receptor molecule, three human NK clones and fresh peripheral blood lymphocytes with NK activity from two patients with a CD16+ lymphocytosis were analyzed for rearrangements and expression of the human Tcr alpha, beta, and gamma genes. Two of the clones displayed distinct rearrangements of their Tcr beta and gamma genes and expressed mature Tcr alpha, beta, and gamma RNA. However, one of the clones and both patient samples displayed marked NK activity but failed to rearrange or express any of their Tcr genes. These findings demonstrate that human natural killer activity is not dependent on Tcr gamma gene rearrangement and expression. In addition, they confirm previous findings concerning the lack of Tcr alpha and beta gene expression in some natural killer cells. Thus, they suggest the existence of additional NK-specific recognition molecules.
Adult↗
Flow cytometry of urine.
Flow cytometry is becoming an increasingly important tool for the evaluation of urine cytology. Although it has established a strong foothold in oncologic DNA analysis, studies of whole cells and cell markers are only in their infancy. In addition, potential diagnostic applications such as those involving binding of E. coli strains to uroepeithelial cells will move flow cytometry from a research tool to a real diagnostic tool.
Biomarkers, Tumor↗
Determination of helper:suppressor T-cell ratios.
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Humans↗
Differential surface marker expression in patients with CD-16+ lymphoproliferative disorders: in vivo model for NK differentiation.
In this study, we report on three patients, each with a CD-16+ lymphoproliferative disorder. Peripheral blood lymphocyte from all three patients were evaluated for lymphocyte morphology, natural killer (NK) function, and surface marker expression. In addition, two-color flow cytometric analysis was performed to determine the phenotype of the CD-16+ cells. Our findings indicate that the presence of increased numbers of CD-16+ cells alone is not a good predictor of NK activity. However, we observed a differential expression of the HLA class II molecules DR and DQ on the CD-16+ cells obtained from these patients that was associated with NK function. Hence, a CD-16+, Leu-7-, Leu-19+ (NKH-1A) and HLA class II+ phenotype did correlate with NK function in contrast to a CD-16+, Leu-7+, Leu-19- (NKH-1A) and HLA class II- phenotype. Of importance was the fact that the CD-16+, HLA class II+ cells did not express CD-25 or TFR, nor did they mediate cytotoxicity against solid tumor targets, suggesting that these CD-16+ cells are not activated. Thus, in contrast to previous studies of NK ontogeny that utilized in vitro activated NK cells, studies of patients with CD-16+ lymphoproliferative disorders may provide an alternative approach for examining NK differentiation.
Adult↗