1. Pre-analytical Laboratory Errors Dr Sami Saeed Associate Professor/HOD Foundation University Medical College Path Lab, Fauji Foundation Hospital, Path Lab, Fauji Foundation Hospital,Rawalpindi Email: drsami@comsats.net.pk

2. Objectives Identify the significant pre-analytical errors that can occur during blood specimen collection and transport Identify the significant pre-analytical errors that can occur during blood specimen collection and transport Explain the various means of pre-analytical error prevention Explain the various means of pre-analytical error prevention List proactive steps to reduce potential pre- analytical errors associated with blood collection and transport List proactive steps to reduce potential pre- analytical errors associated with blood collection and transport

3. Introduction Three phases of laboratory testing: pre-analytical, analytical and post-analytical Three phases of laboratory testing: pre-analytical, analytical and post-analytical Pre-analytical—specimen collection, transport and processing Pre-analytical—specimen collection, transport and processing Analytical—testing Analytical—testing Post-analytical—results transmission Post-analytical—results transmission

4. Pre Analytical Phase Specimen collection, handling and processing Physiological variables such as the effect of lifestyle, age, gender, pregnancy and menstruation Endogenous variables such as drugs etc

5. Pre Analytical Phase Some such as specimen variables can be controlled Knowledge of uncontrollable variables need to be well understood

6. Pre Analytical Phase German Society for Clinical Chemistry and the German Society for Laboratory Medicine proposed comprehensive recommendations on the quality of diagnostic samples, handling of hemolytic, icteric and lipemic samples Choice of anticoagulants to use, optimal sample size and analyte stability in sample matrix for each analyte

7. Recommendations of the German Society for Clinical Chemistry and the German Society for Laboratory Medicine Optimal sample volume: Twice the analytical volume of serum or plasma required for laboratory tests plus the dead volume of sample cup, replicates, and secondary tubes In general, for testing 20 analytes in clinical chemistry, 3 to 4 mL of whole blood is needed to obtain heparinized plasma, while 4 to 5 mL of clotted blood is needed to express serum 2 to 3 mL of EDTA blood and citrated blood is sufficient to perform hematology and coagulation tests

8. Pre-analytical errors Pre (32-75%)- and post-analytical errors are estimated to constitute 90% of errors Pre (32-75%)- and post-analytical errors are estimated to constitute 90% of errors

9. Clinical Chemistry 53:7 1338–1342 2007

10. Types of Errors Patient Identification Patient Identification Phlebotomy Technique Phlebotomy Technique Test Collection Procedures Test Collection Procedures Specimen Transport Specimen Transport Specimen Processing Specimen Processing

11. Patient Identification Errors Errors in correctly identifying the patient are indefensible Errors in correctly identifying the patient are indefensible Reasons for patient identification errors Reasons for patient identification errors Proper positive patient identification procedures not followed Proper positive patient identification procedures not followed Identification bracelet (inpatients) Identification bracelet (inpatients) Asking patients to state their full name (inpatients/outpatients) Asking patients to state their full name (inpatients/outpatients) Patient identification by staff or family member if patient unable to identify him/herself Patient identification by staff or family member if patient unable to identify him/herself

12. Patient Identification Errors Collection tubes labeled with the wrong patient Collection tubes labeled with the wrong patient Collection tubes not labeled at the time of collection Collection tubes not labeled at the time of collection Wrong labels affixed to collection tubes at bedside Wrong labels affixed to collection tubes at bedside Collection tubes incorrectly labeled by someone other than the phlebotomist who collects the specimen Collection tubes incorrectly labeled by someone other than the phlebotomist who collects the specimen

13. Patient Complications Some patient variables that affect blood specimens Some patient variables that affect blood specimens Diet Diet Fasting Fasting Exercise Exercise Obesity Obesity Allergies to alcohol or iodine used to clean venipuncture site Allergies to alcohol or iodine used to clean venipuncture site Use alternative cleanser such as chlorhexidine Use alternative cleanser such as chlorhexidine

14. Patient Identification Errors Specimen tubes unlabeled Specimen tubes unlabeled Requisition or collection tube labels not affixed to tubes Requisition or collection tube labels not affixed to tubes Requisition or collection tube labels in bag containing collection tubes Requisition or collection tube labels in bag containing collection tubes Requisition or collection tube labels rubber- banded to tubes Requisition or collection tube labels rubber- banded to tubes Collection tube labels not affixed to all tubes Collection tube labels not affixed to all tubes Specimen collection tubes labeled insufficiently Specimen collection tubes labeled insufficiently

