Two multicenter prospective randomized controlled phase III clinical trials of hemoglobin-based oxygen carriers (HBOCs) have been completed. In one, HBOC-201 (Hemoglobin glutamer-250 (bovine), Hemopure, HbO2 Therapeutics, Souderton, PA), a polymerized bovine hemoglobin, eliminated allogenic packed erythrocyte transfusion in a substantial proportion of patients undergoing elective orthopedic surgeries (see Appendix A, Supplemental Digital Content 1, https://links.lww.com/AJT/A106 for review of the HEM-0115 study).1–3
The current retrospective analyses used the HBOC-201 HEM-0115 phase III trial database and compared the product to determine whether there was increased transfusion of blood components between groups and differences in coagulation studies.
Specifically, the following questions were examined: In the HBOC-201 HEM-0115 trial1 were more platelets, fresh frozen plasma (FFP), or cryoprecipitate transfused to subjects randomized to HBOC-201 versus allogeneic erythrocytes? The database was queried to determine any differences in the use of platelets, plasma, or cryoprecipitate between allogeneic erythrocytes and HBOC-201 infusion. Formulas were derived to estimate total hemoglobin concentrations [THb] and compared with published data. Morbidity/mortality was compared in the 2 groups because it related to total hemoglobin (Hb) deficit.
METHODS AND MATERIALS
A subset data analysis was performed on all 688 subjects in the HEM-0115 trial to evaluate coagulopathy in subjects randomized to HBOC-201 compared with those randomized to allogeneic erythrocytes to determine whether the HBOC-201 group received additional platelets, FFP, or cryoprecipitate. There were no guidelines or upper limits designated for the use of these 3 blood components in the original study protocol.1 Hb, hematocrit, reticulocyte count, and volumes of intravenous fluids (non-Hb containing plus Hb containing) as possible mechanisms for any changes noted in bleeding or clotting times were queried.
Transfusion of erythrocytes was evaluated in subjects randomized to HBOC-201, determination of adverse events in the HBOC-201 arm versus erythrocyte arm, and documentation of administration of platelets, cryoprecipitate, and albumin (blood product administration) between the 2 arms.
Data were collected as described by Jahr et al.1 Descriptive statistics were used for the evaluation of the data for the current retrospective analysis.
A careful review of the Hb deficit was undertaken, with new data not presented in the original presentation of this study.1 Novel models were created to explain the data and allowed for unique interpretation of the erythropoiesis, based on these models. Specifically, both duration and magnitude of Hb deficit (defined as difference between [THb] and CSL (clinically significant low calculated as 0.8 multiplied on lower end of normal range for [THb])) were evaluated, and a logistical model of features predicting adverse events was developed, using an upper cutoff of 1.37 g/dL for averaged Hb deficit during the treatment (expected addition of plasma Hb from 2 units of HBOC-201 administration in adult). Undertreatment of anemia, age per decade, and pre-existing cardiac disease was evaluated to determine adverse outcomes (see Appendix 2, Supplemental Digital Content 2, https://links.lww.com/AJT/A106 for references and full explanation).
To predict changes in [THb] after any transfusion, the derived earlier formula was applied. To model projected changes in [THb] after bolus administration of any resuscitation fluid, a simplistic 1 compartment model was used assuming the resulting volume in circulation after the bolus as V + v, where V is volume in circulation before administration and v is administered bolus volume. The change in [THb] δ = Δ* v/(V + v) = Δ/(k + 1), where Δ is the difference in [THb] between administered fluid and baseline [THb] and k = V/v. The coefficient k = V/v is important and represents the ratio between starting volume in circulation and volume of infusion. In HEM-0115 for approximately normovolemic patients, V was near 5 L, thus k for 1 unit infusion was approximately 20 and for 2 units infusion was approximately 10. The importance of this tool in transfusion field (especially with HBOCs) is hard to overestimate. Accurate predictions for THb increases promote realistic expectations for anemia treatment using relatively low-concentrated HBOCs and help to avoid fluid overload because of overly aggressive treatment. Moreover, application of this formula provides a clear explanation of issues encountered in pivotal HBOC trials that were conducted without it.
