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Objectives Automated sample delivery and laboratory acceptance systems (PTAS) may influence the hemolysis rate of blood samples due to g-forces, abrupt acceleration, and rapid deceleration. However, quantitative data regarding the rate of hemolysis in PTAS is limited. To fill this void, the effect of a pneumatic tube in combination with an acceptance system (PTAS) on the hemolysis rate was investigated in this study. Methods Lithium heparin plasma tubes were transported from different clinical departments to the hospital’s laboratory (a) by employees or (b) with an automated PTAS and analyzed for the presence of hemolysis based on a hemolysis index (HI) of >25. Hemolysis indices of 68.513 samples were retrieved from the laboratory information system before and after installation of the PTAS and were subjected to statistical analysis. Results A total of 32.614 samples were transported by employees, of which 3.815 samples (11.70%) were hemolytic, and 9.441 out of 35.899 samples delivered by PTAS (26.30%) were hemolytic. After the implementation of the PTAS, hemolysis rates increased in all departments. Conclusions Automated PTAS are associated with increased hemolysis rates. This has implications for routine patient management and should be considered for the transportation of samples used for the determination of hemolysis-sensitive laboratory parameters.

Point-of-care antigen tests (AgTs) for the detection of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) enable the rapid testing of infected individuals and are easy-to-use. However, there are few studies evaluating their clinical use.The present study aimed to evaluate and compare the clinical performance characteristics of various commercial SARS-CoV-2 AgTs.The sensitivity of five AgTs, comprising four rapid antigen tests (RAT; AMP Rapid Test SARS-CoV-2 Ag, NADAL COVID-19 Antigen Rapid Test, CLINITEST Rapid COVID-19 Antigen Test, and Roche SARS-CoV-2 Rapid Antigen Test) and one sandwich chemiluminescence immunoassay (CLIA; LIAISON SARS-CoV-2 Assay), were evaluated in 300 nasopharyngeal (NP) swabs. Reverse transcriptase (RT) polymerase chain reaction (PCR) was used as a reference method.NP swabs were collected from patients admitted to hospital due to COVID-19.Sensitivities of the AgTs ranged from 64.9 to 91.7% for samples with RT-PCR cycle threshold (Ct) values lower than 30 and were 100% for cycle threshold (Ct) values lower than 20. The highest sensitivity was observed for CLINITEST Rapid COVID-19 Antigen Test, and Roche SARS-CoV-2 rapid antigen test. Multivariate analysis using time from symptom onset and the Ct value for AgT sensitivity showed an inverse correlation. Further, the female sex was an independent factor of lower RAT sensitivity.Antigen tests from NP swab samples show high sensitivity in patients with a Ct value20. The best clinical sensitivity can be obtained using AgTs within the first 6 days after symptom onset.

The COVID-19 pandemic has demonstrated the need for massively-parallel, cost-effective tests monitoring viral spread. Here we present SARSeq, saliva analysis by RNA sequencing, a method to detect SARS-CoV-2 and other respiratory viruses on tens of thousands of samples in parallel. SARSeq relies on next generation sequencing of multiple amplicons generated in a multiplexed RT-PCR reaction. Two-dimensional, unique dual indexing, using four indices per sample, enables unambiguous and scalable assignment of reads to individual samples. We calibrate SARSeq on SARS-CoV-2 synthetic RNA, virions, and hundreds of human samples of various types. Robustness and sensitivity were virtually identical to quantitative RT-PCR. Double-blinded benchmarking to gold standard quantitative-RT-PCR performed by human diagnostics laboratories confirms this high sensitivity. SARSeq can be used to detect Influenza A and B viruses and human rhinovirus in parallel, and can be expanded for detection of other pathogens. Thus, SARSeq is ideally suited for differential diagnostic of infections during a pandemic., Massively parallel but cost-effective testing is essential to monitor the spread of pathogenic agents. Here the authors present SARSseq, which uses a dual indexing strategy in a multiplexed RT-PCR reaction to diagnose SARS-CoV-2 at scale.

Summary Background Chronic kidney disease patients show a high mortality in cases of a severe acute respiratory syndrome coronavirus-2 (SARS-CoV‑2) infection. Thus, information on the sero-status of nephrology personnel might be crucial for patient protection; however, limited information exists about the presence of SARS-CoV‑2 antibodies in asymptomatic individuals. Methods We examined the seroprevalence of SARS-CoV‑2 IgG and IgM antibodies among healthcare workers of a tertiary care kidney center during the the first peak phase of the corona virus disease 2019 (COVID-19) crisis in Austria using an orthogonal test strategy and a total of 12 commercial nucleocapsid protein or spike glycoprotein-based assays as well as Western blotting and a neutralization assay. Results At baseline 60 of 235 study participants (25.5%, 95% confidence interval, CI 20.4–31.5%) were judged to be borderline positive or positive for IgM or IgG using a high sensitivity/low specificity threshold in one test system. Follow-up analysis after about 2 weeks revealed IgG positivity in 12 (5.1%, 95% CI: 2.9–8.8%) and IgM positivity in 6 (2.6%, 95% CI: 1.1–5.6) in at least one assay. Of the healthcare workers 2.1% (95% CI: 0.8–5.0%) showed IgG nucleocapsid antibodies in at least 2 assays. By contrast, positive controls with proven COVID-19 showed antibody positivity among almost all test systems. Moreover, serum samples obtained from healthcare workers did not show SARS-CoV‑2 neutralizing capacity, in contrast to positive controls. Conclusion Using a broad spectrum of antibody tests the present study revealed inconsistent results for SARS-CoV‑2 seroprevalence among asymptomatic individuals, while this was not the case among COVID-19 patients. Trial registration number CONEC, ClinicalTrials.gov number NCT04347694

