General Laboratory: Pre-analytical Issues Online Training
Identify Key factors affecting sample quality and corrective action to reduce the occurance of problems. State the impact of underfilled collection tubes on these tests and list three key factors affecting sample stability. This clinical laboratory training qualifies for continuing education units (CEU).
Welcome to the Siemens Healthcare Diagnostics course on Pre-analytical Issues: Sample Quality in an Automated World. This course reviews the basic information and focuses on the necessary key factors in today’s automated environment and was developed by Nils B. Person, Ph.D., FACB Select Next to continue. . At the end of this course, you will be able to: Identify the impact of pre-analytical issues on patient results Identify key factors that affect sample quality for automated analysis Identify corrective actions to reduce occurrence of problems Select Next to continue. Pre-analytical phase covers wide range of topics: Patient condition / preparation Presence of drugs or other interfering substances Phlebotomy technique Sample identification Sample transport Sample preparation Sample storage Select Next to continue. Error frequency and types: a comparison between data from 2006 and 1996 Absolute numbers Relative frequency 1996 1996 Total Tests 40,490 Total Errors 189 Pre-analytical 129 68.2% Analytical 25 13.3% Post-analytical 35 18.5% Error frequency and types: a comparison between data from 2006 and 1996 Absolute numbers Relative frequency 1996 1996 Total Tests 40,490 Total Errors 189 Pre-analytical 129 68.2% Analytical 25 13.3% Post-analytical 35 18.5% Error frequency and types: a comparison between data from 2006 and 1996 Absolute numbers Relative frequency 1996 2006 1996 2006 Total Tests 40,490 51,746 Total Errors 189 160 Pre-analytical 129 99 68.2% 61.9% Analytical 25 24 13.3% 15% Post-analytical 35 37 18.5% 23.1% Pre-analytical issues make up largest percentage of laboratory errors 1996: Plebani and Carraro, Mistakes in a STAT laboratory: Types and frequency, ClinChem 43, 1348-51, 1997 2006: Carraro and Plebani, Errors in a STAT Laboratory: Types and frequencies 10 years later, ClinChem, 53, 1338-42, 2007 Pre-analytical errors impacting sample quality: ~40% of all pre-analytical errors Tube filling error Inappropriate container Mis-handled specimen Specimen collected from IV site ~25% of all errors H3-A61 _________________________________________________________________________________________ H18-A42 _________________________________________________________________________________________ Documents available at www.CLSI.org 1 Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard – Sixth Edition NCCLS Document H3-A6, Wayne, PA: NCCLS; 2007 2 Procedures for the Handling and Processing of Blood Specimens for Common Laboratory Tests; Approved Standard – Fourth Edition NCCLS Document H18-A4, Wayne, PA: NCCLS; 2010 In days gone by... Small workload Fewer specimen labels Sample viewable Specimens spun - delay before analyzed Transferred manually to instrument cup / container Opportunity to check sample quality visually Still had problems with poor quality samples Today... Multiple labels covering almost entire tube Loaded into front end automation almost immediately on arrival Not seen until after spinning Higher volume of work Greater pressure to reduce TAT Sample quality goes unchecked Select Next to continue. What determines sample quality? Correct tube / container Correct collection technique Proper fill volume & mixing Proper handling during transport Adequate clotting time for serum Correct centrifugation Proper handling / storage until analysis Select Next to continue. Many choices From one vendor: 50+ choices for tubes commonly used for chemistry Which is the right one? Assay package insert How many assays will be done? Patient population Time to analysis Turn around time requirements Select Next to continue. Good technique reduces incidence of hemolyzed samples Sequence of collection can impact sample quality Collection tubes need to be filled to correct volume Tubes need to be properly mixed immediately Select Next to