Marco Caputo

Recommendations for detection and management of unsuitable samples in clinical laboratories

Abstract A large body of evidence attests that quality programs developed around the analytical phase of the total testing process would only produce limited improvements, since the large majority of errors encountered in clinical laboratories still prevails within extra-analytical areas of testing, especially in manually intensive preanalytical processes. Most preanalytical errors result from system flaws and insufficient audit of the operators involved in specimen collection and handling responsibilities, leading to an unacceptable number of unsuitable specimens due to misidentification, in vitro hemolysis, clotting, inappropriate volume, wrong container or contamination from infusive routes. Detection and management of unsuitable samples are necessary to overcome this variability. The present document, issued by the Italian Inter-society SIBioC-SIMeL-CISMEL (Society of Clinical Biochemistry and Clinical Molecular Biology-Italian Society of Laboratory Medicine-Italian Committee for Standardization of Hematological and Laboratory Methods) Study Group on Extra-analytical Variability, reviews the major causes of unsuitable specimens in clinical laboratories, providing consensus recommendations for detection and management. Clin Chem Lab Med 2007;45:728–36.

Assessment of beta-cell function during the oral glucose tolerance test by a minimal model of insulin secretion

Objective To characterise the performance of β‐cell during a standard oral glucose tolerance test (OGTT).

Measurement of Urine Relative Density Using Refractometer and Reagent Strips

Abstract The relative density of urine is the ratio of its density to that of water and depends on both the number and weight of solute particles in the sample, while osmolality depends only on the number of solute particles. Water metabolism is regulated by the interaction of the renal medullary countercurrent system with the circulating levels of antidiuretic hormone and thirst. The concentration of solids in urine can be measured by weighing, hydrometry, oscillations of a capillary tube, refractometry and reagent strip. These techniques, interrelated but not identical, are commonly used in hospital laboratories and in clinical wards. We compared the results obtained in 1725 urine samples of inpatients and outpatients using an automated refractometer to those obtained using two visually read dip stick tests. The correlation coefficients (Super Aution analyser vs. Aution Sticks 10 EA, Aution Sticks 10 EA vs. N-Multistix, Super Aution analyser vs. N-Multisticks were 0.663, 0.645 and 0.514, respectively) and the great dispersion of mountain plots demonstrates that different techniques are not interchangeable in the measurement of relative density. Since the results obtained after discarding the samples with pH higher than 7 and those containing glucose or protein were very similar to the ones reported above, the role of these interferents appears negligible in inducing the discrepancy.

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.

PATHFAST NT-proBNP (N-terminal-pro B type natriuretic peptide): a multicenter evaluation of a new point-of-care assay.

Abstract Background: The biochemical determination of cardiac natriuretic peptides, primarily brain natriuretic peptide (BNP) and the amino-terminal fragment of its pro-hormone proBNP (NT-proBNP), are reliable tools for diagnosing cardiac disease, establishing prognosis and evaluating the effectiveness of treatment. These biomarkers have proven to be of particular value in the management of chronic and acute heart failure patients, and in the outpatient and the emergency setting. Methods: A multicenter evaluation was performed to assess the practicability, and the analytical and clinical performance of a new point-of-care testing (POCT) PATHFAST™ NT-proBNP assay. This is an immunochemiluminescent assay using two polyclonal antibodies in a sandwich test format, and performed with a PATHFAST™ automated analyzer. Results: The limit of detection (mean+3 SD of the signal of 20 replicates of the zero calibrator obtained in one run) was 0.535 ng/L. An imprecision study, performed in accordance with the CLSI protocol, showed coefficients of variation of 4.0%–6.4% (within-run imprecision), 0.0%–3.4% (between-run imprecision), 5.5%–7.2% (between-day imprecision), 7.6%–8.9% (total imprecision). The method was linear to 28,755 ng/L. Slopes and intercepts ranged from 0.89 to 0.90 and from 10.96 to 22.85, respectively when lithium-heparin plasma samples (n=100) were used to compare the assay under evaluation with the routine laboratory methods (Dimension RxL®, Stratus® CS). When testing matched samples (n=52), a significant difference was found between the 50th percentile NT-proBNP concentration in K2EDTA whole blood, K2EDTA plasma, lithium-heparin plasma and serum. No significant interference was observed for NT-proBNP in lipemic (tryglicerides up to 28.54 mmol/L), icteric (total and conjugated bilirubin up to 513 and 13 μmol/L, respectively) or hemolyzed (hemoglobin up to 13.50 g/L) samples. The NT-proBNP concentration in a group of 180 healthy donors was significantly influenced by age and gender. In a selected population of patients (n=56) with acute dyspnea admitted to the emergency department, a marked reduction in cardiac natriuretic peptide concentrations was observed in hospitalized patients suffering from heart failure who had a better prognosis compared with those with a poorer prognosis (NT-proBNP mean Δ change, % from –22 to –71 vs. +9 to –11). Conclusions: The satisfactory analytical and clinical performance of the PATHFAST™ NT-proBNP assay, together with its excellent practicability, suggests that it would be a reliable tool in clinical practice, in the emergency setting for point-of-care testing, as well as in the central laboratory. Clin Chem Lab Med 2010;48:1029–34.

