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Clinical significance of recipient antibodies to stem cell donor mismatched class I HLA antigens
Clinical Significance of Recipient Antibodies to Stem Cell Donor Mismatched Class I HLA Antigens Marilyn S. Pollack and David Ririe ABSTRACT: A stem cell transplant candidate whose best-matched related donor had a mismatch for A68 by virtue of the patient’s maternal human leukocyte antigen A/B (HLA-A/B) recombinant haplotype, was referred to Wilford Hall Medical Center for transplantation. She was determined prior to transplant to have a high-titered antibody to that HLA type and to several crossreactive antigens (A2 and A24). When she initially failed to engraft, plasmapheresis was tried three times with no effect on the antibody titer. Subsequently, plasmapheresis followed by intravenous immunoglobulin treatment and retransplant completely eliminated the donor-specific antibody. Engraftment occurred, and several weeks after the ABBREVIATIONS AHG anti-human globulin GVHD graft-versus-host disease IVIG intravenous immunoglobulin NT not tested PRA panel reactive antibody
INTRODUCTION Testing potential allogeneic stem cell recipients for antibodies to potential donors’ mismatched human leukocyte antigen (HLA) antigens has been recommended as the the standard of clinical practice for many years [1–3]. In one recent report on stem cell transplants using partially mismatched donors [4], for example, it was noted that 62% of patients with a positive crossmatch rejected their graft whereas only 6% of patients with a negative crossmatch rejected their grafts. Nevertheless, the actual number of HLA Laboratories performing that testing for stem cell transplant programs is very small (American
From the University of Texas Health Science Center (M.S.P.), and the Wilford Hall Medical Center (M.S.P., D.R.), San Antonio, TX, USA. Address reprint requests to: Dr. Marilyn S. Pollack, Department of Pathology, UTHSCSA, 7703 Floyd Curl Drive, San Antonio, TX 78229; Tel: (210) 567-5698; Fax: (210) 358-0777; E-mail: pollack@uthscsa. edu. Received October 6, 2003; revised December 10, 2003; accepted December 18, 2003. Human Immunology 65, 245⫺247 (2004) © American Society for Histocompatibility and Immunogenetics, 2004 Published by Elsevier Inc.
second transplant antibodies to A68 could no longer be detected although the antibodies to other HLA types were still present. This case report should serve to remind stem cell transplant programs of the importance of checking for the presence of recipient antibodies to donor-mismatched antigens and suggests that patients with such antibodies be treated prior to transplant. Human Immunology 65, 245⫺247 (2004). © American Society for Histocompatibility and Immunogenetics, 2004. Published by Elsevier Inc. KEYWORDS: Stem cell transplantation; HLA antibodies; crossmatch; HLA mismatch; HLA-A68
TX UTHSCSA WHMC
transplant University of Texas Health Science Center at San Antonio Wilford Hall Medical Center
Society for Histocompatibility and Immunogenetics [ASHI] Accreditation Review Board [ARB] data, 2003). It is therefore our intention to remind HLA laboratories of the importance of this testing by reporting a specific case history. METHODS Both the University of Texas Health Science Center at San Antonio (UTHSCSA) and Wilford Hall Medical Center (WHMC) programs will perform stem cell transplants using related donors who have a single mismatched HLA-A, -B, or -DR type at the serological or low resolution DNA typing level. Donors mismatched for an A or B type are checked for the possible additional mismatch of an HLA-C type and donors mismatched for a DR type are checked for the possible additional mismatch of a DQ type. We routinely also check for the presence of recipient antibodies to the donor’s mismatched type(s). The presence of donor specific antibod0198-8859/04/$–see front matter doi:10.1016/j.humimm.2003.12.010
246
M.S. Pollack and D. Ririe
TABLE 1 HLA haplotypes for the case report family Patient
a⫽ d/c⫽ Father: a⫽ b⫽ Mother: c⫽ d⫽ Brother#1: (donor) a⫽ c⫽ Brother#2: b⫽ c⫽ Sister: a⫽ d⫽
A*32, B*35, Cw*04, DRB1*11, DRB3 A*29/, B*07, Cw*07, DRB1*01 A*32, B*35, Cw*04, DRB1*11, DRB3 A*24, B*38, Cw*NT, DRB1*14, DRB3 A*68, B*07, Cw*07, DRB1*01 A*29, B*45, Cw*NT, DRB1*12, DRB3 A*32, B*35, Cw*04, DRB1*11, DRB3 A*68, B*07, Cw*07, DRB1*01 A*24, B*38, Cw*NT, DRB1*14, DRB3 A*68, B*07, Cw*07, DRB1*01 A*32, B*35, Cw*04, DRB1*11, DRB3 A*29, B*45, Cw*NT, DRB1*12, DRB3
The Patient and Brother #1 were HLA typed at WHMC (A, B, and DR) and UTHSCSA (HLA-C) using polymerase chain reaction–sequence-specific primer methods. Results for the father, mother, brother #2, and the sister were provided by the Bill Young Department of Defense Marrow Transplant Program.