continue. ______________________________________________________________________________________________________________ Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard—Sixth Edition 8.10 Step 10: Order of Draw The following order of draw is recommended for both glass and plastic venous collection tubes when drawing multiple specimens for clinical laboratory testing during a single venipuncture. Its purpose is to avoid possible test result error due to additive carryover.50,51 All additive tubes should be filled to their stated volumes (see section 8.9.1). (1) Blood culture tube (2) Coagulation tube (eg, blue closure) (3) Serum tube with or without clot activator, with or without gel (eg, red closure) (4) Heparin tube with or without gel plasma separator (eg, green closure) (5) EDTA tube with or without gel separator (eg, lavender closure, pearl closure) (6) Glycolytic inhibitor (eg, gray closure) Select Next to continue. Additives: ALL Anticoagulants Barrier gels Clot activators (all plastic serum tubes) Surfactants Stopper lubricants (all tubes) Select Next to continue. Common Types: Heparin – works for most, but not all chemistries EDTA – most commonly used for Hematology Citrate – used for Hemostasis Potential impact on sample quality: May interfere with some assays Fill volume and mixing are important Heparin’s effect is temporary & temperature-dependent Tubes stored too warm (>77°F / 25°C) prior to use Select Next to continue. Gel forms barrier between serum/plasma and cells Potential impact on sample quality: Not spun correctly Correct speed – to achieve 1300xg Type of centrifuge – swing rotor vs fixed Correct duration 10 minutes – swing rotor centrifuge 15 minutes – fixed rotor centrifuge Degraded gel Expired tubes or stored too hot (>77°F) prior to use Gel breaks down - floating pieces Leaves oily film Select Next to continue. Promotes Clotting: Glass tube Plastic tubes All plastic serum tubes have clot activators Most common is silica (inert) Rapid clot tubes contain thrombin Added in a medium, usually a surfactant Potential impact on sample quality: Tubes must be well mixed for efficient clotting Unmixed tubes may continue to form fibrin even after centrifugation Select Next to continue. Reduce hemolysis of red cells during filling; facilitate distribution of clotting activators in serum tubes Generally inert, however 2004: trending of patient data indicated shift in assay performance for several immunoassays Investigation traced start of change to first use of new lot of gel serum tubes Further investigation found several immunoassays from multiple manufacturers affected Root cause was surfactant used in tubes Tube manufacturer assisted in studies and changed manufacturing process to prevent recurrence Source: Raffick, Chan, Ruddel, Hortin, Csako, Demosky, Remaley, Immunoassay Interference by a Commonly Used Blood Collection Tube Additive, the Organosilicone Silwet L-720, Clin Chem, 2005, 51, 1874-1882. Select Next to continue. Blood collection tubes: Not chemically inert Any change requires validation Keep tube changes (brand, type, lot, etc.) in mind when investigating problem results Short draws Effectively concentrate tube additives May cause sample quality problems Select Next to continue. Source: Validation and Verification of Tubes for Venous and Capillary Blood Specimen Collection; Approved Standard NCCLS Document GP34-A, Wayne, PA: NCCLS; 2010 Plasma/whole blood tubes: Heparin / EDTA – invert 8-10 times Citrate – invert 3-4 times Delayed mixing allows clotting to begin before anticoagulant is distributed throughout the specimen Mixing is effectively delayed if sample drawn in syringe and transferred to collection tube Serum Tubes: Invert 5 – 8 times for thrombin Evenly distributes clot activator(s) Poor mixing increases clotting time 1 Source: BD Vacutainer Venous Blood Collection Tube Guide, Becton Dickinson, Franklin Lakes, NJ; VS5229-8, 2006 Select Next to continue. Potential impact on sample quality: Time Temperature Handling Source: Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard – Sixth Edition NCCLS Document H18-A4, Wayne, PA: NCCLS; 2010 CLSI H18-A4 Procedures for Handling and Processing Blood Specimens for Common Laboratory Tests