Volume 32, Issue 3

Based on the systemic and life-long need for immunosuppressive drug therapy, patients after organ transplantation suffer from an increased risk of infectious complications and disease due to primary infection or reactivation with the human cytomegalovirus (CMV). In contrast, CMV infections in immunocompetent individuals are usually clinically silent. In recent years, significant advances have been made in the development of novel experimental approaches to quantify antigen-specific T-cells on the single cell level. These assays have been extensively characterised and evaluated as a novel diagnostic measure to analyse the individual immunocompetence towards CMV in a clinical setting. A close correlation was found between decreasing frequencies of CMV-specific T-cells and increasing viral load, whereas CMV-specific T-cells were stable in the absence of viral replication. Due to easy technical performance, limited sample volume and short processing times, the combined monitoring of CMV-specific T-cells and viral load represents a valuable diagnostic tool to identify patients at risk for CMV-related complications after organ transplantation. Zusammenfassung Bei Patienten nach Organtransplantation besteht aufgrund der Notwendigkeit einer systemischen und lebenslangen Gabe von Immunsuppressiva ein erhöhtes Risiko, infektiöse Komplikationen durch eine CMV-Primärinfektion oder -Reaktivierung zu erleiden. Demgegenüber zeigen CMV-Infektionen bei Immungesunden in der Regel einen klinisch unauffälligen Verlauf. In den letzten Jahren wurden neue experimentelle Verfahren zur Quantifizierung Antigen-spezifischer T-Zellen auf Einzelzellniveau entwickelt. Diese Verfahren wurden umfassend als neue Werkzeuge zur Analyse der individuellen CMV-spezifischen Immunkompetenz in der klinischen Routinedia-gnostik charakterisiert und evaluiert. Hier zeigte sich eine signifikante Assoziation zwischen einem Abfall CMV-spezifischer T-Zellen und einer Zunahme der Viruslast, während CMV-spezifische T-Zell-Frequenzen bei nicht nachweisbarer Viruslast stabil waren. Aufgrund der einfachen Handhabung, geringer Probenvolumina und kurzer Verarbeitungszeit stellt das kombinierte Monitoring von CMV-spezifischen T-Zellen und CMV-Viruslast ein wertvolles diagnostisches Werkzeug zur Identifizierung von Risikopatienten nach solider Organtransplantation dar.

Dendritic cells (DCs) start out on every immune reaction. They can initiate or suppress an immune response against antigens. Their diagnostic and therapeutic utilization is a research topic in transplantation medicine. One goal of studies in transplantation-immunology is the identification of transplanted patients, whose immunosuppressive treatment can be reduced or potentially discontinued without an impairment of graft function. The induction of tolerogenic DCs and their immunological monitoring in the patient blood would be an option for the realization of this goal. In experiments with cell cultures and animals, DCs were identified as target cells of immunosuppressive drugs. Clinical studies with transplant recipients showed alterations of DC subpopulations in the patient blood depending on the immunosuppressive drugs and their dosage. Up to now, no DC subpopulation could be identified in the blood of transplant recipients that would allow, prospectively, a discontinuation of the immunosuppressive protocol. Further studies in vitro and in vivo are necessary to define the DC subsets that are relevant for the induction and maintenance of transplant tolerance.

Dendritic cells (DC) start out on every immune reaction. They can initiate or suppress an immune response against antigen. Their diagnostic and therapeutic utilization is a research topic in transplantation medicine. One goal of studies in transplantation-immunology is the identification of transplanted patients, whose immunosuppressive treatment can be reduced or potentially discontinued without an impairment of graft function. The induction of tolerogenic DCs and their immunological monitoring in the patient blood would be an option for the realization of this goal. In experiments with cell cultures and animals, DCs were identified as target cells of immunosuppressive drugs. Clinical studies with transplant recipients showed alterations of DC subpopulations in the patient blood depending on the immunosuppressive drugs and their dosage. Up to now, no DC sub-population could be identified in the blood of transplant recipients that would allow prospectively a discontinuation of the immunosuppressive protocol. Further studies in vitro and in vivo are necessary to define the DC subsets that are relevant for the induction and maintenance of transplant tolerance.

