Immunosuppressive Drug Measurement by Liquid Chromatography Coupled to Tandem Mass Spectrometry: Interlaboratory Comparison in the Korean Clinical Laboratories

Background@#Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is increasingly used for immunosuppressive drug tests. However, most LC-MS/MS tests are laboratory-developed and their agreement is unknown in different Korean laboratories.This interlaboratory comparison study evaluated test reproducibility and identified potential error sources. @*Methods@#Test samples containing three concentrations of tacrolimus, sirolimus, everolimus, cyclosporine, and mycophenolic acid were prepared by pooling surplus samples from patients undergoing routine therapeutic drug monitoring and tested in duplicate in the participating 10 clinical laboratories. Reconstitution and storage experiments were conducted for the commonly used commercial calibrator set. The robust estimators of reproducibility parameters were calculated. Spearman’s rank correlation coefficient (rho, ρ) was used to evaluate the correlation between drugs. Multiple linear regression was used to determine whether the experimental conditions alter the calibration curves. @*Results@#The reproducibility coefficient of variation exceeded 10% only for sirolimus concentrations 1 and 2 (10.8% and 12.5%, respectively) and everolimus concentrations 1 and 2 (12.3% and 11.4%, respectively). The percent difference values showed weak correlations between sirolimus and everolimus (ρ = 0.334, P = 0.175). The everolimus calibration curve slope was significantly altered after reconstitution following prolonged 5°C storage (P = 0.015 for 14 days; P = 0.025 for 28 days); the expected differences at 6 ng/mL were 0.598% for 14 days and 0.384% for 28 days. @*Conclusions@#LC-MS/MS test reproducibility for immunosuppressive drugs seems to be good in the Korean clinical laboratories. Continuous efforts are required to achieve test standardization and harmonization, especially for sirolimus and everolimus.

Recommendations for Liquid Chromatography-Mass Spectrometry in the Clinical Laboratory: Part III. Quality Assurance

The use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in clinical laboratories is increasing and is likely to expand into even more clinical venues in the future. Mass spectrometry is the standard method for analyte identification in the clinical chemistry field; however, differences in mass spectrometry protocols and handling affect the accuracy and reliability of these tests and prevent direct comparisons of results between laboratories. For example, the results of laboratories using LC-MS/MS methods are less likely to be reproducible than those of laboratories using conventional, automated methods. This is due to inadequate handling of the equipment and/or poor quality control after the implementation of the method, which may result in unnecessary medical expenditures or even adverse outcomes for the patients. Unfortunately, guidelines to monitor the accuracy of LC-MS/MS-based clinical tests are still lacking. In general, the quality control methods used in conventional clinical tests could also be applied to LC-MS/MS. However, additional quality control methods specific to LC-MS/MS techniques must be continuously employed to maintain the same quality level achieved during method development and verification. This report is intended to help clinical laboratories that operate LC-MS/MS improve the accuracy and reliability of their testing by providing guidance for quality assurance and improvement, based on a collection of existing guidelines and expert opinions from the literature.

Recommendations for Liquid Chromatography-Mass Spectrometry in the Clinical Laboratory: Part II. Method Validation

The demand for obtaining test results using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for accurate diagnosis in the field of laboratory medicine is expected to increase, but it is still not easy to introduce diagnostic methods using LC-MS/MS into clinical laboratories for many reasons. There are many different methods used to evaluate the performance of LC-MS/MS in clinical laboratories, which have not been standardized to date. Thus, various data have been analyzed and described based on the type of validation method used and the criteria needed to introduce a new test using LC-MS/MS in a clinical laboratory. Relevant data from home and abroad were reviewed to include the minimum number of validation items required and methods of implementation. In general, the items required for a full validation of the quantitative test and various guidelines were used to summarize the following validation items: accuracy, precision, calibration, specificity, ion suppression or improvement, limit of detection, limit of quantification, stability, reference interval, carryover, and dilution integrity. Among these, the first five items mentioned beforehand are essential parameters for LC-MS/MS validation and are presented in numerous guidelines. The other parameters are required for further verification depending on the characteristics of the analysis and the analytes. This recommendation is intended to outline and present the validation methods that should be carried out when introducing new tests in clinical laboratories using LC-MS/MS with reference to the existing guidelines and literature containing expert opinions.

Recommendations for the Use of Liquid Chromatography-Mass Spectrometry in the Clinical Laboratory: Part I. Implementation and Management

Unusually Elevated Serum Insulin Level in a Diabetic Patient during Recombinant Insulin Therapy

Herein, we report a case of unusually elevated serum insulin level as a result of increased anti-insulin antibody (IA)-bound insulin after continuous subcutaneous insulin infusion therapy. Detecting free insulin (unbound IAs) levels after polyethylene glycol pre-treatment could be useful to assess functional insulin levels in diabetic patients receiving insulin therapy. The E170 insulin assay can estimate total insulin (bound IAs and free insulin) levels, but it does not measure the levels of exogenous insulin analogues.

