ORIGINAL ARTICLE 
 

Investigation of whether the acute hemolysis associated with

Rho(D) immune globulin intravenous (human) administration

for treatment of immune thrombocytopenic purpura is

consistent with the acute hemolytic transfusion reaction model

Ann Reed Gaines, Hallie Lee-Stroka, Karen Byrne, Dorothy E. Scott, Lynne Uhl, Ellen Lazarus, and
 

David F. Stroncek
 

BACKGROUND: Immune thrombocytopenic purpura 

and secondary thrombocytopenia patients treated with 

Rho(D) immune globulin intravenous (human; anti-D 

IGIV) have experienced acute hemolysis, which is 

inconsistent with the typical presentation of extravascular hemolysis—the presumed mechanism of action of 

anti-D IGIV. Although the mechanism of anti-D-IGIV– 

associated acute hemolysis has not been established, 

the onset, signs/symptoms, and complications appear 

consistent with the intravascular hemolysis of acute 

hemolytic transfusion reactions (AHTRs). In transfusion 

medicine, the red blood cell (RBC) antigen-antibody 

incompatibility(-ies) that precipitate AHTRs can be 

detected in vitro with compatibility testing. Under the 

premise that anti-D-IGIV–associated acute hemolysis 

results from RBC antigen-antibody–mediated complement activation, this study evaluated whether the 

incompatibility(-ies) could be detected in vitro with a 

hemolysin assay, which would support the AHTR model 

as the hemolytic mechanism. 

STUDY DESIGN AND METHODS: Seven anti-D IGIV 

lots were tested to determine the RBC antibody identities in those lots, including four lots that had been implicated in acute hemolytic episodes. Hemolysin assays 

were performed that tested each of 73 RBC specimens 

against each lot, including the RBCs of one patient who 

had experienced acute hemolysis after anti-D IGIV 

administration. 

RESULTS: Only two anti-D IGIV lots contained RBC 

antibodies beyond those expected. No hemolysis endpoint was observed in any of the hemolysin assays. 

CONCLUSION: Although the findings did not support 

the AHTR model, the results are reported to contribute 

knowledge about the mechanism of anti-D-IGIV– 

associated acute hemolysis and to prompt continued 

investigation into cause(s), prediction, and prevention of 

this potentially serious adverse event. 

ABBREVIATIONS: AHTR(s) = acute hemolytic transfusion 

reaction(s); anti-D IGIV = Rho(D) immune globulin intravenous 

(human); IRB = institutional review board; ITP = immune 

thrombocytopenic purpura. 

From the Office of Biostatistics and Epidemiology, the Office of 

Blood Research and Review, and the Office of Cellular, Tissue 

and Gene Therapies, Center for Biologics Evaluation and 

Research, Food and Drug Administration, Rockville, Maryland; 

the Department of Transfusion Medicine, Clinical Center, 

National Institutes of Health, Bethesda, Maryland; and the Division of Laboratory and Transfusion Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, 

Massachusetts. 

Address correspondence to: Ann Reed Gaines, Office of Biostatistics and Epidemiology (HFM-220), Center for Biologics 

Evaluation and Research, Food and Drug Administration, 1401 

Rockville Pike, Rockville, MD 20852; e-mail: ann.gaines@ 

fda.hhs.gov. 

The article was produced by employees of the United 

States Government and Beth Israel Deaconess Medical Center/ 

Harvard Medical School as part of their official duties. 

The content of this publication does not necessarily reflect 

the views or polices of the U.S. Department of Health and 

Human Services, nor does mention of trade names, commercial 

products, or organizations imply endorsement by the U.S. 

government. 

None of the authors has financial interest in Cangene Corporation, the company whose product was studied in the 

present work, or in any other company(ies) whose products 

were mentioned in this article. This study was not supported by 

research funding from Cangene or any other entity. 

Received for publication August 27, 2008; revision received 

November 9, 2008; and accepted November 20, 2008. 

doi: 10.1111/j.1537-2995.2008.02083.x 

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Volume **,   TRANSFUSION 1
 

 

GAINES ET AL.

