Causes of Preanalytical Interferences on Laboratory Immunoassays - A Critical Review.

The immunoassays methods need avoiding interferences that can influence result interpretation. Main sources of interference arise from either patient status, preparation and physiology or laboratory process and procedures. The aim of this non-systematic critical review is to highlight the preanalytical interferences on laboratory immunoassays. Blood hormone profile changes according with age and depending on sex: these are important variables, mainly in newborn, during both sexual maturation and childbearing. Gonadotropins FSH and LH show a sharp increase with age in females, whereas in males LH appears rather stable. With age both males and females show progressive decay of the hormone profile. Stress causes variations, as it influences GH, prolactin, Cortisol and the total/free ratio of thyroid hormone. Diurnal variations, day of cycle, influence by estrogens on thyroid hormone are relevant for result variability. Paraproteins and autoantibodies can interfere in some assays particularly drug, vitamin D and thyroid hormone. As regards the variables due to sample matrix, and to evacuated tubes components, some additives and anticoagulants have been reported to influence specific assays, e.g. thyroid hormone. Hemolysis, lipemia and bilirubin cause interferences on specific techniques/tests, e.g. ferritin, TSH, Vitamin B12, progesterone and folic acid. Nicotine and cocaine addictions interfere with some hormones. Thus, laboratory professionals should be aware of preanalytical problems particularly important when dealing with the immunoassays, by taking appropriate actions to avoid any relevant interferences.

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.

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.

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

ABSTRACT 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 25 min in the supine position, 20 min in a sitting position and 20 min 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.

Blood transfusions may impair endothelium-dependent vasodilatation during coronary artery bypass surgery.

OBJECTIVE: The hemolytic product free-hemoglobin (fHb) reduces nitric oxide (NO) bioavailability. The present study aims to establish whether administration of different blood transfusions result in increased circulating fHb levels and NO consumption with effects on arterial NO-dependent blood flow in patients undergoing CABG surgery. METHODS: Ninety-five consecutive patients undergoing elective CABG surgery were prospectively divided in four groups based on blood transfusion requirements during surgery: stored blood cells (SBC, n. 21), intraoperative autologous salvaged blood (ASB, n. 25), SBC and ASB (n.22), no transfusion (control, n. 27). Blood samples were collected before and after intervention to analyse plasma levels of fHb and NO consumption. Endothelium-dependent relaxation was assessed in left internal mammary artery (LIMA) rings harvested before chest closure. Peripheral artery tonometry was assessed after intervention. RESULTS: Transfusions with SBC increased plasma fHb (p<0.05). Transfusions of ASB resulted in higher plasma fHb compared to SBC (p<0.01). fHb concentrations directly correlated with NO consumption (r=0.65, p<0.001). Maximal endothelium-dependent relaxation in LIMA was significantly attenuated in SBC and ASB patients compared to control (15.2±3.1% vs 21.1±2.5% vs 43±5.0% respectively; p<0.01). Significant correlations were identified between the aortic pressure wave velocity, plasma fHb concentration and NO consumption (p<0.01). CONCLUSIONS: Intraoperative blood transfusions and particularly autologous salvaged blood impair endothelium-dependent relaxation through NO scavenging by fHb. These findings obtained in vitro and in vivo provide new insights into the adverse relation between blood transfusions and patient outcome.

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.

Aberrant MicroRNA Expression in Patients With Endometrial Cancer.

OBJECTIVE: Current evidence suggests that no single serum biomarker displays satisfactory diagnostic performance in patients with endometrial carcinoma (EC), the most frequent gynecological cancer in developed countries. However, aberrant tissue microRNA (miRNA) expression has been recently described in EC. Therefore, this study aimed to investigate the differential expression of 4 serum miRNAs and their association with CA125 (cancer antigen 125) and HE4 (human epididymis protein 4) in EC patients and in a control population. METHODS: Forty-six consecutive women with EC and 28 matched control subjects without a history of cancer or other diseases were enrolled. Total serum RNA was extracted using mirVana PARIS Kit. TaqMan MicroRNA Assay was used for quantitative real-time reverse transcriptase-polymerase chain reaction on ABI 7500 Sequence Detection System to assess differential miRNAs expression. The relative expression levels of 4 miRNAs (miR-222, miR-223, miR-186, and miR-204) were normalized to miR-16 and calculated using the 2-△Ct approach. RESULTS: Serum levels of miR-186, miR-222, and miR-223 appeared to be significantly higher in patients compared with control subjects (P = 0.004, P = 0.002, and P < 0.0001). Contrarily, serum miR-204 was found to be significantly lower in EC patients (P < 0.0001). The diagnostic performance of miRNAs was found to be significantly better than that of CA125. Among the various biomarker tested, serum miR-204 and HE4 exhibited the best diagnostic performance for discriminating EC patients from control subjects. CONCLUSIONS: These results underpin that the 4 miRNAs that we have investigated are implicated in development and progression of EC, thus opening new avenues in EC diagnostics.

