None

of the IAC patients showed signs of malignant disease Adriamycin nmr (hematological, pancreatico biliary, or other) observed to date (mean follow-up, 16 months; range, 8-20 months). From all newly diagnosed patients, peripheral blood was drawn before the start of treatment with prednisolone (median, 40 mg/day; range, 20-40 mg/day). After 4 and 8 weeks, additional blood samples were collected. Two patients underwent endoscopic retrograde cholangio pancreatography for stent replacement, which allowed the collection of a duodenal papilla biopsy (ampulla of Vater), and paired peripheral blood. Patients included in the PSC control group were selected based on a diagnosis of PSC compliant with the current European Association for the Study of the Liver guidelines16 and including a history of colitis; one patient with elevated serum IgG4 underwent additional short-term prednisolone treatment without biochemical response. Patients included in the malignancy control group had a histologically proven hepatobiliary malignancy (pancreatic cancer or bile duct cancer) (Table 2). (An extended overview of clinical characteristics of all analyzed patients is provided selleck chemical in Supporting Table 2.) Anonymous healthy individuals were age- and sex-matched to the IAC patient group. The study was performed according to the Declaration of Helsinki and was approved

by the local medical ethical committee of the Academic Medical Center (METC10/007). All patients provided written informed consent prior to inclusion in the study. Peripheral blood was collected and stored using PAXGene Blood RNA selleck chemicals tubes according

to the manufacturer’s instructions (catalog #762165, PreAnalytiX, Breda, The Netherlands). Isolation of total RNA was performed using the PAXGene Blood RNA isolation kit (catalog #762174, Qiagen, Venlo, The Netherlands). Biopsies of the duodenal papilla (ampulla of Vater) during endoscopic retrograde cholangio pancreatography were immediately preserved in RNAlater reagent (Qiagen) and stored at −80°C until use. Total RNA was isolated using polytron homogenizer in the presence of STAT60 reagent as described.17 cDNA was synthesized with 250 ng total RNA input using Superscript III RT (Invitrogen Life Technologies, Carlsbad, CA). The linear amplification used in this study was based on the protocol used for T cells and B cells in previous studies.17, 18 In the first step of the protocol, a linear amplification of the complete immunoglobulin repertoire was performed using a primer set covering all functional Vheavy genes of the BCR (Supporting Fig. 4) (primer sequences available on request). The Vheavy-primers contained a primer B sequence required for Amplicon sequencing according to the 454 titanium protocol (version 2010; Roche Diagnostics, Mannheim, Germany).

28 However, Cetuximab purchase these studies had limitations, as usually only one functional readout was applied and CD4+ and CD8+ T-cell responses were not distinguished. Moreover, and importantly, no study until now has addressed the

role of T-cell responses in resolving and chronic HEV infection. Thus, we here aimed to study cellular immune responses in different patient groups including organ transplant recipients with chronic and resolved hepatitis E. We show that (1) strong and multispecific HEV-specific T-cell responses are present in exposed healthy controls and to a lesser extent also in recovered patients after transplantation; (2) that these responses are absent in patients with chronic hepatitis check details E but become detectable after viral clearance; and (3) that HEV-specific T-cell responses can be restored in vitro by blocking the PD-1 or CTLA-4 pathways. However,

a combination of PD-1 and CTLA-4 blockade was not synergistic. CBA, cytometric bead array; CFSE, 5,6-carboxyfluorescein diacetate succinimidyl ester; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; HEV, hepatitis E virus; ICS, intracellular cytokine staining; ORF, open reading frame; PBMC, peripheral blood mononuclear cells; PD-1, programmed death 1; PDL-1, PD-1 ligand 1; Tx, transplantation. Informed consent in writing was obtained from each patient included in this study. The study protocol conformed to the ethical guidelines of the Institutional Review Committee. Immune responses against HEV were studied in a total of 38 subjects including 19 healthy immunocompetent individuals and 19 immunocompromised organ recipients. The immunocompetent group included anti-HEV-positive (“exposed”) subjects (n = 9; median age 32; range 26-66) and anti-HEV-negative this website (“no exposure”) individuals (n = 10; median age 30; range 25-37). The immunocompromised group included transplanted

