Autologous humanized mouse models of iPSC-derived tumors enable characterization and modulation of cancer-immune cell interactions.

Modeling the tumor-immune cell interactions in humanized mice is complex and limits drug development. Here, we generated easily accessible tumor models by transforming either primary skin fibroblasts or induced pluripotent stem cell-derived cell lines injected in immune-deficient mice reconstituted with human autologous immune cells. Our results showed that fibroblastic, hepatic, or neural tumors were all efficiently infiltrated and partially or totally rejected by autologous immune cells in humanized mice. Characterization of tumor-immune infiltrates revealed high expression levels of the dysfunction markers Tim3 and PD-1 in T cells and an enrichment in regulatory T cells, suggesting rapid establishment of immunomodulatory phenotypes. Inhibition of PD-1 by Nivolumab in humanized mice resulted in increased immune cell infiltration and a slight decrease in tumor growth. We expect that these versatile and accessible cancer models will facilitate preclinical studies and the evaluation of autologous cancer immunotherapies across a range of different tumor cell types.

Generation of Complex Syngeneic Liver Organoids from Induced Pluripotent Stem Cells to Model Human Liver Pathophysiology.

The study of human liver pathophysiology has been hampered for decades by the lack of easily accessible, robust, and representative in vitro models. The discovery of induced pluripotent stem cells (iPSCs)-which can be generated from patients' somatic cells, engineered to harbor specific mutations, and differentiated into hepatocyte-like cells-opened the way to more meaningful modeling of liver development and disease. Nevertheless, representative modeling of many complex liver conditions requires the recreation of the interplay between hepatocytes and nonparenchymal liver cells. Here we describe protocols we developed to generate and characterize complex human liver organoids composed of iPSC-derived hepatic, endothelial, and mesenchymal cells. With all cell types derived from the same iPSC population, such organoids reproduce the liver niche, allowing for the study of liver development and the modeling of complex inflammatory and fibrotic conditions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Differentiation of human iPSCs into hepatic progenitor cells (hepatoblasts) Basic Protocol 2: Differentiation of human iPSCs into endothelial progenitor cells Support Protocol 1: Characterization of iPSC-derived endothelial progenitor cells Basic Protocol 3: Differentiation of human iPSCs into mesenchymal progenitor cells Support Protocol 2: Characterization of iPSC-derived mesenchymal progenitor cells Basic Protocol 4: Generation of complex syngeneic liver organoids.

Leveraging interacting signaling pathways to robustly improve the quality and yield of human pluripotent stem cell-derived hepatoblasts and hepatocytes.

Pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs) have shown great potential as an alternative to primary human hepatocytes (PHHs) for in vitro modeling. Several differentiation protocols have been described to direct PSCs toward the hepatic fate. Here, by leveraging recent knowledge of the signaling pathways involved in liver development, we describe a robust, scalable protocol that allowed us to consistently generate high-quality bipotent human hepatoblasts and HLCs from both embryonic stem cells and induced PSC (iPSCs). Although not yet fully mature, such HLCs were more similar to adult PHHs than were cells obtained with previously described protocols, showing good potential as a physiologically representative alternative to PHHs for in vitro modeling. PSC-derived hepatoblasts effectively generated with this protocol could differentiate into mature hepatocytes and cholangiocytes within syngeneic liver organoids, thus opening the way for representative human 3D in vitro modeling of liver development and pathophysiology.

High-throughput assessment of mutations generated by genome editing in induced pluripotent stem cells by high-resolution melting analysis.

BACKGROUND AND AIMS: Genome editing of induced pluripotent stem cells (iPSCs) holds great potential for both disease modeling and regenerative medicine. Although clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 provides an efficient and precise genome editing tool, iPSCs are especially difficult to transfect, resulting in a small percentage of cells carrying the desired correction. A high-throughput method to identify edited clones is required to reduce the time and costs of such an approach. METHODS: Here we assess high-resolution melting analysis (HRMA), a simple and efficient real-time polymerase chain reaction-based method, and compare it with more commonly used assays. RESULTS AND CONCLUSIONS: Our data show that HRMA is a robust and highly sensitive method, allowing the cost-effective and time-saving screening of genome-edited iPSCs. Samples can be prepared directly from 96-well microtiter plates for high-throughput analysis, and amplicons can be further analyzed with downstream techniques for further confirmation, if needed.

