1. CHOICE OF METHODS AND INSTRUMENTS

2. Introduction
Choice of proper instrument or method of analysis is a major preventive quality assurance activity that will affect the quality of the service provided
The process of choosing a new instrument or kit can be divided into two parts:
Method selection
Method evaluation

3. A good quality assurance program has three major aspects

4. Choice of Methods Sources of information Method selection
Method evaluation

5. 1- Sources of Information
You need to have a full information before deciding to buy a new instrument or kit
The best sources of information are:
The laboratory technical literature (Journals)
It will have evaluations and recommendations for different methods and instruments
Use the literature to find the method's application, methodology, and performance characteristics

6. 1- Sources of Information

7. 1- Sources of Information

8. 1- Sources of Information

9. 1- Sources of Information
Conferences & Exhibitions
Vendors come to show their wares with demonstrations and discussions of their product
Word-of-mouth recommendations
From reliable and trustworthy sources
Contact others to find out what they are using and if they are happy or unhappy with their choice

10. 2- Method Selection
The goal of the selection process is to choose a test method that best suits the laboratory's service requirements
The primary consideration for making this selection should be based on the test method's usefulness in providing medically relevant data
The demands on the laboratory are imposed by the users of its services and this information can be determined by communicating with these users
In making the selection among various methods, the desired characteristics should be carefully considered

11. 2- Method Selection
Three classes of characteristics should be considered:
A- The Method's Application Characteristics:
These are the characteristics of a test method that determine whether it can or cannot be implemented in a particular laboratory, include factors such as:
Personnel
Turnaround time
Cost per test
Space and utility requirements
Rate of analysis
Types of specimens
Safety considerations
Run size, materials
Sample volume

12. 2- Method Selection B- Methodology Characteristics:
These contribute to the quality of the method such as:
Chemical sensitivity and specificity,
manner of calibration,
and optimization of the reaction conditions
C- Performance Characteristics:
Those that determine how well the method performs in its practical application. They include:
the method's linear range (also known as the analytical or working range),
its precision,
and its accuracy

13. 3- Method Evaluation
Once an instrument or test kit has been selected as a possible candidate for use, it should be carefully evaluated before making a final commitment
Goals of the evaluation includes:
The determinations to be made in a method evaluation are the imprecision and inaccuracy, which should be compared with the maximum allowable error based on medical criteria

14. 3- Method Evaluation
Imprecision is estimated from studies in which multiple aliquots of the same specimen (with a constant concentration) are analyzed repetitively (Replication Experiment)
Inaccuracy, or the difference between a measured value and its actual value, is due to the presence of a systemic error
Systemic error can be due to constant or proportional error and is estimated from Recovery study and interference study

15. 3- Method Evaluation
Experiments and type of error which can be detected

16. 3- Method Evaluation Total error (TE)
Accept that all lab measurements contain experimental error
Lab error should be:
Smaller than CLIA (or other regulatory) requirement
Consistent with manufacturer’s claims
Compatible with patients’ care
CLIS: Clinical Laboratory Improvement Amendments
The Clinical Laboratory Improvement Amendments of 1988 statute is an amendment to the Public Health Services Act in which Congress revised the federal program for certification and oversight of clinical laboratory testing. Two subsequent amendments were made after The law continues to be cited as CLIA ’88 as named in legislation.

17. 3- Method Evaluation Total error (TE)
Magnitude of Error – TE
TE is the total maximum error of a test as measured in the lab
TE = random + systematic errors
Determined
For each given method
At various medical decision levels

18. 3- Method Evaluation Medical Decision Levels
What are Medical Decision Levels?
Medical Decision Levels (MDL) are the analyte values at which medical professionals can determine whether a patient may be suffering from a certain condition.
The MDL is determined by a consensus of medical professionals and clinical research.
Patients’ test results are compared to the MDL and appropriate diagnoses or medical interventions can be made.

