Abstract

Several acute hemolysis episodes, sometimes lethal, have been recently described after transplantation of allogeneic peripheral blood hematopoietic stem cells (PBHSCs). Hemolysis resulted from the production of donor-derived antibodies (Abs) directed at ABO antigens (Ags) present on recipient red blood cells (RBCs). A multicenter randomized phase III clinical study comparing allogeneic PBHSC transplantation (PBHSCT) versus bone marrow hematopoietic stem cell transplantation (BMHSCT) has been conducted in France. In the course of this study, serum anti-A and/or anti-B Ab titers were compared before the conditioning regimen and on day +30 after transplantation in 49 consecutive evaluable PBHSCT (n = 21) or BMHSCT (n = 28) recipients. PBHSCT resulted in a higher frequency of increased anti-A and/or anti-B Ab titers 30 days after transplantation as compared to BMHSCT: 8 (38%) of 21 versus 3 (11%) of 28 (P = .04). In PBHSCT recipients, increased titers were observed mostly after receiving a minor ABO mismatch transplant: 5 of 7 versus 3 of 14 in the absence of any minor ABO mismatch (P = .05), whereas this was not the case after BMHSCT: 1 of 8 versus 2 of 20. Anti-A and/or anti-B serum Abs detectable at day +30 after PBHSCT were always directed against A and/or B Ags absent both on donor and recipient RBCs. Finally, 3 of 21 PBHSCT versus 0 of 28 BMHSCT recipients developed anti-allogeneic RBC Abs other than ABO (P = .07). Overall, the data strongly suggest that immunohematologic reconstitution differs significantly after granulocyte colony-stimulating factor–mobilized PBHSCT when compared to BMHSCT. Such a difference could contribute to the acute hemolysis described after PBHSCT as well as to distinct alloreactivity after PBHSCT.

Introduction

The use of peripheral blood after granulocyte colony-stimulating factor (G-CSF) mobilization as a source of allogeneic hematopoietic stem cells (HSCs) is being increasingly considered.1-10 The high number of HSCs in such a graft might result in enhanced engraftment and accelerated hematopoietic recovery.6-9 Immune reconstitution and overall alloreactivity after peripheral blood HSC transplantation (PBHSCT) might also be significantly different from what is observed after bone marrow (BM) HSC transplantation (BMHSCT).6-12 Indeed, despite an increase in the number of donor T cells infused with a peripheral blood HSC (PBHSC) graft, the incidence of grade II or greater acute graft-versus-host disease (GvHD) appears to be similar or even less than after BMHSCT. However, a higher risk of chronic GvHD has been reported after allogeneic PBHSCT.7,11,13 

Several cases of severe, potentially lethal cases of acute intravascular hemolysis because of the production of donor-derived antibodies (Abs) directed against ABO antigens (Ags) present on recipient red blood cells (RBCs) and not on donor RBCs (ABO “minor” incompatibility) have been described after PBHSCT14-20(Table 1), thus suggesting an influence of such a type of graft on posttransplantation immunohematologic reconstitution.

Table 1.

Summary of reported cases of hemolysis after minor (± major) ABO-incompatible peripheral blood hematopoietic stem cell transplantation

ReferenceRBC group
donor/
recipient
Gender
donor/
recipient
DiagnosisCD34+/kg
× 106
Donor anti-A
and/or
anti-B Ab
Group of
RBC
transfused
GvHD
prophylaxis
Day of
hemolysis
Hemolysis treatment
(other than transfusion)
and follow-up
Laurencet et al14 O/A F/M Myeloma 5.0 Anti-A: 1/64
anti-B: 1/128 
CSA alone 12 Steroids, forced diuresis
Alive  
Toren et al15 O/A F/M ALL 5.0 Anti-A: 1/32 NR CSA alone Steroids, blood exchange
Alive 
Oziel-Taieb et al16 O/A F/M Myeloma 3.5 Anti-A:
IgG: 1/8;
IgM: 1/8 
CSA alone No treatment reported
Death day 20  
Moog et al17 A/B M/M AML 10 NR CSA alone Forced diuresis
Alive  
Lyding et al18 A/B M/F Erythrophagocytic
lymphohistiocytosis 
11.5 NR CSA and MTX:
d + 1, d + 3,
d + 6 
13 Steroids
Severe acute hemolysis
Death day 48
(multiorgan failure) 
Fitzgerald et al19 O/A F/M ALL NR NR CSA alone Steroids, plasmapheresis
Severe acute hemolysis
Death day 117
(infection + GvHD)  
Salmon et al20 O/A F/M
(mother) 
AML 12.23 Anti-A: 1/2048 O
(from mother) 
CSA alone Steroids, plasmapheresis
Death day 35
(massive gastrointestinal
bleeding + liver failure + GvHD) 
ReferenceRBC group
donor/
recipient
Gender
donor/
recipient
DiagnosisCD34+/kg
× 106
Donor anti-A
and/or
anti-B Ab
Group of
RBC
transfused
GvHD
prophylaxis
Day of
hemolysis
Hemolysis treatment
(other than transfusion)
and follow-up
Laurencet et al14 O/A F/M Myeloma 5.0 Anti-A: 1/64
anti-B: 1/128 
CSA alone 12 Steroids, forced diuresis
Alive  
Toren et al15 O/A F/M ALL 5.0 Anti-A: 1/32 NR CSA alone Steroids, blood exchange
Alive 
Oziel-Taieb et al16 O/A F/M Myeloma 3.5 Anti-A:
IgG: 1/8;
IgM: 1/8 
CSA alone No treatment reported
Death day 20  
Moog et al17 A/B M/M AML 10 NR CSA alone Forced diuresis
Alive  
Lyding et al18 A/B M/F Erythrophagocytic
lymphohistiocytosis 
11.5 NR CSA and MTX:
d + 1, d + 3,
d + 6 
13 Steroids
Severe acute hemolysis
Death day 48
(multiorgan failure) 
Fitzgerald et al19 O/A F/M ALL NR NR CSA alone Steroids, plasmapheresis
Severe acute hemolysis
Death day 117
(infection + GvHD)  
Salmon et al20 O/A F/M
(mother) 
AML 12.23 Anti-A: 1/2048 O
(from mother) 
CSA alone Steroids, plasmapheresis
Death day 35
(massive gastrointestinal
bleeding + liver failure + GvHD) 

RBC indicates red blood cell; Ab, antibody; CSA, cyclosporin A; ALL, acute lymphoid leukemia; NR, not reported; Ig, immunoglobulin; AML, acute myeloid leukemia; MTX, methotrexate; GvHD, graft-versus-host disease.

