1. New Results from a Multi- Center, Prospective Study of Blood Donor Iron Status REDS-II Donor Iron Status Evaluation (RISE) Ritchard G. Cable for the National Heart, Lung, and Blood Institute Retrovirus Epidemiology Donor Study-II (REDS-II)

2. 2 Objectives of Today’s Presentation Briefly review iron depletion in whole blood donors Review RISE Study design and Objectives Present current data from and future analysis plans for the RISE study Assess fingerstick measurement as a proxy for venous hemoglobin Review data from July 2010 BPAC meeting on proposed changes in donor hemoglobin/frequency Discuss implications for blood center practice

3. 3 REDS-II Blood Centers American Red Cross Blood Services, New England Region R. Cable, J. Rios and R. Benjamin American Red Cross Blood Services, Southern Region/ Department of Pathology and Laboratory Medicine, Emory University School of Medicine J.D. Roback Hoxworth Blood Center, University of Cincinnati Academic Health Center R.A. Sacher, S.L. Wilkinson and P.M. Carey Blood Centers of the Pacific, University of California San Francisco, Blood Systems Research Institute E.L. Murphy, B. Custer and N. Hirschler The Institute for Transfusion Medicine D. Triulzi, R. Kakaiya and J. Kiss Blood Center of Wisconsin J.L. Gottschall and A.E. Mast Coordinating Center: Westat, Inc J. Schulman and M. King National Heart, Lung, and Blood Institute, NIH S.A. Glynn, T. Mondoro, and E. Wagner Central Laboratory: Blood Systems Research Institute M.P. Busch and P. Norris Acknowledgements 3

4. 4 Many studies have shown iron depletion (low ferritin) in blood donors. Observational, cross sectional studies have shown –Expected sex differences. –Lower ferritin levels in regular donors, related to donation intensity. –Finch CA, et al. Blood 1977; 50:441-447 –Simon TL, et al. JAMA 1981; 245:2038-2043 –Deferred donors drop out and are not represented as repeat donors. Comprehensive Longitudinal (Cohort) studies are not available. Previous Studies - Blood Donor Iron Status

5. 5 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.

6. 6 RISE Enrollment CohortsStudy Targets Total Enrolled First-time/ Reactivated (no red cell donations past 2 years) Male420407 Female420481 Total840888 FrequentMale750769 Female750768 Total15001537 Total23402425

7. 7 RISE Baseline Enrollment Data Collection Hematologic and iron status markers Iron protein polymorphisms 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

8. 8 Ferritin – Reflects body iron stores – Loss of iron initially leads to a decrease in ferritin (down to ~30 ng/mL). Log (sTfR/ferritin) – A specific measure of red cell iron deficit. With further reduction in body iron, ferritin is progressively reduced (down to <12 ng/mL), the red cell elaborates transferrin receptor into the serum (sTfR). With further iron loss, red cell hemoglobinization is impaired (change in RBC/reticulocyte indices). Iron deficiency anemia - Recognized only when the hemoglobin level drops (usually measured against population norms). Donor deferral occurs at some point, depending on the initial hemoglobin level. Measures of Donor Iron Used in RISE

9. 9 RISE Definitions: 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. 9

10. 10 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 10

11. 11 Effect of Previous 12 month RBC Donation Frequency on Plasma Ferritin at Enrollment 11

12. 12 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) 12 *Adjusted for Gender, Age, Blood Center, Weight, Smoking, Fe+ supplementation, HFE, Menstrual status, Pregnancy

13. 13 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) 13 *Adjusted for Previous RBC donations, Gender, Blood Center, Weight, Smoking, Fe+ supplementation, HFE, Menstrual status, Pregnancy

14. 14 Other Significant Enrollment Variables (other than gender) in the Adjusted Models for AIS and IDE Weight: Reduced odds of AIS and IDE in the heaviest donors. Smoking: Past and current smokers have lower odds for IDE than do donors who never smoked. Self-prescribed iron supplement: Donors taking iron supplements (with or without multivitamins) are slightly less likely to have AIS, OR = 0.7. HFE genotype: Donors homozygous or mixed heterozygous for C282Y and/or H63D are less likely to have IDE. Pre-menopausal or ever pregnant status: Women with either status are more likely to have IDE. Center 14

