Molecules Of Equivalent Soluble Fluorochrome Values Research Articles

Significance of qualitative and quantitative evaluations of anti-HLA antibodies in kidney transplantation

In this study, we retrospectively investigated the relationship between the presence/titers of donor-specific (DSA)/nondonor-specific antibody (NDSA) and the rate of graft rejection after transplantation. The subjects comprised 34 recipients who tested positive by FlowPRA(®) Screening. The recipients were divided into two groups; 22 recipients with DSA and 12 recipients with NDSA, as detected using FlowPRA(®) Single Antigen I and II beads. The antibodies were also quantitatively examined using the molecules of equivalent soluble fluorochrome (MESF) method. Nine of the 22 recipients with DSA (9/22, 40%) developed antibody-mediated rejection (AMR), while none of the 12 recipients with NDSA (0/12, 0%) developed AMR (P < 0.01). In a quantitative analysis of the MESF data, patients with DSA with MESF values of over 3000 frequently showed AMR (8/11, 73%). In contrast, one of the patients with DSA with MESF values of <3000 showed AMR (1/11, 9%). One of the 12 patients (1/12, 8%) with NDSA showed cellular rejection (T-cell-mediated rejection), regardless of the MESF values. In patients with DSA, an MESF value of 3000 may be a useful cutoff value for identifying patients at a high risk for AMR.

Clinical Importance of Anti-Human Leukocyte Antigen-Specific Antibody Concentration in Performing Calculated Panel Reactive Antibody and Virtual Crossmatches

Highly sensitized patients develop multi-human leukocyte antigen (HLA) specific antibodies. This study measures concentrations of anti-HLA antibodies in multispecific sera by converting fluorescence intensity into molecules of equivalent soluble fluorochrome (MESF) units. This was used to determine MESF units required for a positive T and B flow crossmatches (FLXM). MESF values of negative controls and sera from patients devoid of HLA antibodies were measured by FLXM and flow panel reactive antibody (PRA) screening beads. Fluorescence intensity values of anti-HLA specific antibodies determined by FlowPRA single antigen beads of highly sensitized patients were converted into MESF units. In addition, endpoint titers, MESF units, and % PRA of 26 sera were established. MESF analysis accurately predicted the outcomes of 100% of T and B FLXM of sera with strong (MESF units>18,000) donor-specific antibody (DSA). The predictive values of T and B FLXM declined to 95% and 88% with weak DSA (6,000 MESF<10,000). Endpoint titers of sera from highly sensitized patients ranged from 1:512 to 1:8 with corresponding MESF values of 452,596 to 20,000 units. However, there was no statistical difference in PRA values among these sera (95%-100%). We successfully transplanted five patients who had weak donor-specific HLA antibodies (MESF units>2,000). The graft survival at 1 year was 100% and there was no evidence of DSA posttransplant. MESF analysis is both a time and cost efficient way of measuring antibody strength. The strength of the antibody present in the sera of transplant candidates is critical for crossmatch prediction.

Discrepancy in measuring CD4 expression on T‐lymphocytes using fluorescein conjugates in comparison with unimolar CD4‐phycoerythrin conjugates