In addition, Bornhäuser et al21 have described a case of acute hemolysis occurring at day 5 after an unrelated peripheral blood hematopoietic stem cells transplantation with minor and major ABO incompatibility (donor B; recipient A). Immunohematologic assessment at time of hemolysis was not reported.

All cases described concern HLA identical sibling allograft except for Salmon et al.20 In this case, the donor was the recipient's mother who had a major HLA class II mismatch.

A multicenter randomized phase III clinical trial comparing allogeneic BMHSCT to allogeneic PBHSCT has recently been conducted by the Société Française de Greffe de Moelle (SFGM).7 The phenotype and function of immune cells in the HSC grafts as well as the quality of immune reconstitution in patients 30 days after transplantation were evaluated in a cohort of consecutive patients enrolled in the trial.

We have taken advantage of this randomized study to prospectively compare the kinetics of immunohematologic reconstitution after PBHSCT versus BMHSCT. More specifically, and in view of the severe cases of hemolysis recently described, we postulated that the use of PBHSCT might be associated with an accelerated increase in donor-derived anti-RBC Abs.

Patients, materials, and methods

Patients and blood sample collection

Between June 1997 and February 1999, 122 patients were enrolled in a clinical multicenter SFGM phase III randomized study that compared allogeneic PBHSCT versus BMHSCT. The protocol was approved by an ethics committee (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale of Marseille 2) and was conducted with respect to the Helsinki accords for human subject research. All procedures were performed after written, informed consent was given by the donor and the recipient. In June 1998, a prospective immunobiological evaluation was initiated for patients subsequently entered in the study. This evaluation included white blood cell immunophenotyping and immunohematologic assessment of the donor (before G-CSF administration [for PBHSCT recipients] and at the time of HSC graft harvest [for both groups]) as well as of the recipient before and 30 days after transplantation. In addition, cell immunophenotyping was performed on the PBHSC or BM graft. Blood and graft samples were shipped by overnight mail to the Etablissement Français du Sang–Bourgogne/Franche-Comté and were immediately processed or cryopreserved (viable cells and serum). From June 1998 to February 1999, 49 (74%) of 66 consecutive randomized patients (PBHSCT, n = 21; BMHSCT, n = 28) were included in the immunohematologic study. Seventeen patients were not included because of missing (nonharvested) samples.

Clinical study

The clinical study design and results have been previously reported.5 The randomization was stratified by diagnosis and by center to minimize the variations resulting from different practices in terms of supportive care, GvHD prophylaxis, or transfusions policies. Patient and recipient characteristics are described in Table 2. In the PBHSCT arm, donors received 10 μg/kg/d of subcutaneous G-CSF (Filgrastim; Rhône-Poulenc-Rhorer, Montrouge, France) for 5 days. On the 5th day (day −1 of transplantation), the first HSC harvest was performed by apheresis. If the CD34+ cell count in the HSC bag was less than 4 × 106/kg of recipient body weight, a second harvest was performed on the 6th day. G-CSF was administered on the 6th day if a third harvest was necessary at day +1. GvHD prophylaxis comprised cyclosporin A (CSA) (initiated at day −1) and methotrexate (MTX) (15mg/m2 on day +1; 10 mg/m2 on days +3 and +6). The CSA was started intravenously on day −1 at the dosage of 2 to 3 mg/kg/d and was switched to oral formulation as soon as oral intake was satisfactory. The dosage was adapted to whole blood or plasma level and renal function according to each center's practice. No recipient received G-CSF during the immunohematologic study period.

Table 2.

Donor and recipient characteristics

BMHSCT group (N = 28)PBHSCT group (N = 21)P
Age, y, median (range)    
 Recipients 33  (16-50) 35  (21-49) .50  
 Donors 37  (20-63) 33  (21-50) .38  
Sex mismatch (donor/recipient)    
 Female/female (parous donor) 3  (11%) [2] 3  (14%) [1]  
 Female/male (parous donor) 6  (21%) [2] 6  (29%) [6]  
 Male/female 11  (39%) 4  (19%)  
 Male/male 8  (29%) 8  (38%) .50 
Donor pregnancies    
 Nulliparous female 5  (18%) 2  (10%)  
 Parous female 4  (14%) 7  (33%)  
 Male 19  (68%) 12  (57%) .34  
ABO group mismatch    
 Identical 15  (54%) 10  (48%)  
 Minor 8  (29%) 7  (33%)  
 Major 4  (14%) 3  (14%)  
 Minor + major 1  (3%) 1  (5%) .96  
Diagnosis    
 ALL 7  (25%) 2  (9%)  
 AML 10  (36%) 10  (48%)  
 CML 11  (39%) 9  (43%) .48  
Conditioning regimen    
 TBI-EDX 17  (61%) 13  (62%)  
 BUS-EDX 6  (21%) 5  (24%)  
 VP16-EDX-TBI or TAME 5  (18%) 3  (14%) 
BMHSCT group (N = 28)PBHSCT group (N = 21)P
Age, y, median (range)    
 Recipients 33  (16-50) 35  (21-49) .50  
 Donors 37  (20-63) 33  (21-50) .38  
Sex mismatch (donor/recipient)    
 Female/female (parous donor) 3  (11%) [2] 3  (14%) [1]  
 Female/male (parous donor) 6  (21%) [2] 6  (29%) [6]  
 Male/female 11  (39%) 4  (19%)  
 Male/male 8  (29%) 8  (38%) .50 
Donor pregnancies    
 Nulliparous female 5  (18%) 2  (10%)  
 Parous female 4  (14%) 7  (33%)  
 Male 19  (68%) 12  (57%) .34  
ABO group mismatch    
 Identical 15  (54%) 10  (48%)  
 Minor 8  (29%) 7  (33%)  
 Major 4  (14%) 3  (14%)  
 Minor + major 1  (3%) 1  (5%) .96  
Diagnosis    
 ALL 7  (25%) 2  (9%)  
 AML 10  (36%) 10  (48%)  
 CML 11  (39%) 9  (43%) .48  
Conditioning regimen    
 TBI-EDX 17  (61%) 13  (62%)  
 BUS-EDX 6  (21%) 5  (24%)  
 VP16-EDX-TBI or TAME 5  (18%) 3  (14%) 

