Jochem Hoyer

The detection of human cytomegalovirus immediate early antigen in peripheral blood leucocytes

Recently, Van der Bij et al. (1988) reported that active human cytomegalovirus (HCMV) infection could be diagnosed by the detection of HCMV immediate early antigen (IEA) directly in the peripheral blood leucocytes of renal transplant recipients. However, the indirect peroxidase technique used resulted in high background staining due to endogenous peroxidase activity and thus the detection of HCMV-IEA positive leucocytes, which are sometimes present in extremely low numbers, was not always reliable. In an attempt to solve this problem, we have evaluated the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique, immunogold-silver staining (IGSS), and several fixatives. Fixation with acetone: methanol 1:1 in conjunction with the APAAP technique proved to be the most successful method. In 155 blood samples obtained from 44 patients following renal transplantation and from three AIDS patients, the number of positive cells ranged between 1 and 700 out of 400,000 (median 2). In 23 samples from 11 patients (one AIDS patient) at least one positive cell was found. In this series there were no problems with the evaluation since strong positive signals were obtained without any background staining. We therefore recommend the use of this protocol for the rapid and reliable detection of HCMV-IEA in peripheral blood leucocytes.

The long persistence of CMV DNA in the blood of renal transplant patients after recovery from CMV infection.

A total of 30–50% of all renal transplant recipients undergo infections caused by human cytomegalovirus. With the introduction of ganciclovir and foscarnet for specific antiviral therapy there is an increasing demand for diagnostic tools that allow the early and rapid identification of CMV as the causative agent of the observed disease. We and others previously showed the direct detection of pp65 antigen in peripheral blood leukocytes to be an excellent marker for active cytomegalovirus infection. In order to establish whether the detection of CMV DNA by the polymerase chain reaction (PCR) sup

Laparoscopy in renal transplant patients

OBJECTIVES To evaluate the use of laparoscopic techniques in patients with a renal transplant. METHODS Since 1992, 358 patients have undergone urologic laparoscopy at our hospital. Among these, 37 procedures (10.4%) were performed in patients with a renal transplant: 14 bilateral nephrectomies for severe drug-resistant hypertension, 9 marsupializations of symptomatic lymphoceles, 6 renal allograft biopsies in patients with clotting abnormalities, 6 unilateral nephrectomies, and 2 nephroureterectomies for recurrent episodes of pyelonephritis and symptomatic vesicoureteral reflux, respectively. RESULTS Five complications (14%) and three conversions (8%) occurred. Patients who underwent successful laparoscopic operations began mobilization and oral intake on the day after the operation. The hospital stay ranged from 1 to 6 days. In the nephrectomy groups, perioperative urine outputs remained stable and post-operative urine outputs were increased as compared with those in the perioperative period (P < 0.05). Post-operatively, serum creatinine remained stable or improved in patients who underwent nephrectomy or marsupialization of lymphocele. CONCLUSIONS Our results indicate that laparoscopic techniques are safe and effective in the treatment of patients with a renal transplant. Renal allograft function apparently is not affected by laparoscopic procedures.

Monitoring of anti‐HLA class I and II antibodies by flow cytometry in patients after first cadaveric kidney transplantation

While the relevance of pre‐formed anti‐human leukocyte antigen (HLA) antibodies has been studied extensively, the role of anti‐HLA class I and II antibodies produced after cadaveric kidney transplantation is still a matter of discussion. As it has been proposed that they are involved in a considerable number of cases, it should be investigated whether a post‐transplant monitoring is a sensitive parameter for the early diagnosis of acute rejection episodes. Additionally, it has been suggested that antibodies are a major cause for chronic rejection; thus, it would be of interest to correlate antibody detection and graft survival. We retrospectively investigated 59 patients after a first cadaveric kidney transplantation without known anti‐HLA antibodies (complement‐dependent cytotoxicity [CDC] testing). The panel reactivity was determined with a new highly sensitive and specific flow‐cytometric technique (Flow‐PRA Screening Test©, One Lambda, Canoga Park, USA) in sequentially collected serum samples pre‐ and post‐transplant. In patients with acute rejection episodes during the clinical course, the last sample prior to rejection, and in patients without rejection, the last sample prior to discharge, was analyzed. Furthermore, we analyzed 3‐yr graft survival and several clinical parameters such as cold ischemia time (CIT).Twenty‐four of 59 patients (41%) experienced acute rejections during the clinical course. Five of 59 died with a functioning graft within the first 3 yr. Seven of 54 patients, still alive after 3 yr, lost their graft. Anti‐HLA antibodies were detectable in only 7/59 patients and a correlation between antibody positivity and acute rejections (p=0.32 and 0.54 for anti‐HLA class I and II, respectively) could not be identified (sensitivity 12.5 and 8.3%). However, we found a significant correlation between the detection of anti‐HLA class II and graft loss within 3 yr (p=0.005, specificity 97.9%). Additionally, anti‐HLA class II positive patients had significantly longer CIT (p=0.003).Whether the detection of anti‐HLA class II antibodies in the early post‐transplant phase is of great value for the identification of patients at high risk for early graft loss needs additional investigation. However, we found that anti‐HLA antibodies are detectable only in a minority of unsensitized patients and we conclude that flow‐cytometric monitoring with Flow PRA is not a sensitive parameter for the early diagnosis of acute rejection episodes in patients after first cadaveric kidney transplantation.

