Gabriel Lima-Oliveira

Quality Standards for Sample Collection in Coagulation Testing

Preanalytical activities, especially those directly connected with blood sample collection and handling, are the most vulnerable steps throughout the testing process. The receipt of unsuitable samples is commonplace in laboratory practice and represents a serious problem, given the reliability of test results can be adversely compromised following analysis of these specimens. The basic criteria for an appropriate and safe venipuncture are nearly identical to those used for collecting blood for clinical chemistry and immunochemistry testing, and entail proper patient identification, use of the correct technique, as well as appropriate devices and needles. There are, however, some peculiar aspects, which are deemed to be particularly critical when collecting quality specimens for clot-based tests, and these require clearer recognition. These include prevention of prolonged venous stasis, collection of nonhemolyzed specimens, order of draw, and appropriate filling and mixing of the primary collection tubes. All of these important preanalytical issues are discussed in this article, and evidence-based suggestions as well as recommendations on how to obtain a high-quality sample for coagulation testing are also illustrated. We have also performed an investigation aimed to identify variation of test results due to underfilling of primary blood tubes, and have identified a clinically significant bias in test results when tubes are drawn at less than 89% of total fill for activated partial thromboplastin time, less than 78% for fibrinogen, and less than 67% for coagulation factor VIII, whereas prothrombin time and activated protein C resistance remain relatively reliable even in tubes drawn at 67% of the nominal volume.

Influence of a light meal on routine haematological tests.

INTRODUCTION Patient-related variables, such as physical exercise, stress and fasting status are important sources of variability in laboratory testing. However, no clear indications about fasting requirements exist for routine haematological tests, nor has the influence of meals been assessed. METHODS We studied 17 healthy volunteers who consumed a light meal containing a standardized amount of carbohydrates, protein and lipids. Blood was taken for routine haematological tests before the meal and 1, 2 and 4 hours thereafter. RESULTS One hour after the meal, neutrophil count and mean corpuscular haemoglobin (MHC) increased significantly, whereas lymphocyte and monocyte counts, red blood cell distribution width, haematocrit, and mean corpuscular volume decreased significantly. A clinically significant variation was only observed for lymphocytes. Two hours after the meal, a significant increase was observed for neutrophils and MCH, whereas lymphocytes, eosinophils, haemoglobin and haematocrit decreased significantly. Clinically significant variations were recorded for lymphocytes, red blood cells (RBC), haemoglobin, haematocrit and MCH. Four hours after the meal MCH was significantly increased, while lymphocytes, eosinophils, RBC, haemoglobin and haematocrit were significantly decreased. Clinically significant variations were recorded for neutrophils, eosinophils, RBC, hematocrit and MCH. CONCLUSION The significant variation of several haematological parameters after a light meal demonstrates that the fasting time needs to be carefully considered in order to interpret the results of haematological tests correctly.

Impact of the phlebotomy training based on CLSI/NCCLS H03-A6 - procedures for the collection of diagnostic blood specimens by venipuncture.

Introduction: The activities involving phlebotomy, a critical task for obtaining diagnostic blood samples, are poorly studied as regards the major sources of errors and the procedures related to laboratory quality control. The aim of this study was to verify the compliance with CLSI documents of clinical laboratories from South America and to assess whether teaching phlebotomists to follow the exact procedure for blood collection by venipuncture from CLSI/NCCLS H03-A6 - Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture might improve the quality of the process. Materials and methods: A survey was sent by mail to 3674 laboratories from South America to verify the use of CLSI documents. Thirty skilled phlebotomists were trained with the CLSI H03-A6 document to perform venipuncture procedures for a period of 20 consecutive working days. The overall performances of the phlebotomists were further compared before and after the training program. Results: 2622 from 2781 laboratories that did answer our survey used CLSI documents to standardize their procedures and process. The phlebotomists’ training for 20 days before our evaluation completely eliminated non-conformity procedures for: i) incorrect friction of the forearm, during the cleaning of the venipuncture site to ease vein location; ii) incorrect sequence of vacuum tubes collection; and iii) inadequate mixing of the blood in primary vacuum tubes containing anticoagulants or clot activators. Unfortunately the CLSI H03-A6 document does not caution against both unsuitable tourniquet application time (i.e., for more than one minute) and inappropriate request to clench the fist repeatedly. These inadequate procedures were observed for all phlebotomists. Conclusion: We showed that strict observance of the CLSI H03-A6 document can remarkably improve quality, although the various steps for collecting diagnostic blood specimens are not a gold standard, since they may still permit errors. Tourniquet application time and forearm clench should be verified by all quality laboratory managers in the services. Moreover, the procedure for collecting blood specimens should be revised to eliminate this source of laboratory variability and safeguard the quality.

