Triple Red Blood Cell Alloantibody Formation After Bone-Allograft Transplantation


Corresponding author: Lenneke Prinzen,


In this case report, we provide evidence for the possibility of red blood cell alloimmunization after bone-allograft transplantation. Here, we present a 13-year-old boy who received a bone allograft due to impending hip-luxation. Five months later he was shown to have developed three different alloantibodies: anti-D, anti-C and anti-E, which were induced by the bone allograft. Red blood cell alloimmunization is a possible adverse event when a patient is exposed to allogenic red blood cells. These antibodies may cause transfusion reactions when incompatible blood is administered. More importantly, these antibodies may cause severe, or even fatal, hemolytic disease of the fetus or newborn, stretching the importance of preventing antibody formation, especially in young women. This case demonstrates the importance of selecting rhesus phenotype compatible bone allografts.


direct antiglobulin test


dithiothreitol test


hemolytic disease of the newborn


red blood cell.

Case Report

A 13-year-old boy with spastic diplegia, probably caused by hypoxic encephalopathy at birth, had to undergo two surgeries because of impending bilateral hipluxation. The patient's history reveals one prior surgery to the lower limb muscles without blood transfusion. The surgeries were scheduled 5 months apart; during both he received femoral bone allografts. Prior to implantation, the bone-allografts, recovered from living donors and stored at −80°C for at least 6 months, were washed in saline and antibiotics. The allograft used during the first surgery had a size of approximately 75 g in weight and 7 mL in volume.


Blood was collected in ethylenediaminetetraacetate. Routinely, type and screen procedure is performed as follows. ABO and rhesus D bloodgroups are typed automatically (ID DiaClon ABO/D + Reverse Grouping, Biorad, Cressier, Switzerland), as well as the three-cell screening (ID-Diacell, Biorad, Cressier, Switzerland) on Diamed WA Diana (Diamed, Cressier, Switzerland) in a low ionic salt solution (LISS). Eleven-cell ID-Diapanel (Biorad, Cressier, Switzerland) and 16-cell Macropanel (Sanquin, Amsterdam, the Netherlands) were used for further analysis of antibody specificity using the indirect agglutinin test (IAT) in Gel-Test ID-Liss/Coombs cards (Biorad, Cressier, Switzerland). Antibody analysis was also performed using papainized test cells (ID-Diacell P/ ID-panel P, Biorad, Cressier, Switserland) in ID-cards ‘NaCl enzyme test and cold agluttinins’ (Biorad, Cressier, Switzerland), according to the manufacturer's instructions. A direct antiglobulin test (DAT) was performed using Gel-Test ID-Liss/Coombs cards (Biorad, Cressier, Switzerland) according to the manufacturer's instructions. Rhesus phenotype and K-antigen were also determined by the Diamed ID-system using ID-Diaclon Rh-subgroups+K cards (Biorad, Cressier, Switzerland). A dithiothreitol test (DTT) was performed by adding one part of 0.1M DTT to one part of serum followed by a 15-minute incubation at 37°C. This is then analyzed for antibody specificity in LISS and papainized test cells as described above. As a control, non-DTT treated samples were checked for dilution effects by 1:1 dilution with phosphate-buffered saline and analyzed similarly.


Preoperative screening for red blood cell (RBC) antibodies was routinely performed. The patient was typed as bloodgroup 0, rhesus D negative. Initial screening was negative; however prior to the second surgery three RBC antibodies were identified: anti-D (2+ in LISS, 4+ in papainized cells), anti-E (1+ in LISS, 3/4+ in papainized cells) and anti-C (2/3+ in LISS, 4+ in papainized cells). DAT was negative. The patient's rhesus phenotype was ccdee and K negative, confirming that the antibodies were allogenic. We had no documentation of prior transfusions, and since he was born in our hospital, and had always been treated here, the chance of previous allogenic blood transfusion was negligible. This was also confirmed by his mother. Therefore, the only reasonable explanation for immunization was the first bone allograft. This was confirmed by the blood group and rhesus phenotype of the first donor: blood group 0, rhesus phenotype CcDEe and K negative. The second donor had blood group A and was rhesus D positive (rhesus phenotype unknown). Screening was repeated 6 weeks after the second hip surgery in order to confirm the first positive screening and was again positive for anti-D, anti-C and anti-E (similar reaction strengths). The possibility of naturally occurring antibodies (IgM antibodies) had been ruled out for anti-D and anti-C by DTT (14), confirming the IgG isotype. For anti-E this could not be confirmed, due to weaker reaction strengths.


RBC alloimmunization is known to occur during pregnancy or labor due to fetomaternal transfusion, after allogenic blood transfusions, after bone marrow or stem cell transplantation, or transplantation of vascularized organs such as kidneys. Here, we also show that after transplantation of nonvascularized tissue, such as a femoral bone allograft, RBC alloimmunization can take place. Storage conditions and washing procedure make it unlikely that intact RBCs or bone marrow stem cells are still present in the bone allograft. However, antigen-presenting remnants may still be confined inside the allograft. There is no literature available about RBC remnants in frozen bone allografts. However, a unit of fresh frozen plasma, containing fewer than 1 × 108 RBCs, is considered safe with respect to the risk of rhesus D immunization [1]. This implies that more RBC remnants were present in the used bone allograft in this case. This further implies that larger allografts may have a higher risk of alloimmunization, since they likely contain more remnants.