Demographics were no different between the subjects receiving HBOC-201 compared with those randomized to erythrocytes (Table 1). Adverse events (8.47 vs. 5.9) and serious adverse events (SAE) (0.35 vs. 0.25) were higher in the patients receiving HBOC-201 study group as published by Jahr et al.1 Overall, erythrocyte use in those randomized to HBOC-201 was significant (P < 0.0001) (using a T-TEST for mean. 95% confidence limit is approximately Mean ± 2SE, which gives 1.2–1.6 for HBOC-201 arm and 2.9–3.3 for RBC arm) (H arm) versus erythrocyte (R arm). The use of platelets, cryoprecipitate, and albumin was limited to less than 6% of the subjects in both HEM-0115 treatment groups (Table 1) and deemed too low in each group on which to perform any descriptive statistics. The HEM-0115 trial surpassed its primary efficacy end point of eliminating transfusion in 35% of patients and instead did so in excess of 50% of subjects.1
Table 1. -
Allogeneic component use: HBOC-201 and erythrocyte treatment groups in the HEM 0115 study.
||Mean ± SE
||Mean ± SE
||1.4 ± 0.1 (n = 350)
||3.0 ± 0.1 (n = 338)
|Fresh frozen plasma (mL)
||1157 ± 238 (n = 12)
||1058 ± 199 (n = 9)
||661 ± 141 (n = 4)
||323 ± 39 (n = 3)
|Other (mL) (cryoprecipitate, albumin)
||433 ± 285 (n = 3)
||747 ± 127 (n = 3)
Allogeneic component use: HBOC-201 (HBOC-201) and erythrocyte (RBC) treatment groups.
The presented data were collected for n = 4 or n = 3 for either group, which represents approximately 1% of entire population. Making any conclusion about trends on such modest amount of data is not statistically sound. The only valid conclusion is that for 99% of patients, platelet, cryoprecipitate, and albumin usage were not reported in the database. Low total [Hb] and lack of adequate oxygen carrying capacity was found to be an independent predictor of morbidity/mortality caused by it and was never established directly. It is a clinical conclusion based on reported low [THb].
Specific results from the retrospective review
Three hundred fifty patients received HBOC-201 infusion, and 338 patients were randomized to erythrocytes. For the total clinical trial material units administered, most HBOC-201 patients received 5 or less 250 mL/unit infusions with 18.7% receiving 6–10, 250 mL/unit infusions; one patient received 330 g or 11 infusions (Table 1). By contrast, the erythrocyte subjects received 243 ± 9 g Hb on the average, with 78.4% receiving ≥2 units. Administration of HBOC-201 resulted in an expected hematocrit reduction related to the acellular HBOC-201 solution infusions through treatment day 5 (Figures 1, 2); hematocrit values returned to normal at 6 weeks in both groups. With the exception of first infusion (which was 2 units instead of 1 unit for all the following infusions), the average [THb] remained below transfusion trigger even immediately after completed infusion with the exception of the number of 250 mL/unit infusions post-HBOC-201 and produced a 1.44 ± 0.03 g/dL increase in plasma Hb and a 0.39 ± 0.06 g/dL increase in [THb] concentration. As a rule, for all HBOC subjects, the entire number of infused units was numerically 1 bigger than the number of infusions.1
Analysis of the total hemoglobin concentrations [THb] as a function of the number of infusions indicated that patients requiring ongoing HBOC-201 treatment maintained a lower [THb] (P < 0.05) than those given erythrocytes at any preinfusion and postinfusion point. The average [THb] of HBOC-201–treated patients remained below that of the original protocol transfusion trigger (Hb <10.5 g/dL) at a Hb level of 8.85 ± 0.07 g/dL throughout the treatment period, with the exception of the number of 250 mL/unit infusions post–HBOC-201 total Hb (Figure 2). In the original study, 139 subjects whose oxygen transport requirements were unmet by 10 units HBOC-201 by the protocol were transfused erythrocytes.1
A comparison of [THb] and SAEs among study subjects administered less than 10 units of HBOC-201 (n = 211) and those given HBOC-201 plus erythrocytes showed that [THb] was significantly lower (P < 0.01) in those patients whose oxygen transport requirements were unmet by HBOC-201 ([THb] = 8.56 ± 0.10 g/dL) versus those only given HBOC-201 ([THb] =9.11 ± 0.08 g/dL). The incidence of SAE increased (P ≤ 0.0002) in both the erythrocytes and HBOC-201 arms of the study increased with clinical need.4–6
The protocol did not require coagulation data collection during HBOC-201 administration; thus, data for PT and aPTT were collected for approximately only 10% of patients. There was a trend toward increased aPTT in erythrocytes arm (P = 0.33) compared with HBOC-201 (37.8 ± 5.84 vs. 31.9 ± 1.67 seconds, respectively) during HBOC-201 administration. [THb] and mortality rates of 1% in those patients who required <10 HBOC-201 250 mL/unit infusions were identical to those receiving ≤3 units erythrocytes. Five deaths in the HBOC-201 arm occurred in patients older than 80 years compared with 1 in the erythrocyte group. There were an equal number of deaths (n = 5) among patients younger than 80 years receiving either treatment.
