Median Fluorescence Intensity Values Research Articles

Converting an HLA-B27 flow assay from the BD FACSCanto to the BD FACSLyric.

HLA-B27 is a major histocompatibility complex (MHC) class I antigen which exhibits strong association (90%) with ankylosing spondylitis. HLA-B27 detection in patients by flow cytometry is a widely used clinical test, performed on many different flow cytometer models. We sought to develop and validate a test conversion protocol for the HLA-B27 test performed on the BD FACSCanto to BD's newer FACSLyric flow cytometers. The development and validation experiments were performed using anti-HLA-B27*FITC/CD3*PE antibody-stained whole blood patient specimens. The anti-HLA-B27*FITC logarithmic median fluorescence (LMF) results on the BD FACSCanto were converted to median fluorescence intensity (MFI) values on the BD FACSLyric. Clustering of the HLA-B27 positive and negative values, using a 3rd order polynomial equation, resulted in a conversion of the BD FACSCanto cutoff values, negative (<150 LMF) and positive (≥160 LMF), to negative (<4530 MFI) and positive (≥6950 MFI) on the BD FACSLyric. Accuracy was assessed by comparing the flow results obtained on the BD FACSCanto and BD FACSLyric to a molecular PCR based assay. Additional validation parameters (compensation verification, intra- and inter-assay precision, and instrument comparison) were performed per the recommendations outlined in the Clinical and Laboratory Standards Institute (CLSI) H62 guidelines for validation of flow cytometry assays.

Development of a T-cell activation panel using image cytometry in rheumatoid arthritis patient samples

Abstract Rheumatoid arthritis (RA) is an autoimmune disease that results in cartilage damage and bone loss due to chronic inflammation of joints. Treatments for RA include disease-modifying antirheumatic drugs and nonsteroidal anti-inflammatory drugs, as well as the inhibition of activated T-cells. Consequently, there is a need to screen RA patient samples rapidly and effectively to monitor treatment efficacy, disease progression, immune response, and predictive markers. In this work, we developed and demonstrated a multiplex detection method to monitor T-cell activation in a panel consisting of CD3, CD69, CD25, and CD279 (PD-1) surface markers using the Cellaca™ PLX Image Cytometer (Revvity). First, a dosage titration of PHA-P was performed on PBMCs to stimulate T-cell activation. Once stimulated, CD3 positive T-cells were evaluated for CD69, CD25, and the checkpoint molecule CD279. After 24 hours, the percent surface marker positive T-cells and the level of marker expression (median fluorescent intensity values) were used to determine optimal PHA-P dosage. Results showed concentration dependent increases in the expression levels with a calculated EC50 value for PHA-P ranging from 20.16-30.78 µg/mL with a 95% CV based on the CD279 titration curve. The proposed image cytometric surface marker detection method may prove to be an efficient tool to rapidly screen RA patient samples for T-cell activation in the setting of clinical disease treatment or progression.

Double filtration plasmapheresis combined with rituximab for donor-specific antibody desensitization in haploidentical haematopoietic stem cell transplantation.

Donor-specific anti-HLA antibodies (DSA) are a major cause of engraftment failure in patients receiving haploidentical haematopoietic stem cell transplantation (Haplo-HSCT). Double filtration plasmapheresis (DFPP) avoids the unnecessary loss of plasma proteins and increases the efficiency of purification. To investigate the effectiveness of the desensitization protocol including DFPP and rituximab, we conducted a nested case-control study. Thirty-three patients who had positive DSA were desensitized by the protocol and 99 patients with negative DSA were randomly matched as control. The median DSA mean fluorescence intensity values before and after DFPP treatment were 7505.88 ± 4424.38 versus 2013.29 ± 4067.22 (p < 0.001). All patients in DSA group achieved haematopoietic reconstitution and the median neutrophils and platelets engraftment times were 13 (10-21) and 13 (10-29) days respectively. Although the cumulative incidence of II-IV aGVHD (41.4% vs. 28.1%) and 3-year moderate to severe cGVHD (16.8% vs. 7.2%) were higher in DSA cohort than in the control, no statistical significance was observed. The 3-year non-relapse mortality and the overall survival were 6.39% and 72.0%, respectively, in the DSA cohort, which were comparable to the negative control. In conclusion, DFPP and rituximab could be effectively used for desensitization and overcome the negative effects of DSA in Haplo-HSCT.