BMHSCT indicates bone marrow hematopoietic stem cell transplantation; PBHSCT, peripheral blood hematopoietic stem cell transplantation; ALL, acute lymphoid leukemia; AML, acute myeloid leukemia; CML, chronic myeloid leukemia; TBI, total body irradiation (median, 12 Gy [range, 11-13.5]); EDX, cyclophosphamide (120 mg/kg); BUS, busulfan (16 mg/kg per os); VP16, vepeside (20 mg/kg); TAME, TBI + aracytine (12 g/m2) + melphalan (140 mg/m2) + etoposide (60 mg/kg).

Biological samples

Citrate-anticoagulated peripheral blood and serum samples (Vacutainer, Becton Dickinson, Le Pont de Claix, France) were obtained from the BM donors before or at the time of BM harvest, from the PBHSC donors before and after G-CSF mobilization (ie, before the first apheresis), and from recipients before the conditioning regimen and at day +30 after transplantation. The use of citrate as blood anticoagulant was chosen for the sake of homogeneity between both types of harvests, as PBHSCs were collected in a bag containing citrate.

Immunohematologic assessment

Preparation of reagent RBCs.

For anti-A and anti-B titration, 3 suspensions of A1Rh+, BRh+, and ORh reagent RBCs (human reagent red blood cells for the reverse determination of ABO grouping, Sanofi Diagnostics Pasteur, Marnes-la Coquette, France) were used. The reagent RBCs were suspended to a concentration of 40% in a buffered preservative solution. The direct antiglobulin test for each reagent RBC sample was negative. Each preparation of reagent RBCs was washed 3 times in 0.15 M saline solution (9% NaCl). The hematocrit of each suspension was then adjusted to 3% in 0.15 M saline solution.

Serum anti-A and anti-B Ab detection and titration.

The presence of anti-A or anti-B Abs was determined by a standard indirect antiglobulin test. Titration of the Abs was performed by serial 2-fold dilution. All samples from the same donor/recipient combination were studied in a blinded manner and were evaluated concomitantly by the same person. Three series of 10 tubes were prepared for each tested sample. In the first tube of each series, 50 μL of the tested serum was added. Then, serial dilutions were performed in 0.15 M saline solution to 1:512. Secondarily, 50 μL of 3% A1, B, or O reagent RBC suspension was added in the appropriate sera before a 45-minute incubation at 37°C. After 3 washes in 0.15 M saline solution, the supernatant was carefully removed, and 50 μL anti-immunoglobulin (Ig)G antihuman globulin (Sanofi Diagnostic Pasteur, Marnes-la Coquette, France) was added in each tube. Agglutination was assessed macroscopically and then microscopically after centrifugation (125g for 1 minute) and gentle resuspension. The agglutination of at least 50% ABO reagent RBCs was required to be considered positive. The titers of anti-A and/or B Abs corresponded to the reverse of the last positive dilution. An increase of the anti-A and/or anti-B Ab titers between the day preceding the conditioning regimen and day +30 was defined prior to initiating the study as an increase of 2 or more dilutions.

Anti-RBC Ag (other than ABO) allo-Ab.

The low ionic strength solution indirect antiglobulin method and enzymatic technique (DiaMed-ID Micro Typing System, DiaMed AG, Cressier, Switzerland) were used to detect anti-RBC Ag allo-Abs. Detection of Abs was performed on a panel of 3 reagent RBCs (DiaMed-ID Micro Typing System, test cell reagents for antibody screening ID-DiaCell, and ID-DiaCell P). In addition, in case of positivity, identification was performed on a panel of 10 RBCs (reagent red cells; Sanofi Diagnostics Pasteur, Marnes-la Coquette, France). These techniques were performed as described by the manufacturer.

Platelet concentrate and RBC concentrate transfusion.

HSC donor– and recipient–ABO compatible RBC concentrates (RBC-Cs) were transfused when the hemoglobin level was below 8 g/dL. Platelet concentrates (PCs) were administered to treat or prevent hemorrhage when the blood platelet count was below 20 × 109/L. If available, PC with the same ABO group as the HSC donor was administered. If unavailable, PC with an ABO Ag compatible with the HSC donor was preferred over PC ABO Ag incompatible with the HSC donor. Platelet products were never washed or plasma-reduced. These rules were applied on initiation of the conditioning regimen. Donor/recipient blood group as well as the number, time of administration, and blood group of transfused PC and RBC-C were recorded for all recipients. Similarly, administration of intravenous polyvalent immunoglobulins (IVIGs) was documented. In addition, the delay between the last PC transfusion containing anti-A and/or anti-B Abs or the last administration of IVIGs and the day +30 immunohematologic testing was calculated.

Statistical analysis

Continuous variables were compared between the 2 groups by using the Wilcoxon rank-sum test. Qualitative variables were analyzed with a chi-square test, or exact test when expected frequencies were lower than 5. The potential confounding effects of covariables on the relation between source of HSC and increased anti-A and/or anti-B titers in the recipient at day +30 were studied one by one by bivariate analysis (Mantel-Haenszel or exact methods for qualitative variables and exact logistic regression for quantitative variables). Breslow test for homogeneity was used to test whether the type of ABO mismatch was an interaction term in the relation between source of HSC and increased titer of anti-A and/or B Abs.22 

Results

Influence of the HSC graft on anti-allo RBC Ab serum titers after transplantation

The use of a PBHSC graft resulted in a higher frequency of increased anti-A and/or anti-B Ab titers 30 days after transplantation as compared to a BMHSC graft: 8 (38%) of 21 versus 3 (11%) of 28 (P = .04) (Table 3).