Long-term Outcome of Kidney Recipients Transplanted for Aristolochic Acid Nephropathy.

BACKGROUND: Aristolochic acids (AA) are nephrotoxic and carcinogenic. The aim of this study was to assess the long-term outcome of patients with AA nephropathy (AAN) after kidney transplantation. METHODS: Observational study. Patients' characteristics, long-term surveillance and follow-up data, patient and graft survival, as well as outcomes with respect to rejection, cardiovascular complications, infections, and cancers with a focus on urothelial carcinomas, are reported. RESULTS: Twenty patients transplanted for AAN were included. All were submitted to prophylactic bilateral ureteronephrectomy and annual surveillance of the bladder. Median duration of posttransplant follow-up was 12.5 (3-19) years. Time from diagnosis of AAN to renal replacement therapy was relatively short (1 [0-15] years). Immunosuppression consisted of a triple therapy in the majority of patients. Nineteen patients had upper urinary tract multifocal atypia. Eleven patients presented with urothelial carcinomas of the upper tract; 2 of them with additional bladder urothelial carcinomas. Of these 2 patients, one required radical cystectomy. One patient developed a hepatocarcinoma. Patient survival was 100% in AAN patients at 5, 10, and 15 years after transplantation. Graft survival at 5, 10, and 15 years was 95%, 83%, and 75%. CONCLUSIONS: Despite a high prevalence of urothelial carcinoma and the risk of bladder carcinoma, the long-term patient and kidney graft survival is excellent in patients with AAN, provided that prophylactic bilateral ureteronephrectomy and lifelong surveillance of the bladder are performed.

Dedifferentiation and aberrations of the endolysosomal compartment characterize the early stage of nephropathic cystinosis.

Nephropathic cystinosis, a lysosomal storage disease caused by mutations in the CTNS gene encoding the lysosomal cystine transporter cystinosin, is characterized by generalized proximal tubule (PT) dysfunction that progresses, if untreated, to end-stage renal disease. The pathogenesis of defective PT cellular transport in nephropathic cystinosis remains unclear. We characterized a recently generated line of C57BL/6 Ctns mice and analyzed endocytic uptake, lysosome function, and dedifferentiation and proliferation markers using primary cultures of PT epithelial cells derived from Ctns(-/-) and Ctns(+/+) littermates. Metabolic studies revealed that Ctns(-/-) mice show a progressive PT dysfunction characterized by low-molecular-weight (LMW) proteinuria, glucosuria and phosphaturia, before structural damage and in the absence of renal failure. These changes are related to decreased expression of the multi-ligand receptors megalin and cubilin and to increased dedifferentiation (ZONAB transcription factor) and proliferation (PCNA and Cyclin D1) rates. Studies on PT cells derived from Ctns(-/-) kidneys confirmed cystine overload, with accumulation of enlarged, dysfunctional lysosomes and reduced expression of endocytic receptors reflected by decreased uptake of specific ligands. These changes were related to a loss of integrity of tight junctions with a nuclear translocation of ZONAB and increased proliferation, as observed in Ctns(-/-) kidneys. These data reveal that the absence of cystinosin in PT cells triggers aberrations of the endolysosomal compartment, transport defects and an abnormal transcription program in the early stage of nephropathic cystinosis. Insights into the early manifestations of cystinosis may offer new targets for intervention, before irreversible renal damage.

Decreased renal accumulation of aminoglycoside reflects defective receptor-mediated endocytosis in cystic fibrosis and Dent's disease.

The clinical use of aminoglycoside (AG) antibiotics is limited by their renal toxicity, which is caused by drug accumulation in proximal tubule (PT) cells. Clinical studies reported that renal clearance of AG is enhanced in cystic fibrosis (CF) patients, which might reflect the role of CFTR in PT cell endocytosis. In order to assess the role of chloride transporters on the renal handling of AG, we investigated gentamicin uptake and renal accumulation in mice lacking functional CFTR (Cftr ( ∆F/∆F)) or knock-out for the Cl(-)/H(+) exchanger ClC-5 (Clcn5 ( Y/- )). The latter represent a paradigm of PT dysfunction and defective receptor-mediated endocytosis. As compared with controls, Cftr ( ∆F/∆F) and Clcn5 ( Y/- ) mice showed a 15% to 85% decrease in gentamicin accumulation in the kidney, respectively, in absence of renal failure. Studies on primary cultures of Cftr ( ∆F/∆F) and Clcn5 ( Y/- ) mouse PT cells confirmed the reduction in gentamicin uptake, although colocalization with endosomes and lysosomes was maintained. Quantification of endocytosis in PT cells revealed that gentamicin, similar to albumin, preferentially binds to megalin. The functional loss of ClC-5 or CFTR was reflected by a decrease of the endocytic uptake of gentamicin, with a more pronounced effect in cells lacking ClC-5. These results support the concept that CFTR, as well as ClC-5, plays a relevant role in PT cell endocytosis. They also demonstrate that the functional loss of these two chloride transporters is associated with impaired uptake of AG in PT cells, reflected by a decreased renal accumulation of the drug.

