Correlation of Biochemical and Hematological Changes with Graft Failure Following Pig Heart and Kidney Transplantation in Baboons

American Journal of TransplantationVolume 3, Issue 12 p. 1510-1519 Free Access Correlation of Biochemical and Hematological Changes with Graft Failure Following Pig Heart and Kidney Transplantation in Baboons Christoph Knosalla, Christoph Knosalla Transplantation Biology Research Center,Search for more papers by this authorBernd Gollackner, Bernd Gollackner Transplantation Biology Research Center,Search for more papers by this authorLeo Bühler, Leo Bühler Transplantation Biology Research Center,Search for more papers by this authorNicolas J. Mueller, Nicolas J. Mueller Division of Infectious Disease, and theSearch for more papers by this authorStuart Houser, Stuart Houser Department of Pathology, Massachusetts General Hospital/Harvard Medical School, Boston, MASearch for more papers by this authorShamila Mauiyyedi, Shamila Mauiyyedi Department of Pathology, Massachusetts General Hospital/Harvard Medical School, Boston, MASearch for more papers by this authorDavid H. Sachs, David H. Sachs Transplantation Biology Research Center,Search for more papers by this authorSimon C. Robson, Simon C. Robson Center for Immunobiology, Beth Israel-Deaconess Medical Center/Harvard Medical School, Boston, MASearch for more papers by this authorJay Fishman, Jay Fishman Division of Infectious Disease, and theSearch for more papers by this authorHenk-Jan Schuurman, Henk-Jan Schuurman Immerge BioTherapeutics, Cambridge, MASearch for more papers by this authorMichel Awwad, Michel Awwad Immerge BioTherapeutics, Cambridge, MASearch for more papers by this authorDavid K. C. Cooper, Corresponding Author David K. C. Cooper Transplantation Biology Research Center, *Corresponding author: David Cooper, David.Cooper@tbrc.mgh.harvard.eduSearch for more papers by this author Christoph Knosalla, Christoph Knosalla Transplantation Biology Research Center,Search for more papers by this authorBernd Gollackner, Bernd Gollackner Transplantation Biology Research Center,Search for more papers by this authorLeo Bühler, Leo Bühler Transplantation Biology Research Center,Search for more papers by this authorNicolas J. Mueller, Nicolas J. Mueller Division of Infectious Disease, and theSearch for more papers by this authorStuart Houser, Stuart Houser Department of Pathology, Massachusetts General Hospital/Harvard Medical School, Boston, MASearch for more papers by this authorShamila Mauiyyedi, Shamila Mauiyyedi Department of Pathology, Massachusetts General Hospital/Harvard Medical School, Boston, MASearch for more papers by this authorDavid H. Sachs, David H. Sachs Transplantation Biology Research Center,Search for more papers by this authorSimon C. Robson, Simon C. Robson Center for Immunobiology, Beth Israel-Deaconess Medical Center/Harvard Medical School, Boston, MASearch for more papers by this authorJay Fishman, Jay Fishman Division of Infectious Disease, and theSearch for more papers by this authorHenk-Jan Schuurman, Henk-Jan Schuurman Immerge BioTherapeutics, Cambridge, MASearch for more papers by this authorMichel Awwad, Michel Awwad Immerge BioTherapeutics, Cambridge, MASearch for more papers by this authorDavid K. C. Cooper, Corresponding Author David K. C. Cooper Transplantation Biology Research Center, *Corresponding author: David Cooper, David.Cooper@tbrc.mgh.harvard.eduSearch for more papers by this author First published: 06 October 2003 https://doi.org/10.1046/j.1600-6135.2003.00258.xCitations: 36AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract We have explored biochemical and hematologic parameters that might indicate acute humoral xenograft rejection (AHXR) following pig organ transplantation in baboons. Baboons (n = 15) received an immunosuppressive regimen, and underwent a miniature swine or hDAF kidney (Group 1, n = 6) or heart (Group 2, n = 7) transplantation. Control baboons (Group 3, n = 2) received the immunosuppressive regimen without organ transplantation. Blood chemistry and hematologic parameters were measured daily. Baboon and