In the unlikely case the patient had received previous allogenic blood transfusions, a secondary instead of a primary immune response may have been induced by the bone allograft. In a percentage of cases, RBC antibodies are known to become undetectable over time [2], and a secondary response can therefore not be ruled out completely. However, if our patient had ever been transfused, he would have received rhesus D negative blood, as this blood group is virtually always considered when administering packed cells. Rhesus D positive platelet concentrates can be given to rhesus D negative patients, and immunization may occur due to few RBCs present. Despite the fact that the possibility of having received prior transfusions cannot be ruled out completely, we find this very unlikely. According to our patient's file neither packed cells, nor platelet concentrates or fresh frozen plasma was ever administered, and this possibility was also convincingly denied by the patient's mother.

Another source for primary immunization has been described in the literature: previous exposure to microbes may be a risk factor for RBC immunization when subsequently exposed to foreign RBCs. This primary immunization would not be detected serologically in the hematology laboratory, and can therefore not be ruled out in our case. However, it has been implied that this is not very likely in the case of rhesus antibodies [3].

Alloimmunization after blood transfusion is not an uncommon finding in the hematology laboratory. Approximately 2–8% of patients receiving blood transfusions develop alloantibodies [4, 5]. Little is known about RBC alloimmunization after organ transplantation. Presumably, this is not very common in organ-transplant patients due to the use of immunosuppressants. However, in many bone-transplant cases no immunosuppressants are used, as in our patient, which may explain why alloimmunization did occur. This suggests that alloimmunization occurs more often after bone-allograft transplantation; however, patients are usually not screened for RBC antibodies postoperatively, leaving antibody formation unnoticed.

As in this case, RBC antibodies are clinically relevant, and may cause a severe transfusion reaction when given mismatched blood. A similar occurrence in a female patient may, also, complicate future pregnancies as RBC antibodies may cause severe, potentially fatal, hemolytic disease of the newborn (HDN) by crossing the placenta and entering the fetal circulation. HDN may be caused by most rhesus-antibodies, but anti-D is known to bear the highest risk of causing severe HDN, defined as a fetus requiring intrauterine transfusion, a neonate requiring exchange transfusion, or perinatal mortality [6]. Once anti-D immunization has occurred, the risk of severe HDN during pregnancy is 19–25% [6]. HDN due to anti-D in the general population has, in many countries, been greatly reduced due to post- and antenatal anti-D prophylaxis. In the Netherlands, the prevalence of new pregnancy-induced anti-D immunizations was reduced from 3.5% in 1969 before anti-D prophylaxis, to 0.67% with only postnatal prophylaxis, to 0.31% with both ante- and postnatal prophylaxis [6]. More importantly, the prevalence of severe HDN was also found to decrease [6]. Despite the fact that anti-D prophylaxis had remarkably reduced the risk of rhesus D immunization in rhesus D negative pregnant women, the underlying immunological mechanism has not been clarified completely [7]. One hypothesis is that anti-D prophylaxis clears rhesus D positive fetal RBCs from the maternal circulation before immunization occurs [7]. However, whether or not anti-D prophylaxis would prevent anti-D formation in bone-allograft patients remains speculative, especially because it is unlikely that intact donor RBCs circulate after bone-allograft transplantation.

The risk of anti-C or anti-E causing severe HDN is much smaller (<5%), but still present [8]. Other RBC antibodies are also known to cause severe HDN. The risk of anti-c causing severe HDN is 10.2% [8]. K is a highly immunogenic, non-rhesus blood group with a prevalence of only 9% in the Caucasian population, meaning that 91% is able to form alloantibodies when exposed to K-antigen. Anti-K is clinically important: it has a high risk of causing severe HDN (26.3%) [8]. In the Netherlands, c-, E- and K-compatible blood is always given to women of childbearing age or younger to prevent immunization.

Only seven, presumable, cases of RBC alloimmunization after bone allografting have been described in the literature [9-11], all females. All of them were already of childbearing age. Six had denied previous pregnancies; however, this possibility can never be fully excluded. The seventh case was a female with HDN during her first pregnancy due to anti-D, suggesting that she was alloimmunized before gestation [12]. On the other hand, a study of 144 patients with transplanted cancellous bone chips did not show alloimmunization [13]. Among these, 30 out of 37 rhesus D negative patients received bone allografts from rhesus D positive donors. However, no other blood group or additional patient information was presented. Furthermore, the authors acknowledge that only small amounts of bone were transplanted.

We report here the first male patient who developed RBC alloantibodies after bone-allograft transplantation, providing evidence that bone allografting can cause primary RBC alloimmunization. Allogenic bonegrafts are used extensively among different specialties; such as orthopedic and oral surgery, otolaryngology, traumatology and neurosurgery. Concluding, we propose transplanting rhesus and K compatible bone allografts whenever possible, especially when women of childbearing age or younger are concerned, in order to reduce the chance of RBC immunization and thus severe HDN in the future.


The authors acknowledge Mrs. Helma Voets, physician assistant at the Department of Orthopedic Surgery, for her assistance with the donor-related data collection.


No funding was received. The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.