Investigating Vitreous Cytokines in Choroidal Melanoma.

Due to the close relationship between the vitreous and posterior eye layers, the microenvironment of these layers can affect the composition of the vitreous. Molecular analysis of the vitreous may therefore provide important insights into the pathogenesis of chorioretinal diseases. In this study, vitreous cytokines (n = 41) were evaluated to gain further insights into the tumor microenvironment in uveal melanoma (UM) arising from the choroid (CM). Cytokine levels were measured using a bead-based multiplex immunoassay panel in vitreous samples obtained from 32 eyes, including 18 with CM and 14 controls. Median fluorescence intensity values were extracted and used as relative quantification of the cytokine abundance. Vitreous cytokine levels were compared between the CM and non-CM groups and between different prognostic categories within the CM group (classified as having low or high metastatic risk using tumor biopsy-based gene expression profiling). Correlations between vitreous cytokine levels and tumor dimensions were also evaluated. Our analysis revealed twenty-six vitreous cytokines significantly upregulated in CM-affected eyes compared to the control eyes. Within the CM group, six vitreous cytokines showed altered levels (five upregulated and one downregulated) in eyes with high- vs. low-risk tumors. Levels of these six plus several other cytokines showed correlations with the tumor dimensions. In conclusion, our study has uncovered several UM-relevant vitreous cytokines, worthy of follow-up in larger studies as potential candidates for liquid biopsy-based biomarker development and/or new therapeutic targeting.

Diminishment of High PD‐1 Expressing Peripheral Cytotoxic T Lymphocytes in Behavioral and Psychological Symptoms of Alzheimer’s Disease

AbstractBackgroundBehavioral and psychological symptoms of dementia (BPSD) are troublesome in managing Alzheimer’s disease (AD). Growing research strongly suggests the relationship between AD and immunity of peripheral blood. Immunotherapy with blockade of PD‐L1/PD‐1 axis restored cognitive performance in the murine tauopathy model. We analyzed cytotoxic T lymphocyte (CTL) activity or possibility to be regulated, expression of PD‐1 of peripheral blood, and tried to find the correlation between PD‐1 expression and BPSD severity of AD patients.Method16 AD patients meeting NIA‐AA criteria for probable AD dementia (mean age = 80.1, 5 males,11 females) were enrolled in our study. Cognitive function was assessed by clinical dementia rating scale (CDR) and clinical dementia rating scale sum of box (CDR‐SOB). BPSD was evaluated by total neuropsychiatric inventory (NPI) scores. Higher total NPI scores suggested more serious BPSD.Histograms of CTL PD‐1 median fluorescence intensity (MFI) of 16 AD patients were compared head‐to‐head to determine a cutoff MFI value. CTL with high and low PD‐1 expressing populations were seperated. Correlation between total NPI score and CTL PD‐1 expressing population was further analyzed.ResultThe CTL subset located at the peak with the lowest MFI was defined as PD‐1dim CTL, and this subset was positively correlated to total NPI score. The other CTL subset was defined as PD‐1bright CTL, which was negatively correlated to total NPI score. No correlation was found between PD‐1 expression and CDR‐SOB (data not shown)ConclusionOur results revealed that the PD‐1 expression level of CTL decreased with enlarged total NPI scores. Interestingly, CDR‐SOB positively correlated with AD disease progression, yet BPSD could happen in even mild cognitive impairment (MCI) and very early AD stage. Although our study had limitations in sex distribution and small sample size, the results raised concern that CTL might participate in clinical manifestations of BPSD. PD‐1 expressing CTL might not correlate to global dementia progression.Further studies are expected to evaluate how peripheral CTL interplays with inflammatory molecules, excitatory and inhibitory neurons, cerebral blood perfusion, and gating of choroid plexus in the demented brain.

