Solid-Organ Transplantation John A. Lott Department of Pathology, The Ohio State Univetsiiy, Starling-Loving M-368, Columbus, Ohio 43210
Organ transplantationfor the treatment of end-stage failure of the liver, kidneys, and pancreas is now well established. With liver failure, transplantation is the only option; patients with kidney failure can be maintained by chronic hemodialysis, and pancreatic failure can be treated by replacement of both the exocrine and endocrine secretions. Digestive disturbances, weight loss, and debilitation are the primary problems occumng with marked reduction or loss of the exocrine pancreas; diabetes mellitus is the major problem that develops with endocrine pancreatic failure. For liver, and kidney, the surgical methods are now well established. Liver replacements are orthotopic transplants (Le., in the same place as the original organ), whereas the kidney is generally placed extraperitoneally in the iliac fossa in adults and retroperitoneallyin small children. Pancreas transplantation has not been standardized, and current alternative surgical techniques are described here briefly. Worldwide from the beginning of transplantation surgery up to approximately 1990, there were 270 OOO kidney, 14 OOO liver, and 3000 pancreas transplants ( G I ) . Lung transplantation, especially heart-lung transplantation, is becoming more prevalent, although the 5year survival for both the organs and patients is much lower than for kidney transplantation. Recent reviews are available on pediatric heart transplantation (GZ), kidney (G3G5), pediatric liver (G6), adult liver (G7-G9), lung ( G I & G I I ) , pancreatic islets (GIZ), and other organ transplantations (GI3, G I 4 ) . A highly detailed assessment of liver transplantation is available from the US.Department of Health and Human Services (GI5). Complications of transplantation such as infections have been reviewed recently a number of times (GI6-GI9). Reviews on other complications such as malignancy and the toxicity of immunosuppressive agents are available (G4, G20-GZZ). An extensive review exits of the organ distributions of cyclosporine A and its metabolites (G23). The leading impediment of transplantation therapy is the shortage of suitable organs. Many patients die while awaiting a possibly livesaving organ transplantation. For kidneys in the United States in 1993, there were about 25 OOO patients on the waiting list (GZ4). Unfortunately, only about 50%of families give consent for organ donation by their kin. The major medical problems of patients having organ transplantation are acute and chronic rejection and infections with bacterial, fungal, and viral agents, especially with cytomegalovirus (CMV) and much less so with hepatitis C 0.Other significant complications are leakages at anastomoses, thromboses, inflammation (e.g., pancreatitis), bleeding problems, and various malignancies, especially lymphoproliierative diseases. Cyclosporine A (CSA), prednisone, azathioprine, and other antirejection drugs are discussed below. For some of these, the clinical laboratory plays a role because of the need for the assay of blood concentrations of the parent drug and its active metabolites to assure adequate dosing but to minimize overdosing and the attendant undesirable side effects. A new drug, tacrolimus (FK 506), will likely also play an important role in retarding transplant rejection, especially in liver transplantation. Except for transplants between identical twins, and with very rare exceptions,
all patients receiving organ transplantation must take antirejection drugs for the rest of their lives. This review considers the laboratory problems common to all transplantation, e.g., compatibility testing between donor and recipient, and then discusses laboratory issues that are pertinent for the support of patients having kidney, liver, or pancreas transplantation. Rejection, especially chronic rejection, is a common problem with transplantation, and the biopsy remains as the “gold standard” for diagnosing rejection of solid-organ transplants. ROLE OF THE CLINICAL LABORATORY Transplant recipients need postoperative laboratory support to manage acute-care problems such as fluid and electrolyte disturbances, acid/base abnormalities, bleeding and coagulation concerns, and testing for organ function, infections, and rejection. Testing for infections caused by viral, fungal, and bacterial agents and for possible transplant rejection is a critical part of posttransplantation care; the two conditions have a similar clinical