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. 1- 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

5. 1- 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

6. 1- 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

7. 2- 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
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

8. 2- SOURCES OF INFORMATION

9. 2- SOURCES OF INFORMATION

10. 2- SOURCES OF INFORMATION

11. 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
The evaluation process should be logically structured so that a minimum of time and effort is invested to obtain maximum results
Goals of the evaluation includes:
The first 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
Discover if the new method fits into the framework of the laboratory's organization and workload

12. 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

13. Allowable Analytic Error (Ea)
Probably the most important aspect of method evaluation is to determine if the random and systematic error (total error) is less than the Ea
The error that can be tolerated without invalidating the medical usefulness of the test
The Clinical Laboratory Improvement Amendments of 1988 (CLIA 88) have published Eas for an array of clinical tests
physiologic variation, pathologist judgment

14. Allowable Analytic Error (Ea)

15. 3- METHOD EVALUATION
The evaluation should last for no less than one week and no more than 60 days.
Barnett and Westgard recommend a minimum of 20 days for a complete evaluation that includes day-to-day precision studies.
Enough time should be allowed to perform all of the necessary evaluation experiments and to observe the instrument's day-to-day variation.
Too short a time period may result in important data being missed or misrepresented.
Too long an evaluation period may last longer than the expiration date of reagents, calibrators, and controls, and also incorporate long-range instrument variation.

16. A- Instrument and Method Evaluation Steps:
Construct a good evaluation plan and establish a working procedure detailing calibration, operation, and maintenance during the evaluation period.
Acquire enough reagents, calibrators, and controls to last the evaluation period.
The plan should be designed as a series of logical steps so that failure in the preliminary steps will cancel the continuation of the process.

17. A- Instrument and Method Evaluation Steps:
The course that the evaluation process takes will depend on the availability of an acceptable comparative method
If the comparative method has satisfactory and well-documented precision and accuracy, then the evaluation should determine if the new method is as accurate and precise as the old.
If a satisfactory comparative method is not available for comparison, then other experiments should be used to determine the new method's precision and accuracy.
If possible, the entire evaluation should be conducted by a single individual.
Variations between individual techniques and bias can be excluded if one person, conducts all of the experiment.

18. B- Linearity Check
The first step in the Evaluation process is to perform a Linearity Check
It is important to assess the useful analytical range of a laboratory method,
i.e., the lowest and highest test results that are reliable and can be reported
This confirms that the instrument or method is linear over its published working range stated by Manufacturers

19. B- Linearity Check 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

20. B- Linearity Check
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

21. B- Linearity Check
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.
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.

22. Glucose Colorimetric Assay Kit
Cayman Chemicals Co.

25. Experiments and type of error which can be detected

26. C- Replication Experiment
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

27. C- Replication Experiment
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

28. C- Replication Experiment
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

29. C- Replication Experiment
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

30. C- Replication Experiment
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

31. C- Replication Experiment
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

32. D- 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

33. D- 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 (95% of the limit of allowable error) at the medical decision levels

34. D- Recovery Experiment
If it is determined that the allowable error of a value is limited to 95% of that value without any harm or influence on the physician’s course of treatment, then any proportional systematic error greater than 5% at that decision level will judge the method to be unacceptable

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

37. E- Interference Experiment
The interference experiment is performed to estimate the systematic error caused by other materials that may be present in the specimen being analyzed
We describe these errors as constant systematic errors because a given concentration of interfering material will generally cause a constant amount of error regardless of the concentration of the analyte being tested in the specimen
As the concentration of interfering material changes, however, the size of the error is expected to change

38. E- Interference Experiment
The volume of the interferer should be small relative to the original test sample to minimize the dilution of the patient specimen
However the amount of dilution is not as important as maintaining the exact same dilutions for the pair of test samples
Using the test method in question, a sample is analyzed in triplicate to determine a value
A specific amount and concentration of a substance thought or known to be an interfering substance is added to this sample and the sample is then reanalyzed

39. E- Interference Experiment
The difference between the result of the sample with and without the substance is calculated
The sample is repeated with several different concentrations of the interfering substance
The acceptability of this experiment, according to Westagrd, is that the difference between the two values, one with and the other without interfering substance, should be less than the allowable analytical error at that concentration

40. E- Interference Experiment
Interference to be tested
Bilirubin can be tested by addition of a standard bilirubin solution
Hemolysis is often tested by removing one aliquot of a sample, then mechanically hemolyzing or freezing and thawing the specimen before removing a second aliquot.
Lipemia can be tested by addition of a commercial fat emulsion, such as Liposyn (Abbott Laboratories) or lntralipid (Cutter Laboratories)
Additives to specimen collection tubes can be conveniently studied by drawing a whole blood specimen, then dispensing aliquots into a series of tubes containing the different additives

41. E- Interference Experiment
As example, determination of glucose will be used
Creatinine is an interfering substance in this method. By adding creatinine to a sample in measured amounts the amount of interference can be determined
Prepare the sample as follows:
Sample #1, 0.9 ml serum ml deionized water (baseline)
Sample #2, 0.9 ml serum ml of 50 mg/dl creatinine STD
Sample #3, 0.9 ml serum ml of 100 mg/dl creatinine STD
Each sample is prepared and run in triplicate and the results are averaged.