AXL and its corresponding ligand growth arrest-specific 6 (GAS-6) are critically involved in hepatic immunomodulation and regenerative processes. Pleiotropic inhibitory effects on innate inflammatory responses might essentially involve the shift of macrophage phenotype from a pro-inflammatory M1 to an anti-inflammatory M2. We aimed to assess the relevance of the AXL/GAS-6-pathway in human liver regeneration and, consequently, its association with clinical outcome after hepatic resection. Soluble AXL (sAXL) and GAS-6 levels were analyzed at preoperative and postoperative stages in 154 patients undergoing partial hepatectomy and correlated with clinical outcome. Perioperative dynamics of interleukin (IL)-6, soluble tyrosine-protein kinase MER (sMerTK), soluble CD163 (sCD163), and cytokeratin (CK) 18 were assessed to reflect pathophysiological processes. Preoperatively elevated sAXL and GAS-6 levels predicted postoperative liver dysfunction (area under the curve = 0.721 and 0.722; P 0.005) and worse clinical outcome. These patients failed to respond with an immediate increase of sAXL and GAS-6 upon induction of liver regeneration. Abolished AXL pathway response resulted in a restricted increase of sCD163, suggesting a disrupted phenotypical switch to regeneratory M2 macrophages. No association with sMerTK was observed. Concomitantly, a distinct association of IL-6 levels with an absent increase of AXL/GAS-6 signaling indicated pronounced postoperative inflammation. This was further supported by increased intrahepatic secondary necrosis as reflected by CK18M65. sAXL and GAS-6 represent not only potent and easily accessible preoperative biomarkers for the postoperative outcome but also AXL/GAS-6 signaling might be of critical relevance in human liver regeneration. Refractory AXL/GAS-6 signaling, due to chronic overactivation/stimulation in the context of underlying liver disease, appears to abolish their immediate release following induction of liver regeneration, causing overwhelming immune activation, presumably via intrahepatic immune regulation.

BackgroundChronic kidney disease patients show a high mortality in case of a SARS-CoV-2 infection. Thus, to be informed on Nephrology personnel’s sero-status might be crucial for patient protection. However, limited information exists about the presence of SARS-CoV-2 antibodies in asymptomatic individuals.MethodsWe examined the seroprevalence of SARS-CoV-2 IgG and IgM antibodies among health care workers of a tertiary care kidney center during the peak phase of the Covid-19 crisis in Austria using an orthogonal test strategy and a total of 12 commercial nucleocapsid protein or spike glycoprotein based assays as well as Western blotting and a neutralization assay.ResultsAt baseline 60 of 235 study participants (25.5%, 95% CI: 20.4-31.5) were judged to be borderline positive or positive for IgM or IgG using a high sensitivity/low specificity threshold in one test system. Follow-up analysis after about two weeks revealed IgG positivity in 12 (5.1%, 95% CI: 2.9-8.8) and IgM positivity in six (2.6%, 95% CI: 1.1-5.6) in at least one assay. 2.1% (95% CI: 0.8-5.0) of health care workers showed IgG nucleocapsid antibodies in at least two assays. By contrast, positive controls with proven Covid-19 showed antibody positivity among almost all test systems. Moreover, serum samples obtained from health care workers did not show SARS-CoV-2 neutralizing capacity, in contrast to positive controls.ConclusionsUsing a broad spectrum of antibody tests the present study revealed inconsistent results for SARS-CoV-2 seroprevalence among asymptomatic individuals, while this was not the case among Covid-19 patients.Trial registration numberCONEC, ClinicalTrials.gov number NCT04347694

Background Methylenetetrahydrofolate-reductase (MTHFR) deficiency is a rare autosomal recessive disorder affecting intracellular folate metabolism with affection of different organ systems and clinical manifestation usually in childhood. Objective We report on four adult members of a family with MTHFR deficiency presenting with neurological and thromboembolic complications in adulthood. Methods Extensive diagnostic work-up including genetic testing was performed in four adult members. Results The male siblings aged 42 and 32 years presented with various neurological symptoms, and a recent history of deep vein thrombosis. Extensive diagnostic work-up revealed total homocysteine (tHcy) plasma concentrations of 135 μmol/L and 231 μmol/L. and compound heterozygosity for two novel MTHFR gene mutations in exon 2 (c.202C > G, p.Arg68Gly) and intron 10 (c.1632 + 2T > G), and the known polymorphic variant MTHFR c.665C > T (p.Ala222Val, MTHFR 677C > T). Their mother was heterozygous for MTHFR c.1632 + 2T > G and c.665C > T, and a paternal relative was heterozygous for MTHFR c.202.C > G and MTHFR c.665C > T mutation. Both brothers showed partial response to therapy with betaine and multivitamins with clinical improvement. MTHFR activity was determined in fibroblast extracts and was around 4% of the mean control. Cell culture analysis indicated a re-methylation defect due to MTHFR deficiency. Conclusion Severe hyperhomocysteinemia due to two mutations of the MTHFR gene resulted in severe neurological symptoms in adulthood. Vitamin and methionine supplementation stabilize tHcy plasma levels. Severity of clinical manifestation varied greatly between the siblings. Damages to the nervous system may be present for years before becoming clinically manifest.

Background Reference intervals provided by diagnostic test manufacturers should be transferred to clinical laboratories after validation. Although protocols exist, laboratories rarely perform and report on results of validation studies. Methods We validated reference intervals (RIs) of 87 analytes on a Cobas 8000 platform according to standards published by the Clinical and Laboratory Standards Institute (CLSI). Results For 8 analytes, decision limits were provided in the package inserts. Among the 79 RIs subjected to transference validation, 8 were found not valid for transference, including lactate dehydrogenase (LDH) among women, and the following among both sexes: potassium, homocysteine, immunoglobulin E (IgE), free lambda light chain (FLC λ), C3 complement (C3c), folate, and 25-hydroxy vitamin D (25[(OH]D). For LDH, potassium, homocysteine, C3c, folate, and 25(OH)D, RIs or thresholds suitable for transference were available in the literature; however, this was not the case for IgE and FLC λ. Conclusion The present study demonstrates that validation of RIs provided in the manufacturer provided package inserts is indispensable.

Background The multidrug resistance 1 gene (ABCB1) encodes P-glycoprotein (PGP), mainly expressed in the liver and engaged in metabolism of drugs including the immunosuppressant tacrolimus (TAC). ABCB1 single nucleotide polymorphisms (SNP) may significantly alter pharmacokinetics and influence TAC concentrations of kidney transplant recipients (KTR). Methods The genotype distribution of ABCB1 1236C>T, 2677G>T/A and 3435C>T was investigated among 96 Austrian KTR who were converted from cyclosporin to TAC. Dose adjusted TAC trough levels and L/D ratios were assessed at week 1, 2, 4, and 8, and month 3, 12, and 24, and the influence of ABCB1 genotypes on dose adjusted TAC trough levels and level to dose (L/D) ratios were analyzed. Results The genotype distributions for ABCB1 1236C>T were CC 36.4%, CT 5.2%, TT 58.3%, for ABCB1 2677G>T/A GA 2%, GG 63.5%, GT 20.8%, TA 1%, TT 12.5%, and for ABCB1 3435C>T CC 20.8%, CT 7.2%, TT 71.8%. Dose adjusted TAC trough levels and L/D ratios were independent of ABCB1 genotypes except for ABCB1 1236C>T at a single time point (week 2: 0.02599 [CC] vs. 0.05704 [CT] vs. 0.03218 [TT]; p = 0.024). Conclusions Serial analyses of TAC trough levels revealed no significant association with important ABCB1 genotypes among stable long-term Austrian KTR.

