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PubMedDate not supplied

Effects of DTIC, DM-COOK and ICRF-159 on the number of circulating Lewis lung carcinoma cells detected by flow cytometry.

Circulating tumor cells can be detected by means of flow cytometry in the blood of mice bearing i.m. Lewis lung carcinoma. This technique can be applied in the case of aneuploid tumors and does not require either concentration of nucleated cells or other processing of the blood samples. It offers the advantages of simplicity and speed and allows quantitative measurement of the number of circulating tumor cells. It can be applied to the study of the effects of drug treatment on the number of circulating tumor cells, for those drugs which do not cause alterations in the nuclear DNA content of normal diploid blood cells. The number of circulating tumor cells determined by flow cytometry is markedly reduced by treatment with ICRF-159, by dimethyltriazene DM-COOK, and also by its clinically used analog, DTIC.

Animals↗

PubMed2003

Prognostic significance of preoperative DNA flow cytometry in surgically-treated cervical cancer.

OBJECTIVE: To evaluate the prognostic significance of preoperative DNA flow cytometry compared with other clinical and histologic variables in cervical carcinoma. STUDY DESIGN: Sixty-four patients with FIGO Stage Ib-II cervical cancer treated with radical abdominal hysterectomy and systematic pelvic lymphadenectomy were analyzed. The mean follow-up was 3.4 (range 0.3-9.8) years. DNA flow cytometry was performed with fresh tumor tissue. Four biopsies were recut from the surgical specimen within 30 minutes of the operation. The ectocervix was divided into four quadrants and a specimen obtained from each. DNA-low-grade tumors (diploid, near-diploid, tetraploid and near-tetraploid) were distinguished from DNA-high-grade tumors (aneuploid and hypoploid). Carcinomas with more than one non-diploid stem line were considered heterogeneous. An S phase fraction >7% was classified as low, 7% - < 14% as moderate, and > or = 14 as high. DNA ploidy, DNA heterogeneity, S phase fraction and various clinical and histological variables were related to disease-free survival. RESULTS: In the univariate analysis patients with DNA-low-grade carcinomas had significantly better disease-free survival than patients with DNA-high-grade tumors (82% vs 45%, p = 0.021). Carcinomas with an S-phase fraction < 7% were associated with better disease-free survival (0.8) than those with an S-phase fraction 7% - > 14% (0.62) and those with > or = 14% (0.64), but this was not statistically significant. Cox stepwise regression analysis showed DNA-heterogeneity, age, grade, parametrial involvement and extrapelvic metastasis to be independent prognostic factors. CONCLUSION: DNA ploidy and DNA heterogeneity are of prognostic importance in cervical cancer. DNA flow cytometry may be used preoperatively to identify low-risk and high-risk patients within a given stage.

Adult↗

PubMed2000

Studies of Atlantic salmon (Salmo salar L.) blood, spleen and head kidney leucocytes using specific monoclonal antibodies, immunohistochemistry and flow cytometry.

Monoclonal antibodies (mabs) raised against Atlantic salmon serum IgM (C7G7 and G2H3) and isolated peripheral blood leucocytes (PBL) (E3D9, C4B6 and D8B3) were applied in this study. Using immunoenzymehistochemistry, immunofluorescence and flow cytometry, the distribution of mab+ cells in blood, spleen and head kidney from Atlantic salmon were studied. Immunostaining on cytospin preparations and flow cytometry of isolated PBL showed that the Ig+ cells recognised by C7G7 and G2H3 were mononuclear leucocytes (MNL). The cytospin preparations showed some Ig+ cells with strong cytoplasmic staining, most likely plasma cells. The salmon blood neutrophils were the only E3D9+ cells in cytospin preparations of PBL, and E3D9 recognised about 94% of the defined neutrophil fraction in flow cytometry. The reactivities of C4B6 and D8B3 were to a large degree similar in both immunoenzymehistochemistry and flow cytometry, recognising both MNL and blood neutrophils. Immunofluorescence double staining of PBL with C4B6 and D8B3 showed double staining of all mab+ cells and D8B3 was apparently not able to block the binding of C4B6 to PBL. Immunofluorescence double staining of PBL also revealed more E3D9+ than C4B6+ neutrophils. In immunostaining on cryostat sections of spleen and head kidney, staining of cells was observed with all the mabs, the head kidney generally containing more positive cells than the spleen. Some potential applications for immunological studies using these mabs are suggested.