The Food and Drug Administration (FDA) initially 

licensed Rho(D) immune globulin intravenous 

(human; anti-D IGIV) as a lyophilized formulation (then WinRho, currently WinRho SDF;1 

Cangene Corporation, Winnipeg, Manitoba, Canada) in 

March 1995 and as a liquid formulation (WinRho SDF 

Liquid1

) in March 2005. Both formulations (hereinafter 

referred to as WinRho unless otherwise noted) are 

approved for treatment of immune thrombocytopenic 

purpura (ITP) in Rho(D)-positive, nonsplenectomized 

children with acute ITP, children and adults with chronic 

ITP, and children and adults with ITP secondary to human 

immunodeficiency virus (HIV) infection as well as for suppression of Rh isoimmunization.1 WinRho is also used 

“off-label” to an unknown extent for treatment of secondary thrombocytopenia. 

The presumed mechanism of action of WinRho in ITP 

involves extravascular hemolysis of anti-D–sensitized red 

blood cells (RBCs) by splenic macrophages.1 In patients 

who respond therapeutically, this mechanism results 

in decreased splenic sequestration of autoantibodysensitized platelets (PLTs), which results in an increased 

PLT count.1 In what appears contradictory to the presumed extravascular hemolysis mechanism of action and 

its typical clinical and laboratory findings, two cases of 

“acute-onset hemoglobinuria consistent with intravascular hemolysis” were observed during the WinRho ITP 

clinical trials.2 After licensure, additional reports of acute 

hemolysis after WinRho administration for ITP or secondary thrombocytopenia were (and continue to be) submitted to the FDA. 

Most patients treated with WinRho for ITP or secondary thrombocytopenia do not experience signs/symptoms 

of acute hemolysis,1,3-13 and not all who experience signs/ 

symptoms of acute hemolysis experience hemolysisrelated complications14 or require medical intervention 

for any complications experienced.13-16 Nonetheless, the 

acute hemolysis–associated complications that have been 

reported to date include clinically significant anemia, the 

need for RBC transfusion(s), acute or exacerbated renal 

failure, the need for dialysis, disseminated intravascular 

coagulation, and death secondary to these complications.14,17 The complications may occur singly or in combination,14,17 were previously reported in two case series of 

patients,14,17 and are listed in the WinRho professional 

package insert.1 

If the acute hemolysis that occurs in some patients 

treated with WinRho for ITP or secondary thrombocytopenia is consistent with the acute hemolytic transfusion 

reaction (AHTR) mechanism and could be detected in 

vitro with a hemolysin assay, this assay could conceivably 

be used to identify patients at risk for acute hemolysis with 

specific WinRho lots. For such patients, those lots might 

be contraindicated. The hemolysin assay might also 

allow identification of WinRho lots that appear to pose no 

2 TRANSFUSION Volume ** , ** ** 

risk of acute hemolysis and could presumably be safely 

administered. 

Such testing could be performed prior to administration of WinRho, borrowing from the historical precedent 

for the manufacture and distribution of two-vial packaging of an FDA-licensed Rho(D) immune globulin for intramuscular administration—one vial for preadministration 

testing of product and patient RBCs and one vial for 

subsequent patient administration (RhoGAM, then Ortho 

Diagnostic Systems, now Ortho-Clinical Diagnostics, 

Raritan, NJ18). Although what prompted the two-vial packaging that was previously used with RhoGAM was unrelated to either acute hemolysis or ITP, we recognized that 

this packaging precedent could apply to the performance 

of a hemolysin assay as a screening procedure before 

administration of anti-D IGIV for treatment of ITP or secondary thrombocytopenia. 

We report the results of a hemolysin assay study that 

we designed to evaluate whether the acute hemolysis 

associated with WinRho administration for treatment of 

ITP is consistent with the AHTR model. We also report an 

additional case of WinRho-associated acute hemolysis in a 

patient with a history of ITP. 

CASE REPORT

A 51-year-old female with ITP secondary to HIV infection 

and concurrent medical problems that included hepatitis 

C, liver failure, splenomegaly, and insulin-dependent diabetes mellitus was admitted to the hospital for evaluation 

of abdominal pain and thrombocytopenia. She had been 

treated successfully for ITP with steroids in the past. 

