The coefficient of variation (CV) is the normalized expression of the SD, ordinarily articulated as a percentage (CV%). CV% is the most commonly used measure of dispersion in laboratory medicine. CV% is expressed without units (except percentage), thus making it possible to compare data sets that use different units. The computation formula is:
CV SD
x % 100
where CV% 5 coefficient of variation expressed as a percent- age; SD 5 standard deviation; and
x
5 meanCHAPTER 5 Quality Assurance in Hematology and Hemostasis Testing
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VALIDATION OF A NEW OR MODIFIED ASSAY
All new laboratory assays and all assay modifications require
validation.14 Validation is an activity comprised of procedures
to determine accuracy, specificity, precision, limits, and linear- ity.15 The results of these procedures are faithfully recorded and
made available to on-site assessors upon request.16
Accuracy
Accuracy is the measure of agreement between an assay value
and the theoretical “true value” of its analyte (Figure 5-1). Some statisticians prefer to define accuracy as the magnitude of error separating the assay result from the true value. By comparison, precision is the expression of reproducibility or dispersion about the mean, often expressed as SD or CV%, as discussed in a subsequent section, “Precision.” Accuracy is easy to define but difficult to establish and maintain; precision is relatively easy to measure and maintain.
For many analytes, laboratory professionals employ primary
standards to establish accuracy. A primary standard is a material
of known, fixed composition that is prepared in pure form, often by determining its mass on an analytical balance. The practitioner dissolves the weighed standard in an aqueous solution, prepares suitable dilutions, calculates the anticipated concentration for each dilution, and assigns the calculated concentrations to assay outcomes. For example, he or she may obtain pure glucose, weigh 100 mg, dilute it in 100 mL of buffer, and assay an aliquot of the solution using photometry. The resulting absorbance would then be assigned the value of 100 mg/dL. The practitioner may repeat this procedure using a series of four additional glucose solutions at 20, 60, 120, and 160 mg/dL to produce a five-point standard curve. The curve may be reassayed several times to generate means for each concentration. Standard curve generation is automated, how- ever laboratory professionals retain the ability to generate curves manually when necessary. The assay is then employed
Target Dispersion Value Frequency Gaussian, accurate, and precise Target Value Frequency Gaussian, inaccurate, but precise Target Value Frequency Gaussian, accurate, but imprecise Target Value Frequency
Gaussian, but inaccurate, and imprecise
Figure 5-1 The values generated by repeated assays of an analyte are graphed as a frequency distribution. Incremental values are plotted on the horizon-
tal (x) scale and number of times each value was obtained (frequency) on the vertical (y) scale. In this example, the values are normally distributed about their mean (symmetric, Gaussian distribution). Results from an accurate assay generate a mean that closely duplicates the reference target value. Results from a precise assay generate small dispersion about the mean, whereas imprecision is reflected in a broad curve. The ideal assay is both accurate and precise.
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PART I Introduction to Hematologyon human serum or plasma, with absorbance compared with the standard curve to generate a result. The matrix of a primary standard need not match the matrix of the patient specimen; the standard may be dissolved in an aqueous buffer, whereas the test specimen may be human serum or plasma.
To save time and resources, the laboratory professional may employ a secondary standard, perhaps purchased, that the vendor has previously calibrated to a primary standard. The secondary standard may be a preserved plasma preparation at a certified known concentration. The laboratory professional merely thaws or reconstitutes the secondary standard and in- corporates it into the test series during validation or revalida- tion. Manufacturers often match secondary standards as closely as possible to the test specimen’s matrix, for instance, serum to serum, plasma to plasma, and whole blood to whole blood. Primary and secondary standards are seldom assayed during routine patient specimen testing, only during calibration or when the assay tends to be unstable.
Regrettably, in hematology and hemostasis, where the ana- lytes are often cell suspensions or enzymes, there are just a hand- ful of primary standards: cyanmethemoglobin, fibrinogen, factor VIII, protein C, antithrombin, and von Willebrand factor.17 For
scores of analytes, the hematology and hemostasis practitioner relies on calibrators. Calibrators for hematology may be preserved human blood cell suspensions, sometimes supplemented with microlatex particles or nucleated avian red blood cells (RBCs) as surrogates for hard-to-preserve human white blood cells (WBCs). In hemostasis, calibrators may be frozen or lyophilized plasma from healthy human donors. For most of these analytes, it is impossible to prepare “weighed-in” standards; instead, calibra- tors are assayed using reference methods (“gold standards”) at se- lected independent expert laboratories. For instance, a vendor may prepare a 1000-L lot of preserved human blood cell suspension, assay for the desired analytes within their laboratory (“in- house”), and send aliquots to five laboratories that employ well- controlled reference instrumentation and methods. The vendor obtains blood count results from all five, averages the results, compares them to their in-house values, and publishes the aver- ages as the reference calibrator values. The vendor then distrib- utes sealed aliquots to customer laboratories with the calibrator values published in the accompanying package inserts. Vendors often market calibrators in sets of three or five, spanning the range of assay linearity or the range of potential clinical results.
As with secondary standards, vendors attempt to match their calibrators as closely as possible to the physical proper- ties of the test specimen. For instance, human preserved blood used to calibrate complete blood count (CBC) ana- lytes generated by an automated cell counter is prepared to closely match the matrix of fresh anticoagulated patient blood specimens, despite the need for preservatives, refrig- eration, and sealed packaging. Vendors submit themselves to rigorous certification by governmental or voluntary stan- dards agencies in an effort to verify and maintain the validity of their products.
The laboratory practitioner assays the calibration material using the new or modified assay and compares results with the vendor’s published results. When new results parallel published results within a selected range, for example 610%,
the results are recorded and the assay is validated for accuracy. If they fail to match, the new assay is modified or a new refer- ence interval and therapeutic range is prepared.
Medical laboratory professionals may employ locally collected fresh blood from a healthy donor as a calibrator; however, the process for validation and certification is labori- ous, so few attempt it. The selected specimens are assayed using reference instrumentation and methods, calibration val- ues are assigned, and the new or modified assay is calibrated (adjusted) from these values.
New or modified assays may also be compared to reference methods. A reference method may be a previously employed, well-controlled assay or an assay currently being used by a neighboring laboratory. Several statistics are available to com- pare results of the new or modified assay to a reference method, including the Student’s t-test, analysis of variance (ANOVA), linear regression, Pearson correlation coefficient, and the Bland-Altman plot.