porcine cytomegalovirus were monitored. In Groups 1 and 2, organ grafts survived for up to 29 days. A plasma fibrinogen of <80 mg/dL on 2 consecutive days, and a serum lactate dehydrogenase of >600 U/L and aspartate transaminase of >300 U/L, were associated with the development of AHXR in both heart and kidney grafts. In Group 1, a decrease in platelet count of >150 000/μL within 3 days, or a count of <50 000/μL, were associated with AHXR. In Group 2, a creatine phosphokinase of >500 U/L was associated with graft failure. In Group 3, no abnormalities were observed. The possibility that porcine CMV may play a role in graft injury could not be excluded. Noninvasive parameters were identified that have predictive potential for AHXR. Monitoring of these might enable therapeutic intervention to reverse rejection. Introduction Acute humoral xenograft rejection (AHXR) is currently the major immunological barrier to successful discordant xenogeneic transplantation (Tx) in pig-to-nonhuman primate models. Although hyperacute rejection can be overcome either by the use of organs from pigs transgenic for one or more regulators of complement activation, e.g. human decay accelerating factor (hDAF) (1, 2), or by immunoadsorption of anti-Galα1–3Gal (Gal) antibody (Ab) from the potential recipient before organ Tx (2-4), increasing immunoglobulin deposition on the vascular endothelium of the transplanted organ appears to be related to the development of AHXR (5). Although elicited anti-Gal and antinonGal Abs play a significant role in the development of AHXR, we have previously reported evidence indicating that when the elicited Ab response has been successfully prevented by therapy with an anti-CD154 monoclonal antibody (mAb), natural anti-Gal Ab alone can lead to this complication (6). Our previous studies indicated that pig kidney Tx in baboons may be associated with a fall in the level of plasma fibrinogen (6). We hypothesize that this is a consequence of endothelial cell activation. Consumptive coagulopathy (CC) may develop in these cases (6). We found no definitive correlation between the extent of histopathological features of AHXR and the development of CC, as CC could occur in the presence of mild or moderate histopathologic changes. The development of CC was also associated with a decrease in platelet count, an abrupt terminal increase in prothrombin time, and in activation of porcine cytomegalovirus (PCMV) in some animals (7). However, AHXR could occur in the absence of the development of CC and, in such cases it could only be monitored by biopsy and histological examination of the transplanted kidney. As one native kidney was left in situ in our studies, changes in serum creatinine or blood urea nitrogen could not be followed as indicators of graft dysfunction. Changes in these two parameters are associated with graft dysfunction and rejection (8). When heterotopic heart Tx is performed in the pig-to-nonhuman primate model, no simple serum marker has been reported as an indicator for AHXR, and so we have monitored the blood for various biochemical and hematological parameters in an effort to assess their association with graft dysfunction. We here report our retrospective observations in animals that underwent an identical immunosuppressive regimen, and show that changes in certain parameters, when taken together, are indicative for kidney or heart graft failure well in advance of significant histopathological changes. The exact cause of the systemic changes remains uncertain. We also asked whether these changes could be associated with the immunosuppressive therapy being administered rather than with the presence of the transplanted pig organ. In baboons receiving the same immunosuppressive regimen, but not receiving a pig organ, none of these biochemical or hematological changes was seen. We have attempted to quantify these changes and believe that they may allow the opportunity for modification of therapy before graft failure develops. Materials and Methods Animals Baboons (Papio anubis, n = 15) of known ABO blood group and of body weight 8–17 kg (Biological