Sample management for clinical biochemistry assays: Are serum and plasma interchangeable specimens?

The constrained economic context leads laboratories to centralize their routine analyses on high-throughput platforms, to which blood collection tubes are sent from peripheral sampling sites that are sometimes distantly located. Providing biochemistry results as quickly as possible implies to consolidate the maximum number of tests on a minimum number of blood collection tubes, mainly serum tubes and/or tubes with anticoagulants. However, depending on the parameters and their pre-analytical conditions, the type of matrix - serum or plasma - may have a significant impact on results, which is often unknown or underestimated in clinical practice. Importantly, the matrix-related effects may be a limit to the consolidation of analyses on a single tube, and thus must be known by laboratory professionals. The purpose of the present critical review is to put forward the main differences between using serum and plasma samples on clinical biochemistry analyses, in order to sensitize laboratory managers to the need for standardization. To enrich the debate, we also provide an additional comparison of serum and plasma concentrations for approximately 30 biochemistry parameters. Properties, advantages, and disadvantages of serum and plasma are discussed from a pre-analytical standpoint - before, during, and after centrifugation - with an emphasis on the importance of temperature, delay, and transport conditions. Then, differences in results between these matrices are addressed for many classes of biochemistry markers, particularly proteins, enzymes, electrolytes, lipids, circulating nucleic acids, metabolomics markers, and therapeutic drugs. Finally, important key-points are proposed to help others choose the best sample matrix and guarantee quality of clinical biochemistry assays. Moreover, awareness of the implications of using serum and plasma samples on various parameters assayed in the laboratory is an important requirement to ensure reliable results and improve patient care.

Joint EFLM-COLABIOCLI Recommendation for venous blood sampling

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.

Estimating the intra- and inter-individual imprecision of manual pipetting.

BACKGROUND: Despite the importance of manual pipetting of fluids such as water, solutions, buffers, reagents, or biological samples in daily laboratory practice, the intra- and inter-individual imprecision of this activity has not been recently described in scientific publications. METHODS: Twenty laboratory operators were randomly enrolled for this study. Imprecision of manual pipetting was estimated by asking each laboratory professional to dispense 1 mL, 100 µL or 10 µL of distilled water for 10 consecutive times with three certified pipettes into a 50-mL plastic container placed into a gravimetric balance. The weight of the water dispensed was systematically recorded for each of the 10 repeated attempts, and the inter- and intra-operator imprecision was finally calculated and expressed as coefficient of variation (CV%). RESULTS: The mean intra-individual imprecision was 5.7% (range, 0%-11.8%) for pipetting 10 µL, 0.8% (range, 0.4%-1.9%) for pipetting 100 µL, and 0.2% (range, 0.1%-0.5%) for pipetting 1 mL. Overall, the mean inter-individual imprecision was 8.1% for pipetting 10 µL, 1.1% for pipetting 100 µL and 0.4% for pipetting 1 mL. A significantly inverse correlation was found between intra-individual pipetting imprecision and the amount of water dispensed (r = -0.80; p<0.001). No significant correlation was observed between individual pipetting performance and sex, age, qualification, and years of experience in the laboratory. CONCLUSIONS: The results of this study show that manual pipetting is plagued by a considerable intra- and inter-individual imprecision, which is inversely correlated with the amount of fluid dispensed.

Pre-analytical phase management: a review of the procedures from patient preparation to laboratory analysis.

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.

The impact of fist clenching and its maintenance during venipuncture on routine hematology testing.