Hemoglobin deficit in HEM-0115 study
Hb deficit (or lack of adequate oxygen carrying capacity) duration indicated not just the effects around a single Hb concentration value but the total period of anemia (see Appendix A2, Supplemental Digital Content 3, https://links.lww.com/AJT/A106).4–6 In the HEM-0115 trial, the average Hb deficit in patients randomized to HBOC-201 was 1.31 ± 0.06 g/dL; in patients randomized to erythrocytes, it was 0.45 ± 0.03 (P < 0.05). In a logistical model of features predicting adverse events, including all subjects in the HEM-0115 study using Hb deficit upper limit cutoff of 1.37 g/dL, undertreatment of anemia (P = 0.035), age per decade (P = 0.046), and pre-existing cardiac disease (P = 0.004) were predictors of cardiac adverse events. Under the conditions of the HEM-0115 trial, increased [THb] after erythrocyte transfusion was 4.4 times greater than after a unit of HBOC-201. This finding of comparative efficacy is in close agreement with the Hb deficit modeling and validates these predictions. In the HEM-0115 trial, the average [THb] at which the HBOC-201 loading dose of 500 mL of 13 g/dL began was 8.85 + 0.07 g/dL. Infusion of a single unit of HBOC-201 (32.5 g Hb) produced a 0.63 + 0.03 g/dL increase in plasma Hb and a 0.18 + 0.03 g/dL increase in [THb]. A single unit of PRBC (32.5 g Hb) increased [THb] by 0.87 + 0.07 g/dL. Infusion of the loading dose of 2 units of HBOC-201 produced a 1.44 + 0.03 g/dL increase in plasma Hb and a 0.39 + 0.06 g/dL increase in [THb]. In this case, average [THb] represents starting [THb] before treatment (averaged by entire arm).
In the presence of HBOC-201 in the circulation, [THb] represents both RBC Hb and free Hb dissolved in plasma. To calculate hematocrit (HCT) as HCT [%] = [THb] (in g/dL) × 3 is not valid because of plasma volume expansion by acellular HBOC-201. Plasma Hb measurement represents free Hb of HBOC-201 dissolved in plasma of volume V × (1−HCT [%]/100), where V is circulatory volume. The numerical relationship between these measurements taken together was approximated as [THb] [g/dL] = HCT [%]/3 + Plasma Hb [g/dL] × (1−HCT [%]/100), where HCT [%]/3 = RBC Hb [g/dL]. This theoretical relationship was evaluated with data including all 3 measurements made simultaneously to permit calculation of any of 3 measurements if 2 others were collected. It was the difference in administrated solution volume (v) distributed over new larger volume V + v. To model projected changes in [THb] after bolus administration of any resuscitation fluid, a simplistic 1 compartment model was used assuming the resulting volume in circulation after the bolus V + v, where V is volume in circulation before administration and v is administered bolus volume. The change in [THb] δ = Δ v/(V + v) = Δ/(k + 1), where Δ is difference in [THb] between administered fluid and baseline [THb] and k = V/v.
