PREANALYTIC PHASE IN IMMUNOCHEMISTRY

PREANALYTIC PHASE IN IMMUNOCHEMISTRYDidem Barlak KetiDepartment of Biochemistry, Faculty of Medicine, Erciyes University, KayseriIn spite of the improvements in laboratory medicine, the pre-analytical phaseis still the main responsible for laboratory errors. Immunoassay is an importantpart of the diagnostic process. Because of the relatively low concentrations ofanalyte being measured and the complexities of the antigen-antibody interaction,this technique is relatively susceptible to interferences. There are many possiblereasons for false results to be obtained during an immunoassay procedure.Interferences in immunoassay fall in to two broad categories: analyte-independentand analyte-dependent.Analyte-dependent interferences○Cross reacting substances (lack of specificity)○Endogenous antibodies Antireagent antibodies (heterophile, HAAA, RF) Antianalyte antibodies-autoantibody (macro complexes)○Hook effectAnalyte-independent interferences○ Inadequate centrifugation with microclots○ Hemolysis, lipemia, icterus○ Specimen collection tubes, transport, stability and storage○ Disease statesThe ways of becoming aware of possible interferences and the investigation themDiscordant resultsClinical interactionHigh index of suspicionExclude pre-analytical problemsRepeat analysis on another instrument from a different manufacturerTreatment with heterophilic blocking reagentsPEG precipitationSerial dilutionsCheck using a different matrix e.g. urine for hCGSelective removal of immunoglobulinsChromatographyTandem-mass spectrometryFalsely high or falsely low results due to interferences endogenous to the specimenpresent a particular risk to patient care because they (a) are not detectable bynormal laboratory quality control procedures, (b) are reproducible within the testsystem, (c) are often clinically plausible and (d) are relatively rare.The mechanism of interference and its severity depend both on assay design(two-site; one step) and on the nature of the interfering antibody.It is important to recognize that interfering antibodies may be present onlytransiently in a patient’s serum, and that their characteristics and reactivity mayvary, such that no immunoassay can be considered to be completely robust to allpossible interference. Therefore it is important to inform clinicians and activatethe consultation process between the departments.References1. Greg Warda, Aaron Simpsonb, Lyn Boscatoc, Peter E. Hickman. Theinvestigation of interferences in immunoassay. Clin Biochem 2017; 50: 1306- 1311.2. Catharine M Sturgeon and Adie Viljoen. Analytical error and interference inimmunoassay: minimizing risk Ann Clin Biochem 2011; 48: 418-432.3. Ellen Anckaert and Johan Smitz, Interferences in ImmunoassaysJohanSchiettecatte, Laboratory Clinical Chemistry and Radioimmunology, BelgiumPREANALYTIC PHASE IN ADVANCED SYSTEMSFehime Benli AksungarAcıbadem University, School of Medicine, Department of BiochemistryAcıbadem Labmed Clinical Laboratories, Advanced Tests and MetabolismSection, IstanbulDiagnostic medical branches such as clinical biochemistry, clinical microbiology,pathology and radiology, keep pace with technology in a faster way than othermedical areas. In the last 15 years, with the emergence of electrospray ionisation(ESI) method especially for the ionization of molecules, mass spectrometryfinds place in routine laboratories. All analyse methods have three commonstages: Isolating the particular analyte from a complex matrix, determiningthe concentration and reporting the result in proper units. Mass spectrometers(MS) determine analyte concentrations more accurately than the other systems.Especially therapeutic drug concentrations, biologic amins and steroid hormonmeasurements are more sensitive in MS measurements. Today, in routinelaboratories, small molecule (metabolite) analyzes are shifting towards massspectrometers. High-precision measurement of modern MS systems means thatchemical contamination is also measured. Hence, preanalytical errors can leadto serious errors in these systems. In the clinical laboratories, in addition to thepre-analtytical phase of routine biochemistry and hormone systems, preanalyticalphase of MS systems must be evaluated and unfortunately the process has to bere-examined from the very beginning. Sample collecting time, sex, age, fastingstate, sample types, tubes and sample containers should be re-examined for MSsystems. The preferred sample type for blood analysis in MS systems is plasma.In addition, dried blood spot (DBS) is accepted to be an alternative sample for MSmeasurements. Plasma and serum metabolic profiles are different. Metabolism ofthe cells in serum continues until coagulation has occurred. In particular, plateletsare active from the moment they are removed out from the body, and secretemany metabolites during coagulation eg. lipids and proteases. It is important thatthe plazma or whole blood can be placed directly in ice.Preanalytic stage standardization in MS systems:1. Before validating the method, evaluation should be done for any contaminationfrom water, equipment used: tubes, pipette tips2. Hemolytic specimens must be handled carefully and clarify the interactionwith the analyte3. As soon as samples are collected, precautions must be taken to quickly startthe cooling process4. Cells from plasma/serum should be separated as quickly as possible andsamples should be transferred to secondary tubes5. Samples to be stored for analysis should be stored at -20 ° C and then at -80 ° C6. Repeated thawing-freezing is not acceptable7. A standardized / validated SOP should be prepared for sample pretreatmentprior to analysis8. An SOP is also required for each new method, containing sample collection,separation, transport, storage and sample preparation steps.Metal analyzes have been performed by Atomic Absorption Spectrometry (AAS)or Atomic Emission Spectrometry (AES) methods from the early 1900’s. In thelast 20 years, Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) hasbeen developed to measure simultaneous and highly precise measurement ofmultiple heavy metals in a single run Whole blood, serum, urine and CSF heavymetals can be analyzed by these systems. Contamination may be a problemduring sample collection and analysis. Hence special equipment and training arerequired for the sample collection</p

