1. Predicting Iron Deficiency in Blood Donors  REDS-II Donor Iron Status Evaluation (RISE)  Ritchard G. Cable*, Simone A. Glynn, Joseph E. Kiss, Alan E. Mast, Whitney R. Steele, Edward L. Murphy, David J. Wright, Ronald A. Sacher, Jerry L. Gottschall, Vibha Vij, Toby L. Simon  *American Red Cross Blood Services, Northeast Division  for the National Heart, Lung, and Blood Institute Retrovirus Epidemiology Donor Study-II (REDS-II)

2. REDS-II and RISE The Retrovirus Epidemiology Donor Study-II (REDS-II) is a multi- center research program funded by the National Heart, Lung and Blood Institute to conduct studies on the safety and adequacy of the blood supply.  REDS-II Participating U.S. Blood Centers  Blood Center of Wisconsin  ARC New England Region  Emory University/ARC Southeast Region  University Cincinnati/Hoxworth BC  Institute For Transfusion Medicine  UCSF/Blood Centers of the Pacific/Blood Systems Research Institute (BSRI)  Coordinating Center – Westat  Central Laboratory – BSRI The REDS-II Donor Iron Status Evaluation (RISE) is a longitudinal study involving the six REDS-II centers which is designed to evaluate the effects of blood donation intensity on iron and hemoglobin status. 2

3. RISE Study Design Two cohorts of blood donors were recruited and enrolled in the RISE study –A first time and reactivated donor cohort, with no whole blood/red cell donations in the previous two years –A frequent donor cohort, with ≥2 (F) or ≥ 3 (M) donations in the past year Enrolled donors agreed to donate frequently for the 15-24 month study period. Iron status and related variables were evaluated at baseline and at the end of the study. In addition donation outcomes were recorded for all visits, and additional iron measures were performed on interim visits of: - First time/Reactivated donors - Donors with hemoglobin deferrals - Selected female repeat donors 3

4. RISE Baseline Enrollment Data Collection Hematologic and iron status markers: –Fingerstick hemoglobin or hematocrit –HemoCue venous hemoglobin –Plasma ferritin, sTfR (soluble transferrin receptor) Iron protein polymorphisms: –Transferrin polymorphism G277S –HFE (hemochromatosis) polymorphisms C282Y and H63D Donor Questionnaire Data: –Donation History: Lifetime; Past 1,2 years –Smoking: Lifetime, recent –Dietary Iron Consumption –Use of multivitamin/multiminerals and iron supplements –Aspirin use –For women only: Menstrual status/nature of periods; Detailed pregnancy history 4

5. RISE Enrollment CohortsStudy Targets Total Enrolled First-time/ Reactivated (no red cell donations past 2 years) Male420407 Female420481 Total840888 FrequentMale750769 Female750768 Total15001537 Total23402425 5

6. RISE Enrollment Measures * Post donation Hgb samples adjusted to pre values using conversion formula: Pre vHb(g/dL) = Post vHb + 0.8423 -(0.002035 x Weight (lbs) Medians (2.5%-97.5% Range) Cohort (n) Hemoglobin* g/dL Ferritin ng/mL sTfR mg/L Log (sTfR/Fer) FemaleFT/RA (481) 13.3 (11.5-15.2) 37 (9-175) 2.7 (1.7-4.8) 1.8 (1.1-2.6) Fqnt (769) 13.2 (11.4-15.2) 19 (5-68) 3.1 (1.8-6.6) 2.2 (1.5-3.0) MaleFT/RA (407) 15.1 (12.8-17.4) 108 (29-430) 2.7 (1.6-4.4) 1.4 (0.8-2.1) Fqnt (768) 14.5 (12.0-16.6) 25 (6-117) 3.1 (1.8-8.0) 2.1 (1.3-3.1)

7. Absent Iron Stores (AIS) and Iron Deficient Erythropoiesis (IDE) Absent iron stores (AIS) is defined as plasma ferritin < 12 ng/mL -This is a relatively specific finding, correlating in other studies with absent bone marrow iron stores Iron deficient erythropoiesis (IDE) is defined as log(sTfR/ferritin) above the 97.5 th percentile for first time/reactivated male donors (negligible risk of IDE). -This has been shown to best correlate with other measures of IDE -For the RISE data set this correlates with log(sTfR/ferritin) ≥ 2.07 7

8. Donors with Absent Iron Stores (AIS) and/or Iron Deficient Erythropoiesis (IDE) at Enrollment GenderDonor Status AIS % ferritin <12 µg/L IDE % Log (sTfR/F) ≥ 2.07 FemalesFT/RA 6.524.6 Fqnt 27.066.1 MalesFT/RA 02.5 Fqnt 16.448.7 8