Myelodysplastic syndromes (MDS) are myeloid neoplasms defined by peripheral cytopenia, dysplasia of myeloid cells and an increased risk to transform to secondary acute myeloid leukemia (AML). Although several laboratory and molecular risk factors and scoring systems have been established, little is known about genetic features contributing to disease evolution, AML development, and survival. Accumulating evidence suggests that iron metabolism and transfusion frequency are important prognostic determinants in MDS. It has also been described that patients with MDS frequently display HFE gene variants. However, little is known about the clinical impact of HFE variants. We examined the HFE status by restriction fragment length polymorphism (RFLP) analysis covering 2 common variants (H63D and C282Y) in 167 patients with MDS (observation-period: 1992-2019) and in 494 healthy Austrian controls. The median age of our MDS patients at diagnosis was 69 years (range 27-85 years) with a f:m ratio of 1:1.13. In the MDS cohort the international prognostic scoring systems, IPSS and IPSS-R, were applied, and in 94 patients a ´myeloid mutation´ screen was performed by next-generation sequencing (NGS). In all patients, serum ferritin levels were recorded at diagnosis and during follow up. Variant HFE species (H63D and/or C282Y) were detected in 63/167 patients with MDS (38.4%) and in 170/494 controls (34.4%). Median ferritin levels at diagnosis were slightly higher in HFE-mutated patients (409 mg/dL; range: 23-7415 mg/dL) compared to non-mutated patients (346.5 mg/dL; range: 10-5450 mg/dL) (p=0.62). During the first 2 years of transfusion therapy, HFE-mutated patients showed a slightly faster increase in serum ferritin levels compared to non-mutated patients. However, again, the difference was not significant, and patients with massive iron overload were found in both cohorts. When examining French-American-British (FAB) subgroups of MDS, the percentage of patients withHFE variants was higher in the group with refractory anemia, RA (22/53=41.5%) or RA with ring sideroblasts, RARS (17/39=43.9%) than in the group of RA with excess of blasts, RAEB (16/46=34.8%) or RAEB in transformation (5/17=29.4%). In CMML, 5/12 patients (41.7%) were found to carry a HFE variant. Clear differences were also observed between subgroups of MDS patients classified by World Health Organization (WHO) criteria. The prevalence of HFE mutations was 50.0% (13/26) in patients with ring sideroblasts (RS) and multilineage dysplasia (MDS-RS-MLD), 50.0% in MDS-MLD (6/12), 43% (7/18) in MDS with RS and single-lineage dysplasia (MDS-RS-SLD), 42.9% (12/28) in patients with excess of blasts-1 (MDS-EB1), 38.9% (7/18) in MDS-SLD, 21.4% (6/28) in MDS-EB2, and 20% (2/10) in MDS with isolated 5q-. When splitting patients according to the IPSS and IPSS-R, patients at highest risk were found to have a lower prevalence of HFE variants (IPSS high: 20%; IPSS-R very high: 15.4%) compared to other risk groups (IPSS low: 34.9%, int-1: 42.0%, int-2: 31.0%; and IPSS-R very low: 33.3%, low: 38.5%, int: 36.8%, high: 42.9%). Next, we examined correlations between HFE variants and somatic myeloid mutations in our MDS patients (n=94). No significant correlations were found when comparing expression of HFE variants with somatic mutations in ASXL1, ASXL1, SF3B1, SRSF2, TET2, DNMT3A, RUNX1, NRAS, TP53, IDH2 or EZH2 detected by NGS. Finally, we examined the HFE status in the context of clinical end points. In these studies, we found a clear difference in AML-free survival between HFE-mutated and non-mutated patients, although the difference did not reach statistical significance (p=0.089) (Figure 1A). No differences in overall survival (OS) were found when comparing patients with or without HFE variants (Figure 1B). In conclusion, the HFE variants H63D and C282Y are frequently detected in Austrian patients with MDS. These variants are more commonly detectable in low risk FAB patients and correlate roughly with higher ferritin levels at diagnosis and during transfusion therapy. In addition, HFE-mutated MDS patients have a slightly better AML-free survival. However, the correlation is weak. Therefore, we currently do not recommend screening for HFE gene variants at diagnosis in patients with MDS unless ferritin levels are excessively high or increase very rapidly during transfusion suggesting the possibility of a concomitant primary hemochromatosis. Disclosures Valent: Blueprint: Research Funding; Celgene: Honoraria; Deciphera: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Sperr:Novartis: Honoraria; Celgene: Consultancy, Honoraria.

In this non-randomized extension study of a randomized controlled trial we converted 87 stable long-term kidney transplant recipients (KTR) from either ciclosporin (CSA, n = 28) or tacrolimus (TAC, n = 59) to TAC modified release (TAC MR4) to study the characteristics of TAC trough levels after conversion with the primary endpoint graft function after 12 months. TAC MR4 consumption was calculated by level-to-dose ([ng/mL]/[mg/d]) and concentration-to-dose ([mg/kg])/d) ratios. Influences of ABCB1 single nucleotide polymorphisms (2677G>T/A, 1236C>T, 3435C>T) on TAC metabolism were studied. Graft function of KTR converted from CSA to TAC MR4 significantly declined over 12 months, and remained unchanged after conversion from TAC to TAC MR4. Conversion from CSA to TAC MR4 resulted in supra therapeutic- and conversion from TAC to TAC MR4 in low trough levels. We could not find associations of ABCB1 genotypes and TAC MR4 trough levels. Adverse events and errors with TAC/TAC MR4 intake were common. In stable long-term KTR conversion from TAC to TAC MR4 is feasible. For conversion from CSA we suggest a rate of 1:40 for a rough estimation of TAC MR4 target doses. Trial registration PEP Study: Ethics committee N° 393/2004, EudraCT 2004-004209-98. PEP-X Study: Ethics committee amendment application N° 154/01/2008. ClinicalTrials.gov NCT03751332.

