Accurate and reproducible enumeration of T-, B-, and NK lymphocytes using the BD FACSLyric 10-color system: A multisite clinical evaluation.

Clinical flow cytometry is a reliable methodology for whole blood cell phenotyping for different applications. The BD FACSLyric™ system comprises a flow cytometer available in different optical configurations, BD FACSuite™ Clinical software, and optional BD FACS™ Universal Loader. BD FACSuite Clinical software used with BD™ FC Beads and BD CS&T Beads enable universal setup for performance QC, instrument control, data acquisition/storage, online/offline data analysis, and instrument standardization. BD Biosciences sponsored the clinical evaluation of the BD FACSLyric 10-color configuration at seven clinical sites using delinked and de-identified blood specimens from HIV-infected and uninfected subjects to enumerate T-, B-, and NK-lymphocytes with the BD Multitest™ reagents (BD Multitest IMK kit and BD Multitest 6-color TBNK). Samples were analyzed on the BD FACSLyric system with BD FACSuite Clinical software, and on the BD FACSCanto™ II system with BD FACSCanto clinical software and BD FACS 7-Color Setup beads. For equivalency between methods, data (n = 362) were analyzed with Deming regression for absolute count and percentage of lymphocytes. Results gave R2 ≥0.98, with slope values ≥0.96, and slope ranges between 0.90-1.05. The percent (%) bias values were <10% for T- and NK cells and <15% for B- cells. The between-site (n = 4) total precision was tested for 5 days (2 runs/day), and gave %coefficient of variation below 10% for absolute cell counts. The stability claims were confirmed (n = 186) for the two BD Multitest reagents. The reference intervals were re-established in male and female adults (n = 134). The analysis by gender showed statistically significant differences for CD3+ and CD4+ T-cell counts and %CD4. In summary, the BD FACSLyric and the BD FACSCanto II systems generated comparable measurements of T-, B-, and NK-cells using BD Multitest assays.

In vitro evaluation of di(2-ethylhexyl)terephthalate-plasticized polyvinyl chloride blood bags for red blood cell storage in AS-1 and PAGGSM additive solutions.

BACKGROUND: Di(2-ethylhexyl)phthalate (DEHP) makes polyvinyl chloride flexible for use in blood bags and stabilizes the red blood cell (RBC) membrane preventing excessive hemolysis. DEHP migrates into the blood product and rodent studies have suggested that DEHP exposure may be associated with adverse health effects albeit at high dosages. Although structurally and functionally similar to DEHP, di(2-ethylhexyl)terephthalate (DEHT; or Eastman 168 SG [Eastman Chemical Company]) is metabolically distinct with a comprehensive and benign toxicology profile. This study evaluated RBC stability in DEHT-plasticized bags with AS-1 and PAGGSM compared to conventional DEHP-plasticized bags with AS-1. STUDY DESIGN AND METHODS: Thirty-six whole blood units were collected into CPD solution, leukoreduced, centrifuged, and divided into RBCs and plasma. To limit donor-related variability, three ABO-identical RBCs were mixed together and then divided equally and stored among the three different plasticizer and additive solution combinations. RBCs from 12 trios were analyzed for a standard panel of in vitro variables on Day 0 and after storage. RESULTS: No individual bag on Day 42 exceeded the US 1.0% hemolysis criteria. While hemolysis during storage was higher in the DEHT bags, the PAGGSM RBCs were close to the control RBCs (0.38% vs. 0.32%, respectively). ATP retention was higher than 70% and potassium levels were similar regardless of plasticizer. Additional RBC variables exhibited some significant differences but were not viewed as clinically important. CONCLUSION: DEHT/PAGGSM provides similar hemolysis protection to that of DEHP/AS-1. Although hemolysis values with DEHT and AS-1 are higher than that of DEHP, DEHT is a potential DEHP alternative.

Human HLA-B27 typing using the BD™ HLA-B27 kit on the BD FACSVia™ system: A multicenter study.