Numerous methods for quantitative fluorescence calibration (QFC) have been developed to quantify receptor expression on lymphocytes. However, the results from the use of these different QFC methods vary considerably in the literature. To better identify the causes of these discrepancies, we measured CD4 expression using FITC and phycoerythrin (PE) conjugates to stain CYTO-TROL Control Cells and T-lymphocytes in whole blood and isolated cell preparations. We further examined pH of the cellular microenvironment as a cause of discordant results obtained with the FITC conjugate. Calibration with Quantibrite PE-labeled microspheres and the use of unimolar CD4-PE conjugates provided direct measurement of the antibody bound per cell value (ABC) for CD4 expression on normal T-lymphocytes. Calibration for CD4-FITC monoclonal antibody (Mab) labeled CYTO-TROL Control Cells and normal T-lymphocytes was based on molecules of equivalent soluble fluorochrome (MESF) as determined by FITC-labeled microspheres traceable to NIST RM 8640. The MESF value for CD4-FITC Mab was determined that enabled the conversion of the MESF values obtained for CYTO-TROL cells to ABC. We investigated the likely pH change in the fluorescein microenvironments within FITC-labeled Mab and cells stained with FITC-labeled Mab using a pH sensitive indicator. The mean ABC value for T-lymphocytes prepared from fresh whole blood using CD4-PE conjugate (48,321) was consistent with previous results, and it was much higher than the mean ABC using CD4-FITC Mab (22,156). The mean ABC value for CYTO-TROL cells using CD4-PE conjugate (43,090) was also higher than that using CD4-FITC conjugate (34,734), although the discrepancy was not as great. Further studies suggested the discrepancy in CYTO-TROL results may be accounted for by the low pH of the membrane microenvironment, but the greater discrepancy in T-lymphocytes could not be fully explained. CD4 expression on fresh normal whole blood samples and CYTO-TROL cells can be consistently quantified in ABC units using Quantibrite PE quantification beads and unimolar CD4-PE conjugates. Quantification with CD4-FITC conjugate is not as consistent, but may be improved by the use of CD4 T-cells as biological calibrators. This approximation is valid only for surface receptors with consensus ABC values measured by different QFC methods serving as biological standards.

Comparison of fluorescein and phycoerythrin conjugates for quantifying CD20 expression on normal and leukemic B‐cells

Numerous methods for quantitative fluorescence calibration (QFC) have been developed to quantify receptor expression on lymphocytes as potential disease biomarkers. CD20 expression in B-cell chronic lymphocytic leukemia (B-CLL) is one of the best examples of such a biomarker, but results from the use of different QFC methods vary considerably. We measured CD20 expression on normal and B-CLL B-cells, using FITC and PE conjugates from the same monoclonal antibody (Mab). As a biological control and calibrator, we also measured CD4 expression on T-cells with FITC and PE Mab. Calibration curves were constructed using the CLSI (formerly NCCLS) consensus guidelines for QFC. Calibration with QuantiBRITE PE-labeled microspheres and the use of unimolar PE conjugates provided direct measurement of antibody bound per cell (ABC) for CD4 and CD20. Calibration for FITC conjugates was based on molecules of equivalent soluble fluorochrome (MESF), as determined by NIST RM 8640 microsphere standards. These MESF values were then converted to ABC, using the CD4 T-cell as a biologic calibrator, to normalize FITC and PE results for CD20 expression. On normal B cells, the mean ABC value for unimolar CD20-PE conjugate was 143,500 (CV +/- 19.1%). The mean ABC value for B-CLL B-cells stained with the same conjugate was 21,700 (CV +/- 42.0%). Using the CD4 T-cell as a biologic calibrator for FITC conjugate, the mean ABC value for CD20-FITC on normal B-cells was 199,300. CD20-FITC staining on B-CLL cells was generally too weak for accurate quantification. On normal T-cells, the mean ABC value for CD4 unimolar PE conjugate was (36,800 +/- 10.4)%, and it did not differ significantly in CLL samples. The expression of CD20 on normal and B-CLL lymphocytes can be quantified in ABC units using unimolar CD20-PE conjugates. In addition, CD4 expression on T-cells can be used as a biological calibrator to quantify CD20-FITC ABC, with reasonable agreement between the two conjugates with different fluorochromes. Issues regarding the accuracy of MESF microsphere calibrators and effective F/P ratios for FITC conjugates will require additional laboratory studies.

Quantification of EGFP expression on Molt-4 T cells using calibration standards.

