The Italian External Quality Assessment (EQA) program on urinary sediment by microscopy examination: a 20 years journey.

OBJECTIVES: In spite of the introduction of automated systems for urinary sediment analysis, microscopy examination remains the gold standard, and it is more than ever important to perform it with a good and reliable quality. External Quality Assessment (EQA) programs on urinary sediment are rare. The present paper provides an analysis of results from 2001 to date of the EQA Italian program which involves today 230 laboratories. METHODS: The program includes four surveys per year. Participants are asked the identification and clinical associations of urinary sediment particles, shown as phase contrast microscopy images in the website of the Center of Biomedical Research (CRB) (2 surveys), and the diagnosis of clinical cases presented by both images and a short clinical history (2 surveys). The results of each survey are then scored and commented. In 20 years, 298 images were presented: 90 cells (9 types), 23 lipids (5 types), 87 casts (21 types), 53 crystals (14 types), 22 microorganisms (5 types), and 23 contaminants (9 types). Moreover, 27 clinical cases, covering a wide spectrum of conditions with different degrees of complexity, were presented to participants. RESULTS: Identification: among urinary particle categories, the correct identification rate (obtained for each particle from the sum of correct + partially correct answers) was very high for micro-organisms (mean ± SD: 96.2 ± 3.5%), high for lipids (88.0 ± 11.8%) and crystals (87.0 ± 16.5%) followed, in decreasing order, by cells (82.1 ± 15.9%), casts (81.8 ± 14.8%), and contaminants (76.7 ± 22.1%). Clinical associations (n=67): the rate of correct answers was 93.5 ± 5.7% ranging from 75.0 to 100% for all but one clinical association (i.e., acute glomerulonephritis: 55.4%). Clinical cases: throughout surveys, due to the overall rate of particle misidentification, only 59.8 ± 17.1%, (range 32.5-88.7%) of participants achieved access to clinical diagnosis. Of these, 88.7 ± 10.6% (range 59.9-99.3%) were able to indicate the correct diagnosis. CONCLUSIONS: Our program can be used as a tool to improve the identification of urine particles and the knowledge of their clinical meaning and to encourage specialists of laboratory medicine to correlate urinary findings with other laboratory data and the clinical history, an aspect that improves the value of the day by day work.

[PHYSICAL, CHEMICAL AND MORPHOLOGICAL URINE EXAMINATION: RECOMMENDATIONS FOR THE POST ANALYTICAL PHASE FROM THE INTERDISCIPLINARY URINALYSIS GROUP (GIAU)].

With these recommendations the Interdisciplinary Urinalysis Group (GIAU) aims to stimulate the following aspects : improvement and standardization of the post analytical approach to physical, chemical and morphological urine examination (ECMU); emphasize the value added to ECMU by selection of clinically significant parameters, indication of analytical methods, of units of measurement, of reference values; improvement of interpretation of dip stick urinalysis with particular regard to the reconsideration of the diagnostic significance of the evaluated parameters together with an increasing awareness of the limits of sensitivity and specificity of this analytical method. Accompanied by the skills to propose and carry out in-depth investigations with analytical methods that are more sensitive and specific;increase the awareness of the importance of professional skills in the field of urinary morphology and their relationships with the clinicians. through the introduction, in the report, of descriptive and interpretative comments depending on the type of request, the complexity of the laboratory, the competence of the pathologist;implement a policy of evaluation of the analytical quality by using, in addition to traditional internal and external controls, a program for the evaluation of morphological competence. The hope is to revalue the enormous potential diagnostic of ECMU, implementing a urinalysis on personalized diagnostic needs that each patient brings with it.

[Physical, chemical and morphological urine examination guidelines for the Analytical Phase from the Intersociety Urinalysis Group]. / Esame fisico, chimico e morfologico delle urine proposta di linee guida per la fase analitica del Gruppo Intersocietario Analisi delle Urine (GIAU).

