Hemoglobin A2 and Heterogeneous Diagnostic Relevance Observed in Eight New Variants of the Delta Globin Gene.

BACKGROUND: Hemoglobin A (Hb A) (α2ß2) in the normal adult subject constitutes 96-98% of hemoglobin, and Hb F is normally less than 1%, while for hemoglobin A2 (Hb A2) (α2δ2), the normal reference values are between 2.0 and 3.3%. It is important to evaluate the presence of possible delta gene mutations in a population at high risk for globin gene defects in order to correctly diagnose the ß-thalassemia carrier. METHODS: The most used methods for the quantification of Hb A2 are based on automated high performance liquid chromatography (HPLC) or capillary electrophoresis (CE). In particular Hb analyses were performed by HPLC on three dedicated devices. DNA analyses were performed according to local standard protocols. RESULTS: Here, we described eight new δ-globin gene variants discovered and characterized in some laboratories in Northern Italy in recent years. These new variants were added to the many already known Hb A2 variants that were found with an estimated frequency of about 1-2% during the screening tests in our laboratories. CONCLUSIONS: The knowledge recognition of the delta variant on Hb analysis and accurate molecular characterization is crucial to provide an accurate definitive thalassemia diagnosis, particularly in young subjects who would like to ask for a prenatal diagnosis or preimplantation genetic diagnosis.

[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.

The Sleeping Remnant. Effect of Roux-En-Y Gastric Bypass on Plasma Levels of Gastric Biomarkers in Morbidly Obese Women: A Prospective Longitudinal Study.

BACKGROUND: Morpho-functional modifications of the gastric remnant after Roux-en-Y gastric bypass (RYGB) have not been completely defined, due to its inaccessibility for bioptic mapping. The aim of the study is to evaluate such modifications using Gastropanel®, a non-invasive blood test cross-checking four gastric biomarkers, able to provide a snapshot of mucosa conditions. SUBJECTS AND METHODS: Twenty-four women undergoing RYGB were prospectively enrolled. Gastropanel® parameters (pepsinogens, Gastrin-17 and immunoglobulins against Helicobacter pylori), biometrical/clinical data were collected preoperatively and at 6-months follow-up. RESULTS: All parameters showed significant reduction (p < 0.05). Pepsinogen I reduction correlated with BMI percent decrease. CONCLUSIONS: The exclusion of food transit is responsible for significant drop in gastric output, hardly representing a risk factor in the remnant carcinogenesis, being unexposed to alimentary carcinogenic agents.

[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.

Cylindruria.

The morphological analysis of urine sediment is an essential part of urinalysis and casts are important elements of urinary sediment. Their shape is typically cylindrical, with extremities often rounded. Casts form within the renal tubules and are made of Tamm-Horsfall glycoprotein (THG). Under some physiological or pathological conditions THG fibrils aggregate giving rise to casts, whose formation is favoured by a number of factors including high urine osmolality and/or low pH. Casts can be found in normal subjects, in non-renal conditions, such as fever, dehydration, and acute heart failure as well as in virtually all renal diseases. Casts can be classified on the basis of their morphology as hyaline, granular, waxy, fatty, cellular (leukocytic, erythrocytic, epithelial), containing crystals or microorganisms, pigmented and mixed. As the correct identification of casts is crucial for an accurate and timely diagnosis of renal disorders, laboratory professionals should be trained to identify and classify casts properly.

The role of neutrophil gelatinase-associated lipocalin (NGAL) in cerebrospinal fluids for screening of acute bacterial meningitis.

BACKGROUND: Acute bacterial meningitis is a rare but extremely severe disease. The aim of this study was to investigate whether neutrophil gelatinase-associated lipocalin (NGAL) is present and measurable in cerebrospinal fluid (CSF) and if its assessment may be useful for identifying patients with bacterial meningitis. METHODS: Eligible specimens were all consecutive CSFs of patients with suspect acute bacterial meningitis that were referred from the Unit of Infectious Diseases for routine chemical and morphological analysis over a three months period. CSF measurements consisted in NGAL, glucose, and total protein concentrations, along with cell count and differential. RESULTS: Eighty eight CSFs were received throughout the study period, 58 (66%) with CSF findings compatible with bacterial meningitis. The values of white blood cells (WBC), polymorphonuclear (PMN) and mononuclear (MONO) leukocytes, red blood cells (RBC), total proteins, and NGAL were significantly increased in positive CSFs, whereas that of glucose did not significantly differ. A significant correlation was found between CSF concentration of NGAL and CSF values of PMN, WBC, RBC and total proteins, but not with that of glucose and MONO. The concentration of NGAL in CSF showed an area under the curve (AUC) of 0.94 for identifying positive CSFs, with specificity and sensitivity of 1.00 and 0.741 at a diagnostic threshold of 13 ng/mL. CONCLUSIONS: NGAL is present in CSF of patients with bacterial meningitis and its measurement may be helpful for identifying positive CSFs.