Askorbik Asidi Ölçen veya Dirençli olan Stripler: Hangisi Tercih Edilmeli?

Amaç: Askorbik asidin bazı testler (glukoz, kan ve lökosit esteraz) üzerine etkisini; interferansa dirençlistriplerle, askorbik asit düzeyini ölçen stripleri karşılaştırarak değerlendirmektir.Gereç ve Yöntem: Ardışık 10055 hastaya ait idrar sonuçlarını retrospektif olarak inceledik. Askorbikasidi negatif olan ve farklı düzeylerde glukoz veya kan ya da lökosit esteraz içeren örneklerden idrarhavuzları oluşturuldu. İdrar örneklerine son konsantrasyon 10, 25, 50, 100, 200 ve 400 mg/dL olacakşekilde askorbik asit eklenerek her iki striple analiz gerçekleştirildi.Bulgular: 10055 idrar örneğinin % 9.2’sinde askorbik asit pozitif olarak belirlendi. Hastaların %70’indeaskorbik asit 10 mg/dL düzeyindeydi. Askorbik asidi pozitif olan idrar numunelerinin %50’sinin acilservisten geldiği ve hastaların %51’inin çocuk olduğu belirlendi. Askorbik asit interferansına dirençlistriplerin; glukoz için ≤100 mg/dL’de negatif interferans göstermediği, ancak kan testinin askorbik asitinterferansından etkilenmekle birlikte negatifleşmediği tespit edildi. Askorbik asidi ölçen stripler; lökositesteraz, kan ve glukoz için sırasıyla &gt;50 mg/dL, ≥10 mg/dL ve ≥25 mg/dL askorbik asit düzeylerindenegatif interferans gösterdi.Sonuç: Askorbik asidin 10 mg/dL’lik konsantrasyonu, strip kan testini etkilemek için yeterli olup, acilşartlarında vitamin C alımı kesildikten sonra test tekrarı uygun olmadığından; askorbik aside dirençlistrip kullanımı tercih edilebilir. Bununla birlikte bu striplerin dirençli olduğu askorbik asitkonsantrasyonlarını belirlemek gereklidir.Anahtar Sözcükler: Askorbik asit; interferans; idrar strip&nbsp;</p

Holotranskobalamin ve Vitamin B12 Düzeyleri Arasındaki Uyumun Değerlendirilmesi

Amaç: Total vitamin B12 eksikliği olan hastalarda; holotranskobalamin (holoTC) düzeylerini analizederek total vitamin B12 ve holoTC ölçüm sonuçları arasındaki uyumu değerlendirmektir.Gereç ve Yöntem: Serum total vitamin B12 düzeyi düşük çıkan hasta numunelerinden (n=98) rastgeleseçilen serum örneklerinde holoTC düzeyi, Architect 2000i analizöründe kemilüminesan mikropartikülimmünoassay yöntemiyle analiz edildi. HoloTC eksikliği için &lt;47 pg/mL (&lt;35 pmol/L) cut-off değerolarak tanımlandı. HoloTC düzeyi bu değerin altında olanlar total vitamin B12 düzeyiyle uyumlu, budeğerin üstünde olanlar ise uyumsuz olarak değerlendirildi.Bulgular: Serum total vitamin B12 ve holoTC için ortalama ± standart sapma sırasıyla 147 ± 29 ve39.29 ± 18.60 pg/mL olarak tespit edildi. HoloTC ve total vitamin B12 düzeyleri arasında anlamlı zayıfbir korelasyon gözlendi (r =0.249, p=0.013). Regresyon denklemi; holoTC = 15.91 + 0.158 totalvitamin B12 şeklinde belirlendi. Total vitamin B12 düzeyi düşük olan 98 hastanın %26’sının holoTCdüzeyi yeterliydi.Sonuç: HoloTC’nin, hastanın kliniği doğrultusunda total B12 yerine kullanımı veya refleks test olarakrutine dahil edilmesi hastaların tanı ve takibinde yararlı olabilir.Anahtar sözcükler: Holotranskobalamin; vitamin B12; vitamin B12 eksikliği</p

CAN PLASMA OR SERUM BE USED INTERCHANGEABLY FOR DIFFERENT IMMUNOCHEMICAL ANALYTES?