Assuming that patients were hypovolemic (V0 = 4.5 L) (this is probably not a valid assumption because fluid overload was a concern in the HEM-0115 study), the change (Δ) in [THb] resulting from the loading dose of 2 units [500 mL] and starting [THb] = 8 could be calculated as follows: k = V/v = 4.5/0.5 = 9 so δ = Δ/10, and since Δ = 13−8 = 5 g/dL, then δ = 0.5 g/Dl, and resulting [THb] after 500 mL of HBOC-201 was 8.5 g/dL.
Attempts to increase [THb] further would require more HBOC-201 leading to fluid overload. In comparison, administration of 500 mL of erythrocytes (in reality, erythrocytes have 300–350 mL volume, the remaining being acellular components) with Hb concentration of 24 g/dL increased [THb] by (24–8)/10 = 1.6 g/dL resulting in [THb] of 9.6 g/dL and will not (barring emergencies) require additional transfusion. On average, this was acceptable, but if patients with high and low needs for increased [THb] were separated, the [THb] in patients administered erythrocytes compared with HBOC-201 changed considerably, as demonstrated by a subset of patients with major hemorrhage with [THb] = 7 g/dL and hypovolemia (V = 4.5 L) at the first infusion. Administration of 3 units of HBOC-201 in the first hour would increase [THb] by (13–7)/(4.5/0.75 + 1) = 6/7 = 0.85 g/dL; thus, these patients, even after receiving the maintenance dose of 3 HBOC-201 units per day, would never attain [THb] = 8 g/dL.
In addition, after 4–5 days (with 3–4 days still left until rebound of native red blood cell production), the HEM-0115 trial patients would exceed the protocol permitted 10 units HBOC-201 and would be required to cross-over to erythrocytes. In comparison, their counterparts receiving erythrocytes would likely be transfused 2 units (approximately 750 mL) of erythrocytes on first day resulting in increased [THb] of (24−7) (4.5/0.75 + 1) = 17/7 = 2.5 g/dL to attain Hb of 9.5 g/dL. The patients with [THb] below 8 g/dL in comparison with 9.5 g/dL for 4–5 days would probably not create an imbalance in mortality. However, if 30% of this subpopulation was older than 70 years with significant cardiac medical history, an increase in morbidity and adverse events, including mortality, would be expected and could be enough to derail a safety trial.
Once the current investigation began, it became clear that the incidence of coagulopathy in both arms of the HBOC-201 study was low, and expected as such, since elective orthopedic joint replacements rarely have this complication, and the data from that portion of this study were not interpretable.
However, in 24 subjects who received 6 or more units of HBOC-201 (1500 mL +) in the first 24 hours of treatment, all eventually crossed over to receive erythrocyte, and all but 1 survived, suggesting that with large volumes of HBOC-201, survival was improved, but there may have been issues with coagulation because of massive infusion of noncoagulation factors. Three subjects had coagulopathy reported events: thrombocytopenia, prothrombin concentrations decreased based on laboratory investigations, and coagulopathy disorders. All had onset in the first day, which confirms that dilutional coagulopathy after significant blood loss, and receiving 2–2.5 L of HBOC-201 and many other fluids.
In reference to PT and aPTT values in the HEM-0115 study, they were collected before administration of HBOC-201, within 1 day after HBOC-201 administration and at the 6-week follow-up. Lower abnormal values, suggesting hypercoagulopathy, are rare (around 15% for aPTT and 5% for PT, with more high abnormalities, suggesting hypocoagulopathy observed noted around 40% for aPTT and around 60% for PT). In addition, PT was measured in 2 formats: in seconds at some sites and as an INR at others, making comparison difficult.