Evaluation of analytical performance of the Pathfast® cardiac troponin I

Abstract Background: Cardiac troponins are considered the cornestore for risk stratification and diagnosis of patients whit acute coronary syndrome (ACS). Following Clinical Laboratory Standards Institute (CLSI) guidelines, we assessed the analytical performances of the Pathfast® (Mitsubishi, Japan) cTnI method. Methods: We evaluated different sample types. Control materials and lithium heparin plasma pools were used to determine: limit of blank (LoB), limit of detection (LoD), imprecision and linearity. The effects of potential endogenous interfering substances and the possibility of falsely increased cardiac troponin I (cTnI) concentrations attributable to the presence of heterophilic antibodies (HA), rheumatoid factor (RF) and human anti-mouse antibodies (HAMA) in high concentrations were evaluated. The 99th percentile limit of the cTnI value distribution was determined from 320 Caucasian reference individuals. Results: No significant differences were found when cTnI concentrations of 40 lithium-heparin plasma samples were compared with the matched values of K2-EDTA plasma, whole blood and serum samples. The LoB and the LoD of the cTnI method were 0.0048 and 0.0066 μg/L, respectively. cTnI mean values from 0.66 to 6.0 μg/L showed a total CV% from 6.0 to 6.4. cTnI at a concentration of 0.02 μg/L was associated with a total CV of 9.6%. The method gave a linear response for cTnI concentrations within the measurement range. In six of 12 samples containing HA, a positive interference was demonstrated. The 99th percentile limit of the cTnI distribution in the reference population was 0.013 μg/L. Conclusions: The data indicate that the cTnI Pathfast method may be suitable for helping clinicians in the management of patients with ACS. Clin Chem Lab Med 2009;47:829–33.

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.

Likelihood ratios and Fagan's nomogram : valuable but underrated tools for in vitro latex sensitization assessment

The relevance of latex allergy has greatly increased in the last decade especially in health workers and in children with spina bifida. Serological testing has been proposed in the diagnosis since history can be inconclusive and skin and provocative testing can induce severe adverse reactions. We evaluated positive likelihood ratios (LRpos) and the Fagans nomogram in the diagnosis of latex allergy. Thirty patients with positive clinical history, who showed positive results in the skin prick test and in glove-exposure test were compared to 36 blood donors with negative clinical history, who showed negative results in the skin prick test and in glove-exposure test. LRpos was calculated at four different cutoff concentrations of IgE specific to latex measured with a totally automated analyzer. LRs appear better compared to the traditional classes in the reporting of IgE and, coupled with the Fagans nomogram which allows the calculation of post-test probability, could improve laboratory testing in latex allergy.