Kidney transplantation is a well established procedure in the treatment of patients suffering from chronic renal failure. Despite high standards, post-operative complications are common. In addition to transplant rejection, viral, bacterial and fungal infections are of relevance. Frequently, clinical examination and clinical chemistry cannot establish requested diagnoses in a timely manner. Here, we propose the inclusion of natural killer (NK) cells and natural killer T (NKT) cells into flow cytometric immunomonitoring of patients undergoing kidney transplantation. Patients were followed from the pre-operative to the 28th post-operative day. Using flow cytometry, we investigated NK and NKT cells and their functional markers, NKp30, NKp44, NKp46, CD16 and CXCR3. Both cell populations were influenced by transplantation. Results were independent of the origin of the graft (cadaveric or living donor) and of the immunosuppressive protocol used. However, NKT cells were influenced by induction therapies and indicated organ loss. We conclude that detection of these cell types should be added to immunomonitoring panels in solid organ transplanted patients.

Notwithstanding high clinical standards, renal transplantation, as the established procedure for treating terminal renal failure, involves the risk of numerous complications, especially in the postoperative period. Rejection episodes and bacterial, viral as well as fungal infections are of particular relevance. Frequently their timely clinical as well as laboratory identification and the resulting therapeutic consequences are too imprecise. This paper describes a practice-oriented concept for immunological monitoring within 28 days of observation following transplantation, including natural killer cells (NK) and natural killer T cells (NKT cells). This monitoring process focuses on flow cytometric identification of the functional markers NKp30, NKp44, NKp46, CD16 and CXCR3 on NK and NKT cells. Both cell populations were influenced by the therapy. While there was hardly any difference between living and cadaver donors and while no significant differences were found between the immunosuppressants, NKT cell findings in particular appear to make a diagnostic use of these cells possible while the graft is being monitored. The development of these cells following renal transplantation shows the changes typical under standard immunosuppression and permits the identification of postoperative complications.