presentation: malaise, fever, etc. The proper diagnosis of rejection or infection plays an important role in diagnosis. If rejection is suspected, then the regimen of antirejection drugs is usually stepped up and new agents may be added. If in fact the patient has an infection rather than rejection, the increased antirejection therapy may exacerbate the infection and lead to graft and patient loss. The reverse case also obtains: if infection is diagnosed but the case is really rejection, reducing the antirejection drugs to fight the believed infection may also lead to graft loss. HIA Compatibility Testing. The major human histocompatibility complex (MHC), commonly called the human leukocyte antigen (HLA) complex, is coded by the nucleotides on chromosome 6. There are three classes of HLA antigens;when present, they occur on cell membranes: class I or HLA-A, HLA-B, and HLA-C; class I1 or HLA-DP, HLA-DQ, and HLA-DR; and class 111, which contains proteins including those of the classic and alternate complement pathways, 21-hydroxylase, and tumor necrosis factor. /?-2 microglobulin is part of the class I HLA molecules and its gene locus in on chromosome 15 (GZ5). HLA class I antigens are expressed on all nucleated cells: they also occur on platelets. The HLA-DR, -DQ, and -DP (class II) antigens are expressed on B lymphocytes, monocyte-macrophages, and dendritic cells (G3). Prior to assay, a concentration or isolation step of the B cells is usually performed by using a nylon wool column to retain the B cells and elute the T cells; the B cells are then brought out with another eluent to permit testing for class I1 antigens. In typing for HLA compatibility, lymphocytes from the donor and serum from the recipient are used; the typical test uses rabbit complement and then determines microlymphocytotoxicitywith and without complement by a technique that is described elsewhere (GZ6). The HLA cross-match test can be made more sensitive by adding antihuman globulin to the incubation mixture. If the test is positive with antihuman globulin present, the likelihood of graft rejection is about 5%greater than if the test is negative. With an HLA mismatch, lysis of the lymphocytes occurs. By staining the lymphocytes with vital dyes, the proportion of dead cells can be estimated by light microscopy. A positive mismatch is revealed by a large fraction of dead cells. Analytical Chemistty, Vol. 67, No. 12, June 15, 1995
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Flow Cytometry. Flow cytometry makes possible the measurement of low concentrations of both complement-king and non-complement-king antibodies. This technique is more sensitive than the standard lymphocyte cytotoxicity cross-match test in predicting graft rejection (G27431). Ogura et al. (G32) showed that flow cytometry was clearly superior to a standard T-cell cross-match test. Of 84 patients, 10 had a positive T-cell cross match and 20 of the 84 had a positive flow cytometry test. At one month post transplantation, 3 of 10 patients with a positive T-cell test lost their graft, while 17 of 74 with a negative T-cell test had lost their graft, suggesting that the T-cell test gave some false negative results. In an earlier report by the same group, they found that patients with a negative flow test had a l-year graft survival rate of 82%vs a 75%rate for those with a positive flow test. The patients with a positive lymphocyte cross-match test also had a lower graft survival rate. It appears that patients with a negative flow test do better, and given the scarcity of suitable kidneys, preference should be given to patients with a negative flow test (G33). Others also reported that flow is superior to the cytotoxicity assay and that flow cytometry alone is enough to make a go, no-go transplantation decision in about 80% of patients ((234). The development of antibodies to the donor’s T and B lymphocytes can be determined with a sensitive flow cytometric assay; their presence was a harbinger of clinical kidney rejection (G35). Others made similar observations: four patients with antiB-cell antibodies specific against the class I1 HLA antigen, HLADR or HLA-DQ, showed hyperacute rejection in three of these patients and acute rejection in one (G36). Thus a test for antibodies against donor B-cell class I1 HLA antigens should be part of a cross match. Of considerable importance in the use of flow cytometry is the 97% sensitivity and 88% specificity in discriminating between patients with late (>2 years) acute renal allograft rejection and other causes of graft