However, due to concern for immunosuppression, among 

other steroid-related side effects, her hematologist elected 

to treat her ITP with WinRho SDF. She received 27.7 mg per 

kg WinRho SDF (Lot 0240501), and within 1 hour of 

administration, she experienced severe rigors, back pain, 

headache, and chills. She developed hemoglobinuria, as 

evidenced by “tea-colored” urine that was collected the 

following day and yielded a urine reagent strip reading 

of “large” blood and a microscopic examination 

showing 0 RBCs per high-power field. She subsequently 

developed acute renal failure, presumed secondary to 

hemoglobinuria-induced acute tubular necrosis. Her 

serum creatinine level peaked at 2.7 mg per dL 2 days after 

WinRho SDF, increased from a baseline of 0.9 mg per dL; 

however, she did not undergo dialysis. By 3 days after 

WinRho SDF, her hemoglobin (Hb) level had decreased 

3.7 mg per dL, from a baseline of 11.2 to 7.5 g per dL, and 

she was transfused with 1 unit of RBCs 6 days after 

WinRho SDF. She was subsequently discharged from the 

hospital 12 days later in stable condition without apparent 

sequelae. 

Pre–WinRho SDF testing revealed that her RBC phenotype was A; D+C+E-c-e+; K-; Jk(a-b+); Fy(a+b+); 
 

 

 

 

 

 

 

 

 

 

 

 

M-N+S+s+; Le(a+); P1+. She had a negative direct antiglobulin test (DAT) but a positive indirect antiglobulin test 

(IAT) that revealed anti-c and anti-M due to alloimmunization from previous blood transfusions. Four hours 

after WinRho SDF, her DAT was 3+ positive for antiimmunoglobulin G (IgG) but negative for anti-C3; an acid 

eluate revealed anti-D. Her IAT remained unchanged 

when tested against c– and M– RBCs. At 4 months after 

WinRho SDF, her DAT remained weakly positive for antiIgG but negative for anti-C3, although no eluate was 

performed. Her IAT test results again revealed anti-c and 

anti-M. Also after WinRho SDF, her RBCs were genotyped 

and tested for Rh antigen density. The results of that 

testing revealed her genotype as R1R1 (DCe/DCe) and 

her antigen density “as expected for a [D antigen] 

homozygote.” 

MATERIALS AND METHODS

Institutional review board clearances

The National Institutes of Health Institutional Review 

Board (IRB) approved the testing of blood specimens from 

normal blood donors for this study. The Beth Israel Deaconess Medical Center IRB approved the testing of blood 

specimens obtained from the patient who experienced 

acute hemolysis after administration of WinRho SDF. The 

patient gave verbal consent, in accordance with local IRB 

policy, for blood specimens to be collected for this study. 

Testing procedures

Overview

This study consisted of two separate sets of testing procedures. We tested each of the seven WinRho lots for RBC 

antibodies. We then performed hemolysin assays in which 

we tested each of those WinRho lots against each of 73 

RBCs (i.e., 511 hemolysin assays). 

WinRho lots tested

We sought to include both WinRho lots that had been 

implicated in cases of acute hemolysis and those that had 

not been reported as having been associated with acute 

hemolysis. We tested seven lots of WinRho for RBC antibodies, which consisted of four purposively selected lots 

that had been implicated in three separate cases of acute 

hemolysis (one patient received a dose pooled from two 

lots) and three nonimplicated lots that were selected by 

convenience sampling (i.e., availability). WinRho SDF lots 

were 0240501, 1460402, 1460501, 1470403, and 1480410; 

WinRho SDF Liquid lots were 0110501 and 0120502. 

Three of the WinRho SDF lots had passed their expiration dates at the time of testing. Implicated Lot 1480410 

was 1 month beyond its dating period; nonimplicated Lots 

MECHANISM OF ANTI-D-IGIV ACUTE HEMOLYSIS

1460402 and 1470403 were 7 and 8 months beyond their 

dating periods, respectively. All of the lots beyond their 

expiration dates were lyophilized and would not have 

been expected to exhibit significant declines in antibody 

titers or integrity between the expiration and testing 

dates.19,20 The remaining WinRho SDF lots were within 

their dating periods, as were both WinRho SDF Liquid lots 

included in this study. 

WinRho is not equivalent to serum, and we had no 

frame of reference for determining what WinRho dilutions 

should be used in this study, other than wanting to replicate the in vivo environment (i.e., “physiologic” conditions) as much as possible in the hemolysin assays. If 

RBCs were hemolyzed by WinRho under conditions that 

reflected physiologic conditions, we believed that this 

finding could provide important clues as to the mechanism of the acute hemolysis that occurs in vivo. 