Background : T-cell activation markers in peripheral blood are helpful to detect acute rejection in liver transplantation (LTX) patients with immunosuppression by Cyclosporin A or Tacrolimus. Immunosuppression including basiliximab may influence the diagnostic value of T-cell markers. Methods : In the present study, 80 LTX patients receiving immunosuppressive treatment based on induction with basiliximab were monitored as well as clinical examination with conventional (liver enzymes, cholesterol, bilirubin, prothrombin time, leukocyte count and differentiation) and immunologic markers [soluble interleukin (IL)-2 receptor, IL-8, lymphocyte subpopulations] during the first 3 weeks after transplantation. In total, 15 patients suffered from acute rejection, 34 patients had no complications, and 31 patients suffered from other complications including infections, poor graft function or renal failure. Results : Cholesterol, prothrombin time and CD3+/CD4–/CD8– lymphocytes allowed for discrimination between the group with acute rejection and patients suffering from other complications. Furthermore, significant time-dependent changes between both groups were found for alanine amino transferase (ALT), soluble IL-2 receptor (sIL-2R), eosinophils, lymphocytes and CD3+/CD4–/CD8– lymphocytes. In contrast, receiver operating characteristic (ROC) curve analysis for diagnostic relevance revealed only a Youden index >0.5 for prothrombin. When combining cholesterol, lymphocyte count, sIL-2R and time courses of ALT, eosinophils and sIL-2R, diagnostic efficacy could be maximized to a Youden index of 1.0. Conclusion : Despite significant differences between several single parameters, diagnostic application of the laboratory results requires a combination of a broad panel including the analysis of time courses to distinguish groups suffering from acute rejection or other complications. The complexity of this approach may limit its application in clinical practice. Zusammenfassung Einführung : Bei Patienten nach Lebertransplantation und Immunsuppression mit Cyclosporin A oder Tacrolimus ist die Bestimmung von T-Zell-Aktivierungsmarkern zur Erkennung akuter Rejektionen hilfreich. Die immunsuppressive Therapie mit Basiliximab kann die diagnostische Wertigkeit von T-Zell-Markern beeinflussen. Methoden : Achtzig Leber-transplantierte Patienten, deren Immunsuppression auf einer Induktionstherapie mit Basiliximab basierte, wurden in die Studie eingeschlossen. Während der ersten drei Wochen nach Transplantation wurden neben klinischen Kriterien konventionelle Laborparameter (Leberenzyme, Cholesterol, Bilirubin, Prothrombinzeit, Blutbild mit Leukozytendifferenzierung) und immunologische Parameter (löslicher IL-2 Rezeptor, IL-8 und Lymphozytensubpopulationen) untersucht. Fünfzehn Patienten erlitten eine akute Rejektion, 34 Verläufe waren komplikationslos und 31 Patienten wiesen im Verlauf andere Komplikationen auf wie Infektionen, schlechte Transplantatfunktion oder akutes Nierenversagen. Ergebnis : Mit den Parametern Cholesterol, Prothrombinzeit und CD3+/CD4–/CD8– Lymphozyten kann zwischen den Gruppen mit akuter Rejektion und der Patientengruppe mit anderen Komplikationen differenziert werden. Zusätzlich zeigt die Kinetik der Parameter ALT (Alanin-Aminotransferase), sIL-2R (löslicher Interleukin-2 Rezeptor), Eosinophilenzahl, Lymphozytenzahl und CD3+/CD4–/CD8– Lymphozyten signifikante Unterschiede zwischen diesen Gruppen. Im Gegensatz dazu zeigt die Analyse der ROC (receiver operating characteristic) Kurven nur eine diagnostische Relevanz für die Prothrombinzeit (Youden Index >0.5). Die Kombination der Parameter Cholesterol, Lymphozytenzahl, sIL-2R und die Verläufe von ALT, Eosinophilenzahl und sIL-2R konnte die diagnostische Effizienz deutlich verbessern (Youden Index 1.0). Schlussfolgerungen : Obwohl einzelne Parameter signifikante Unterschiede zwischen den Patientengruppen mit akuter Rejektion bzw. anderen Komplikationen nach Lebertransplantation aufweisen, ist für die klinische Diagnostik des Einzelfalles die Kombination mehrerer Parameter erforderlich.

Fecal occult blood testing (FOBT) is the most widely prescribed screening test for colorectal cancer (CRC) because of its simplicity, non-invasiveness, and demonstrated mortality benefits. However, guaiac-based fecal blood tests (G-FOBT) suffer from poor sensitivity, a limited ability to detect early lesions, and low population compliance. These limitations have prompted many innovations in stool testing. Preliminary data on fecal proteins including calprotectin and tumor-M2-PK show better performance characteristics compared to G-FOBT. Nevertheless, these tests also suffer from low sensitivity in detecting early lesions and poor specificity, leading to high costs for follow-up of false-positive tests. Recently developed immunological tests (I-FOBT) demonstrate significantly higher sensitivity and specificity. I-FOBTs use antibodies specific for human hemoglobin and are therefore, unlike G-FOBT, not affected by diet or drug administration, leading to improved patient partipication in screening for CRC. At present, I-FOBTs seem to be the most cost-effective approach to non-invasive CRC screening. Fecal DNA analysis opens up a new field for early detection of colorectal neoplasia. Small trials of multitarget assays demonstrate a CRC sensitivity of 62–91% and adenoma sensitivity of 26–73%. The specificity of these assays is high, ranging from 93% to 100%. At present, the major drawback of fecal DNA testing compared to other fecal CRC screening tests is its high cost.