15. Phlebotomy Errors Phlebotomy is a highly complex skill requiring expert knowledge, dexterity and critical judgment Phlebotomy is a highly complex skill requiring expert knowledge, dexterity and critical judgment It is estimated that one billion venipunctures are performed annually in the U.S It is estimated that one billion venipunctures are performed annually in the U.S Phlebotomy errors may cause harm to patients or result in needlestick injury to the phlebotomist Phlebotomy errors may cause harm to patients or result in needlestick injury to the phlebotomist

16. Phlebotomy Technique Errors Phlebotomy technique is important Phlebotomy technique is important Ensures test result validity Ensures test result validity Minimizes trauma to patient Minimizes trauma to patient Minimizes potential for phlebotomist injury Minimizes potential for phlebotomist injury Reduces recollections Reduces recollections Vein selection essential for successful venipuncture Vein selection essential for successful venipuncture Three veins in antecubital fossa in order of selection (1) median cubital (2) cephalic (3) basilic Three veins in antecubital fossa in order of selection (1) median cubital (2) cephalic (3) basilic

17. Phlebotomy Technique Errors Venous Access Difficulties Venous Access Difficulties Obstructed, hardened, scarred veins Obstructed, hardened, scarred veins Veins difficult to locate Veins difficult to locate Use of Alternative sites Use of Alternative sites Top of hand/Side of wrist Top of hand/Side of wrist Vein Collapse Vein Collapse Use of appropriate needle size Use of appropriate needle size Smaller evacuated collection tube Smaller evacuated collection tube

18. Phlebotomy Technique Errors Site Selection Site Selection Avoid sites with IV Avoid sites with IV Use alternative arm or draw below IV to avoid contamination/dilution from IV Use alternative arm or draw below IV to avoid contamination/dilution from IV Document arm if IV Document arm if IV Mastectomy—avoid site due to lymphostasis Mastectomy—avoid site due to lymphostasis Infection risk/alteration in body fluids and blood analytes Infection risk/alteration in body fluids and blood analytes Edematous areas — avoid due to accumulation of body fluids Edematous areas — avoid due to accumulation of body fluids Possible contamination/dilution of specimen Possible contamination/dilution of specimen

19. Phlebotomy Technique Errors Tourniquet Application Tourniquet Application Tourniquet tied too close to the venipuncture site can cause hematoma Tourniquet tied too close to the venipuncture site can cause hematoma Veins may not become prominent if tourniquet is tied too high (more than 3 to 4 inches above venipuncture site ) Veins may not become prominent if tourniquet is tied too high (more than 3 to 4 inches above venipuncture site ) Tourniquet left on longer than one minute can result in hemoconcentration, affecting some test results Tourniquet left on longer than one minute can result in hemoconcentration, affecting some test results Tourniquet should be released as soon as needle is in the lumen of the vein and blood flow established Tourniquet should be released as soon as needle is in the lumen of the vein and blood flow established

20. Phlebotomy Technique Errors Cleansing of venipuncture site Cleansing of venipuncture site Thorough cleaning with alcohol Thorough cleaning with alcohol Allow alcohol to dry completely to avoid stinging sensation upon needle entry and hemolysis of sample Allow alcohol to dry completely to avoid stinging sensation upon needle entry and hemolysis of sample Samples such as blood cultures should be collected using iodine to cleanse site to ensure sterility of sample Samples such as blood cultures should be collected using iodine to cleanse site to ensure sterility of sample Recollection rate for blood cultures ranges due to contamination is as high as 50% in hospitals with increased costs, patient overtreatment Recollection rate for blood cultures ranges due to contamination is as high as 50% in hospitals with increased costs, patient overtreatment

21. Test Collection Errors Order of Draw Order of Draw Order of draw affects the quality of the sample and can lead to erroneous test results due to contamination with the additive from the previous blood collection tube Order of draw affects the quality of the sample and can lead to erroneous test results due to contamination with the additive from the previous blood collection tube Hemolysis Hemolysis Blood collected insufficient to amount of additive in tube, Blood collected insufficient to amount of additive in tube, Traumatic venipuncture Traumatic venipuncture Blood collected from area with hematoma Blood collected from area with hematoma Vigorous shaking of tubes after collection Vigorous shaking of tubes after collection Milking the site when collecting capillary samples and blood collected using a small diameter needle. Milking the site when collecting capillary samples and blood collected using a small diameter needle.