patients who developed chronic hepatitis E (n = 7; median age 49; range 34-66) and transplanted patients who resolved HEV infection (n = 12; median age 53; range 27-69). Out of these 12 patients, three subjects acquired HEV infection post transplantation, one subject before transplantation, and no information on the time of HEV acquisition was available for the remaining patients. Presence of antibodies (IgG) against HEV was tested by using a commercially available enzyme-linked immunosorbent assay kit (Abbott Laboratories, North Chicago, IL) according to the manufacturer’s instructions. All study subjects were negative for hepatitis B surface antigen (HBsAg) and anti-HCV except one transplant recipient with resolved HEV infection (LTxR2), being anti-HCV positive with serum HCV RNA levels of 1 Mio IU/mL. The presence of HEV RNA was confirmed by both qualitative and quantitative real-time polymerase chain reaction (PCR). All assays were performed as previously described. 29, 30 For details, please see Supporting Information.

28 However, selleck inhibitor these studies had limitations, as usually only one functional readout was applied and CD4+ and CD8+ T-cell responses were not distinguished. Moreover, and importantly, no study until now has addressed the

role of T-cell responses in resolving and chronic HEV infection. Thus, we here aimed to study cellular immune responses in different patient groups including organ transplant recipients with chronic and resolved hepatitis E. We show that (1) strong and multispecific HEV-specific T-cell responses are present in exposed healthy controls and to a lesser extent also in recovered patients after transplantation; (2) that these responses are absent in patients with chronic hepatitis Selleck Roxadustat E but become detectable after viral clearance; and (3) that HEV-specific T-cell responses can be restored in vitro by blocking the PD-1 or CTLA-4 pathways. However,

a combination of PD-1 and CTLA-4 blockade was not synergistic. CBA, cytometric bead array; CFSE, 5,6-carboxyfluorescein diacetate succinimidyl ester; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; HEV, hepatitis E virus; ICS, intracellular cytokine staining; ORF, open reading frame; PBMC, peripheral blood mononuclear cells; PD-1, programmed death 1; PDL-1, PD-1 ligand 1; Tx, transplantation. Informed consent in writing was obtained from each patient included in this study. The study protocol conformed to the ethical guidelines of the Institutional Review Committee. Immune responses against HEV were studied in a total of 38 subjects including 19 healthy immunocompetent individuals and 19 immunocompromised organ recipients. The immunocompetent group included anti-HEV-positive (“exposed”) subjects (n = 9; median age 32; range 26-66) and anti-HEV-negative selleck screening library (“no exposure”) individuals (n = 10; median age 30; range 25-37). The immunocompromised group included transplanted

patients who developed chronic hepatitis E (n = 7; median age 49; range 34-66) and transplanted patients who resolved HEV infection (n = 12; median age 53; range 27-69). Out of these 12 patients, three subjects acquired HEV infection post transplantation, one subject before transplantation, and no information on the time of HEV acquisition was available for the remaining patients. Presence of antibodies (IgG) against HEV was tested by using a commercially available enzyme-linked immunosorbent assay kit (Abbott Laboratories, North Chicago, IL) according to the manufacturer’s instructions. All study subjects were negative for hepatitis B surface antigen (HBsAg) and anti-HCV except one transplant recipient with resolved HEV infection (LTxR2), being anti-HCV positive with serum HCV RNA levels of 1 Mio IU/mL. The presence of HEV RNA was confirmed by both qualitative and quantitative real-time polymerase chain reaction (PCR). All assays were performed as previously described. 29, 30 For details, please see Supporting Information.

Hansen Background and aim The recently identified interferon lambda 4 (IFNL4) gene harbors the dinucleotide variant rs368234815-TT/ΔG, a genetic marker of outcome check details to IFN-based therapy of HCV infection in high linkage disequilibrium (LD) with the rs12979860-C/T polymorphism. The IFNλ-4

protein, which can be generated only by carriers of the rs368234815-ΔG allele, is thought to counteract IFN responsiveness by inducing a weak expression of interfer-on-stimulated genes. Three nonsynonymous variants of the IFNL4 gene (rs73555604, rs142981501 and rs117648444) could affect the IFNλ-4 protein. We aimed to explore whether IFNL4 polymorphisms impact on response to IFN-based treatment in the setting of chronic hepatitis B (CHB). Methods IFNL4 gene was sequenced by Sanger method on genomic DNA extracted from whole blood of 126 HBeAg-negative CHB patients treated with either standard or pegylated-IFN-α and followed-up