dysfunction such as infection, immunosuppressant drug toxicity, arteriopathy, or chronic rejection. Flow analysis also has the advantage of predicting successful antirejection therapy within a few days whereas the conventional T-cell test requires 1-3 weeks before they are predictors (G37). Others also found that flow was more specific than the standard cytotoxicity cross-matching techniques (G30) or was too sensitive and gave false-positive results (G38, G 3 9 ) . Mahoney et al. (G31) found that flow cytometric cross matching was a better predictor of a renal allograft loss at < 2 months after surgery in patients receiving a first and especially for those receiving a second cadaveric kidney who had a negative cross match by the standard complement-kation cytotoxicity test. The consensus is that flow analysis is more sensitive than the lymphocytotoxicity test and that patients with a negative flow cross match do better and have a longer graft survival (G40). DNATesting. Molecular techniques to compare the genotype of the donor and recipient are starting to be used. Opelz et al. found that the graft survival rate was 87%when the recipient and donor were HLA-DR identical by both the cytotoxicity and DNA tests; graft survival decreased to 69%when the HLA-DR cytotoxicity test was negative but the molecular test showed DNA inequalities between the donor and host (G41). The HLA genes are highly polymorphic, and there are dozens of recognized HLA specificities. With the exception of identical twins, the likelihood of finding two immunogenetically identical individuals is essentially zero. The presence of donor-speciiic class 418R
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I HLA antibodies in the recipient’s serum effectively prohibits the use of an organ from that donor; 80-9G% of those having such a positive test will have an acute or hyperacute transplant rejection (G42). Graft survival is inversely related to how many HLA mismatches are present. The current policy in many centers is that renal transplantation recipients must be phenotypically identical with the donor for the HLA-A, -B, and -DR antigens (G43). The rejection phenomenon begins when the recipient’s T lymphocytes recognize HLA proteins that are expressed on the surface of the antigen-presenting cells, i.e., on the graft T-cell activation follows by an unknown process. Here, the recipient’s activated T lymphocytes will attack the transplanted organ. Rejection can be largely blunted with immunosuppressive drugs that inhibit T cells; however, rejection is inevitable, and the greater the mismatch, the shorter is the life of the transplanted organ. Investigators have found donor-antigen-specific cytotoxic T lymphocytes and antibodies to the donor HLA antigens during or just before an episode of clinically demonstrable rejection. The mechanism of the antiallograft response of the host is described in detail elsewhere (G3). Infections. The commonest and most serious posttransplantation infection is by CMV; it threatens the survival of both the graft and the patient. The consensus is that a CMV infection alone does not lead to graft rejection. CMV infects the endothelium; it is the interface between the transplanted tissue and the recipient’s immune system. Although expression of HLA class I1 antigens on endothelial cells is a hallmark of vascular rejection, CMV does not directly induce these antigens on infected endothelial cells; in fact, CMV renders endothelial cells refractory to HLA-DR induction by certain agents (G44). Sherlock et al. (G45) made similar observations in kidney transplant recipients. They found active CMV infection in 11of 18 patients who rejected their grafts and also in 13 of 18 patients who did not reject. Furthermore, an active CMV infection was found in 8 of 15 patients who developed anti-donor lymphocyte antibodies and in 12 of 17 who did not develop such antibodies. In their patients, there was no statistically significant association between CMV infection and rejection nor between CMV infection and the development of antibodies to the donor’s lymphocytes. The most serious CMV infections are temporally associated with the most intense immunosuppression therapy that typically occurs within the first several months following transplantation. CMV infections present a spectrum of disorders ranging from minimal disease such as malaise and fever to severe forms that include pancreatitis, hepatitis, gastrointestinal bleeding, multisystem organ failure, and death (G46). Our current knowledge of CMV has a number