Pre-analytical Problems Research Articles

Are shortened aPTT values always to be attributed only to preanalytical problems?

It has been recognized that shortened activated partial thromboplastin time (aPTT) may be caused by various preanalytical conditions. As coagulation Factor VIII is included in the invitro intrinsic coagulation cascade measured by aPTT, we hypothesized that the shortened aPTT could be a result of elevated FVIII activity. We aimed to inspect the connection of elevated FVIII with shortened aPTT, and the possible effect inflammation has on routine laboratory parameters. 40 patients from various hospital departments with aPTT measurement below the lower limit of the reference interval (<23.0 s) were included in the study. To compare the obtained results with aPTT measurements in the non-inflammatory state, samples from 25 volunteers (laboratory personnel) were collected. White blood cell count, C-reactive protein, aPTT, and FVIII values were measured in the control group. Only two samples among 40 patients with shortened aPTT (5 %) were clotted. Out of the remaining 38, 26 had FVIII activity above 150 % (upper limit of a reference interval), median value of 194 % (IQR: 143-243 %). Seven samples in the control group had shortened aPTT results (36 %). However, all coagulation samples were clot and hemolysis-free. Multiple regression identified only FVIII activity as an independent variable in predicting aPTT values (p=0.001). Our results support the thesis that shortened aPTT is rarely a consequence of preanalytical problems. Elevated FVIII activity causes shortened aPTT, not only in theinflammatory state but also in individuals with concentration of inflammatory markers within reference intervals.

Observations from the Proteomics Bench.

Many challenges in proteomics result from the high-throughput nature of the experiments. This paper first presents pre-analytical problems, which still occur, although the call for standardization in omics has been ongoing for many years. This article also discusses aspects that affect bioinformatic analysis based on three sets of reference data measured with different orbitrap instruments. Despite continuous advances in mass spectrometer technology as well as analysis software, data-set-wise quality control is still necessary, and decoy-based estimation, although challenged by modern instruments, should be utilized. We draw attention to the fact that numerous young researchers perceive proteomics as a mature, readily applicable technology. However, it is important to emphasize that the maximum potential of the technology can only be realized by an educated handling of its limitations.

Flow Cytometric Analysis of Bone Marrow Particle Cells for Measuring Minimal Residual Disease in Multiple Myeloma.

Simple SummaryThe hemodilution of bone marrow aspirates is the main factor affecting the reliability of the determination of the minimal residual disease (MRD) resulting in multiple myeloma (MM). A bone marrow particle cell (BMPL) enrichment assay may be applied to overcome the problems caused by the hemodilution of bone marrow aspirates and to improve the rate of myeloma cell detection. Assessment of BMPL samples could decrease the false-negative rate of the MRD assessed with routine bone marrow aspirates. This study validated that the BMPL was superior to the routine bone marrow aspirate samples in the MM and MRD analysis.Minimal residual disease (MRD) is one of the most relevant prognostic factors in patients with multiple myeloma (MM). However, the hemodilution of bone marrow (BM) aspirates, the most common preanalytical problem, is known to affect MRD detection. In the present study, we analyzed a preanalytical method for routine BM aspirates and a bone marrow particle cell (BMPL) enrichment assay and validated it as a reliable preanalytical method for flow cytometric MRD determination. A total of 120 BM samples were taken from 103 MM patients consecutively recruited; 77 BM samples had BMPL enrichment analysis and 99 BM samples were routinely analyzed. Then, the two different samples from patients with MM were sent for MRD detection using an eight-color flow cytometry. Our data showed that assessment of the BMPL enrichment samples attenuated the overestimation of MRD-negative assessed in the routine BM samples, which was mainly caused by hemodilution. In conclusion, the BMPL enrichment assay is a functional and practical preanalytical method for flow cytometric MRD analysis.