for 11 years (range 1-17) post-treatment. Results The distribution BI 2536 price of the rs368234815 genotype (48% for TT/ TT, 40% for ΔG/TT and 12% for ΔG/ΔG) matched that of the rs12979860 variant (LD r2=0.98). Sustained response rates to IFN were not significantly different between the 62 carriers of the rs368234815-TT/TT (IFNλ-4 eliminating) genotype and the 64 carriers of the rs368234815-ΔG (IFNλ-4 generating) allele (31% vs 17%, p=0.079). Because of the exclusive association between the nonsynonymous variant identified in our cohort, the Pro70Ser rs117648444-C/T polymorphism, and carriers of the IFNλ-4 generating allele, patients were stratified into rs117648444-CC (IFNX-4 wild-type, n=45) and rs117648444-CT/TT (IFNX-4 mutated, n=19)

genotypes. Given the similar rates of sustained response in the IFNλ-4 mutated and IFNλ-4 eliminating genotypes carriers (37% vs 32%, p=ns), these two patients groups were combined together (n=81). Sustained see more responses among these 81 patients, who either could not produce IFNλ-4 (n=62) or produced a mutated protein (n=19), were significantly higher than those in the 45 IFNX-4 wild-type subjects (33.3% vs 9%, OR=4.8, 95%CI 1.6-15.0, p=0.006). In a multivariate analysis including all the canonical pretreatment predictors of response, the combination of IFNλ-4 mutated and IFNλ-4 eliminating genotypes strongly predicted not only a sustained response (OR=5.33, 95%CI 1.7-16.8, p=0.004) but also off-treatment HBsAg sero-clearance (HR=4.3, 95%CI 1.5-12.3, p=0.007). Pretreatment serum HBV-DNA was the only other independent predictor of HBsAg loss (HR=0.61, 95%CI 0.43-0.87, p=0.007).

Hansen Background and aim The recently identified interferon lambda 4 (IFNL4) gene harbors the dinucleotide variant rs368234815-TT/ΔG, a genetic marker of outcome Sorafenib to IFN-based therapy of HCV infection in high linkage disequilibrium (LD) with the rs12979860-C/T polymorphism. The IFNλ-4

protein, which can be generated only by carriers of the rs368234815-ΔG allele, is thought to counteract IFN responsiveness by inducing a weak expression of interfer-on-stimulated genes. Three nonsynonymous variants of the IFNL4 gene (rs73555604, rs142981501 and rs117648444) could affect the IFNλ-4 protein. We aimed to explore whether IFNL4 polymorphisms impact on response to IFN-based treatment in the setting of chronic hepatitis B (CHB). Methods IFNL4 gene was sequenced by Sanger method on genomic DNA extracted from whole blood of 126 HBeAg-negative CHB patients treated with either standard or pegylated-IFN-α and followed-up

for 11 years (range 1-17) post-treatment. Results The distribution Sirolimus ic50 of the rs368234815 genotype (48% for TT/ TT, 40% for ΔG/TT and 12% for ΔG/ΔG) matched that of the rs12979860 variant (LD r2=0.98). Sustained response rates to IFN were not significantly different between the 62 carriers of the rs368234815-TT/TT (IFNλ-4 eliminating) genotype and the 64 carriers of the rs368234815-ΔG (IFNλ-4 generating) allele (31% vs 17%, p=0.079). Because of the exclusive association between the nonsynonymous variant identified in our cohort, the Pro70Ser rs117648444-C/T polymorphism, and carriers of the IFNλ-4 generating allele, patients were stratified into rs117648444-CC (IFNX-4 wild-type, n=45) and rs117648444-CT/TT (IFNX-4 mutated, n=19)

genotypes. Given the similar rates of sustained response in the IFNλ-4 mutated and IFNλ-4 eliminating genotypes carriers (37% vs 32%, p=ns), these two patients groups were combined together (n=81). Sustained selleck kinase inhibitor responses among these 81 patients, who either could not produce IFNλ-4 (n=62) or produced a mutated protein (n=19), were significantly higher than those in the 45 IFNX-4 wild-type subjects (33.3% vs 9%, OR=4.8, 95%CI 1.6-15.0, p=0.006). In a multivariate analysis including all the canonical pretreatment predictors of response, the combination of IFNλ-4 mutated and IFNλ-4 eliminating genotypes strongly predicted not only a sustained response (OR=5.33, 95%CI 1.7-16.8, p=0.004) but also off-treatment HBsAg sero-clearance (HR=4.3, 95%CI 1.5-12.3, p=0.007). Pretreatment serum HBV-DNA was the only other independent predictor of HBsAg loss (HR=0.61, 95%CI 0.43-0.87, p=0.007).