Suitability of a transport box for blood sample shipment over a long period

BACKGROUND Safety transport boxes are increasingly used to ship laboratory specimens but there is little information on their capacity to maintain suitable transportation temperatures. MATERIALS AND METHODS Inner temperature was assessed using a commercially available transport box during an 8-h transportation period in the heat. RESULTS Temperature stability was unsatisfactory during approximately 64% of the transportation time (i.e., from 125 to 450 min). CONCLUSIONS Transport boxes might be unsuitable for shipping specimens over long periods.

Transillumination: a new tool to eliminate the impact of venous stasis during the procedure for the collection of diagnostic blood specimens for routine haematological testing

Introduction:  The collection of diagnostic blood specimens for routine haematological testing (RHT) is traditionally performed with tourniquet. However, the transillumination devices based on cold near‐infrared LEDs have been formerly proposed as a valuable tool for identifying reliable venous accesses, especially in patients with difficult or small veins, such as children. This study was aimed to evaluate whether a transillumination device can advantageously replace the use of the tourniquet during the procedure for collection of blood specimens for RHT and thereby eliminating the discomfort and risk of spurious results caused by excessive or prolonged venous stasis.

Influence of a Regular, Standardized Meal on Clinical Chemistry Analytes

Background Preanalytical variability, including biological variability and patient preparation, is an important source of variability in laboratory testing. In this study, we assessed whether a regular light meal might bias the results of routine clinical chemistry testing. Methods We studied 17 healthy volunteers who consumed light meals containing a standardized amount of carbohydrates, proteins, and lipids. We collected blood for routine clinical chemistry tests before the meal and 1, 2, and 4 hr thereafter. Results One hour after the meal, triglycerides (TG), albumin (ALB), uric acid (UA), phosphatase (ALP), Ca, Fe, and Na levels significantly increased, whereas blood urea nitrogen (BUN) and P levels decreased. TG, ALB, Ca, Na, P, and total protein (TP) levels varied significantly. Two hours after the meal, TG, ALB, Ca, Fe, and Na levels remained significantly high, whereas BUN, P, UA, and total bilirubin (BT) levels decreased. Clinically significant variations were recorded for TG, ALB, ALT, Ca, Fe, Na, P, BT, and direct bilirubin (BD) levels. Four hours after the meal, TG, ALB, Ca, Fe, Na, lactate dehydrogenase (LDH), P, Mg, and K levels significantly increased, whereas UA and BT levels decreased. Clinically significant variations were observed for TG, ALB, ALT, Ca, Na, Mg, K, C-reactive protein (CRP), AST, UA, and BT levels. Conclusions A significant variation in the clinical chemistry parameters after a regular meal shows that fasting time needs to be carefully considered when performing tests to prevent spurious results and reduce laboratory errors, especially in an emergency setting.

Elimination of the venous stasis error for routine coagulation testing by transillumination.