Analysis of Factors Affecting Waiting Time for Results of Clinical Laboratory Examinations at Medan Haji General Hospital

The purpose of this study was to analyze the factors that affect the waiting time for clinical laboratory examinations at RSU Haji Medan in 2021. This study is a mixed method. Using a total sampling of 17 people, namely all laboratory workers, the main informants in this study were clinical pathology doctors, room coordinators, clinical laboratory analysts at Haji Medan General Hospital (RSU) totaling 3 people and patients totaling 3 people. 1 additional informant, namely the Management section at RSU Haji Medan. The quantitative results are that there is a relationship between the qualifications of laboratory personnel having a value of p = 0.000, at the facility p = 0.044, there is no relationship between the transportation of specimens, the value of p = 0.099, there is a relationship between pre-analytical, analytical and pre-analytical problems. post analytic p value = 0.022, there is no electrical stability relationship p value = 0.235. Based on the results of research according to information and informants about the qualifications of officers running smoothly. have to send the sample to a private laboratory. Based on these results, it was concluded that the waiting time for laboratory results at RSU Haji has reached the standard that is in accordance with the minimum service standards at the Haji General Hospital in Medan City.

Heritable platelet disorders: an enigma even guidelines can't unravel.

Heritable platelet disorders: an enigma even guidelines can't unravel.

FC 057SALIVARY POTASSIUM FOLLOWS PLASMA POTASSIUM DYNAMICS IN HEMODIALYSIS PATIENTS

Abstract Background and Aims Hyperkalemia is common in patients on hemodialysis (HD) and in cardiorenal patients on RAAS inhibitors. Frequently, hyperkalemia is a leading reason to withdraw potentially lifesaving therapy in cardiorenal patients. Out-patient monitoring of plasma K+ levels due to preanalytical problems has proven problematic. The present pilot-study examined salivary K+ levels and kinetics based on plasma K+ measurements in HD patients employing a novel class of genetically-encoded fluorescent potassium-ion indicators, the GEPIIs (Bischof et al. Nat. Commun. 2017). HD patients represent a unique population in whom significant K+ derangements and rapid K+ level changes predictably occur and where these phenomena can be safely investigated. Method K+ assessments were performed in healthy individuals (n=20) and HD patients (n=30). Study-related procedures were approved by the local Institutional Review Board. Healthy individuals were recruited on a voluntary basis and provided only saliva samples. HD patients were recruited from our HD unit. Blood and saliva samples for K+ assessment were collected simultaneously during three consecutive HD sessions (HD1-3) prior to and directly after each session (pre/post HD). Blood was drawn in a standardized procedure directly from the HD access line and plasma K+ was measured immediately using standardized ion-selective electrodes (ISE). Saliva samples were gathered in a standardized procedure using a commercially available collection device (SuperSALTM). Of note, gaining adequate amounts of saliva from chronic HD patients, who frequently suffer from xerostomia, has proven challenging in some cases. The K+ content of saliva samples was measured by ISE and GEPII-technique. For the latter, samples were mixed with purified GEPIIs and were inserted into a fluorescent plate-reader. Probes were illuminated at 430 nm and emission light were collected at 475 nm and 525 nm, respectively. The ratio of the fluorescent intensities (F535/F480) after appropriate calibration is a direct measure of the K+ concentration sensed by GEPIIs. Results K+ determination in saliva samples using the GEPII-technique and ISE showed a strong agreement ((Figure 1A), Bias 0,71; 95% limits of agreement from -2.79 to 4.40). Pre-dialytic [K+]saliva of HD patients compared to healthy individuals, was higher (40,64±1,50 vs. 23,15±0,76 mmol/l, p&amp;lt;0.05). As expected, each HD session (HD1-3) led to a significant reduction in [K+]plasma, which is followed by a similar, significant reduction of [K+]saliva (Figure 1B). Dynamics of plasma and salivary [K+] showed a very similar pattern: strong reduction during a HD session followed by a marked increase in the dialysis-free period until the next session 48-72h later (Figure 1C). Although basal [K+]saliva shows individual variations, [K+]Saliva and [K+]plasma exhibited a tendency of linear association (Figure 1D). Correlation analysis in each HD session (HD1-3 pre/post) revealed however no or weak correlation of pre- and post-dialytic saliva and plasma K+ values (Figure 1E). Conclusion The GEPII-technique is an easy to use, reliable and suitable method for salivary K+ determination in healthy individuals and in HD patients with accuracy and precision comparable to that of ISE. Despite heterogeneous baselines, changes of [K+]saliva represent a sensitive marker of K+ derangements as well as hyper- and normokalemia in HD patients. Although we observed that [K+]saliva dynamically follows [K+]plasma , an exact quantification - most likely due to the low number of cases per HD sessions in this pilot-study – was not possible. Additionally, how closely [K+]saliva tracks [K+]plasma in patients with hypokalemia was not addressed in this study. To confidently answer whether [K+]saliva measurement can potentially be used in the care of patients at increased risk of hyperkalemia, further studies in a larger number of patients need to be conducted.