Successful Deceased Donor Kidney Transplantation in a Highly Sensitized Patient after Reclassification of Unacceptable Antigens Based on HLA Epitope Analysis

For patients with end stage renal disease, kidney transplant offers significant survival and quality-of-life advantages compared with dialysis. But for patients seeking transplant who are highly sensitized, waiting times have traditionally been long and options limited. We present the case of a 34-year-old hypersensitized female who underwent renal retransplantation. Histocompatibility tests revealed a calculated panel-reactive antibody of 99.53% with multiple antibodies against class I and II human leucocyte antigens and an eplet analysis was performed. The donor’s potential unacceptable antigens were re-defined and the calculated panel-reactive antibody decreased to 88.38%. After one month the patient received a deceased-donor kidney transplant. Complement dependent cytotoxicity crossmatch was negative; virtual crossmatch and flow cytometry crossmatch with historical serum were positive. High-dose intravenous immunoglobulin and rituximab were added to the thymoglobulin-based induction immunosuppression. Three donor-specific antibodies were detected and plasmapheresis was performed. Renal allograft biopsy revealed no manifestations of rejection. Repeated testing observed a decrease in donor-specific antibodies median fluorescence intensity values. Four months post-transplant, the patient remained with normal graft function without proteinuria. She is receiving a standard maintenance immunosuppression regime with prednisolone, mycophenolate mofetil and tacrolimus. The careful discussion among the transplantation center and histocompatibility laboratory in association with intense immunosuppression and close laboratory monitoring allowed a successful human leukocyte antigen-incompatible deceased donor kidney transplantation in the most critical phase for the occurrence of humoral rejection. It is noteworthy that the new histocompatibility and immunogenetics methodologies provide a more affirmative and comprehensive assessment of mismatch acceptability.

CD20 Expression as a Possible Novel Prognostic Marker in CLL: Application of EuroFlow Standardization Technique and Normalization Procedures in Flow Cytometric Expression Analysis.

Simple SummaryUp to 50% of patients with chronic lymphocytic leukemia relapse within four years after anti-CD20-directed treatment with a need for a subsequent treatment, which can potentially include anti-CD20 agents again. To retrospectively study the influence of CD20 expression in CLL patients receiving anti-CD20 directed therapy on therapy response, we designed and evaluated a bead-based normalization approach for the analysis of CD20 expression levels to reduce variation of flow cytometric measurements due to technical issues. Normalization significantly reduced the variability of median fluorescence intensities of fluorochrome-conjugated beads without artificially rendering the instruments’ performances, and longitudinal biological variations of marker expression based on MFI values could be robustly assessed. In a cross-trial comparison, strong MRD response correlated with the CD20 expression level before therapy started in patients receiving Ofatumumab, but not in patients receiving Obinutuzumab.Background: CD20 expression is a controversial issue regarding response prediction to anti-CD20 therapy in chronic lymphocytic leukemia (CLL). Methods: Median fluorescence intensities (MFIs) of standard fluorescence beads from the daily calibration of flow cytometers according to EuroFlow protocols were used to establish a normalization approach to study CD20 expression on CLL cells. CD20 MFI was retrospectively assessed prior to and during treatment from flow cytometric measurements of peripheral blood in patients with different depths of molecular response in the four phase-II CLL2-BXX trials (BIG; BAG; BIO; BCG; N = 194) administering either Obinutuzumab or Ofatumumab in combination with targeted agents. Results: No significant difference was observed between the normalized and measured MFIs of CD19 and CD20 on CLL cells. During treatment, CD20 expression levels on CLL cells did not significantly differ between the four investigated different treatment schemes, but a strong molecular response to Ofatumumab seemed to correlate with higher CD20 expression prior to therapy. Conclusions: Standardized staining and instrument monitoring enable a robust assessment of longitudinal biological variations of marker expression based on MFI values. Obinutuzumab showed a higher proportion of patients with a strong MRD response independent from initial CD20 expression, whereas high pre-therapeutic CD20 expression levels seem to correlate with a profound response to Ofatumumab.