Table 3.

Comparison of anti-A and/or anti-B antibody at day +30 after bone marrow hematopoietic stem cell transplantation or peripheral blood hematopoietic stem cell transplantation

BMHSCT recipients
N = 28
PBHSCT recipients
N = 21
P
Increased anti-A and/or anti-B Ab    
 Yes (N = 11) 3  (11%) 8  (38%)  
 No (N = 38) 25  (89%) 13  (62%) .04  
ABO identical    
Increased anti-A and/or anti-B Ab    
 Yes (N = 4) 1  (7%) 3  (30%)  
 No (N = 21) 14  (93%) 7  (70%)  
ABO minor mismatch alone    
Increased anti-A and/or anti-B Ab   .103-150 
 Yes (N = 6) 1  (13%) 5  (71%)  
 No (N = 9) 7  (87%) 2  (29%)  
ABO major (± minor) mismatch    
Increased anti-A and/or anti-B Ab    
 Yes (N = 1) 1  (20%)  
 No (N = 8) 4  (80%) 4  (100%)  
Presence of anti-RBC Ag allo-Ab (other than A, B Ag)    
 Yes (N = 3) 0  (0%) 3  (14%)  
  (anti-Cw, anti-e, anti-E)  
 No (N = 46) 28  (100%) 18  (86%) .07 
BMHSCT recipients
N = 28
PBHSCT recipients
N = 21
P
Increased anti-A and/or anti-B Ab    
 Yes (N = 11) 3  (11%) 8  (38%)  
 No (N = 38) 25  (89%) 13  (62%) .04  
ABO identical    
Increased anti-A and/or anti-B Ab    
 Yes (N = 4) 1  (7%) 3  (30%)  
 No (N = 21) 14  (93%) 7  (70%)  
ABO minor mismatch alone    
Increased anti-A and/or anti-B Ab   .103-150 
 Yes (N = 6) 1  (13%) 5  (71%)  
 No (N = 9) 7  (87%) 2  (29%)  
ABO major (± minor) mismatch    
Increased anti-A and/or anti-B Ab    
 Yes (N = 1) 1  (20%)  
 No (N = 8) 4  (80%) 4  (100%)  
Presence of anti-RBC Ag allo-Ab (other than A, B Ag)    
 Yes (N = 3) 0  (0%) 3  (14%)  
  (anti-Cw, anti-e, anti-E)  
 No (N = 46) 28  (100%) 18  (86%) .07 

Ag indicates antigen; for other abbreviations, see Tables 1 and2.

F3-150

Breslow test for homogeneity.

In addition, 3 (14%) of 21 PBHSCT versus 0 of 28 BMHSCT recipients developed an anti-RBC (other than ABO) Ab, detected at day +30 and absent before the conditioning regimen (P = .07) (Table3).

With one exception, anti-A and/or anti-B Abs with increased titers at day +30 after PBHSCT or BMHSCT was always directed against A and/or B Ags absent both on donor and recipient RBCs. One patient, after an ABO minor and major incompatible (A into B) BMHSCT, had increased antidonor (anti-A) Ab titers at day +30. Interestingly, this patient had received anti-A–containing PC 12 hours prior to blood sample harvesting, suggesting passive transmission. In all other patients, the last PC transfusion was performed at least 48 hours before sample harvesting.

To verify that the absence of measurable Ab titers against donor and/or recipient was not due to in vitro absorption on the RBCs present in the tube during sample transportation, we compared Ab titers in the serum of posttransplantation blood samples processed (serum separation) immediately or after a 24-hour delay as in our study. Ab titers were identical in both cases (data not shown), thus eliminating any in vitro artifact.

Donor anti-allo RBC Ab serum titers before and after G-CSF administration

Comparison of anti-A or anti-B titers in the donor before and after G-CSF treatment was performed in 19 of 21 donors. There were no significant changes in Ab titers for 17 of 19 donors. One donor (blood group O female with no history of pregnancy) had decreased anti-A Ab titers (256 to 64) and one donor (blood group O female with a history of 4 pregnancies) had increased anti-B Ab titers (32 to 128). In all donors, no anti-allo RBC (other than ABO) Abs were found either before or after G-CSF treatment.

Potential confounding factors

Both BMHSCT and PBHSCT donor/recipient groups were compared for a number of parameters that might have influenced the characteristics of immunohematologic reconstitution after transplantation. As detailed in Tables 2 and 4, age, previous pregnancies, ABO mismatch between HSC donor and recipient, diagnosis, conditioning regimen, donor anti-A and/or anti-B Ab titers, episodes of RBC-C transfusion, passive transfusion of anti-A and/or anti-B Ab with PC, ABO Ag compatibility of transfused PC, administration of IVIG, and delay between the last transfusion of an anti-A– and/or anti-B–containing blood product and immunohematologic assessment were parameters that did not differ significantly between both groups. In contrast, the number of episodes of transfused PC was significantly lower in the PBHSCT group than in the BMHSCT group (3 [1-18] versus 6 [3-33]; P = .03) (Table 4). After adjustment for each of these potential confounding variables in bivariate analyses, the use of a PBHSC graft remained significantly associated with increased anti-A and/or anti-B Ab titers 30 days after transplantation.

Table 4.

Immunohematologic characteristics and transfusion parameters

BMHSCT donors
N = 21
PBHSCT donors4-150
N = 20
P
Anti-A Ab titers, median (range) 2  (0-512) 8  (0-256) .38 
Anti-B Ab titers, median (range) 8  (0-64) 24  (0-512) .24  
 
