MANAGING PRE-ANALYTICAL FACTORS Dr AE Zemlin 2nd Biennial Congress of the African Federation of Clinical Chemistry HUQAS Session 28th-30th September 2011

Introduction Laboratory analytes subject to several sources of variation Biological variation (within and between person variation) Pre-analytical variation (patient status, specimen collection and transport, specimen processing and delivery to the instrument) Analytical variation (bias and imprecision) Postanalytical variation (reporting of results) Also prepre and postpost

BIOLOGICAL VARIATION TOTAL VARIATION PRE- ANALYTICAL VARIATION Total variation is the sum of all errors, I.e. every step in the process of obtaining that result, from patient preparation to the eventual reporting of the result. These include biological variation, pre-& post- analytical variation as well as the analytical variation. PRE- ANALYTICAL VARIATION ANALYTICAL VARIATION POST- ANALYTICAL VARIATION

Introduction (2) Each laboratory is composed of multiple departments Each department has numerous tests Each test has specific requirements

Introduction (3) Clinicians relying more and more on biochemical markers for early detection, diagnosis, and prognosis of patients Accuracy of the clinical laboratory is an important component of quality patient care

Biological variation NON RANDOM Lifespan Daily rhythms Monthly rhythms Seasonal rhythms Biological variation is exactly what it says, variation of biological characteristics. It becomes important when assessing lab results, lack of consideration for this characteristic may lead to inappropriate interpretations and hence perhaps incorrect management.There are two types of biological variation, the first being the non- random BV which is the predictable form of BV.

NON-RANDOM BIOLOGICAL VARIATION LIFESPAN This includes the ageing process. Neonates have lower creatinine and urea and higher Hb and FFA levels than adults; while pregnant women have lower urea and uric acid levels and non-pregnant women. The graph shows the changes associated with age of 2 analytes: cholesterol and urea increase with age. Fraser CG. Biological Variation: From principles to practice. 2001

NON-RANDOM BIOLOGICAL VARIATION DAILY RHYTHMS Daily rhythms exist for certain analytes. E.g. cortisol is highest in the morning and lowest at midnight. Thus when testing for levels of these analytes, the rhythm must be taken into account. Standardised timing and reference ranges at specific times are crucial in order to derive adequate information. Prof JS Koeslag. “Are the fat soluble hormones really hormones?” Http://academic.sun.ac.za/medphys/hormone.htm

NON-RANDOM BIOLOGICAL VARIATION MONTHLY RHYTHM The monthly cycle of the female reproductive hormones is a good example of a monthly rhythm. Here, again, it is important to develop good reference ranges for each point during the cycle. Samples should be taken at specific times. E.g. progesterone should be taken at day 21 when assessing ovulation. So again, knowledge of the expected cyclical behaviour and differences in values are important.

Biological variation RANDOM Within subject Between subject

WITHIN-SUBJECT BIOLOGICAL VARIATION (CVI) Random fluctuation around a homeostatic set point This graph illustrates the within-individual variation of 4 analytes testing in the same individual over a period of 4 years. You can see that there is fluctuation around a set point for each of these analytes.

BIOLOGICAL VARIATION (CVI) (%) ANALYTE BIOLOGICAL VARIATION (CVI) (%) ABSOLUTE Arbitrary Set Point Variation Sodium (s) 0.7 140 138 142 Potassium 4.8 4.3 3.9 4.9 Calcium 1.9 2.25 2.21 2.29 Creatinine 88 81 95 Albumin 3.1 45 42 48 Cholesterol 6.0 4.0 3.5 4.5 The within-subject or intra-individual biological variation has been ascertained for various analytes. These can be accessed on the internet (Westgard website). Here are a few examples. Sodium has a rather tight within individual biological variation of 0.7%. Thus, if we chose an arbitrary set point for sodium of 140 in a patient, the values can vary between 138 and 142 in this patient at any given time point. http://www.westgard.com/biodatabase1.htm

1 2 3 4 60 63 66 62 SERUM CREATININE CONCENTRATION IN A HEALTHY MAN OVER A 14 DAY INTERVAL Serum creatinine was determined 4 times over a period of 14 days in a healthy male. The intra-individual biological variation is apparent (with a set point of approx 60). The same process was performed on 9 other healthy males. Apparent from this data are the different set points for each individual. This is the between subject or inter individual biological variation.