9. Effect of previous 12 month RBC donation frequency on plasma ferritin at enrollment 9

10. Effect of previous 12 month RBC donation frequency on donor hemoglobin at enrollment

11. Adjusted Odds Ratios for AIS and IDE at Enrollment by 2 Year Red Cell Donation Frequency Number of RBC units in 24 months prior to enrollment AIS Ferritin<12 Adjusted ORs (95% CI) IDE Log (R/F) ≥ 2.07 Adjusted ORs (95% CI) p value <0.001 FT: 0 donations 1.0 RA: 0 Donations 0.5 (0.2-1.2)1.5 (0.9-2.4) R: ≤ 4 donations 5.3 (2.8-10.1)14.0 (8.6-22.7) R: 5-6 donations 12.5 (6.4-24.6)24.0 (14.3-40.5) R: 7-9 donations 13.5 (6.8-26.6)32.3 (19.2-54.5) R: 10+ donations 18.9 (9.0-39.6)50.5 (28.4-89.9) 11

12. Adjusted Odds Ratios for AIS and IDE at Enrollment by Age Age (in yrs) AIS Ferritin<12 Adjusted* ORs (95% CI) IDE Log (R/F) ≥ 2.07 Adjusted* ORs (95% CI) MaleFemaleMaleFemale p value0.31<0.00010.01<0.0001 <200.4 (0.0-3.3)3.1 (1.0-9.6)1.1 (0.7-5.9)4.9 (1.9-12.5) 20-291.8 (0.8-4.2)3.9 (2.0-7.6)2.3 (1.2-4.3)3.1 (1.8-5.4) 30-391.5 (0.7-3.2)1.6 (0.9-3.0)1.7 (1.0-2.9)1.7 (1.0-2.8) 40-491.0 50-591.6 (0.9-2.9)1.0 (0.6-1.7)1.4 (0.9-2.2)0.8 (0.5-1.2) 60+0.9 (0.5-1.9)0.7 (0.3-1.3)1.2(0.8-2.0)0.8 (0.5-1.4) 12

13. Adjusted Odds Ratios for AIS and IDE at Enrollment by Gender and Weight AIS Ferritin<12 Adjusted* ORs (95% CI) IDE Log (R/F) ≥ 2.07 Adjusted* ORs (95% CI) Gender p value <0.003<0.0001 Male 1.0 Female 1.8 (1.2-2.6)2.8 (2.1-3.7) Weight (in lbs) p value 0.00010.04 <150 1.3 (0.9-1.9)1.1 (0.8-1.5) 150-174 1.0 175-199 0.9 (0.6-1.3)0.9 (0.7-1.2) 200+ 0.5 (0.3-0.8)0.7 (0.5-0.9) 13

14. Other Significant Enrollment Variables in the Adjusted Models for AIS and IDE Smoking: Past and current smokers have lower odds for IDE than do donors who never smoked. The same is not found for AIS. This result may be related to changes in sTfR measurement in smokers, rather than to iron status. Self-prescribed iron supplement: Donors taking iron supplements (with or without multivitamins) are slightly less likely to have AIS, OR = 0.7 (and a similar NS tendency for IDE). HFE genotype: Donors homozygous or mixed heterozygous for C282Y and/or H63D are less likely to have IDE (and a similar NS tendency for AIS). No impact of heterozygosity for either gene. Pre-menopausal or ever pregnant status: Women with either status are more likely to have IDE (and a similar NS tendency for AIS). Center: At one center the odds of AIS or IDE were ~2 times the odds of reference center 14

15. Preliminary Analysis of RISE Longitudinal Data The RISE study data collection phase ended in January 2010. Analysis of the enrollment data is complete and In Press. Analysis of the longitudinal data is ongoing, and the following is a preliminary analysis of the data, prepared for this meeting. 15

16. RISE Longitudinal – Frequency of Return by Cohort FT/RA Females FT/RA Males Frequent Females Frequent Males Enrolled at Baseline 481407769768 Donors w/ no follow-up visits 102 (21.2%) 119 (29.2%) 30 (3.9%) 19 (2.5%) Donors w/ one or more follow- up visits 379 (78.8%) 288 (70.8%) 739 (96.1%) 749 (97.5%) Average return visits* 3.33.15.45.5 Average time in study* 429 days440 days516 days524 days 16 * For donors with one or more return visits