Analysis of the Clinical and Epidemiological Meaning of Screening Test for SARS-CoV-2: Considerations in the Chronic Kidney Disease Patients during the COVID-19 Pandemic

The COronaVIrus Disease 19 (COVID-19) pandemic is an emerging reality in nephrology. In a continuously changing scenario, we need to assess our patients’ additional risk in terms of attending hemodialysis treatments, follow-up peritoneal dialysis, and kidney transplant visits. The prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-20 infection in the general population plays a pivotal role in estimating the additional COVID-19 risk in chronic kidney disease (CKD) patients. Unfortunately, local prevalence is often obscure, and when we have an estimation, we neglect the number of asymptomatic subjects in the same area and, consequently, the risk of infection in CKD patients. Furthermore, we still have the problem of managing COVID-19 diagnosis and the test’s accuracy. Currently, the gold standard for SARS-CoV-2 detection is a real-time reverse transcription-polymerase chain reaction (rRT-PCR) on respiratory tract samples. rRT-PCR presents some vulnerability related to pre-analytic and analytic problems and could impact strongly on its diagnostic accuracy. Specifically, the operative proceedings to obtain the samples and the different types of diagnostic assay could affect the results of the test. In this scenario, knowing the local prevalence and the local screening test accuracy helps the clinician to perform preventive measures to limit the diffusion of COVID-19 in the CKD population.

An initiative to assess the quality of Tanzanian cervical cancer specimens for HPV and telomerase detection

The aim of our study was to assess the quality of Tanzanian cervical cancer specimens, evaluating telomerase alterations and human papilloma virus (HPV) infection in relation to histopathological characteristics since these biomarkers are not routinely analyzed. Thirty-two Tanzanian women with invasive cervical cancer were included in the study. Histopathological classification and all the analyses on tissue, including TERT immunohistochemistry, were performed at IRST IRCCS (Meldola, Italy). HPV typization was performed by pyrosequencing. FHACT™ was used to identify chromosomal aberrations. Nonparametric ranking statistics were used. The majority (75 %) of the cases analyzed were squamous carcinoma, while 12.5 % were adenocarcinoma. The presence of HPV infection was confirmed in 26/27 (96.3 %) cases. A high percentage of patients (88 %) were infected with HPV16 of whom 12 (44.4 %) with African type 1, and 4 (14.8 %) with African type 2. TERT expression evaluated in the entire case series showed a median H-score of 130 (range 3−270), with only one negative case. 88 % of the FISH-evaluable samples showed an amplification of the chromosomal region 3q26 (TERC) and/or 5p15, and 20q13, associated with a higher median expression of TERT (P = 0.0226). Despite pre-analytical problems in terms of sample fixation, we showed that the search for biomarkers such as HPV and telomerase is feasible in Tanzanian tissue. These markers could be important risk-stratification tools in this population.

Managing Deviating EQA Results: A Survey to Assess the Corrective and Preventive Actions of Medical Laboratories Testing for Oncological Biomarkers.