The Effects of Anti-insulin Antibodies and Cross-reactivity with Human Recombinant Insulin Analogues in the E170 Insulin Immunometric Assay / 대한진단검사의학회지

BACKGROUND: Insulin assays are affected by varying degrees of interference from anti-insulin antibodies (IAs) and by cross-reactivity with recombinant insulin analogues. We evaluated the usefulness of the E170 insulin assay by assessing IA effects and cross-reactivity with 2 analogues. METHODS: Sera were obtained from 59 type 2 diabetes patients receiving continuous subcutaneous insulin infusion and 18 healthy controls. Insulin levels were determined using an E170 analyzer. To investigate the effects of IAs, we performed IA radioimmunoassays, and analyzed the differences between directly measured insulin (direct insulin) and polyethylene glycol (PEG)-treated insulins (free, IA-unbound; total, IA-bound and unbound insulin). We performed in-vitro cross-reactivity tests with insulin aspart and insulin glulisine. RESULTS: In IA-positive patients, E170 free insulin levels measured using the E170 analyzer were significantly lower than the direct insulin levels. The mean value of the direct/free insulin ratio and IA-bound insulin, which were calculated as the difference between total and free insulin, increased significantly as endogenous IA levels increased. The E170 insulin assay showed low cross-reactivities with both analogues (< 0.7%). CONCLUSIONS: IAs interfered with E170 insulin assay, and the extent of interference correlated with the IA levels, which may be attributable to the increase in IA-bound insulin, and not to an error in the assay. The E170 insulin assay may measure only endogenous insulin since cross-reactivity is low. Our results suggest that the measurement of free insulin after PEG pre-treatment could be useful for beta cell function assessment in diabetic patients undergoing insulin therapy.

Determination of Carbohydrate-deficient Transferrin Levels by Using Capillary Electrophoresis in a Korean Population / 대한진단검사의학회지

BACKGROUND: Carbohydrate-deficient transferrin (CDT) levels have rarely been determined in an Asian population. We evaluated the analytical performance of a test for measuring CDT levels by using capillary electrophoresis (EP). METHODS: We determined the precision of CDT measurement by using capillary EP and nephelometry and compared the CDT values obtained using both the methods. We included healthy control subjects, abstinent patients with liver disease, and individuals consuming varying amounts of alcohol. RESULTS: The CDT measurement by using capillary EP were correlated well with those CDT measurement by using nephelometry, N Latex CDT assay, Y=0.5706X+1.581, R=0.930. The results obtained from both methods showed good qualitative agreement with each other (kappa coefficient=0.61). Genetic variants of transferrin isoforms were detected in 4.1% of the tested population. Both the CDT and gamma-glutamyl transpeptidase (GGT) levels in the abstinent patients with liver disease were significantly higher than those in healthy abstinent individuals (0.9% vs. 0.5%, 109.5 mg/dL vs. 28.5 mg/dL, respectively), but the difference in CDT values in the 2 groups was less pronounced for the CDT values. Individuals who had a mean daily alcohol intake of more than 60 g/day showed significantly higher CDT levels than those who had a mean daily alcohol intake of less than 60 g/day (1.9% vs. 0.7%, P=0.03). CONCLUSIONS: The CDT test using capillary EP showed good performance, and this method has several advantages such as automation and detection of variant forms. Thus, CDT can be a more useful marker than GGT for monitoring alcohol abstinence, especially in patients with liver disease.

Differences in Circulating Dendritic Cell Subtypes in Pregnant Women, Cord Blood and Healthy Adult Women

Different subtypes of dendritic cells (DC) influence the differentiation of naive T lymphocytes into T helper type 1 (Th1) and Th2 effector cells. We evaluated the percentages of DC subtypes in peripheral blood from pregnant women (maternal blood) and their cord blood compared to the peripheral blood of healthy non pregnant women (control). Circulating DC were identified by flow cytometry as lineage (CD3, CD14, CD16, CD19, CD20, and CD56)-negative and HLA-DR-positive cells. Subtypes of DC were further characterized as myeloid DC (CD11c+/CD123+/-), lymphoid DC (CD11c-/CD123+++) and less differentiated DC (CD11c-/CD123+/-). The frequency of DC out of all nucleated cells was significantly lower in maternal blood than in control (P<0.001). The ratio of myeloid DC/lymphoid DC was significantly higher in maternal blood than in control (P<0.01). HLA-DR expressions of myeloid DC as mean fluorescence intensity (MFI) were significantly less in maternal blood and in cord blood than in control (P<0.001, respectively). The DC differentiation factors, TNF-alpha and GM-CSF, released from mononuclear cells after lipopolysaccharide stimulation were significantly lower in maternal blood than in control (P<0.01). The distribution of DC subtypes was different in maternal and cord blood from those of non-pregnant women. Their role during pregnancy remains to be determined.