 BMHSCT recipients
N = 28 
PBHSCT recipients
N = 21 
 
 
PC transfusions    
Episodes, median (range) 6  (3-33) 3  (1-18) .03  
At least one PC transfusion ABO Ag incompatible with the HSC donor    
 Yes (N = 26) 10  (36%) 9  (43%)  
 No (N = 23) 18  (64%) 12  (57%) .61  
At least one PC transfusion ABO Ag incompatible with both the HSC donor and the recipient    
 Yes (N = 10) 6  (21%) 4  (19%)  
 No (N = 39) 22  (79%) 17  (81%) 
Plasma containing anti-A Ab    
 Yes (N = 35) 19  (68%) 16  (76%)  
 No (N = 14) 9  (32%) 5  (24%) .52  
 Delay,4-151 d, median (range) 13  (1-40) 17  (2-33) .56  
Plasma containing anti-B Ab    
 Yes (N = 47) 27  (96%) 20  (95%)  
 No (N = 2) 1  (4%) 1  (5%) 1  
 Delay,4-151 d, median (range) 11  (2-40) 17  (2-23) .17  
RBC-C transfusions    
 Episodes, median (range)  3  (0-12) 3  (0-8) .34  
Patients receiving IVIG    
 Yes (N = 23) 17  (61%) 9  (43%)  
 No (N = 26) 11  (39%) 12  (57%) .22 
 Delay,4-151 d, median (range)  6  (1-11) 5  (1-9) .33 
BMHSCT donors
N = 21
PBHSCT donors4-150
N = 20
P
Anti-A Ab titers, median (range) 2  (0-512) 8  (0-256) .38 
Anti-B Ab titers, median (range) 8  (0-64) 24  (0-512) .24  
 
 BMHSCT recipients
N = 28 
PBHSCT recipients
N = 21 
 
 
PC transfusions    
Episodes, median (range) 6  (3-33) 3  (1-18) .03  
At least one PC transfusion ABO Ag incompatible with the HSC donor    
 Yes (N = 26) 10  (36%) 9  (43%)  
 No (N = 23) 18  (64%) 12  (57%) .61  
At least one PC transfusion ABO Ag incompatible with both the HSC donor and the recipient    
 Yes (N = 10) 6  (21%) 4  (19%)  
 No (N = 39) 22  (79%) 17  (81%) 
Plasma containing anti-A Ab    
 Yes (N = 35) 19  (68%) 16  (76%)  
 No (N = 14) 9  (32%) 5  (24%) .52  
 Delay,4-151 d, median (range) 13  (1-40) 17  (2-33) .56  
Plasma containing anti-B Ab    
 Yes (N = 47) 27  (96%) 20  (95%)  
 No (N = 2) 1  (4%) 1  (5%) 1  
 Delay,4-151 d, median (range) 11  (2-40) 17  (2-23) .17  
RBC-C transfusions    
 Episodes, median (range)  3  (0-12) 3  (0-8) .34  
Patients receiving IVIG    
 Yes (N = 23) 17  (61%) 9  (43%)  
 No (N = 26) 11  (39%) 12  (57%) .22 
 Delay,4-151 d, median (range)  6  (1-11) 5  (1-9) .33 

PC indicates platelet concentrate; HSC, hematopoietic stem cell; RBC-C, red blood cell concentrate; IVIG, intravenous polyvalent immunoglobulin (Sandoglobulin; laboratoires Sandoz, Reuil Malmaison, France) median dose, 250 mg/kg (range, 50-500); for other abbreviations, see Tables 1, 2, and 3.

F4-150

Before administration of granulocyte colony-stimulating factor.

F4-151

Delay between the last transfusion of passive Ab with PC or IVIG administration and day + 30 samples.

Analysis of risk factors for increased Ab titers after PBHSCT

Minor ABO incompatible transplantation.

In PBHSCT recipients, increased titers were observed mostly in the context of a minor ABO mismatch only (O into A or B or AB and A or B into AB) transplant: 5 of 7 versus 3 of 14 in the absence of any minor ABO mismatch (P = .05) (Table 3). This was not the case after BMHSCT: 1 of 8 in the presence of a minor ABO mismatch versus 2 of 20. The interaction test between type of transplant and type of compatibility on the occurrence of increased Ab titers after transplantation approached significance (P = .10).

Donor anti-A and/or anti-B allo-Ab serum titers before or after G-CSF treatment.

The presence of high (> 32) anti-A and/or anti-B serum Ab titers in the donor, before as well as after G-CSF administration, tended to be associated with increased anti-A and/or anti-B titers in the recipient at day +30 after transplantation (P = .09).

Other factors

Donor sex and previous pregnancies did not differ between recipients who had increased or similar Ab titers after PBHSCT (data not shown).

Discussion

The use of a PBHSC allogeneic graft is being increasingly considered, and several randomized studies comparing a PBHSC graft to a BMHSC graft have been performed.5-10 In addition to the absence of general anesthesia for the donor, accelerated hematopoietic reconstitution as well as reduced transfusion requirements6-9 could be associated with the use of such a source of hematopoietic stem cells. However, several cases of severe, potentially lethal, cases of immune-mediated acute hemolysis have been described after allogeneic PBHSCT14-20 (Table 1). These hemolysis episodes occurred 8 to 14 days after transplantation and are associated with the recognition of residual recipient RBCs by donor-derived Abs. A similar case of severe acute hemolysis has recently been described after nonmyeloablative conditioning regimen and PBHSCT (J. Barrett, personal communication, December 7, 1999).

ABO incompatibility between donor and recipient is not considered as a major obstacle to allogeneic HSC transplantation. ABO incompatibility is said to be major when the recipient has Abs recognizing ABO antigens present on donor RBCs.23,24 In this situation, acute hemolysis, which may occur at the time of HSC graft infusion, may be prevented by using an RBC-depleted graft. In the case of a recipient with high anti-A and/or anti-B Ab titers, a possible later complication is a delayed onset of erythropoiesis after transplantation, associated with an increased RBC-C transfusion requirement after transplantation. ABO incompatibility is termed “minor” when donor-derived anti-ABO Abs can recognize one or several ABO Ags present on recipient erythrocytes. An immune-mediated hemolysis may occur either at the time of allograft transfusion by passive transfer of Abs (this complication is easily prevented by washing the HSC graft), or subsequently, most often 1 to 3 weeks after transplantation, concomitantly to the initiation of donor-derived immune reconstitution. In the latter case, hemolysis is in relation with Ab production by donor-derived lymphocytes. Several risk factors have been associated with the occurrence of delayed hemolysis: T-cell depletion,25 use of FK 506,26 absence of MTX to prevent GvHD,27,28 or use of BMHSCT from an unrelated donor.27,28 

In our study, PBHSCT recipients increased anti-A and/or anti-B Ab titers at day +30 after transplantation more frequently than did BMHSCT recipients (P = .04). Importantly, this result persisted after adjustment for each potential confounding factor, such as age, ABO mismatch, transfusion practices, IVIG administration, and donor anti-A and/or anti-B titers, known to possibly affect immunohematologic reconstitution after transplantation.