Blood pressure load, proteinuria and renal function in pre-hypertensive children.

It is as yet unclear whether blood pressure load (BPL) can affect renal function in pre-hypertensive children. We have studied 250 children, with a mean age of 9.12 +/- 3.28 years, with the aim of assessing if pre-hypertension in children can indeed affect renal function. The study cohort consisted of 146 children with pre-hypertension (group P) and a control group of 104 children with normal blood pressure (group C). All children were tested for orthostatic proteinuria, an exclusion criterion, glomerular filtration rate (GFR), and proteinuria, and ambulatory blood pressure monitoring was performed. Based on the BPL, group P was further subdivided into group P1 (BPL 40%, high BPL). We found that GFR was reduced in pre-hypertensive children (90.74 +/- 48.69 vs. 110.32 +/- 20.30 ml/min per 1.73 m(2), p < 0.0001) and that proteinuria was increased (145.36 +/- 110.91 vs. 66.84 +/- 42.94 mg/m(2) per 24 h; p < 0.0001). However, mean values were still within normal limits. A comparison of the group with high BPL and that with low BPL revealed that the former had relatively reduced GFR (79.15 +/- 42.04 vs. 96.78 +/- 51.20 ml/min per 1.73 m(2); p < 0.006) and increased proteinuria (198.29 +/- 142.17 vs. 118.31 +/- 80.07 mg/m(2) per 24 h; p < 0.036). In comparison to the reference values of the normal population, the GFR was reduced and proteinuria was increased in the group with high BPL. Based on our results, pre-hypertension in children with high BPL seems to be associated with reduced GFR and increased proteinuria. A reasonable doubt remains that the patients with higher proteinuria and larger reduction of GFR may harbor an as yet unknown subclinical renal condition responsible for the onset of pre-hypertension. Therefore, children with even mildly elevated BP are at risk of developing renal damage and should change their lifestyle to prevent further increases in BP.

Relationship between global end-diastolic volume and cardiac output in critically ill infants and children.

OBJECTIVE: The objective of this study was to investigate possible correlations between the preload index global end-diastolic volume (GEDV) and the indexes of cardiac function, cardiac index, and stroke volume index in critically ill pediatric patients. The aim was to evaluate whether GEDV may help in the decision-making process concerning volume loading. DESIGN: Prospective clinical study. SETTING: Pediatric intensive care unit of the Bambino Gesù Children's Research Hospital. PATIENTS: Seventy patients, 40 male and 30 female, mean age 62 +/- 41 months (range 5-156 months), divided into six groups: group A, hemorrhagic shock, ten cases; group B, head injury, 21 cases; group C, septic shock, ten cases; group D, encephalitis, ten cases; group E, respiratory failure, nine cases; group F, cardiogenic shock, ten cases. INTERVENTIONS: All patients received volumetric hemodynamic monitoring following initial resuscitation and every 4 hrs thereafter or whenever a hemodynamic deterioration was suspected. During the cumulative in-hospital stay, a total 1,184 sets of measurements were done. MEASUREMENTS AND MAIN RESULTS: Findings are consistent with a statistically significant linear correlation of GEDV with cardiac index and stroke volume index in hemorrhagic shock (group A) (R2 = .647, p < .0001; R2 = .738, p < .0001) and cardiogenic shock (group F) (R2 = .645, p < .0001; R2 = .841, p < .0001). CONCLUSIONS: GEDV may potentially be a useful guide to treatment in preload-dependent conditions, such as hemorrhagic and cardiogenic shock. In the other groups where there is little relationship between preload and cardiac function indexes, the influence of non-preload-dependent mechanisms on cardiac output is certainly more significant.