Resources, Houston, TX, or Mannheimer Foundation, Homestead, FL) were used as recipients. A Massachusetts General Hospital (MGH) MHC-inbred miniature swine (n = 6) of blood group O, 1.5–4 months old, 6.5–40 kg body weight (Charles River Laboratories, Wilmington, MA) or Large White/Landrace crossbreed pigs transgenic for hDAF (n = 7) (Novartis Pharma/Harlan, Madison, WI) of blood group O, 1.5–3.5 months old, weighing 9–35 kg, served as donors of kidneys or hearts. All experiments were conducted according to the NIH Guidelines for Care and Use of Laboratory Animals and were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care. Surgical procedures Anesthesia, intravascular line placements in pigs and baboon, kidney or heart excision in pigs, splenectomy, and kidney or heart Tx in baboons have been described in detail previously (4,9). In the baboons that underwent kidney Tx, one kidney was left in situ to allow the potential for survival and follow up in the event of the need for donor organ excision for rejection. At the time of graft excision, cuffs of a pig's renal artery and vein remained in the wall of the baboon's aorta and inferior vena cava, respectively, and a remnant of the pig's ureter remained in the bladder wall. Heterotopic heart Tx was performed through a midline abdominal incision. Graft function was monitored by daily palpation, and recorded by using a four-grade classification system with grade 3, representing excellent graft function, and grade 0, representing cessation of contractions. After excision of a rejected heart graft, remnants of pig aorta and pulmonary artery remained in the baboon. Extracorporeal immunoadsorption in baboons Anti-Gal Ab was depleted from the baboon's circulation by the perfusion of plasma through immunoadsorption columns containing synthetic Galα1–3Galβ1–4Glc-X-Y (αGal type 6 trisaccharide, Alberta Research Council, Edmonton, Alberta, Canada), as described previously (10-12). Conditioning regimen in baboons All baboons in Groups 1–3 (see later) underwent splenectomy on day –8, received horse antihuman thymocyte globulin (ATGAM, Upjohn, Kalamazoo, MI) 50 mg/kg/day i.v. on days –3, –2 and –1, underwent extracorporeal immunoadsorption of anti-Gal Ab on days –3, –2 and –1, and received thymic irradiation (700 cGy) on day –1 (4,10). All baboons also received mycophenolate mofetil by a continuous i.v. infusion (at approximately 110 mg/kg/day) from day –8 to maintain a whole blood level of 3–6 μg/mL (13). Cobra venom factor at approximately 100 units/kg/d i.v. was given from day –1 until graftectomy to maintain the CH50 at 0% (14). Prostacyclin (PGI2; 20 ng/kg/min by continuous i.v. infusion for 14 days) and methylprednisolone (2 mg/kg × 2 daily i.v. for 7 days, reducing weekly to 0.25 mg/kg daily at 28 days) were started on day 0. Murine antihuman CD154 mAb (5C8; ATCC, Rockville, MD, and prepared by Immerge BioTherapeutics, Cambridge, MA) (20 mg/kg/i.v.) therapy was started on day –1 or 0 (two doses) and administered on alternate days until graft excision or recipient death (6). Anti-CD154 mAb levels were measured and maintained >300 μg/mL. Heparin (10–20 U/kg/h by continuous i.v. infusion) was started on day 2 and continued until graftectomy. Cyclophosphamide was administered at a total dose of 60–80 mg/kg/i.v. in the perioperative period between days –6 and 4. After Tx, when the white blood cell count increased to >3000/mm3 or signs of rejection were evident, additional doses of cyclophosphamide (5–20 mg/kg) were administered. Pig kidney Tx (and unilateral native nephrectomy, Group 1) or pig heterotopic heart Tx (Group 2) was performed on day 0. Prophylactic ganciclovir (5 mg/kg/day) was administered to five baboons in Group 2, beginning on day –5 in three animals and on day 4 in two animals. In Group 3, no organ was transplanted. Prophylactic ganciclovir was given in one of these control subjects, starting on day –5. Monitoring and supportive therapy Blood chemistry, including troponin T levels, blood cell counts, international normalized ratio and partial thromboplastin time (PTT), fibrinogen, and fibrin