Laboratories testing predictive biomarkers in lung and colorectal cancer are advised to participate in external quality assessment (EQA) schemes. This study aimed to investigate which corrective actions were taken by laboratories if predetermined performance criteria were not met, to ultimately improve current test practices. EQA participants from the European Society of Pathology between 2014 and 2018 for lung and colorectal cancer were contacted, if they had at least one analysis error or test failure in the provided cases, to complete a survey. For 72.4% of 514 deviating EQA results, an appropriate action was performed, most often including staff training (15.2%) and protocol revisions (14.6%). Main assigned persons were the molecular biologist (40.0%) and pathologist (46.5%). A change in test method or the use of complex techniques, such as next-generation sequencing, required more training and the involvement of dedicated personnel to reduce future test failures. The majority of participants adhered to ISO 15189 and implemented suitable actions by designated staff, not limited to accredited laboratories. However, for 27.6% of cases (by 20 laboratories) no corrective action was taken, especially for pre-analytic problems and complex techniques. The surveys were feasible to request information on results follow-up and further recommendations were provided.

Pièges d’interprétation de l’hémogramme automatisé sur analyseur XN Sysmex : paramètres érythrocytaires

Medical interpretation of a complete blood count (CBC) requires the respect of the pre-analytical rules and a knowledge from the biologist of the analytical system at his disposal as well as a regular literature review. Pre-analytical problems, interferences or technological limitations may lead to wrong results and misinterpretation of the CBC.This paper presents various pitfalls to avoid for the analysis of red blood cells according to our experience acquired with the XN series hematology analyzers (Sysmex).

Defining and Managing the Preanalytical Phase With FMECA: Automation and/or "Human" Control.

Our scope is to provide methodological elements on how to manage effectively the preanalytical phase in the laboratory testing process, by objectively measuring the risk connected to the phases handled by man with respect to those managed by machines. Preanalytical errors account for most of the mistakes related to laboratory testing and can affect patient care. Hence, it is necessary to manage the risk connected to the preanalytical phase, as required by certification and accreditation bodies. The risk assessment discloses the steps at greater risk and gives indications to make decisions. We have reviewed the state of art in the automation of the preanalytical phase, addressing needs and problems. We have used the proactive risk assessment methodology FMECA (Failure Mode, Effects, and Criticality Analysis) to identify the most critical phases in our preanalytical process and have calculated the risk associated. The most critical phases were the human controlled ones. In particular, the highest risk indexes were associated to manual acceptance of test orders, identification of the patients, tube labeling, and sample collection. Automation in the preanalytical phase is fundamental to replace, support, or extend the human contribution. Nevertheless each organization is different about workloads and competencies, so the most suitable management must be tailor-made in each context. We present a method by which each organization is able to find its best balance between automation and human contribution in the control of the preanalytical phase.

Pièges d’interprétation de l’hémogramme automatisé sur analyseur XN Sysmex : paramètres plaquettaires

Medical interpretation of a complete blood count (CBC) requires the respect of the pre-analytical rules and a perfect knowledge from the biologist of the analytical system at his disposal as well as a regular literature review. Pre-analytical problems, interferences or technological limitations may lead to wrong results and misinterpretation of the CBC.This paper presents various pitfalls to avoid for the analysis of the platelets according to our experience acquired with the XN series hematology analyzers (Sysmex).

Hemolyzed Specimens: Major Challenge for Identifying and Rejecting Specimens in Clinical Laboratories.

Pre-analytical quality in clinical chemistry testing is as important as analytical and post-analytical quality. The most prevalent pre-analytical interference and a major source of error producing unreliable laboratory test results is hemolysis of blood samples. In vitro hemolysis may be due to the blood withdrawal technique or sample handling whereas in vivo hemolysis can originate from acquired, hereditary, or iatrogenic conditions and is not technique dependent. Interpreting in vivo or in vitro hemolysis requires clinicians to supply reliable clinical history and findings. Even then, to reject or release the result with interpretation is still under debate. Thus, hemolyzed specimens are a serious pre-analytical problem calling for well-designed and strictly implemented laboratory guidelines. The aim of this non-systematic review (addressed to healthcare professionals) was to highlight the challenges in identifying and rejecting hemolysis specimens.