In addition, cells that were deficient for LRP1 proved approximately 50% less efficient than their LRP1-expressing counterparts in the uptake and

degradation of FVIII [33,53,54]. Similar results were obtained by blocking cellular LRP1 with its universal inhibitor receptor-associated protein (RAP). Thus, it became apparent that LRP1 participates in the uptake and transport of FVIII to intracellular degradation pathways. However, a message that could easily be overlooked from these experiments is that the absence of LRP1 resulted in but a partial inhibition of FVIII degradation, strongly indicating that alternative pathways contributing to FVIII catabolism should exist. Nevertheless, a vast amount of data has been produced showing that the contribution of LRP1 to FVIII catabolism is of in vivo relevance. These include experiments using Omipalisib mice with a

conditional induced deletion of the LRP1 gene, which resulted in increased plasma levels of FVIII in these mice [55]. In addition, the mean residence time of intravenously administered FVIII was prolonged 1.5-fold, from 2.5 to 4 h. A number of epidemiological studies revealed that LRP1 IWR-1 chemical structure modulates FVIII plasma levels also in humans [56–59]. So far, two distinct LRP1 polymorphisms (LRP1/D2080N and LRP1/A217V) have been suggested to be associated with up to 20% higher FVIII plasma levels [57,58]. The underlying mechanism of how these polymorphisms affect FVIII levels remains to be elucidated. Despite the

see more proven physiological relevance of LRP1 in FVIII clearance, a number of issues still remain unclear. For instance, LRP1 is known for its large spectrum of structurally and functionally unrelated ligands, with more than 50 ligands currently being identified [60]. It is unknown however, if and how these other ligands affect LRP1-dependent clearance of FVIII. Another point relates to the fact that LRP is able to assemble into heterologous receptor complexes. Examples hereof include platelet-derived growth factor (PDGF) receptor in smooth muscle cells, N-methyl-d-aspartate (NMDA) receptor in neurons and β2-integrins on leukocytes [61–63]. It cannot be excluded therefore that part of the LRP-mediated effects are indirect, in that LRP1 affects the function of other receptors. Direct evidence for this possibility is currently lacking. However, it has been shown that LRP1 is able to modulate FVIII catabolism in concert with other receptors. For instance, Bovenschen et al. [64] demonstrated that LRP1 regulates FVIII levels in a coordinated fashion with LDL receptor, illustrated by a synergistic increase in plasma levels and survival of FVIII in mice with a combined LRP1/LDL receptor deficiency [64]. Of note, also other members of the LDL receptor family are able to recognize FVIII, such as vLDL receptor and megalin [65–67].

In LT patients exhibiting SF ≥365 μg/L and TFS <55%, an overall survival of 54.5% in comparison to 74.8% in the remaining group was observed and confirmed in the validation cohort (28.6% versus 72%). These data indicate that with TFS below 55% the elevation of SF is associated with a higher risk of post-LT mortality. Ferritin is also an acute phase protein elevated in response to immune-mediated and infectious stimuli, which may thus represent

a surrogate marker for a general predisposition for morbidity and mortality. In our click here study, c-reactive protein levels were compared and found to be lower in the group in which SF correlated well with overall recipient survival (Table 4). Generally, elevated SF need not be linked to c-reactive protein levels in acute

phase responses.41, 42 In addition, advanced liver diseases can contribute to a low c-reactive protein level response by reduced hepatic protein synthesis. In patients treated with interferon alpha-2b decreased c-reactive protein and significant elevations of SF were reported.43 This indicates a differential activation of acute Proteases inhibitor phase markers such as c-reactive protein and SF, which is likely to be responsible for high SF, i.e., in adult-onset Still’s disease29, 30 and other conditions. In patients undergoing hemodialysis and those with metabolic syndrome, elevated SF without elevations of TFS44, 45 has been observed, and SF levels have been associated with inferior prognosis.19, 21 Therefore, SF and TFS are not only markers for iron overload but can indicate an activation of acute phase and possibly other mechanisms35, 36 that influence mortality. In our study cohorts, liver biopsy material was not available to correlate histological iron load with the biochemical data. However, an analysis of the National Health and Nutrition Examination Survey (NHANES) 1999-2002 reported that even modest