WinRho SDF lots were reconstituted with the 

manufacturer-supplied diluent according to the professional package insert instructions before testing. WinRho 

SDF Liquid lots were tested as distributed. All lots were 

subsequently diluted with phosphate-buffered saline 

(PBS; Cellgro, Mediatech, Inc., Herndon, VA) to both 

1-in-10 and “physiologic” dilutions. The 1-in-10 dilution 

was tested to detect low-titer RBC antibodies that might 

be diluted beyond the limits of detection in the physiologic dilutions. Physiologic dilution was defined and 

calculated to approximate the in vivo dilution of a recommended initial 50 mg per kg dose of WinRho in the blood 

volume of an average adult patient. We chose to test a 

physiologic dilution under the assumption that this dilution would approximate the RBC antibody titers at which 

acute hemolysis occurs in vivo after WinRho administration. The physiologic dilutions of WinRho lots ranged from 

1 in 40 to 1 in 100, depending on the labeled international 

unit (IU) dosage of anti-D in the vials. 

All lots were tested for RBC antibodies by an IAT 

method using column agglutination (MTS anti-IgG, Micro 

Tying Systems, Inc., Pompano Beach, FL) and a tube technique using polyethylene glycol additive (PeG, Immucor 

Gamma, Gamma Biologicals, Inc., Houston, TX) and antiIgG anti-human globulin (Ortho-Clinical Diagnostics). 

Tests to differentiate anti-G from anti-D and anti-C were 

not performed. 

RBCs tested

We sought to include RBCs that varied in both ABO blood 

group and Rh antigen phenotypes. The rationale was that 

patients who have experienced acute hemolysis after 

WinRho administration have ranged across ABO blood 

groups and Rh phenotypes. We used 73 RBC specimens 

in our study, which included RBCs from normal blood 

donors (n = 40), commercially available RBC reagents 

(n = 32), and RBCs from the patient who experienced 

acute hemolysis after WinRho SDF administration (n = 1) 

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GAINES ET AL.

and from patient “facsimiles” (n = 4, from among the 40 

normal blood donor and 32 reagent specimens). 

The normal blood donor RBCs were selected by convenience sampling, which we presumed would yield a 

range of both ABO blood groups and Rh phenotypes/ 

genotypes. The reagent RBCs were selected by purposive 

sampling to provide an array of Rh phenotypes/genotypes 

that we presumed corresponded to different D and other 

Rh antigenic densities,21,22 which has been proposed as a 

potential factor in whether acute hemolysis occurs after 

administration of WinRho.13,21-25 The patient facsimiles 

were selected by purposive sampling to express the 

equivalent non-ABO RBC antigen–positive and antigennegative phenotype as the patient. Two facsimiles were of 

the same ABO blood group as the patient; two were of 

another ABO blood group. 

The normal blood donor specimens were collected in 

ethylenediaminetetraacetate (EDTA) and tested within 7 

days of collection. The ABO blood groupings and Rh 

typings for these specimens were excerpted from blood 

donor records. The reagent RBCs (Immucor, Inc., Norcross, GA; and Ortho-Clinical Diagnostics, Inc.) were 

washed with pH 7.4 PBS before testing but were otherwise 

tested as supplied by the manufacturers. If the Rh genotype of a reagent RBC was not specifically noted by the 

manufacturer, we assigned the most probable genotype 

based on the reported Rh phenotype. 

We deliberately opted not to use enzyme-treated 

RBCs in the hemolysin assay to more closely approximate 

in vivo conditions. Had we tested enzyme-treated RBCs, 

we would have been unsure that we could generalize our 

findings to physiologic conditions. Furthermore, although 

we realized that using enzyme-treated RBCs may have 

increased the sensitivity of the hemolysin assay, that was 

not the purpose of this study. 

The ABO blood groups of the normal blood donor and 

reagent RBCs were as follows: O (n = 32), A (n = 27), and B 

(n = 13). The Rh types of these RBCs included the following 

distribution: R1R1 (DCe/DCe; n = 16), R1

wR1 (DCwe/DCe; 

n = 3), R2R2 (DcE/DcE; n = 15), R1R2 (DCe/DcE; n = 7), R1r 

(DCe/dce; n = 16), rr (dce/dce; n = 3), RoRo (Dce/Dce; 

n = 6), R2r” (DcE/dcE; n = 1), R1Rz (DCe/DCE; n = 2), R2Rz 

(DcE/DCE; n = 2), and R=NR2 (D(C)(e)/DcE; n = 1). 

