Red Cell In Vivo Recovery and Survival Studies Richard J. Davey, MD The Methodist Hospital, Houston TX Blood Products Advisory Committee May 1, 2008

Evolution of Labeling and Evaluation Criteria 1947: Ross proposes 70% red cell survival at 24 hours as a minimum recovery 1947: Ross proposes 70% red cell survival at 24 hours as a minimum recovery 1950: Gray and Sterling describe 51 Cr as a red cell label 1950: Gray and Sterling describe 51 Cr as a red cell label 1967: Fisher describes 99m Tc as a red cell label 1967: Fisher describes 99m Tc as a red cell label 1985: Minimum survival raised to 75% following FDA workshop 1985: Minimum survival raised to 75% following FDA workshop 1989: AuBuchon refines use of 111 In as a red cell label 1989: AuBuchon refines use of 111 In as a red cell label 1998: Minimum survival ≥ 75%: SD ≤ 9% based on historical data 1998: Minimum survival ≥ 75%: SD ≤ 9% based on historical data 2004: Current criteria based on historical data, BPAC 2004: Current criteria based on historical data, BPAC

Characteristics of an Ideal Red Cell Radiolabel Minimal preparative manipulation of red cells Minimal preparative manipulation of red cells Specific for red cells Specific for red cells Nontoxic to the cell Nontoxic to the cell Nontoxic to the recipient Nontoxic to the recipient No metabolism by red cells No metabolism by red cells No elution from the cell No elution from the cell Radioactive half-life appropriate for the duration of the study Radioactive half-life appropriate for the duration of the study No relabeling of other cells No relabeling of other cells

Characteristics of Commonly Used Red Cell Radiolabels Characteristic 51 Cr 99m Tc 111 In Half-life27.7 days6.0 hours2.8 days Elution1% day4% hour4% day Major γ-photon320 keV140 keV 173,247 keV emissions emissions Suitable for imaging NoYesYes

Technical Steps for Labeling Red Cells with 51 Cr A suitable sample of red cells stored under experimental condition is chosen. For autologous studies, A suitable sample of red cells stored under experimental condition is chosen. For autologous studies, ~ 15ml is used. ~ 15ml is used. An amount of 51 Cr is used to insure minimum adequate counts throughout the study (~ 4000 counts/sample: 5 to 20 μCi) An amount of 51 Cr is used to insure minimum adequate counts throughout the study (~ 4000 counts/sample: 5 to 20 μCi) After incubation with test red cells, unbound 51 Cr is removed After incubation with test red cells, unbound 51 Cr is removed An appropriate standard is prepared for blood volume determination An appropriate standard is prepared for blood volume determination Labeled red cells are infused into a peripheral vein and exact volume of infused material is determined Labeled red cells are infused into a peripheral vein and exact volume of infused material is determined

Technical Steps for Labeling Red Cells with 51 Cr Samples are drawn from opposite arm. Several early samples are drawn for 100% survival determination. Samples are drawn from opposite arm. Several early samples are drawn for 100% survival determination. Samples counted in duplicate and corrected for elution. Samples counted in duplicate and corrected for elution.

Early Loss of Labeled, Stored Red Cells After Infusion

Zero-Time Determination by Direct Red Cell Mass Measurement ( 99m Tc) and by Back-Extrapolation ( 51 Cr)

Partial List of Red Cell Products Evaluated Using Red Cell Recovery Studies New plastics and plasticizers New plastics and plasticizers New anticoagulant/preservatives New anticoagulant/preservatives Filtration devices and leukoreduced products Filtration devices and leukoreduced products Irradiated red cells Irradiated red cells “Undercollected” red cell units “Undercollected” red cell units Pathogen inactivation procedures Pathogen inactivation procedures Enzymatic removal of A and B antigens Enzymatic removal of A and B antigens

Effect of Prestorage Irradiation on 24-hour Posttransfusion Red Cell Recovery Storage Interval 21 Days 1 28days 1 42 Days 2 Storage Interval 21 Days 1 28days 1 42 Days 2 Radiation dose 20 Gy 20 Gy 30 Gy Radiation dose 20 Gy 20 Gy 30 Gy Control Irrad. Cont. Irrad Cont. Irrad. Control Irrad. Cont. Irrad Cont. Irrad. Potassium (mEq/L) ATP (μM/g Hb) RBC 24 hr recovery (%) Friedman et al. Transfusion 1991;31:50S 2. Davey et al. Transfusion 1992:32:525-8

Evolution of Labeling and Evaluation Criteria 1947: Ross proposes 70% red cell survival at 24 hours as a minimum recovery 1947: Ross proposes 70% red cell survival at 24 hours as a minimum recovery 1950: Gray and Sterling describe 51 Cr as a red cell label 1950: Gray and Sterling describe 51 Cr as a red cell label 1967: Fisher describes 99m Tc as a red cell label 1967: Fisher describes 99m Tc as a red cell label 1985: Minimum survival raised to 75% following FDA workshop 1985: Minimum survival raised to 75% following FDA workshop 1989: AuBuchon refines use of 111 In as a red cell label 1989: AuBuchon refines use of 111 In as a red cell label 1998: Minimum survival ≥ 75%: SD ≤ 9% based on historical data 1998: Minimum survival ≥ 75%: SD ≤ 9% based on historical data 2004: Current criteria based on historical data, BPAC 2004: Current criteria based on historical data, BPAC