Given the simplicity of the method and how it can be applied, as well as proof that it lowers the mortality rate, fecal occult blood testing (FOBT) is currently the most commonly used screening method for colorectal cancer (CRC). However, the test suffers from poor sensitivity, particularly with respect to detecting early stages, as well as low acceptance among the population. Preliminary data on detecting calprotectin and tumour-M2-PK in the stool indicated that a better screening performance could be expected. But these tests also suffer from low sensitivity in detecting early stages and from poor specificity, thus limiting the usefulness of the tests as a result of high follow-up costs. Recently developed immunological tests (I-FOBT) demonstrate significantly increased sensitivity and specificity. I-FOBTs use antibodies specific to human hemoglobin and are therefore not affected by diet and drugs, leading to improved patient partipication. At present, I-FOBTs seem to be the most cost-effective approach for non-invasive screening. The detection of tumour-DNA in the stool opens up a new era in early diagnosis of colorectal cancer. Small trials have pointed to a very high sensitivity of these methods: 62–91% for colorectal cancer and between 26% and 73% for adenomas, with a very high level of specificity (93–100%). The major drawback of this type of testing, compared with other screening tests available today, is its high cost.

Test strips are commonly used in rapid screening for drugs of abuse in clinical applications and forensic situations, but the operator does not necessarily have a toxicological qualification. Pharmacological and toxicological knowledge is necessary for interpretation of the results and for technical understanding of the test system (e.g., cutoff values, cross-reactivity). Possible false-negative results have to be considered and strategies for the detection of sample manipulation should be established. Quality controls are indispensable in ensuring results are authentic and reproducible, and proper documentation is mandatory to achieve traceability. Analysis using chromatographic methods should be included to confirm positive test-strip results. National and international regulations for decision limits should also be considered.

Test strips for detecting narcotics are being used more frequently – ranging from clinical issues to forensically relevant cases. Analyses are not always done by staff trained in toxicology. Pharmacological and toxicological knowledge is necessary for the interpretation of the results as well as the technical understanding of the test system (e.g., cut-off values, cross reactivity). False negative results have to be considered possible when interpreting results. Suitable measures should be taken to rule out sample manipulation to the widest extent possible. To ensure the accuracy and reproducibility of analytical results, quality checks will have to be carried out. Analytical results will be reproducible only when backed up by the necessary documentation. Further diagnosis involving chromatographic methods should be carried out to differentiate and confirm positive findings. National and international rules and regulations regarding decision limits should be observed.

No Abstract available

The aim of our prospective study, on 246 consecutive outpatients with suspected acute venous thromboembolism (VTE), was to evaluate the actual performance of four D-dimer assays (D-Dimer plus, Stratus CS, Innovance, Siemens Medical Solutions Diagnostics, and Tina-quant D-Dimer, Roche Diagnostics) for exclusion of VTE. Analysis was initiated after false-negative results in three patients with proven VTE were observed in routine diagnostics. Diagnostic sensitivity, specificity, negative and positive predictive values of the D-dimer tests were calculated based on recommended cut-off levels and on upper limits of corresponding 95% confidence intervals (95% CI). VTE was confirmed in 46 patients (18.7%) by imaging techniques. The D-dimer plus assay yielded seven false-negative results (nine considering 95% CI) in 46 patients. A sensitivity of 84.8% and 80.4% and a negative predictive value of 93.8% and 93.0%, respectively, was achieved. In contrast, the alternative tests showed no false negative results (sensitivity and negative predictive value 100%) and a higher D-dimer result to cut-off ratio. Detection of performance deficiencies of established diagnostic assays requires close cooperation with the clinician and further evaluation by the medical laboratory in accordance with the requirements of DIN EN ISO 15189.

The aim of our prospective study on 246 consecutive outpatients with suspected acute venous thromboembolism (VTE) was to evaluate the actual performance of four D-dimer assays (D-dimer plus, Stratus CS, Innovance, Siemens Medical Solutions Diagnostics, and Tina-quant D-dimer, Roche Diagnostics) for exclusion of VTE. Analysis was initiated after false negative results in three patients with proven VTE were observed in routine diagnostics. Diagnostic sensitivity, specificity, negative and positive predictive values of the D-dimer tests were calculated based on recommended cut-off levels and on upper limits of corresponding 95% confidence intervals (95% CI). VTE was confirmed in 46 patients (18.7%) by imaging techniques. The D-dimer plus assay yielded seven false negative results (nine considering 95% CI) in 46 patients. A sensitivity of 84.8% and 80.4% and a negative predictive value of 93.8% and 93.0%, respectively, was achieved. In contrast, the alternative tests showed no false negative results (sensitivity and negative predictive value 100%), and a higher D-dimer result to cut-off ratio. Detection of performance deficiencies of established diagnostic assays requires close cooperation with the clinician and further evaluation by the medical laboratory in accordance with the requirements of DIN EN ISO 15189.