degradation products were measured daily by standard methods. Washed irradiated red blood cells from AB-matched baboon donors were administered to maintain the hematocrit >20%. Erythropoietin was administered to some baboons at a dose of 100 units/kg subcutaneously ×1–2 weekly; this did not appear to affect outcome. Thrombocytopenia of <10 000 platelets/mm3 was corrected by the transfusion of fresh-washed irradiated baboon platelets. All baboons received daily prophylactic cefazolin sodium (500 mg/day i.v.) or levofloxacin (100 mg/day i.v.) throughout the period of immunosuppressive therapy. Surveillance blood cultures were drawn twice weekly, and specific antibiotic therapy was initiated when indicated. Quantitative, real-time polymerase chain reaction analyses of blood using primers for cytomegalovirus (CMV) that were baboon- or pig-specific were performed in six baboons × 3 weekly (Group 2, n = 4; Group 3, n = 2), on the organ after graftectomy (n = 9), and on the native baboon tissues at necropsy (n = 9). Assays for detection of baboon anti-Gal antibody and of antibody directed against porcine nonGal determinants Details of these methods have been reported previously (10, 11). IgM and IgG Ab reactive with Gal type 6 and type 2 were determined by ELISA, and antipig IgM and IgG by flow cytometric analysis. Histopathology, immunohistopathology, and ultrastructure of tissue biopsies Tissues were fixed in 10% formalin and paraffin-embedded. Five-micron sections of tissue were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff for light microscopy. Immunohistochemical staining for immunoglobulins (IgM and IgG), complement, and fibrin was performed on frozen sections; details have been reported previously (5). For electron microscopy studies, tissues were fixed in 2.5% glutaraldehyde in phosphate buffer (pH 7.4) and postfixed with 1% osmium tetroxide, and embedded in Epon 812. Ultrathin sections were stained with lead citrate and examined with a Phillips 301 electron microscope. Measurement of porcine and baboon CMV DNA Total DNA extracted from tissue or peripheral blood mononuclear cells was quantified as described previously (14). Statistical analyses Sensitivity, specificity, positive predictive value, and negative predictive value were calculated for biochemical and hematological parameters. Confidence intervals for sensitivity, specificity, and positive and negative predictive values were computed as an exact binomial 95% confidence interval, using SPSS 9.0 for Windows (SPSS Inc., Chicago, IL). Experimental groups Group 1 (n = 6) consisted of baboons that underwent either MGH (n = 3) or hDAF kidney (n = 3). Tx. Group 2 (n = 7) underwent heterotopic Tx of either MGH (n = 3) or hDAF (n = 4) hearts. Group 3 (n = 2) were subjected to the same induction and maintenance immunosuppressive therapy as Groups 1 and 2, but did not receive an organ transplant. Results Graft survival Pig kidney (Group 1) and heart (Group 2) grafts survived for up to 29 and 28 days, respectively. Human decay accelerating factor kidneys survived longer than MGH kidneys (median survival 29 vs. 7 days); failure of the MGH kidneys resulted from several different causes (Table 1), whereas failure of the hDAF kidneys uniformly resulted from AHXR. There was little difference in survival of the heart grafts from these two sources (median 27 vs. 24 days) (Table 1). Acute humoral xenograft rejection was the prime reason for termination of the experiment in seven cases (Table 1), in all cases confirmed by histopathology. In six cases, the experiment was terminated through death of the baboon from infection (n = 3) or other complications (Table 1) [pulmonary thrombosis (n = 1), necrosis of the distal ureter (without signs of AHXR in the kidney graft) (n = 1), heart graft failure of uncertain cause (cardiac distension with no definite histopathologic features of AHXR) (n = 1)]. Consumptive coagulopathy developed in four cases (Table 1); it proved fatal in two cases, and in one the baboon was euthanized. Table 1. Causes of graft failure and recipient death in Groups 1 and 2 Group (Baboon #) Donor