P859: ANTI-CD38 NANOBODY JK36 ALLOWS RELIABLE MRD DETECTION IN DARATUMUMAB TREATED MULTIPLE MYELOMA PATIENTS

Background: The introduction of immunotherapies such as daratumumab, a CD38 monoclonal antibody has improved survival in multiple myeloma (MM), however a few considerations on minimal residual disease (MRD) assessment by flow cytometry have been raised and deserve our attention. In flow cytometry CD38 is frequently used as a gating marker of plasma cells (PCs), but daratumumab can hampering myeloma cell detection. A strategy to overcome this interference is to staining PCs with anti-CD38 nanobody JK36. Nanobodies are single variable domain antibody fragments (VHH) that often expose a long complementarity-determining region 3 (CDR3), feature that allows anti-CD38 nanobody JK36 recognizing a cryptic epitope not masked by anti-CD38 therapies. Aims: To compare the median fluorescence intensity (MFI) of CD38 multiepitope (ME) antibody and anti-CD38 nanobody in bone marrow samples from MM patients not treated with daratumumab. Secondly, to compare flow cytometric MRD data obtained using CD38-ME and anti-CD38 nanobody from daratumumab treated MM patients. Methods: Samples were obtained from 10 MM patients not treated with daratumumab to compare the MFI of CD38-ME vs CD38-nanobody at baseline and after incubation with daratumumab at a concentration of 10 nM for 60 min at room temperature. Marrow samples were stained using panel CD38/CD56/CD19/CD138/CD45 with variable CD38, CD38-ME vs anti-CD38 nanobody. Data were analyzed using Kaluza 2.1.1 software (Beckman Coulter). Moreover, 17 bone marrow samples were processed using bulk lysis and subsequently stained using the MM-MRD EuroFlow 8-color antibody combination panel and tubes 1 and 2 with variable CD38, CD38-ME vs anti-CD38 nanobody. Samples were acquired on CytoFlex flow cytometer (Beckman Coulter) and data were analyzed using Infinicyt 2.0 software (Cytognos). Statistical analysis was performed using SPSS version 24, verifying the assumption of normality of the variable, pairwise comparisons were carried out using the T-tests for related samples. Results: The MFI values of CD38 ME on samples from daratumumab not treated patients was higher than samples after incubation with daratumumab (p=0.003). For CD38 nanobody, no significant differences were observed for the MFI values of samples, independent of daratumumab incubation (p=0,139) (figure 1A). The percentage of loss of MFI were higher in the CD38 ME group (-36,32%) than in the CD38 nanobody group (-0,35%). Regarding 17 patients treated with anti-CD38 therapies, 14 were treated with daratumumab and 3 with isatuximab. Sixteen samples were MRD positive with a sensitivity of 10-5 or 10-6 by both approaches. As shown in figure 1B, CD38-ME and CD38 nanobody both showed high and comparable MFI on PCs from patients treated with anti-CD38 therapies. Image:Summary/Conclusion: The anti-CD38 nanobody JK36 construct has lower coefficient of variation than CD38 ME antibody. It is a viable alternative to CD38 ME antibody to gate PCs by flow cytometry allowing reliable MRD detection to identify PCs in the presence of anti CD38 biologics. Nanobody technology open new avenues in the detection capability of MRD flow cytometry studies in the era of immunotherapy.

CD86 occupancy in belatacept-treated kidney transplant patients is not associated with clinical and infectious outcomes

The CD86 occupancy assay has been developed to measure the number of CD86molecules unbound to belatacept, but its association with clinical outcomes has not been assessed yet. All kidney transplant patients switched to belatacept in our center between 2016 and 2018 were included. Blood samples were collected before each infusion for 1year to assess CD86 occupancy by CD86 antibody cytometry staining on the surface of CD14+ monocytes. Results were expressed as the median fluorescence intensity (MFI) value of CD86staining. At each infusion, the MFIDay of infusion /MFIDay 0 ratio was calculated. Forty-one patients were consecutively included. After every 2-week infusion period, CD86MFI ratio dropped from 1.00 to 0.73 [0.57-0.98], p=.07. However, this ratio progressively increased to 0.78 [0.53-1.13] at 1year, which was not statistically different from pre-switch ratio, p=.4. Over the first year, the MFI ratio coefficient of variation was 31.58% [23.75-38.31]. MFI ratio was not different between patients with or without opportunistic infections: 0.73 [0.60-0.88] versus 0.80 [0.71-1.00], p=.2, or between patients with or without EBV DNAemia, p=.2. Despite previous in vitro results, the CD86 occupancy assay suffers from a high intra-individual variability and does not appear to be relevant to clinical outcomes.

Phage-Host Interaction Analysis by Flow Cytometry Allows for Rapid and Efficient Screening of Phages.