22. Order of Specimen Collection Blood culture tube Coagulation tube (citrate) Serum tube (with or without clot activator or gel separator) Heparin (with or without gel separator) EDTA Oxalate/ Fluoride NCCLS (CLSI) H3-A5 standard, 2003

23. Test Collection Errors Timing of Collection Timing of Collection Timed Draws Timed Draws Therapeutic Drug Monitoring Therapeutic Drug Monitoring Peak and trough collection times Peak and trough collection times Basal State Collections Basal State Collections Fasting requirements—no food or liquid except water Fasting requirements—no food or liquid except water Specimens affected by time of day, for example, cortisol Specimens affected by time of day, for example, cortisol

24. Test Collection Errors Collection tube not completely filled Collection tube not completely filled Example— Incomplete filling results in specimen dilution and erroneous Prothrombin and aPTT test results Example— Incomplete filling results in specimen dilution and erroneous Prothrombin and aPTT test results

25. Test Collection Errors Capillary Collections—finger stick or heel stick Capillary Collections—finger stick or heel stick Appropriate site Appropriate site Heel stick—sides of the bottom surface of the heel Heel stick—sides of the bottom surface of the heel Finger stick—third or fourth fingers, perpendicular to fingerprint lines on fleshy pads on finger surface Finger stick—third or fourth fingers, perpendicular to fingerprint lines on fleshy pads on finger surface Warming—Warm before collection to increase capillary blood flow near skin surface Warming—Warm before collection to increase capillary blood flow near skin surface Cleaning—cleanse site with alcohol and allow to air dry Cleaning—cleanse site with alcohol and allow to air dry

26. Capillary Collections Massaging site to increase blood flow Massaging site to increase blood flow Milking site can cause hemolysis or tissue fluid contamination Milking site can cause hemolysis or tissue fluid contamination Finger sticks—roll fingers toward fingertip at 1 st finger joint several times Finger sticks—roll fingers toward fingertip at 1 st finger joint several times Heel sticks—gently squeeze infant’s heel before performing puncture. Heel sticks—gently squeeze infant’s heel before performing puncture. Perform puncture while firmly squeezing finger or heel Perform puncture while firmly squeezing finger or heel Wipe away first two drops of blood Wipe away first two drops of blood Ensure that full blood drop wells up each time Ensure that full blood drop wells up each time

27. Capillary Collections Avoid touching capillary collection tube or micro collection tube to skin or scraping skin surface Avoid touching capillary collection tube or micro collection tube to skin or scraping skin surface Contaminates puncture site Contaminates puncture site Blood may become hemolyzed Blood may become hemolyzed Mixing micro collection tubes with additive frequently to avoid micro clots Mixing micro collection tubes with additive frequently to avoid micro clots Collecting tubes with additives first Collecting tubes with additives first Protecting tubes for bilirubin from light Protecting tubes for bilirubin from light

28. Specimen Transport Errors Timing Timing Some specimens must be transported immediately after collection, for example Arterial Blood Gases Some specimens must be transported immediately after collection, for example Arterial Blood Gases Specimens for serum or plasma chemistry testing should be centrifuged and separated within two hours Specimens for serum or plasma chemistry testing should be centrifuged and separated within two hours

29. Transport Errors Temperature Temperature Specimens must be transported at the appropriate temperature for the required test Specimens must be transported at the appropriate temperature for the required test On ice—ABGs, Ammonia On ice—ABGs, Ammonia Warmed --98.6 degrees (37 C), cryoglobulins Warmed --98.6 degrees (37 C), cryoglobulins Avoid temperature extremes if transported from via vehicle from other collection site Avoid temperature extremes if transported from via vehicle from other collection site Transport Container Transport Container Some samples need to be protected from light, for example, bilirubin Some samples need to be protected from light, for example, bilirubin Transport in leak-proof plastic bags in lockable rigid containers Transport in leak-proof plastic bags in lockable rigid containers

30. Physiological Pre Analytical Variables TIME Increased/Decreased Cortisol Toward evening and midnight Glucose tolerance test values Afternoon SEASON Increased/Decreased Summer Vitamin-D, Triidothyronine 20% Winter Total cholesterol (slight)Triglycerides MENSTRUATION Increased/Decreased Serum iron and phosphate Cholesterol, lowest at ovulation CAFFEINE Increased/Decreased cAMP Free fatty acids Free ionic calcium Plasma renin and catecholamine SMOKING Increased/Decreased Plasma Epinephrine Carboxyhemoglobin, Hemoglobin, RBC, WBC, MCV, HDL-C

32. Tests Referred 01 st Jan 2013-30 th June 2013 OPDWards Chemical Pathology1,25,9321,26,887 Hematology79,84081,257 Microbiology32,15930,541 Total2,37,9312,38,685