of unresolved issues. Many normal individnals harbor the CMV virus; it remains latent, and why transplantation and (or) antirejection drugs activate the virus is unknown. There is a big clinical difference between a serologically positive CMV test and overt CMV disease, and current laboratory testing technology cannot distinguish between the two. Also, better antiviral drugs are needed to treat a fulminant CMV infection, although gancyclovir is generally effective (G19). It is not possible to diagnose a CMV infection solely on the basis of clinical findings, because the signs and symptoms of organ rejection and CMV infection are similar. Currently, the laboratory diagnosis of CMV relies primarily on the culture of the virus on fibroblasts by a shell vial procedure; the results are generally available in 24-48 h (G47). Other less widely used methods are
the polymerase chain reaction (PCR) to identify the presence of sequences of the CMV genome in serum; the disadvantages of PCR are cost, complexity, long turnaround times, and difficulty in some cases of interpreting the results. The PCR test is nearly always positive if leukocytes are present, which does not necessarily mean that the patient is going to develop active CMV disease. Other tests on serum include serological tests for CMV-speci6c IgM or IgG antibodies; their disadvantage is that antibody synthesis takes at least 1-2 weeks after infection, and no antibody formation at all may occur in immunosuppressed patients. In situ hybridization has been used to identify CMV infection in tissue biopsies (G48).Another test is the CMV-specific lymphocyte proliferation test (G49).The currently used standard test for CMV in tissue biopsies is an overlay with peroxidase-labeled antibodies to CMV that generates a chromophore followed by light or fluorescence microscopy. Ideally, the test for CMV should be sensitive, speciiic, and available on a short turnaround-timebasis. Marsano et al. (G50) compared the culture of the virus by a shell vial procedure with testing for IgM antibodies to CMV. Of 35 patients with active CMV infections, 31 showed positive viral cultures and 29 had detectable IgM antibodies to CMV. They claimed that, after solidorgan transplantation, the determination of CMV with the viral culture technique can give a result earlier and with better accuracy. Others (G51)described a rapid immunocytochemical test for CMV that is based on the reaction of CMV antigens in peripheral polymorphonuclear cells with a mixture of monoclonal antibodies. The monoclonals react with the CMV immediate early antigen (IEA) present on the leukocytes, and the results are available within 4 h. The test has excellent sensitivity but almost no speciiicity for CMV disease, revealing one of the difticulties with CMV testing; Le., patients may have viremia without obvious clinical infection. But asymptomatic patients with a strongly positive test may be candidates for CMV prophylaxis with an antiviral agent like gancyclovir to prevent a lifethreatening condition. Steinhoff et al. (G52)found an association of an increased urine firmicroglobulin concentration with a positive IEA test in patients with renal transplantation; the firmicroglobulin test may be of value in the early diagnosis of CMV infection. Use of Organs from Patients with HepatitisC (HCV). The transplantation of organs from donors having hepatitis B to a recipient without the disease is clearly contraindicated; all patients become infected with hepatitis B posttransplantation; chronic active hepatitis is likely, and survival is reduced (G53,G54). With hepatitis C 0 - p o s i t i v e organs, it is acceptable to use these in special circumstances. Most cases with “non-A,non-B” hepatitis have HCV, and reasonably reliable tests now exist for HCV. A HCV infection of parenteral origin becomes chronic in 50-60% of patients and cirrhosis develops in about 20% of these. HCV appears to be as important as hepatitis B as a cause of chronic liver disease and hepatocellular cancer, especially in Japan. Most patients with chronic HCV have only mild symptoms such as fatigue (G55). The first-generation ELISA test for HCV detects antibodies to a recombinant HCV antigen (~100)from the nonstructural region of HCV (G56).We now know that this test has poor sensitivity. In the older literature, a HCV infection developing in a c100negative patient has been attributed to unknown or sporadic causes of HCV (G57).A more likely explanation is that the test
