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.

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.

This document provides a joint recommendation for venous blood sampling of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group for Preanalytical Phase (WG-PRE) and Latin American Working Group for Preanalytical Phase (WG-PRE-LATAM) of the Latin America Confederation of Clinical Biochemistry (COLABIOCLI). It offers guidance on the requirements for ensuring that blood collection is a safe and patient-centered procedure and provides practical guidance on how to successfully overcome potential barriers and obstacles to its widespread implementation. The target audience for this recommendation are healthcare staff members directly involved in blood collection. This recommendation applies to the use of a closed blood collection system and does not provide guidance for the blood collection with an open needle and syringe and catheter collections. Moreover, this document neither addresses patient consent, test ordering, sample handling and transport nor collection from children and unconscious patients. The recommended procedure is based on the best available evidence. Each step was graded using a system that scores the quality of the evidence and the strength of the recommendation. The process of grading was done at several face-to-face meetings involving the same mixture of stakeholders stated previously. The main parts of this recommendation are: 1) Pre-sampling procedures, 2) Sampling procedure, 3) Post-sampling procedures and 4) Implementation. A first draft of the recommendation was circulated to EFLM members for public consultation. WG-PRE-LATAM was also invited to comment the document. A revised version has been sent for voting on to all EFLM and COLABIOCLI members and has been officially endorsed by 33/40 EFLM and 21/21 COLABIOCLI members. We encourage professionals throughout Europe and Latin America to adopt and implement this recommendation to improve the quality of blood collection practices and increase patient and workers safety.

Introduction Scope of the guidance Disclaimer Methodology I. Pre-sampling General considerations on appropriate mode of communication with the patient Patient position Step 1. Patient identification (1C) Step 2. Verify patient is fasting and properly prepared (1B) Step 3. Obtain supplies required for venous blood collection (2C) Step 4. Labeling and/or identifying tubes (1C) II. Sampling Step 5. Put on gloves (1C) Step 6. Apply tourniquet (1A) Step 7. Select venepuncture site (1B) Step 8. Clean sampling site (1B) Step 9. Puncture the vein (1A) Step 10. Drawing blood into the first tube (1A) Step 11. Release the tourniquet (1A) Step 12. Gently invert the tubes once immediately after collection (1B) Step 13. Draw additional tubes following the recommended order of draw (1B) Step 14. Remove the needle from the vein and ensure the safety mechanism is activated (1A) Step 15. Dispose of the needle (1A) Step 16. Bandage the puncture site (1C) Step 17. Tell the patient to apply gentle pressure and do not bend the arm (1C) Step 18. Invert all tubes at least 4 more times (1B) Step 19. Remove gloves (1A) III. Post sampling Step 20. Advise the patient to rest for 5 min (1B) IV. Implementation of the guidelines Potential barriers and challenges Framework for a successful implementation of this recommendation Conclusions References

The pre-analytical phase encompasses all the procedures before the start of laboratory testing. This phase of the testing process is responsible for the majority of the laboratory errors, since the related procedures involve many sorts of non-laboratory professionals working outside the laboratory setting, thus without direct supervision by the laboratory staff. Therefore, either correct organization or management of both personnel and procedures that regard blood specimen collection by venipuncture are of fundamental importance, since the various steps for performing blood collection represent per se sources of laboratory variability. The aim of this (non-systematic) review addressed to healthcare professionals is to highlight the importance of blood specimen management (from patient preparation to laboratory analyses), as a tool to prevent laboratory errors, with the concept that laboratory results from inappropriate blood specimens are inconsistent and do not allow proper treatment nor monitoring of the patient.

MicroRNAs (miRNAs) hold great promise in cancer research. The use of appropriate reference miRNAs for normalization of qPCR data is crucial for accurate expression analysis. We present here analysis and verification of current data, proposing a workflow strategy for identification of reference miRNAs in colorectal cancer (CRC). We performed a systematic review of studies aimed to identify stable reference miRNAs in CRC through high-throughput screening. Among the candidate miRNAs selected from the literature we excluded those predicted to target oncogenes or tumor suppressor gene. We then assessed the expression levels of the remaining candidates in exosomes, plasma and tissue samples from CRC patients and healthy controls. The expression stability was evaluated by box-plot, ∆Cq analysis, NormFinder and BestKeeper statistical algorithms. The effects of normalisers on the relative quantification of the oncogenic miR-1290 was also assessed. Our results consistently showed that different combinations of miR-520d, miR-1228 and miR-345 provided the most stably expressed reference miRNAs in the three biological matrices. We identified suitable reference miRNAs for future miRNA expression studies in exosomes plasma and tissues CRC samples. We also provided a novel conceptual framework that overcome the need of performing ex novo identification of suitable reference genes in single experimental systems.

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In tro duc tion: Tour niquet due ve nous sta sis can al ter bo th con cen tra tion and/or ac ti vi ty of se ve ral blood ana lytes, but is ra re ly re gar ded as an is sue of la bo ra to ry va ria bi li ty. To over co me the prob lem tran sil lu mi na tion de vi ces (TD) ha ve been pro po sed for a sta si s-free phle bo to my. In this pa per the use of a TD in pla ce of tour niquet du ri ng blood col lec tion has been eva lua ted. Ma te ria ls and met ho ds: Blood was col lec ted from 250 vo lun tee rs di vi ded in fi ve ho mo ge nous grou ps of tour niquet ti mes (G1: 30 sec, G2: 60 sec, G3: 90 sec, G4: 120 sec, G5: 180 sec) and com pa red to blood ob tai ned usi ng TD. All sam ples we re ana lyzed for glu co se (GLU), total pro tein (TP), albumin (ALB), trig lyce ri des (TRIG), potas sium (K), sodium (NA), phos pha te (PHOS), cal cium (CA), alka li ne phos pha ta se (ALKP) and mag ne sium (MG). Re sul ts: In res pe ct of TD, G1 did not show sta tis ti cal ly sig ni fi ca nt in crea ses in all cli ni cal che mis try tes ts; G2 showed in crea ses for GLU, TP, ALB, TRIG, K, CA, MG and ALKP. G3 and G4, showed no sig ni fi ca nt in crea se on ly for PHOS. G5 showed sig ni fi ca nt in crea ses in all the tes ts eva lua ted. Mo reo ver, cli ni cal ly sig ni fi ca nt va ria tio ns we re ob ser ved for TP, ALB, K and CA in G2 to G5; for NA in G3 to G5; for MG in G4 and G5; for GLU, TRIG, ALKP on ly in G5. Con clu sio ns:The se re sul ts sup po rt the ap pli ca tion of TD in blood col lec tion for rou ti ne cli ni cal che mis try la bo ra to ry tes ts, sug ges ti ng its use shou ld be mo re diff u sed. Key wor ds: phle bo to my and blood col lec tion; pre ana lyti cal pha se; qua li ty ma na ge me nt system in cli ni cal la bo ra to ry; tran sil lu mi na tion; ve nous sta sis.