Plasma Leptin in Patients at Intermediate to High Cardiovascular Risk With and Without Type 2 Diabetes Mellitus.

BACKGROUND: A number of clinical studies have demonstrated that leptin concentrations are related to the metabolic disturbances that constitute the metabolic syndrome (MetS) and to diabetes mellitus (DM). AIM: To investigate possible determinants of leptin concentrations in a sample of patients at high cardiovascular (CV) risk carrying two or more features of the MetS and to investigate if any difference exist between at risk patients with or without DM. METHODS: Serum leptin concentrations were measured in 60 consecutive male patients affected by at least two CV risk factors which belong to the National Cholesterol Education Program/Adult Treatment Panel III (NCEP/ATP III) definition of MetS: 30 patients affected by type 2 DM (T2DM) and 30 nondiabetic patients (non-T2DM). Nineteen healthy subjects were included in the study as a control group (HC). RESULTS: Leptin was significantly higher in patients carrying two or more features of the MetS compared with HC (P = 0.02). Stratifying MetS patients for DM, we found that leptin level was higher in non-T2DM patients (7.8 ng/ml), intermediate in T2DM (6.2 ng/ml), and lower in HC (4.6 ng/ml). In MetS patients, a positive correlation was found between leptin and waist, triglycerides, and number of MetS criteria. After stratification for T2DM, the correlations were still significant in the non-T2DM but not in the T2DM group. CONCLUSIONS: In our sample of moderate-to-high-risk patients, leptin level is positively associated with waist circumference and triglycerides but only in non-T2DM patients. Our data suggest that diabetic subjects could modulate leptin production in a different way compared with patients carrying other MetS-related anomalies.

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.

Abnormal gel flotation caused by contrast media during adrenal vein sampling.

INTRODUCTION: During adrenal venous sampling (AVS) procedure, radiologists administer a contrast agent via the catheter to visualize the proper catheter position. MATERIALS AND METHODS: A patient with primary aldosteronism diagnostic-hypothesis was admitted for AVS. A venogram was performed toconfirm the catheter's position with 2mL of Iopamidol 300 mg/mL. Samples were collected with syringe connected to a hydrophilic coated catheter by low-pressure aspiration from each of the four collection sites: inferior vena cava in the suprarenal portion, inferior vena cava in the infrarenal portion, left adrenal vein, and right adrenal vein; then immediately transferred from syringe to tubes with gel separator. All tubes were centrifuged at 1200 x g for 10 minutes. RESULTS: At the end of centrifugation process, primary blood tubes containing blood from inferior vena cava and left adrenal vein exhibited the standard gel separator barrier, while tubes from right adrenal vein showed abnormal flotation of gel separator. The radiologist confirmed the usage of 2.6 mL instead of 2.0 mL of Iopamidol 300 mg/mL. This iodinated contrast media, with 1.33 g/cm3 of density, was used close to the right adrenal vein due to some difficulty to access it. CONCLUSION: The abnormal flotation of gel separator in samples taken from right adrenal vein can be explained by the usage of the iodinatedcontrast media. We suggest using plain-tubes (without gel separator) for AVS in order to avoid preanalytical nonconformities. Moreover, a blood volume equivalent to twice the catheter extension should be discarded to eliminate residual contrast media before collection of samples for laboratory assays.

Aging: a portrait from gene expression profile in blood cells.

The availability of reliable biomarkers of aging is important not only to monitor the effect of interventions and predict the timing of pathologies associated with aging but also to understand the mechanisms and devise appropriate countermeasures. Blood cells provide an easily available tissue and gene expression profiles from whole blood samples appear to mirror disease states and some aspects of the aging process itself. We report here a microarray analysis of whole blood samples from two cohorts of healthy adult and elderly subjects, aged 43±3 and 68±4 years, respectively, to monitor gene expression changes in the initial phase of the senescence process. A number of significant changes were found in the elderly compared to the adult group, including decreased levels of transcripts coding for components of the mitochondrial respiratory chain, which correlate with a parallel decline in the maximum rate of oxygen consumption (VO2max), as monitored in the same subjects. In addition, blood cells show age-related changes in the expression of several markers of immunosenescence, inflammation and oxidative stress. These findings support the notion that the immune system has a major role in tissue homeostasis and repair, which appears to be impaired since early stages of the aging process.