ies to class I or class II antigens are determined using a standard flow cytometry crossmatch test (BD FACScan; Becton Dickinson, Mountain View, CA, USA) using actual donor cells or surrogate cells mismatched for the same type as the donor’s mismatch [5]. Test lymphocytes (2.5⫻105) are incubated with 25 l of test serum and stained with FITC conjugated anti-human IgG (FC receptor specific) and either PE conjugated anti-CD3 or anti-CD19 antibodies for gating on T cells or B cells, respectively. By comparison with testing multiple samples of “normal” human sera, a channel shift of ⬎ 20 or ⬎ 30 is considered a positive crossmatch for T cells or B
cells, respectively. Channel shifts within 15 channels of these cutoffs are considered to be “weakly positive.” If antibodies are detected, their specificity is further defined using panels of cells and/or antigens with a standard antiglobulin enhanced (AHG) serological test [5] and/or a standard flow cytometry antibody identification test using antigen coated beads [6], as appropriate.
RESULTS The family’s HLA typing results for this case report are listed in Table 1 (only the patient and donor were typed for HLA-C). The donor is clearly mismatched to the recipient for the single antigen/allele A68 by virtue of the patient’s inherited maternal A/B recombination. The patient’s pretransplant serum had antibodies to the mismatched antigen and to several crossreactive antigens and was crossmatch positive with cells from surrogate donors sharing those mismatches (Table 2, line 1, donor cells not available for direct testing). The patient was transplanted using Busulfan (3.2 mg/kg) over 4 days and Cytoxan (120 mg/kg) over 2 days as the pretransplant conditioning regimen. She received a dose of 9.04⫻106 CD34 positive cells collected after a standard peripheral blood stem cell stimulation protocol utilizing G-CSF at 10 g/kg for 5 consecutive days. Cyclosporin and Methotrexate were used for graft-versushost disease (GVHD) prophylaxis Three weeks after transplant, her white blood cell
TABLE 2 Flow cytometry and PRA test results before and after transplant #1 and transplant #2 Sample date
Clinical status
6/25/03 7/8/03
Pretransplant #1 19 Days post ⫽ TX #1
7/13/03
24 Days post ⫽ TX #1, immediately postplasmapheresis X3 32 Post⫽TX #1, preplasmapheresis ⫹ IVIG 3 Days post⫽TX #2, immediately postplasmapheresis ⫹ IVIG X4 9 Days post⫽TX #2; 6 days postplasmapheresis ⫹ IVIG X4 13 Days post⫽TX #2; 10 days postplasmapheresis ⫹ IVIG X4 20 Days post⫽TX #2; 17 days postplasmapheresis ⫹ IVIG X4 86 Days post⫽TX #2; 83 days postplasmapheresis ⫹ IVIG X4
7/21/03 7/25/03 7/31/03 8/4/03 8/11/03 10/16/03
PRA & AGH titer (antibody specificities)
Flow crossmatch shift/result A68 mismatcha
Flow crossmatch shift/result A2 mismatcha
84%, (␣ A2,23,24,68) 57%, ␣ A68 Titer ⬎ 1:80 (␣ A2,23,24,68) NT
119-Positive 123-Positive
115-Positive 114-Positive
130-Positive
NT
␣ A68 titer ⬎ 1:80
157-Positive
NT
NT
33-Weak Positive
NT
37%, (␣ A2,24)
12-Negative
NT
29%, (␣ A2)
7-Negative
NT
NT
5-Negative
110-Positive
0%-no antibodies detected
NT
NT
a Surrogate donors mismatched to the patient for A68 or A2, respectively, but not for any other major types (surrogate “recipient” cells matched to the recipient for all major types were negative in all cases [not shown]). Abbreviations: AGH ⫽ anti-human globulin; IVIG ⫽ intravenous immunoglobulin; NT ⫽ not tested; TX ⫽ transplantation.