was falsely negative. The second-generationELISA test detects antibody to recombinant HCV antigens from the c100, c200, and c20 sections of the nonstructural and core regions of the virus. A second-generationrecombinant immunoblot assay (RIBA) detects antibody to four recombinant HCV antigens: 51-1,c100, c33, and c22; all are from the nonstructural and core regions of HCV (G58). The detection of HCV RNA by PCR is currently the final arbiter for the presence of HCV antigens (G59).The details of the PCR assay are elsewhere (G56).The PCR test is costly and time consuming, and the diagnosis of HCV can be made in most patients with a positive RIBA test together with positive liver function tests and abnormal tissue pathology (G58). Given the extreme shortage of transplantable organs, is it acceptable to perform an orthotopic liver transplant with an HCV-positiveorgan into an HCV-positive or -negative recipient? Shah et al. (G54) concluded that the procedure is acceptable,and that there is “no increased risk for the development of HCV post OLT...”. A similar conclusion was reached by others (G60)for renal transplantation. Obviously, patients should be told of this risk owing to the -17% incidence of chronic liver disease associated with the transplantation of a HCV-seropositive kidney (G61). A survey of centers performing heart and (or) lung transplantation regarding the use of HCV seropositive donors revealed varying practices. Ten centers would accept a heart or lung from an HCV-positive donor, and 17 would only transplant same into an HCV-seropositive recipient or into a critically ill patient as a lifesaving procedure. Twelve centers indicated that HCV seropositive organs were always unacceptable (G62). There appears to be a trend toward a national protocol much like that of the New England Organ Bank owing to the unacceptably high prevalence of liver disease in recipients of HCVpositive organs, such organs are used only for lifesaving transplantation, Le., heart, heart-lung, or liver, and they are not to be used in kidney or pancreas transplants into HCV-negative recipients. The evidence is clear that HCV can be transmitted by organ transplantation, sometimes with disastrous results (G63).Wherever possible, testing for HCV should be by PCR for the viral RNA owing to the superior sensitivity of PCR as compared to the serological tests. Antirejection Drugs. The contemporary greater success in the transplantation of solid organs is attributable in large part the emergence of better immunosuppressive drugs; these include azathioprine, brequinar sodium, CSA, cyclosporine G, deoxyspergualine, rapamycin, glucocorticoids (e.g., prednisone), leflunomide, mizoribine (bredinin), mofetil (RS61443), mycophenolic acid, and tacrolimus (FK506)(G64).Tacrolimus and rampamycin inhibit the phosphatase calcineurin and thereby inhibit transcrip tional activation of the interleukin-2 gene. These drugs also inhibit the protein kinases that are important signaling mediators in Tcell activation (G65).CSA is widely used, and tacrolimus is a new drug. Both are actually precursors of the agents that become active only when bound to specific membranes of the cyclophilinor tacrolimus-binding protein receptor complex; both are extremely potent inhibitors of T-lymphocyte activation (G66). Evidence is accumulating that old and new immunosuppressive drugs permit the establishmentof donor-derived multilineage cell chimerism following transplantation; i.e., the transplanted cells and the host cells exist compatibly without a rejection reaction. This phenomenon explains the occasional patients who can wean themselves off all antirejection drugs without a rejection phenomAnalytical Chemistry, Vol. 67, No. 72, June 15, 1995
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enon; such patients are quite rare. Fontes et al. (G67) reported that, in liver, kidney, and heart transplantation, tolerance to donor cells could be induced in 17 of 36 patients by the infusion of donor bone marrow followed by conventional immunosuppression with tacrolimus and prednisone. This interesting approach may determine the direction of future approaches to solid-organ transplantation. The primary action of rapamycin and leflunomide appears to be inhibition of the effects of cytokines and growth factors on B, T, and some nonimmune cells. B and T cells are more sensitive than somatic cells to the reduced concentration of purines and pyrimidines as caused by mizoribine, mycophenolic acid, and bequinar sodium. Note that nucleotide depletion causes a break in the synthesis of DNA and the glycosylation of adhesion molecules