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.

Plasma Expression Levels of Circulating miR-21 are not Useful for Diagnosing and Monitoring Colorectal Cancer.

BACKGROUND: Recent evidence suggests that microRNAs play an important role in cancer diagnostics. We assessed plasma microRNA-21 levels in patients with colorectal cancer (CRC) at different stages and in patients with benign polyps. METHODS: Plasma levels of miR-21 were assessed by quantitative reverse transcription polymerase chain reaction assay in plasma samples of 76 CRC patients and in 20 patients with benign polyps. Differences between groups were evaluated with Mann-Whitney and Kruskal-Wallis tests. RESULTS: No significant differences of miR-21 plasma levels were observed between CRC patients and subjects with benign polyps (p > 0.05). Also, no significant differences were found between CRC patients with advanced (III-IV) or early cancer stages (I-II) (p > 0.05). CONCLUSIONS: These results do not support the hypothesis that circulating miR-21 expression is increased in adenoma-carcinoma-advanced carcinoma sequence. Accordingly, plasma miR-21 assessment does not appear to be a useful biomarker for diagnosing and staging CRC.

Robotic assisted radical prostatectomy accelerates postoperative stress recovery: Final results of a contemporary prospective study assessing pathophysiology of cortisol peri-operative kinetics in prostate cancer surgery.

OBJECTIVE: To investigate the effects of prostate cancer (PCa) surgery on the stress system and to identify potential independent factors associating with stress recovery. METHODS: The design of the study was prospective and PCa surgery included robot assisted radical prostatectomy (RARP) or retropubic radical prostatectomy (RRP). Between February 2013 to December 2014, 315 consecutive patients were evaluated. The effects of PCa surgery on the stress system were measured by cortisol serum levels before and after surgery on postoperative day (POD) 0, 1, 3, 5 and 45. Cortisol variations in the population and subpopulation (RARP vs. RRP) of patients were investigated by statistical methods. Factors associating with stress recovery were assessed by simple linear regression (SLR) and multiple linear regression (MLR) analysis. RESULTS: RARP was performed in 75.9% of cases. In the patient population, there were wide serum cortisol perioperative variations. PCa surgery triggered the stress system which immediately (POD 0) responded by cortisol overproduction which induced the negative feedback mechanism that started on POD 1, continued on POD 3, was still ongoing on POD 5 and completely settled on POD 45 (stress recovery). In the subpopulation of patients, significantly lower cortisol serum levels were detected on POD 3-5 in RARP cases in whom cortisol levels were close to preoperative levels (stress recovery) on POD 5. Independent predictive factors of serum cortisol on POD 5 (stress recovery) were preoperative cortisol (p = 0.02), cortisol levels on POD 3 (p < 0.0001) and RARP (p = 0.03) in which the association was negative (stress recovery faster than RRP). CONCLUSION: Our study shows that PCa surgery immediately (POD 0) triggers the stress system which respond by overproduction of cortisol which induces the negative feedback mechanism that starts on POD 1, is still ongoing on POD 5, but is completely settled on POD 45. Moreover, after surgical trauma, our study gives evidence that the RARP procedure associates with stress recovery faster than RRP. Further confirmatory studies are required.

Birth season predicts the values of red blood cell distribution width (RDW) in adulthood.

BACKGROUND: Recent evidence suggests that red blood cell distribution width (RDW), a simple measure of anisocytosis, may predict the risk of adverse clinical outcomes in both the general population and in patients with severe pathologies. Since it was also shown that the birth season influences the lifetime disease risk, this study was aimed to investigate whether an association may exist between adult RDW values and birth season. METHODS: The study population consisted in healthy Caucasian blood donors aged 18 or older, undergoing routine laboratory testing before regular blood donation. RESULTS: Overall, 6122 healthy blood donors were included in this study (median age 41 years; 1807 women and 4315 men). Age, sex, mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) but not hemoglobin and hematocrit were found to be independent predictors of RDW. When the study population was classified according to birth season, a significant difference was found for RDW values, but not for age, sex, hemoglobin, hematocrit, MCV and MCH. Subjects born in spring exhibited RDW values generally higher compared to those born in other seasons, reaching statistical significance when compared to those born in summer and winter. In particular, subjects born in spring had a 33% (p=0.014) higher probability of displaying increased RDW values in adulthood compared to those with summer birth. CONCLUSIONS: Despite additional studies that are needed to confirm these original findings, the evidence that a significant link exists between birth season and adult anisocytosis provides a plausible explanation for the association between birth season and lifetime disease risk.

