Pre analytical variables affecting laboratory results

Pre analytical variables affecting laboratory results, Pre-analytical, variables, laboratory results, Pre analytical variables, total testing process, controllable & non-controllable factors

1. PRE-ANALYTICAL VARIABLES AFFECTING LABORATORY RESULTS DR. OFONMBUK UMOH 1

2. OUTLINE INTRODUCTION CLASSIFICATION OF PRE-ANALYTICAL VARIABLES OVERVIEW OF VARIABLES AND THEIR EFFECTS CONCLUSION REFERENCES 2

3. INTRODUCTION Laboratory services play a pivotal role of clinical decision making process. Effective laboratory service is the amalgamation of precision, accuracy and promptness. Recently, Johns Hopkins University School of Medicine in Baltimore made headlines when they estimated that medical error is the third leading cause of death in the United States. While patient safety remains a struggle in many areas of healthcare, laboratory medicine has been a leader in reducing error, with an estimated total error rate of 0.33%, the lowest in diagnostic medicine. 3

4. …introduction The classic paradigm of Total Testing Process beginning with Ordering of test to Reporting of test results, encompasses the Pre-analytical, Analytical and Post-analytical phases of testing. Of the laboratory-associated errors, pre-analytical errors currently accounts for up to 75% of all mistakes. While the likelihood of variation in any of these three phases is not negligible, the vast majority of laboratory variation emerges from the many factors affecting laboratory specimens prior to testing. 4

5. …introduction Sadly, since most activities in the pre-analytical phase are neither performed entirely in the clinical laboratory nor under the control of laboratory personnel, they are harder to monitor and improve. Besides, most laboratories often leave pre-analytical activities to healthcare personnel who have little to no formal training in laboratory medical practice. 5

6. CLASSIFICATION OF PRE-ANALYTICAL VARIABLES Pre-analytical processes includes all the steps that occur from test ordering until right before sample analysis. Pre-analytical variables is classified into; Controllable factors and Non-controllable factors 6

7. CONTROLLABLE FACTORS REQUEST & TEST SAMPLE FACTORS PHYSIOLOGICAL FACTORS POSTURE/RECUMBENCY CIRCADIAN VARIATIONS LIFESTYLE FACTORS FOOD & FASTING STATE HERBAL PREPARATIONS RECREATIONAL SUBSTANCES PHYSCAL FITNESS, EXERCSE AND TRAINING TRAVELS DIET STIMULANTS DRUGS 7

8. NON-CONTROLLABLE FACTORS AGE SEX RACE ENVIRONMENTAL MEDICAL CONDITIONS / COMORBIDITY 8

9. REQUEST & TEST SAMPLE FACTORS include: Test ordering, Patient identification Patient preparation Site Selection & Preparation Tourniquet Application & Time Venipuncture Technique… Use of appropriate sample bottles Order of draw 9

10. Correct Specimen Volume Proper sample Handling and Specimen Processing Serum or Plasma Samples ? Centrifugation Proper handling of blood samples Stability and storage for Whole Blood, Serum and Plasma 10

11. Test ordering Test requisition forms should contain a minimum of patient’s name, Age, Sex, Hospital Number, Location of patient, Clinical diagnosis and brief clinical information, if necessary. Ordering of the wrong test. This could be due to: Confusion over tests with similar names (such as 25-hydroxyvitamin D versus 1,25-dihydroxyvitamin D), Transcription errors and misinterpreted verbal orders, which occur when physicians do not place test orders themselves. 11

12. Patient identification Identification bracelet for in-patients, Positive Vocal identification (spell their name correctly) from the conscious & alert, or identification by staff/family member; for the unconscious. Label sample collection tubes with a minimum of Patients full name, proposed test, Date/time of collection, and the Laboratory processing serial number. 12

13. Patient preparation Patient should be informed, and given a brief education on the test to be performed. If pertinent for the test, it should be verified that the patient is fasting. Ideally, a patient should’ve remained in the same position for at least 30mins before a specimen is collected, and should be in the same likely position for the next similar specimen, e.g. supine if an inpatient, or sitting if outpatient. 13

14. Site selection Ante cubital and median cubital veins are the most suitable for use in a conscious patient. Basilic, cephalic or dorsal dorsum veins of the hands should be secondary sites for consideration – as they are prone to frequent trials and errors, thereby increasing stress and tourniquet time. Swab with alcohol – based antiseptic in cotton wool or gauze and allow to air-dry. Using the same / similar vascular site for serial sample is advised. Minor difference occurs from use of different vascular location. 14