Antibodies to Stem Cell Donor Class I Antigens
count was still less than 0.1/mm3 and she not only remained platelet and red blood cell transfusion dependent, but failed to respond to random donor platelets due to the persistance of her anti-HLA antibodies (Table 2, line 2). She was subsequently transfused with platelets from donors selected to lack her target antigens and treated with plasmapheresis on 3 consecutive days. When no change in her antibody activities was observed following that treatment (Table 2, lines 3 and 4), she was treated with fludara for 4 days at a dose of 30 mg/m2, then again with plasmapheresis, this time for 4 consecutvie days and this time with an intravenous infusion of immunoglobulin (0.5 g/kg) following each plasmapheresis treatment (total intravenous immunoglobulin [IVIG] ⫽ 2 g/kg). Although no sample was tested for residual antibody activity at that time, after the first of these plasmapheresis ⫹ IVIG treatments, she was given a total dose of 17.41⫻106 CD34 positive cells (12.98⫻106 collected following a second standard peripheral blood stem cell stimulation protocol utilizing granulocyte macrophage– colony-stimulating factor at 10 g/kg for 5 consecutive days and 4.43⫻106 that had been stored frozen in reserve from the previous collection). Cyclosporin alone was used for GVHD prophylaxis. Surrogate crossmatch testing 3 days post-transplantation #2 (TX#2; immediately after the last plasmapheresis ⫹ IVIG treatment) indicated that the anti-donor specific antibody had almost disappeared from the circulation (Table 2, line 5). Within 1 week of the IVIG treatment and second transplant, she showed signs of engraftment with 100% donor chimerism detected after 3 weeks (data not shown). Although the patient’s antibodies to the crossreactive antigen, HLA-A2, continued to be detected for several weeks after transplant (Table 2, lines 6 – 8), these also disappeared after several more weeks (Table 2, line 9). DISCUSSION The initial engraftment failure this patient experienced indicates that preformed anti-donor class I HLA antibodies can eliminate enough of the donor’s differentiating stem cells to preclude engraftment, even though the most primitive stem cells do not express HLA antigens. Although testing potential allogeneic stem cell recipients for antibodies to potential donors’ mismatched HLA antigens has been recommended as the the standard of clinical practice for many years [1– 4], the actual number of HLA laboratories performing that testing for HLA mismatched stem cell transplant donors is very small (ASHI ARB data 2003). This case report should serve to remind HLA laboratories of the importance of this testing. We can also suggest that when such antibodies are
247
found to be present in high enough titer to cause a strongly positive crossmatch, consideration should be given to pretransplant plasmapheresis with IVIG treatment at the time of transplant; plasmapheresis alone seemed to be ineffective, at least in the three daily treatment schedule used for this patient at WHMC. The observation that the weakly positive crossmatch detected after the four treatments did not prevent engraftment (Table 2, line 5) provides the basis for suggesting that such treatment might not be necessary for patients with weak anti-donor antibodies. The incidental finding that the donor-specific antibody (to A68) disappeared while the antibody to the crossreactive antigen A2 remained detectable for several weeks might reflect one of the proposed mechanisms of action IVIG, namely that antibodies are still specifically bound to the target (donor) cells but the IVIG antibodies to Fc receptors prevent the antibody binding cells from being eliminated. As chimerism became complete, the residual recipient B cells all disappeared, causing the (expected) disappearance of all recipient-derived antibodies. ACKNOWLEDGMENTS
The authors wish to thank Evan Ray, Jeffrey Hoskins, Laura McNeish, Julie Forman, and Brook Holt for their excellent technical support for this study and the Bill Young Department of Defense Marrow Transplant Program for referring this patient and for providing the HLA typing results for the other family members.
REFERENCES 1. Anasetti C: Effect of HLA compatibility on engraftment of bone marrow transplants in patients with leukemia or lymphoma. New Engl J Med 320:197, 1989. 2. Pollack MS: Histocompatibility testing for bone marrow/ stem cell transplantation of matched and partially mismatched donors. In: Guidelines for Clinical Histocompatibility Practice. Lenexa, KS: ASHI, 49⫺53, 1999. 3. Hurley CK, Wade JA, Oudshoorn M, Middleton D, Kukuruga D, Navarrete C, Christiansen F, Hegland J, Ren E-C, Anderson I, Cleaver SA, Brautbar C, Raffoux C: A special report: Histocompatibility Testing guidelines for hematopoietic stem cell transplantation using volunteer donors. Hum Immunol 60:347, 1999. 4. Mickelson EM, Petersdorf E, Anasetti C, Martin P, Woolfrey A, Hansen JA: HLA matching in hematopoietic cell transplantation. Hum Immunol 61:92, 2000. 5. ASHI Laboratory Manual, 2nd Edition. Lenexa, KS: American Society for Histocompatibility and Immunogenetics, 1990. 6. Flow PRA I Screening Test Product Insert. Canoga Park, CA: One Lambda, Inc.