The BD FACSVia™ system is a novel flow cytometer with improved workflow efficiencies. To evaluate the HLA-B27 application developed on the BD FACSVia system utilizing the BD™ HLA-B27 kit, we conducted a concordance study at three centers to compare with the BD FACSCalibur™ system. Prepared donor samples (n = 594) were analyzed on both the BD FACSVia and BD FACSCalibur for the HLA-B27 assay. Adjudication of HLA-B27 discordant results was performed using the reverse sequence-specific oligonucleotide (rSSO) DNA typing method (LABType® SSO, One Lambda). On the BD FACSVia system 80 B27 positive, 499 B27 negative and 15 "Inconclusive" samples were observed. The corresponding BD FACSCalibur results were 73 B27 positive, 502 B27 negative and 19 "gray zone" samples. The overall concordance of HLA-B27 determination was 98% between the two systems with seven more positives identified on BD FACSVia as compared to BD FACSCalibur. The equivocal zone between positive and negative on BD FACSVia (named "Inconclusive") and on BD FACSCalibur (named "gray zone") is due to antibody cross reactivity of HLA-B27 clone GS145.2. One negative sample verified with the rSSO DNA method was reported as HLA-B27 positive by the BD FACSVia system leading to a false positive result. Our study demonstrated concordance results between the BD FACSVia system and BD FACSCalibur. Intersite reproducibility of BD HLA-B27 assay remained within the limits of acceptability. © 2018 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

Comparison of Flow Cytometric Methods for the Enumeration of Residual Leucocytes in Leucoreduced Blood Products: A Multicenter Study.

The BD FACSVia™ System features novel designs in hardware, software, and instrument QC. We compared the performance of the BD FACSVia System using the BD Leucocount™ kit with the BD FACSCalibur™ flow cytometer. Leucoreduced platelet (PLT, n = 252) and red blood cell (RBC, n = 278) specimens were enrolled at four sites. Each specimen was stained in four tubes using the BD Leucocount kit reagents and acquired on the two systems. BD Leucocount Control cells (high and low) were used to evaluate the inter-site reproducibility on the BD FACSVia System at three sites over 20 days. Deming regression and Bland-Altman analysis were performed to determine the WBC absolute counts on the BD FACSVia System vs. the BD FACSCalibur system. Assay accuracy for the range of 0-350 WBCs/µl was adequate. For samples with <25 WBCs/µl, the bias with 95% limits of agreement was 0.136 (-1.897 to 2.169) WBC/µl for PLTs (n = 184) and 0.170 (-2.025 to 2.365) WBC/µl for RBCs (n = 193). For inter-site reproducibility, the CV% was 6.46% (upper 95% CI 7.16%) for the PLT high control and 9.49% (10.52%) for the PLT low control. The CV% was 7.51% (8.32%) for the RBC high control and 10.76% (11.92%) for the RBC low control. The BD FACSVia System reported equivalent results of WBC absolute counts for leucoreduced PLT and RBC samples compared to the BD FACSCalibur system. The inter-laboratory reproducibility of the BD FACSVia System met study specifications. © 2018 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC.

CD4 Lymphocyte Enumeration and Hemoglobin Assessment Aid for Priority Decisions: A Multisite Evaluation of the BD FACSPresto™ System.

BACKGROUND: The BD FACSPresto™ system uses capillary and venous blood to measure CD4 absolute counts (CD4), %CD4 in lymphocytes, and hemoglobin (Hb) in approximately 25 minutes. CD4 cell count is used with portable CD4 counters in resource-limited settings to manage HIV/AIDS patients. A method comparison was performed using capillary and venous samples from seven clinical laboratories in five countries. The BD FACSPresto system was assessed for variability between laboratory, instrument/operators, cartridge lots and within-run at four sites. METHODS: Samples were collected under approved voluntary consent. EDTA-anticoagulated venous samples were tested for CD4 and %CD4 T cells using the gold-standard BD FACSCalibur™ system, and for Hb, using the Sysmex® KX-21N™ analyzer. Venous and capillary samples were tested on the BD FACSPresto system. Matched data was analyzed for bias (Deming linear regression and Bland-Altman methods), and for concordance around the clinical decision point. The coefficient of variation was estimated per site, instrument/operator, cartridge-lot and between-runs. RESULTS: For method comparison, 93% of the 720 samples were from HIV-positive and 7% from HIV-negative or normal subjects. CD4 and %CD4 T cells venous and capillary results gave slopes within 0.96-1.05 and R2 ≥0.96; Hb slopes were ≥1.00 and R2 ≥0.89. Variability across sites/operators gave %CV <5.8% for CD4 counts, <1.9% for %CD4 and <3.2% for Hb. The total %CV was <7.7% across instrument/cartridge lot. CONCLUSION: The BD FACSPresto system provides accurate, reliable, precise CD4/%CD4/Hb results compared to gold-standard methods, irrespective of venous or capillary blood sampling. The data showed good agreement between the BD FACSPresto, BD FACSCalibur and Sysmex systems.

