July 2011
Feature Story

William Check, PhD

It’s hardly eHarmony or Match.com, but transplantation services have made great strides in creating close-to-perfect matches. From more refined histocompatibility testing methods to flow cytometry for crossmatching to post-transplant antibody monitoring, laboratories are finding new ways to support this critical service.

“There have been dramatic changes in histocompatibility testing methods for transplant patients,” says Kathryn Tinckam, MD, MMSc, co-director of the histocompatibility laboratory in the Laboratory Medicine Program, University Health Network, Toronto, and assistant professor, Department of Laboratory Medicine and Pathobiology, University of Toronto. Historically, serological methods have been used for HLA typing of donors and recipients. Dr. Tinckam calls these methods “fairly crude” and says they can’t define small differences in human leukocyte antigen molecules on the surface of endothelial cells in the organ that often result in large immunological reactions. “Now we’ve moved into DNA-based methods,” says Dr. Tinckam, who is also a staff physician in the UT Division of Nephrology and Multi-Organ Transplant Program. “We are able to define many important HLA alleles, so we get a much better description of immune compatibility between donors and recipients.”

In crossmatching, too, an important change has taken place. “Crossmatching used to be done by complement-dependent cellular cytotoxicity, which can be both insensitive and nonspecific,” Dr. Tinckam says. “Now we do most of our crossmatching by flow cytometry, which is able to detect low-titer antibodies that can have a detrimental impact but that were not detectable by the older method.”

James L. Wisecarver, MD, PhD, professor of pathology and microbiology and director of clinical laboratories at the University of Nebraska Medical Center, says that as typing has moved to molecular methods, new HLA alleles have been identified. Quantitative molecular tests for viruses have also been brought onboard, including assays for members of the herpesvirus family—cytomegalovirus, Epstein-Barr virus, herpes simplex virus, and varicella-zoster virus. In liver transplantation, quantitative molecular tests for hepatitis B and C viruses are performed routinely. “In renal transplant patients we are very interested in quantitating BK virus,” Dr. Wisecarver adds.

Anatomic pathology of transplantation has undergone less change but still fills critical functions. The surgical pathologist must be able to evaluate biopsies of many organs. Moreover, transplant pathology “has some of the closest interactions with clinical teams in all of AP,” says Geoffrey A. Talmon, MD, assistant professor of pathology and microbiology at the University of Nebraska Medical Center. This is one reason why Dr. Talmon calls the anatomic pathology of transplantation “without a doubt one of the most enjoyable areas in pathology.”

At the University of Nebraska Medical Center, surgeons perform transplants of bone marrow, heart, liver, kidney, pancreas, and small bowel. For small bowel, they do isolated and cluster transplants. In a cluster transplant, small intestine, liver, and pancreas are transplanted together along with the biliary tract. These are mainly done in children born with gastroschisis or necrotizing enterocolitis.

At the front end of organ transplants, HLA testing is typically done. For solid organs, Dr. Wisecarver says they usually run both serologic and low-level molecular analyses. “In certain settings serologic testing is not terribly specific,” he says. “It can’t call all antigen splits.”

For bone marrow transplantation they perform high-level molecular typing, which gets down to the actual allele. “Typing for bone marrow transplantation needs to be more specific because you are taking away the patient’s immune cells,” Dr. Wisecarver explains. Without a close match there is an increased risk of graft-versus-host disease. “Graft-versus-host disease is still an issue in stem cell transplantation,” Dr. Wisecarver says, but adds, “A bit of a mismatch can be a good thing.” With an exact match the risk of recurrence is higher. With an inexact match the patient gets the benefit of a graft-versus-leukemia effect.

In many instances the clinician wants to know if the recipient has antibodies against HLA antigens, Dr. Wisecarver says. “So the HLA lab monitors for those and determines their specificities. We are asking whether the recipient has anything in their serum that could damage the donor organ.” Once a specific antibody has been identified, that specificity is listed on the UNOS register and the patient would not be a candidate for an organ bearing that antigen.

With the greater specificity now available regarding a recipient’s antibodies, that information can be used to do a virtual crossmatch against a potential donor organ. Virtual crossmatching is a computer-based process in which a potential recipient’s anti-HLA antibodies are compared with a potential donor’s HLA antigens to predict whether there will be a reaction. “Virtual crossmatching is relatively new, only one or two years old,” Dr. Wisecarver says. “Some studies have found it to be 90 percent to 95 percent accurate.” Even so, he adds, “We still like to do an actual crossmatch on a sample of fresh recipient serum just before the transplant.” This is particularly true for pancreas and kidney transplants.