In the PBHSCT group, the increase in anti-A and/or anti-B titers after transplantation was associated with minor ABO-incompatible transplantation: 5 (71%) of 7 versus 3 (21%) of 14 in the absence of minor ABO incompatibility (P = .05), which was not the case after BMT: 1 (13%) of 8 versus 2 (10%) of 20. The increased anti-A and/or B serum Ab production after PBHSCT could significantly contribute to enhanced susceptibility to hemolysis, particularly in the case of minor ABO donor/recipient incompatibility. Indeed, as previously mentioned, all reported cases of severe hemolysis after PBHSCT involved a minor ABO mismatch transplantation. In this situation, an accelerated production of antirecipient RBC Abs by donor-derived-B lymphocytes could result in acute hemolysis of the residual recipient RBCs or transfused donor-incompatible RBC-C.

Anti-A and/or anti-B Abs with increased titers at day +30 were almost always directed against A and/or B Ags absent on both donor and recipient RBCs. On the contrary, Abs directed against recipient A and/or B Ags remained undetectable at day +30. Several mechanisms might account for the absence of such Abs. We have eliminated the possibility that in vitro absorption during the 24-hour transport-related conservation of whole blood played a significant role. Low-titer antirecipient RBC Abs produced in vivo might be absorbed in vivo on the RBC Ags present on residual recipient (or transfused) RBCs as well as on the A and/or B Ags present on extra-hematopoietic cells such as endothelial cells.29 In such cases, measurable antirecipient RBC Ab serum titers would be found only when Ab titers increase significantly as in the observed cases of hemolysis (Table 1) or much later on completion of immune reconstitution and disappearance of circulating recipient RBCs.

In addition to minor ABO-incompatible PBHSCT, transfusion of donor-incompatible RBCs as well as the absence of MTX (in the presence of CSA) have been associated with the occurrence of acute hemolysis in most cases. In our study, all patients received MTX, and guidelines, requiring that RBC-C transfusion be both donor and recipient compatible or at least donor compatible for the Rh and Kell Ags, were enforced.30 Indeed, significant hemolysis was observed in none of the recipients of the SFGM trial.

The absence of MTX (in the presence of CSA) has been previously reported as a risk factor for delayed hemolysis.27,28 MTX is cytotoxic for B lymphocytes.31 The immunosuppressive effects of CSA are T-cell specific,32,33 and the use of such a drug, in the absence of MTX, could result in enhanced B-cell proliferation and T-cell–independent Ab response such as anti-RBC IgM isotype Ab production. In addition, because of the mechanisms by which CSA exerts its immunosuppressive action (inhibition of nuclear factor of activated T cells [NFAT] translocation to the nucleus),34 such a drug should be unable to control ongoing T-cell–dependent immune response. This hypothesis implies that transplantation from a donor with high anti-recipient RBC Ab titers might be associated with a higher risk of posttransplantation hemolysis. Interestingly, anti-A and/or anti-B Ab titers after G-CSF administration in the HSC donors of recipients who increased anti-A and/or anti-B Ab titers at day +30 tended to be higher. Such a finding needs be confirmed on a larger number of patients. Nevertheless, the presence of high Ab titers in a putative minor ABO-mismatched HSC donor is a parameter that could contribute to the choice of the HSC graft source as well as the type of posttransplantation immunosuppression.

Several quantitative and qualitative differences in a PBHSC graft versus a marrow graft could contribute to accelerating immunohematologic reconstitution after PBHSCT. The increased number of B cells, T cells, monocytes, and CD34+ cells present in the PBHSC grafts as compared to the BM grafts35 could result in accelerated immune-reconstitution with the production of anti-recipient Abs early after transplantation at a time when significant numbers of recipient RBCs are still present. Indeed, we have determined that circulating T cells (CD4+, CD8+ as well as CD3+, CD4, and CD8) and B cells are present in higher numbers 30 days after PBHSCT than after BMHSCT.36 In addition, G-CSF has been reported to directly enhance in vitro immunoglobulin production by activated B cells.37 G-CSF–induced TH2 cytokine profile of the T cells present in the graft could also contribute to enhancing post-PBHSCT Ab responses.38,39 In this respect, analysis of anti-HLA Ab immunization in our cohort of recipients has demonstrated that a PBHSCT is associated with a significantly increased incidence of de novo anti-HLA immunization.40 The frequency of interferon (IFN)-gamma–producing T cells as well as the capacity of producing IFN-gamma at single cell level are indeed reduced in a PBHSC graft versus a BM graft.35 Reduced tumor necrosis factor-alpha and interleukin (IL)-12 production41and increased IL-10 production42 have been attributed to G-CSF exposure and could also contribute to the observed increase in Ab titers after PBHSCT.

ABO compatibility in the context of marrow transplantation has not been conclusively associated with overall outcome.43However, minor ABO-compatible BMT might be associated with an increased risk of GvHD.44 Indeed, the immune activation following the binding of anti-AB Abs to their target Ags (on residual host or transfused RBCs), or extra-hematopoietic cells such as endothelial cells might initiate an alloreactive immune response.45 In view of the accelerated immunohematologic reconstitution we have evidenced after PBHSCT, the issue of ABO incompatibility and posttransplantation alloreactivity clearly needs now to be carefully examined in the setting of PBHSCT. In our study, minor ABO-incompatible PBHSCT was not significantly associated with an increased incidence or severity of acute or chronic GvHD (data not shown).

In conclusion, we observed, in a randomized setting, that the use of G-CSF–mobilized PBSHC was associated with accelerated erythrocyte immunohematologic reconstitution. Larger studies with a longer follow-up will be necessary to confirm this finding. Such an early increase in donor-derived anti-RBC Abs at a time when significant numbers of recipient RBCs are still present can result in severe hemolysis after transplantation. Stringent immunohematologic assessment and transfusion practices should reduce such a risk.30Increased numbers of B and T lymphocytes in the graft, G-CSF-induced functional alterations, and accelerated cellular immune reconstitution could contribute to such findings. Further studies should clarify the mechanisms involved. Overall, different HSC sources are associated with distinct immune reconstitution patterns, including immunohematologic reconstitution.