Influence of 17q gain and promoter polymorphisms on mRNA expression of somatostatin receptor type 2 in neuroblastoma.

BACKGROUND: Neuroblastoma, the most frequent solid extracranial tumor in children, is characterized by a wide spectrum of clinical behaviours. We previously reported that high expression of somatostatin receptor type-2 (sst2) mRNA is associated to increased overall and event free survival. Several genetic abnormalities are detected in neuroblastomas, frequently involving balanced and/or unbalanced gain on the long arm on chromosome 17, the same region containing sst2 gene. METHODS: In this study we detected balanced and/or unbalanced 17q gain in 50 neuroblastomas. Since two polymorphisms in sst2 promoter (-57 C>G and -83 A>G) were previously described as responsible for an in vitro reduction of sst2 mRNA expression, promoter sequencing was also performed in the same samples. The results were compared to sst2 mRNA expression, measured by real-time RT-PCR. RESULTS: The frequency of 17q gain (14/50 neuroblastomas) was significantly associated to sst2 mRNA over-expression (Fischer's exact test: p=0.0012). The sst2 expression was significant higher both in balance and unbalance 17q amplifications (ANOVA: p=0.04). Conversely, we found a reduction of sst2 mRNA in neuroblastomas with -57 C>G promoter polymorphism (ANOVA: p=0.03). CONCLUSION: We highlighted that 17q gain and promoter polymorphisms can play a role into the regulation of sst2 expression in neuroblastomas.

EQUAL-qual: a European program for external quality assessment of genomic DNA extraction and PCR amplification.

BACKGROUND: Despite the rapid transition into routine clinical practice of molecular techniques based on PCR, external quality assessment (EQA) is still not widely available. The European Union and European Communities Confederation of Clinical Chemistry have supported the EQUAL project as a series of 3 different EQA programs for the assessment of molecular methods independently from analytes. We present the results from the EQUAL-qual program designed to evaluate the analytical aspects of DNA analysis by means of a conventional qualitative PCR experiment. METHODS: The EQUAL-qual program provided DNA, blood samples, and primer sets to participant laboratories to assess DNA extraction and PCR amplification. We have developed statistical procedures to identify laboratories performing poorly in DNA extraction (quality and quantity), PCR efficiency, and data interpretation after electrophoresis. RESULTS: An application to participate was obtained from 213 laboratories (from 25 countries), and 175 (82%) of laboratories submitted results for assessment. Questionable results in terms of quality and/or quantity of DNA derived from blood extractions were returned by 27% of laboratories (46 of 166). PCR efficiency showed high variability, with 3% of laboratories (5 of 163) showing a consistently low rate of amplification and 10% (18 of 175) not reporting the expected number of bands of the amplified targets. CONCLUSIONS: The results showed considerable variability in all phases of the experiment. The approach confirms the validity of EQA as a method for evaluating analytical aspects of PCR-based tests.

Renal transplant in methylmalonic acidemia: could it be the best option? Report on a case at 10 years and review of the literature.

Methylmalonic acidemia (MMA) is an inborn error of organic acid metabolism. Patients with severe disease develop many complications despite treatment; often, the disease progresses to severe damage of the central nervous system or to end-stage renal disease (ESRD). When medical treatment is ineffective, liver, kidney, or combined liver and kidney transplantation is advocated. At present, there are no definite guidelines as for the organ to be transplanted, and results are inconsistent. We report on a 27-year-old woman with MMA MUT0. The clinical symptoms developed at age 4 months. She progressed to ESRD and received a kidney transplant in November 1996 at age 17 years. One hundred and twenty months after transplant, renal function is normal; although urinary levels of methylmalonic acid are above normal limits, no episodes of metabolic decompensation have been observed after transplantation. Although liver is the major site of methylmalonyl-CoA mutase activity, this case and similar ones in the literature suggest that the smaller mutase activity present in the transplanted kidney may be sufficient to ensure partial correction of the metabolism of organic acids sufficient to prevent the onset of episodes of metabolic decompensation. It is worth investigating whether kidney transplant can be a safer and more satisfactory alternative to liver transplantation in cases of MMA unresponsive to medical treatment although urine MMA excretion remains significantly elevated.