OP-060CAN PLASMA OR SERUM BE USED INTERCHANGEABLY FORDIFFERENT IMMUNOCHEMICAL ANALYTES?Didem Barlak Keti, Sabahattin MuhtarogluDepartment of Medical Biochemistry, Erciyes University, Kayseri, TurkeyOBJECTIVES: Studies revealing the difference in plasma and serum test resultsfor immunochemical analytes are limited and the results are also contradictory.The aim of this study was to evaluate whether there is a difference between serumand plasma for 10 immunochemical analytes.MATERIALS and METHODS: Total of 30 healthy volunteers were includedin the study. Blood samples were collected in clot-activator with gel (Vacuette)and containing lithium heparin tubes with barrier (Barricor). Serum and plasmawere obtained by centrifugation at 2000 g for 10 minutes. Hemolysis indexwas lower than 20 in serum and plasma samples. Paired t test was used forstatistical analysis. Bias% results were compared with the desirable specification(B%) obtained from the Ricos database for clinically significant. Vitamin B12(B12), ferritin, folate, free triiodothyronine (FT3), free thyroxine (FT4), insulin,ProBNP, parathyroid hormone (PTH), thyroid stimulating hormone (TSH) andvitamin D levels analysed on a Roche COBAS 8000 device.RESULTS: There was a statistically significant difference for B12, ferritin, folate,insulin, proBNP, PTH, vitamin D between serum and plasma. Only, plasma PTHlevels were clinically significant higher than serum. PTH levels were stable upto 8 hours at room temperature in plasma with lithium heparin. However, insulinlevels remained stable up to 8 hours at room temperature in serum.CONCLUSIONS: Plasma cannot be used instead of serum for PTH analysis.Therefore serum reference range is not suitable for this analyte. Unlike insulin,PTH levels are more stable in plasma with lithium heparin at room temperature.Keywords: Plasma, Serum, Lithium Heparin, Parathyroid Hormone, Insulin</p

Reliability of Friedewald formula in patients with type 2 diabetes mellitus and its relation to lipid profile in diabetes regulation

Introduction: Many laboratories utilize Friedewald formula (FF) to analyze LDL cholesterol levels of patients including diabetes mellitus (DM). Therefore, it is essential to consider the coherence of results acquired by FF and direct measurement. The number of studies that investigated the effect of lipid parameters, especially TG/ HDL cholesterol ratio, on the difference between the two methods is limited. The study was designed to compare LDL cholesterol values obtained by using FF with direct measurement, and to evaluate the relationship between diabetes regulation and lipid profile. Material and Methods: In the cross-sectional study, 529 type 2 DM patients and 1703 non-DM subjects were divided into four groups regarding TG concentrations. Unlike other studies, the study focuses on direct LDL (DLDL) cholesterol levels obtained with the help of different DLDL cholesterol kits (n=20). The correlations were implemented between HbA1c and lipid profiles. Results: It was determined that the bias% was over 10% in 24% of patients with 100-199 mg/dL TG levels. The parameter revealed that the most significant difference and the strongest correlation with HbA1c was TG/HDL cholesterol ratio in patients with type 2 DM. Conclusions: In patients with type 2 DM, even if it was TG <200 mg/dL, LDL calculated with FF should be evaluated together with the TG/HDL cholesterol ratio. Otherwise, direct measurement can be recommended. This ratio is related to diabetes regulation and may be used to monitor patients.

The Effect of Hemolysis and Storage Conditions on Insulin Stability

O-013The Effect of Hemolysis and Storage Conditions on Insulin StabilityDidem Barlak Keti̇, Sabahattin MuhtarogluDepartment of Medical Biochemistry, Erciyes University, Kayseri, TurkeyOBJECTIVES:Biochemical or spectrophotometric measurements are known tobe more affected by hemolysis when compared to immunochemical analysis.This situation can often lead to less consideration on immunochemical assays.Threshold values at which hemolysis affects immunochemical tests are indicatedin our kit inserts, but there is no value related to insulin. Therefore, the aim ofthis study is to determine the hemolysis threshold for insulin and the effect ofstorage conditions on serum insulin stability.MATERIALS and METHODS:Serum pools were formed from the samplesof the routine laboratory. Serum samples of equal volume were transferred toseven tubes. The tubes were designed as only serum in the first tube, serum+ assay diluent in the second tube, and serum + hemolysate in the 3-7 tubeswhich correspond to 50,100, 200, 400 and 800, respectively hemolysis index. Inaddition, insulin levels were measured in the patient samples with &lt;20 (n = 10),20-50 (n=10), 50-100 (n=10) and 100-200 (n=10) hemolysis index immediatelyand after 8 hours at room temperature.RESULTS:Negative bias was detected as 10% in the samples with below 200mg/dL hemolysis index which were analysed immediately after centrifugation.Negative bias was determined as &lt;10%, 27.6% and 29.5% in the samples &lt;20,20-50 and 50-100 hemolysis index, respectively which stayed for 8 hours atroom temperature.CONCLUSIONS:Hemolysis index should be considered when reporting insulinlevels. Insulin analysis is not suitable for hemolysed serum samples that havewaited 8 hours at room temperature.Keywords: Hemolysis, insulin, stability</p