elevations of SF were associated with reduced cardiovascular fitness in young male subjects,46 and that SF may represent a morbidity-associated parameter. Against this background, the finding that elevated SF in addition to lower levels of TFS are predictive for mortality and morbidity may not indicate systemic iron overload. One limitation of this retrospective study is that no selleck products measurements of iron metabolism parameters were performed or were available after LT, which should be studied in future analyses to observe whether elevated SF persists after LT in patients with decreased survival. In addition, it may be of interest to reanalyze the pretransplant situation in other studies17 to assess whether there is also a difference between patients with high or low TFS and elevated SF regarding mortality on the waiting list. This may contribute to potential pre-LT therapeutic strategies. In conclusion, we show that SF elevations before LT predict an increased mortality following LT. This risk is highest in patients with SF ≥365 μg/L and TFS <55%, which was identified as an independent parameter.

Further, RT-PCR analysis with specific primers for Tomato chlorosis virus (ToCV) heat shock protein 70, for TICV heat shock protein 70 and for TICV minor capsid protein was positive for TICV in all tested samples. No signals were obtained with primers for ToCV. Phylogenetic analysis showed that the Bulgarian sequence of Hsp70 and a sequence of Greek isolate clustered together having the highest resampling score. Regarding CPm, the Bulgarian isolate was more relevant to the French isolate. The obtained results from phylogenetic analysis supported the idea of a close relationship

between the Bulgarian and Greek isolates. “
“In April 2010, a severe occurrence of Stewart’s wilt on Dracaena sanderiana plants was observed in greenhouses in Seongnam, Gyeonggi Province, South Korea, with an incidence of 35-50%. Being imported plants, little Cobimetinib price was known about the pathogens associated with D. sanderiana. Symptoms included chlorosis, wilting and leaf

blight on the leaf surfaces. Physiological analysis, pathogenicity tests, sequencing and phylogenetic analysis Doxorubicin chemical structure of the 16S rRNA gene revealed that the pathogen was the bacterium Pantoea stewartii. To the best of our knowledge, this is the first report on bacterial wilt caused by P. stewartii on D. sanderiana. “
“Employing known susceptible and resistant genotypes and pure bacterial inoculum (0.1 OD; 108 CFU/ml−1), five different inoculation methods were tried to assess the response of tomato genotypes to Ralstonia solanacearum. This included seed-soaking inoculation, seed-sowing followed by inoculum drenching, or at 2-week stage through petiole-excision inoculation, soaking of planting medium with inoculum either directly or after imparting seedling root-injury. Seed-based inoculations or mere inoculum drenching at 2 weeks did not induce much disease in seedlings.

Petiole inoculation induced 90–100% mortality in susceptible checks but also 50–60% mortality in normally resistant genotypes within click here 7–10 days. Root-injury inoculation at 2-week seedling stage appeared the best for early and clearer distinction between resistant and susceptible lines. The observations suggest a role played by the root system in governing genotypic resistance to the pathogen. Direct shoot inoculation is to be adopted only for selecting highly resistant lines or to thin down segregating populations during resistance breeding. “
“Globodera rostochiensis is one of the most important plant parasitic nematodes, worldwide. As a quarantine pest of solanaceous crops, the species is often subjected to the morphological and genetic analysis as well as biological tests. They constitute the basis for this nematode detection and control. This paper presents the results of the study on variability of 16 populations of golden potato cyst nematode from Poland – one of the important potato producers – using molecular techniques.