Laparoscopic treatment of lymphoceles in patients after renal transplantation

Postoperative lymphoceles after renal transplantation appear in up to 18% of patients, followed by individual indisposition, pain or impaired graft function. Therapeutic options are percutaneous drainage, needle aspiration with sclerosing therapy, or internal surgical drainage by conventional or laparoscopic approach. The laparoscopic procedure offers short hospitalisation time and quick postoperative recovery. From 1993 to 1997, 16 patients underwent laparoscopic fenestration of a post‐renal transplant lymphocele, and were presented in a retrospective analysis. Three patients have had previous abdominal surgery. Following preoperative ultrasound and CT scan, 16 patients underwent laparoscopic drainage after drainage and staining of the lymphocele with methylene blue. No conversion was necessary. Mean operation time was 42 min, no intraoperative complications were seen. Oral nutrition and immunosuppression were continued on the day of surgery, and patients were discharged between the 2nd and 5th (median hospital stay 3.3 d) day after surgery. No recurrence was evident in a follow‐up time of 15–54 months (median 31.4 months). Renal function remained unchanged in all patients postoperatively.

A longitudinal prospective study of cytomegalovirus pp65 antigenemia in renal transplant recipients

Cytomegalovirus (CMV)-encoded pp65 antigen in peripheral blood leukocytes (CMV antigenemia) was investigated in 1017 serial samples from 64 patients for 16 weeks after renal transplantation in a prospective study. In 110 samples from 24 patients, at least one antigen-positive leukocyte was identified. The median number of stained cells was 4 (range 1–1000) per 4×105 leukocytes. Twenty-one of 24 patients with serological signs of an active CMV infection were antigen-positive (sensitivity 87.5%), whereas 3 patients with antigenemia did not show serological signs of infection during the observation period (specificity 92.5%). Positive results were obtained 19 days (median) before serological response and 9 days (median) before the onset of CMV syndrome. The sensitivity in defining a CMV syndrome was 100% (n=8). In all patients who presented with CMV syndrome, antigenemia was present prior to the onset of symptoms or on the same day. In contrast, serological monitoring rendered the diagnosis of CMV infection possible at the onset of clinical symptoms in only two of eight patients. We conclude that (1) insufficient results obtained with the CMV antigenemia assay by other investigators are mainly due to technical problems that can easily be overcome by the protocol presented and (2) the detection of CMV pp65 antigen in peripheral blood leukocytes is an excellent tool for rapid and early diagnosis of CMV infection.

A nondirected kidney donation and its consequences: personal experience of a transplant surgeon.

Until 1996, living kidney donation had been neglected in Germany. To encourage potential donors and to convince them that nephrectomy is not too risky for a healthy person, the author decided to set an example with a personal nondirected donation. This donation, controversially discussed in the public domain, nevertheless succeeded in raising the percentage of living donors in Germany from 4% to 20% of renal transplants. In 1997, the new German Transplant Act prohibited nondirected donation. With the aim of allowing nondirected donation as an act of Christian charity, a plaint had been submitted to the Constitutional Court against that law on the basis that it violated several fundamental rights, but it was dismissed because of strong medical opposition. The author experienced some unpleasant professional consequences of his act but, nevertheless, is convinced that he had taken the right step when deciding to give a kidney to a stranger.

β2-Microglobulinuria as an early sign of cytomegalovirus infection following renal transplantation

The frequency of cytomegalovirus infection was studied in a prospective study of 106 kidney recipients. The detection of cytomegalovirus-immediate-early-antigen and cytomegalovirus-immunoglobulin (IgM) antibodies in serum was used as the reference method and showed that 23.6% (25/106) of all patients were infected. In addition, four urinary proteins (IgG and transferrin as glomerular markers and α1-microglobulin and β2-microglobulin as tubular markers) were quantitatively measured in 24-h urine samples from all of the patients using an immunoluminometric assay (ILMA). In all cytomegalovirus infection cases a pronounced but isolated increase of urinary β2-microglobulin excretion was observed. In 20 of 25 infected patients, the p2-microglo-bulinuria occurred 1–21 days (median 5.0) earlier than the appearance of the cytomegalovirus-immediate-early-antigen in blood. Thus, it can be seen that the quantitative measurement of β2-microglobulin in urine is useful for the early detection of cytomegalovirus infection following renal transplantation.