Animals↗

PubMed1997

Thyrotropin receptor autoantibodies in serum are present at much lower levels than thyroid peroxidase autoantibodies: analysis by flow cytometry.

Using Chinese hamster ovary (CHO) cells that express high numbers of TSH receptor (TSHR) on their surface, we studied the feasibility of detecting directly by flow cytometry the binding of autoantibodies in patients' sera to the native TSHR. After using a serum (BBI) with high potency in the TSH binding inhibition (TBI) assay to establish the protocol, we studied an additional 38 sera: 10 without TBI activity (1-4.2% inhibition), 10 with moderately high TBI values (17.3-39.4% inhibition), 10 with high TBI levels (52-95.1% inhibition), 4 from normal individuals without autoimmune thyroid disease, and 4 from patients with systemic lupus erythematosus. We observed that a number of sera, including some without thyroid autoantibodies, contain antibodies against unknown antigens on CHO cells. Preadsorption with untransfected CHO cells before addition to the TSHR-10,000 cells eliminated or greatly reduced this nonspecific background. None of the sera from normal individuals, subjects with negative TBI values, or patients with systemic autoimmunity generated a positive signal on flow cytometry with TSHR-10,000 cells relative to the signal on untransfected cells. Remarkably, only 4 of 21 TBI-positive sera (including BBI) unequivocally recognized the TSHR on flow cytometry. In contrast, when thyroid peroxidase (TPO) autoantibodies in the same sera were studied using CHO cells overexpressing TPO on their surface, all 20 sera with TPO autoantibodies clearly elicited positive net fluorescence relative to untransfected cells. Study of the potent serum, BBI, revealed similar fluorescence (approximately 250 U) for TPO autoantibodies and TSHR autoantibodies at dilutions of 1:1000 and 1:10, respectively. Thus, by flow cytometry, the titer of TPO autoantibodies in the BBI serum is about 100-fold higher than that for TSHR autoantibodies in the same serum. In conclusion, the present data provide the strongest support for the idea that TSHR autoantibodies in the sera of patients with autoimmune thyroid disease are present at much lower levels than are TPO autoantibodies. This finding has important implications for the diagnostic detection of TSHR autoantibodies and for understanding the pathogenesis of Graves' disease.

Animals↗

PubMed1980

Determination of rat mast cells by flow-cytometry.

Counting of rat mast cells and assessment of degranulation were performed by flow-cytometry using a cytograph. It was shown that the number of mast cells in a total cell population may be obtained rapidly and reproducibly following toluidine blue staining. A dose-response for degranulation by compound 48/80 was performed and it was shown that azure A staining (in 40% sucrose) discriminates degranulated from non-degranulated mast cells, while trypan blue staining discriminates between cytotoxic and non-cytotoxic degranulation. Values obtained by flow-cytometry and microscopic evaluation were in good agreement; assessment by flow-cytometry provides a convenient new approach to counting mast cells and assessing their degree of degranulation.

Animals↗

PubMed1992

Clinical application of flow cytometry for DNA analysis of solid tumors.