We wish to acknowledge the contribution of the following BMT centers: Angers: N. Ifrah, S. François, N. Piard; Besançon: J. Y. Cahn; Bordeaux: J. M. Boiron, B. Daze; Grenoble: F. Garban, F. Chenais; Hôtel-Dieu (Paris): B. Rio, M. F. Fruchart; Lille: J. P. Jouet, P. Renom; Lyon: M. Michallet; Nancy: F. Witz; Nantes: N. Milpied, B. David; PitiéSalpétrière (Paris): L. Sutton, A. Verdier; Saint-Etienne: C. Oriol; Toulouse: M. Attal, C. Payen, F. Roubinet; Institut Gustave Roussy (Villejuif): J. H. Bourhis, J. L. Pico; Poitiers: A. Sadoun, C. Giraud; Robert Debré (Paris): M. Duval, F. Sellami and the help of F. Bassompierre (Direction Régionale de la Recherche Clinique, Paris).

Supported by grant 005610 from L'Etablissement Français des Greffes, grant 9552 from L'Association pour la Recherche sur le Cancer, and grant 99004035 from La Fondation de France.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

References

1
Goldman
J
Peripheral blood stem cells for allografting.
Blood.
85
1995
1413
1415
2
Bensinger
WI
Weaver
CH
Appelbaum
FR
et al
Transplantation of allogeneic peripheral blood stem cells mobilized by recombinant human granulocyte colony-stimulating factor.
Blood.
85
1995
1655
1658
3
Körbling
M
Przepiorka
D
Huh
YO
et al
Allogeneic blood stem cell transplantation for refractory leukemia and lymphoma: potential advantage of blood over marrow allografts.
Blood.
85
1995
1659
1665
4
Schmitz
N
Dreger
P
Suttorp
M
et al
Primary transplantation of allogeneic peripheral blood progenitor cells mobilized by filgrastim (granulocyte colony-stimulating factor).
Blood.
85
1995
1666
1672
5
Schmitz
N
Bacigalupo
A
Hasenclever
D
et al
Allogeneic bone marrow transplantation vs filgrastim-mobilised peripheral blood progenitor cell transplantation in patients with early leukaemia: first results of a randomised multicentre trial of the European Group for Blood and Marrow Transplantation.
Bone Marrow Transplant.
21
1998
995
1003
6
Vigorito
AC
Azevedo
WM
Marques
JF
et al
A randomised, prospective comparison of allogeneic bone marrow and peripheral blood progenitor cell transplantation in the treatment of haematological malignancies.
Bone Marrow Transplant.
22
1998
1145
1151
7
Blaise
D
Kuentz
M
Fortanier
C
et al
Randomized trial of bone marrow versus lenograstim-primed blood cell allogeneic transplantation in patients with early-stage leukemia: a report from the Societe Francaise de Greffe de Moelle.
J Clin Oncol.
18
2000
537
546
8
Powles
R
Mehta
J
Kulkarni
S
et al
Allogeneic blood and bone-marrow stem-cell transplantation in haematological malignant diseases: a randomised trial.
Lancet.
355
2000
1231
1237
9
Bensinger
W
Martin
P
Clift
R
et al
A prospective, randomised trial of peripheral blood stem cells or marrow for patients undergoing allogeneic transplantation for hematologic malignancies [abstract].
Blood.
94
1999
368a
10
Durrant
ST
Morton
AJ
A randomised trial of filgrastim stimulated donor marrow versus peripheral blood for allogeneic transplantation: increased extensive chronic graft versus host disease following peripheral blood transplantation [abstract].
Blood.
94
1999
608a
11
Urbano-Ispizua
A
Solano
C
Brunet
S
et al
Allogeneic peripheral blood progenitor cell transplantation: analysis of short-term engraftment and acute GVHD incidence in 33 cases.
Bone Marrow Transplant.
18
1996
35
40
12
Ottinger
HD
Beelen
DW
Scheulen
B
Schaefer
UW
Grosse-Wilde
H
Improved immune reconstitution after allotransplantation of peripheral blood stem cells instead of bone marrow.
Blood.
88
1996
2775
2779
13
Storek
J
Gooley
T
Siadak
M
et al
Allogeneic peripheral blood stem cell transplantation may be associated with a high risk of chronic graft versus host disease.
Blood.
90
1997
4705
4709
14
Laurencet
FM
Samii
K
Bressoud
A
et al
Massive delayed hemolysis following peripheral blood stem cell transplantation with minor ABO incompatibility.
Hematol Cell Ther.
39
1997
159
162
15
Toren
A
Dacosta
Y
Manny
N
Varadi
G
Or
R
Nagler
A
Passenger B-lymphocyte-induced severe hemolytic disease after allogeneic peripheral blood stem cell transplantation [letter].
Blood.
87
1996
843
844
16
Oziel-Taieb
S
Faucher-Barbey
C
Chabannon
C
et al
Early and fatal immune haemolysis after so-called ‘minor’ ABO-incompatible peripheral blood stem cell allotransplantation.
Bone Marrow Transplant.
19
1997
1155
1156
17
Moog
R
Melder
C
Prumbaum
M
Muller
N
Schaefer
UW
Rapid donor type isoagglutinin production after allogeneic peripheral progenitor cell transplantation.
Beitr Infusionsther Transfusionsmed.
34
1997
150
152
18
Lyding
S
Nürnberger
W
Bandi
G
et al
ABO-bidirectional incompatibility in PBSCT: a case of acute severe hemolysis [abstract].
Bone Marrow Transplant.
23
1999
S45
19
Fitzgerald
JM
Conn
JS
Proctor
SJ
Severe haemolysis complicating the rapid engraftment of a minor ABO mismatched peripheral stem cell allogeneic transplant [abstract].
10th International Symposium on Autologous PBSCT;
September 1999
Mulhouse
France
20
Salmon
JP
Michaux
S
Hermanne
JP
et al
Delayed massive immune hemolysis mediated by minor ABO incompatibility after allogeneic peripheral blood progenitor cell transplantation.