Folate metabolism is an important target for drug therapy. Drug-induced inhibition of folate metabolism often causes an elevation of plasma total homocysteine (tHcy). Plasma tHcy levels are influenced by several nongenetic (e.g. folate intake, age, smoking) as well as genetic factors. Over the last decade, several countries have implemented a nationwide folate fortification program of all grain products. This investigation sought to determine the impact of folate fortification on the relative contribution of environmental and genetic factors to the variability of plasma tHcy.Two cohorts were compared in this study, one from the United States (with folate fortification, n=281) and one from Austria (without folate fortification, n=139). Several environmental factors as well as previously identified gene variants important for tHcy levels (MTHFR C677T, MTHFR A1298C, MTRR A66G) were examined for their ability to predict plasma tHcy in a multiple linear regression model.Nongenetic, environmental factors had a comparable influence on plasma tHcy between the two cohorts (R: approximately 0.19). However, after adjusting for other covariates, the tested gene variants had a substantially smaller impact among patients from the folate-fortified cohort (R=0.021) compared with the nonfolate-fortified cohort (R=0.095). The MTHFR C677T polymorphism was the single most important genetic factor. Male sex, smoking, and folate levels were important predictors for nonfolate-fortified patients; age was for folate-fortified patients.Population wide folate fortification had a significant effect on the variability of plasma tHcy and reduced the influence of genetic factors, most importantly the MTHFR 677TT genotype, and may be an important confounder for a personalized drug therapy.

Eur J Clin Invest 2011; 41 (6): 579–583 Abstract Background Over the years, the demonstration and confirmation of cell-free DNA in the circulation has increasingly been recognized as a valuable diagnostic tool. Likewise, it has been known for some time that DNA structures that are targeted by auto-antibodies play a central role in systemic lupus erythematosis (SLE) and that DNA-antibody complexes in the circulation are one of the hallmarks of SLE. Investigating whether and to what degree fluctuations in free plasma DNA levels in patients with SLE might correspond to disease severity was therefore the goal of this investigation. Methods Blood from 13 patients with SLE and from 13 healthy controls was taken and analysed for the presence of anti-dsDNA, anti-ssDNA, anti-nucleosome, anti-histone antibodies as well as for cell-free DNA concentrations. For each patient, the SLE disease activity index (SLEDAI) was calculated. Results As demonstrated herein, compared to healthy subjects, cell-free DNA plasma levels in patients with SLE were significantly increased and so were anti-dsDNA, anti-ssDNA, anti-histone and anti-nucleosome antibodies. Furthermore, a statistically significant correlation was noted between cell-free DNA and anti-histone antibodies in patients with SLE. However, no correlation was noted between disease activity and anti-dsDNA, anti-ssDNA and anti-nucleosome antibody concentrations. Surprisingly, and more important in the context of this study, there was no correlation between cell-free DNA levels and SLEDAI scores. Conclusions The presented data seem to exclude measuring free plasma DNA as an inexpensive, simple and quick tool to assess disease activity in patients with SLE. Further studies on a larger patient population would be needed to confirm our results.

The KIT mutation D816V is associated with autonomous growth of mast cells (MC) and is detectable in most patients with systemic mastocytosis (SM), including cases with associated hematologic non‐MC‐lineage disease (AHNMD). Recently, KIT D816V was reported to be expressed in patients with acute myeloid leukemia (AML). However, it was not clarified whether these patients have co‐existing occult SM. We investigated neoplastic cells in 101 patients with AML for expression of KIT D816V. In 7/101 patients (6.9%), KIT D816V was detectable. After a thorough histologic, molecular, and biochemical analysis, all 7 cases were found to have an associated SM, leading to the final diagnosis SM‐AML. Microdissected tryptase+ MC displayed KIT D816V in all patients tested, whereas CD34+ blasts exhibited KIT D816V in only 2/4 patients. In one AML patient, SM without KIT D816V was detected. In all other patients, no associated SM was found, and leukemic blasts were negative for KIT D816V. In summary, our data show that KIT D816V in AML is highly associated with co‐existing SM (SM‐AML). Moreover, our data show that AML blasts may lack this transforming target‐mutant, which may be important when considering the use of KIT D816V‐targeting drugs for treatment of patients with KIT D816V‐positive AML.

Objectives Oxidation of vitamin B12 by nitrous oxide leads to the inactivation of methionine synthase resulting in elevated plasma total homocysteine concentrations. Methionine synthase reductase is the only human enzyme that is able to reverse the oxidation of vitamin B12, which also occurs naturally by reactive oxygen species. A common polymorphism in methionine synthase reductase, MTRR 66A>G, is associated with reduced enzyme activity. Thus, we hypothesized that patients with this gene variant develop higher plasma total homocysteine concentrations after nitrous oxide anesthesia than wild-type patients. Methods In this follow-up investigation of a previous gene association study, we prospectively included 140 healthy individuals undergoing elective surgery under general anesthesia that included 66% nitrous oxide. Peak postoperative plasma total homocysteine was the main outcome variable and was measured within 2 h after the end of anesthesia. The MTRR 66A>G genotype was determined after completion of the study. The association between genotype and peak postoperative total homocysteine was modeled with a general linear model. Results No association between MTRR 66A>G and immediate postoperative plasma total homocysteine after nitrous oxide anesthesia was detected. All three groups, stratified by genotype (MTRR 66AA, AG, GG), shared similar baseline characteristics and increases in plasma total homocysteine. The average increase in plasma homocysteine was 2.4 mumol/l (+28%) in all three groups indicating the expected inactivation of methionine synthase by nitrous oxide through oxidation of vitamin B12, but no genetic effect. Conclusion In conclusion, this study showed that the MTRR 66A>G gene variant is not associated with peak elevated postoperative plasma total homocysteine after nitrous oxide anesthesia. Whether the gene influences the rate of recovery of methionine synthase remains to be determined.

