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.

Application of unsupervised machine learning to identify areas of blood product wastage in transfusion medicine.

BACKGROUND: Wastage of blood products can be a significant cost to blood banks. However, the cause of wastage is often complex and makes it difficult to determine wastage-associated factors. Machine learning techniques may be useful tools to investigate these complex associations. We investigated whether unsupervised machine learning can identify patterns associated with wastage in our blood bank. MATERIALS AND METHODS: Data on red blood cells, platelets and frozen products were obtained from the laboratory information system of the Central Zone Blood Transfusion Services at Nova Scotia Health Authority. A total of 879 532 transactions were analysed by association rule mining, a type of machine learning algorithm. Associations with lift scores greater than 25 and with clinical relevance were flagged for further examination. RESULTS: Association rule mining returned a total of 3355 associations related to wastage. Several notable associations were identified. For example, certain wards were associated with wastage due to thawing unused frozen products. Other examples included association between smaller blood banks and evening work shifts with product wastage due to excess time outside the laboratory or returning products with high temperatures. CONCLUSION: This paper demonstrates the effective use of unsupervised machine learning for the purpose of investigating wastage in a large blood bank. The use of association rule mining was able to identify wastage factors, which can help guide quality improvement initiatives. This technique can be automated to provide rapid analysis of complex associations contributing to wastage and could be utilized in modern blood banks.

A contemporary description of patients' estimated blood losses from diagnostic phlebotomy in a census of hospital episodes from a Canadian tertiary care center.

BACKGROUND: Phlebotomy for diagnostic testing is among the commonest hospital procedures, but hospital-wide surveys of all inpatients characterizing blood draw volumes have not been published. The objectives were to characterize the daily blood volumes drawn for diagnostic testing from patients discharged from a Canadian tertiary care center, describe the daily distributions of phlebotomy volumes across service locations, and describe changes in hemoglobin (Hb) and transfusion across service locations. STUDY DESIGN AND METHODS: Data were obtained on all patients discharged between 2012 and 2014 using linked discharge abstract and laboratory data. Cumulative daily blood volume and draw frequency were reported by service and days since admission. Changes in Hb and red blood cell (RBC) transfusion rates were reported for nontransfused and transfused patients. RESULTS: Data were included on 59,715 subjects. Mean daily estimated blood loss varied from 8.5 ± 6.5 mL/day onward to 27.2 ± 20.0 mL/day in the intensive care unit (ICU; p < 0.001). Phlebotomy volumes were highest on the first day of admission and declined thereafter (p < 0.001). For nontransfused individuals in the first week of admission, Hb levels decreased by the highest percentage in the ICU. The rate of RBC unit transfusion was highest in the ICU (232.4 units/1000 patient-days; 95% confidence interval, 225.8-239.2; p < 0.0001 compared with all other locations). CONCLUSION: Considerable variation was observed in estimated blood loss due to diagnostic phlebotomy across different services within one teaching hospital. Thi information is foundational for planning interventions to minimize estimated blood loss from phlebotomy.

The successful implementation of an automated institution-wide assessment of hemoglobin and ABO typing to dynamically estimate red blood cell inventory requirements.

BACKGROUND: Blood bank inventories must balance adequate supply with minimal outdate rates. The day-to-day practice of ordering red blood cell (RBC) inventory usually involves manually comparing current inventory levels with predetermined thresholds calculated from historical usage and ordering the difference. To date, there have been no published methods for ordering RBC inventory based on laboratory characteristics of admitted patients. STUDY DESIGN AND METHODS: We designed and implemented a blood ordering algorithm to provide a more accurate measure of predicted RBC utilization in our institution. Cerner Command Language (Cerner Millennium) was used to extract and combine historical RBC unit usage, current inventory levels, and system-wide hematology values and blood groups. This report contains a suggested order based on current inventory, historical inventory data, ABO group, and the current "anemia index" for the institution. RESULTS: The mean daily total RBC inventory was significantly reduced after implementation (401.7 units vs. 309.0 units, p < 0.05). There was a significant reduction in monthly RBC outdates in this period (19.1 vs. 8.1, p < 0.05). The age of RBCs at time of transfusion was reduced as well. CONCLUSION: We developed a novel algorithm that automatically generates a suggested RBC inventory order using real-time hospital-wide survey of patient ABO typing, hematology values, and historical data. After implementation of the algorithm we demonstrated a significant reduction in daily inventory levels and RBC outdate rates.