in the immune cells (G68). Tacrolimus was discovered in 1984 in a fungus; it is a potent and selective anti-T-lymphocyte agent with actions similar to that of CSA. Unlike CSA, it has a hepatotrophic effect leading to active growth of normal liver tissue; this likely accounts for its success in liver allografts. Testing for Cyclosporine A and Tacrolimus. Monitoring of serum concentrations of antirejection drugs such as CSA and tacrolimus is carried out to assure adequate serum concentrations and to avoid overdosing. Like CSA, tacrolimus is nephrotoxic, and careful blood concentration monitoring is necessary during its use (G69). The clinical response for both drugs does not correlate well with the administered dose; concentrations in blood must be known to optimize treatment. Also, the range of drug concentrations is narrow to give adequate immunosuppression and to minimize nephrotoxicity. A review on the monitoring of CSA and specific recommendations for the assay of the drug in whole blood is elsewhere (G70). Assays for CSA include monoclonal immunoassays (G71), radioimmunoassays, and high-pressure liquid chromatography. Fluorescence polarization immunoassay is the most widely used procedure for both CSA and tacrolimus. KIDNEY AND (OR) PANCREAS TRANSPLANTATION
End-stage kidney disease as a complication of diabetes mellitus is a common indication for renal and pancreas transplantation in the same patient; the procedure is maturing into a widely accepted practice for the control of uremia and diabetes. The surgical technique in widest use is pancreaticoduodenocystostomywhereby the pancreatic exocrine duct is lead into the urinary bladder via a small section of donor duodenum (G72). An earlier surgical technique of performing pancreatic allografts included occluding the pancreatic duct with, for example, latex to cause atrophy of the acinar pancreas and thereby stop the exocrine secretions. This sought to avoid the problems of ducting the pancreatic juice to the small intestine, large intestine, or urinary bladder; however, this approach has been replaced in most centers by urinary bladder drainage of pancreatic fluid owing to the better graft survival and a reduced incidence of acute pancreatitis (G73). Nevertheless, the routing of the exocrine flow or the obliteration of the exocrine pancreas is still under debate in the literature (G72, G74). Secchi et al. (G75) occluded the pancreatic duct with neoprene in one patient to produce atrophy of the acinar pancreas and then transplanted a segment of the pancreas. They also transplanted the whole, unmodified pancreas into eight patients 420R
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with ileal drainage of the pancreatic juice. Both techniques gave satisfactory results, although the patients receiving the entire gland had a better glucose tolerance. Others have performed pancreas transplantation with systemic pancreatic venous or portal drainage of the pancreatic vein. The patients with systemic drainage showed higher insulin concentrations in blood, the consequences of which are unclear (G76). Another group made similar observations: pancreas transplantation with portal or systemic venous drainage showed higher insulin concentrations than did normals; the cause was ascribed to a possible side effect of the immunosuppression drugs (G77). Transplantation of human islets, e.g., infusion of cells into a portal vein, is still an experimental procedure, and the functionality is unacceptably short (G78). The graft survival for kidneys obtained from cadavers is only slightly lower than that for kidneys from living donors, assuming good HL4 matching in both. Typical postoperative problems are rejection, infections, bleeding, leaking at the anastomoses, pancreatitis and other kinds of inflammation, vascular thrombi, hematuria, and death (G79). Pancreas transplant patients require close biochemical monitoring for possible transplant failures and for the metabolic disturbances owing to the profound loss of HC03- in patients with urinary bladder drains of the exocrine secretions. If the renal allograft is functioning well, then the renal synthesis of HC03can usually keep up with the urinary loss. If the serum creatinine rises, the patient is then usually in a negative HC03- balance and must receive -25 g/day of HC03- parenterally if the HC03- falls below 16 mmol/L or -3 g/day by mouth if the HC03- is between 17 and 21 mmol/L. With renal dysfunction, hyperchloremic acidosis can be severe with C1- values of >110 mmol/L and HC03- concentrations of