With these guidelines the Intersociety Urinalysis Group (GIAU) aims to stimulate the following aspects: Improvement and standardization of the analytical approach to physical, chemical and morphological urine examination (ECMU). Improvement of the chemical analysis of urine with particular regard to the reconsideration of the diagnostic significance of the parameters that are traditionally evaluated in dipstick analysis together with an increasing awareness of the limits of sensitivity and specificity of this analytical method. Increase the awareness of the importance of professional skills in the field of urinary morphology and the relationship with the clinicians. Implement a policy of evaluation of the analytical quality by using, in addition to traditional internal and external controls, a program for the evaluation of morphological competence. Stimulate the diagnostics industry to focus research efforts and development methodology and instrumental catering on the needs of clinical diagnosis. The hope is to revalue the enormous diagnostic potential of 'ECMU, implementing a urinalysis on personalized diagnostic needs for each patient. Emphasize the value added to ECMU by automated analyzers for the study of the morphology of the corpuscular fraction urine. The hope is to revalue the enormous potential diagnostic of 'ECMU, implementing a urinalysis on personalized diagnostic needs that each patient brings with it.

The Italian External Quality Assessment (EQA) program on urinary sediment: results of the period 2012-2015.

BACKGROUND: Manual microscopy still represents the gold standard for urinary sediment (US) examination. We report the results obtained in the period 2012-2015 by the EQA Italian program on US, which today involves about 260 laboratories. METHODS: The program includes four surveys per year. In two surveys, participants are asked to supply identification and clinical association of US particles. In two other surveys, they are asked to supply the diagnosis of clinical cases, presented with images, some key laboratory findings and a short clinical history. Sixty-six images of US particles (21 cells, 2 lipids, 21 casts, 10 crystals, 3 microorganisms, 15 contaminants) and seven clinical cases were presented. RESULTS: The correct identification rate for each category of particles, in decreasing order, was: micro-organisms (mean±SD: 92.4%±4.5%), lipids (92.0%±1.8%), casts (82.8%±8.8%), crystals (79.4%±29.8%), cells (77.3%±13.5%), and contaminants (70.9%±22.2%). For 13 particles, a correct clinical association was indicated by 91.5%±11.7% of participants, while it was 52.7% for particles associated with urinary tract infection. For clinical cases, due to a high rate of particles misidentification, only 44.3%±10.1% of participants achieved access to clinical diagnosis, which was then correctly indicated by 92.5%±5.3% of them. CONCLUSIONS: The results of the EQA program confirm that, while some US particles are well known in terms of identification, clinical association and clinical meaning, others particles still are not, and this represents an important reason to encourage EQA programs on US.

Does flow cytometry have a role in preliminary differentiation between urinary tract infections sustained by gram positive and gram negative bacteria? An Italian polycentric study.

BACKGROUND: Urine culture is the most frequently requested test for a Microbiology Lab. A reliable screening tool would be of paramount importance both to clinicians and laboratorians, provided that it could get fast and accurate negative results in order to rule-out urinary tract infection (UTI). MATERIALS AND METHODS: We evaluated 1907 consecutive urine samples from outpatients. Culture was performed on chromogenic agar with 1µL loop, using 10(5)CFU/mL as a limit of positive growth. Using Sysmex Uf-1000i analyzer we evaluated bacteria forward scatter (B_FSC) and fluorescent light scatter (B_FLH) in a preliminary discrimination step for UTI caused by Gram+ or Gram- bacteria. RESULTS: We got 512 positive samples. A mono-microbial infection was observed in 490 samples; two bacterial strains were isolated in 22 samples, so 534 bacterial strains were found: 392 Gram-, 133 Gram+ and 9 yeasts. Comparing Gram+ and Gram- bacteria we observed a statistically significant difference for B_FSC but not for B_FLH. In this application experimental cut-off value for B_FSC was 25ch. Using this cut-off to perform a presumptive identification of UTI sustained by Gram-+ bacteria, we observed a SE 0.68, SP 0.84. CONCLUSION: Our data although preliminary suggest that B_FSC could be useful in presumptive exclusion of UTI caused by Gram-positive bacteria.

Gender's equality in evaluation of urine particles: Results of a multicenter study of the Italian Urinalysis Group.