Process mining is an underutilized clinical research tool in transfusion medicine.

BACKGROUND: To understand inventory performance, transfusion services commonly use key performance indicators (KPIs) as summary descriptors of inventory efficiency that are graphed, trended, and used to benchmark institutions. STUDY DESIGN AND METHODS: Here, we summarize current limitations in KPI-based evaluation of blood bank inventory efficiency and propose process mining as an ideal methodology for application to inventory management research to improve inventory flows and performance. RESULTS: The transit of a blood product from inventory receipt to final disposition is complex and relates to many internal and external influences, and KPIs may be inadequate to fully understand the complexity of the blood supply chain and how units interact with its processes. Process mining lends itself well to analysis of blood bank inventories, and modern laboratory information systems can track nearly all of the complex processes that occur in the blood bank. CONCLUSION: Process mining is an analytical tool already used in other industries and can be applied to blood bank inventory management and research through laboratory information systems data using commercial applications. Although the current understanding of real blood bank inventories is value-centric through KPIs, it potentially can be understood from a process-centric lens using process mining.

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.

Process Mining Uncovers Actionable Patterns of Red Blood Cell Unit Wastage in a Health Care Network.

Packed red blood cell transfusions are integral to the care of the critically and chronically ill patient, but require careful storage and a large, coordinated network to ensure their integrity during distribution and administration. Auditing a Transfusion Medicine service can be challenging due to the complexity of this network. Process mining is an analytical technique that allows for the identification of high-efficiency pathways through a network, as well as areas of challenge for targeted innovation. Here, we detail a case study of an efficiency audit of the Transfusion Medicine service of the Nova Scotia Health Administration Central Zone using process mining, across a period encompassing years prior to, during, and after the acute COVID-19 pandemic. Service efficiency from a product wastage perspective was consistently demonstrated at benchmarks near globally published optima. Furthermore, we detail key areas of continued challenge in product wastage, and suggest potential strategies for further targeted optimization.

Blood Demand Forecasting and Supply Management: An Analytical Assessment of Key Studies Utilizing Novel Computational Techniques.

Use of data-driven methodologies in enhancing blood transfusion practices is rising, leveraging big data, machine learning, and optimization techniques to improve demand forecasting and supply chain management. This review used a narrative approach to identify, evaluate, and synthesize key studies that considered novel computational techniques for blood demand forecasting and inventory management through a search of PubMed and Web of Sciences databases for studies published from January 01, 2016, to March 30, 2023. The studies were analyzed for their utilization of various techniques, and their strengths, limitations, and areas for improvement. Seven key studies were identified. The studies focused on different blood components using various computational methods, such as regression, machine learning, hybrid models, and time series models, across different locations and time periods. Key variables used for demand forecasting were largely derived from electronic health record data, including clinical related predictors such as laboratory test results and hospital census by location. Each study offered unique strengths and valuable insights into the use of data-driven methods in blood bank management. Common limitations were unknown generalizability to other healthcare settings or blood components, need for field-specific performance measures, lack of ABO compatibility consideration, and ethical challenges in resource allocation. While data-driven research in blood demand forecasting and management has progressed, limitations persist and further exploration is needed. Understanding these innovative, interdisciplinary methods and their complexities can help refine inventory strategies and address healthcare challenges more effectively, leading to more robust, accurate models to enhance blood management across diverse healthcare scenarios.

Using Interactive Visual Analytics to Optimize Blood Products Inventory at a Blood Bank.