Recently, phages have become popular as an alternative to antibiotics. This increased demand for phage therapy needs rapid and efficient methods to screen phages infecting specific hosts. Existing methods are time-consuming, and for clinical purposes, novel, quick, and reliable screening methods are highly needed. Flow cytometry (FC) allows a quick differentiation and enumeration of bacterial cell populations and has been used to assess in vitro the activity of antimicrobial compounds. In this work, we propose FC as a rapid and reliable method to assess the susceptibility of a bacterial population to phage infection. For that, the interaction of phages vB_PaeM_CEB_DP1 and vB_PaeP_PE3 with Pseudomonas aeruginosa PAO1 was characterized by FC. Synchronous infection assays were performed, and samples were collected at different time points and stained with SYTO BC and PI before analysis. Part of the collected samples was used to characterize the expression of early, middle, and late genes by qPCR. Both FC and qPCR results were correlated with phage propagation assays. Results showed that SYTO BC median fluorescence intensity (MFI) values increased in the first 25 min of PE3 and DP1 infection. The increase of fluorescence is due to the expression of phage genes observed by qPCR. Since SYTO BC MFI values increase with gene expression, it allows the determination of host susceptibility to a phage in a short period of time, avoiding false positives caused by lysis from without. In conclusion, this method may allow for a quick and high-throughput real-time screening of different phages to a specific host, which can be crucial for a quick phage selection in clinical practice.

Prozone phenomenon in pretransplant testing: An interesting conundrum involving solid-phase and cell-based assays.

Human leukocyte antigen (HLA) is a major determinant in deciding upon solid organ histocompatibility. Donor-specific anti-HLA antibodies (Donor-specific anti-HLA antibodies (DSAs)) are always a contraindication for solid organ transplantation, and identification of DSA becomes very crucial before transplantation to provide long-term graft survival. For identification of DSA, usually, either cell-based or HLA bead-based assay is being used in laboratories. However, both cell-based and bead-based assays have certain limitations. One such common limitation is "prozone effect," which can give false-negative results. Here, we would like to present a small pilot study to analyze the effect of the prozone phenomenon in the cell-based and HLA bead-based assays and its utility in histocompatibility testing. In a series of four experiments, cell-based assay, flow cytometric cross-match (FCXM), and HLA bead-based flow cytometric panel reactive antibodies (PRAs) were performed. Single-antigen bead (SAB) testing was conducted as a first experiment on four known positives samples for anti-HLA antibody-antibodies. In the second experiment, these four samples were pooled together (called pooled sera in the text) and tested for FCXM and PRA. In the third experiment, known commercially available positive control sera were mixed with pooled positive sera (positive control sera + pooled sera) to prepare, what we have called "positive concoction" in the text. In the fourth experiment, the positive concoction was diluted serially (1:2, 1:4, 1:8, and 1:16) and FCXM and PRA were performed again to analyze and compare the prozone effect. Pooled sera did not have the expected median fluorescence intensity (MFI) values in FCXM assay, whereas the PRA was showing >90% positivity. In positive concoction, the MFI of FCXM assay was observed to be declining; however, PRA values remained almost constant. Dilutions of the pooled sera showed that MFI values of FCXM assays were increased suddenly after dilution. The highest MFI values were observed in 1:4 dilution of the sera, and then, it declined gradually, but the PRA values remained almost constant even after serial dilutions. In our experimental findings, it was clear that cell-based assay (FCXM) was more severely affected by the prozone, whereas solid-phase (flow PRA) assay remained resistant to prozone.

High-Throughput Multiplex SARS-CoV-2 IgG Microsphere Immunoassay for Dried Blood Spots: A Public Health Strategy for Enhanced Serosurvey Capacity.