Background Mutations in the methylenetetrahydrofolate reductase (MTHFR) gene (677C>T, 1298A>C) cause elevated plasma homocysteine concentrations and have been linked to fatal outcomes after nitrous oxide anesthesia. This study tested the hypothesis that patients with common MTHFR 677C>T or 1298A>C mutations develop higher plasma homocysteine concentrations after nitrous oxide anesthesia than wild-type patients. Methods In this prospective, observational cohort study with blinded, mendelian randomization, the authors included 140 healthy patients undergoing elective surgery. All patients received 66% nitrous oxide for at least 2 h. The main outcome variable, plasma total homocysteine, and folate, vitamin B12, and holotranscobalamin II were measured before, during, and after surgery. After completion of the study, all patients were tested for their MTHFR 677C>T or 1298A>C genotype. Results Patients with a homozygous MTHFR 677C>T or 1298A>C mutation (n = 25) developed higher plasma homocysteine concentrations (median [interquartile range], 14.9 [10.0-26.4] microm) than wild-type or heterozygous patients (9.3 [7.5-15.5] microm; n = 115). The change in homocysteine after nitrous oxide anesthesia was tripled in homozygous patients compared with wild-type (5.6 microm [+60%] vs. 1.8 microm [+22%]). Only homozygous patients reached average homocysteine levels considered abnormal (> 15 microm). Plasma 5-methyl-tetrahydrofolate concentrations increased uniformly by 20% after nitrous oxide anesthesia, indicating the inactivation of methionine synthase and subsequent folate trapping. Holotranscobalamin II concentrations remained unchanged, indicating no effect of nitrous oxide on vitamin B12 plasma concentrations. Conclusions This study shows that patients with a homozygous MTHFR 677C>T or 1298A>C mutation are at a higher risk of developing abnormal plasma homocysteine concentrations after nitrous oxide anesthesia.

Summary Patients with systemic mastocytosis (SM) may acquire an associated hematologic non–mast cell (MC)–lineage disease (AHNMD). In most cases, a myeloid neoplasm is diagnosed, whereas the occurrence of a lymphoproliferative disease is an extremely rare event. We report on a patient with indolent SM associated with small lymphocytic lymphoma (SLL). The patient presented with lymphadenopathy, maculopapular exanthema, and elevated serum tryptase. The bone marrow biopsy showed focal MC aggregates together with SLL. As assessed by immunostaining, neoplastic MC were found to exhibit CD117 and CD25 but did not display CD5 or CD20, whereas SLL cells were found to coexpress CD5 and CD20 but did not express MC antigens. The KIT mutation D816V was detected in sorted CD34 + cells and unfractionated marrow cells but not in CD5 + SLL cells, confirming the coexistence of 2 distinct neoplasms.

Hyperhomocysteinemia has occasionally been reported in patients with phenylketonuria (PKU) and B-vitamin deficiency. In our study total homocysteine (tHcy) and B-vitamins were measured in treated PKU patients and healthy controls. In the patients, dietary parameters and genetic polymorphisms affecting the Hcy pathway were investigated to identify parameters modulating tHcy. A case control study including 37 PKU patients and 63 healthy controls was conducted. t -Tests for independent samples were used to test between groups. Multiple regressions with tHcy as dependent variable were calculated. Hardy–Weinberg expectations were tested against the observed distribution of genotypes applying the Chi-square goodness-of-fit method. THcy concentrations were not significantly different ( p =0.059) while folate and cobalamin (Cbl) concentrations were significantly higher in PKU patients compared to controls. However, 29.7% of patients had tHcy concentrations >97th centile. THcy did not vary with age nor correlate with folate and Cbl concentrations probably due to high saturatory levels. The presence of genetic polymorphisms had no impact on tHcy. In conclusion, in PKU patients treated with amino acid mixtures enriched with B-vitamins, tHcy is not significantly higher than in healthy controls, but tHcy concentrations exceed the 97th centile in about one third of patients. Even higher B-vitamin saturation may be required to further decrease tHcy concentrations and factors generally influencing tHcy such as betaine are to be investigated in PKU patients in the future.

Systemic mastocytosis (SM) is a clonal disease of mast cells (MC) and their progenitors.[1][1] The hallmark of the disease is the multifocal accumulation of MC in one or more extracutaneous organs. The clinical course and prognosis in SM vary among patients.[1][1],[2][2] Based on WHO criteria, 4

In a group of patients with systemic mastocytosis (SM), marked and sustained eosinophilia is detectable (SM-eo).Although the molecular defect has been defined in some cases, little is known about the impact and clinical correlates of eosinophilia.In a cohort of 63 patients with SM, we identified 9 with permanent eosinophilia (1500/microL). According to the World Health Organization classification, 2 had indolent SM, 1 had smoldering SM, 2 had SM with associated chronic eosinophilic leukemia (SM-CEL), and 4 had aggressive SM.SM-eo was found to be associated with a significantly reduced probability of overall and event-free survival compared with SM without eosinophilia (P.05). In the 2 patients with SM-CEL, a CHIC2 deletion was found. By contrast, no KIT mutation at codon 816 was detectable in these patients. In the other patients with SM-eo, KIT D816V was demonstrable. The 2 patients with SM-CEL had cardiomyopathy, whereas other organ systems remained largely unaffected. By contrast, in all other patients with SM-eo, organopathy, if recorded, affected the bone marrow, liver, or/and skeletal system, but not the heart, even when eosinophilia persisted for many years.The biochemical basis of eosinophilia in SM is variable and predictive for the type of organopathy.In SM eosinophilia is of prognostic significance but is not a final diagnosis and is not invariably associated with cardiomyopathy. The latter might be restricted to cases with an associated primary eosinophilic disorder (SM-CEL).

Although a classification for mastocytosis and diagnostic criteria are available, there remains a need to define standards for the application of diagnostic tests, clinical evaluations, and treatment responses. To address these demands, leading experts discussed current issues and standards in mastocytosis in a Working Conference. The present article provides the resulting outcome with consensus statements, which focus on the appropriate application of clinical and laboratory tests, patient selection for interventional therapy, and the selection of appropriate drugs. In addition, treatment response criteria for the various clinical conditions, disease-specific symptoms, and specific pathologies are provided. Resulting recommendations and algorithms should greatly facilitate the management of patients with mastocytosis in clinical practice, selection of patients for therapies, and the conduct of clinical trials.