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.

Evaluation of the fully automated hematological analyzer Sysmex XE-5000 for flow cytometric analysis of peritoneal fluid.

Although microscopy still represents the gold standard for cytometric analysis of peritoneal fluids, automated flow cytometry may improve throughput and accuracy. We evaluated the performance of total nucleated cell (TNC), white blood cell (WBC), polymorphonuclear cell (PMN), and mononuclear cell (MONO) counts of Sysmex XE-5000 on peritoneal fluids. The imprecision was excellent, being always lower than 11%, whereas linearity studies yielded correlation coefficients of 1.00 for all parameters. The carryover was always lower than 0.2%. The comparison between XE-5000 and microscopic analysis of 117 ascitic fluids yielded correlation coefficients always greater than 0.96, with mean biases <11/µL. The diagnostic accuracy versus manual microscopy was greater than that of XE-2100, especially at thresholds for septic ascites (100 versus 98% for ≥500 WBC/µL; 98 versus 93% for ≥250 PMN/µL). The correlation with manual microscopy for macrophages and mesothelial cell count was also higher for XE-5000 than for XE-2100 (0.63 versus 0.55). The results of this evaluation show optimal performance of XE-5000 for routine analysis of ascitic fluids, which are combined with the advantages of automated analysis such as high throughout, shortened turnaround time, no need of sample preparation and trained staff, reduced sample volume, and less likelihood of transcriptional errors.

Assessment of neutrophil gelatinase-associated lipocalin and lactate dehydrogenase in peritoneal fluids for the screening of bacterial peritonitis.

BACKGROUND: Manual microscopy remains the gold standard for enumeration and classification of nucleated cells in peritoneal fluids, especially for diagnosing bacterial peritonitis. However, this approach carries several drawbacks, so that the use of simple and automated tests may be a viable option for initial screening of peritoneal fluids. MATERIALS AND METHODS: Neutrophil gelatinase-associated lipocalin (NGAL), lactate dehydrogenase (LDH), proteins and glucose were assessed in peritoneal fluids from patients with new onset nonmalignant ascites, along with nucleated cell count and differentiation. RESULTS: One hundred and eleven specimens were analyzed, 26 of which (23%) with polymorphonuclear leukocyte (PMN) count≥250/µL, thus compatible with bacterial peritonitis. The median concentration of LDH and NGAL was 3.4 and 3.7-fold higher in samples with ≥250 PMN/µL. The concentration of proteins was also higher in samples with ≥250 PMN/µL, whereas that of glucose was lower. The PMN count significantly correlated with peritoneal fluid values of LDH (r=0.859), NGAL (r=0.774) and proteins (r=0.268), but not with glucose (r=-0.069). The area under ROC curve was 0.88 for LDH, 0.89 for NGAL and 0.94 for their combination (both tests positive), whereas that of proteins and glucose was 0.80 and 0.71, respectively. Sensitivities and specificities were 0.81 and 0.87 for LDH≥227 U/L, 0.96 and 0.75 for NGAL≥120 ng/mL, 0.77 and 0.95 for their combination. The agreement with PMN count was 0.86 for LDH, 0.80 for NGAL, and 0.91 for their combination. CONCLUSIONS: These results suggest that assessment of NGAL in peritoneal fluids, especially in combination with LDH, may be a reliable approach for screening of bacterial peritonitis in patients with new onset nonmalignant ascites.

Evaluation of white blood cell count in peritoneal fluid with five different hemocytometers.

OBJECTIVES: Evaluation of automated flow cytometric analysis of white blood cell (WBC) count in peritoneal fluids. METHODS: One hundred peritoneal fluids were analyzed with manual microscopy, Sysmex XE-2100 and XE-5000, Siemens Advia 2120, Mindray BC-6800, Abbott Sapphire. RESULTS: High correlations (0.978 to 0.999) and modes biases (-132 to 80 WBC/mm(3)) were found. Agreement at septic peritonitis cutoff ranged between 96% and 99%. CONCLUSIONS: These hemocytometers display acceptable performance for WBC screening in peritoneal fluids.

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.