BACKGROUND: Prevention of a disturbance of the blood vessel allows phlebotomists to collect a blood specimen by venipuncture that will truly mirror the patient condition. This study was aimed to evaluate the impact of repeated fist clenching and maintenance of the fist during blood collection by venipuncture for routine hematology testing. METHODS: Blood were collected from 16 healthy volunteers with two separate sequential procedures, entailing standard venipuncture with hand opened throughout blood collection, or clenching the fist six times before venipuncture and maintaining the fist until completion of blood collection. The parameters tested included red blood cell (RBC) count, hemoglobin, hematocrit, mean corpuscular volume (MCV), RBC distribution width, white blood cell count and differential, including neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cells, platelet count, mean platelet volume, and reticulocytes. The results were reported as median and interquartile range. The comparison of data obtained with the two different venipuncture procedures (i.e., with or without fist clenching and closed hand) was performed with Wilcoxon-Mann-Whitney ranked-pairs test. The degree of statistical significance was set at P<.01. RESULTS AND CONCLUSION: Fist clenching and maintenance during blood collection for routine hematology testing was effective to increase the MCV by 1.2% (P<.001). All others hematological parameters were not significantly biased by fist clenching, though hematocrit, neutrophils, eosinophils, and reticulocytes displayed mindful of trends. We hence advise patients against clenching their fist before blood collection for hematology testing.

The order of draw: myth or science?

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.

Quality impact on diagnostic blood specimen collection using a new device to relieve venipuncture pain.

A new device called Buzzy(®) has been recently presented that combines a cooling ice pack and a vibrating motor in order to relieve the venipuncture pain. The aim of this study was to evaluate the impact of Buzzy(®) use during diagnostic blood specimen collection by venipuncture for routine immunochemistry tests. Blood was collected from 100 volunteers by a single, expert phlebotomist. A vein was located on the left forearm without applying tourniquet, in order to prevent any interference from venous stasis, and blood samples were collected using a 20-G straight needle directly into 5 mL vacuum tubes with clot activator and gel separator. In sequence, external cold and vibration by Buzzy(®) was applied on the right forearm-5 cm above the chosen puncture site-for 1 min before venipuncture and continued until the end of the same procedure already done in the left forearm. The panel of tests included the following: glucose, total cholesterol, HDL-cholesterol, triglycerides, total protein, albumin, c-reactive protein, urea, creatinine, uric acid, alkaline phosphatase, amylase, AST, ALT, g-glutamyltransferase, lactate dehydrogenase, creatine kinase, total bilirubin, phosphorus, calcium, magnesium, iron, sodium, potassium, chloride, lipase, cortisol, insulin, thyroid-stimulating hormone, total triiodothyronine, free triiodothyronine, total thyroxine, free thyroxine and haemolysis index. Clinically significant differences between samples were found only for: total protein, albumin and transferrin. The Buzzy(®) can be used during diagnostic blood specimens collection by venipuncture for the majority of the routine immunochemistry tests. We only suggest avoiding this device during blood collection when protein, albumin and transferrin determinations should be performed.

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.

Can chewing gum be another source of preanalytical variability in fasting outpatients?

INTRODUCTION: In the daily laboratory practice, there are patients coming to blood collection sites chewing sugar-free gum, considering it irrelevant to laboratory tests. The aim of this study was to evaluate whether a sugar-free chewing gum can interfere with laboratory tests. METHODS: We studied 22 healthy volunteers. After a 12-hour overnight fasting, the first blood sample was collected between 8:00 and 8:30 a.m. Then, immediately after the first venous blood collection, the subjects started chewing the gum (declared sugar-free) for 20 min. Subsequent venous blood samples were collected at 1, 2, and 4 hours after chewing the gum. Significant differences between samples were assessed by the Wilcoxon ranked-pairs test. RESULTS: Among all the results, statistically significant differences (p < 0.05) between basal and × hours after chewing sugar-free gum were observed for the following parameters: cortisol, insulin, C-peptide, triglycerides, uric acid, urea, amylase, alanine aminotransferase, lipase, creatine kinase, total bilirubin, direct bilirubin, phosphate, iron, potassium, thyroid stimulating hormone, red blood cell count, hematocrit, hemoglobin, mean cell volume, red cell distribution width, white blood cell count, lymphocytes, neutrophils, and eosinophils; whereas, coagulation tests were not impacted by chewing sugar-free gum. CONCLUSIONS: We recommend instructing the patients to avoid the use of chewing gum before blood collection for laboratory tests.

Rejection of hemolyzed samples can jeopardize patient safety.