Proposta di una checklist per il prelievo di sangue venoso

RiassuntoIl prelievo venoso rappresenta una procedura inevitabile per ottenere campioni biologici per l’esecuzione dei test di laboratorio. Malgrado la pratica della flebotomia sia sovente considerata semplice e scevra da complicazioni e complicanze, essa causa la maggior parte degli errori di laboratorio, determinando inaccuratezza dei risultati se eseguita con imperizia, negligenza e scarsa professionalità. Si è quindi ritenuto opportuno provvedere alla redazione di un documento nella forma semplificata di checklist, composta da un semplice ma esaustivo elenco di attività da svolgere o da verificare da parte del prelevatore, al fine di prevenire i principali errori di prelievo. Nell’intento dei redattori e delle Società italiane di Medicina di Laboratorio, questa sintetica checklist rappresenta uno strumento modulabile e potenzialmente adattabile ai differenti contesti locali, diffondibile in maniera facile e graduale, supportata da evidenze scientifiche e dal consenso di esperti, redatta con il contributo di professionisti di diversi contesti sanitari, aderente alle best practice e che richiede risorse minime per essere implementata. È ragionevole supporre che questo strumento sia in grado di sostenere sia i cambiamenti di sistema sia i cambiamenti dei comportamenti individuali, rafforzando gli standard per la sicurezza di operatori e pazienti, contrastando i possibili fattori di fallimento. Auspichiamo, inoltre, che la checklist possa essere adottata dalle strutture sanitarie in cui si renda necessaria la raccolta di campioni di sangue venoso, adattandola alle caratteristiche dell’organizzazione locale.SummaryThe collection of venous blood is essential to obtain biological samples for performance of laboratory testing. Although there is widespread perception that this practice is simple and free of complications and side effects, it is undeniable that the vast majority of laboratory errors arises from ignorance, incompetence or negligence during venipuncture. It has hence become advisable to edit a document in simplified form of checklist, consisting of a concise but comprehensive list of activities to complete or verify, in order to prevent the onset of errors during venous blood collection. In the intention of authors and of the Italian societies of laboratory medicine, this synthetic checklist is a modular tool, adaptable to different local contexts, it can be easily and gradually implemented, is supported by scientific evidence and consensus of experts, has been created with the support of different healthcare professionals, is adherent to the best practices and requires minimal resources for implementation. It is reasonable to assume that this checklist may be able to withstand system and individual changes, strengthening the standards for safety of both operators and patients, limiting potential failure patterns. We also hope that the checklist may be implemented in all healthcare facilities where routine venous blood collection is performed, after adaptation to suit characteristics of local organization.

Raccomandazioni di consenso SIBioC-SIMeL per la rilevazione e gestione dei campioni emolizzati e utilizzo dell’indice di emolisi

RiassuntoLa presenza di emolisi in un campione biologico è causata principalmente da anemia emolitica o emolisi in vitro. La seconda circostanza è conseguente ad attività inappropriate per la raccolta e il trattamento del campione biologico che possono inficiare l’attendibilità dei risultati di molti esami di laboratorio. L’emolisi è valutabile mediante la determinazione dell’emoglobina libera, il cui limite è 20 mg/L nel plasma e 50 mg/L nel siero. L’emolisi si rende visivamente palese quando la concentrazione di emoglobina libera supera 300 mg/L. Poiché i campioni emolizzati sono la causa più frequente di non conformità dei campioni biologici nei laboratori clinici, con prevalenza prossima al 3% di tutti i campioni ricevuti, queste raccomandazioni di consenso sono state redatte specificatamente per assistere i professionisti di laboratorio nella rilevazione e gestione dei campioni emolitici. In sintesi, l’approccio raccomandato si basa su: (i) rilevazione e quantificazione sistematica dell’emolisi mediante ispezione visiva e successiva determinazione dell’indice di emolisi in tutti i campioni con emolisi visibile; (ii) immediata notifica al reparto della presenza di emolisi del campione secondo modalità definite localmente; (iii) soppressione di tutti i test influenzati dalla presenza e/o grado di emolisi; (iv) richiesta tempestiva di un secondo campione sul quale eseguire gli esami precedentemente soppressi.SummaryThe presence of haemolysis in a biological blood sample is mainly a result of haemolytic anaemia or haemolysis in vitro. Another cause may be inappropriate handling at the time of collection and processing of the sample, and this may affect the reliability of the test results. Haemolysis is assessed by quantification of free haemoglobin, whose limit is 20 mg/l in plasma and 50 mg/l in serum. Haemolysis can be observed visually when the concentration of free haemoglobin exceeds 300 mg/l. Since haemolysis is the most frequent reason for a biological sample to be considered unsuitable for processing in the clinical laboratory, with a prevalence approaching 3% of all samples received, these consensus recommendations have been drafted specifically to assist laboratory professionals in the detection and management of haemolysed specimens. The recommended approach can be summarized as follows: (1) systematic detection and quantification of haemolysis by visual inspection and subsequent quantification of the haemolysis index in all samples with visually detectable haemolysis; (2) immediate notification to the referring department of the presence of haemolysis in the sample, as locally determined; (3) suspension of all tests affected by the presence and/or the degree of haemolysis detected; and (4) timely request for a second sample to allow the tests previously suspended to be performed.