**4.5. Stability**

*4.2.2. Clinical specificity*

**4.3. Sensitivity of PNH assays**

lower levels should be validated in each laboratory.

*4.3.1. Analytical sensitivity*

*4.3.2. Functional sensitivity*

*4.3.3. Clinical sensitivity*

The clinical specificity or the ability to exclude abnormal specimen defined by true negatives/ true negatives + false positives should be determined by assay of a series of samples and scoring for abnormality in comparison to a suitable reference method, such as clinical diagnosis

The analytical sensitivity of PNH assays is determined by the limit of blank (LoB) defined by the highest apparent signal detected in replicates of a sample containing no measurand and the limit of detection (LoD) defined by the lowest level of measurand that can be reliably distinguished from the LoB [28]. LoB for PNH assays could be determined by measuring a few replicates of a few negative specimens run over a few separate days and calculating the mean and standard deviation (SD) according to: LoB = mean of blank +1.645 SD of blank, assuming that 95% of negative values will be below this limit. Typically, the LoB for wellestablished PNH assays is <0.001% (<10 PNH phenotypes out of 1,000,000 acquired events). LoD for PNH assays is closely related but usually greater than LoB and could be determined by measuring a few replicates of a few negative specimens run over a few separate days and calculating the mean and SD according to: LoD = mean of blank + 2SD (3SD) of blank or by measuring a few replicates of a few low positive specimens run over a few separate days and calculating the SD according to: LoD = LoB + 1.645 SD of low positive. Alternatively, target LoD could be estimated by measuring a few replicates of a few low positive specimens run over a few separate days and calculating the reproducibility (inter-assay imprecision) expressed as coefficient of variation (CV%) or by confirming that no more than 5% of the values for a target LoD fall beyond the LoB. The generally accepted smallest number of events required to reproducibly detect a PNH population and determine LoD is 20 PNH events,

The functional sensitivity of PNH assays is determined by the limit of quantification (LoQ), which is the lowest level of measurand that can be reliably detected at predefined levels of bias and imprecision [28]. LoQ is usually greater than LOD and for PNH assays could be determined by measuring a few replicates of a few positive (near the expected LoQ) specimens run over a few separate days and calculating the reproducibility (inter-assay imprecision) expressed as CV%, which should be acceptable at levels below 10%. The generally accepted smallest number of events required to reproducibly quantify a PNH population and

determine LoQ is 50 PNH events, lower levels should be validated in each laboratory.

The clinical sensitivity or the ability to detect an abnormal specimen and distinguish from normal specimens defined by true positive/true positive + false negative should be determined

[27]. The clinical specificity of well-established PNH assays is usually >99%.

12 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

The validation of specimen, processed specimen and reagent stability has been reviewed in Section 2 [13, 16, 23].