pig (days) Graft survival (cause of recipient death) Cause of graft failure Group 1 B69-254 MGH 6 Minimal AHXR (infection) B129-23 MGH 13 AHXR/CC (bleeding)1 B133-59 MGH 7 Distal ureter necrotic/perforated (euthanized) B117-63 hDAF 28 AHXR/CC (bleeding)1 B182-323 hDAF 29 AHXR (necrotizing pancreatitis)1 B69-169 hDAF 29 AHXR/CC (survived)1 Group 2 B116-16 MGH 21 No AHXR(infection) B69-126 MGH 26 AHXR (euthanized)1 B69-134 MGH 27 AHXR (euthanized)1 B69-210 hDAF 19 No AHXR (pulmonary thrombosis) B69-321 hDAF 28 AHXR/CC (euthanized)1 B69-714 hDAF 15 Graft failure of uncertain cause (euthanized)1 B69-171 hDAF 28 No AHXR (infection) AHXR = acute humoral xenograft rejection, CC = consumptive coagulopathy. 1Indicates those experiments in which changes were seen in systemic parameters. Changes in biochemical and hematological parameters There was a steady reduction in hematocrit and hemoglobin during the course of all experiments in Groups 1–3, which was considered to be related to frequent blood withdrawal and the effect of immunosuppressive therapy on the bone marrow. Serum bilirubin remained normal in all cases in all groups, suggesting that there was no significant hemolysis. In both Groups 1 and 2, following the initial surgery, there was a temporary increase in lactate dehydrogenase (LDH), aspartate aminotransaminase (AST), and creatine phosphokinase (CPK). Increases in these parameters were more marked in the Group 2 baboons, some of which developed a temporary rise in troponin T as a result of reversible ischemic injury to the transplanted pig heart. Plasma fibrinogen and platelet count fell during the first week postTx in most baboons in all groups, as a result of induction therapy with cyclophosphamide. Group 1 Biochemical parameters. An increase in LDH to >600 U/L occurred in four baboons, and rose to a mean of 1144 (± 800) before graft failure, which was determined by the development of CC or histological features of AHXR; a sustained increase of >600 U/L was associated with the development of AHXR on histological examination (Table 2 and Figure 1). The increase to >600 U/L occurred at a mean of 6 (±2) days before final graft failure. Serum creatinine was not predictive, as one native kidney remained in situ (see Methods). Changes in other biochemical parameters, e.g. AST (Figure 2) and CPK, were not associated with graft failure (Table 2). Table 2. Sensitivity, specificity, positive predictive value, and negative predictive value of biochemical and hematological parameters predicting graft failure after porcine kidney transplantation in baboons (Group 1) Parameter Sensitivity (95%CI) Specificity (95%CI) PPV (95%CI) NPV (95%CI) LDH (>600 U/L) 100% 100% 100% 100% (51–100%) (34–100%) (51–100%) (34–100%) Fibrinogen (decrease >80 mg/dL over 3 days, or to 100% 100% 100% 100% <80 mg/dL on 2 consecutive days) (51–100%) (34–100%) (51–100%) (34–100%) PLT (decrease >150 000/μL over 3 days, or to 100% 100% 100% 100% <50 000/μL) (51–100%) (34–100%) (51–100%) (34–100%) AST (>300 U/L) 0% 100% 0% 33% (0–49%) (34–100%) (0–100%) (70–97%) CPK (>500 U/L) 0% 100% 0% 33% (0–49%) (34–100%) (0–100%) (70–97%) CI95%= binomial 95% confidence interval, LDH = lactate dehydrogenase, PLT = platelet count, AST = aspartate aminotransaminase, CPK = creatine phosphokinase. Figure 1Open in figure viewerPowerPoint Changes in lactate dehydrogenase (in U/L) in baboons in which pig organ failure developed; (A) pig kidney recipients (Group 1); (B) pig heart recipients (Group 2); and (C) control baboons: no organ graft (Group 3). Massachusetts General Hospital pig organs (—), and human decay accelerating factor pig organs (–). Figure 2Open in figure viewerPowerPoint Changes in aspartate transaminase (in U/L) in baboons in which pig organ failure developed; (A) pig kidney recipients (Group 1); (B) pig heart recipients (Group 2); and (C) control baboons: no organ graft (Group 3). Massachusetts General Hospital pig organs (—), and human decay accelerating factor pig organs (–). Hematological parameters. Excluding the initial transient reduction (associated with cyclophosphamide therapy), a