Recent developments of flow cytometry (FCM) technology which make multiple correlative biological measurements on normal and neoplastic cells is affecting areas of diagnostic pathology as well as research fields, and a general understanding of FCM techniques is essential for pathologists. Today, FCM DNA measurements of tumors also becomes routine in the clinical and/or pathological laboratory for aid in cancer diagnosis and cancer treatment. It can also contribute to diagnosis of tumors as a supplemental method to conventional histopathology, and DNA ploidy and the percentage of S-phase fraction are considered as complementary prognostic parameters independent of the stage of disease. This article reviews clinical applications of flow cytometry focusing on the DNA measurements of solid tumors, and related practical issues, such as the methodology for nuclear DNA measurement, interpretation of DNA histograms and the relationship of DNA ploidy and S-phase fraction to clinical and pathological features of human solid tumors.

Aneuploidy↗

PubMed2004

Monitoring hyperproliferative disorders in human skin: flow cytometry of changing cytokeratin expression.

BACKGROUND: Monitoring dynamics of different cell populations in solid tissues using flow cytometry has several limitations. The interaction and changes in epidermal subpopulations in hyperproliferative skin disorders such as psoriasis, a very common chronic inflammatory skin disease, may, however, elucidate the role of different cell populations in the pathogenesis and the effect of therapy in these disorders. We describe a new, simple method for identifying and quantifying different epidermal subpopulations in hyperproliferative skin disorders and an in vivo model for epidermal hyperproliferation by using six-parameter assessment and three-color flow cytometry. METHODS: Epidermal single-cell suspensions were derived from normal human skin before and after standardized injury and from untreated and treated psoriatic lesions. Samples were stained with anti-cytokeratins 6 (K6) and 10 (K10), anti-vimentin, and 4',6-diamidino-2-phenylindole. With three-color flow cytometry, different epidermal subpopulations were further defined by six different parameters. RESULTS: Correlations were found for mild psoriasis and K10(+)K6(-) cells and for moderate psoriasis and K10(-)K6(+) cells. Treatment of mild psoriasis resulted in a 70% increase of the K10(+)K6(-) cells and an 18% decrease of the K10(-)K6(-) cells, whereas treatment of more severe psoriasis resulted in a 77% increase of the K10(+)K6(-) cells and a 33% decrease of the K10(-)K6(+) cells. The tape-stripping model showed changes in suprabasal keratin expression before proliferative activity. CONCLUSIONS: The present flow cytometric assay permits simultaneous quantification of epidermal differentiation, inflammation, proliferation, and hyperproliferation-associated keratinization and provides information on pathogenesis and therapy of hyperproliferative skin disorders.

Antibodies↗

PubMed1996

Prediction of relapse of pediatric acute myeloid leukemia by use of multidimensional flow cytometry.

BACKGROUND: Most patients receiving therapy for acute myeloid leukemia (AML) enter an interval in which leukemic blast cells cannot be detected by light microscopy (i.e., morphologic remission). However, many of these patients experience a subsequent relapse. Multidimensional flow cytometry, which allows the discrimination of antigens expressed on normal and malignant cells, can detect small numbers of cancer cells in bone marrow or peripheral blood specimens. This technique enables the detection of one leukemic blast cell among 10(3) to 10(2) normal regenerating hematopoietic cells. PURPOSE: We determined whether the presence of residual leukemic blast cells, identified in the bone marrow of pediatric patients with AML by use of multidimensional flow cytometry, would be predictive of subsequent leukemic relapse. METHODS: Multidimensional flow cytometry was performed on 205 marrow specimens collected throughout the course of treatment from 39 patients who had achieved morphologic remission. The analyses employed monoclonal antibodies directed against CD45 in combination with mixed pairs of monoclonal antibodies directed against 10 other antigens. A time-varying Cox regression analysis that controlled for sample time intervals, age, sex, morphologic classification of disease, and white blood cell count at diagnosis was used to relate the multidimensional flow cytometric results to the risk of relapse after achieving remission. Reported P values are two-sided. RESULTS: Thirty-five of the 39 patients had bone marrow specimens available from the time that first morphologic remission was achieved. Leukemic blast cells were detected in the specimens from 19 (54%) of these 35 patients. Twenty-five of the 35 patients did not receive an allogeneic (i.e., from a different genetic background) bone marrow transplant during first morphologic remission, and 13 of 14 with residual leukemic cells experienced a relapse at a median time of 153 days after diagnosis (range, 48-863 days). Nine of the 11 patients who did not receive an allogeneic bone marrow transplant and lacked evidence of leukemic blast cells at first morphologic remission relapsed at a median time of 413 days after diagnosis (range, 321-794 days). Among the 10 individuals who received an allogeneic bone marrow transplant during first morphologic remission, five were positive for leukemic blast cells and five were negative; one of these patients (positive for leukemic blast cells) experienced a relapse 265 days after diagnosis, and three others died of transplant-related complications. The estimated risk of relapse during intervals of multidimensional flow cytometric positivity (i.e., intervals of remission for which the immediately preceding cytometry measurement was positive) was 2.8 times greater than that during negative intervals (95% confidence interval = 1.1-7.0; P = .02). CONCLUSIONS AND IMPLICATIONS: Multidimensional flow cytometry identifies residual leukemia in more than half of the patients with AML who are in morphologic remission. The detection of leukemic blast cells in these patients by multidimensional flow cytometry is predictive of a more rapid relapse.