Appropriate measurement of the glomerular filtration rate (GFR) is important for the assessment of renal function. This paper reviews the methods used to assess GFR in clinical trials of enzyme replacement therapy (ERT) in patients with Fabry disease, which include inulin clearance, 24-hour creatinine clearance, chromium ethylene diamine tetraacetate ( 5 1 Cr-EDTA) clearance and cystatin C concentrations. GFR has also been estimated using calculations based on creatinine clearance (the Cockcroft-Gault formula) and the Modification of Diet in Renal Disease (MDRD) equation. Analysis of the results of these studies shows that there are striking discrepancies between estimated and measured GFR. For example, the MDRD equation overestimates GFR in patients with Fabry disease who have normal renal function. In addition, cystatin C has been shown to be of limited use for measuring renal function during ERT, because it is influenced by other factors such as age, gender and weight. Conclusion: The use of exact methods, such as inulin clearance, 1 2 4 I-iothalamate, 9 9 mTc-DTPA, 5 1 Cr-EDTA and iohexol, appears to be mandatory for a robust evaluation of the effects of ERT on GFR in patients with Fabry disease.

Background: The effect of serum prolactin levels on the response to erythropoiesis-stimulating agents (ESA) in patients on chronic hemodialysis is unknown. Methods: We included 111 stable hemodialysis patients in this study and examined the association of serum prolactin concentrations with the response to ESA. Accordingly we implemented an ESA index as a measure of therapeutic efficacy. The two outcomes of this cohort study were the association of prolactin concentrations with response to ESA, both at baseline (cross-sectional component) and after 1-year of follow-up (prospective component), and the presence of macroprolactin. Results: Two male patients, but none of the female patients, had serum prolactin concentrations within the reference range. Following precipitation with polyethylene glycol (PEG), 17 males (25.4%) and 9 females (20.6%) had serum prolactin concentrations within the reference range. Females had somewhat higher levels than males: 39.8 (IQR 32.3–64.5) versus 27.8 (IQR 23.4–47.8) ng/ml (p = 0.003). The ratio of prolactinPEG/prolactinNative was greater than 0.60 in 103 patients (92.8%), thus excluding significant amounts of macroprolactin. From uni- and multivariate analyses we did not find associations of serum prolactin concentrations with a response to ESA either at baseline or at follow-up. Conclusions: From this prospective study we provide evidence that elevated serum prolactin levels are not related to the presence of macroprolactin in chronic hemodialysis patients. Furthermore, serum prolactin concentrations are not associated with the response to erythropoietic therapy in these individuals.

Clinical implications of subclinical hypothyroidism (SCH) are still matter of intense debate, resulting in the controversial discussion whether subclinical hypothyroidism should be treated. We performed a cohort study to evaluate the impact of subclinical hypothyroidism on vascular and overall mortality. Between 02/1993 and 03/2004, a total of 103,135 persons attending the General Hospital Vienna with baseline serum thyrotropin (TSH, thyroid-stimulating hormone) and free thyroxin (fT4) measurements could be enrolled in a retrospective cohort study. Subclinical hypothyroidism was defined by elevated TSH ranging from 4.5 to 20.0 mIU/L and normal fT4 concentration (0.7–1.7 ng/dL). Overall and vascular mortality as primary endpoints were assessed via record linkage with the Austrian Death Registry. A total of 80,490 subjects fulfilled inclusion criteria of whom 3934 participants (3.7 %) were classified as SCH (868 males and 3066 females, median age 48 years). The mean follow-up among the 80,490 subjects was 4.1 years yielding an observation period of 373,301 person-years at risk. In a multivariate Cox regression model adjusted for age and gender TSH levels showed a dose-dependent association with all-cause mortality. The association between SCH and overall or vascular mortality was stronger in men below 60 years compared to older males or females. Our data support the hypothesis that SCH might represent an independent risk factor for overall and vascular mortality, especially in men below 60 years. Whether this group would benefit from replacement therapy should be evaluated in interventional studies.

Background: Patients with mastocytosis may suffer from severe hypotension after wasp or bee stings. In these patients, no specific IgE is detectable, but they usually have skin lesions and an elevated serum tryptase level. Methods: We report on 6 patients who were referred to our department because of severe hypotension following bee or wasp stings without cutaneous lesions. Results: In 3 patients, the baseline serum tryptase level was elevated (26, 36, and 67 ng/ml, respectively), and investigation of their bone marrow revealed systemic mastocytosis (SM). In the remaining 3 patients, serum tryptase levels were Conclusions: All patients with unexplained hypotension after hymenoptera stings should undergo a thorough investigation for major and minor SM criteria regardless of the tryptase level or presence of skin lesions, in order to diagnose or exclude SM or a related subdiagnostic condition (1 or 2 minor SM criteria) tentatively termed monoclonal mast cell activation syndrome.

Background: Hyperlipidemia is highly prevalent in kidney transplant recipients, but the prognostic significance for mortality and allograft survival in these patients has not been established sufficiently. Methods: We prospectively enrolled 733 kidney transplant recipients between 1996 and 1998. Clinical information was collected and blood was drawn for laboratory evaluation. Information on the previous transplantation procedures and organ donor were obtained from the Eurotransplant Foundation database. We used the Austrian Dialysis and Transplantation Registry for follow-up. Using multivariate proportional hazard regression, independent relations of fasting plasma triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol levels to risk for death from any cause and risk for kidney allograft loss were examined. Results: During a median follow-up of 6.1 years, 154 patients died and 260 kidney allografts were lost. After careful multivariate adjustment, there were no significant associations between TG and TC levels and patient mortality. Patients in the highest quartile of TG and TC levels had no difference in risks for mortality compared with patients in the lowest quartile of these parameters (hazards ratio, 0.81; 95% confidence interval, 0.51 to 1.28; hazards ratio, 0.68; 95% confidence interval, 0.42 to 1.10, respectively). Similarly, no associations were found with allograft loss. Further analysis of associations between high-density lipoprotein cholesterol or low-density lipoprotein cholesterol categories and patient mortality or kidney allograft loss did not show associations. Conclusion: Elevated levels of TC or its subfractions and elevated TG levels are not associated with increased risk for patient mortality or allograft loss in these kidney transplant recipients.