INTRODUCTION Testing potential allogeneic stem cell recipients for antibodies to potential donors’ mismatched human leukocyte antigen (HLA) antigens has been recommended as the the standard of clinical practice for many years [1–3]. In one recent report on stem cell transplants using partially mismatched donors [4], for example, it was noted that 62% of patients with a positive crossmatch rejected their graft whereas only 6% of patients with a negative crossmatch rejected their grafts. Nevertheless, the actual number of HLA Laboratories performing that testing for stem cell transplant programs is very small (American
From the University of Texas Health Science Center (M.S.P.), and the Wilford Hall Medical Center (M.S.P., D.R.), San Antonio, TX, USA. Address reprint requests to: Dr. Marilyn S. Pollack, Department of Pathology, UTHSCSA, 7703 Floyd Curl Drive, San Antonio, TX 78229; Tel: (210) 567-5698; Fax: (210) 358-0777; E-mail: pollack@uthscsa. edu. Received October 6, 2003; revised December 10, 2003; accepted December 18, 2003. Human Immunology 65, 245⫺247 (2004) © American Society for Histocompatibility and Immunogenetics, 2004 Published by Elsevier Inc.
second transplant antibodies to A68 could no longer be detected although the antibodies to other HLA types were still present. This case report should serve to remind stem cell transplant programs of the importance of checking for the presence of recipient antibodies to donor-mismatched antigens and suggests that patients with such antibodies be treated prior to transplant. Human Immunology 65, 245⫺247 (2004). © American Society for Histocompatibility and Immunogenetics, 2004. Published by Elsevier Inc. KEYWORDS: Stem cell transplantation; HLA antibodies; crossmatch; HLA mismatch; HLA-A68
TX UTHSCSA WHMC
transplant University of Texas Health Science Center at San Antonio Wilford Hall Medical Center
Society for Histocompatibility and Immunogenetics [ASHI] Accreditation Review Board [ARB] data, 2003). It is therefore our intention to remind HLA laboratories of the importance of this testing by reporting a specific case history. METHODS Both the University of Texas Health Science Center at San Antonio (UTHSCSA) and Wilford Hall Medical Center (WHMC) programs will perform stem cell transplants using related donors who have a single mismatched HLA-A, -B, or -DR type at the serological or low resolution DNA typing level. Donors mismatched for an A or B type are checked for the possible additional mismatch of an HLA-C type and donors mismatched for a DR type are checked for the possible additional mismatch of a DQ type. We routinely also check for the presence of recipient antibodies to the donor’s mismatched type(s). The presence of donor specific antibod0198-8859/04/$–see front matter doi:10.1016/j.humimm.2003.12.010
246
M.S. Pollack and D. Ririe
TABLE 1 HLA haplotypes for the case report family Patient
a⫽ d/c⫽ Father: a⫽ b⫽ Mother: c⫽ d⫽ Brother#1: (donor) a⫽ c⫽ Brother#2: b⫽ c⫽ Sister: a⫽ d⫽
A*32, B*35, Cw*04, DRB1*11, DRB3 A*29/, B*07, Cw*07, DRB1*01 A*32, B*35, Cw*04, DRB1*11, DRB3 A*24, B*38, Cw*NT, DRB1*14, DRB3 A*68, B*07, Cw*07, DRB1*01 A*29, B*45, Cw*NT, DRB1*12, DRB3 A*32, B*35, Cw*04, DRB1*11, DRB3 A*68, B*07, Cw*07, DRB1*01 A*24, B*38, Cw*NT, DRB1*14, DRB3 A*68, B*07, Cw*07, DRB1*01 A*32, B*35, Cw*04, DRB1*11, DRB3 A*29, B*45, Cw*NT, DRB1*12, DRB3
The Patient and Brother #1 were HLA typed at WHMC (A, B, and DR) and UTHSCSA (HLA-C) using polymerase chain reaction–sequence-specific primer methods. Results for the father, mother, brother #2, and the sister were provided by the Bill Young Department of Defense Marrow Transplant Program.