Acute Disease↗

PubMed2004

Potential pitfalls of comparative measurements of reticulocytes with flow cytometry and microscopy in prematures and infants.

Reticulocyte counting by flow cytometry (Bayer H*3, ADVIA 120) in blood of prematures, infants and children > 1 year of age was compared with microscopic counting under research conditions (9000 counted red blood cells per slide). While in children >1 year a good concordance of both methods was observed, 2.3-2.4-fold higher values were obtained in neonates by microscopy (Brilliant Cresyl Blue stain, 0.5%). However, another laboratory found good agreement between H*3-counting and microscopy in samples also obtained from neonates using the same methods. Despite very similar results for all age groups in comparative flow cytometry measurements in both laboratories, counting of smears from neonates differed, showing an approximately 2.3-fold larger amount of reticulocytes in our laboratory. The reason for these observations was a greater enlargement (1250-fold) used routinely in our laboratory compared with 800-fold in the other one. Thus very mature reticulocytes frequently found in neonates could only be detected using a 1250-fold enlargement. Similarly, the low concentration of the colouring matter used in the H*3 (0.0005% oxazin or 0.001% ADVIA 120) is obviously not sufficient for detection of mature reticulocytes. Therefore, it is important to consider this phenomenon and to standardise microscopic enlargement, especially for comparisons in multicentre studies.

Child↗

PubMed1997

Diagnostic accuracy of urinary cytology, and deoxyribonucleic acid flow cytometry and cytology on bladder washings during followup for bladder tumors.

PURPOSE: We evaluated the diagnostic accuracy of cytology on urine and bladder washings, and deoxyribonucleic acid (DNA) flow cytometry on bladder washings compared to cystoscopy during followup for bladder tumors. MATERIALS AND METHODS: A cross-sectional analysis was performed on 166 patients with a history of bladder tumor for the initial visit when cytology and DNA flow cytometry results were available for urine and bladder washing samples. There were 114 bladder tumor events and 52 negative controls (normal cystoscopy). A prospective study was conducted among the 66 patients with no tumor detected by cystoscopy at the initial visit and for whom the 3 test results were available. The patients were followed from this visit until tumor recurrence or the end of the followup period. Cytological examination was considered positive only when interpreted as malignant. Suspicious examinations were considered negative for the purpose of our study. RESULTS: Sensitivity and specificity of urinary cytology were 59 and 85%, respectively. Sensitivity was increased to 66% using bladder washing cytology whereas specificity was moderately decreased to 83%. The sensitivity and specificity of DNA aneuploidy were 45 and 87%, respectively. Inclusion within the aneuploid category of hyperdiploid samples (defined as more than 7% of cells in the S + G2M phase) increased sensitivity to 77%, while specificity decreased to 42%. Kaplan-Meier analysis of tumor-free survival in patients initially free of bladder cancer at cystoscopy showed an inability of DNA flow cytometry to predict recurrence, whereas positive urine or bladder washing cytology predicted a shorter time to recurrence (log-rank test p <0.01). CONCLUSIONS: These results show that DNA flow cytometry and cytology on bladder washing samples add little information to that obtained from voided urine cytology.