INTRODUCTION: In vitro hemolysis is the primary cause of sample/test rejection by the laboratory. CASE REPORT: A 10-year-old, admitted with an asthma attack in the emergency-room, medicated with albuterol sulphate (intravenous bronchodilator that could induce hypokalemia), needed laboratory test monitoring. The physician prescribed the technical-nurse to perform blood sampling for: complete blood count, electrolytes, glucose, and blood gas analysis-within 30min after therapy. Samples were delivered to laboratory with a note "I had difficult to locate an appropriate access to perform the blood collection". LABORATORY RESULTS: Glucose: 4.77 mmol/L. Complete blood count revealed discreet eosinophilia 0.13x109/L, and thrombocytopenia 18x109/L. However, platelet clumps were observed in peripheral blood smear. Blood gas analysis was unreported, laboratory informed that sample had micro clots.Electrolytes: laboratory did not report the results; sample hemolyzed. 0.9 g/L of free hemoglobin is the cut-off defined by the laboratory; the sample presented 2.3 g/L of free hemoglobin. 3.9 mmol/L of potassium was the unreported result vs 2.1 mmol/L in the new sample.Briefly, the laboratory technician was trained to hide potassium results on hemolyzed sample due to the potential overestimation. Even if the hemolyzed sample presented a potassium value close to the lower reference range value (3.5-5.1 mmol/L), reporting the potassium result could allow the physician starting proper therapy to revert the hypokalemia by albuterol sulfate. CONCLUSION: The laboratory should be aware of the clinical patient conditions and of the related physician needs, before hiding results. Therefore, both the laboratory and the clinic personnel should communicate in order to guarantee the patient safety.

Preanalytical aspects on short- and long-term storage of serum and plasma.

Following an ordered clinical chemistry plasma/serum test, ideally the venous blood specimen is adequately collected at a health care facility, then swiftly transported to and readily handled, analyzed and sometimes interpreted at a clinical chemistry laboratory followed by a report of the test result to the ordering physician to finally handle the result. However, often there are practical as well as sample quality reasons for short- or long-term storage of samples before and after analysis. If there are specific storage needs, the preanalytical handling practices are specified in the laboratory's specimen collection instructions for the ordered test analyte. Biobanking of specimens over a very long time prior to analysis includes an often neglected preanalytical challenge for preserved quality of the blood specimen and also involves administrative and additional practical handling aspects (specified in a standard operating procedure - SOP) when demands and considerations from academic, industry, research organizations and authorities are included. This short review highlights some preanalytical aspects of plasma/serum short- and long- term storage that must be considered by clinicians, laboratory staff as well as the researchers.

Breakfast can Affect Routine Hematology and Coagulation Laboratory Testing: An Evaluation on Behalf of COLABIOCLI WG-PRE-LATAM.

Laboratories worldwide perform both hematological and coagulation testing on patients avoiding fasting time. In 2017, the Latin America Confederation of Clinical Biochemistry (COLABIOCLI) commissioned the Latin American Working Group for Preanalytical Phase (WG-PRE-LATAM) to study preanalytical variability and establish guidelines for preanalytical procedures to be applied by clinical laboratories and health care professionals. This study, on behalf of COLABIOCLI WG-PRE-LATAM, aims to evaluate the effect of the breakfast on routine hematology and coagulation laboratory testing. We studied 20 healthy volunteers who consumed a breakfast containing a standardized amount of carbohydrates, proteins, and lipids. We collected blood specimens for routine hematology and coagulation laboratory testing before breakfast and 1, 2, and 4 hours thereafter. Significant differences between samples were assessed by the Wilcoxon ranked-pairs test. Statistically significant differences ( p < 0.05) between basal and 4 hours after the breakfast were observed for red blood cells, hemoglobin, hematocrit, mean corpuscular volume, white blood cells, neutrophils, lymphocytes, monocytes, mean platelet volume, and activated partial thromboplastin time. In conclusion, the significant variations observed in several hematological parameters, and activated partial thromboplastin time due to breakfast feeding demonstrate that the fasting time needs to be carefully considered prior to performing routine hematological and coagulation testing to avoid interpretive mistakes of test results, and to guarantee patient safety. Therefore, COLABIOCLI WG-PRE-LATAM encourages laboratory quality managers to standardize the fasting requirements in their laboratory, i.e., 12 hours.