42. E- Interference Experiment
Results:
Amount of glucose measured
Amount of Creatinine added
Interference
Sample # 1
100 mg/dl
0 mg/dl
Sample # 2
104 mg/dl
5 mg/dl
+ 4 mg/dl
Sample # 3
111 mg/dl
10 mg/dl
+11 mg/dl

43. E- Interference Experiment
Calculations:
Amount of creatinine added
Amount of Interference
Interference = Conc. Measured (test) — Conc. Measured (baseline)
Interference = mg/dl — mg/dl = 4 mg/dl
Conc. Added =
STD. Conc. X
ml STD. Added
ml of serum+ ml of STD
Conc. Added =
50 mg/dl X
0.1 ml
= 5 mg/dl
0.1 ml ml

44. E- Interference Experiment
Interpretation:
The experiment shows that increasing amounts of creatinine will result in an increasing positive interference
The acceptable level of recovery and accuracy will vary depending on the analyte and the concentration
In this case, further investigating is recommended

45. F- Comparison Of Methods
The comparison of methods is a procedure that determines accuracy and precision by comparing the test method to a method of known precision and accuracy
The comparative method can be either a recognized reference method (is one that has a well documented low level of imprecision and inaccuracy) or a method of analysis that the laboratory wishes to replace.
Choose a method that is well documented and widely accepted
Check the comparative method carefully during the experiment to ensure that it is performing properly
Run controls and use proper procedures in performing the comparison on both methods

46. F- Comparison Of Methods
When using a known reference method, all of the observed differences between the methods can be attributed to the new method.
When the comparative method is the one that is being used and the bias and analytical error is known, then part of the observed analytical error noted between the two methods can be attributed to the comparative method, with the remaining error belonging to the test method
The difference between the two methods at medical decision levels should be less than the allowable analytical error for that concentration of the analyte

47. F- Comparison Of Methods
This experiment involves simultaneously analyzing split samples on both the test and comparative method
Only patient samples should be used for evaluation between methods
Lyophilized, aqueous, or ethylene glycol-based control samples have slightly different physical properties than fresh human serum or plasma and may react differently in different systems
Westgard recommends that each sample be run in duplicate, at different analytical runs
This is done in order to check on the validity of the experimental observations and to detect random errors, such as mixing up of samples and short sampling

48. F- Comparison Of Methods
The replicates should be analyzed the same day on the same instrument and within as short a time as possible
The difference between the duplicate measurements should be less than or equal to the determined between-run precision
If the differences are grater than this, suspect an error
Analyze a minimum of 40 samples, 5 samples a day for 8 days
Select a variety of concentrations so that the entire linear or working range of the new method will be represented
This requires the pre-selection of samples but comparing samples only in the " normal " range can cover analytical differences between the methods at medical decision levels

49. F- Comparison Of Methods
Reduce opportunities for bias and mistakes to occur:
Limit the number of technologists participating in the experiment.
Try to run samples on both instruments at the same time to limit the effects of sample deterioration.
Use acceptable techniques in preparing reagents and samples for analysis.

50. F- Comparison Of Methods
There are several ways of approach in evaluating the comparison between two methods:
Plot the results on a scattergram
Linear regression analysis
The paired t-Test
The F-Test
The correlation of coefficient

51. F- Comparison Of Methods
One of the easiest and most visually studied is to plot the results on a scattergram, draw a best straight line through the points, and determine the slope and intercept of the line
The slope and the intercept offer a fair evaluation of the agreement between the methods
Data should be plotted on the scattergram on a daily basis and inspected for outliers so that original samples can be reanalyzed as needed
If the technologist waits until all of the samples are analyzed there will be no opportunity to investigate outliers or to reanalyze samples

52. F- Comparison Of Methods
Plot the test method on the y-axis and the reference or comparative method on the x-axis.
A best fit straight line can be drawn through the plotted points
A visual inspection of the line and the points around it can give a preliminary assessment of the agreement between the methods

53. F- Comparison Of Methods
Perfect correlation (Hypothetical) Slope (b) = 1 Y intercept (a) = 0 Correlation coefficent (r) = 1