OBJECTIVE: We performed a multicenter study to calculate the upper reference limits (URL) for urine particle quantification in mid-stream samples by using automated urine analyzers. DESIGN & METHODS: Two laboratories tested 283 subjects using a Sysmex UF-100, two other laboratories tested 313 subjects using Sysmex UF-1000i, whereas two other laboratories tested 267 subjects using Iris IQ®200. RESULTS: The URLs of UF-100 in females and males were 7.8/µL and 6.7/µL for epithelial cells (EC), 11.1/µL and 9.9/µL for red blood cells (RBC), 10.2/µL and 9.7/µL for white blood cells (WBC), and 0.85/µL and 0.87/µL for cylinders (CAST). The URLs of UF-1000i in females and males were 7.6/µL and 7.1/µL for EC, 12.2/µL and 11.1/µL for RBC, 11.9/µL and 11.7/µL for WBC, and 0.88/µL and 0.86/µL for CAST. The URLs of Iris IQ®200 in females and males were 7.8/µL and 6.6/µL for EC, 12.4/µL and 10.1/µL for RBC, 10.9/µL and 9.9/µL for WBC, and 1.1/µL and 1.0/µL for CAST. CONCLUSION: The URLs obtained in this study were comparable to the lowest values previously reported in the literature. Moreover, no gender-related difference was observed, and analyzer-specific upper reference limits were very similar.

Pediatric reference values for urine particle quantification by using automated flow cytometer: results of a multicenter study of Italian urinalysis group.

OBJECTIVES: The purpose of this Italian multicenter study was to define pediatric upper reference values for urine particle quantification by using automated flow cytometry. DESIGN AND METHODS: Four hospital-based clinical laboratories participated in this multicenter investigation, which included a total study population of 161 Italian children aged from 1 to 12years. Two laboratories used Sysmex UF-100 and analyzed 86 children, whereas the other two used Sysmex UF-1000i and analyzed 75 subjects. Particle quantification included the analysis of white blood cells (WBC), red blood cells (RBC), squamous epithelial cells (EC), transitional epithelial cells (TC), casts (CAST) and bacteria (BACT). RESULTS: The upper reference values in subjects tested with the Sysmex UF-100 were 9.7WBC/µL, 10.1RBC/µL, 7.5EC/µL, 2.5TC/µL, 0.7CAST/µL and 3090BACT/µL, whereas the upper reference values in subjects tested with the Sysmex UF-1000i were 10.5WBC/µL, 8.3RBC/µL, 7.2EC/µL, 2.9TC/µL, 0.7CAST/µL and 48BACT/µL. No statistically significant differences between genders were found in the value distribution of any of the parameters tested. Similarly, no statistically significant differences were observed between the two urine analyzers, except for BACT. CONCLUSIONS: Automated analysis of urine particles appears a suitable means to optimize the workflow of routine urinalysis of children specimens. The upper reference limits for pediatric subjects obtained in this study were comparable to those previously reported in the literature, with no significant differences between genders and analyzers.

Mid-stream vs. first-voided urine collection by using automated analyzers for particle examination in healthy subjects: an Italian multicenter study.

BACKGROUND: In analogy with other areas of laboratory diagnostics, the pre-analytical phase is the leading source of variability also in urinalysis. We carried out a multicentric study for comparing results obtained from first-voided and mid-stream urine samples. METHODS: Each of the six hospital-based clinical laboratories participating to this study recruited 50 healthy subjects among laboratory staff and/or their relatives. Two consecutive samples of the first morning micturition were collected by vacuum system, the first from the first-void and the second from the mid-stream. Routine urinalysis was performed using dip-stick automated analyzers for chemical examination and automated analyzers for formed particle examination (Sysmex UF-100, Sysmex UF-1000i and Iris iQ-200). RESULTS: Counts of epithelial cells (EC), erythrocytes (ERY) and leukocytes (LEU) but not for cylinders (CAS) were significantly higher in the first-voided samples. A significantly higher count of EC, ERY and LEU was also observed between females and males in first-voided samples, whereas no significant difference could be found in mid-stream samples. Health related analyzer specific upper reference limits (URL) were CAS≤1, EC≤5, ERY≤19, Leu≤13 for UF-100; CAS≤1, EC≤4, ERY≤15, Leu≤11 for UF-1000i; CAS≤1, EC≤4, ERY≤18, Leu≤10 for iQ200. The overall prevalence of subjects with cellular elements count exceeding URL was also higher in first-voided than in mid-stream samples. CONCLUSIONS: Mid-stream urine was confirmed as the most appropriate sample, since the presence of contaminating elements, such as bacteria, analytes and formed particles are minimized.