Although the pathogenesis of HIV infection is independent of age, specific considerations have to be taken into account for the treatment of infants, children and adolescents. In addition to more cautious drug therapy, it is proposed to follow up disease progression by repeated measurement of CD4 lymphocyte percentage (CD4%). Unfortunately, this requires complicated, expensive analytical techniques and complex sample preparation protocols. This is one of the reasons why treatment of the increasing number of children suffering from HIV/AIDS in developing countries remains a challenge. We evaluated a new and simplified no lyse no wash protocol to measure CD4% on a volumetric flow cytometer. Eight blood samples of healthy and HIV-infected individuals were sent to five German hospital laboratories (centres B–F) and were measured by their flow cytometric in-house techniques. The results were compared to those of centre A using the volumetric technique with the new sample preparation protocol. Comparative data with established protocols and flow cytometric techniques showed good correlations for the determination of leukocytes and for lymphocytes. Comparing CD4% results of centre A (new protocol and volumetric measuring technique) with the highest and lowest values of centres B–F by Bland-Altman analysis yielded a bias of –1.02% (SD 3.16) and 4.27% (SD 3.72) over the whole range (4.01%–70.56%), respectively. A comparably simple three-step protocol as introduced for absolute CD4+ cell count can be used to calculate CD4% only by the addition of two antibodies (CD4 and CD45). The cost per test is in the range of €2.50 and therefore far below current prices of €15.00–€40.00. Zusammenfassung Obwohl die Pathogenese der HIV-Infektion unabhängig vom Alter ist, müssen besondere Umstände für die Behandlung von Neugeborenen, Kindern und Heranwachsenden berücksichtigt werden. Neben einer vorsichtigeren Medikation sollte der Verlauf der Krankheit durch die wiederholte Messung der CD4-Zellen relativ zu den Lymphozyten (CD4%) verfolgt werden. Leider wird hierfür eine komplizierte, teure Messtechnik und ein komplexes Protokoll zur Probenaufbereitung benötigt. Das ist auch ein Grund dafür, dass die steigende Anzahl der Kinder, die in Entwicklungsländern an HIV/AIDS leiden, nicht behandelt werden kann. Wir evaluierten ein neues und einfaches durchflusszytometrisches Protokoll für die CD4%-Messung, welches auf eine Erythozytenlyse verzichtet. Acht Blutproben von gesunden und HIV-infizierten Individuen wurden an fünf deutsche Krankenhauslabore geschickt und mit deren eigener Standardtechnik (Zentrum B–F) gemessen. Die Ergebnisse wurden mit denen von Zentrum A verglichen, das die volumetrische Technik mit dem neuen Probenaufbereitungsprotokoll einsetzt. Der Vergleich der Daten mit denen etablierter durchflusszytometrischer Techniken und Protokolle zeigte gute Korrelationen für die Leukozyten- und für die Lymphozytenzahl. Der Vergleich der Daten von Zentrum A mit dem höchsten und niedrigsten Messwert der anderen Zentren B-F mittels Bland-Altman-Analyse ergab einen systematischen Fehler von –1,02% (Standardabweichung 3,16) für die höchsten Messwerte und 4,27% (Standardabweichung 3,72) für die niedrigsten Werte über den gesamten Messbereich (4,01%–70,56%). Mit einem vereinfachten Drei-Schritt-Protokoll zur Absolutzählung von CD4-Zellen über die Zugabe von nur zwei Antikörpern (CD4 und CD45) lässt sich der CD4%-Wert zuverlässig bestimmen. Die Kosten pro Test belaufen sich auf etwa €2,50 und liegen deutlich unter den aktuellen Preisen von €15,00–€40,00.

No Abstract available

No Abstract available