Collection tube manufacturer recommends1: Time from drawing of blood not arrival in lab 5 minutes for Thrombin tubes 30 minutes for gel barrier tubes 60 minutes for serum Slow-clotting samples need additional clotting time: Renal patients Patients on anticoagulant therapy Pregnancy samples Potential impact on sample quality: Fibrin forming above the clot or gel that may impact analysis Source: 1 BD Vacutainer Venous Blood Collection Tube Guide, Becton Dickinson, Franklin Lakes, NJ; VS5229-8, 2006 Select Next to continue. Two basic types of centrifuge: Fixed angle rotor Swing rotor Correct force or RCF (1100-1300 xg) RCF ≠ RPM Correct RPM depends on centrifuge Adequate time Tubes without Gel Barrier: 10 minutes Tubes with Gel Barrier: Swing rotor: 10 minutes Fixed rotor: 15 minutes Temperature control Fixed angle Swing rotor Source: Support Bulletin DV-1118A, Siemens Healthcare Diagnostics, 2010 Select Next to continue. Potential impact on sample quality: Fixed angle not best choice for gel tubes Speed / RCF: Too slow – gel barrier does not work well Too fast – affects gel performance / leaves cells / hemolysis Time Too short – affects gel performance / leaves cells Temperature: too warm can impact quality Stopping too fast Potential impact on sample quality: Re-suspending Plasma - micro clots / platelets* Serum - fibrin formed after centrifugation Analyte Stability Time & Temperature Adequate volume of sample Samples from remote sites *Source: Dimeski, Badrick, Flatman, Ormiston, ClinChem, 50, 2391-2, 2004 Select Next to continue. 1 cm Reason Adequately clotted? Re-suspended? Stored refrigerated / frozen Potential impact on sample quality: NOT in collection tube Transfer to secondary tube to centrifuge Select Next to continue. Troponin Biomarker of choice for Acute Coronary Syndrome Becoming standard of care for evaluating acute chest pain Newer assays detect much lower concentrations Troubled by discordant results Typically initial result elevated Repeat testing same or new sample does not confirm Not method-specific 2005 FDA Safety Tips for Laboratorians August 2005 1.Roberts, Colate, De, Holmstrom, Narlock, Apple, ClinChem, 43, 860-861, 1997 2.Wenk, ClinChem, 44, 2554, 1998 3.Nosanchuk, ClinChem, 45, 714, 1999 4.Beyne, Vigier, Bourgoin, Vidaud, ClinChem, 46, 1869-1870, 2000 5.Hawkins, ClinChem, 49, 1226-1227, 2003 Select Next to continue. Critical Care application Often ordered STAT Strong pressure for rapid results Automated systems Rapid processing / primary tube sampling Assay sensitivity Troponin assays more susceptible than most Newer assays have very low detection limit – increases potential Perfect set up for pre-analytical problems Select Next to continue. Fibrin is a highly cross linked protein, “very sticky” Can form after centrifugation Serum: Specimens processed too quickly Specimens from patients who clot slowly Plasma Tubes not mixed well immediately after filling Heparin plasma stored in refrigerator Not always readily visible Clots begin as fibrin strands Strands float in serum / plasma – often near surface Can form micro-clots Potential impact on analysis: Strands can coat sample probe Can cause carryover of extra serum/plasma Can occlude probe tip preventing accurate sampling Strands in reaction tube Can potentially bind to assay antibodies Can produce “false” linkage to resemble bound Troponin Added to analyte present can “look” abnormal Select Next to continue. Initial result does not correlate with clinical situation Repeat analysis does not agree with initial result Results from new sample do not agree with initial result To confirm look at original collection tube Fine white strands or small clots may be visible in serum or plasma White pellet of fibrin forms if serum or plasma is placed in secondary tube and given a long spin Select Next to continue. Collection Correct tube & draw order Fill completely Mix immediately Appropriate transport Processing Adequate clotting time for serum Effective centrifugation Careful handling Note: Careful attention to pre-analytical factors not only improves reliability of patient results, but also will improve instrument reliability and reduce maintenance Select Next to continue.