Pièges d’interprétation de l’hémogramme automatisé sur analyseur XN Sysmex : paramètres leucocytaires

Medical interpretation of a complete blood count (CBC) requires the respect of the pre-analytical rules and a perfect knowledge from the biologist of the analytical system at his disposal as well as a regular literature review. Pre-analytical problems, interferences or technological limitations may lead to wrong results and misinterpretation of the CBC. Various pitfalls to avoid for the analysis of the leukocytes according to our experience acquired with the XN series hematology analyzers (Sysmex) are presented in this article.

Evaluation of hemolysis effect on hemoglobin measurement by capillary electrophoresis

BackgroundHemoglobin (Hb) is a hemeprotein with two linked pairs of globin chains. Each chain is connected to a heme residue in its center. Hemoglobinopathies are divided into quantitative and qualitative defects in globin synthesis. Hemolysis is a pre-analytical problem that reduces quality of sample for measurement of many analytes.MethodsBlood samples from 311 female and 189 male subjects with normal and abnormal Hb electrophoresis patterns were studied. Three milliliters of whole blood was obtained from all subjects and transferred into EDTA tubes. To analyze hemolysis, 1.5 ml of blood from each tube was aliquoted and frozen, and the remaining blood was stored at 4 °C for 24 h. Hemoglobin was measured in both hemolyzed and non-hemolyzed samples by capillary electrophoresis.Results and discussionData was analyzed with linear regression. The results were linear for the lower limit of detection of 9, 0.5, and 0.1% up to at least 99.5, 6.6, and 99.4% for HbA, HbA2, and HbF, respectively. Method comparison demonstrated good agreement between non-hemolyzed and hemolyzed conditions for hemoglobin measurement.ConclusionUse of hemolyzed samples had no effected on hemoglobin measurements.

Fallstricke bei der Diabetesdiagnostik: Wird zu lax mit Laborwerten umgegangen?

AbstractThe diagnosis of diabetes is associated with pre-analytical and analytical problems. Fasting glucose (FG), oral glucose tolerance test (oGTT) and HbA1c have advantages and shortcomings and have no equal diagnostic validity. oGTT is the most sensitive test, but its reproducibility is rather poor (CV ± 15 %). FG detects only 70 – 80 % of overt diabetes. FG is falsified by inappropriate blood sampling, intra-individual fluctuations and mistakes with the oGTT. HbA1c despite IFCC-standardization, but with a tolerable coefficient of variation of ± 18 % in round robin tests and use of not commutable control material is not easy to interpret. HbA1c analysis shows also interferences and is therefore of limited diagnostic value. Its threshold value of ≥ 6.5 % (≥ 48 mmol/mol Hb) is based on consensus and not on evidence. The diagnostic effort (FG and/or oGTT + HbA1c) with serious consequences is minimal invasive, reasonable and cheap. It prevents over- and underdiagnosis.

Complications métaboliques en insuffisance rénale chronique : hyperphosphatémie, hyperkaliémie et anémie

Metabolic complications of chronic kidney disease (CKD) are frequent; the aims of this review are to present a 2018 update for hyperkalemia, hyperphosphatemia and anemia.Hyperkalemia is defined by a plasma level above 5.0 mmol/L, after ruling out pre-analytical problems such as hemolysis. It is frequent in CKD, most often due to drugs and notably renin/ angiotensin blockers. Chronic hyperkalemia is deleterious, with an increased risk of mortality. Therapeutic strategies to decrease the incidence and severity of hyperkalemia are therefore crucial in nephrology: experts recommend to maintain the renin/angiotensin blockers as long as possible, whilst associating diuretics and potassium binders.There are apparent discrepancies between optimal protein intake and decreased phosphate intake in CKD; this is even more important in dialysis since protein decrease is associated with denutrition and subsequent increased risk of mortality. Nutritional phosphate intake from vegetables are less absorbed; in contrast, phosphate additives are almost completely absorbed in the gastro-intestinal tract. These “hidden” intake may increase the total daily phosphate intake by 1 000 mg. As such in addition to optimized dialysis, phosphate binders should be used but compliance may be challenging on the long-term. Educational programs focused on phosphate are also mandatory in CKD patients.“Absolute” iron deficiency is less frequent than “functional” iron deficiency in CKD patients: both require the use of iron supplementation, and the latter may benefit from additional erythropoietin stimulating agents (ESA) when hemoglobin is below 10 g/dL. Intravenous iron is more efficient to correct iron deficiency both in pre-dialysis and dialysis especially in patients with chronic deficiency. Last generation intravenous preparations have largely demonstrated their safety. One indication of iron supplementation one should not forget in nephrology is the patient with moderate CKD and heart failure since the expected benefits are multiple, notably in terms of quality of life, renal function and functional capacity.Cet article fait partie du numéro supplément Innovations en Néphrologie réalisé avec le soutien institutionnel de Vifor Fresenius Medical Care Renal Pharma.