Blood products and their derivatives are perishable commodities that require an efficient inventory management to ensure both a low wastage rate and a high product availability rate. To optimize blood product inventory, Blood Transfusion Services (BTS) need to reduce wastage by avoiding outdates and improving availability of different blood products. We took a blood product lifecycle approach and used advanced visualization techniques to design and develop a highly interactive web-based dashboard to audit retrospective data and consequently, to identify and learn from procedural inefficiencies based on analysis of transactional data. We present pertinent scenarios to show how the blood transfusion staff can use the dashboard to investigate blood product lifecycles so as to probe transition sequence patterns that led to wastage as a means to discover causes of procedural inefficiencies in the BTS.

Using Interactive Visual Analytics to Optimize in Real-Time Blood Products Inventory at a Blood Bank.

Blood products and their derivatives are perishable commodities that require an efficient inventory management to ensure both a low wastage rate and a high product availability rate. To optimize blood product inventory, blood transfusion services need to reduce wastage by avoiding outdates and improve availability of different blood products. We used advance visualization techniques to design and develop a highly interactive real-time web-based dashboard to monitor the blood product inventory and the on-going blood unit transactions in near-real-time based on analysis of transactional data. Blood transfusion staff use the dashboard to locate units with specific characteristics, investigate the lifecycle of the units, and efficiently transfer units between facilities to minimize outdates.

Comprehensive survey of red blood cell unit life cycle at a large teaching institution in eastern Canada.

BACKGROUND: Recent blood shortages and the potential clinical impact of red blood cell (RBC) age highlight the need to understand blood supply delivery. This study addresses the characteristics and mechanics of RBC unit trafficking and storage across the transfusion service, previously undescribed in the literature. STUDY DESIGN AND METHODS: This retrospective qualitative institutionwide survey assessed the comprehensive RBC life cycle within Capital District Health Authority in Nova Scotia, Canada, during 2007. RESULTS: A total of 15,930 unique RBC units were received from the supplier with 98.6% having mean age of 12.5 days. The mean ages on receipt, transfusion, and time spent in the transfusion system before transfusion were 12, 19.6, and 7.5 days, respectively. Of 12,298 units, 9689 (78.8%) remained within the blood transfusion services (BTSs), while 2609 (21.2%) migrated having been returned a mean of 1.26 times from locations outside BTS (SD, 0.56 times), the latter spending a mean of 18.4 hours outside the BTS. Stationary units had mean age at dispense and time spent in the transfusion system of 19.2 and 6.9 days, respectively, compared to migratory units at 21.3 and 9.9 days, respectively (p < 0.005). A total of 5.2% (636/12258) of units were discarded; 423 of 636 (66.5%) were due to "unit expiry." CONCLUSION: Quantification of our institutional RBC life cycle highlighted key areas for intervention, such as the reduction of unit migration, especially D- units, as this increased RBC age at transfusion. In the era of limited resources, this method could identify areas where effort should be directed to address unnecessary wastage and aging of RBC units.

Novel web-based real-time dashboard to optimize recycling and use of red cell units at a large multi-site transfusion service.

BACKGROUND: Effective blood inventory management reduces outdates of blood products. Multiple strategies have been employed to reduce the rate of red blood cell (RBC) unit outdate. We designed an automated real-time web-based dashboard interfaced with our laboratory information system to effectively recycle red cell units. The objective of our approach is to decrease RBC outdate rates within our transfusion service. METHODS: The dashboard was deployed in August 2011 and is accessed by a shortcut that was placed on the desktops of all blood transfusion services computers in the Capital District Health Authority region. It was designed to refresh automatically every 10 min. The dashboard provides all vital information on RBC units, and implemented a color coding scheme to indicate an RBC unit's proximity to expiration. RESULTS: The overall RBC unit outdate rate in the 7 months period following implementation of the dashboard (September 2011-March 2012) was 1.24% (123 units outdated/9763 units received), compared to similar periods in 2010-2011 and 2009-2010: 2.03% (188/9395) and 2.81% (261/9220), respectively. The odds ratio of a RBC unit outdate postdashboard (2011-2012) compared with 2010-2011 was 0.625 (95% confidence interval: 0.497-0.786; P < 0.0001). CONCLUSION: Our dashboard system is an inexpensive and novel blood inventory management system which was associated with a significant reduction in RBC unit outdate rates at our institution over a period of 7 months. This system, or components of it, could be a useful addition to existing RBC management systems at other institutions.