Spectra Optia granulocyte apheresis collections result in higher collection efficiency of viable, functional neutrophils in a randomized, crossover, multicenter trial.

BACKGROUND: Granulocyte transfusion from healthy donors is used in the treatment of patients with granulocyte function defects, or transient neutropenia and severe bacterial or fungal infections resistant to maximal antimicrobial treatment. STUDY DESIGN AND METHODS: This study evaluated the performance and safety of the newly developed granulocyte collection protocol of the Spectra Optia in a prospective, multicenter, open-label, randomized, paired crossover trial compared with the COBE Spectra apheresis system in a population of 32 evaluable healthy subjects. All subjects received granulocyte-colony-stimulating factor and dexamethasone before collection. RESULTS: Granulocyte procedures from Spectra Optia apheresis procedures had an approximately 23% higher polymorphonuclear (PMN) collection efficiency (CE) than the COBE Spectra collections (mean, 53.7% vs. 43.2%; p < 0.01), while the platelet CE (10.9% vs. 10.8%, respectively) and hematocrit (7.4% vs. 7.4%) were comparable between collections on both devices. Spectra Optia collections generated a higher total PMN yield per liter of blood processed than those produced by the COBE Spectra (with means of 8.6 × 10(10) vs. 6.8 × 10(10) , respectively). Granulocyte viability was more than 99% with both devices, and chemotaxic and bacterial killing activities of circulating versus collected granulocytes were similarly preserved. Fewer operator adjustments were required with Spectra Optia and there was no significant difference in the number or intensity of adverse events between instruments. CONCLUSION: CE of the granulocyte collection procedure with the Spectra Optia was approximately 10 percentage points higher than with the COBE Spectra, required less operator involvement, and is safe for clinical implementation.

Evaluation of the Spectra Optia apheresis system for mononuclear cell (MNC) collection in G-CSF mobilized and nonmobilized healthy donors: results of a multicenter study.

The Spectra Optia apheresis system is a newer centrifugation-based device that in comparison with the COBE Spectra includes features that enhance procedure automation and usability. In this FDA-approved three-center two-arm observational study we characterized the performance of the Spectra Optia for collection of MNCs and CD34+ cells from nonmobilized and granulocyte-colony stimulating factor (G-CSF) mobilized healthy donors, respectively. There were a total of 15 evaluable subjects in each arm. Key performance indicators included collection efficiency of MNCs/CD34+ cells, product purity and cellular viability. For nonmobilized donors, median MNC collection efficiency, platelet collection efficiency, product hematocrit and granulocyte contamination were 57%, 12%, 4%, and 1.7%, respectively. For mobilized donors, median MNC collection efficiency, CD34+ cell collection efficiency, platelet collection efficiency, product hematocrit and granulocyte contamination were 61%, 77%, 19%, 4%, and 15%, respectively. Average WBC viability in the mobilized products was 99%. There was one severe (grade 3) adverse event related to citrate toxicity. This study demonstrates that the Spectra Optia can be used for safe and efficacious collection of MNCs, and results obtained are in line with expectations on collection efficiency and product characteristics. Adverse events were limited to those that are well documented in the stem-cell mobilization and leukapheresis process. As of the time of this writing, FDA 510(k) approval for use of the Spectra Optia device for MNC collection was achieved in the US based partly on the results of this study.

In vitro and in vivo quality of leukoreduced apheresis platelets stored in a new platelet additive solution.

