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Anemia is common in critically ill patients and may be exacerbated through phlebotomy-associated iatrogenic blood loss. Differences in phlebotomy practice across patient demographic characteristics, clinical features, and practice environments are unclear. This investigation provides a comprehensive description of contemporary phlebotomy practices for critically ill adults.This is an observational cohort study of adults ≥18 years of age requiring intensive care unit (ICU) admission between January 1, 2019, and December 31, 2019, at a large academic medical center. Descriptive statistics were utilized to summarize all phlebotomy episodes throughout hospitalization, with each phlebotomy episode defined by unique peripheral venous, central venous, or arterial accesses for laboratory draws, exclusive of finger sticks. Secondarily, financial costs of phlebotomy and the relationships between phlebotomy practices, hemoglobin concentrations, and red blood cell (RBC) transfusions were evaluated.A total of 6194 patients were included: 59% were men with a median (interquartile range) age of 66 (54-76) years and median ICU and hospital durations of 2.1 (1.4-3.9) and 7.1 (4.3-11.8) days, respectively. The median number of unique laboratory draws was 41 (18-88) throughout hospitalization, with a median volume of 232 (121-442) mL, corresponding to 5.2 (2.6-8.8) draws and 29 (19-43) mL per day. Waste (ie, discard) volume was responsible for 10.8% of total phlebotomy volume. Surgical patients had a higher number of phlebotomy episodes and greater total phlebotomy volumes compared to nonsurgical patients. Phlebotomy practices differed across ICU types, with the greatest frequency of laboratory draws in the cardiac surgical ICU and the greatest daily phlebotomy volume in the medical ICU. Across hospitalization, ICU environments had the greatest frequency and volumes of laboratory draws, with the least intensive phlebotomy practice observed in the general hospital wards. Patients in the highest quartile of cumulative blood drawn experienced the longest hospitalizations, lowest nadir hemoglobin concentrations, and greatest RBC transfusion utilization. Differences in phlebotomy practice were limited across patient age, gender, and race. Hemoglobin concentrations declined during hospitalization, congruent with intensity of phlebotomy practice. Each 100 mL of phlebotomy volume during hospitalization was associated with a 1.15 (95% confidence interval [CI], 1.14-1.17; P.001) multiplicative increase in RBC units transfused in adjusted analyses. Estimated annual phlebotomy costs exceeded $15 million (approximately $2500 per patient admission).Phlebotomy continues to be a major source of blood loss in hospitalized patients with critical illness, and more intensive phlebotomy practices are associated with lower hemoglobin concentrations and greater transfusion utilization.