Case-control time course study of urinary leukocyte and bacterial counts in patients with acute urinary tract infections responsive and not responsive to antibacterial therapy.

BACKGROUND: Urinary tract infections (UTIs) are usually diagnosed by urine particle detection and urine bacterial culturing, but an empiric treatment is often prescribed before the microbiological results are known. The aim of this study was to assess whether quantification of bacteriuria and leukocyturia could predict the outcome of empiric antibiotic therapy. METHODS: By using a Sysmex UF-100 analyser, we performed a case-control study in which bact channel counts (BCCs) and leukocyte (WBC) quantification were compared in urine samples obtained from responsive patients (RPs) and non-responsive patients (NRPs) at diagnosis (t0) and 24h (t24) after the start of empiric antibacterial treatment. RESULTS: BCCs decreased significantly from t0 (median: 30,000 E6/L) to t24 (median: 700 E6/L; p<0.001) in RPs but not in NRPs (median at t0 31,000 E6/L, and 23,000 E6/L at t24; p>0.05). Similarly, WBC counts were reduced in RPs at t24 relative to the initial values (p<0.001) but not in NRPs. A reduction of 70% in the BCC count or 60% in the WBC count of paired samples was useful for discriminating between RPs and NRPs. CONCLUSIONS: Our results suggest that quantitative time course analysis of urine BCCs and WBCs can be used to predict the success of first-line empiric treatment for UTIs.

Urine particle evaluation: a comparison between the UF-1000i and quantitative microscopy.

BACKGROUND: The study of urine particles plays a key role in the diagnosis of kidney diseases. In this study, the authors evaluated the correlation between the UF-1000i and quantitative manual microscopy. METHODS: A total of 214 untreated urine samples were studied using the Sysmex UF-1000i and compared with results obtained from quantitative manual microscopy using the Fuchs-Rosenthal counting chamber. RESULTS: Using Pearson statistics, we observed satisfactory correlation between the UF-1000i and quantitative microscopy: for red blood cells (RBCs) r was 0.98, for white blood cells (WBCs) r was 1.00, for epithelial cells (EC) r was 0.96, and for casts r was 0.69. Using linear regression statistics, we also observed satisfactory correlation between the UF-1000i and quantitative microscopy: for RBCs R(2) was 0.95, for WBCs R(2) was 0.99, for EC R(2) was 0.92, and for casts R(2) was 0.48. CONCLUSIONS: In our experience, automated urine particle analysis performed using the Sysmex UF-1000i analyzer is sufficiently precise and improves the workflow in a routine laboratory. Precision was satisfactory and concordance with the reference method is good for RBC, WBC and EC; for casts microscopic observation is required for flagged samples to discriminate hyaline from pathologic casts.

Cutoff values for bacteria and leukocytes for urine flow cytometer Sysmex UF-1000i in urinary tract infections.

Because urinary tract infections (UTIs) are a quite common disease, the gold standard for diagnosing UTIs is still bacterial culture, although a large percentage of samples are negative: unnecessary cultures can be reduced by means of an effective screening test. The analytic performance of a new urine cytometer, the UF-1000i, has been tested on 1463 urine samples submitted to our laboratory for culture. Bacteria and leukocyte counts have been compared by means of the UF-1000i with colony-forming unit (CFU) quantification on citrate lactose electrolytes deficient agar to assess the best cutoff values. By using quantitative cultures and considering as positive a sample with 10 x 10(5) CFU/mL, 546 positive samples (37%) were observed. If compared with 10 x 10(5) CFU/mL, the cutoff values obtained were 125 bacteria/microL and 40 leukocytes/ microL, respectively. Analytic parameters such as sensitivity, specificity, positive predictive value, negative predictive value, and correctly classified incidence were satisfactory. Based on the results obtained in this study, when using the UF-1000i analyzer for a screening test for UTI, a cutoff value of 40 white blood cells/microL should be adopted. The cutoff value for bacteria should be 125/microL for those clinical conditions in which 10 x 10(5) CFU/mL indicates a positivity.

Performance of automated platelet quantification using different analysers in comparison with an immunological reference method in thrombocytopenic patients.