19. 3- Method Evaluation Medical Decision Levels
Test
Units
Reference
Interval
Decision Levels 
METABOLITES 1 2 3
Bilirubin
mg/dL
1.4
2.5 20
Cholesterol 90
240
260
Creatinine
0.6
1.6 6
Glucose
60-95 45
120
180
Iron
ug/dL
50-165 50
220
400
Triglycerides
20-180 40
150
Urea Nitrogen (BUN)
8-26 26
Uric acid 8
10.7

20. 3- Method Evaluation Allowable Total Error (TEa)
Probably the most important aspect of method evaluation is to determine if the random and systematic errors (total error) are less than the TEa
Total Analytical Error < Total Allowable Error
The error that can be tolerated without invalidating the interpretation of a test result.
The Clinical Laboratory Improvement Amendments of (CLIA 88) have published Eas for an array of clinical tests
physiologic variation, pathologist judgment

21. 3- Method Evaluation Allowable Total Error (TEa)
TEA is the total error permitted by CLIA, based on:
Medical requirements
Best available analytical method
Compatible with proficiency testing expectations

22. 3- Method Evaluation Allowable Total Error (TEa)

23. 3- Method Evaluation Analytical Measurement Range (AMR)
Range of analyte where results are proportional to the true concentration of analyte in the sample
Range over which the test can be performed without modification (no dilutions or concentrations)
Determined by Linearity Check
The manufacturer defines the AMR.
It is the laboratory’s responsibility to verify it.

24. 3- Method Evaluation Analytical Measurement Range (AMR)
Maximum Dilution/Concentration (formerly Clinically Reportable Range – CRR):
Range of analyte values which are clinically significant
Can be reported following modification (such as dilutions)
CRR is typically wider than AMR.
Values greater than or less than the CRR are reported as greater than (>) or less than (<).
Dilution or concentration protocols must be documented for each analyte.

25. 3- Method Evaluation Analytical Measurement Range (AMR)
Analyte
Ref Range
AMR
Max Dilution
CRR
Albumin
mg/dL
None
ALT
13-69 IU/L
6-1000 15
1.0-15,000.0
Cholesterol
mg/dL
50-325 3
50-975 CK
IU/L
100
Actual result
Creatinine
mg/dL 2
Potassium
meq/L
CRR should always be wider than the reference range.

26. 3- Method Evaluation Analytical Measurement Range (AMR)
How to perform linearity check:
An aqueous or protein-based standard of high concentration is diluted to obtain a series of samples of lesser but known concentration.
The range of samples should cover both ends of the method’s range of linearity.
Analyze the samples and compare the results to the expected values by constructing a scatter plot.
Place the expected value on the x-axis and the obtained value on the y-axis and draw a line of agreement.

27. 3- Method Evaluation Analytical Measurement Range (AMR)
Example of linearity experiment (glucose method with a published range from mg/dl)
A known glucose standard of 500 mg/dl is diluted (as shown in the figure) with deionized water
All dilutions should be made from a stock sample avoiding serial dilutions
Dilution errors are magnified in serial dilutions
Use volumetric glassware and good pipetting technique when preparing the dilutions

28. 3- Method Evaluation Analytical Measurement Range (AMR)
Analyze each sample in triplicate and average the results.
Accept the method's linearity if the line of agreement has a slope of ± 0.03 and an intercept of zero plus or minus the within-run precision of the method.
If the within-run precision of the method is not known, it will be determined in the next series of experiments; consequently this part of the evaluation may not be completed until the replication experiments are completed.
Investigate pipetting errors as the probable cause of a nonlinear line by repeating the experiment with a freshly diluted series of samples.

29. 3- Method Evaluation Analytical Measurement Range (AMR)
If the method is not linear over its published range, stop and investigate the problem before any further experiments are carried out.
When a method loses linearity on either end of its range the line will start to curve.