For the patient who experienced acute hemolysis, we 

excerpted the RBC phenotyping, DAT, and IAT results of her 

pre-, 4-hour post-, and 4-month post–WinRho SDF specimens from her medical records. Another set of 4-month 

post–WinRho SDF blood specimens was collected for this 

study. These specimens were collected in EDTA or without 

anticoagulant, respectively, and were tested within 7 days 

of collection. The 4-month post–WinRho SDF time frame 

was selected under the presumption that this blood specimen would be representative of her indigenous RBC population, without the potentially confounding influence of 

residual transfused RBCs. 

Hemolysin assay

We used a standardized hemolysin assay26 that is designed 

to detect the presence of IgM and IgG RBC antibodies that 

are capable of binding complement and initiating 

complement-dependent hemolysis. We used this assay for 

testing each of the seven WinRho lots against each of the 

73 normal blood donor, reagent, patient, and patient facsimile RBCs. RBCs were tested as 10 to 20 percent concentrations suspended in 0.9 percent (wt/vol) isotonic saline 

(Fisher Diagnostics, Fisher Scientific Co., Middletown, 

VA). We performed the assay at what approximated an in 

vivo physiologic dilution by diluting the WinRho lots 

in fresh Group AB serum according to the following 

calculation: 

(50μg WinRho 1kg [recommended initial dose 


of WinRho])×(1kg

 4 mL [plasma volume of 

an average adult]) ×(m

0

L [IUs of anti-D per vial]).
 

We selected fresh group AB serum as the test system 

diluent because it lacked confounding ABO blood group 

antibodies and provided a source of complement required 

for complement-dependent antigen-antibody–mediated 

hemolysis. We collected the group AB serum as white 

blood cells (WBCs) and plasma by an apheresis procedure 

using anticoagulant citrate-dextrose solution Formula A. 

The WBC product was issued for other research purposes, 

and the plasma was centrifuged at 4900 ¥ g for 6.5 minutes 

shortly after collection, 100 mL of which was transferred 

to a 600-mL bag. Approximately 100 mL of thrombin and 

1 mL of 10 percent CaCl2 were added to the plasma; the 

bag was mixed vigorously and placed into a 2 to 8°C monitored refrigerator overnight. The bag was centrifuged the 

next day at 4900 ¥ g for 6.5 minutes and the supernatant 

(serum) was transferred to a 300-mL bag. Serum was then 

aliquoted into 2-mL vials and placed into a monitored 

-30°C freezer. Serum was kept at -30°C until the day of 

testing, when it was removed from the freezer, thawed at 

20 to 24°C, and used in the test system shortly after 

thawing. 

In performing the hemolysis assays, we added 1 drop 

of the RBC suspension to 200 mL of WinRho diluted in 

group AB serum. We mixed and incubated the tubes for 1 

hour at 37°C and then mixed, centrifuged at 1000 RCF 

(1000 ¥ g) for 2 minutes, and examined the tubes for the 

hemolysin assay endpoint of macroscopically visible 

supernatant hemolysis.

26 We suspended the RBCs for both 

the positive and the negative controls in group AB serum 

and followed the mixing, incubation, mixing, centrifugation, and examination procedure that was used with the 

hemolysin assays. For a positive control, we used group O 

penicillin-coated RBCs, coated according to a standardized procedure,26 and an IgG anti-penicillin. The antipenicillin was well-characterized donor plasma that had 

demonstrated 3+ agglutination with penicillin-coated 

4 TRANSFUSION Volume ** , ** ** 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

MECHANISM OF ANTI-D-IGIV ACUTE HEMOLYSIS

AHTRs involve RBC antigen-antibody– 

TABLE 1. RBC antibody identities in WinRho lots tested mediated hemolysis. In most ITP 

WinRho lots tested RBC antibodies patients treated with WinRho, the 

WinRho lot Expiration date 1-in-10 dilution Physiologic dilution “expected” hemolysis is presumed 

0110501 July 2007 Anti-D, -C, -Jsa

* Anti-D, -C attributable to IgG anti-D,1 which 0120502 September 2007 Anti-D, -C, -A, -B, -Jka Anti-D, -C, -A, -B 

0240501 January 2008 Anti-D, -C, -A Anti-D, -C typically causes extravascular hemolysis23,27,28 1460402 April 2006 Anti-D, -C Anti-D, -C in D+ patients. In both text1460501 March 2007 Anti-D, -C, -A Anti-D books and the literature, extravascular 1470403 February 2006 Anti-D, -C, -A, -B Anti-D, -A 

1480410 November 2006 Anti-D, -C Anti-D, -C hemolysis has traditionally been char­

* Antibody could not be ruled out. 