Impact of an Andean breakfast on biochemistry and immunochemistry laboratory tests: an evaluation on behalf COLABIOCLI WG-PRE-LATAM.

INTRODUCTION: In Andean countries, specifically in Ecuador, a food transition in the population has been observed because of economic growth. The Working Group for Preanalytical Phase in Latin America (WG-PRE-LATAM) of the Latin America Confederation of Clinical Biochemistry (COLABIOCLI) was established in 2017, and its main purpose is to study preanalytical variability and establish guidelines for preanalytical procedures in order to be implemented by clinical laboratories and healthcare professionals in Latin America. The aim of this study on behalf of COLABIOCLI WG-PRE-LATAM was to evaluate whether an Andean breakfast can interfere with routine biochemistry and immunochemistry laboratory tests. MATERIALS AND METHODS: We studied 20 healthy volunteers who consumed an Andean breakfast containing a standardized amount of carbohydrates, proteins and lipids. We collected blood specimens for laboratory tests before breakfast and 1, 2, and 4 hours thereafter. Significant differences between samples were assessed by the Wilcoxon ranked-pairs test. RESULTS: The Andean breakfast statistically (P ≤ 0.05), modified the results of the following tests: triglycerides, insulin, cortisol, thyroid stimulating hormone, free thyroxine, total protein, albumin, urea, creatinine, lactate dehydrogenase, alkaline phosphatase, amylase, lipase, total bilirubin, direct bilirubin, iron, calcium, phosphorus, magnesium, and uric acid. CONCLUSIONS: Andean breakfast can influence the routine biochemistry and immunochemistry laboratory tests and might expose patient safety to some risks. Therefore, the COLABIOCLI WG-PRE-LATAM calls attention and highlights that the fasting time needs to be carefully considered when performing blood testing in order to prevent spurious results and thus, reduce laboratory errors.

[Joint EFLM-COLABIOCLI recommendation for venous blood sampling]. / Recommandations communes EFLM-COLABIOCLI relatives au prélèvement sanguin veineux.

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.

Dark Chocolate Intake Acutely Enhances Neutrophil Count in Peripheral Venous Blood.

Beside the well-established impact on decreasing the risk of cardiovascular diseases (1), recent attention has been paid to the relationship between cocoa-containing foods and the immune system (2), showing that dark chocolate consumption enhances the systemic defense against bacterial (3) and viral (4) infections. Hence, the current study aimed at investigating the acute effect of dark chocolate intake on peripheral blood leukocytes.

Preanalytical Nonconformity Management Regarding Primary Tube Mixing in Brazil.

BACKGROUND: The multifaceted clinical laboratory process is divided in three essential phases: the preanalytical, analytical and postanalytical phase. Problems emerging from the preanalytical phase are responsible for more than 60% of laboratory errors. This report is aimed at highlighting and discussing nonconformity (e.g., nonstandardized procedures) in primary blood tube mixing immediately after blood collection by venipuncture with evacuated tube systems. METHODS: From January 2015 to December 2015, fifty different laboratory quality managers from Brazil were contacted to request their internal audit reports on nonconformity regarding primary blood tube mixing immediately after blood collection by venipuncture performed using evacuated tube systems. RESULTS AND CONCLUSIONS: A minority of internal audits (i.e., 4%) concluded that evacuated blood tubes were not accurately mixed after collection, whereas more than half of them reported that evacuated blood tubes were vigorously mixed immediately after collection, thus magnifying the risk of producing spurious hemolysis. Despite the vast ma jority of centers declaring that evacuated blood tubes were mixed gently and carefully, the overall number of inversions was found to be different from that recommended by the manufacturer. Since the turbulence generated by the standard vacuum pressure inside the primary evacuated tubes seems to be sufficient for providing solubilization, mixing and stabilization between additives and blood during venipuncture, avoidance of primary tube mixing probably does not introduce a major bias in tests results and may not be considered a nonconformity during audits for accreditation.

Patient posture for blood collection by venipuncture: recall for standardization after 28 years.