Robotic-assisted radical prostatectomy is less stressful than the open approach: results of a contemporary prospective study evaluating pathophysiology of cortisol stress-related kinetics in prostate cancer surgery.

The objective of this study was to investigate the effects of prostate cancer (PCA) surgery on the stress system and to identify potential independent factors associating with stress recovery. The design of the study was prospective and PCA surgery included robot-assisted radical prostatectomy (RARP) or retro pubic radical prostatectomy. Between February and December 2013, 151 consecutive patients were evaluated. The effects of PCA surgery on the stress system were measured by cortisol serum levels before and after surgery on post-operative day (POD) 0, 1, 3, 5 and 45. Statistical methods were applied. RARP was performed in 71% of cases. PCA surgery triggered the stress system which immediately (POD 0) responded by cortisol overproduction which induced the negative feedback mechanism that started on POD 1, continued on POD.

Venous stasis and whole blood platelet aggregometry: a question of data reliability and patient safety.

The assessment of platelet function by multiple electrode aggregometry (MEA) (Multiplate, Roche Diagnostics GmbH) is common in laboratory hematology. As regards the ISO 15189:2012 international standard, appropriate use of laboratory equipment requires appropriate pre-examination activities (e.g., blood collection). Venous stasis can influence several blood analytes, but the tourniquet time is rarely regarded as a source of variability. Aim of the present study was to evaluate the impact of venous stasis on platelet function by MEA. A total of 6 ml of blood was collected from 20 volunteers into two 3.0 ml Hirudin vacuum tube (Roche Diagnostics GmbH), and subjected to two procedures: procedure 1 (no stasis) - collection after localization of forearm vein by subcutaneous tissue transilluminator device without tourniquet; procedure 2 (stasis) - collection after localization of vein by prior 60 s tourniquet application. Samples were processed on Multiplate, for: ADP-test (without prostaglandin E1), ADP HS-test (with prostaglandin E1), ASPI-test, COL-test, RISTO H-test (high concentration, 0.77 mg/ml), RISTO L-test (low concentration, 0.20 mg/ml), and TRAP-test. The significance of the differences between samples was assessed by Wilcoxon ranked-pairs test. Surprisingly, the results of ADP HS-test, ASPI-test, COL-test, and RISTO H-test appeared unbiased by venous stasis. RISTO L-test, ADP-test, and TRAP-test were significantly biased; the mean percent difference between stasis and no stasis were -7.2% (P = 0.040), -28.4% (P = 0.015), and 1.1% (P = 0.031), respectively. In conclusion, the tourniquet should be avoided when assessing platelet function by MEA.

In vitro diagnostic company recalls and medical laboratory practices: an Italian case.

INTRODUCTION: In vitro human diagnostic (IVD) company recalls are a common practice aimed to either minimize a potential error or eliminate an existing failure. In this case report, we aim to provide a critical analysis of a recent IVD recall and to provide a practical framework about what to do when an IVD company recalls product(s) based on the International Organization for Standardization--ISO 15189:2012 standard. CASE REPORT: In 2014, Abbott Laboratories® (Green Oaks, IL) published an urgent field safety notice regarding a product recall (Architect Intact parathyroid hormone (PTH) Assay List Number 8K25) with immediate action required. The IVD company explained the reasons for the recall as follows: i) Abbott has confirmed that a performance shift in the Architect Intact PTH assay has the potential to generate falsely elevated results on patient samples; ii) results generated with impacted lots may demonstrate a positive shift relative to those generated with previous reagent and/or calibrator lots. This issue may also impact established Architect Intact PTH reference ranges; iii) the magnitude of shift averages approximately 13% to 45%; iv) Abbott Architect Intact PTH controls do not detect the shift; and v) all current reagent, calibrator, and control inventory are impacted. The recall could have resulted in ~40,000 inaccurate laboratory tests reported by 18 laboratories from Italy (Lombardy region). CONCLUSION: IVD company recalls have a serious impact on the patient safety and require a thorough investigation and responsible approach to minimize the possible damage. Medical laboratories accredited according to the ISO 15189 standard have procedures in place to manage such situations and ensure that patient safety is maintained when such recalls are issued.