RBCs in previous testing and hemolyzed penicillin-coated 

RBCs when a source of complement was added (see 

above). For a negative control, we used group O unsensitized RBCs. 

RESULTS

RBC antibody identities in WinRho lots

In Table 1, we list the RBC antibodies identified in the 

WinRho lots. We confirmed the presence of anti-D in all 

lots at both the 1-in-10 and the physiologic dilutions. In 

five lots, we detected other RBC antibodies in the 1-in-10 

dilutions that were not detected in the physiologic dilutions. However, in only two lots did we detect RBC antibodies other than the expected anti-D, anti-A, anti-B, 

anti-C, and anti-E,1 and we detected those other RBC antibodies in only the 1-in-10 dilutions. 

Hemolysin assays

We observed no hemolysis endpoints with any of the 

hemolysin assays in which each of the seven WinRho lots 

was tested against each of the 73 normal blood donor, 

reagent, patient, and patient facsimile RBCs. We observed 

a hemolysis endpoint in the positive control and no 

hemolysis endpoint in the negative control. Because the 

positive control showed a hemolysis endpoint, we concluded that the test system indirectly validated that there 

were adequate amounts of complement and calcium ions 

in our converted plasma/complement source and that the 

anti-penicillin was not diluted beyond the limits of detection of our hemolysin assay. The negative control yielded 

the expected result, which we interpreted as further validation of our test system. 

DISCUSSION

Mechanism of acute hemolysis in AHTRs

compared to anti-D IGIV

Both the presumed mechanism of action of WinRho in the 

treatment of ITP and the established mechanism of 

acterized by delayed onset, indirect 

hyperbilirubinemia, and urobilinogenuria and seldom results in morbidity or 

clinical sequelae.23,26,27,29,30 In contrast, 

the hemolysis in AHTRs is generally due to IgM anti-A or 

anti-B and typically results in complement-mediated 

intravascular hemolysis in correspondingly antigenpositive patients.23,26-30 That hemolysis is generally 

characterized by acute onset, hemoglobinemia (and 

hemoglobinuria), and potentially life-threatening complications of shock, renal failure, and disseminated intravascular coagulation.23,26,27,29,30 

The mechanism of acute hemolysis associated with 

WinRho administration has not yet been definitively 

established as “intravascular hemolysis” in terms of RBC 

antigen-antibody–mediated complement activation, or 

any other mechanism(s) of hemolysis,1,14,17 and remains 

unexplained. If attributable to anti-D, the mechanism of 

the acute hemolysis is perplexing, given the generally 

non–complement-fixing behavior of anti-D;23,28-30 the relatively limited number of D antigens per RBC;23,24 the distance between these antigens, which exceeds the span of 

IgG anti-D;23,24 and the lack of complement activators in 

WinRho.2,19 

However, WinRho contains multiple RBC antibodies1,31 that, at least in theory, could collectively sensitize a 

critical mass of RBC antigens in correspondingly antigenpositive patients, precipitate RBC antigen-antibody– 

mediated complement activation, and present with the 

time of onset, signs/symptoms, and complications of 

AHTRs.14,17,28 RBC antibodies contained in WinRho include 

high-titer IgG anti-D as well as low-titer IgG anti-A, anti-B, 

anti-C, and anti-E, all of which can be passively acquired.1 

The manufacturer quantitatively assays these antibodies 

before release of lots for market distribution to ensure 

compliance with FDA-recommended specifications. 

WinRho may also contain other low-titer RBC IgG antibodies (e.g., anti-Fya

, anti-Jka),31 some of which have been 

implicated in AHTRs.23,27,29,32 These other RBC antibodies 

exhibit lot-to-lot variability in identities and titers31 and 

can likewise be passively acquired,31 but are not routinely 

assayed before lot release for market distribution. 