BACKGROUND: Rapidly available and accurate platelet counts play an important role in the evaluation of haemorrhagic status and in assessing the need for platelet transfusions. We, therefore, evaluated platelet counting performance of haematology analysers using optical, impedance and immunological methods in thrombocytopenic patients. MATERIALS AND METHODS: We considered 99 patients with a platelet (plt) count under 50 x 10(9) plt/L. We compared the platelet counts obtained using ADVIA 2120 (optical method), Cell-Dyn Sapphire (optical, impedance and immunological methods with CD61) and a reference, double staining (CD41+CD61) immunological method. RESULTS: The platelet counts of all the considered methods showed good correlation with those of the reference method, despite an overestimation in platelet quantification. The degree of inaccuracy was greater for platelet counts under 20 x10(9) plt/L. CONCLUSIONS: Clinicians who use platelet thresholds below 20 x10(9) plt/L for making clinical decisions must be aware of the limitations in precision and accuracy of cell counters at this level of platelet count. Inaccurate counts of low platelet numbers could create problems if attempts are made to reduce the threshold below 20 x 10(9) plt/L.

[Thrombophilia risk factors evaluation in a group of Italian patients with deep venous thrombosis]. / Valutazione del rischio trombofilico in una coorte di pazienti con trombosi venosa profonda.

UNLABELLED: Venous thrombosis usually results from coexistence of multiple genetic and acquired risk factors with a trigger condition. In this study the authors report their experience in a cohort of Italian patients with previous venous thrombosis. MATERIAL AND METHODS: We considered 292 consecutive patients. Each patient was studied by using a panel of functional and genetic tests to detect some of the most relevant thrombophilia risk factors. RESULTS: The single most frequent thrombophilia risk factor was activated C protein resistance due to FV Leiden. Tests for anti phospholipids auto antibodies showed reactivity in 62 subjects. CONCLUSION: The great majority (80%) of patients showed almost one thrombophilia risk factor. Presence of multiple risk factors was demonstrated in 128 (44%) patients.

Immunoassay of anti-thyroid autoantibodies: high analytical variability in second generation methods.

The use of highly sensitive immunometric methods in clinical laboratories to assay anti-thyroid antibodies has progressively expanded in recent years but it is not known whether the new techniques have improved the analytical variability connected with the preceding methodologies. The Italian Society of Laboratory Medicine Study Group on Autoimmune Diseases conducted a collaborative study with the biomedical industry to evaluate the degree of standardization of the new analytical procedures. Twelve companies agreed to participate in the study on the search for anti-thyroglobulin (anti-Tg) and anti-thyroperoxidase (anti-TPO) antibodies in nine sera from patients with autoimmune thyroiditis, and in six sera from patients with non-autoimmune thyroid disease; ten immunometric and three immunofluorescence methods were employed. Agreement of qualitative results was close to 90% for anti-Tg and 97% for anti-TPO, with no important differences between the methods; variability of the quantitative results, expressed as CV% of absolute (in lU/ml) and relative (in cut-off concentration multiples) values was 93.9% and 102.3%, respectively, for anti-Tg, and 75.5% and 62.9%, respectively, for anti-TPO. These findings show that despite the progressive improvement in the analytical techniques, the variability between methods for the assay of anti-Tg and anti-TPO is still unexpectedly high, and probably due to several factors such as uncertainty in defining the positive cutoff concentration, absence of adequate international reference preparations, modality of autoantigen purification, and analytical variability in the assay procedures.

Guidelines for the laboratory use of autoantibody tests in the diagnosis and monitoring of autoimmune rheumatic diseases.

The Italian Society of Laboratory Medicine Study Group on the Diagnosis of Autoimmune Diseases has generated a series of guidelines for the laboratory diagnosis and monitoring of systemic autoimmune rheumatic diseases intendedfor the use of clinical pathologists and laboratory physicians. These guidelines are based on a systematic review of published works and expert panel discussion and consist of 13 recommendations for antinuclear antibodies, anti-double-stranded native DNA, and antinuclear specific antibodies. To improve analytic performances and help select the most appropriate test for specific autoantibodies, as well as provide education and guidance in the use of these tests, special emphasis is placed on laboratory methods.