Predictive significance of absolute lymphocyte count and morphology in adults with a new onset peripheral blood lymphocytosis.

AIMS: Lymphocytosis is commonly encountered in the haematology laboratory. Evaluation of blood films is an important screening tool for differentiating between reactive and malignant processes. The optimal lymphocyte number to trigger morphological evaluation of the smear has not been well defined in the literature. Likewise, the significance of lymphocyte morphology has not been well studied and there are no consensus guidelines or follow-up recommendations available. We attempt to evaluate the significance of lymphocyte morphology and to define the best possible cut-off value of absolute lymphocyte count for morphology review. METHODS: 71 adult patients with newly detected lymphocytosis of 5.0×10(9)/L or more were categorised to either a reactive process or a lymphoproliferative disorder. We performed statistical analysis and morphology review to compare the difference in age, gender, lymphocyte count and morphological features between the two groups. Receiver operating characteristic analysis was performed to determine an optimal lymphocyte number to trigger morphology review. RESULTS: Lymphoproliferative disorders are associated with advanced age and higher lymphocyte count. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy of lymphocyte morphology as a screening test were 0.9, 0.59, 0.60, 0.58 and 0.71, respectively. The optimal cut-off of lymphocyte number for morphology review was found to be close to 7×10(9)/L. CONCLUSIONS: We found a moderate interobserver agreement for the morphological assessment. 'Reactive' morphology was very predictive of a reactive process, but 'malignant' morphology was a poor predictor of a lymphoproliferative disorder.

Temporal approach to hematological test usage in a major teaching hospital.

In the era of slowly increasing laboratory budgets, identifying usage patterns can be an important first step in decreasing unnecessary laboratory testing. From January 1, 1998, through December 27, 2002, we studied inpatient laboratory utilization at the University of Alberta Hospital, a large Canadian teaching hospital (665 inpatient beds with an almost 100% continuous occupancy rate). The daily numbers of complete blood cell count (CBC), CBC with differential (CBCD), international normalized ratio, activated partial thromboplastin time, vitamin B12, and red cell folate (FOL) tests were correlated with regularly occurring hospital events, such as weekends, statutory holidays, and resident changeover dates. Testing for hematological monitoring (CBC, CBCD) at our institution has been increasing at an average of 2% to 6% per year with a recent (2001 onward) rate of increase in hematological diagnostic testing (vitamin B12 and FOL) of close to 10%. As expected, laboratory usage was statistically significantly higher on weekdays compared with weekends. During the weekdays, ordering frequencies on Monday and Friday were shown to be statistically significantly higher than on the other days. A nadir occurred on Thursday and on the weekend. This finding was postulated to be due to a combination of attending physician unfamiliarity and defensive testing. Resident changeovers did not contribute to an increase in Monday laboratory testing, as was initially thought, nor did holidays. We believe that we can use our findings to decrease laboratory use. First, weekend or midweek use might become the appropriate standard for frequency of testing. Second, we must alert attending physicians (and residents who are soon to be attending physicians) about their subliminal need to increase testing during changeovers.

Trp-999 of beta-galactosidase (Escherichia coli) is a key residue for binding, catalysis, and synthesis of allolactose, the natural lac operon inducer.