The HEM-0115 trial demonstrated that HBOC-201, with a Hb concentration of 13 g/dL in comparison with erythrocytes, provided management of hemorrhage of up to 3 units for elective orthopedic surgery without the use of erythrocytes or significant differences in mortality or SAEs. HBOC-201 with Hb, a concentration equivalent to whole blood, can be used safely to avoid up to 3 units of erythrocytes for elective surgery (see Appendix 1, Supplemental Digital Content 4, https://links.lww.com/AJT/A106).1
Almost 70% of reticulocyte counts were collected before day 1 of this study, and <1% were collected between day 1 and day 25 because design reticulocyte counts were not required to be collected during this period. Approximately 30% of remaining reticulocyte data were collected at the 6-week follow-up when any effects because of HBOC-201 would not be apparent, suggesting that if there was a benefit, it would not be expected to be demonstrated at 6 weeks, when normal homeostatic mechanisms would be operational.
There were 3 classes of patients in whom HBOC-201 use should be limited unless whole blood or erythrocytes are unavailable. These were patients older than 80 years (those patients older than 80 years had worse outcomes than their younger cohorts with HBOC-201, either because of more pre-existing comorbidities, increased incidence of myocardial events, or possible fluid overload from excessive volume replacement concurrently with HBOC-201), patients with volume overload, or those whose [THb] was undertreated.1 Physicians should monitor patient volume status to avoid fluid overload. Regarding undertreatment, patients with pre-existing cardiac disease may be more susceptible to the decreased Hb concentrations and hematocrit that comes with administering HBOCs.7,8 However, when a patient is undergoing elective orthopedic surgery and is younger than 80 years, using up to 10 units of HBOC-201 may avoid red blood cell transfusion according to the Maximum Surgical Blood Order Schedule.1
Hemodilution of oxygen-carrying capacity concentration, when excess non-Hb containing fluids are administered before HBOC-201, should be avoided. A point-of-care test of Hb should be made before administration of HBOC-201 or erythrocytes because the Hb transfusion threshold in the HEM-0115 trial of <10–10.5 g/dL was too lenient for current transfusion practices.8,9 Over several days, the HEM-0115 trial showed that adverse events were related to decreased [THb] values in the HBOC-201 arm versus the erythrocyte arm.
In conclusion, for the HEM-0115 study, most orthopedic surgery patients treated with HBOC-201 avoided erythrocyte transfusions. HBOC-201 provided a temporary oxygen bridge for adequate oxygen transport until generation of new endogenous erythrocytes restored adequate tissue oxygenation, and there was no difference noted between the group receiving erythrocytes and HBOC-201 regarding the usage of platelets, FFP, or cryoprecipitate. In addition, the data available showed no predilection for reticulocyte formation with the use of HBOC-201; however, the study design precluded accurate assessment of this study.
Dilution by volume expanders, as discussed by Spahn, et al 10 unrelated to this particular study, supports what is demonstrated in Table 1. The effect of volume expanders and the volume of crystalloids infused during transportation (600 mL) in the control arm would likely have minimal impact on the circulating blood volume in 20 minutes because of crystalloid extravasation.
It is clear that rapid massive infusions of any solution without coagulation properties may lead to dilutional coagulopathy. This has not been documented with HBOC-201 either as a suspected cause or an expected outcome. There is not a single study in HBOC-201 in all the randomized controlled studies, that anemia treatment permits study of dilutional coagulopathy, let alone the direct intrinsic effects of HBOC-201 free [Hb] on coagulation.
In theory, 2 units of HBOC-201 transfused at an Hb of 8 g/dL should increase Hb concentration by 0.5 g/dL if the starting volume is 4.5 L in a single compartment model. In addition, with a starting of Hb near 9 g/dL, there would be a theoretical increase in Hb concentration of 0.4 g/dL. If the starting volume increases to 4.75 L, the expected change in Hb concentration will be lower, around 0.37. With a starting volume of 5 L, the increase in [THb] will decrease further to 0.44 g/dL for a starting [THb] of 8 g/dL and 0.35 g/dL with a starting [THb] of 9 g/dL. Statistically speaking, there is good agreement for HBOC-201's loading dose in the HEM-0115 study.