Cross-Sectional Studies↗

PubMed1996

The role of DNA flow cytometry in borderline malignant ovarian tumors.

BACKGROUND: The role of flow cytometric DNA analysis in predicting prognosis of patients with borderline malignant ovarian tumors has been controversial. METHODS: Fifty cases of patients with borderline malignant ovarian tumors were analyzed by histology and by flow cytometry on paraffin embedded tissue. Multiple tissue blocks and serial sections were analyzed for each tumor. The results of DNA analysis were correlated to clinicopathologic data. RESULTS: DNA aneuploidy was demonstrable in 4 cases (8%) when the most atypical section was analyzed. The overall rate of aneuploidy was 14% if additional blocks and serial sections were studied. Two patients died from tumor. One of the two patients had an initial diagnosis of Stage IIc mucinous borderline tumor with DNA indices (DI) of 1.12, 1.42, and 2.04. She had a recurrence in the contralateral ovary 1 year later (DI = 1.83), and a second frankly malignant recurrence diffusely in peritoneum (DI = 1.89). The other patient had an initial diagnosis of Stage IIIc mucinous borderline ovarian tumor with pseudomyxoma peritonei. DNA diploidy was obtained in all of the samples from the primary tumor. An aneuploid peak (DI = 1.28) was demonstrated in only one serial section of the peritoneal implants. Of the other 5 patients who had aneuploid histograms but were disease-free, the DNA indices were 1.35, 1.14/1.18, 1.15, 1.20, and 1.31 and were demonstrable only in either 1 or 2 of the blocks or unproven on serial sections. All patients with diploid-peridiploid tumors were alive and disease free. CONCLUSIONS: Reproducible DNA aneuploidy of high DI may be predicting a poor outcome, whereas the significance of inconsistently reproducible aneuploidy of low DI remains to be determined. Further studies of prospective DNA analysis with adequate sampling are necessary to define the role of flow cytometry in patients with borderline malignant ovarian tumors.

Adolescent↗

PubMed1994

Direct flow cytometry of anaerobic bacteria in human feces.

We describe a flow cytometry method for analysis of noncultured anaerobic bacteria present in human fecal suspensions. Nonbacterial fecal compounds, bacterial fragments, and large aggregates could be discriminated from bacteria by staining with propidium iodide (PI) and setting a discriminator on PI fluorescence and by exclusion of events with large forward scatter. Since anaerobic bacteria, which account for over 99.9% of all fecal bacteria, die during sample preparation, a fixation step was not necessary. A second aim of this study was to investigate the technical possibility of measurement of in vivo IgA coating of fecal anaerobic bacteria as well as their bacterial size. Fecal samples of 22 healthy human volunteers were analyzed. The fluorescence distribution of IgA-coated bacteria labeled with fluorescein isothiocyanate (FITC)-anti-Hu-IgA had overlap with noncoated bacteria. However, with match region subtraction, detection of low levels of specific FITC fluorescence on IgA-coated bacteria was achieved. The median bacterial two-dimensional surface area was 1.0 microns2. To validate flow cytometry data, all samples were analyzed with an image analysis system as well. With this new method, a rapid evaluation of fecal flora with high sensitivity for specific FITC fluorescence is possible without culturing.

Adult↗

PubMed1996

Flow cytometry for rapid assessment of viability after exposure to a quaternary ammonium compound.