Background. Lower concentrations of endothelial progenitor cells (EPCs) maybe associated with increased cardiovascular risk. EPC counts and their correlates have not yet been studied in kidney transplant recipients (KTR). Methods. We cross-sectionally studied EPC counts in 105 middle-aged KTR (mean estimated glomerular filtration rate 45.2 ml/min/1.73m 2 ; range: 5.4 to 117.5). Using univariate and multivariate linear regression assuming a gamma distribution of the outcome, we examined the associations between counts of cultured EPCs and traditional cardiovascular disease risk factors (hypertension, diabetes, hyperlipidemia, smoking), kidney function, and immunosuppressive agents, amongst others. Results. The median count of cultured EPCs was 34 cells per high-power field (interquartile range: 19 to 64), comparable to healthy individuals. From multivariate analyses, we found independent inverse associations between counts of cultured EPCs and body mass index, mean arterial pressure, and history of cardiovascular disease. Statin use was associated with greater EPC counts, whereas patients receiving azathioprine or angiotensin II receptor treatment had lower EPC counts (all P

The pathogenesis of hypertensive nephropathy is multifactoral and in addition to BP, other factors contribute to the development of this renal pathology and its progression to end-stage renal disease. These include genetic predisposition and increased pleasure level of homocysteine-intermediate protein catabolism product known to induce kidney injury. The 677C --T polymorphism in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene is associated with elevated homocysteine level in the general population, and therefore it has been hypothesized to be a risk factor for the development of renal failure in the course of essential hypertension.In this case-control, cross-sectional study the frequency of the MTHFR 677C --T and the 1298A --C polymorphism was compared between patients with hypertension-related chronic renal failure (n = 90), patients with essential hypertension without kidney injury (n = 90), and healthy individuals (n = 90) who were matched for age and gender. In addition, the influence of these polymorphisms on homocysteine concentration in individuals with essential hypertension was examined.The frequency of the MTHFR 677 TT genotype did not differ between groups (4.5%, 12.3%, and 11.1%, respectively). Patients with hypertension and the 677TT genotype showed significantly higher homocysteine levels as compared to individuals having CC and CT. In the multivariate correlation analysis the MTHFR 677TT genotype (P.01; beta = 0.27), age (P.001; beta = 0.33), and body mass index (P.01; beta = 0.3) were independent predictors for total homocysteine level.Plasma homocysteine levels in individuals with essential hypertension is affected by the MTHFR 677C --T polymorphism. However, we did not prove the hypothesis that MTHFR 677C --T influences the risk of development of renal failure in the course of hypertension.

In most cases, the diagnosis of systemic mastocytosis (SM) is based on histomorphologic evaluation of the bone marrow. We analyzed mast cell (MC) infiltration patterns in 57 cases of SM and 31 cases of mast cell hyperplasia (MCH). Tryptase immunohistochemical analysis was used for MC detection and CD25 to distinguish neoplastic from normal MCs. The following infiltration patterns were found: I, diffuse interstitial; II, focal, dense; III, focal, dense with an additional diffuse component, located preferentially around focal infiltrates; IV, focal, dense with an additional diffuse component evenly distributed throughout; and V, diffuse, dense. In 29 cases of MCH, MCs formed the type I pattern. The majority of SM cases exhibited patterns II to V; type IV was the most frequent (n = 36). Type V was seen in 3 cases of MC leukemia and 1 case of smoldering SM. In 1 case of SM, type I infiltration was found; the SM diagnosis was based on 3 minor SM criteria. Our data show that the infiltration pattern in SM correlates with the disease subtype and should be recognized as an important aspect in the histomorphologic evaluation of the bone marrow.

Background: It is unknown whether traditional cardiovascular disease risk factors influence the number of endothelial progenitor cells (EPCs) and whether numbers of EPCs correlate with endothelial function in patients with end-stage renal disease. Methods: In a cross-sectional study of 38 peritoneal dialysis patients, we examined numbers of circulating CD34 + /KDR + /CD133 + cells, CD34 + hematopoietic stem cells, and EPCs cultured from peripheral blood. We also assessed conventional cardiovascular disease risk factors, such as history of vascular disease, diabetes, hypercholesterolemia, hypertension, and smoking. We determined endothelial function by measurement of endothelium-dependent and endothelium-independent reactivity of forearm resistance arteries by using strain-gauge plethysmography. Results: Numbers of EPCs cultured from peripheral blood and forearm blood flow reactivity did not differ between erythropoietin-treated peritoneal dialysis patients and healthy individuals. A history of vascular disease was associated with number of cultured EPCs, but other cardiovascular disease risk factors showed no association. Furthermore, there was no association of endothelial-dependent and endothelial-independent forearm blood flow reactivity with EPCs in peritoneal dialysis patients. Conclusion: In this first study of EPCs in peritoneal dialysis patients, we found an association between history of vascular disease and EPCs, but no association of EPCs with endothelial function or other cardiovascular disease risk factors.

Cobalamin deficiency is a common finding. In the elderly the prevalence is 10-20%, but only 5-10% of these are clinically symptomatic. Typical clinical symptoms include macrocytic anemia, neuropsychiatric symptoms and glossitis. In many cases this triad is lacking, however. The serum cobalamin assay is the best first line test, but the results must be carefully interpreted, since a normal level does not exclude deficiency. Markers of cobalamin activity, such as serum homocysteine or methylmalonic acid may be helpful in this situation. The main cause of cobalamin deficiency is atrophic gastritis. It is either caused by an autoimmune process which leads to achlorhydria and severe intrinsic factor deficiency ("classical pernicious anemia") or by atrophic gastritis from other causes, in particular helicobacter pylori infection. In the latter cases the lack of gastric acid does not allow separation of cobalamin from proteins, but intrinsic factor, although low, is sufficient for cobalamin protection (food cobalamin malabsorption). Helicobacter pylori eradication may cure some of these patients. While in food cobalamin malabsorption syndrome small doses of oral cobalamin are effective, parenteral therapy or high oral doses are required for treatment of pernicious anemia. While almost all patients respond hematologically, only half of the patients with neurological signs, and a small minority of psychiatric patients respond to treatment. Patients with pernicious anemia and atrophic gastritis have a greatly increased long-term risk for gastric carcinoids.