Trp-999 is a key residue for the action of beta-galactosidases (Escherichia coli). Several site specific substitutions (Phe, Gly, Tyr, Leu) for Trp-999 were made. Each substitution caused greatly decreased affinities for substrates and inhibitors that bind in the "shallow" mode, while the affinities of inhibitors that bind in the "deep" mode were not decreased nearly as much. This shows that Trp-999 is important for binding in the shallow mode. The residue is also very important for binding glucose to galactosyl-beta-galactosidase (as a transgalactosidic acceptor). Substitution greatly diminished the affinity for glucose. Substitutions also changed the activation thermodynamics and, subsequently, the rates of the catalytic reactions. The enthalpies of activation of the glycolytic bond cleavage step (galactosylation, k(2)) became less favorable while the entropies of activation of that step became more favorable as a result of the substitutions. Differing magnitudes of these enthalpic and entropic effects with ONPG as compared to PNPG caused the k(2) values for ONPG to decrease but to increase for PNPG. The enthalpies of activation for the common hydrolytic step (degalactosylation, k(3)) increased while the entropies of activation for this step did not change much. As a result, k(3) became small and rate determining for each substituted enzyme. The substitutions caused the rate constant (k(4)) of the transgalactosidic acceptor reactions with glucose (for the formation of allolactose) to become much larger and of the same order of magnitude as the normally large rate constants for transgalactosidic acceptor reactions with small alcohols. This is probably because glucose can approach with less restriction in the absence of Trp-999. However, since glucose binds very poorly to the galactosyl-beta-galactosidases with substitutions for Trp-999, the proportion of lactose molecules converted to allolactose is small. Thus, Trp-999 is also important for ensuring that an appropriate proportion of lactose is converted to allolactose.

Complete blood count reference interval diagrams derived from NHANES III: stratification by age, sex, and race.

BACKGROUND: Comprehensive, up-to-date "health-associated" reference interval studies of North American populations are uncommon. The third US National Health and Nutrition Examination Survey (NHANES III) was concluded in 1994 and yielded important reference interval data. OBJECTIVE: To obtain health-associated Coulter counter reference interval data from NHANES III according to age, sex, and race. METHODS: Of the 29,314 civilian noninstitutionalized US citizens who participated in NHANES III, approximately 25,000 had a complete blood count, red cell distribution width (RDW), platelet count, and automated white blood cell (WBC) differential determined on a Coulter S-Plus Jr. To determine health-associated reference intervals, we used the following exclusion criteria: pregnancy, breast feeding, obesity (body mass index [BMI] >40 and >35 for females and males, respectively), diastolic blood pressure >100 mm Hg, any smoking, any drinking of alcohol, recent treatment for anemia, creatinine level >2.5 mg/dL, glucose level >126 mg/dL, excessive thinness (BMI <8), recent surgery or hospitalization, or having antibodies to hepatitis viruses A, B, or C. The Coulter counter data (hemoglobin, hematocrit, red blood cell count, mean corpuscular volume (MCV), mean cell hemoglobin concentration (MCHC), MCH, WBC count, platelet count, granulocyte count, monocyte count, lymphocyte count, RDW, platelet distribution width, and mean platelet volume) were separated into 6 sex/racial categories (female non-Hispanic white, female non-Hispanic black, female Mexican American, male non-Hispanic white, male non-Hispanic black, and male Mexican American) and 9 age groupings (10-14, 14-18, 18-25, 25-35, 35-45, 45-55, 55-65, 65-75, and >75 years). RESULTS: There was a high exclusion rate; for example, of the 20,685 individuals with measured hemoglobin levels, 12,688 (61.3%) were excluded. Percentile estimates could be derived accurately for almost all of the female age/sex categories. A few of the male Mexican American and non-Hispanic black categories contained observations for ages 45 to 75 years. CONCLUSIONS: There are age-dependent trends for many of the tests, notably in RDW, MCMV, platelet count, and granulocyte and lymphocyte percentages. Sex-dependent changes involved hemoglobin values, and race-related trends centered around mononuclear and lymphocyte percentages, hematocrit, MCHC, MCH, and hemoglobin. This study reveals the potential for using data mining of large samples to yield potentially useful reference ranges.