15. Tourniquet time Tourniquet – while used to dam venous pool to facilitate easy location of a vein for venipuncture, application for longer than 1min begins to induce hemoconcentration. It mimics the effect of change from lying to standing position. Venipuncture technique Appropriately sized needles should be used, to lessen the possibility of hemolysis. Aim at successful venipuncture and blood draw with at most 2 attempts. No puncturing of skin before the alcohol used for skin asepsis has evaporated. 15

16. Correct sample bottles & sample volume Use of appropriate sample bottle as indicated for the test to be carried out. Collect adequate sample quantity as labelled on tube, unless in peculiar situations, e.g. neonates. Order of Draw Blood Culture Serum tubes Heparin tubes EDTA tubes Fluoride oxalate tubes 16

17. Proper sample handling and specimen processing No vigorous shaking with blood sample, to avoid massive hemolysis. Gentle rocking of the sample tube to mix, for anticoagulant tubes. (…) Sample collected into one type of tube should never be transferred into another tube. If blood collection site is distant from the laboratory, specimen should be collected into evacuated tubes containing thixotropic polymer gel and should be centrifuged on site. The gel forms an effective barrier between the separated serum / plasma and cells, so that no leakage of cellular constituents into the supernatant above the gel. 17

18. Serum or Plasma ? Generally, plasma allows for more rapid processing of specimen for clinical chemistry tests. But anticoagulants may interfere with some analytical methods. E.g. K+ and phosphate. Also, note that the type of urine specimen needed for different tests can be quite different. For e.g. the first morning urine specimen is usually the most concentrated, and appropriate for microscopic examination, while a collection of 24hr urine is appropriate for quantitative measurement. 18

19. Storage Within the laboratory, if specimen cannot be tested timely, it should be stored under appropriate conditions until testing can take place. For example, whole blood sample collected into Fluoride – Oxalate bottle and separated as soon as possible can last as long as 3 days at room temperature, or up to 21 days when frozen. 19

20. Prolonged bed rest: Prolonged bed rest can dramatically affects body constituents. Plasma volume and ECF volume decrease within few days Hematocrit increase by up to 10%. Creatine kinase also increases from muscle release. There is reduction in plasma protein concentration. Protein-bound constituents decreases. Serum potassium, reduce by 0.5mmol/L. Hydrogen ion excretion decreases – (!)Skeletal muscle metabolism. PHYSIOLOGICAL FACTORS 20

21. Calcium: Hypercalcemia, hypercalciuria, from bone resorption. The amplitude of circadian variation of cortisol is reduced with prolonged immobilization. As a result, some hospitalized patients should delay certain tests until after they leave the hospital and resume normal activity. When an individual becomes active after a period of bedrest, longer than 3 weeks is required before calcium balance is achieved. 21 …Prolonged bed rest:

22. 10% reduction in blood volume, equivalent to approximately 600-700mls. Resultant increase in plasma proteins by approx. 8-10%. 5 – 15% increase in concentration of protein-bound molecules Increase secretion of catecholamines, aldosterone, angiotensin II, Renin and ADH (Vasopressin). Increase in Heart Rate, Systolic & Diastolic blood pressures Posture / Recumbency In an adult, a change from lying to an upright position, results in: 10mins to happen & 30mins to revert back, on switching posture. 22

23. Decrease in GFR…, Decrease in urinary pH Reduced excretion of bicarbonate as Hydrogen ions are exchanged for Sodium. Increase in K+: 0.2 – 0.3 mmol/L Increases in: Calcium 3%, Cholesterol 7%, TG 6%, IgG 7%, IgA 7%, IgM 5%, Amylase 6% ALP 7%, ALT 7%, AST 5%, Albumin 9%, Thyroxine 11% 23

24. Exercise: Blood pressure, increase, Arterial pH is reduced Blood glucose, increased by up to 2folds (M), but hypoglycemia in prolonged strenuous exercise Lactate, urates, increase approx. 2folds, Strenuous exercise: increases plasma renin by 400% Cortisol secretion is stimulated, the normal diurnal variation may be abolished. Urinary free cortisol excretion and plasma concentration of cortisol, aldosterone, growth hormone and prolactin are increased. 24