The preanalytical phase is responsible for more than two-thirds of all errors attributed to the clinical laboratory [1–4] and there are only a few routine procedures for the detection of nonconformities in this field of activity [5,6]. In this phase the procedures involving phlebotomy, critical to the obtainment of diagnostic blood specimens, are poorly studied as regards the major sources of errors and the procedures related to quality control process [7,8]. The collection of diagnostic blood specimens for routine coagulation tests are traditionally performed by phlebotomists using a tourniquet [9]. The Clinical and Laboratory Standards Institute (CLSI) recommends the use of the tourniquet for localizing suitable veins for ≤60 s. When performing specimen collection for diagnostic purposes such an interval of time both allows easy localization of vein paths and concomitantly circumvents possible problems due to excess venous stasis [10–12]. Although the venous stasis can influence the concentration and/or the activity of several blood analytes, the tourniquet time is rarely regarded as a potential source of laboratory variability [13–17]. Reportedly, the mean tourniquet application times by phlebotomists were 98 s in public laboratories and 70 s in private laboratories respectively, thus raising some issues about proper specimen collection [18]. The use of transillumination devices, based on cold near infrared light-emitting diodes (LEDs) whose lightsare absorbed by intra-erythrocyte hemoglobin flowing along the veins, has been initially proposed in order to ease the vein puncture in children [19]. The efficacy of palm transillumination for establishing venous access in small infants has already been assessed [20]. Moreover, the transillumination has been proposed for mapping veins to be cannulated prior to ambulatory phlebotomy because it allows accurate visualization of the vein course [21]. Reliability in coagulation testing is pivotal to the appropriate diagnosis and treatment of patients with hemostasis disturbances [17,22]. In this context some preanalytical details/procedures appear critical such as a) adequate fasting time before blood collection [23], b) use of appropriate tubes [24–26] and additives [27], c) appropriateness of blood collection, storage and centrifugation[28–30], and d) strict conformity to the recommendations regarding tourniquet time. Clinical laboratory results are estimated to be able to influence 60% to 70% of medical decisions and thus affect diagnostic outcome and/or patient treatments [31], such as oral anticoagulant therapy employed in patients at risk of thrombosis [32,33] or blood transfusion components prescription [34] recommended in bleeding patients with disseminated intravascular coagulation (DIC) and prolonged

Preanalytical management: serum vacuum tubes validation for routine clinical chemistry

Introduction The validation process is essential in accredited clinical laboratories. Aim of this study was to validate five kinds of serum vacuum tubes for routine clinical chemistry laboratory testing. Materials and methods: Blood specimens from 100 volunteers in five diff erent serum vacuum tubes (Tube I: VACUETTE®, Tube II: LABOR IMPORT®, Tube III: S-Monovette®, Tube IV: SST® and Tube V: SST II®) were collected by a single, expert phlebotomist. The routine clinical chemistry tests were analyzed on cobas® 6000 module. The significance of the diff erences between samples was assessed by paired Student’s t-test after checking for normality. The level of statistical significance was set at P < 0.005. Finally, the biases from Tube I, Tube II, Tube III, Tube IV and Tube V were compared with the current desirable quality specifications for bias (B), derived from biological variation. Results and conclusions: Basically, our validation will permit the laboratory or hospital managers to select the brand’s vacuum tubes validated according him/her technical or economical reasons, in order to perform the following laboratory tests: glucose, total cholesterol, high density lipoprotein-cholesterol, triglycerides, total protein, albumin, blood urea nitrogen, uric acid, alkaline phosphatise, aspartate aminotransferase, gamma-glutamyltransferase, lactate dehydrogenase, creatine kinase, total bilirubin, direct bilirubin, calcium, iron, sodium and potassium. On the contrary special attention will be required if the laboratory already performs creatinine, amylase, phosphate and magnesium determinations and the quality laboratory manager intend to change the serum tubes. We suggest that laboratory management should both standardize the procedures and frequently evaluate the quality of in vitro diagnostic devices.