BETWEEN SUBJECT BIOLOGICAL VARIATION (CVG) Different homeostatic set points in different individuals MAN 1 2 3 4 60 63 66 62 103 99 110 107 88 85 93 86 125 120 115 118 5 75 83 78 6 92 98 90 96 7 70 68 71 8 105 9 72 81 74 10 77 Serum creatinine was determined 4 times over a period of 14 days in a healthy male. The intra-individual biological variation is apparent (with a set point of approx 60). The same process was performed on 9 other healthy males. Apparent from this data are the different set points for each individual. This is the between subject or inter individual biological variation. SERUM CREATININE CONCENTRATIONS (uM) IN 10 HEALTHY MEN OVER A 14 DAY INTERVAL

Pre-analytical errors Any factor which occurs prior to analysis and affects the result of a pathology test There is a perception that errors occur mainly in the analytical phase due to instrument malfunction ….. Most occur in extra-analytical phases (Lippi et al) Pre-analytical errors constitute > 60% of important pathology errors

Potential pre-analytical errors Test requesting Patient preparation Timing of collection Patient identification Collection process Sample transport and handling

Impact of pre-analytical phase on laboratory test outcomes Pre-analytical phase is complex and labour intensive Many steps in specimen collection Potential for error increases as number of steps increase

Effects of pre-analytical errors Minor Major Detected in laboratory Need to recollect Inconvenience for patient / doctor Increases TAT Wasted effort Error not detected Result accepted Patient wrongly treated Detrimental to patient outcome

Test requesting Prepre: clinician needs to choose correct test Form must be correctly filled in – all relevant information Clinician must order correct test on correct patient Writing must be legible Information must be correctly transcribed

Patient preparation / Timing Fasting: glucose, TG Special diet: OGTT, 5-HIAA Time of day: bone markers, UAE Drugs Time since last dose: digoxin Time since change of dose: phenytoin Time in menstrual cycle: progesterone

Patient identification Confirm – minimum of 2 positive identifiers Attached wrist band Specimen and form must have same identification Bar coded scanning device that prints labels at bed side Radiofrequency identification (RFID) – chip follows sample from order through analysis - would be ideal

Sample collection Ensure correct tube – fill completely Drip arm EDTA (ethylene diamine tetra-acetic acid) Di-potassium EDTA – beware carryover NaF for glucose Drip arm Prolonged tourniquet Skin preparation Fist clenching Haemolysis – most common error Thrombocytosis Tourniquet – not > 1 min

Handling of tubes post collection Invert – 3 – 10 times If not, improper clotting or anticoagulation Do not shake - haemolysis Protect from light if necessary Chill or warm Avoid excessive heat or cold – haemolysis and deterioration of analytes Never expose whole blood to dry ice - haemolysis

Transportation As efficiently as possible Pneumatic tube Rapid May cause haemolysis Correct temperature

Sample delivery Delays “Add on’s” Before centrifugation After centrifugation At room temperature In the fridge “Add on’s”

Sample processing Specimen separation REGISTRATION Centrifuging Aliquotting REGISTRATION Delivery to departments

Sample processing – pre-analytical issues For serum samples – allow complete clot formation to occur Centrifuge properly to prevent haemolysis Centrifuge only once Fibrin clot in test tube

Sample processing – pre-analytical issues (2) Haemolysis Prolonged tourniquet Alcohol swab Small bore needle Tissue trauma Occlusion of needle lumen by vein wall Large bore needle and syringe causing increased pressure with plunger Shaking of tube Freezing red blood cells for transport Excessive heat during transport Prolonged contact of serum or plasma with cells

Shared specimens Prioritize – perform stat test first Split into appropriate volume aliquots Divide into proper containers Store at correct temperature Deliver promptly to designated areas Notify staff if urgent

Impact of pre-analytical errors Occur too frequently! Specimen rejection and recollection - TAT Delay or inaccuracy in diagnosis and treatment Possible compromise in patient safety

CONCLUSION Emphasis on laboratory errors changed – now focuses on pre- analytical Greater care needs to be taken in this phase Staff involved in this phase often unaware of the importance of their duties (clinicians and pre-analytical staff) Our duty to perform audits and use these results to educate the relevant staff DO NO HARM!

Any Questions???