Enhanced green fluorescent protein (EGFP) is used extensively to assess gene expression on cells; however, quantification of this expression by flow cytometry has been limited by the unavailability of calibration standards. Thus, we characterized the response of an experimental set of EGFP calibration standards to environmental changes and then quantitate the expression of EGFP, in molecules of equivalent soluble fluorochrome (MESF) units, of a transfected Molt-4 T cell line by flow cytometry. Characterization of the EGFP standards: EGFP standards were equilibrated in suspension solutions having a pH range of 5.0-9.0, temperatures of 37-80 degrees C, and osmolalities of 100-600 mOsm/kg. Quantification of EGFP on cells: For transfections, Molt-4 T cells were incubated with two different concentrations (0.2 microg and 0.4 microg) of pEGFP-N2 vector and the EGFP expression was quantified after 48 h by flow cytometry using the EGFP standards and by the cytofluor technique using a standard curve of known EGFP solutions. The fluorescence intensity of the EGFP standards increased from pH 5.0 to 9.0 and remained relatively constant from 37 degrees C to 65 degrees C, and from 100 to 600 mOsm/kg. After transfection, the expression of the populations with high and low EGFP expression averaged 8,098 +/- 584 MESF and 3,808 +/- 375 MESF respectively. No significant differences were observed after comparing the MESF values obtained by flow cytometry and the values obtained by Cytofluor technique (high: 8,791 +/- 492 MESF; low: 4,082 +/- 398 MESF). Our data demonstrate the feasibility of using calibration standards to quantify EGFP expression on cells. Our results emphasize the importance of monitoring the effects of environmental changes in the fluorescence intensity of both standards and samples when quantifying the expression of EGFP on living cells.

Flow cytometry measurement of GM-CSF receptors in acute leukemic blasts, and normal hemopoietic cells.

A quantitative analysis of expression levels of GM-CSF receptors was performed by flow cytometry in different disease categories, ie AML (n = 72), ALL (n = 18), and MDS (n = 12), as well as 12 healthy volunteers, using three different unconjugated GM-CSF/R monoclonal antibodies (McAbs) (HGM-CSFR (CD116), M5D12, 4B5F5), and appropriate standards. By using the reference HGM-CSFR McAb, in healthy subjects we found detectable levels of GM-CSF/R on blood monocytes (mean MESF (molecules of equivalent soluble fluorochrome)/cell: 36.1 x 10[3]), neutrophils (mean MESF/cell: 7.4 x 10[3]), bone marrow (BM) myelo-monocytic precursors (MESF range for the myeloid component, ie promyelocytes, myelocytes, metamyelocytes: 11.7-40.5 x 10[3], and for the monocytic lineage: 25.7-69.2 x 10[3]), and in two distinct subsets of BM CD34+ progenitor cells (GM-CSF/R dim: 2.5 x 10[3] MESF/cell, GM-CSF/R bright (10% of the total number of CD34 cells: 22.0 x 10[3] MESF/cell). In these subjects, there was no correlation between the expression levels of GM-CSF/R and CFU (CFU-GM, CFU-GEMM, BFU-E) colony production. Among the AML samples, M5D12 McAb was positive in 33%, 4B5F5 McAb in 90%, and HGM-CSF/R McAb in 78% of the cases examined (range of MESF/cell for the HGM-CSFR McAb: 0.9 x 10[3]-106.7 x 10[3]). The highest MESF values were seen in the M5 FAB subvariety (mean: 39.4 x 10[3]), where all the patients tested (n = 20) showed a strong positivity for the HGM-CSFR McAb. On the contrary, all ALL samples were GM-CSF/R negative except in two patients, who displayed a dim GM-CSF/R positivity (My+ALL: 1.3 x 10[3] MESF/cell; pro-B ALL: 1.0 x 10[3] MESF/cell). In most (>70%) M1 FAB subtypes, GM-CSF/R+ blasts co-expressed CD34low, HLA-DRhigh, CD33, CD38 antigens, and had little or no capacity to form CFU-GM colonies. GM-CSF/R+ blasts from the M5 FAB category were also positive for CD14, CD11c, CD33 and CD87. Furthermore, the number of GM-CSF/R expressed by leukemic cells from five out of 72 (7%) AML patients was above the highest values seen in normal samples (>69.2 x 10[3] MESF/cell), allowing the possibility of using this marker for the monitoring of the minimal residual disease (MRD) in a subset of AML. Cell culture studies aimed at evaluating GM-CSF receptor modulation following AML blast exposure to rhGM-CSF showed two distinct patterns of response; in the first group (6/10 cases) rhGM-CSF down-modulated GM-CSF receptors, whereas in the second group (4/10 cases), rhGM-CSF treatment was associated with either an increase or no change in the number of GM-CSF/R. In conclusion, cellular GM-CSF/R expression was variable and ranged from undetectable (ALL and a minority of AML) to very high intensities in M5 AML, and were also documented in some M0 AML, thus suggesting the concept that GM-CSF/R detection may be of help in lineage assignment of undifferentiated forms. Since the number of GM-CSF/R on AML blasts may be modulated after GM-CSF treatment, it can be postulated that the clinical use of GM-CSF in this disease may be optimized by a dynamic analysis of the number and the affinity status of GM-CSF-R in blasts and normal hemopoietic cells.