ABSTRACTEarly in the pandemic when diagnostic testing was not widely available, serosurveys played an important role in estimating the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in different populations. Dried blood spots (DBS), which can be collected in nonclinical settings, provide a minimally invasive alternative to serum for serosurveys. We developed a Luminex-based SARS-CoV-2 microsphere immunoassay (MIA) for DBS that detects IgG antibodies to nucleocapsid (N) and spike subunit 1 (S1) antigens. The assay uses a 384-well plate format and automated liquid handlers for high-throughput capacity. Specificity was assessed using a large collection of prepandemic DBS and well-characterized sera. Sensitivity was analyzed using serology data from New York State SARS-CoV-2 serosurvey testing and matched diagnostic test results. For DBS, the specificity was 99.5% for the individual N and S1 antigens. Median fluorescence intensity (MFI) values for DBS and paired sera showed a strong positive correlation for N (R2 = 0.91) and S1 (R2 = 0.93). Sensitivity, assessed from 1,134 DBS with prior laboratory-confirmed SARS-CoV-2 infection, ranged from 83% at 0 to 20 days to 95% at 61 to 90 days after a positive test. When stratified using coronavirus disease 2019 (COVID-19) symptom data, sensitivity ranged from 90 to 96% for symptomatic and 77 to 91% for asymptomatic individuals. For 8,367 health care workers reporting detailed symptom data, MFI values were significantly higher for all symptom categories. Our results indicate that the SARS-CoV-2 IgG DBS MIA is sensitive, specific, and well-suited for large population-based serosurveys. The ability to readily modify and multiplex antigens is important for ongoing assessment of SARS-CoV-2 antibody responses to emerging variants and vaccines.IMPORTANCE Testing for antibodies to SARS-CoV-2 has been used to estimate the prevalence of COVID-19 in different populations. Seroprevalence studies, or serosurveys, were especially useful during the early phase of the pandemic when diagnostic testing was not widely available, and the resulting seroprevalence estimates played an important role in public health decision making. To achieve meaningful results, antibody tests used for serosurveys should be accurate and accessible to diverse populations. We developed a test that detects antibodies to two different SARS-CoV-2 proteins in dried blood spots (DBS). DBS require only a simple fingerstick and can be collected in nonclinical settings. We conducted a robust validation study and have demonstrated that our test is both sensitive and specific. Furthermore, we demonstrated that our test is suitable for large-scale serosurveys by testing over 56,000 DBS collected in a variety of community-based venues in New York State during the spring of 2020.

Dynamic and Temporal Transcriptomic Analysis Reveals Ferroptosis-Mediated Antileukemia Activity of S-Dimethylarsino-Glutathione: Insights into Novel Therapeutic Strategy

Dynamic and Temporal Transcriptomic Analysis Reveals Ferroptosis-Mediated Antileukemia Activity of S-Dimethylarsino-Glutathione: Insights into Novel Therapeutic Strategy

The effect of Mass Drug Administration for trachoma on antibodies to Chlamydia trachomatis pgp3 in children

A serologic test for antibodies to chlamydia may be a useful tool for trachoma surveillance. However, little is known about the longitudinal stability of antibody status, especially following Mass Drug Administration (MDA), which is critical to understanding serostatus in trachoma-endemic areas. A longitudinal cohort of 1908 children ages 1–9 years in Tanzania from 50 communities were followed at baseline and for 6 months after MDA. They were evaluated for clinical trachoma, conjunctival swabs were tested for chlamydial infection using GeneXpert platform, and blood spots were collected on filter paper and dried to test for antibodies to Chlamydia trachomatis pgp3 using the Luminex platform. 6.3% of children in the study had infection, and coverage with MDA was 97%. 670 (35%) were sero-positive for pgp3 antibodies at baseline, and 4.0% of these seroreverted to negative following MDA. Of those seronegative at baseline, 3.6% seroconverted. The individual change in log median fluorescence intensity(MFI-BG) values was -0.15 overall (p < .001). Seroconversion rates were lower following MDA and seroreversion rates were slightly higher compared to rates in this same cohort in the absence of MDA. MDA has a small effect on reduction of MFI-BG.

Flow cytometry expression pattern of CD44 and CD18 markers on feline leukocytes.

The paucity of specific feline antibodies for flow cytometry (FC) is an ongoing challenge. Flow cytometrists must extrapolate information from relatively few markers. We evaluated the expression pattern of the panleukocyte markers CD18 and CD44 on leukocyte (white blood cell, WBC) subclasses in the peripheral blood (PB) of 14 healthy cats. The degree of expression of CD18 and CD44 was calculated as the ratio between the median fluorescence intensity (MFI) value of antibody-stained cells and autofluorescence. All samples were acquired with the same cytometer with constant photomultiplier setting and compensation matrices. Both molecules were expressed at higher levels on monocytes, intermediate levels on polymorphonuclear cells (PMNs), and lower levels on lymphocytes. CD18-MFI discriminated well among the 3 populations, whereas CD44-MFI mostly overlapped between monocytes and PMNs. However, CD44-MFI had a lower intra-population variability. Evaluation of CD18 and CD44, together with morphologic parameters, was useful for discriminating among WBC subclasses in healthy cats. This information may be helpful for future studies given that an increase in CD18-MFI may indicate reactive changes, whereas fluctuations in CD44-MFI may suggest neoplasia.