17. RISE Longitudinal – Visits/Data points of RISE donors 12,695 Visits ( 11,381 donations – 10% were double RBC) were made by RISE donors during the course of the study –2,425 Enrollment Visits (no deferrals) –8,936 Interim Visits –1,334 Final Visits 1,213 Deferrals –945 Hemoglobin Deferrals (84% in female, 16% in male) –268 Other Deferrals 101 were Sample Only Final Visits

18. RISE Longitudinal Testing Data All 2425 enrollment visits have venous hemoglobin and iron measures. Of 8936 interim visits, 6692 have venous hemoglobin and 2714 iron measures. Of 1334 final visits, 1320 have venous hemoglobin and 1310 have iron measures. Of the 945 hemoglobin deferrals, 482 have venous hemoglobin and 463 iron measures. 18

19. RISE Longitudinal – Development of model to predict hemoglobin deferral A model was developed to predict hemoglobin deferral at any return visit during RISE using only the kinds of information that would routinely be known to the blood center. The model included: gender, age, race, weight, donations in last 24 months, days since last RBC donation, last donation type (1-2RBCs), blood center. A repeated measures logistic regression model was developed. The model accounts for multiple measurements on the same donor. 19

20. Odds Ratio95% CI Black vs. White1.91.2-3.2 Hispanic vs. White1.60.9-2.7 Asian vs. White1.20.7-2.2 Other vs. White1.70.8-3.4 Female, 40-49, 150-174lbs vs. Male, 40-49, 150-174lbs 7.53.6-15.8 AgeMaleFemale <30 vs. 40-490.70.2-2.31.51.1-2.1 30-39 vs. 40-490.90.3-2.71.10.8-1.5 50-59 vs. 40-491.80.9-3.70.90.7-1.1 60+ vs. 40-491.70.8-3.60.70.5-0.9 Preliminary Model Predicting Hgb/Hct Deferral - Demographics 20

21. Odds Ratio95% CI Days since last donation 8-<12 vs. 32+ weeks2.31.6-3.2 12-<16 vs. 32+ weeks2.01.4-3.0 16-<20 vs. 32+ weeks1.51.0-2.3 20-<24 vs. 32+ weeks1.30.8-2.1 24-<32 vs. 32+ weeks0.90.6-1.4 Preliminary Model Predicting Hgb/Hct Deferral – Donation Characteristics 21

22. Donation Interval and Hemoglobin Deferral Rate 22

23. Summary of Hgb/Hct Deferral Model Of 9901 return donation visits during RISE, 9.5% were hemoglobin deferrals. This model predicted the odds of Hgb/Hct deferral at any return visit following the enrollment visit (only accepted donors were enrolled). The following variables were significant: Days since last donation, Race/ethnicity, Gender, Age in women, Blood Center. The following variables were not significant : Weight, Number of RBC units given in the preceding 24 months, Previous donation a WB or double red cell.

24. Conclusions from Enrollment Data Frequent whole blood and red cell donors have a high prevalence of iron deficiency. Ferritin levels decrease with increasing donation frequency. This decrease is more marked in men. Donation intensity, gender, weight, and age are important independent predictors of AIS/IDE. Reducing the allowable frequency of blood donation is likely to reduce the prevalence of iron deficiency among frequent blood donors, as might implementing routine iron supplementation. 24

25. Preliminary Conclusions from Longitudinal Data The most significant predictors of hemoglobin deferral appear to be: –Fewer Days since last RBC donation ▪But not number of RBC donations in the last two years –Female Gender –Black Race –Younger Age in women 25

26. Planned RISE Analyses related to prediction of Hgb deferral and iron stores Development of additional models to examine predictors of hemoglobin deferral and measures of iron stores Including: - Models predicting iron stores which focus on the information that would be routinely known to the center at the time of donor presentation (gender, age, etc.) - Expanded models for hemoglobin deferral and iron stores including the above variables plus information on use of iron supplements, smoking, menstrual and pregnancy status, HFE genotype and iron status at enrollment 26

27. Planned RISE Analyses Related to Use of Laboratory Measures to predict iron stores Identification of optimal laboratory measures that predict the development of iron depletion and hemoglobin deferral at subsequent visits –Development of models to assess the utility of ferritin, sTfR, hemoglobin and selected red cell and reticulocyte indices (such as CHr) to predict iron depletion and hemoglobin deferral at subsequent visits –Examination of within donor and between donor correlation among iron stores and selected laboratory measures, for example the association of CHr and ferritin 27

28. Analysis Needed in the Future - Evaluating the impact of various donation guidelines Utilize information from the analyses just described to project the impact of various blood donation guidelines on the iron status of blood donors, potential for hemoglobin deferral, and the adequacy of the blood supply