15. 15 RISE Longitudinal Study – 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* 2.62.94.45.2 Average time in study* 429 days440 days516 days524 days 15 * For donors with one or more return visits

16. 16 12,695 total RISE donor visits 2,425 Enrollment Visits (no deferrals) 10,270 follow-up visits 8956 Donations 945 Hemoglobin Deferrals (84% Female, 16% Male) 268 Other Deferrals 101 Sample Only Visits RISE Longitudinal Study – Visits/Data Points of RISE donors 16

17. Statistical Methods - Modeling Repeated measures logistic regression models were developed to predict AIS and IDE at all visits Similar models were developed to predict hemoglobin deferral at any return visit. –Enrollment visits not included in model since only accepted donors were enrolled These models account for multiple measurements on the same donor. 17

18. 18 Variables Included in the Model Predicting Hemoglobin Deferral at any Return Visit Variables significant in the adjusted model: Female Gender Black Race Younger Age in Women, not Men Blood Center Time since last RBC donation Variables not significant: Iron supplements Weight Smoking Menstrual status HFE status Previous donation a WB or double red cell Number of RBC units in previous 24 months 18

19. Adjusted Odds Ratio95% CI Black vs. White2.11.3-3.5 Hispanic vs. White1.61.0-2.7 Asian vs. White1.20.7-2.3 Other vs. White1.70.9-3.5 Female vs. Male6.33.8-10.5 AgeMaleFemale <30 vs. 40-490.90.2-3.21.71.2-2.4 30-39 vs. 40-490.90.3-2.91.10.8-1.5 50-59 vs. 40-491.90.9-4.00.90.7-1.3 60+ vs. 40-491.60.8-3.50.70.5-1.1 Demographic Variables Predicting Hemoglobin Deferral 19

20. Effect of Donation Interval on Hemoglobin Deferral: Adjusted ORs (vs. ≥ 26 weeks) Mean ORs for all donations are represented by Gray squares and CI bars. Blue diamonds represent mean ORs for female donations only. Insufficient male deferrals to analyze by week

21. 21 Variables Included in the Models Predicting AIS and IDE at any Visit Significant Variables: Female Gender and Younger Age in Women Time since last RBC donation Number of RBC units in previous 24 months Smoking Taking Iron Blood Center Variables minimally or not significant: Race Weight Menstrual status Previous donation a WB or double red cell HFE status 21

22. Significant Variables Predicting AIS 22 Adjusted Odds Ratio95% CI RBCs donated in last 2 yrs <4 donations vs none2.51.6 - 3.9 4-6 donations vs none9.25.6-15.0 7-9 donations vs none6.44.0-10.1 10+ donations vs none8.65.3-14.0 Iron Supplements Takes supplemental iron vs. None 0.60.5-0.7 Smoking Current vs. Never0.60.4-0.9 Past vs. Never0.80.6-1.0 Gender Female vs. Male2.91.9-4.7

23. Effect of Donation Interval on Iron Stores (AIS): Adjusted ORs (vs ≥ 26 weeks) Mean ORs for all donations are represented by Gray squares and CI bars. Blue diamonds represent mean ORs for female donations only. Insufficient male AIS to analyze by week

24. 24 Summary of Hemoglobin Deferral and AIS Models The most significant predictors of hemoglobin deferral were: –Fewer weeks since last RBC donation ▪But not number of RBC donations in the last two years –Black race –Female Gender –Younger age in women –Blood Center Significant predictors of severe iron depletion (AIS) were similar, except for race. Other predictors of AIS were: –RBC Donations in the last two years –Smoking and iron supplements had a protective effect Hemoglobin deferral was more likely if a donor attempted to donate within 14 weeks of his/her last donation and AIS more likely within 19- 20 weeks. 24