25. Snapshot 25

26. Circadian variation: Cortisol: highest around 0600 and 0800 hours Renin & Aldosterone: maximum renin activity occurs in the morning during sleep. Minimum in the afternoon. Plasma aldosterone concentration shows a similar pattern. Growth Hormone: secretion is increased threefold to fivefold from its minimum around afternoon & night, to its maximum between midnight and waking. Thyroid Stimulating Hormone: TSH is maximum between 0200 and 0400, and minimum between 1800 and 2200…varying between 50 – 200% 26

27. LIFESTYLE Plasma urea conc. doubles, within 4days of change from a normal diet to a high protein diet, along with increase in urinary excretion. Serum cholesterol, phosphate, urates and NH3 conc also increase concomitantly. A high-fat diet, increases serum TG concentration, but depletes the nitrogen pool because of Nitrogen requirement for excretion of ammonium ions to maintain acid-base homeostasis. A high-carbohydrate diet decreases the serum concentrations of LDL-cholesterol, triglycerides, cholesterol, and protein The effects of a meal may be long lasting. For e.g. ingestion of a protein-rich meal in the evening may cause increases in concentration of serum urea nitrogen, phosphorus, and urate that are still apparent 12 hours later. Diet: 27

28. Food & Fasting state: Food ingestion is a significant source of pre-analytical variability. This effect varies based on the analyte and the time between meal ingestion and blood collection. For e.g , glucose and TGs significantly increase after meals with high carbohydrates and fat, respectively. An overnight fasting period of 10 to 14 hours prior to blood collection is optimal for minimizing variations. Glucagon and insulin secretions are stimulated by a protein meal, and insulin is also stimulated by carbohydrate meals. In response to a meal, the stomach secretes hydrochloric acid, causing a reduction in the plasma chloride concentration. 28

29. Venous blood from the stomach contains an increased amount of bicarbonate. This condition reflects a mild metabolic alkalosis (“alkaline tide”) and increased PCO 2 . The metabolic alkalosis is sufficient to reduce serum-free ionized calcium by 0.2 mg/dL (0.05 mmol/L). Caffeine ingestion stimulates adrenal medulla: increases plasma epinephrine by 2-3folds, increases plasma cortisol: free cortisol,, so much so great that the normal diurnal variation of plasma cortisol may be suppressed. Ingestion of two cup of coffee may increase the plasma free fatty acid concentration, glycerol, total lipids and lipoproteins by as much as 30%. 29 …Food & Fasting state

30. Malnutrition: Plasma protein – reduced . Complement C3, retinol binding globulin, prealbumin and transferrin decrease more rapidly . Plasma cortisol – increased , from free cortisol moiety , and decreased metabolic clearance. T3, T4 – reduced because of reduced TBG and Prealbumin . Vegetarianism Total lipids and phospolipids concentration – reduced to 2/3rd of normal diet . In strict vegetarians, the LDL-cholesterol concentration may be 37% less and the HDL-cholesterol concentration 12% less than in nonvegetarians. The cholesterol : HDL-cholesterol ratio is decreased. Urinary pH is usually higher in vegetarians than in meat-eaters Vitamin B12 & folate – reduced . 30

31. Travel Travel across several time zones affects the normal circadian rhythm. Five days is required to establish a new stable diurnal rhythm after travel across 10 time zones. Changes in laboratory test results are attributable to altered pituitary and adrenal function. Urinary excretion of catecholamines – increased for 2days. Serum cortisol – reduced. During a 20-hour flight, serum glucose and TG concentration increases, while glucocorticoids is stimulated. During a prolonged flight, fluid and sodium retention occurs, but urinary excretion return to normal after 2days. 31

32. Smoking: 32

33. Alcohol: Glucose metabolism: inebriation level induces transient hyperglycemia (20-50%) that inhibits gluconeogenesis, then results in hypoglycemia and ketonemia, as alcohol gets metabolized to acetaldehyde and acetate High lipids, as Liver TG formation is increased (up to 0.23mmol/L), and there is impaired removal of VLDL & Chylomicrons from circulation . AST and ALT activities – may be increased by 250% and 60% respectively. GGT, Cortisol, Lactate & Urate – all increased. 33