Again, this may relate to allocation policy, as patients with T3 lesions are not given priority for deceased donation. It is also important

to note that the non-LDLT group did contain significantly more patients with HCV, fewer with cholestatic liver disease, and more racial diversity. Although these factors were adjusted for in the model, the power to detect differences is impacted as the numbers decrease. In addition, although this was a multicenter study the results of individual centers were not reported. A center effect has been reported to have a major impact on outcomes with liver transplantation.4 Although it is likely all centers within A2ALL are highly experienced at both living donor and deceased donor transplant, it is not known whether these results could be applied to all centers. In addition, there are marked geographic differences in MELD

threshold for access to deceased donor transplant, as MI-503 nmr well as in the quality of deceased donor allografts.5, 6 These differences would likely augment or mitigate the survival benefit of living donor transplant, depending on the donor service area and region of the transplant center. Finally, and most essentially, this was not a randomized trial of all patients wait-listed for liver transplantation, but rather of a selected group of patients deemed appropriate candidates for LDLT by their transplant centers. Thus, centers must still consider what is best for each and every individual patient on their wait list based on factors that may impact outcome at their center. If there is a very short anticipated wait time to DDLT based on factors such as HCC MELD exception selleck chemical selleck or favorable blood type, then a survival benefit to LDLT compared to DDLT likely will not be present. For individual patients, other factors that impact the decision to proceed to LDLT beyond the

potential for a survival benefit, such as uncontrolled encephalopathy, refractory ascites, and intractable pruritus must also be carefully considered. In the future, information regarding validated quality of life outcomes following LDLT versus prolonged time on the wait list and/or DDLT would provide exceptionally helpful additional guidance to assist in discussion with patients and families regarding timing and donor options for liver transplantation. “
“In the most recent American Association for the Study of Liver Diseases hepatitis B virus (HBV) guidelines, Drs. Lok and McMahon recommend as “prudent” “to test all human immunodeficiency virus (HIV)-infected persons for both hepatitis B surface antigen (HBsAg) and antibody to hepatitis B core antigen (anti-HBc) and if either is positive, to test for HBV DNA”.1 I think that the recommendation of testing for HBV DNA in HIV-infected patients with isolated anti-HBc antibodies should be better supported by clinical evidence. HBV DNA testing is an expensive test, and unless there is a well-defined clinical benefit, it is difficult to justify it.

Again, this may relate to allocation policy, as patients with T3 lesions are not given priority for deceased donation. It is also important

to note that the non-LDLT group did contain significantly more patients with HCV, fewer with cholestatic liver disease, and more racial diversity. Although these factors were adjusted for in the model, the power to detect differences is impacted as the numbers decrease. In addition, although this was a multicenter study the results of individual centers were not reported. A center effect has been reported to have a major impact on outcomes with liver transplantation.4 Although it is likely all centers within A2ALL are highly experienced at both living donor and deceased donor transplant, it is not known whether these results could be applied to all centers. In addition, there are marked geographic differences in MELD

threshold for access to deceased donor transplant, as Cilomilast datasheet well as in the quality of deceased donor allografts.5, 6 These differences would likely augment or mitigate the survival benefit of living donor transplant, depending on the donor service area and region of the transplant center. Finally, and most essentially, this was not a randomized trial of all patients wait-listed for liver transplantation, but rather of a selected group of patients deemed appropriate candidates for LDLT by their transplant centers. Thus, centers must still consider what is best for each and every individual patient on their wait list based on factors that may impact outcome at their center. If there is a very short anticipated wait time to DDLT based on factors such as HCC MELD exception LDE225 price this website or favorable blood type, then a survival benefit to LDLT compared to DDLT likely will not be present. For individual patients, other factors that impact the decision to proceed to LDLT beyond the

potential for a survival benefit, such as uncontrolled encephalopathy, refractory ascites, and intractable pruritus must also be carefully considered. In the future, information regarding validated quality of life outcomes following LDLT versus prolonged time on the wait list and/or DDLT would provide exceptionally helpful additional guidance to assist in discussion with patients and families regarding timing and donor options for liver transplantation. “
“In the most recent American Association for the Study of Liver Diseases hepatitis B virus (HBV) guidelines, Drs. Lok and McMahon recommend as “prudent” “to test all human immunodeficiency virus (HIV)-infected persons for both hepatitis B surface antigen (HBsAg) and antibody to hepatitis B core antigen (anti-HBc) and if either is positive, to test for HBV DNA”.1 I think that the recommendation of testing for HBV DNA in HIV-infected patients with isolated anti-HBc antibodies should be better supported by clinical evidence. HBV DNA testing is an expensive test, and unless there is a well-defined clinical benefit, it is difficult to justify it.