BACKGROUND: Platelets (PLTs) stored in additive solutions (PASs) may reduce the risk of several plasma-associated adverse transfusion reactions such as allergic reactions and potentially transfusion-associated lung injury. The objective of this study was to determine the in vitro characteristics and the in vivo radiolabeled recovery and survival of apheresis PLTs (APs) stored in a new PAS and compare the latter to Food and Drug Administration (FDA) criteria. STUDY DESIGN AND METHODS: Hyperconcentrated APs were collected from healthy subjects in a paired crossover study comparing PAS (35% plasma) and 100% plasma-stored APs (Part 1) up to 7 days and, in Part 2, to determine the in vivo recovery and survival of PAS stored AP at 5 days compared to fresh PLT controls. In vitro and in vivo assays were performed following standard methods. RESULTS: Sixty-six and 25 evaluable subjects successfully completed Parts 1 and 2, respectively. pH for PAS AP was maintained above 6.6 for 5 days of storage. P-selectin values were consistent with published values for commonly transfused PLT products. The PAS in vivo PLT recovery (54.3 ± 8.1%) was 86.7% of the fresh control, and survival (6.4 ± 1.3 days) was 78.0% of the fresh control, both meeting the FDA performance criteria. CONCLUSION: APs stored in PAS with 35% plasma carryover maintained pH over 5 days of storage and met current FDA criteria for radiolabeled recovery and survival. The use of PAS for storage of single-donor PLTs in clinical practice represents an acceptable transfusion product that reduces the volume of plasma associated with PLT transfusion.

Absence of acute adverse in vitro effects on aged AS-1 red blood cells and thawed plasma after prolonged exposure to 13.56-MHz radio energy.

BACKGROUND: There is growing interest in radio frequency identification (RFID) technology for tracking blood products to improve productivity and safety in the transfusion medicine supply chain. We conducted a limited study to assess the temperature and biologic effects after extreme exposure to 13.56-MHz RF radiation on aged red blood cells (aRBCs) nearing their 42-day life and three types of thawed plasma (TP). STUDY DESIGN AND METHODS: Using a Food and Drug Administration-approved limit test protocol, test units of both aRBCs and three types of TP were subjected to high levels of RF energy for an extended duration to assess worst-case effects compared to minimally exposed control units. Three replications were performed for each product type. RESULTS: Hemolysis after 23 to 25 hours of RF energy exposure was less than 0.3% for all test and control aRBC units and well within the 1% or less acceptance criterion. Both biologic test and temperature increase results were within acceptance criteria and consistent with earlier tests on 6- to 9-day RBCs, with no detectable acceleration in cellular degradation of aRBCs. Nine different plasma coagulation factors were evaluated and, with one explainable exception, all showed less than 20% change in their measured test versus control values, meeting the acceptance criteria. The relative temperature increase between test and control units never exceeded the 1.5°C acceptance criterion for RBCs and 4°C for plasma. CONCLUSION: Use of 13.56-MHz RFID technology is unlikely to have any significant temperature or biologic effects on aRBC and plasma units under normal operating conditions.

Automated collection of double red blood cell units with a variable-volume separation chamber.

BACKGROUND: Automated collection of blood components offers multiple advantages and has prompted development of portable devices. This study sought to document the biochemical and hematologic properties and in vivo recovery of red cells (RBCs) collected via a new device that employed a variable-volume centrifugal separation chamber. STUDY DESIGN AND METHODS: Normal subjects (n = 153) donated 2 units of RBCs via an automated blood collection system (Cymbal, Haemonetics). Procedures were conducted with wall outlet power (n = 49) or the device's battery source (n = 104). Units were collected with or without leukoreduction filtration and were stored in AS-3 for 42 days. The units were assessed via standard biochemical and hematologic tests before and after storage, and 24 leukoreduced (LR) and 24 non-LR RBCs were radiolabeled on Day 42 with Na(2)(51)CrO(4) for autologous return to determine recovery at 24 hours with concomitant determination of RBC volume via infusion of (99m)Tc-labeled fresh RBCs. RESULTS: Two standard RBC units (targeted to contain 180 mL of RBCs plus 100 mL of AS-3) could be collected in 35.7 +/- 2.0 minutes (n = 30) or 40.3 +/- 2.7 minutes for LR RBCs (n = 92). An additional 31 collections were conducted successfully with intentional filter bypassing. RBC units contained 104 +/- 4.1 percent of their targeted volumes (170-204 mL of RBCs), and LR RBCs contained 92 percent of non-LR RBCs' hemoglobin. All LR RBCs contained less than 1 x 10(6) white blood cells. Mean hemolysis was below 0.8 percent (Day 42) for all configurations. Adenosine triphosphate was well preserved. Mean recovery was 82 +/- 4.9 percent for RBCs and 84 +/- 7.0 percent for LR RBCs. CONCLUSIONS: The Cymbal device provided quick and efficient collection of 2 RBC units with properties meeting regulatory requirements and consistent with good clinical utility.