34. Drugs: Opiates – increased pancreatic enzymes Oral contraceptives – reduces HDL & increases LDL Thiazides - Hypokalemia, hypercalcemia, Hyperglycemia, hyperuricemia, prerenal azotemia, increased LDL-Cholesterol, total cholesterol and TGs Phenytoin – Reduces calcium and phosphate concentration (with risk of osteomalacia ), increases ALP activity and induces synthesis of Bilirubin conjugating enzymes in the liver. Also cause hyperglycemia and glycosuria. Barbiturates induce the hepatic cytochrome P450 enzyme system, which could interfere with the metabolism and subsequent plasma level of co-administered drugs. 34

35. NON-CONTROLLABLE FACTORS After puberty, the serum activities of ALP, ALT, AST, CK, and aldolase are greater in men than in women. Serum iron is low during a woman’s fertile years, and her plasma ferritin may be only one third of the concentration in men. Total cholesterol and LDL-cholesterol concentrations are typically higher in men than in women. Creatinine clearance is greater in men than in women. Sex: 35

36. Age: Newborn and jaundice: In infants, even in the absence of disease, the concentration of bilirubin rises after birth because of enhanced erythrocyte destruction. Newborn and low Blood glucose: RBC count in childhood lower than in adults. Childhood and Plasma Proteins. 36

37. Race: Total Proteins & Albumin: total serum protein concentration is known to be higher in blacks than in whites. This is largely attributable to a much higher γ-globulin. However, Serum albumin is typically less in blacks than in whites. In black men, serum IgG is often 40% higher and serum IgA may be as much as 20% higher than in white men Hemoglobin concentration is lower in blacks The activity of CK and LD is usually much higher in both black men and women than in whites. 37

38. Environmental: Altitude and Hemoglobin/blood gases: In individuals living at a high altitude, blood hemoglobin and hematocrit are greatly increased because of reduced atmospheric PO 2 . Erythrocyte 2,3-diphosphoglycerate is also increased, and the oxygen dissociation curve is shifted to the right….with increased erythrocyte production Plasma and urine concentrations of catecholamine are increased with increased altitude, up to twofold, largely caused by increase in norepinephrine secretion. Urinary Creatinine concentration and clearance are decreased as a result. 38

39. Medical conditions: Obesity: Serum concentrations of cholesterol, triglycerides, and β-lipoproteins are positively correlated with obesity. HDL-C reduces, LDL-C increases, Cortisol increases. Increased plasma concentration of acute phase reactants. Serum iron and transferrin concentrations are low. Pregnancy: Blood volume increases by about 2600 – 3500ml. Total protein reduces, mostly Albumin. Cholesterol and TG increases. Urine volume increases by up to 25%. Increased fibrinogen concentration. 39

40. Stress: Anxiety stimulates increased secretion of aldosterone, angiotensin, catecholamines, cortisol, prolactin, renin, growth hormone, TSH, and antidiuretic hormone. Plasma cholesterol, fibrinogen, glucose, insulin and lactate are increased by a large degree Plasma concentrations of albumin are decreased by up to 5%. Fever provokes lipolysis and glycogenolysis with hyperglycemia, which stimulates insulin secretion. Fever is often associated with a respiratory alkalosis caused by hyperventilation. 40

41. SUMMARY OF 41

42. CONCLUSION Major advancements in methodologies, automation and analytical instrumentation have helped increase accuracy over the last decade. To help reduce pre-analytical errors, vis -a- vis laboratory-associated errors, laboratory medicine professionals should also expand their focus to what is happening outside of the laboratory that can affect the outcome of test results. 42

43. THANK YOU 43

44. REFERENCES Tietz Clinical Chemistry and Molecular Diagnostics, 5 th Ed Bishops Clinical Chemistry – Principles, Techniques, Correlations, 7 th Ed. Makary MA, Daniel M. Medical error-the third leading cause of death in the us. BMJ 2016;353:i2139. Carraro P, Plebani M. Errors in a stat laboratory: Types and frequencies 10 years later. Clin Chem 2007;53:1338-42. Bonini P, Plebani M, Ceriotti F, Rubboli F. Errors in laboratory medicine. Clin Chem 2002;48:691-8. Standardization IOf . Iso 15189:2012: Medical laboratories: Particular requirements for quality and competence. Vol. Geneva, Switzerland, 2012. Young DS. Effects of preanalytical variables on clinical laboratory tests. 3rd ed. Washington, DC: AACCPress , 2007. 44