Donor-specific B Cell Memory in Alloimmunized Kidney Transplant Recipients: First Clinical Application of a Novel Method.

HLA-specific memory B cells may contribute to the serum HLA antibody pool upon antigen reexposure. The aim of this pilot study was to investigate the presence of concurrent donor-specific memory B cell-derived HLA antibodies (DSA-M) in renal allograft recipients with pretransplant donor-specific HLA antibodies (DSA) and its association with occurrence of antibody-mediated rejection (AMR) using a recently developed method. Twenty patients with Luminex single antigen bead (SAB) assay-defined DSA but negative complement-dependent cytotoxicity crossmatches were enrolled. Plasma samples and peripheral blood mononuclear cells were collected at 3 timepoints (pretransplant, mo 6, mo 12). We analyzed IgG-purified and concentrated culture supernatants from polyclonally activated peripheral blood mononuclear cells using SAB assays and compared HLA antibody profiles with same day plasma results. Plasma SAB analysis revealed 35 DSA in 20 patients pretransplant. DSA-M were detected in 9 of 20 (45%) patients and for 10 of 35 specificities (29%). While median mean fluorescence intensity values of DSA with concurrent DSA-M (5877) were higher than those of DSA without DSA-M (1476), 3 of 6 patients with AMR and low mean fluorescence intensity DSA (<3000) had DSA-M. Overall, pretransplant DSA/DSA-Mpos allograft recipients showed a higher incidence of biopsy-proven (sub)clinical AMR (P = 0.032) and a higher extent (g≥1 + ptc≥1) of microvascular inflammation (67% vs 9%, P = 0.02). In 17 patients (28 DSA) with posttransplant analyses, persisting DSA posttransplant had more often DSA-M (6/12; 50%) than nonpersisting DSA (2/16; 13%). Assessment of DSA-M might be a novel tool to supplement serum HLA antibody analysis for pretransplant risk stratification in patients with DSA.

Extending flow cytometry sample stability by freezing lysed whole blood for clinical monitoring of Treg.

Aim: A major challenge for flow cytometry assays supporting clinical trials is postcollection sample stability. Here we present an approach that could mitigate the stability issue while preserving sample integrity and cellular markers, especially when enumerating rare populations such asTregs. Materials & methods: Stability was evaluated using whole blood stored at roomtemperatureand lysed whole blood stored at -80°C. Results: Freezing of lysed whole blood preserved sample integrity and prolonged sample stability for Tregpercentage, absolute cell countand median fluorescent intensity values to 11versus 3 days at room temperature storage. Conclusion: Frozen storage of lysed whole blood can extend sample stability, improve data qualityand facilitate sample batch processing during clinical study sample analysis.

An initial investigation of serum cytokine levels in patients with gadolinium retention.

ObjectiveTo determine whether individuals with proposed gadolinium deposition disease (GDD) have elevated serum levels of pro-inflammatory and pro-fibrotic cytokines, and whether specific cytokines are correlated with certain symptoms.Materials and MethodsTwenty-four participants recruited between May 2016 and June 2017 met GDD diagnostic criteria. The 64 control subjects provided serum samples before prophylactic flu vaccination. Serum cytokine levels were obtained with Luminex serum cytokine assay using eBiosciences/Affymetrix human 62-plex kits. Wilcoxon rank-sum tests were performed to assess the difference between the median fluorescence intensity values for the participants and the control group. Generalized linear models were built to evaluate the association between each cytokine of interest and selected participant symptoms.ResultsSerum levels of 14 cytokines, including nine pro-inflammatory cytokines, were statistically significantly elevated compared to controls (p ≤ 0.05). Hypotheses regarding pro-fibrotic cytokines and cytokine links to specific symptoms' intensity were not confirmed.ConclusionThe statistically significantly elevated cytokines may be markers of susceptibility to GDD or agents of symptom induction. These findings suggest that individuals developing symptoms characteristic of GDD after a contrast-assisted magnetic resonance imaging should be studied to investigate whether gadolinium retention and elevated cytokines may be related to their symptoms.