Associations between MTHFR 1793G>A and plasma total homocysteine, folate, and vitamin B 12 in kidney transplant recipients. Background Currently, no evidence is available on the putative associations between a novel single nucleotide polymorphism of the 5,10-methylenetetrahydrofolate reductase gene MTHFR 1793G>A and plasma levels of vitamin B 12 , folate, or total homocysteine (tHcy). Methods In a cross-sectional study of 730 kidney allograft recipients, patients were categorized by MTHFR 1793G>A genotype. In univariate and multivariate linear regression models that allowed the outcome variables vitamin B 12 , folate, and tHcy plasma levels to follow a gamma distribution, we tested for possible associations of allelic variants of MTHFR 1793G>A and these three dependent variables. As hypothesized in previous work, we specifically evaluated possible effect modification between the MTHFR 1793G>A and 1298A>C mutations on these outcomes. Results The allele frequency for MTHFR 1793G>A was 0.052. Heterozygosity ( N = 72) or homozygosity ( N = 2) for MTHFR 1793G>A was not independently associated with plasma levels of vitamin B 12 ( P = 0.33) or tHcy ( P = 0.70), but a borderline association with higher folate concentrations was detected (Δfolate = 1.91 nmol/L) (95% CI -0.03 to 3.86 nmol/L) ( P = 0.05). Further, we found strong and significant positive interactions between the MTHFR 1793G>A and 1298A>C mutations on vitamin B 12 concentrations. Conclusion Higher folate concentrations in kidney transplant recipients with MTHFR 1793GA or 1793AA and markedly higher concentrations of vitamin B 12 in patients with combined MTHFR 1793G>A and 1298A>C mutations may contribute to the survival advantage that has been postulated for such patients showing these genotypes.

Background Recently, a new mutation of the 5,10-methylenetetrahydrofolate reductase (MTHFR) encoding gene was first described (1793G > A). Only few reports have studied the prevalence of this polymorphism, especially in combination with other MTHFR mutations (677C > T, 1298A > C). Methods We cross-sectionally identified the novel MTHFR 1793G > A polymorphism in 730 kidney transplant recipients. MTHFR 677C > T and 1298A > C were also assessed and the frequency of each was described individually as well as in cross-tabulation with the other MTHFR genotypes. The expected number of patients for each MTHFR genotype combination was calculated and contrasted with the observed numbers. Fisher's exact test was used for statistical inference. Results The allelic frequency of MTHFR 1793G > A was 0·052. Seventy-two patients (9·9%) were heterozygous and two patients (0·3%) were homozygous. From the cross-tabulations, we identified 53 patients (expected: 33·6) with the MTHFR 1298AC/1793GA genotype and 17 patients (expected: 6·7) with the MTHFR 1298CC/1793GA genotype. Furthermore, we found two patients with double homozygosity for MTHFR 1793G > A and MTHFR 1298A > C (MTHFR 1793AA/1298CC genotype). The frequencies of these genotype combinations were substantially larger than could be expected (P A and MTHFR 1298A > C genotypes, possibly owing to a mutually stabilizing effect on MTHFR enzyme activity.

C-reactive protein (CRP) is a risk factor for cardiovascular outcomes and mortality in the general population. To date, there are no prospective studies of the association between CRP and mortality or allograft loss in kidney transplant recipients (KTR). In 1995, 438 consecutive KTR were enrolled in this prospective study. Important information on demographic, clinical and immunological characteristics was collected at baseline, and CRP was measured using standard methods. Patients were then followed-up for a median 7.8 years. Time-to-event analyses (univariate and multivariate Cox proportional hazards regression models) were used to study the main outcomes: all-cause mortality and kidney allograft loss, defined as the earlier of return to dialysis, re-transplantation, or death. From univariate analyses, we found that CRP >or=0.5 mg/dL was associated with a 83% greater mortality risk compared with lower levels of this inflammatory marker [hazard ratio (HR) = 1.83; 95% confidence interval (CI): 1.23-2.72; p = 0.003]. After multivariate adjustment, patients with a CRP >or=0.5 mg/dL had a 53% higher mortality risk compared with patients whose CRP was below that threshold (HR = 1.53; 95% CI: 1.01-2.31; p = 0.04). No associations between CRP and the risk of kidney allograft loss were detected. Furthermore, we were not able to detect any effect modification between CRP and body mass index on the outcomes under study. We conclude that CRP predicts all-cause mortality, but not allograft loss in stable KTR.

Dose-dependent effect of parenteral iron therapy on bleomycin-detectable iron in immune apheresis patients. Background Iron deficiency and anemia are commonly encountered in patients with autoimmune diseases undergoing immune apheresis. This makes erythropoietin and iron substitution necessary in most patients. However, intravenous iron therapy may result in an increase of potentially toxic nontransferrin-bound iron. Methods We examined the effect of 50 mg or 100 mg of iron (III) sucrose on bleomycin-detectable iron (BDI) in immune apheresis patients. Six patients with autoimmune disorders and normal kidney function were enrolled. Before and after the injection of 50 mg or 100 mg of iron (III) sucrose, BDI was measured in serum samples at five different time points. Results There was no BDI traceable before injection of iron (III) sucrose. BDI was present in serum of all patients after the administration of 100 mg of iron (III) sucrose in concentrations up to 0.49 μmol/L. In contrast, only one patient showed BDI at a concentration of 0.16 μmol/L after the administration of 50 mg of iron (III) sucrose. Conclusion We conclude that if parenteral iron is administered after apheresis treatment, despite the equal tolerability, use of 50 mg of iron (III) sucrose is superior to 100 mg of iron (III) sucrose in avoiding the formation of potentially toxic nontransferrin-bound iron.

Effects of TCN2 776C>G on vitamin B 12 , folate, and total homocysteine levels in kidney transplant patients. Background Controversy exists regarding the possible associations between a single nucleotide polymorphism of the transcobalamin II encoding gene ( TCN2 776C>G) and plasma levels of vitamin B 12 , folate, or total homocysteine. Methods In a cross-sectional study of 732 kidney allograft recipients, patients were categorized by TCN2 776C>G genotype. In univariate and multivariate linear regression models that allowed the outcome variables vitamin B 12 , folate, and total homocysteine plasma levels to follow a gamma distribution, we tested for possible associations of allelic variants of the TCN2 776C>G gene and these three dependent variables. Results The allele frequency for TCN2 776C>G was 0.46. Heterozygosity or homozygosity for TCN2 776C>G was not associated with plasma levels of vitamin B 12 (776CG, P = 0.22; 776GG, P = 0.89), folate (776CG, P = 0.91; 776GG, P = 0.84), or total homocysteine (776CG, P = 0.11; 776GG, P = 0.33) even after adjustment for several possible confounders. Conclusion We conclude from this largest study on the subject thus far that there are no associations between allelic variants of TCN2 776C>G and plasma vitamin B 12 , folate, or total homocysteine plasma levels in kidney transplant patients.