25. 25 Assessment of Fingerstick Hemoglobin Nearly all blood donor qualification schemes depend on capillary sampling of the donor’s finger. Earstick sampling produces values that are higher than venous values. Fingerstick sampling more closely approximates venous hemoglobin. –Woods et al. Transfusion 2001 The RISE study determined fingerstick Hgb/Hct in routine blood center practice along with simultaneous venous hemoglobin, allowing comprehensive assessment of the accuracy of fingersticks. 25

26. 26 Donor Qualification Methods at REDS-II Centers Blood Center Hemoglobin or Hematocrit Device AHematocritHematastat BHematocritHematastat CHematocritHematastat DHematocritUltraCrit EHemoglobinHemoCue B FHemoglobin HemoCue Donor Checker 26

27. 27 Fingerstick/Venous Hemoglobin Values in 2425 RISE Donors, by Gender and Iron Status Females with AIS (n=239) FS V V V V Females with No AIS (n=1011) Males with AIS (n=126) Males with No AIS (n=1049) 10 11 12 13 14 15 16 17 18 19 20 Hemoglobin g/dL FS and Venous Groups

28. 28 Frequency of Donors Passing FS Qualification Method but with Venous Hgb < 12.5 g/dL Iron StatusFemaleMale AIS Ferritin < 12 ng/mL 46.4% (n=239) 25.4% (n=126) No AIS Ferritin ≥ 12 ng/mL 18.4% (n=1011) 1.8% (n=1049) 28

29. 29 Conclusion - Fingerstick/Venous Analysis FS Hgb or Hct is a reasonable estimate of venous Hgb for male donors without iron depletion. However in females and all donors with absent iron stores, FS overestimates venous Hgb values. This is a significant limitation of fingerstick testing which should be considered in setting hemoglobin requirements. 29

30. 30 Four Questions posed by FDA What would be the impact of changing the minimum predonation Hemoglobin from 12.5 g/dL… 1.…to 13.0 g/dL for men? …to 13.5 g/dL for men? 2. …to 12.0 g/dL for women? What would be the impact of changing the interdonation interval from 8 weeks (about 6-7 times per year) ….. 3.….to 12 weeks (about 4-5 times per year) for men? 4.….to 16 weeks (about 3-4 times per year) for women? 30

31. 31 ARC Balance Sheet Minimum Hemoglobin CurrentProposed Difference in Donations Percent Gain or Loss Men 12.5 13.0- 146,790 - 4% 13.5- 386,781- 10% Women12.0 +198,200 + 5% Donation Interval (Frequency) Men ~ 6 -7 ~4-5 (4.33) - 157,378- 5% Women ~ 3-4 (3.25) - 261,494- 7% This and previous slide courtesy of Anne Eder, American Red Cross 31

32. Some Other Options to Manage Donor Iron Monitor donor Iron stores (ferritin?) and alter donation requirements for donors with significant iron depletion –All donors –Donors at high risk of iron depletion –Implement monitoring programs as a condition of frequent donation? Increase donor iron intake with iron supplements –Blood Center provided versus donor acquired pills –All donors or donors at greater risk –Implement Iron programs as a condition of frequent donation?

33. 33 Planned or In-progress RISE Analyses Related to Laboratory Measures Identify optimal laboratory measures that predict the development of iron depletion and hemoglobin deferral. –Examine within donor and between donor correlation among selected laboratory measures of iron stores. –Develop models to assess the utility of ferritin, sTfR, as well as newer iron measures (hepcidin, and selected red cell/reticulocyte indices) to predict iron depletion and hemoglobin deferral at subsequent visits. 33

34. 34 Further Actions - Evaluating the Impact of Various Donation Guidelines Utilize information from RISE and other REDS-II analyses 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. Engage policy makers in understanding and utilizing REDS-II, RISE and other available data to develop future donor management strategies and eligibility guidelines. AABB Committee FDA workshop in Fall 2011 34