The use of flow cytometry to rapidly assess the viability of Pseudomonas spp. and Staphylococcus spp. after exposure to a quaternary ammonium compound (QAC) was investigated using rhodamine 123 (Rh 123), Stain A (LIVE Stain) accumulating in viable but not in dead cells (Live/Dead Baclight bacterial viability kit, Molecular Probes Inc., Eugene, OR, USA), and Sytox green (Molecular Probes) accumulating in dead but not viable cells. Staining conditions were optimized for each stain. The fraction of viable cells after exposure to benzalkonium chloride was determined by using the three staining techniques and colony counts on agar medium. For all Staphylococcus spp. tested there was a high correlation the methods based on flow cytometry and colony counts irrespective of which stain was used. Although viable, all Pseudomonas spp. tested accumulated Rh 123 poorly and about 30% failed to accumulate LIVE stain as well. However, the correlation between colony counts and Sytox green labelling of Pseudomonas spp. was high. Our results indicate that flow cytometry together with live or dead cell labelling can be used to study the bactericidal effect of QACs. The methods based on LIVE stain and Sytox green were simpler and less time consuming than Rh 123 labelling. Only Sytox green could be used with all strains of Staphylococcus and Pseudomonas tested.

Anti-Bacterial Agents↗

PubMed1982

Heterogeneity of HLA-A,B, Ia-like, and melanoma-associated antigen expression by human melanoma cell lines analyzed with monoclonal antibodies and flow cytometry.

With the use of monoclonal antibodies, indirect immunofluorescence, and flow cytometry, human melanoma cell lines (Colo 38, M-16, and M-21) were examined for the quantitative level of expression of HLA-A,B antigens, Ia-like antigens, and two human melanoma-associated antigens (MAA) referred to as 280K and 94K MAAs. Each of the melanoma cell lines examined showed tremendous heterogeneity with regard to antigen expression. A detailed study of the heterogeneity of the four antigens listed revealed that variation in cell size could, in part, account for the large differences in antigen expression observed. Cell surface density of HLA-A,B antigens, Ia-like antigens, and the 280K and 94K MAAs remained relatively constant over a wide range of cell sizes that were examined, with the exception that small melanoma cells showed a slightly lower mean surface density of MAA expression than did large cells. A novel method was used to detect the expression of the two MAAs as a function of the Colo 38 cell cycle using dual-laser beam flow cytometry. The results of these experiments show that both the 280K MAA and the 94K MAA are differentially expressed during various stages of the cell cycle, with each antigen being maximally detected during G2 + M.

Antibodies, Monoclonal↗

PubMed1985

Comparison of the supravital DNA dyes Hoechst 33342 and DAPI for flow cytometry and clonogenicity studies of human leukemic marrow cells.

Human leukemic bone marrow cells were studied by flow cytometry and a colony-forming assay. Two supravital DNA dyes, Hoechst 33342 (H33342) and 4',6-diamidino-2-phenyl indole dihydrochloride (DAPI), were compared in terms of DNA histograms by flow cytometry and toxicity to cells by colony-forming assay. Initially, Chinese hamster ovary (CHO) cells were used, and the optimal staining conditions for the two dyes were determined: 30 min exposure to 10 micrograms/ml at 37 degrees C for H33342 and at 23 degrees C for DAPI. DAPI demonstrated DNA profiles with better coefficients of variation for Go/G1 cells than did H33342. This difference was consistently shown in four additional mammalian cell lines and bone marrows freshly obtained from five patients, four of which were leukemic. Both dyes, in the optimal staining conditions, can suppress the growth of CHO cells with H33342 more toxic than DAPI. In experiments on three leukemic bone marrows, H33342 was shown to be more toxic than DAPI in terms of colony-forming capability. Although there is considerable variation in the degree of the toxicity between different cases, more than 50% of leukemic colony-forming cells can survive after DAPI staining. These data indicate that DAPI is preferable to H33342 for use with human leukemic cells because the staining technique required is less stringent; there is a more homogenous staining of the DNA, and there is less cytotoxicity induced. Supravital staining of DNA with DAPI and viable sorting by flow cytometry should be reasonably possible for functional studies such as colony formation after sorting.