33. Total Number of Errors OPD and Wards: 196405 (41%) OPD and Wards: 196405 (41%)

34. The Errors! Type of errorInpatientsOutpatients Hemolyzed sample Clotted sample Incorrect sample No Label or ID Insufficient sample Incomplete Info (blood group) Empty tube Total 67566 53700 33764 16725 7608 5862 7664 192889 828 1015 456 857 208 - 152 3516

35. Causes, Probable (?) Improper mixing Improper mixing Labeling by junior/untrained staff Labeling by junior/untrained staff Sample ordering system operated by Nursing staff Sample ordering system operated by Nursing staff Sample transport to lab by wards boys/ayas Sample transport to lab by wards boys/ayas Lack of knowledge about sampling requirements in PGT’s Lack of knowledge about sampling requirements in PGT’s

36. Review of the literature on laboratory errors Sector of the laboratory Lapworth and Teal Clinical chemistry Goldschmidt and Lent Whole laboratory Nutting et al. Primary care Plebani and Carraro Stat laboratory Stahl et al. Whole laboratory Hofg ä rtner and Tait Molecular genetic tests onsite survey (2 laboratories) Molecular genetic tests questionnaire (101 sent, 42 respondents) Preana- lytical phase 31.6%53%55.6%68.2%75%44%60% Analytical phase 31.6%23%13.3% 16%31%19% Postana- lytical phase 30.8%24%30%18.5%9%12.5%15% Multiple phases 6 %12.5%6%

37. Types of preanalytical errors at the Laboratory of San Raffaele Hospital, Italy Type of errorInpatientsOutpatients Hemolyzed sample Insufficient sample Incorrect sample Clotted sample Incorrect identification Lack of signature (blood group) Empty tube Lack or wrong compilation of the accompanying module Sample not on ice Tube broken in the centrifuge Test not reserved Urine not acidified Open container Module without signature Urine volume not indicated Total 8494 3256 1824 792 287 266 238 120 75 57 31 24 20 14 5 15,503 256 102 289 80 2 8 6 36 13 792

38. Error Prevention Phlebotomy Education Phlebotomy Education Phlebotomists should undergo thorough on-the-job training under the supervision of a senior phlebotomist Phlebotomists should undergo thorough on-the-job training under the supervision of a senior phlebotomist Continuing Education Continuing Education Phlebotomists should participate in regular educational competency assessments (written and observational) Phlebotomists should participate in regular educational competency assessments (written and observational) Phlebotomy Staffing Phlebotomy Staffing Adequate staffing to maintain collection standards Adequate staffing to maintain collection standards Technology Technology Use of barcode scanners for patient identification Use of barcode scanners for patient identification

40. Recognition of Pre analytical Variables Causing Changes in Laboratory Results A 55-year-old man was hospitalized with a serum potassium of 6.9 mmol/L on a non- hemolyzed sample obtained in an outpatient clinic. All other laboratory tests were normal. During hospitalization serum potassium values ranged from 3.9 - 4.5 mmol/L (normal 3.5 - 5.0 mmol/L).

41. Recognition of Pre analytical Variables Causing Changes in Laboratory Results In OPD, blood was collected with the application of tourniquet and fist clenching In the ward, blood was collected through an in- dwelling catheter Cause of pseudohyperkalemia was repeated fist clenching during tourniquet application intended to make the veins prominent The contraction of forearm muscles causes release of potassium. This effect can lead to a 1-2 mmol/L increase in potassium with as much as 2.7 mmol/L increase

42. Recognition of Pre analytical Variables Causing Changes in Laboratory Results Abnormal laboratory findings in a 43 year old male: Alkaline phosphatase 5 IU/L (normal 45-115 IU/L), calcium 0.5 mmol/L (normal 2.1 - 2.6 mmol/L) and potassium 22.0 mmol/L (normal 3.5-5.0 mmol/L) on a non-hemolyzed sample.

43. Recognition of Pre analytical Variables Causing Changes in Laboratory Results Plasma was obtained from blood collected in a tri potassium EDTA tube EDTA chelated magnesium and zinc required for the activity of alkaline phosphatase; EDTA also chelated calcium leading to its gross underestimation Potassium in EDTA was responsible for raised K + to a physiologically impossible level.

44. Discussion How are pre-analytical errors prevented in your laboratory? How are pre-analytical errors prevented in your laboratory? What do you do to prevent human error? What do you do to prevent human error? What systems does your hospital use to prevent errors by non-laboratory staff collecting blood? What systems does your hospital use to prevent errors by non-laboratory staff collecting blood? What pro-active improvements would reduce the number of pre-analytical errors? What pro-active improvements would reduce the number of pre-analytical errors?

45. Questions?