Dr. Tinckam, of UHN, notes that improved specificity identification of potentially organ-damaging HLA antibodies has been especially valuable to those patients for whom it is most difficult to find organs—individuals who are found to have antibodies to many potential donors. About 30 percent of people on transplant waiting lists have detectable antibodies to HLA antigens at the time they are listed, she says. Some highly sensitized patients have antibodies to greater than 80 percent of randomly selected donors. “Unfortunately, with serological antibody detection methods we weren’t able to say with certainty to which HLA antigens in those donors these antibodies were directed,” Dr. Tinckam says. That made choosing organs a challenge and meant testing many donors for these recipients before a suitable HLA type could be identified.

In the past decade, methods were developed in which HLA antigens are covalently bound to solid-phase platforms such as microbeads. With these reagents, and using flow cytometry platforms, it is possible to detect antibodies to specific HLA proteins with high sensitivity and specificity. “Now, instead of saying a patient has antibodies to, for example, 60 percent of the population, we can say that patient has antibodies to a defined list of HLA antigens,” Dr. Tinckam says. “This information, in combination with excellent typing methods, allows us more precisely to select a donor to whom a recipient with preformed antibodies will not have a humoral reaction.” Transplanting an organ to which the recipient has HLA antibodies greatly lowers graft survival—from about 85 percent at five years post-transplant, when the recipient does not have antibodies, to as low as 30 percent when antibodies are present. “The current evidence demonstrates that when we are able to identify a donor to whom the recipient does not have donor-specific HLA antibodies,” Dr. Tinckam says, “we can offer that recipient the same graft survival as someone who has no antibodies at all. So, if used correctly, this testing improves both access to transplantation and graft survival.” Their goal, she adds, is to have all recipients living with the same graft survival as that of unsensitized patients.

When it appears unlikely that a suitable donor can be found, clinicians may perform desensitization by treating the recipient with immunosuppressive agents before transplantation to reduce antibody titers. “This strategy blunts the immune reaction,” Dr. Tinckam says. “In the HLA lab we can identify the antibody present in the recipient and quantitate it repetitively, so that we can define the moment when transplantation can proceed.”

Monitoring immunosuppression is also important, of course, and Dr. Wisecarver says his department used the need to monitor immunosuppressive drugs to justify the purchase of a mass spectrometry instrument. “We find that to be a very cost-effective way to measure those drugs and to generate data in the timeframe that clinicians need,” he says. “We got away from immunoassays. Mass spectrometry is much cheaper and you can measure multiple drugs in one run.” They adjusted the work schedule to run the instruments seven days per week. Measuring for cyclosporin, tacrolimus, sirolimus, and eve­roli­mus, they do between 160 and 200 assays per day on Monday and Tuesday, when reference samples arrive, and 100 to 120 on each of the other days.

In the University of Toronto transplantation program, the same argument is used to justify the choice of immunoassays to monitor immunosuppressive drugs. The menu of drugs monitored is similar to that in the University of Nebraska program—cyclosporin, tacrolimus, sirolimus, mycophenolic acid, and everolimus, says Paul M. Yip, PhD, clinical biochemist and assistant professor in the UT Laboratory Medicine Program and the Department of Laboratory Medicine and Pathobiology. “Most drugs get dosed early in the morning,” says Pui-Yuen Wong, PhD, a UT clinical biochemist who is now retired from clinical service. “We promise the clinicians they will get results by 3 or 4 o’clock in the afternoon so they can adjust doses in the evening.” That mandates a turnaround time of three to four hours. “When you have 200 samples coming in at the same time, that dictates an automated method,” Dr. Wong says.

Drug monitoring can be done by commercial immunoassays and by in-house assays, which include HPLC and mass spectrometry. “There are pros and cons to each of those methods,” Dr. Yip says. “We used to do in-house assays. Now we use commercial immunoassays because the automated platform allows us to handle a large volume with a fast turnaround time.”

In the Toronto program, Oyedele A. Adeyi, MB, BS, assistant professor of laboratory medicine and pathobiology and staff physician, performs the surgical pathology on liver and small intestine biopsy specimens. Liver biopsies can be done every six to 12 months by protocol or for cause when liver dysfunction is suspected, usually because of elevated liver enzymes or elevated bilirubin, or both, or when other measures of functional abnormalities are seen.