plasma fibrinogen level of <80 mg/dL on 2 consecutive days, or a decrease >80 mg/dL over 3 days, was associated with AHXR (Table 2 and Figure 3) and occurred 6 (±6) days before the need to excise the graft. The platelet count also fell in five of six baboons initially, but a subsequent reduction of >150 000/μL within a period of 3 days, or a fall to an absolute count of <50 000/μL, was associated with impending AHXR, and occurred 6 (±2) days before graft failure (Table 2 and Figure 4). Partial thromboplastin time and the international normalized ratio remained stable over the entire course, but increased acutely to values of >150 s and 17, respectively, when CC occurred. Fibrinogen degradation products showed a variable pattern, but were high (>160 μg/mL) when CC occurred. Figure 3Open in figure viewerPowerPoint Changes in plasma fibrinogen (in mg/dL) in baboons in which pig organ failure developed; (A) pig kidney recipients (Group 1); (B) pig heart recipients (Group 2); and (C) control baboons: no organ graft (Group 3). Massachusetts General Hospital pig organs (—), and human decay accelerating factor pig organs (–). Figure 4Open in figure viewerPowerPoint Changes in platelet count (/μL) in baboons in which pig organ failure developed; (A) pig kidney recipients (Group1); (B) pig heart recipients (Group 2); and (C) control baboons: no organ graft (Group 3). Massachusetts General Hospital pig organs (—), and human decay accelerating factor pig organs (–). Group 2 Biochemical parameters. An increase of LDH to >600 U/L occurred in five baboons, beginning 15 days (± 8 days) before cessation of contractions, and continued to rise to a maximum mean level of 3471 (±2303) U/L (Table 3 and Figure 1). The maximum LDH was higher in those baboons that received MGH pig hearts than hDAF hearts. In two baboons, an increase in AST (to >300 U/L) was associated with AHXR (4 and 7 days, respectively, before graft failure), and in two it was associated with infection or pulmonary thrombosis (Table 3). The rate of increase of LDH or AST (Figure 2) correlated with the speed of development of graft failure. An increase in CPK to >500 U/L occurred in two cases in association with AHXR, and in one when graft failure occurred for uncertain cause, 10 (±3) days before graft failure (Table 3). Measurement of human CPK MB isoenzymes showed normal parameters in all cases, and appears not to reflect injury in pig hearts, which has been confirmed by other studies in pigs at our center. Table 3. Sensitivity, specificity, positive predictive value, and negative predictive value of biochemical and hematological parameters predicting graft failure after porcine heart transplantation in baboons (Group 2) Parameter Sensitivity (CI95%) Specificity (CI95%) PPV (CI95%) NPV (CI95%) LDH (>600 U/L) 100% 50% 60% 100% (44–100%) (15–85%) (23–88%) (34–100%) Fibrinogen (decrease >80 mg/dL over 3 days 75% 100% 100% 25% or to <80 mg/dL) (30–95%) (44–100%) (44–100%) (5–70%) AST (>300 U/L) 67% 50% 50% 67% (21–94%) (15–85%) (15–85%) (21–94%) CPK (>500 U/L) 67% 50% 50% 67% (21–94%) (15–85%) (15–85%) (21–94%) PLT (decrease 150 000/μL over 3 days 0% 100% 0% 50% or to <50 000/μL) (0–56%) (51–100%) (0–100%) (25–84%) PTT 100% 0% 43% 0% (44–100%) (0–49%) (16–75%) (0%–100%) PTT = partial thromboplastin time, CI95%= binomial 95% confidence interval, LDH = lactate dehydrogenase, PLT = platelet count, AST = aspartate aminotransaminase, CPK = creatine phosphokinase. Troponin T rose significantly immediately after heart Tx as a result of the ischemic injury experienced during Tx (confirmed by alloTx studies), but returned to baseline level within 3–4 days. It showed no increase during the period when LDH and AST were rising, but only began to rise 2 days before functional graft failure from AHXR. This terminal rise in troponin T correlated with a deterioration in contractility of the transplanted heart as detected by palpation, particularly when AHXR was severe (confirmed by histopathology) when the level rose to >2 mg/dL. Hematological parameters. A steady decrease in fibrinogen to 80–110 mg/dL over the first week