BACKGROUND: Although data about the effect of posture on routine hematological testing were published 28 years ago, this pre-analytical issue has not been standardized so far. This study was planned to evaluate whether postural changes influence the results of hematology testing. METHODS: A complete blood count was performed in 19 healthy volunteers after 25min in the supine position, 20min in a sitting position and 20min stationary standing in an upright position. RESULTS: The change from supine to sitting position caused clinically significant increases in the hemoglobin, hematocrit and red blood cell count. Furthermore, the change from supine to standing caused clinically significant increases in the hemoglobin, hematocrit, red blood cell, leukocyte, neutrophil, lymphocyte, basophil and platelet counts, and mean platelet volume, and that from sitting to standing caused clinically significant increases in hemoglobin, hematocrit, and red blood cell, leukocyte, neutrophil and lymphocyte counts. CONCLUSION: The results of this investigation provide further support to the notion that effort should be made to achieve widespread standardization in the practice of phlebotomy, including patient posture.

Estimation of the imprecision on clinical chemistry testing due to fist clenching and maintenance during venipuncture.

OBJECTIVES: An experimental study was planned to assess the influence on routine clinical chemistry parameters of fist making prior to, and maintenance during, venipuncture. DESIGN AND METHODS: Blood was collected from 16 healthy volunteers with two separate sequential procedures, entailing standard venipuncture with hand opened throughout blood collection, or clenching the fist 6 times before venipuncture and maintaining the fist until completion of blood collection. After separation of lithium-heparin plasma at vacuum tubes with gel separator, 28 routine clinical chemistry parameters and serum indices were measured on Roche Cobas 6000 〈c501〉 module. RESULTS: Fist clenching and maintaining were associated with significant variations of 8/26 (31%) analytes tested. Specifically, aspartate aminotransferase (+2.3%), calcium (+2.2%), chloride (+1.0%), creatine kinase (+2.0%), magnesium (+2.3%), potassium (+13.4%), and sodium (+0.7%) increased, whereas phosphate (-5.0%) decreased. All variations except aspartate aminotransferase and creatine kinase exceeded the quality specifications for desirable imprecision. A remarkable increase of free hemoglobin in plasma (i.e., +28.2%) was also observed. The ratio of plasma potassium was significantly associated with that of plasma CK (r=0.55; p=0.029), but not with variations of other analytes. No significant correlation was observed between the ratio of free hemoglobin and those of other analytes. CONCLUSIONS: The results of our investigation demonstrate that repeated clenching and maintenance of fist during venipuncture may trigger acute variations of several routine clinical chemistry parameters, which may be attributable to muscle contraction, hemolysis or both. Accordingly, venipuncture should be performed avoiding fist clenching and maintenance.

Mixing of thawed coagulation samples prior to testing: Is any technique better than another?

OBJECTIVE: Thus study was aimed to investigate whether the mixing technique could influence the results of routine and specialized clotting tests on post-thawed specimens. METHODS: The sample population consisted of 13 healthy volunteers. Venous blood was collected by evacuated system into three 3.5mL tubes containing 0.109mmol/L buffered sodium citrate. The three blood tubes of each subject were pooled immediately after collection inside a Falcon 15mL tube, then mixed by 6 gentle end-over-end inversions, and centrifuged at 1500g for 15min. Plasma-pool of each subject was then divided in 4 identical aliquots. All aliquots were thawed after 2-day freezing -70°C. Immediately afterwards, the plasma of the four paired aliquots were treated using four different techniques: (a) reference procedure, entailing 6 gentle end-over-end inversions; (b) placing the sample on a blood tube rocker (i.e., rotor mixing) for 5min to induce agitation and mixing; (c) use of a vortex mixer for 20s to induce agitation and mixing; and (d) no mixing. The significance of differences against the reference technique for mixing thawed plasma specimens (i.e., 6 gentle end-over-end inversions) were assessed with paired Student's t-test. The statistical significance was set at p<0.05. RESULTS AND CONCLUSION: As compared to the reference 6-time gentle inversion technique, statistically significant differences were only observed for fibrinogen, and factor VIII in plasma mixed on tube rocker. Some trends were observed in the remaining other cases, but the bias did not achieve statistical significance. We hence suggest that each laboratory should standardize the procedures for mixing of thawed plasma according to a single technique.