FDA Requirements for Approval of Red Blood Cell Products Total 20 or more evaluable 24-hour recoveries Total 20 or more evaluable 24-hour recoveries Minimum two laboratories Minimum two laboratories Sample mean ≥ 75% Sample mean ≥ 75% Sample standard deviation ≤ 9% Sample standard deviation ≤ 9% One-sided 95% lower confidence limit for the proportion of successes must be >70% with success threshold for individual recovery ≥ 75%. One-sided 95% lower confidence limit for the proportion of successes must be >70% with success threshold for individual recovery ≥ 75%. A successful trial can have no more than 3 of 24 RBC recoveries below 75% A successful trial can have no more than 3 of 24 RBC recoveries below 75% Hemolysis < 1% at end of storage Hemolysis < 1% at end of storage Comparison, control vs. test: ATP, 2,3 DPG, pH, glucose, lactate Comparison, control vs. test: ATP, 2,3 DPG, pH, glucose, lactate

The Problem With “Old” Blood

Mechanisms of Morbidity and Mortality: The Red Cell Storage Lesion Storage Lesion Decreased 2,3 DPG Decreased 2,3 DPG Increased rigidity Increased rigidity High PA I level Poor O High PA I level Poor O 2 transport High CD 40 Inflammation High CD 40 Inflammation NO depletion Thrombosis NO depletion Thrombosis Increase in IL-10 Increase in IL-10 Decrease in TNF-α Decrease in TNF-α

Depletion of 2,3 DPG

Loss of RBC Deformability

Storage Depletes RBC Nitric Oxide Bioactivity

Outcomes: Liberal vs. Restrictive Transfusions Outcome Measure Restrictive (n=418) Liberal (n=420) p value (Hb: ) (Hb: ) Death (# / %) 30 day 78 (18.7)98 (23.3) day 78 (18.7)98 (23.3)0.11 ICU 56 (13.4)68 (16.2) 0.29 ICU 56 (13.4)68 (16.2) 0.29 Hospital 93 (22.2) 118 (28.1) 0.05 Hospital 93 (22.2) 118 (28.1) 0.05 Length of stay – days ICU 11.0 ± ± ICU 11.0 ± ± Hospital 34.8 ± ± Hospital 34.8 ± ± Hebert PC, et al. New Engl J Med 1999;340:409.

Factors Associated With Postoperative Morbidity Post-CABG Variable Odds Ratio Upper CL Lower CL p value Variable Odds Ratio Upper CL Lower CL p valueTransfusion PRBC, units <.0001 PRBC, units <.0001Preoperative Higher BMI <.0001 Higher BMI <.0001 Abnormal LVEF <.0001 Abnormal LVEF <.0001 Peripheral vasc. disease Peripheral vasc. disease Previous stroke Previous stroke “Perioperative RBC transfusion is the single factor most reliably associated with postoperative morbid events” Koch GC et al. Crit Care Med 2006;34;1608

Hypothesis Hypothesis Serious complications and mortality after cardiac surgery may increase when red cell units are transfused after they have been stored for more than 2 weeks Serious complications and mortality after cardiac surgery may increase when red cell units are transfused after they have been stored for more than 2 weeks Patients underwent coronary-artery bypass grafting, cardiac-valve surgery, or a combination of the two procedures Patients underwent coronary-artery bypass grafting, cardiac-valve surgery, or a combination of the two procedures 6002 patients studied from June 30, 1998 to January 30, patients studied from June 30, 1998 to January 30, 2006 All serious morbid events evaluated All serious morbid events evaluated When blood was ordered for transfusion, the blood bank provided the oldest available matching unit for each request (FIFO) When blood was ordered for transfusion, the blood bank provided the oldest available matching unit for each request (FIFO) Median time of storage was 15 days Median time of storage was 15 days

Conclusions Patients given older blood: Patients given older blood: Greater in-hospital mortality (2.8% vs. 1.7%, P < 0.004) Greater in-hospital mortality (2.8% vs. 1.7%, P < 0.004) More likely to need prolonged ventilatory support (9.7% vs. 5.6%, P<0.001) More likely to need prolonged ventilatory support (9.7% vs. 5.6%, P<0.001) More likely to have renal failure (2.7% vs. 1.6%, P < 0.003) More likely to have renal failure (2.7% vs. 1.6%, P < 0.003) Increased sepsis (4.0% vs. 2.8%, P < 0.01) Increased sepsis (4.0% vs. 2.8%, P < 0.01) Increased multisystem organ failure (0.7% vs. 0.2%, P < 0.007) Increased multisystem organ failure (0.7% vs. 0.2%, P < 0.007)

Red Cell Recoveries in Clinical Trials: Where Should We Set the Bar? Technical labeling procedures are well established and performed by skilled laboratories Technical labeling procedures are well established and performed by skilled laboratories Prudent to be cautious in liberalizing threshold value Prudent to be cautious in liberalizing threshold value Do not want to set bar so high as to lead to unnecessary trial failures. (67.3% historical success rate for liquid 75% threshold value) Do not want to set bar so high as to lead to unnecessary trial failures. (67.3% historical success rate for liquid 75% threshold value) Do not want to set the bar so low as to have almost 100% success. (99.9% historical success for liquid 67% threshold) Do not want to set the bar so low as to have almost 100% success. (99.9% historical success for liquid 67% threshold) Alternatives: Alternatives: 70% threshold value = 95.5% historical success (liquid storage) 70% threshold value = 95.5% historical success (liquid storage) % threshold value? % threshold value? Davey RJ. Editorial. Transfusion. In press.