occurred in all baboons. A further decrease to <80 mg/dL, sustained for 2 consecutive days [which occurred 11 (±2) days before graft failure], was associated with the development of AHXR (Table 3 and Figure 3). In one baboon, a fall in platelet count of >150 000/μL within a period of 3 days occurred as a result of Pseudomonas aeruginosa sepsis, but was not seen when graft rejection occurred. No such decrease occurred in any other baboon (Table 3 and Figure 4). An increase in PTT over time occurred in all animals, but was not associated with the development of AHXR or graft failure (Table 3). Other parameters showed variable levels, but were not associated with the development of AHXR. In those animals in Groups 1 and 2 that survived after successful excision of the graft for AHXR with CC (n = 2), all biochemical and hematological parameters normalized within 48 h, and remained within normal limits thereafter, with follow up for >150 days. Group 3 After initial falls in fibrinogen and platelet count (associated with the induction therapy), no abnormalities in biochemical and hematological parameters were observed during the 4- or 6-week period of immunosuppressive therapy (Figures 1 and 3). Correlation of changes in combined parameters with graft failure The three parameters that most closely correlated with graft failure after kidney Tx (Group 1) were LDH (increase), fibrinogen (decrease), and platelet count (decrease), and those after heart Tx (Group 2) were LDH (increase), fibrinogen (decrease), and AST (or CPK) (increase). In an attempt to quantify the changes occurring in these three parameters, the changes in each baboon of each group were scored as in Table 4; the mean sum of the scores was correlated with clinical outcome. The minimum score was 0 (indicating no deterioration in any of the three parameters), and the maximum that could be achieved was 3 (indicating a significant change in all three parameters). Table 4. Scoring system of biochemical and hematological changes occurring during graft failure Parameter Score LDH >600 U/L 1 <600 U/L 0 Fibrinogen Decrease >80 mg/dL over 3 days, 1 or to <80 mg/dL on 2 consecutive days No decrease >80 mg/dL over 3 days, 0 or to <80 mg/dL on 2 consecutive days Platelets Decrease >150 000/μL over 3 days, 1 or to <50 000/μL No fall >150 000/μL over 3 days, 0 or to <50 000/μL AST >300 U/L 1 <300 U/L 0 Group 1: In baboons that developed AHXR (n = 4), the mean score began increasing approximately 2 weeks before graft failure, with the maximum possible score of 3.0 being reached 3 days before graft failure (Figure 5). In contrast, in baboons that did not develop AHXR (n = 2), the score increased only terminally to a mean of 0.3 (not shown). Figure 5Open in figure viewerPowerPoint Mean prediction score in baboon recipients of pig kidneys (Group 1; —) and pig hearts (Group 2; –) in which acute humoral xenograft rejection developed over time prior to graft failure (n = 4 in each group). When graft failure did not develop, but the experiment was terminated for other reasons, the score did not rise >0.5 (not shown). Group 2: In baboons that developed graft failure (n = 4) (i.e. cessation of contractions, including three with AHXR and one without conclusive histopathologic evidence of AHXR), the score began increasing approximately 2 weeks before graft failure. The maximum score of 3.0 was reached 4 days before termination of the experiment (Figure 5). The increase in score began several days before any decrease in cardiac contractions, as determined by palpation. In baboons without graft failure (n = 3), the increase in score began later and was far less marked, reaching a maximum score of only 0.5 (not shown). Group 3: There was no increase in score greater than the baseline of 0 in either baboon (not shown). Anti-Gal and anti-nonGal antibody responses In all baboons in Groups 1–3, whenever anti-CD154 mAb therapy was being administered, anti-Gal Ab remained at low levels. There was no obvious correlation between anti-Gal IgM or IgG levels and the biochemical and hematological changes described earlier. No Ab to nonGal pi