Laboratory Diagnostics and Quality of Blood Collection / Laboratorijska Dijagnostika I Kvalitet Uzimanja Uzoraka Krvi

Summary Diagnostic blood samples collected by phlebotomy are the most common type of biological specimens drawn and sent to laboratory medicine facilities for being analyzed, thus supporting caring physicians in patient diagnosis, follow-up and/or therapeutic monitoring. Phlebotomy, a relatively invasive medical procedure, is indeed critical for the downstream procedures accomplished either in the analytical phase made in the laboratory or in the interpretive process done by the physicians. Diagnosis, management, treatment of patients and ultimately patient safety itself can be compromised by poor phlebotomy quality. We have read with interest a recent article where the authors addressed important aspects of venous blood collection for laboratory medicine analysis. The authors conducted a phlebotomy survey based on the Clinical and Laboratory Standard Institute (CLSI) H03-A6 document (presently replaced by the GP41-A6 document) in three government hospitals in Ethiopia to evaluate 120 professionals (101 non-laboratory professionals vs. 19 laboratory professionals) as regards the venous blood collection practice. The aim of this mini (non-systematic) review is to both take a cue from the above article and from current practices we had already observed in other laboratory settings, and discuss four questionable activities performed by health care professionals during venous blood collection. We refer to: i) diet restriction assessment; ii) puncture site cleansing; iii) timing of tourniquet removal and; iv) mixing specimen with additives Krotak sadrzaj Uzorci krvi za dijagnostiku uzeti pomoću flebotomije najčeštić su od svih bioloških uzoraka koji se uzimaju i šalju u medicinske laboratorije na analizu, time se pruža podrška nadleinim lekarima u postavljanju dijagnoze, pradenju i/ili terapijskom nadzoru bolesnika. Flebotomija, kao relativno irrvazivna medicinska procedura, zaista je presudna za postupke koji slede bilo u analitiCkoj fazi u laboratory ili u procesu interpretacije koji obavljaju lekari. Loš kvalitet flebotomije može kompromitovati postavljanje dijagnoze, upravljanje pacijentom, njegovo lečenje i najzad bezbednost pacijenta. Sa zanimanjem smo nedavno pročitali članak u kom se autori bave vainim aspektima uzimanja uzoraka venske krvi za medicinske laboratorijske analize. Autori su sproveli anketu o flebotomiji zasnovanu na dokumentu H03-A6 (danas ga zamenjuje dokument GP41-A6) Instituta za kliniCke i laboratorijske standarde (IKLS) u tri vladine bolnice u Etiopiji da bi ispitali 120 zaposlenih (101 nije bio laboratorijski radnik, dok 19 jesu bili laboratorijski radnici) o praksi uzimanja uzoraka venske krvi. Cilj ovog mini (nesistematičnog) pregleda je osvrt na sugestije iz pomenutog Clanka kao i na trenutne prakse koje smo vet primetili u drugim laboratorijama, i uz to kratka diskusija o Cetiri problematične aktivnosti koje prilikom uzimanja uzoraka venske krvi obavljaju zdravstveni radnici. Ovo se odnosi na: i) procenu restrikcija u ishrani; ii) čišdenje mesta punkcije; iii) vreme uklanjanja poveske i iv) mešanje uzoraka sa aditivima

The order of draw: myth or science?

Abstract Background: The potential for cross-contamination of additives among evacuated blood tubes has led to the development of the order of draw. This practice, however, is mainly based on scarce, anecdotal, and mostly outdated literature data. Therefore, the goal of this investigation was to definitely establish whether or not the indication of a specific order of draw is still justified. Methods: The study population consisted of 57 outpatients referred to the outpatient oral anticoagulant (OA) clinic of the Academic Hospital of Verona and 58 healthy volunteers enrolled from the laboratory personnel. In OA outpatients, one serum tube was collected immediately after needle insertion, followed by a buffered sodium citrate tube and another serum tube. In the healthy volunteers, one serum tube was collected immediately after needle insertion, followed by a potassium-ethylenediaminetetraacetic acid (K2-EDTA) tube and another serum tube. After separation, the serum was tested for potassium, sodium, calcium, magnesium, and phosphorus in the first and second serum tubes. Results: No significant difference could be observed between the first and the second serum tubes for any of the parameters. The bias calculated with Bland-Altman plots did not achieve statistical significance when the serum tube was collected after either a K2-EDTA or a sodium citrate tube. Conclusions: According to our data, revision of national and supranational recommendations on blood collection by venipuncture should consider that the order of draw exerts a negligible effect on sample quality, and this aspect should no longer be considered a quality criterion when evaluating the performance of phlebotomists.