Regrettably, neither volume nor concentration of transfused erythrocytes was reported in the HEM-0115 study. In some cases, washed erythrocytes had Hb concentrations close to 30 g/dL, whereas other nonwashed erythrocytes had Hb concentrations of 22–26 g/dL or even lower. In addition, approximately 2%–3% of transfusions were administered with whole blood. Therefore, more is known about the exact amount of HBOC-201 infused in the study compared with erythrocytes. In addition, in the modelling, it did not make sense to use 9 g/dL as the starting Hb in HEM-0115 modeling because updated transfusion practice requires lower Hb concentrations for transfusion to be deemed necessary.10,11
A single-site analysis from the previous HEM-0115 trial assessed platelet function in patients before and after transfusion of erythrocytes or infusion of HBOC-201.12 The analysis using a PFA-100 (Platelet Function Analyzer-100; Seimens Healthcare Diagnostics, Inc, Tarrytown, NY) determined that those using HBOC-201 did not require more blood products than the erythrocyte group. There was a significant difference found in the “after transfusion” period between the HBOC-201 and the erythrocyte group, where the HBOC-201 group had increased cEPI and cADP. An increase in these measurements can correlate with an increased risk for bleeding.12 This increase, however, was reversed about 1 HBOC-201 half-life on “day 1 After transfusion.” One HBOC-201 half-life is 19–24 h, so day 1 measurement will deal with only half of infused HBOC-201 in circulation; thus, effects will be halved as well. For example, if the subject was infused 0.5 L of HBOC-201 at day 1, only 0.25 L will remain. If excess volume was 0.25 (with start at 4.75), there will be no excess volume at the day.1
The increased cEPI may be explained by the lower level of Hb concentration found in the study in patients given HBOC-201 versus erythrocytes. It is also possible that the increase in cEPI and cADP was due to hemodilution of the HBOC-201 blood product and not the HBOC-201 itself. There was no statistically significant change in cEPI or cADP measurements from “before transfusion” and baseline.12
Hemoglobin 02 Therapeutics LLC (Southerton, PA) acquired the intellectual property of Biopure Corp from the previous owner of the HBOC-201. HBOC-201 is approved for human use in South Africa and Russia, and a related product, HBOC-200 (Oxyglobin), is FDA and European Union approved for canine anemia. Recently, a trauma trial was initiated in South Africa using HBOC-201 and a freeze-dried plasma derivative to assist with coagulation during acute trauma that was funded by the US Department of Defense. (https://www.prnewswire.com/news-releases/hemopure-to-be-evaluated-in-groundbreaking-clinical-study-of-trauma-patients-in-the-prehospital-setting-sponsored-by-the-united-states-department-of-defense-and-coordinated-by-stellenbosch-university-300746278.html (accessed on July 19, 2020)).
In summary, HBOC-201 was studied in elective orthopedic patients and did not result in any evidence of coagulopathy, although it could not be specifically evaluated. HBOC-201 continues to be administered to patients for compassionate use both in countries where it is approved and as an alternative to erythrocytes when either they are unavailable or refused.2,3,6–8,11
The authors thank the other members of the Hemopure HEM 0115 study team who participated in collection of data for the original Biopure subset analysis: K. Tseuda, E. Vandermeersch, and D. L. Bourke. The Hemopure HEM 0115 methods, some results, and table in this manuscript are derived in part from the abstract presented at the 2002 AABB Annual Meeting: J. P. Williams, K. Tseuda, E. Vandermeersch, D. L. Bourke, and J. S. Jahr. Transfusion requirements in the phase III study of HBOC-201 (Hemopure, Hemoglobin glutamer-250, Bovine), a bovine hemoglobin-based blood substitute. Transfusion 2002; 42: S49-030I.
The authors also thank Drs. Colin Mackenzie, Greg Dube’, and Jessica Riley for their support in preparing this manuscript; their input and feedback were invaluable. The authors especially thank Arkadiy Pitman, MS, the statistician who participated in the original HEM-0115 study and also this manuscript and was extraordinarily helpful in ensuring accuracy of the data and explanations of the many complex issues/formulas presented in this manuscript. The authors thank Rachel Jahr for her graphic editing.
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