Thus, the complete inventory of RBC antibody identities and titers in WinRho lots in market distribution is 

unknown, as is the extent of variation among lots. Given 

the diversity in patient RBC antigen phenotypes, the 

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GAINES ET AL.

potential lot-to-lot variability in RBC antibody identities 

and titers could explain why only some patients experience acute hemolysis with seemingly random WinRho 

lots.14 Similarly, the potential lot-to-lot variability could 

explain why a given patient experiences an uneventful 

administration with a WinRho lot yet experiences acute 

hemolysis with another WinRho lot administered on 

another occasion.14,17 

Explanations for the negative results of the study

The negative result for the hemolysin assay that was performed with RBCs from the patient who experienced 

acute hemolysis and the WinRho SDF lot administered to 

her did not support the premise that her acute hemolysis 

was consistent with the AHTR model. If, however, the 

AHTR model is the mechanism of the WinRho-related 

acute hemolysis, future research could consider other 

variables that were beyond the scope of, and not 

addressed in, this study. Other technical variables could 

include modifications of the hemolysin assay procedure 

used in this study (e.g., other dilutions of the WinRho lots, 

use of enzyme-treated RBCs) or use of another procedure. 

Although few of the case series patients who experienced 

WinRho-associated acute hemolysis had been recently 

transfused, we cannot rule out that recent transfusion 

history may account for the acute hemolysis of some 

patients. Recent transfusion with ABO-incompatible 

plasma from PLTs22,30,33 could sensitize patient RBCs with 

IgG and/or complement,26,33,34 which, in conjunction with 

passively acquired RBC antibodies from WinRho,23 might 

initiate RBC antigen-antibody–mediated complement 

activation. 

If the AHTR model is not the mechanism of the 

WinRho-related acute hemolysis, future research could 

investigate other immune-mediated and alternative 

mechanisms of hemolysis. The role of complement regulatory proteins CD55 (decay accelerating factor), CD59, 

and complement receptor type 1 (CR1, CD35) that 

may inactivate complement on RBCs and prevent 

complement-mediated hemolysis23,26,33-36 might warrant 

further evaluation. Although not often cited in textbooks 

and the literature, extravascular hemolysis has been associated with hemoglobinemia and hemoglobinuria in 

what has been termed an “acute extravascular hemolytic 

transfusion reaction.” 23,26,30 By this mechanism, the acute 

hemolysis of anti-D–sensitized RBCs is due to splenic 

macrophage lysozymes and/or fragmentation of anti-D– 

sensitized RBCs during phagocytosis by splenic macrophages with release of free Hb into the circulation.23,26,30 

Another potential immune-mediated mechanism of 

RBC hemolysis that could be considered involves cellmediated immune hemolysis by “armed macrophages,” in 

which bound antibody reacts with nonsensitized RBCs 

6 TRANSFUSION Volume ** , ** ** 

that possess the corresponding antigen and leads to more 

extensive hemolysis than would be seen with untreated 

macrophages alone.26,37,38 Finally, the possibility of circulating lymphocyte-mediated cytotoxicity cannot be 

excluded as a cause of, or contributory factor to, the acute 

hemolysis.26,39 

Regardless of the mechanism(s) by which acute 

hemolysis may occur after WinRho administration, other 

patient characteristics (e.g., degree of splenomegaly) 

examined14,15,17,40-48 beyond those previously could be 

evaluated as potential predictors of, and factors related to, 

the acute hemolysis. In terms of other characteristics of 

WinRho composition, environmental conditions of 

WinRho storage, or anomalies of WinRho reconstitution 

or administration, we assess as unlikely that these could 

be implicated as causative factors in the acute hemolysis 

in the absence of lot-related clusters of cases. 

Limitations of this study

We acknowledge several notable limitations of this study. 

We are unaware of any reference(s) or data that cite or 

discuss the sensitivity, specificity, or predictive power of 

the hemolysin assay used in this study for detecting 

RBC antigen-antibody–mediated complement activation. 

Neither can we judge the predictive power of the hemolysin assay as an in vitro corollary to in vivo physiologic 

conditions. We recognize that anti-D-IGIV–associated 

acute hemolysis could, in fact, model the AHTR mechanism but that the hemolysin assay used in this study did 

not possess the power to test our premise. We tested only 

1-in-10 and physiologic dilutions of WinRho. Although 

other dilutions of WinRho could have been included, we 

elected to test only the 1-in-10 and physiologic dilutions, 

given that the purpose of our study was to determine the 

mechanism by which WinRho causes acute hemolysis, not 

if WinRho could be made to hemolyze RBCs. 

We tested only seven WinRho lots for RBC antibody 

identities (although four of those lots had been implicated 

in cases of acute hemolysis). We have no means to assess 

how representative the RBC antibody identities and titers 

of those lots were when compared to other WinRho lots. 