Transfusion.
39
1999
824
827
21
Bornhäuser
M
Ordemann
R
Paaz
U
et al
Rapid engraftment after allogeneic ABO-incompatible peripheral blood progenitor cell transplantation complicated by severe hemolysis.
Bone Marrow Transplant.
19
1997
295
297
22
Breslow
NE
Day
NE
Statistical methods in cancer research. Vol 1: The analysis of case-control studies.
1990
International Agency for Research on Cancer
Lyon: France
23
Klumpp
TR
Immunohematologic complications of bone marrow transplantation.
Bone Marrow Transplant.
8
1991
159
170
24
Petz
LD
Immunohematologic problems associated with bone marrow transplantation.
Transfus Med Rev.
1
1987
85
100
25
Hazlehurst
GR
Brenner
MK
Wimperis
JZ
Knowles
SM
Prentice
HG
Haemolysis after T-cell depleted bone marrow transplantation involving minor ABO incompatibility.
Scand J Haematol.
37
1986
1
3
26
Greeno
EW
Perry
EH
Ilstrup
SJ
Weisdorf
DJ
Exchange transfusion the hard way: massive hemolysis following transplantation of bone marrow with minor ABO incompatibility.
Transfusion.
36
1996
71
74
27
Hows
J
Beddow
K
Gordon-Smith
E
et al
Donor-derived red blood cell antibodies and immune hemolysis after allogeneic bone marrow transplantation.
Blood.
67
1986
177
181
28
Gajewski
JL
Petz
LD
Calhoun
L
et al
Hemolysis of transfused group O red blood cells in minor ABO-incompatible unrelated-donor bone marrow transplants in patients receiving cyclosporine without posttransplant methotrexate.
Blood.
79
1992
3076
3085
29
Oriol
R
Tissular expression of ABH and Lewis antigens in humans and animals: expected value of different animal models in the study of ABO-incompatible organ transplants.
Transplant Proc.
19
1987
4416
4420
30
Lapierre
V
Kuentz
M
Tiberghien
P
for the Société Française de Greffe de Moelle
Allogeneic peripheral blood hematopoietic stem cell transplantation: homogenization of red blood cells immuno-hematological assessment and transfusion practice.
Bone Marrow Transplant.
25
2000
507
512
31
Rosenthal
GJ
Weigand
GW
Germolec
DR
Blank
JA
Luster
MI
Suppression of B cell function by methotrexate and trimetrexate: evidence for inhibition of purine biosynthesis as a major mechanism of action.
J Immunol.
141
1988
410
416
32
Krönke
M
Leonard
WJ
Depper
JM
et al
Cyclosporin A inhibits T-cell growth factor gene expression at the level of mRNA transcription.
Proc Natl Acad Sci U S A.
81
1984
5214
5218
33
Schreiber
SL
Crabtree
GR
The mechanism of action of cyclosporin A and FK 506.
Immunol Today.
13
1992
136
142
34
Flanagan
WM
Corthesy
B
Bram
RJ
Crabtree
GR
Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A.
Nature.
29;352
1991
803
807
35
Tayebi H, Kuttler F, Saas P, et al. Effect of granulocyte-colony stimulating factor (G-CSF) mobilisation on phenotypical and functional properties of immune cells. Exp Haematol. In press.
36
Tayebi
H
Tiberghien
P
Ferrand
C
et al
Allogeneic peripheral blood stem cell transplantation results in weaker alterations of early T-cell compartment homeostasis than bone marrow transplantation.
Bone Marrow Transplant.
27
2001
167
175
37
Morikawa
K
Miyawaki
T
Oseko
F
Morikawa
S
Imai
K
G-CSF enhances the immunoglobulin generation rather than the proliferation of human B lymphocytes.
Eur J Haematol.
51
1993
144
151
38
Pan
L
Delmonte
J
Jr
Jalonen
CK
Ferrara
JL
Pretreatment of donor mice with granulocyte colony-stimulating factor polarizes donor T lymphocytes toward type-2 cytokine production and reduces severity of experimental graft-versus-host disease.
Blood.
86
1995
4422
4429
39
Hartung
T
Döcke
WD
Gantner
F
et al
Effect of granulocyte colony-stimulating factor treatment on ex vivo blood cytokine response in human volunteers.
Blood.
85
1995
2482
2489
40
Lapierre
V
Tayebi
H
Hartmann
O
et al
G-CSF-mobilized peripheral blood hematopoietic stem cell transplantation is associated with an increased incidence of anti-HLA immunization [abstract].
Blood.
92
1999
140a
41
Boneberg
EM
Hareng
L
Gantner
F
Wendel
A
Hartung
T
Human monocytes express functional receptors for granulocyte colony-stimulating factor that mediate suppression of monokines and interferon-gamma.
Blood.
95
2000
270
276
42
Mielcarek
M
Graf
L
Johnson
G
Torok-Storb
B
Production of interleukin-10 by granulocyte colony-stimulating factor-mobilized blood products: a mechanism for monocyte-mediated suppression of T-cell proliferation.
Blood.
92
1998
215
222
43
Bensinger
WI
Buckner
CD
Thomas
ED
Clift
RA
ABO-incompatible marrow transplants.
Transplantation.
33
1982
427
429
44
Bacigalupo
A
Hows
J
Gordon-Smith
EC
et al
Bone marrow transplantation for severe aplastic anemia from donors other than HLA identical siblings: a report of the BMT Working Party.
Bone Marrow Transplant.
3
1988
531
535
45
Benjamin
RJ
Antin
JH
ABO-incompatible bone marrow transplantation: the transfusion of incompatible plasma may exacerbate regimen-related toxicity.
Transfusion.
39
1999
1273
1274

Author notes

Valérie Lapierre, Unité de Médecine Transfusionnelle et d'Hémovigilance, Institut Gustave Roussy; 39 rue Camille Desmoulins, 94805 Villejuif Cedex; e-mail: lapierre@igr.fr.