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

2. 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

3. 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

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

5. 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

6. 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.

7. 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

8. 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?”

9. 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.

10. Biological variation
RANDOM
Within subject
Between subject

11. 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.

12. 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.

13. 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.

14. 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

15. 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

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

18. 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

19. 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

20. 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

21. 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

22. 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

23. 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

25. 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

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

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

28. Sample processing Specimen separation REGISTRATION
Centrifuging
Aliquotting
REGISTRATION
Delivery to departments

29. 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

30. 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

31. 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

32. 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

33. 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!

34. Any Questions???