Bcl-2 Expression in Acute Myeloblastic Leukaemia: Relationship with Autonomous Growth and CD34 Antigen Expression

The bcl-2 gene encodes a mitochondrial protein that inhibits the onset of apoptosis induced by growth factor withdrawal or cytotoxic agents. Using quantitative flow cytometry and expressing bcl-2 levels as the number of molecules of equivalent soluble fluorochrome (MESF) per cell, we have shown that bcl-2 protein expression in the blast cells from patients with acute myeloblastic leukaemia (AML) is heterogeneous, but not related to FAB type. The blast cells from AML patients with the capacity to grow and survive autonomously in vitro were found to have higher bcl-2 MESF values than those that were dependent upon exogenous growth factors. We have previously reported that the blast cells from 70% of AML patients exhibit autonomous growth and autocrine growth factor production in vitro and that this has been shown to be an important indicator of poor prognosis in AML. High bcl-2 expression has also been associated with a low complete remission rate and poor survival in AML.In the patients whose blast cells exhibited autonomous growth, neutralisation of endogenous GM-CSF resulted in down-regulation of bcl-2 protein, whereas in blast cells from patients whose cells proliferated only in the presence of added growth factors, incorporation of recombinant human (rh) GM-CSF in the culture media resulted in up-regulation of bcl-2. Because CD34 positivity has been reported as another indicator of poor prognosis in AML, we compared bcl-2 expression in cases of CD34 positive AML, CD34 negative AML and CD34 positive normal bone marrow cells. Bcl-2 was found to be strongly expressed on the CD34+ normal bone marrow cells. The blast celb from CD34+ AML patients expressed significantly higher bcl-2 levels than CD34- AML patients. In five cases of CD34+ AML, the bcl-2 levels were determined on purified CD34+ and CD34- blast cell populations. The CD34+ blast cells were found to express significantly higher bcl-2 levels compared with the CD34- blast cells. Our data would suggest that quantification of bcl-2 in AML blast cells may be useful as a prognostic indicator in AML.

Flow cytometric HLA-B27 typing using CD3 gating and molecules of equivalent soluble fluorochrome (MESF) quantitation.

The determination of HLA-B27 (B27) status is important in the diagnosis of ankylosing spondylitis, Reiter's disease, and other arthropathies. Flow cytometric (FCM) typing of B27 is a relatively new method which allows for rapid turnaround time and low cost. However, different leukocyte populations may manifest significant variation in staining intensity with B27 antibodies. Therefore, gating utilizing only light scatter properties of cells may lead to high readings in some B27-negative samples. Fluorescence gating on CD3+ cells were postulated as a means to eliminate these anomalies. Furthermore, quantitative FCM measurements, as afforded through use of molecules of equivalent soluble fluorochrome (MESF) units, can minimize day-to-day and instrument-to-instrument variabilities in fluorescence measurements. We compared CD3 gating to light scatter gating and MESF analysis on 123 specimens in a 4-month period and found: 1) CD3 gating gave the lowest nonspecific B27 antibody binding among B27-negative subjects; 2) there was no significant difference in MESF values between CD3 gating and light scatter gating of B27-positive samples; 3) there was a wide range of B27 antibody binding fluorescence intensities among B27-positive subjects; 4) identification of patients with low B27 expression may require the use of CD3 gating in order to avoid costly confirmation testing; 5) fluorescent standard beads are stable for routine use in a clinical flow cytometry laboratory.