ies to class I or class II antigens are determined using a standard flow cytometry crossmatch test (BD FACScan; Becton Dickinson, Mountain View, CA, USA) using actual donor cells or surrogate cells mismatched for the same type as the donor’s mismatch [5]. Test lymphocytes (2.5⫻105) are incubated with 25 l of test serum and stained with FITC conjugated anti-human IgG (FC receptor specific) and either PE conjugated anti-CD3 or anti-CD19 antibodies for gating on T cells or B cells, respectively. By comparison with testing multiple samples of “normal” human sera, a channel shift of ⬎ 20 or ⬎ 30 is considered a positive crossmatch for T cells or B
cells, respectively. Channel shifts within 15 channels of these cutoffs are considered to be “weakly positive.” If antibodies are detected, their specificity is further defined using panels of cells and/or antigens with a standard antiglobulin enhanced (AHG) serological test [5] and/or a standard flow cytometry antibody identification test using antigen coated beads [6], as appropriate.
RESULTS The family’s HLA typing results for this case report are listed in Table 1 (only the patient and donor were typed for HLA-C). The donor is clearly mismatched to the recipient for the single antigen/allele A68 by virtue of the patient’s inherited maternal A/B recombination. The patient’s pretransplant serum had antibodies to the mismatched antigen and to several crossreactive antigens and was crossmatch positive with cells from surrogate donors sharing those mismatches (Table 2, line 1, donor cells not available for direct testing). The patient was transplanted using Busulfan (3.2 mg/kg) over 4 days and Cytoxan (120 mg/kg) over 2 days as the pretransplant conditioning regimen. She received a dose of 9.04⫻106 CD34 positive cells collected after a standard peripheral blood stem cell stimulation protocol utilizing G-CSF at 10 g/kg for 5 consecutive days. Cyclosporin and Methotrexate were used for graft-versushost disease (GVHD) prophylaxis Three weeks after transplant, her white blood cell
TABLE 2 Flow cytometry and PRA test results before and after transplant #1 and transplant #2 Sample date
Clinical status
6/25/03 7/8/03
Pretransplant #1 19 Days post ⫽ TX #1
7/13/03
24 Days post ⫽ TX #1, immediately postplasmapheresis X3 32 Post⫽TX #1, preplasmapheresis ⫹ IVIG 3 Days post⫽TX #2, immediately postplasmapheresis ⫹ IVIG X4 9 Days post⫽TX #2; 6 days postplasmapheresis ⫹ IVIG X4 13 Days post⫽TX #2; 10 days postplasmapheresis ⫹ IVIG X4 20 Days post⫽TX #2; 17 days postplasmapheresis ⫹ IVIG X4 86 Days post⫽TX #2; 83 days postplasmapheresis ⫹ IVIG X4
7/21/03 7/25/03 7/31/03 8/4/03 8/11/03 10/16/03
PRA & AGH titer (antibody specificities)
Flow crossmatch shift/result A68 mismatcha
Flow crossmatch shift/result A2 mismatcha
84%, (␣ A2,23,24,68) 57%, ␣ A68 Titer ⬎ 1:80 (␣ A2,23,24,68) NT
119-Positive 123-Positive
115-Positive 114-Positive
130-Positive
NT
␣ A68 titer ⬎ 1:80
157-Positive
NT
NT
33-Weak Positive
NT
37%, (␣ A2,24)
12-Negative
NT
29%, (␣ A2)
7-Negative
NT
NT
5-Negative
110-Positive
0%-no antibodies detected
NT
NT
a Surrogate donors mismatched to the patient for A68 or A2, respectively, but not for any other major types (surrogate “recipient” cells matched to the recipient for all major types were negative in all cases [not shown]). Abbreviations: AGH ⫽ anti-human globulin; IVIG ⫽ intravenous immunoglobulin; NT ⫽ not tested; TX ⫽ transplantation.