We tested only 73 RBCs in the hemolysin assays, 40 (55%) 

of which were from normal blood donors, who were 

otherwise healthy, and 32 (44%) of which were reagent 

RBCs. Whether there were relevant qualitative differences 

between these RBCs and the RBCs of ITP or secondary 

thrombocytopenic patients is indeterminate. If the AHTR 

model is applicable, but given the low estimated reporting 

rates for WinRho-associated acute hemolysis,14,17 we 

would have needed to test an impracticably large number 

of normal blood donor and reagent RBCs to randomly 

encounter an RBC phenotype/genotype and WinRho lot 

RBC antibody combination that would have yielded a 

hemolysis endpoint in our hemolysin assay. 
 

 

 

 

MECHANISM OF ANTI-D-IGIV ACUTE HEMOLYSIS

We were able to test RBCs from only a single patient 

who experienced acute hemolysis after WinRho SDF 

administration for ITP. Had it been feasible, we would 

have elected to test RBCs from the patient that had been 

collected immediately before and/or at the time of her 

acute hemolytic episode. We cannot be certain whether 

relevant qualitative differences existed in her RBCs at the 

time that she received WinRho SDF and 4 months afterward. In fact, there was a qualitative difference in that her 

pre-WinRho SDF DAT was negative while her 4-month 

post–WinRho SDF DAT was weakly positive (presumably 

due to anti-D but not confirmed). However, the significance, if any, of that difference cannot be assessed. 

Neither can we be certain whether there were relevant 

qualitative differences between her RBCs and the patient 

facsimile RBCs tested. Of note, the WinRho SDF lot implicated in her acute hemolytic episode contained no other 

RBC antibodies beyond those expected to be present. Also 

of note, she had experienced acute hemolysis postWinRho SDF, yet her RBCs were only of an antigen density 

“as expected for a [D antigen] homozygote.” 

The other two patients who had experienced acute 

hemolysis after administration of the other purposively 

sampled WinRho lots had died. Consequently, we were 

unable to obtain blood samples from those patients for 

testing in this study or locate RBC phenotype data in their 

medical records that would have allowed for patient facsimile testing. Testing of RBCs from other patients who 

have experienced acute hemolysis post-WinRho administration may be informative in future research. 

We tested only WinRho because it was the only 

anti-D IGIV that was FDA-licensed for treatment of ITP 

at the time that this study was initiated. However, cases 

of acute hemolysis involving other brands of anti-D 

IGIVs administered for treatment of ITP or secondary 

thrombocytopenia have been reported in clinical studies49 and in the literature.15,43,50 After we initiated our 

study, the FDA licensed Rhophylac (ZLB Bioplasma AG, 

Bern, Switzerland) in February 2004 for suppression of 

Rh isoimmunization49 and in March 2007 for treatment 

of chronic ITP in Rho(D)-positive, nonsplenectomized 

adults.49 Other anti-D IGIVs that are produced by other 

manufacturers are licensed in other countries. Future 

research into the mechanism of the anti-D-IGIV– 

associated acute hemolysis should include other brands 

of anti-D IGIV as well. 

Summary

Based on the negative results obtained with the hemolysin 

assay used for this study, we doubt that the acute hemolysis after WinRho SDF administration in the patient whose 

RBCs were tested is consistent with the AHTR model. Just 

as more than one mechanism may account for PLT 

responses in anti-D IGIV–treated ITP patients, multiple 

mechanisms may contribute to anti-D-IGIV–associated 

acute hemolysis and could account for the acute hemolysis experienced by the patient whose RBCs were tested as 

well as for differences among patients. Understanding the 

mechanism(s) of the acute hemolysis might allow us to 

develop contraindications or strategies for anti-D IGIV 

treatment of ITP and secondary thrombocytopenia 

patients that could minimize the risk of acute hemolysis– 

associated morbidity and mortality. Any and all potential 

causes or mechanisms of anti-D-IGIV–associated acute 

hemolysis warrant further evaluation with targeted 

studies unless and until other patient risk factors or predictive tests for the acute hemolysis can be identified. 

ACKNOWLEDGMENTS

The authors express their appreciation to the patient who experienced acute hemolysis after WinRho SDF administration and 

who agreed to provide us with blood specimens for this study. The 

authors would also express our appreciation to Robert Wise and 

M. Miles Braun, Office of Biostatistics and Epidemiology, and 

Susan Abbondanzo, Office of Blood Research and Review, Center 

for Biologics Evaluation and Research, FDA, for their review of 

the manuscript. The authors declare no competing financial 

interests. 

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