Flow Cytometric Immunophenotyping Distinguishes Lymphoplasmacytic Lymphoma from Marginal Zone Lymphoma

Introduction: Flow cytometric (FC) immunophenotyping is an essential tool for the diagnosis of mature B cell lympho-proliferative disorders (B-LPDs). The most common CD5-negative/CD10-negative B-LPDs are hairy cell leukemia, lymphoplasmacytic lymphoma and marginal zone lymphoma. Although HCL has unique clinicopathologic characteristics, LPL and MZL are often difficult to distinguish by standard diagnostic methods. MYD88 L265P mutations are found in 90% of LPL and 8-10% of MZL, although the finding of this mutation may increase the suspicion of LPL, it is by no means definitive since they are also found in MZL. The distinction between LPL and MZL is of increasing clinical importance given treatment options and prognosis may differ. The Euroflow eight color B-LPD panel provides detailed information on the phenotype of clonal B-cells and has not previously been evaluated as a tool to distinguish these subtypes of B-LPD. Methods: A retrospective study of patients who were diagnosed with CD5-negative/CD10-negative B-LPD from January 2011 to October 2017 was conducted. Patients with HCL and diffuse large B cell lymphoma were excluded. Data collected included the FC immunophenotype, clinical findings, MYD88 mutational status, treatment and outcome. Data were obtained from the FC lab database at the National University Health System (NUHS) Hematology laboratory. Clinical data were obtained from the electronic patient medical records. A clinicopathologic diagnosis of either LPL, MZL or "indeterminate between LPL/MZL" was assigned to the patients based on consensus at the multidisciplinary tumor board. The FC phenotype (based on raw median fluorescence intensity values) of patients assigned to each diagnostic category were then compared. Hierarchical clustering using Gower's distance of variables and Ward's D linkage method were used to determine if the FC immunophenotyping patterns can distinguish LPL and MZL. Results: A total of 22 patients' data was included in the analysis. We found that the LPL cases formed a discrete cluster, while MZL cases were more widely dispersed (figure 1). LPL cases showed negative expression of CD 39, CD 43 and CD 95, while these markers are expressed in a significant proportion of MZL cases. In contrast, CD31 was expressed in the majority of LPL but not the MZL cases. CD 200 and CD38 were found to be variably expressed in both LPL and MZL cases. It is noteworthy that CD 27 was also not consistently expressed across all the cases in our study. There were also no significant differences in terms of surface IgM expression. Conclusions: Our study showed that the eight color flow cytometry has potential as a tool to differentiate LPL and MZL. The lack of a discrete clustering of the MZL cases in our analysis may be explained by the fact that MZL is a more heterogeneous entity compared to LPL. Positivity for CD31 and negativity for CD39, CD95 and CD43 appear to be features of LPL. MZL in contrast show positive expression of CD39, CD95 and CD43 and are negative for CD31. Earlier studies using four color FC suggested that CD200 may have value as a discriminative marker between LPL and MZL. Our data using eight color FC does not show the same discriminative power for CD200. The absence of consistent CD39 and CD27 expression is unusual as LPL and MZL are both post germinal centre B cell neoplasms in which the expression of these antigens is expected. We propose that FC immunophenotyping should be evaluated prospectively as an adjunct to standard clinicopathologic evaluation to distinguish LPL and MZL. Figure 1 Disclosures No relevant conflicts of interest to declare.

VS38 Identifies Myeloma Cells With Dim CD38 Expression and Plasma Cells Following Daratumumab Therapy, Which Interferes With CD38 Detection for 4 to 6 Months.

We report our institutional experience using VS38 to evaluate plasma cells by flow cytometry. Flow cytometry data were reanalyzed to compare plasma cell percentages between the standard panel and VS38 panel. Natural killer (NK) and plasma cell CD38 median fluorescence intensity (MFI) values were calculated. Our cohort included 63 specimens from 38 patients. Twenty-six had received daratumumab (monoclonal anti-CD38 therapy) between less than 1 month and 17 months prior. For NK and plasma cells, CD38 MFI values were suppressed for 0 to 4 months and started to increase 4 to 6 months after last exposure. There was no significant difference in clonal plasma cell percentage calculated by the VS38 and standard panels; however, identification and quantification using the VS38 panel were easier. VS38 is a viable alternative to bright CD38 to identify plasma cells and particularly helpful in myeloma cases with dim CD38 and after daratumumab. Daratumumab interference with CD38 identification persists 4 to 6 months after the last exposure.