A Case of Delayed Hemolytic Transfusion Reaction in a Patient with Anti-c, Anti-E, and Anti-Jk(b) / 대한수혈학회지

Delayed hemolytic transfusion reaction (DHTR) due to multiple red blood cell (RBC) alloantibodies has rarely been reported in Korea. We report a case of DHTR in a patient with anti-c, anti-E, and anti-Jk(b). A 45-year-old man visited the emergency room with flame burn injury over 61% of his entire body. He received six units of packed RBCs and three units of fresh frozen plasma during the operation for excision and glycerol-preserved allografting. His hemoglobin (Hb) level gradually decreased from 13.5 g/dL on the operation day to 7.8 g/dL on the 11th postoperative day in spite of receiving three and two additional units of packed RBCs on the 8th and 9th postoperative days, respectively. His laboratory data was total bilirubin/direct bilirubin 15.9/11.4 mg/dL, lactate dehydrogenase 983 IU/L, haptoglobin 5.93 mg/dL and plasma hemoglobin 8.0 mg/dL. The urinalysis revealed hemoglobinuria, and the peripheral blood film showed moderate spherocytosis. Both the direct and indirect antiglobulin tests were positive, and the follow-up antibody identification test showed anti-c, anti-E, and Jk(b). His Hb levels increased after he was transfused with two units of packed RBCs without c, E, and Jk(b) antigens. This is a case of DHTR due to alloimmunization, which developed within a short interval after the patient had received multiple transfusions.

Current Status of External Quality Assessment of Syphilis Test in Korea / 대한진단검사의학회지

BACKGROUND: Current status of external quality assessment (EQA) of laboratory tests for syphilis in Korea was analyzed to find out the problems that should be improved in the future. METHODS: Based on the data from the external quality assessment program performed twice a year by the Immunoserology Subcommittee of the Korean Association of Quality Assurance for Clinical Laboratory from the year 2004 to 2006, discordance rates were analyzed according to the test method and commercial kit used. RESULTS: Among the laboratories participating in the EQA program for syphilis test, about 90% of them used non-treponemal tests and about 55% treponemal tests. The non-treponemal tests included RPR (rapid plasma reagin) and VDRL tests used in 88% (363/412) and 11% (45/412), respectively, of the laboratories. The discordance rates were 2.2% for RPR test and 3.6% for VDRL. For the treponemal tests, Treponema pallidum hemagglutination assay (TPHA) was used in 60-76% and Immunochromatography assay (ICA) in about 30% of the laboratories in 2006. A high discordance rate of over 10% was reported in both TPHA and in ICA methods, possibly due to a low titer (1:1 in VDRL) of EQA samples in 2005. Analysis of the accumulated data from year 2004 to 2006 showed that the discordance rates of TPHA, ICA, and FTA-ABS were 4.6%, 3.7%, and 2.7%, respectively. CONCLUSIONS: For syphilis tests, RPR test, TPHA, and ICA are mainly used in Korea. A high discordance rate is still reported in TPHA and ICA, especially when testing samples with a low titer. Further analysis of data and education of laboratory personnel are needed for the improvement of the EQA program.

A Living Donor Liver Transplantation after Therapeutic Plasmapheresis in a Patient with Positive HLA Crossmatch / 대한수혈학회지

A positive HLA crossmatch in cadevaric liver transplantation is relatively acceptable, but in living donor liver transplantation (LDLT) using relatively small sized grafts, the rejection rates were higher in positive crossmatchcases than in negative cases, as described in several previous reports. We report a case of LDLT performed with therapeutic plasmapheresis, in a recipient with a positive HLA crossmatch to donor before transplantation. The patient was a 56-year-old male patient with liver cirrhosis (UNOS status IIA, MELD score 28) caused by chronic hepatitis B. The HLA crossmatch results were 1:2 and 1:8 positive for NIH-CDC (complement dependent cytotoxicity) and AHG-CDC, respectively. The flow cytometric crossmatch (FCXM) was also positive (T-MFI ratio 9.0 and B-MFI ratio 3.4). With 5 cycles of preoperative therapeutic plasmapheresis, the HLA crossmatch converted to negative and liver transplantation was performed. The liver function of the patient was well maintained for 5 months, without any sign of hyperacute or acute rejection. However, the patient eventually died from suddenly occurred infection-associated hemophagocytic syndrome at 5 months after surgery. Therapeutic plasmapheresis can be considered as one of therapeutic options for LDLT patients with a positive HLA crossmatch to donor.