Renal and cardiovascular effects of angiotensin-converting enzyme inhibitor plus angiotensin II receptor antagonist therapy in children with proteinuria.

OBJECTIVE: We investigated whether the combination of an angiotensin-converting enzyme inhibitor and an angiotensin II type 1 receptor antagonist offers better control of proteinuria and cardiovascular parameters without causing adverse side effects. METHODS: We enrolled 10 children (mean age: 12.3 +/- 4.06 years) with proteinuria resulting from chronic renal diseases of various causes. The study consisted of 2 phases, 3 months each, for an overall 6-month observation time. During phase 1 (3 months), each child was assigned randomly to treatment with either an angiotensin-converting enzyme inhibitor or an angiotensin II type 1 receptor antagonist alone. During phase 2, each child was advanced to combination therapy with the addition of an angiotensin II type 1 receptor antagonist or an angiotensin-converting enzyme inhibitor, respectively. Renal function tests, echocardiography, and 24-hour ambulatory blood pressure monitoring were performed at the beginning of the study (time 0), at 3 months (time 1), and at 6 months (time 2). RESULTS: At time 2, proteinuria (change: -80.21 +/- 10.75%), interventricular septum index (change: -13.63 +/- 18.64%), posterior wall of the left ventricle index (change: -30.71 +/- 20.32%), and left ventricular mass index (change: -28.33 +/- 24.44%) were reduced significantly, compared with time 0 and time 1. No untoward side effects were detected during the study. CONCLUSIONS: In the short term, the combination of angiotensin-converting enzyme inhibitors and angiotensin II type 1 receptor antagonists for children with proteinuria of renal origin reduced proteinuria significantly, compared with baseline or either drug alone. Furthermore, echocardiographic studies gave evidence of reduction of left ventricular hypertrophy. Additional studies are needed to evaluate long-term results.

Bivariate statistical approach to evaluate laboratory performance by analysis of standard curves in an External Quality Assurance program for quantitative assays based on real-time PCR with Taq-Man probes.

Recently a revolutionary technique for quantitative PCR determination was introduced in diagnostic laboratories. To determine the influence of technical variability on the reliability of the quantitative assay, it is crucial to use External Quality Assurance (EQA) programs. An EQA program was developed in Italy to check the analytical performance of real-time PCR procedures based on Taq-Mantrade mark probes. This article suggests a new statistical approach to discriminate, using a bivariate technique, laboratory performance that appears to be questionable, by separately considering the two main features of the standard curve: analytical sensitivity and efficiency. Furthermore, specific indexes to evaluate the impact of these two features on the determination of the initial number of molecules are given to help to improve the assay procedure.

Early evidence of lymphoproliferative disorder: post-transplant monitoring of Epstein-Barr infection in adult and pediatric patients.

Transplant patients are at high risk of post-transplant lymphoproliferative disorder (PTLD). A strong correlation between Epstein-Barr virus (EBV) and PTLD is observed in pediatric patients with primary infection after transplant. Because many patients have responded to reversal of immunosuppressive therapy, an early identification of EBV is essential for the reduction of immunosuppression and/or introduction of antiviral therapy to prevent PTLD. Polymerase chain reaction (PCR) is a specific and sensitive method to identify EBV DNA in blood. The aim of our study was to establish a protocol for monitoring EBV infection in transplanted patients for early identification those at high risk of PTLD. Viral presence in peripheral blood leukocytes (PBL) and serum samples was revealed by Nested PCR; positive specimens were quantified with Real Time PCR (RT-PCR). DNA in PBL was observed in 12 cases and 6 showed EBV in sera. Quantitative analysis showed a wide range of EBV DNA copies in leukocytes that were higher than in sera. Two patients displayed high viral load values in both PBL and sera associated with clinical evidence of PTLD. Our data suggest that the study of the EBV load represents an essential approach in the diagnosis of PTLD and the analysis of serum samples could provide useful information in the post-transplant monitoring of high-risk patients.

Prognostic value of somatostatin receptor subtype 2 expression in colorectal cancer.

