I would like you to take the responsibility for creating an Excel file to calculate all the necessary statistics related to the serology validation.
It needs to include calculation areas specific to Linearity, Limit of Detection, Precision (QC & Cut-off), Reference Range and Comparison testing.
It would be good if it could also include a “Front page” that summarizes the results from each area.
• A minimum of 20 replicate determinations on at least two levels of control materials are recommended to estimate the imprecision or random error of the method. Should also use a sample to focus on precision at cut-off level with assays that use a cut-off.
• Can be done using QC or patient samples.
Ø Method Comparison (Accuracy)
• A minimum of 40 patient specimens should be analyzed by the new method (test method) and an established method (comparison method) to estimate the inaccuracy or systematic error of the method.
• A minimum of 5 specimens with known or assigned values should be analyzed in triplicate to assess the reportable range. Can use two samples (Negative & Positive) and perform a dilution series of 5 samples
• One Low and One High sample assayed in triplicate as a batch to assess Negative and Positive carry-over between adjacent samples.
Ø Analytical Sensitivity (Limit of Detection)
• Generally, a “blank” specimen and a specimen “spiked” with the amount of analyte in the manufacturer’s claim for the limit of detection are each analyzed 20 times. Only applicable to Quantitative assays.
Ø Specificity (interfering substances)
• Common interferences, such as lipemia, hemolysis, and elevated bilirubin are usually tested, along with potential interferences that are specific to the test and methodology. Recovery experiments are used to test competitive interferences, such as the possible effects of proteins and metabolics in the specimens.
o Qualitative: Negative & Positive QC.
o Quantitative: ≥3 levels – low, medium & high concentration samples.
o Perform measurement 4 times per day (2 am & 2 pm) for 5 days.
o Calculate CV% for intra- and inter-batch runs, as well as all results (Total).
Ø Cutoff Precision
o 3 patient samples – Cutoff and 10% above & below C/O.
o Perform measurement 2 times per day for 10 days.
o Calculate % and perform statistical evaluation.
Ø Method Comparison
o Qualitative assays: 10 Negative & 10 Positive
o Qualitative assays (based on C/O or titre): 40 patient samples comprising 15 Negative, 15 positive & 10 Equivocal
o Quantitative assays: 40 patient samples (across AMR of assay).
o Assay each sample and compare between results from comparative method.
o Use a Negative sample and a High positive sample, indicative of either end of AMR. Can be QC or patient sample.
o Create dilution cascade with 5 samples, as follows:
1. Undiluted Negative sample
2. 3 parts Negative + 1 part Positive
3. 2 parts negative + 2 parts Positive
4. 1 part Negative + 3 parts Positive
5. Undiluted Positive sample
o Perform each measurement in duplicate (use average result for calculations).
o Applicable to Quantitative assays only.
Ø Reference Range Verification
o 20 samples from “normal, healthy” patients.
o Measure each sample and compare percentage results to “Expected” results.
o Use 3 samples – 1 Negative, 1 High positive (within linear range) & 1 around C/O.
o Can be QC or patient sample.
o Assay samples in the order of Med – High – Low – Med – Med – Low – Low – High – High – Med
o Measurements are made in above order as a single batch repeated over 5 days.
o Applicable to systems that do not use disposable pipettes.
o Use a blank sample (QC, Calibrator or patient) and a sample at the manufacturer defined LOD.
o Assay each sample 20 times.
o Calculate LOB = Mean(blank) +1.645(sd(blank))
o Calculate LOD = LOB+1.645(SD(low conc sample))
o Applicable to Quantitative assays only.
o Can use manufacturer recommendations. No need to perform additional experiments.
Ø Quality Control Material
• Assayed or unassayed Commercial materials.
Ø Patient Samples
• Samples from other institutions with known results.
Ø “Spiked” samples
• Patient samples with Negative (or low concentrations) of analyte that are mixed with samples of known Positive (or High concentrations).