Antibodies to Stem Cell Donor Class I Antigens
count was still less than 0.1/mm3 and she not only remained platelet and red blood cell transfusion dependent, but failed to respond to random donor platelets due to the persistance of her anti-HLA antibodies (Table 2, line 2). She was subsequently transfused with platelets from donors selected to lack her target antigens and treated with plasmapheresis on 3 consecutive days. When no change in her antibody activities was observed following that treatment (Table 2, lines 3 and 4), she was treated with fludara for 4 days at a dose of 30 mg/m2, then again with plasmapheresis, this time for 4 consecutvie days and this time with an intravenous infusion of immunoglobulin (0.5 g/kg) following each plasmapheresis treatment (total intravenous immunoglobulin [IVIG] ⫽ 2 g/kg). Although no sample was tested for residual antibody activity at that time, after the first of these plasmapheresis ⫹ IVIG treatments, she was given a total dose of 17.41⫻106 CD34 positive cells (12.98⫻106 collected following a second standard peripheral blood stem cell stimulation protocol utilizing granulocyte macrophage– colony-stimulating factor at 10 g/kg for 5 consecutive days and 4.43⫻106 that had been stored frozen in reserve from the previous collection). Cyclosporin alone was used for GVHD prophylaxis. Surrogate crossmatch testing 3 days post-transplantation #2 (TX#2; immediately after the last plasmapheresis ⫹ IVIG treatment) indicated that the anti-donor specific antibody had almost disappeared from the circulation (Table 2, line 5). Within 1 week of the IVIG treatment and second transplant, she showed signs of engraftment with 100% donor chimerism detected after 3 weeks (data not shown). Although the patient’s antibodies to the crossreactive antigen, HLA-A2, continued to be detected for several weeks after transplant (Table 2, lines 6 – 8), these also disappeared after several more weeks (Table 2, line 9). DISCUSSION The initial engraftment failure this patient experienced indicates that preformed anti-donor class I HLA antibodies can eliminate enough of the donor’s differentiating stem cells to preclude engraftment, even though the most primitive stem cells do not express HLA antigens. Although testing potential allogeneic stem cell recipients for antibodies to potential donors’ mismatched HLA antigens has been recommended as the the standard of clinical practice for many years [1– 4], the actual number of HLA laboratories performing that testing for HLA mismatched stem cell transplant donors is very small (ASHI ARB data 2003). This case report should serve to remind HLA laboratories of the importance of this testing. We can also suggest that when such antibodies are
247
found to be present in high enough titer to cause a strongly positive crossmatch, consideration should be given to pretransplant plasmapheresis with IVIG treatment at the time of transplant; plasmapheresis alone seemed to be ineffective, at least in the three daily treatment schedule used for this patient at WHMC. The observation that the weakly positive crossmatch detected after the four treatments did not prevent engraftment (Table 2, line 5) provides the basis for suggesting that such treatment might not be necessary for patients with weak anti-donor antibodies. The incidental finding that the donor-specific antibody (to A68) disappeared while the antibody to the crossreactive antigen A2 remained detectable for several weeks might reflect one of the proposed mechanisms of action IVIG, namely that antibodies are still specifically bound to the target (donor) cells but the IVIG antibodies to Fc receptors prevent the antibody binding cells from being eliminated. As chimerism became complete, the residual recipient B cells all disappeared, causing the (expected) disappearance of all recipient-derived antibodies. ACKNOWLEDGMENTS
The authors wish to thank Evan Ray, Jeffrey Hoskins, Laura McNeish, Julie Forman, and Brook Holt for their excellent technical support for this study and the Bill Young Department of Defense Marrow Transplant Program for referring this patient and for providing the HLA typing results for the other family members.
REFERENCES 1. Anasetti C: Effect of HLA compatibility on engraftment of bone marrow transplants in patients with leukemia or lymphoma. New Engl J Med 320:197, 1989. 2. Pollack MS: Histocompatibility testing for bone marrow/ stem cell transplantation of matched and partially mismatched donors. In: Guidelines for Clinical Histocompatibility Practice. Lenexa, KS: ASHI, 49⫺53, 1999. 3. Hurley CK, Wade JA, Oudshoorn M, Middleton D, Kukuruga D, Navarrete C, Christiansen F, Hegland J, Ren E-C, Anderson I, Cleaver SA, Brautbar C, Raffoux C: A special report: Histocompatibility Testing guidelines for hematopoietic stem cell transplantation using volunteer donors. Hum Immunol 60:347, 1999. 4. Mickelson EM, Petersdorf E, Anasetti C, Martin P, Woolfrey A, Hansen JA: HLA matching in hematopoietic cell transplantation. Hum Immunol 61:92, 2000. 5. ASHI Laboratory Manual, 2nd Edition. Lenexa, KS: American Society for Histocompatibility and Immunogenetics, 1990. 6. Flow PRA I Screening Test Product Insert. Canoga Park, CA: One Lambda, Inc.