Protocol biopsies usually are processed over­night. “For those, there is hardly any reason to call the gastroenterologist or the hepatologist,” Dr. Adeyi says. Ultra-rush biopsies are a different story. They are frequently performed for cause and, as such, processed for reading within three to four hours, after 60 to 90 minutes of fixation. “We don’t necessarily sign out those specimens immediately,” Dr. Adeyi says. “What is most important and determines the immediate response is the verbal discussion between the pathologist and the treating hepatologist.”

Having adequate tissue is critical. “One of the worst things one could do is to call rejection on an inadequate biopsy,” Dr. Adeyi says. In addition to H&E, he uses trichrome stain on all livers and may order other stains as well, “depending on what we see.”

Acute rejection occurs most often in the first week to six months post-transplant. In an early biopsy, one taken in the first three months, Dr. Adeyi also looks for evidence of technical complications, such as problems with anastomoses of the bile duct or vessels. He also thinks of opportunistic infection because immunosuppression is highest in the early period.

Dr. Adeyi notes that about half of all liver transplants are done for HCV infection, and aggressive recurrence of HCV develops in a small proportion of them at some point after transplantation. “It is critical to be able to differentiate recurrent HCV from its mimics, such as rejection,” Dr. Adeyi says. “We don’t want to call rejection when the patient really has recurrent hepatitis C.” The clinician would unnecessarily increase the dose(s) of immunosuppressive drugs and worsen the infection.

In biopsies taken at six months to one year, opportunistic infection is not as common as in the early days. HCV recurrence becomes more prominent at this time, though most patients have a smoldering, slowly developing form of recurrence. This can be managed by regulating levels of immunosuppressive drugs, though many patients will ultimately require specific antiviral treatment sometime down the line. An aggressive form with fibrosis and cholestatic hepatitis, however, tends to pursue a rapid course to liver failure within weeks to a few months of onset.

Breakthrough rejection after six months looks similar to early acute rejection, but after two to three years it may look different. Some other conditions that are confusing early on are not usually a problem after six months. Inflammation in the portal tract, for instance, can be due to recurrent HCV or rejection. “In most cases it is not very difficult to tell which it is,” Dr. Adeyi says.

At any time during the post-transplant period, Dr. Adeyi considers the possibility of opportunistic Epstein-Barr virus infection, which leads to a kind of lymphoma called post-transplant lymphoproliferative disease. Dr. Adeyi calls it a “true lymphoma,” most often B-cell type, and says it could present as any other B-cell lymphoma outside the transplant setting, including the kind of Burkitt’s lymphoma found in parts of Africa. “If you catch it in the early stage, lowering immunosuppression will usually work,” he says.

Also in the later post-transplant period, chronic rejection becomes a consideration. Both acute and chronic rejection are immune-mediated injuries. Acute rejection is more often a cell-mediated process, whereas chronic rejection could have multifactorial contributors and modulators, including immune- and nonimmune-mediated factors. For example, patients who experienced CMV infection are more likely to develop chronic rejection than CMV-negative patients. Generally, however, antibody-mediated rejection is less of a problem in liver transplants than in kidney, heart, and pancreas. “It is very rare to see pure antibody-mediated rejection in liver transplants,” Dr. Adeyi says. “We believe that chronic rejection [in the liver] is a combination of many things, including nonimmune injury over time, perhaps biliary strictures, and perhaps CMV infection.” In chronic rejection there is less inflammation, but the small bile ducts become atrophic and progressively disappear.

For small bowel biopsies, the questions are the same as for the liver—rejection, infection, and vascular problems. “We can tell injury by looking at the tissue architecture,” Dr. Adeyi says. “Architecture is the first thing to go.” With small bowel transplants, elective biopsies are done more frequently in the very early stages. One reason is that, whereas changes in plasma enzyme levels can give an indication of problems for liver transplants, no such test exists for the small intestine. A second reason is that the small bowel is more sensitive to immune injury than the liver, which Dr. Adeyi calls “more forgiving than other organs.” As a result, asymptomatic patients may be biopsied many times in the first days and weeks after small bowel transplantation.

The pathology findings must be put into the context of other changes in the patient, Dr. Adeyi says. “It is important to discuss the case with the clinician even before you get the biopsy. At the end of the case,” he says, “my job is to look at all the tissue changes, put that into context, and give advice on what to do next.” Fortunately, he says, “In our center we have access to the transplant electronic medical record. Relations between the transplant pathologists and transplant physicians are very good and in many cases personal.”