Pitfalls in the Diagnosis of Diabetes: Are we too Lax with Laboratory Parameters?

The diagnosis of diabetes is associated with pre-analytical and analytical problems. Fasting glucose (FG), oral glucose tolerance test (oGTT) and HbA1c have advantages and shortcomings and have no equal diagnostic validity. oGTT is the most sensitive test, but its reproducibility is rather poor (CV± 15 %). FG detects only 70 - 80 % of overt diabetes. FG is falsified by inappropriate blood sampling, intra-individual fluctuations and mistakes with the oGTT. HbA1c despite IFC- standardization, but with a tolerable coefficient of variation of ± 18 % in round robin tests and use of not commutable control material is not easy to interpret. HbA1c analysis shows also interferences and is therefore of limited diagnostic value. Its threshold value of ≥ 6.5 % (≥ 48 mmol/mol Hb) is based on consensus and not on evidence. The diagnostic effort (FG and/or oGTT+HbA1c) with serious consequences is minimal invasive, reasonable and cheap. It prevents over- and underdiagnosis.

Preanalytical and analytical challenges in gas chromatographic determination of cholesterol synthesis and absorption markers

IntroductionCholesterol homeostasis disruption contributes to the development of different pathologies. Non-cholesterol sterols (NCSs) serve as cholesterol synthesis markers (desmosterol and lathosterol), and cholesterol absorption surrogate markers (campesterol, stigmasterol and β-sitosterol). The study aimed to resolve certain new pre-analytical and analytical problems and ensure a reliable and validated method. Materials and methodsMethod optimization, validation and stability studies were executed in human serum and plasma. Freeze-thaw cycles were done with and without antioxidant. Gas chromatography-mass spectrometer (GC-MS) was used for NCSs confirmation and plasticizer identification, while GC-flame ionization detector (GC-FID) was used for NCSs quantitation. ResultsIntra- and inter-assay variabilities for all NCSs were 2.75–9.55% and 5.80–7.75% for plasma and 3.10–5.72% and 3.05–10.92% for serum, respectively. Recovery studies showed satisfactory percentage errors for all NCSs: 93.4–105.7% in plasma and 87.5–106.9 in serum. Derivatized samples were stable up to 7days at −20°C and derivatization yield was affected by presence of plasticizers. Fatty acid amids were identified as interfering plastic leachates. Statistically different NCSs concentrations were observed after the 1st freeze-thaw cycle, in antioxidant-free samples, and after the 4th cycle in antioxidant-enriched samples. ConclusionsAll of the in-house procedures proved to be useful for minimizing the preanalytical and analytical variations, as proven by the validation results.

Case report of unexplained hypocalcaemia in a slightly haemolysed sample

The case presented highlights a common pre-analytical problem identified in the laboratory that was initially missed. It concerns a young, generally healthy adult patient with no significant medical history and no significant family history. They presented with common flu like symptoms to their primary care clinician who considered this was most likely a viral problem that would pass with time. The clinician, however, did some routine bloods to reassure the patient despite a lack of clinical indication. When the sample was analysed the sample was haemolysed with strikingly low calcium. This led to the patient being called into hospital for urgent repeat investigations, all of which turned out to be within normal ranges. On further investigation the original sample was found to be contaminated. This result would normally have been flagged but was missed due to the complication of haemolysis.