Acute Disease↗

PubMed1999

Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate.

BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications.

Alkaline Phosphatase↗

PubMed1996

No advantage of flow cytometry crossmatch over complement-dependent cytotoxicity in immunologically well-documented renal allograft recipients.

The effect of flow cytometry crossmatches on clinical outcome was studied retrospectively in two groups of immunologically well-documented patients who had received transplants with a negative complement-dependent cytotoxicity crossmatch. The first group consisted of 114 consecutive renal allograft recipients, and the second group consisted of 76 immunologically at-risk recipients. Flow cytometry crossmatches were performed with current and historic sera. In group 1, positive flow cytometry (FC) crossmatches were shown in 15/114 (13%) recipients. Rejection occurred in 8/15 (53%) FC-positive versus 41/99 (41%) FC-negative recipients. The 1-year graft survival rate was 80% for FC-positive patients and 87% for FC-negative patients. Sixty-seven patients were nonsensitized patients; 4 of them had a positive FC crossmatch but no rejection episodes, graft loss, or patient loss. Of 47 retransplanted and/or sensitized recipients, 11 had a positive FC crossmatch. Rejection treatment was needed in 8/11 (73%) FC-positive patients compared with 19/36 (53%) FC-negative patients. Their 1-year graft survival rates were 73% and 81%. None of these differences reached statistical significance. Group 2 consisted of 76 at-risk recipients; 37 were retransplant patients and 39 were sensitized first-transplant patients. Twenty-one (28%) patients showed a positive FC crossmatch. Rejection episodes did not differ between the FC-positive (48%) and FC-negative patients (46%). There was no difference in 1-year graft survival rate (76% vs. 80%) or in 1-year patient survival rate (100% vs. 95%). We conclude that FC crossmatches in our patient group are not superior to the classical complement-dependent cytotoxicity crossmatches with regard to clinical outcome. On the contrary, transplantation with a mandatory negative FC crossmatch would have excluded 28% of the recipients from transplantation, who in fact are doing well.

Adult↗

PubMed1995

Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies.

Recently, there have been several reports demonstrating improvements in the flow cytometric detection of intracellular cytokines. These advances, although significant, have not yielded techniques that have easily been translated into broad use. To address this issue, we have coupled a fixation and permeabilization method with the use of directly labelled monoclonal anti-cytokine antibodies, providing both improved signal and simpler staining. The kinetics of in situ cytokine production in both CD4 and CD8 cells are shown for IL-2, IL-4, IL-5 and IFN-gamma. Based on these data, 6 h was chosen for optimal detection of this combination of cytokines. We show the specificity of this technique by blocking cytokine staining using a molar excess of recombinant cytokine. Additionally, unlabelled anti-cytokine antibodies are demonstrated to block specific staining of labelled antibody, providing an objective means to place statistical markers. Using such controls, we routinely detected as few as 0.1% false positive cells, allowing the flow cytometric detection of IL-5, which is below the threshold of detection of published methods. To further prove the specificity of staining, we stained using two anti-IL-5 mAbs known to recognize different epitopes and demonstrate that the same cells stain with both antibodies. Without permeabilization we could detect a fraction of cells with low intensity staining for cytokine. This staining was further examined using differential two color staining for intracellular and extracellular cytokine, clearly demonstrating no cells staining exclusively for extracellular cytokine, confirming a lack of passive transfer of cytokine to nearby cells. We show that cytokine flow cytometry is useful in examining the increased IL-5 production characteristic of eosinophilic states and that IL-5 production is limited to the CD27 negative subpopulation. These data illustrate the unique capability of cytokine flow cytometry to correlate cytokine expression with cell surface phenotype without cell separation. In summary, using directly conjugated anti-cytokine antibodies, cytokine flow cytometry becomes a specific and versatile technique for the assessment of complex cytokine production phenotypes in fresh ex vivo T cell subpopulations.

Antibodies↗