At the University of Nebraska, the anatomic pathologist may be called in even before transplantation in the case of a liver infected with HCV, Dr. Talmon says. “We are more often being asked to evaluate the amount of fibrosis or scar, which can impact whether that organ is used,” he says. If the liver is transplanted and liver enzymes rise, the baseline exam can help determine whether there is additional damage, which can help differentiate rejection from recurrent HCV.

When distinguishing between rejection and recurrent infection, Dr. Talmon looks at the character of the inflammatory infiltrate in the portal areas. “If I see eosinophils, I lean more toward rejection,” he says. A diagnosis of rejection is also favored when there is quite a bit of bile duct damage. “Definitely the presence of vascular injury, which you don’t see in hepatitis C reactivation, makes me think rejection,” Dr. Talmon adds.

Disease of the small bowel is one of Dr. Talmon’s research interests, and one of the things he likes about AP of small bowel transplants is that he sees rare diseases more frequently than he would otherwise. An example: microvillus inclusion disease, a defect that causes problems in small intestinal epithelial cells. “Children with this condition present at birth with diarrhea refractory to all treatments,” he says. “Pretty much the only way they survive is with intestinal transplantation.”

Dr. Talmon also likes the degree of interaction with other pathologists that occurs in AP of transplants. “Coordinating with HLA, clotting, matching, antibody screens—they’re all central to what we do,” he says. Outside pathology, he cites the weekly multidisciplinary case conferences with the kidney and liver/small bowel services. “For any case that is not a negative biopsy, surgeons, tissue typers, and the medical director discuss organ match at placement in conjunction with our histologic findings and specific antibodies.”

The work of the microbiology laboratory—direct detection of infections—starts with pre-transplantation screening of donors and recipients, says Tony Mazzulli, MD, FRCPC, deputy chief microbiologist and infectious disease specialist in the Department of Microbiology of UHN and Mt. Sinai Hospital in Toronto.

The odds of finding unsuspected infection depends on donor type. The transplant team can interview living donors, and those at high risk for infectious disease can often be weeded out before they enter the donor pool, he says. As a result, unknown and unexpected infectious disease is uncommon in this group of donors. “With organ or tissue donors, who are usually in an ICU setting and can’t speak for themselves, we would be relying on the initial medical history from the family or in the medical record, which wouldn’t rule out unknown risk factors,” Dr. Mazzulli says. “In this context, testing has a slightly higher chance of revealing something unexpected.”

If a potential donor is found to be infected with cytomegalovirus, the donor wouldn’t necessarily be excluded from the donor pool, but it is important to know about the infection to manage the recipient. Dr. Mazzulli cites the example of a donor with a CMV-positive liver going into a CMV-negative recipient. “The recipient may get prophylactic antiviral therapy and be monitored weekly with qPCR for active CMV,” he says. “That can allow intervention before symptoms appear.”

Post-transplantation, the key role of the microbiology laboratory is to help diagnose complications, such as unexplained fever, that may be due to infection. “We normally test across a broad spectrum of infectious agents,” Dr. Mazzulli says. If donor and recipient are negative for viruses, post-transplantation testing looks for bacterial infection of the lung, urinary tract, or blood as well as fungal opportunistic infections. “Depending on the time of year, respiratory illness in a transplant patient may be due to influenza and related viruses.” Because of immunosuppression, even the normally benign flu can be a serious complication.

Any time rejection occurs in a solid organ transplant or graft-versus-host disease occurs in a stem cell transplant, seeking an underlying viral illness, such as CMV or EBV, is one of the two most important parts of the workup, along with asking whether the patient is adequately immunosuppressed. Only a thorough workup can answer these questions, Dr. Mazzulli says, add­ing, “There are no virus-specific signs of rejection, nor are there any special immune tests for rejection.”

Dr. Tinckam, of UHN, sees the next advance in transplantation histocompatibility testing as doing increased antibody testing post-transplant. “Some people who had no anti-HLA anti­bodies pre-transplant develop them after transplantation,” she says, and doing so is associated with worse graft survival. “One of our big challenges is chronic organ damage, in which the allograft is lost over three to five years,” she says. “Those processes can look quite different [from acute rejection]. If antibody develops and they get acute rejection, we know how to treat it. But if antibody develops and the patient seems OK, it’s not so clear what to do.” Dr. Tinckam says there is considerable ongoing research on how to prevent chronic damage: “It’s a really dynamic area right now. There are a number of clinical trials with antibody monitoring. And some treatments are being tested on those who develop antibodies.” Perhaps this work will tie in the development of antibodies after transplantation with chronic damage and lead to effective preventive measures, adding another clinically valuable service to lab support of transplantation services.