The use of intracellular dyes to create a multiplexed flow cytometry-based red blood cell phenotyping assay.

BACKGROUND AND OBJECTIVES: Flow cytometry can be used to phenotype red blood cell antigens, allowing for high-throughput testing while using low reagent volumes. This article utilizes intracellular dyes to pre-label red blood cells to further multiplex flow cytometry-based red blood cell antigen phenotyping. MATERIALS AND METHODS: Red blood cells were pre-labelled using the intracellular dyes V450 and Oregon Green. These dyes are detected fluorescently via flow cytometry. Four combinations of intracellular staining were used to allow four patient or donor red blood cells to be analysed in a single test well. Antigen phenotyping was then performed via flow cytometry using a previously described method. RESULTS: The intracellular dyes showed uniform staining when measured in mean fluorescence intensity and allowed the red blood cells to be clearly distinguished from one another. The presence or absence of red blood cell antigens was determined with 100% accuracy. CONCLUSION: The use of intracellular dyes allowed a fourfold increase in the throughput of our previously described flow cytometry-based red blood cell antigen phenotyping method. The described method allows up to 48 patients to be simultaneously phenotyped using a single 96-well microplate. Furthermore, additional fluorescent dyes could potentially increase the throughput exponentially.

Development of multiplexed flow cytometry-based red blood cell antibody screen and identification assays.

BACKGROUND AND OBJECTIVES: The purpose of this study was to develop a high-throughput method of performing red blood cell antibody screens and identification by utilizing flow cytometry and intracellular dyes to allow a multiplexed assay where three-cell screens can be performed in a single test well and 11-cell panels in three test wells. MATERIALS AND METHODS: Reagent red blood cells were labelled using Violet Proliferation Dye 450 (V450) and Oregon Green fluorescent dyes, which bind intracellular proteins to allow up to four cells to be interrogated in a single test well. Sixteen 3-cell screen panels and ten 11-cell identification panels were tested using sera with known antibody specificity. Antibody binding was detected using secondary anti-immunoglobulin G and anti-immunoglobulin M fluorescently labelled antibodies. RESULTS: Intracellular dyes allowed clear separation of the different screen and identification panel test cells. Three distinct populations of V450+, Oregon Green+ and negative for both stains were demonstrated in the screening panel and an additional double positive for V450 and Oregon Green was utilized to include a fourth cell in the identification panel testing to increase throughput. A total of 158 screen or identification panel RBC/serum combinations were tested against different known antibodies, and expected results were obtained with 100% concordance. CONCLUSION: This study demonstrates the successful development of a high-throughput multiplexed flow cytometry-based red cell antibody screen and identification panel assays. This method could be implemented in clinical laboratories to complement existing antibody detection methods. The multiplexing enabled via intracellular staining could be utilized to further augment other flow cytometry-based transfusion assays.

Validation of a flow-cytometry-based red blood cell antigen phenotyping method.

BACKGROUND AND OBJECTIVES: Current manual and automated phenotyping methods are based on visual detection of the antigen-antibody interaction. This approach has several limitations including the use of large volumes of patient and reagent red blood cells (RBCs) and antisera to produce a visually detectable reaction. We sought to determine whether the flow cytometry could be developed and validated to perform RBC phenotyping to enable a high-throughput method of phenotyping using comparatively miniscule reagent volumes via fluorescence-based detection of antibody binding. MATERIALS AND METHODS: RBC phenotyping by flow cytometry was performed using monoclonal direct typing antisera (human IgM): anti-C, -E, -c, -e, -K, -Jka , -Jkb and indirect typing antisera (human IgG): anti-k, -Fya , -Fyb , -S, -s that are commercially available and currently utilized in our blood transfusion services (BTS) for agglutination-based phenotyping assays. RESULTS: Seventy samples were tested using both flow-cytometry-based-phenotyping and a manual tube standard agglutination assay. For all the antigens tested, 100% concordance was achieved. The flow-cytometry-based method used minimal reagent volume (0.5-1 µl per antigen) compared with the volumes required for manual tube standard agglutination (50 µl per antigen) CONCLUSION: This study demonstrates the successful validation of flow-cytometry-based RBC phenotyping. Flow cytometry offers many benefits compared to common conventional RBC phenotyping methods including high degrees of automation, quantitative assessment with automated interpretation of results and extremely low volumes of reagents. This method could be used for high-throughput, low-cost phenotyping for both blood suppliers and hospital BTS.

Implementation of a novel real-time platelet inventory management system at a multi-site transfusion service.

BACKGROUND: Blood platelets (PLTs) are a valuable commodity. Management of their inventory has implications both for patient care and for the cost of health care delivery. There are a variety of different methods of managing PLT inventory currently in practice and multiple theoretical models aimed at improving PLT inventory metrics. In this study we evaluate the ability of a novel electronic dashboard system that monitors and displays both PLT inventory and patient data to improve transfusion metrics at a quaternary health care center. STUDY DESIGN AND METHODS: The Capital District Health Authority is a quaternary health care center that transfuses approximately 2500 PLT units annually. To improve PLT discard rates a novel, low-overhead system that interfaces with the laboratory information system and displays real-time data between transfusion sites on PLT inventory and orders was implemented in November 2011. This study examines the transfusion quality metrics data from the 24 months before and after implementation. RESULTS: A significant reduction in mean monthly PLT outdate rate was observed after the implementation of the PLT dashboard suite from 24.5% (n = 24, SD ± 6.4%) to 15.1% (n = 24, SD ± 6.4%; p < 0.001). PLT age at time of transfusion was also reduced from 3.60 days (n = 4796, SD ± 0.97 days) to 3.46 days (n = 4881, SD ± 1.00 days; p < 0.001). CONCLUSIONS: This study describes the implementation of a novel PLT dashboard suite. This suite significantly reduced PLT outdate rates at our institution over the 48-month study period.