30. Glucose Colorimetric Assay Kit
Cayman Chemicals Co.

33. 3- Method Evaluation Precision/ Replication Experiments
Part of the process of verifying or validating a method to confirm that it is suitable for use is an assessment of precision.
By this we mean the closeness of agreement between independent results of measurements obtained under specified conditions.
It is solely related to the random error of measurements and has no relation to trueness/accuracy
Determined by Replication Experiments

34. 3- Method Evaluation Precision/ Replication Experiments
This evaluation experiment is used to demonstrate a test method's precision and random error
Three different replication studies are performed:
within-run,
within-day,
and day-to-day
Choose three samples that have the same matrix or physical qualities as the patient samples that the method will be analyzing
The three should represent the low, normal, and high physiological concentrations of the analyte in question

35. 3- Method Evaluation Precision/ Replication Experiments
For most chemical constituents, lyophilized control serum or some similar stable material should be used for the day- to-day replication experiments
Though there are some differences between prepared controls and patient specimens, the control material will remain stable over the 20-day period and chances of obtaining it in the desired concentrations and quantities are greater than using only patient samples

36. 3- Method Evaluation Precision/ Replication Experiments
The within-run replication experiment measures precision or the lack of it caused by random error within an analytical run
Each sample is analyzed a minimum of 20 times within a single analytical run
The within-day replication experiment measures the amount of random error between runs that occurs within a single day
Each of the three samples is analyzed a minimum of 20 times throughout the day in several analytical runs
The day-to-day replication experiment measures the amount of random error inherent in the method from day-to-day
Analyze each sample daily for a minimum of 20 consecutive days

37. 3- Method Evaluation Precision/ Replication Experiments
For each of the replication experiments, calculate a mean, mode, standard deviation (s), and coefficient of variation (CV) for each sample
The greater the imprecision of the method, the larger the standard deviation will be
If the distribution of the values is due to random chance, then it should have a normal or Gaussian distribution with the mean equal to the mode
If the distribution is skewed as the result of some random error, then the mode and the mean will not be equal

38. 3- Method Evaluation Precision/ Replication Experiments
A method lacks precision if it has a large coefficient of variation and there is a substantial difference between the mean and the mode
Such random error is unacceptable and potentially dangerous to the patient
Ninety-five percent of the values should fall within two standard deviations of the mean
One in 20 samples will be beyond the 2s limits
If more than 1 sample in 20 or 2 in 40 are beyond 2s for unexplained reasons (other than acknowledged errors in pipetting, short sampling, or some other obvious problem) the method should be discarded as having too great a degree of random error and thus is potentially dangerous to the patient

39. 3- Method Evaluation Precision/ Replication Experiments
The acceptable CV will vary, depending on the medical significance of the analyte that the method is measuring
The within-run and within-day replication experiments are optimistic evaluations of the method’s precision but are useful in determining if the method is performing properly
The day-to-day replication experiment is a better indication of the method’s overall precision

40. 3- Method Evaluation Precision/ Replication Experiments
What amount of random error is allowable, based on CLIA criteria?
Short term: 0.25 of allowable total error
Long term: 0.33 of allowable total error
The judgment on acceptability depends on what amount of analytical error is allowable without affecting or limiting the use and interpretation of a test result

41. 3- Method Evaluation Precision/ Replication Experiments

42. 3- Method Evaluation Recovery Experiment
The recovery experiment is performed to estimate the proportional systematic error in the absence of a reliable comparative or reference method
This is the type of error whose magnitude increases as the concentration of analyte increases
The error is often caused by a substance in the sample matrix that reacts with the sought for analyte and therefore competes with the analytical reagent
If the recovery is different from 100%, proportional error exists

43. 3- Method Evaluation Recovery Experiment
A patient sample is spiked with varying amounts of pure standard and then analyzed
A baseline sample and samples with concentration at the medical decision levels should be prepared
After analyzing the samples in triplicate and averaging the values for each, the expected results are compared to the actual results and the degree of proportional systematic errors defined as the percent of the added analyte recovered
Westagrd states that the limits of acceptability occur when the percentage of error is less than the determined allowable analytical error at the medical decision levels

45. 3- Method Evaluation Recovery Experiment
Interpretation
This method has a 90% recovery rate or a 10% proportional systematic error based on this experiment
The acceptable level of recovery or accuracy will vary, depending on the analyte and the concentration

46. 3- Method Evaluation Recovery Experiment
Let us evaluate proportional error at 11 mg/dL
10/100*11= 1.1 mg/dl
A proportional error of 1.1 mg/dL is clearly more than the CLIA total error limit of 1 mg/dL at 11 mg/dL,
Hence the conclusion is that recovery is unacceptable.