Flow cytometric HLA‐B27 typing using CD3 gating and molecules of equivalent soluble fluorochrome (MESF) quantitation

The determination of HLA-B27 (B27) status is important in the diagnosis of ankylosing spondylitis, Reiter's disease, and other arthropathies. Flow cytometric (FCM) typing of B27 is a relatively new method which allows for rapid turnaround time and low cost. However, different leukocyte populations may manifest significant variation in staining intensity with B27 antibodies. Therefore, gating utilizing only light scatter properties of cells may lead to high readings in some B27-negative samples. Fluorescence gating on CD3+ cells were postulated as a means to eliminate these anomalies. Furthermore, quantitative FCM measurements, as afforded through use of molecules of equivalent soluble fluorochrome (MESF) units, can minimize day-to-day and instrument-to-instrument variabilities in fluorescence measurements. We compared CD3 gating to light scatter gating and MESF analysis on 123 specimens in a 4-month period and found: (1) CD3 gating gave the lowest nonspecific B27 antibody binding among B27-negative subjects; (2) there was no significant difference in MESF values between CD3 gating and light scatter gating of B27-positive samples; (3) there was a wide range of B27 antibody binding fluorescence intensities among B27-positive subjects; (4) identification of patients with low B27 expression may require the use of CD3 gating in order to avoid costly confirmation testing; (5) fluorescent standard beads are stable for routine use in a clinical flow cytometry laboratory. © 1996 Wiley-Liss, Inc.

Coordinator's report on the flow cytometric analysis of the Rh antibodies.

One hundred and four IgG monoclonal antibodies with specificity within the Rh Blood Group System were evaluated by flow cytometry as part of the Third International Workshop. Standardisation of data to permit interlaboratory comparison of antibody binding was achieved by adherence to a standard red blood cell staining protocol, defined control cells and a standard FITC-labelled antibody. In addition, calibration standards were provided to permit the calculation of Molecules of Equivalent Soluble Fluorochrome (MESF) values from the mean channel fluorescence. For the majority of anti-D antibodies mean MESF values obtained with D positive cells were far higher than with the negative controls (D negative cells), with D variants having intermediate although very varied MESFs. In general MESF values obtained with non anti-D antibodies were less than for the anti-D antibodies although some of the anti-E antibodies were very strong. The highest MESF values were obtained with an anti-CD antibody.

Comparative quantitative expression of bcl-2 by normal and leukaemic myeloid cells.

Expression of the bcl-2 oncoprotein by AML blasts has previously been demonstrated to be heterogenous with high levels of bcl-2 expression being associated with a low complete remission rate and poor survival. We have quantified bcl-2 expression in AML blasts in relation to expression of the CD34 antigen and in comparison to CD34-positive cells from normal bone marrow. When expressed as molecules of equivalent soluble fluorochrome (MESF) per cell. AML blast cell bcl-2 expression varied from 11.1 to 99.9 x 10(3) (median 39.4 x 10(3), n = 56) with 28.5% of patients expressing high MESF values (> 50 x 10(3)) and 16% of patients expressing low MESF values (< 20 x 10(3), the remainder expressing intermediate values. There was no significant difference between intensity of bcl-2 expression and FAB classification in the de novo AML cases; and there was no significant differences between de novo and secondary AML cases. Blasts from CD34+ AML patients expressed significantly higher levels of bcl-2 (mean MESF 43.6 x 10(3), n = 36) than CD34- AML patients (mean MESF 31.7 x 10(3), n = 19). In five cases of CD34+ AML, bcl-2 expression was determined on purified CD34+ and CD34- blast cell populations. In all cases CD34+ blasts were found to express significantly higher bcl-2 MESF values compared to the CD34- fraction. Purified CD34+ cells from normal bone marrow consistently expressed high levels of bcl-2 (MESF > 75 x 10(3), n = 4), which was comparable to that found on CD34+ AML cells. Our results suggest that the poor prognosis previously associated with AML blasts expressing the CD34 antigen may in part be related to high expression of bcl-2. Also the ability to measure bcl-2 in AML blasts quantitatively by flow cytometry and to categorize patients into discrete groups may be of value as a prognostic indicator in AML.