The clinical relevance of the somatostatin receptor subtype 2 (sst2) is well defined in neuroendocrine tumors but it is still a matter of debate whether its expression may have a role also in other tumors not arising from the neuroectoderm. We investigated the prognostic value of the expression levels of sst2 mRNA in a consistent group of patients affected by colorectal cancer. Survival analysis of cancer-related death showed that patients with a high sst2 mRNA expression had an unfavourable outcome (p=0.037) and a significantly shorter disease-free survival (p=0.008). Surprisingly, our findings suggest that sst2 gene overexpression is a feature of colorectal tumors that have a negative outlook; in addition, it may allow additional insight into conventional therapeutic approaches for more aggressive tumors, whose prognosis needs to be improved.

An Italian program of external quality control for quantitative assays based on real-time PCR with Taq-Man probes.

Quantitative real-time PCR techniques are increasingly being used for the measurement of nucleic acids in research applications as well as in the clinical laboratory. It is therefore important that external quality control programs (EQA) are implemented for the evaluation of the analytical aspects common to molecular tests based on quantitative PCR. The aim of this study was the development of an Italian program of external quality control for quantitative assays based on real-time PCR with Taq-Mantrade mark probes to compare the analytical performance of 42 laboratories. Participants were provided with a set of reagents (cDNA for reference curve preparation, primers-probe mix and three unknown samples) and requested to perform a conventional assay using the master mix employed in their laboratories. The quantitative results in unknown samples were analyzed. The results of our study showed clear heterogeneity in performance. Two of the 42 laboratories provided results indicating contamination during the experiment, whereas six did not provide values for at least one of the six standard points. Only 12 laboratories gave results that were both precise and accurate for all the samples tested. Regarding imprecision, 17 laboratories appeared to deviate in at least one result, whereas inaccuracy showed an inverse dose-dependent trend. Finally, 12 laboratories were not able to measure the sample with the lowest concentration. Ten of these laboratories were equipped with the same instruments. The results of this first round of analytical EQA of real-time PCR-based methods seem to indicate high variability among laboratories carrying out the same experimental protocol. These findings could have implications for any assay based on this type of technique. This survey demonstrates the importance of experimental EQAs of methodological proficiency testing. Our approach has proved useful for comparing the analytical aspects shared by all diagnostic laboratories applying quantitative assays for the measurement of nucleic acids based on the use of Taq-Mantrade mark probes and real-time platforms.

Tankyrase, a positive regulator of telomere elongation, is over expressed in human breast cancer.

Tankyrase promotes telomere elongation by interaction with the telomeric protein binding factor TRF1, a negative regulator of telomere extension. We measured tankyrase mRNA by real-time RT-PCR in 66 breast cancers and in paired normal tissues. Results were compared with hTERT mRNA expression. The levels of tankyrase in breast cancers were significantly higher in comparison to normal tissues (P<0.0001) and significantly related to the status of progesterone receptors. No relationship was found between tankyrase and hTERT mRNA expression in breast cancers. According to our results, tankyrase expression appeared up regulated in breast cancers.

External quality assurance program for PCR amplification of genomic DNA: an Italian experience.

BACKGROUND: External quality assurance (EQA) programs for diagnostic tests based on nucleic acid amplification have not been widely implemented in clinical laboratories and remain limited to few tests. Development of specific EQA programs based on application-based proficiency testing for any diagnostic molecular target is challenging. Development of EQA trials based on methodologic proficiency testing and directed to the evaluation of analytical aspects common to the majority of PCR-based tests may be valuable. METHODS: We developed an EQA program for evaluation of DNA extraction and amplification and analysis of products after PCR. Participants received a package containing primers and reference materials to evaluate three specific controls for, respectively, DNA extraction (quality and quantity), PCR performance (specificity and efficiency), and interpretation of results after electrophoresis. Each participant was asked to return to the organizers a form with their numerical results and an aliquot of all amplified samples for joint evaluation. RESULTS: Results varied in all phases of the experimental procedure: preamplification, amplification, and post-PCR interpretation. To give a general estimation on the quality of performances for each laboratory, we designed a score scheme in which the results of any specific action were evaluated on the basis of the distribution around the median consensus values. The maximum possible score was 84. On the basis of total score obtained by each laboratory, we created a qualitative ranking list that provided the final interpretation of results as excellent (>63 points; n = 4 laboratories), good (53-63 points; n = 13), sufficient (42-52 points; n = 15), poor (31-41 points; n = 3), and not acceptable (<31 points; n = 4). CONCLUSIONS: This survey demonstrates the importance of EQA trials based on methodologic proficiency testing directed to evaluation of analytical aspects common to the majority of PCR-based tests.