*4.2.1. Analytical specificity*

The analytical specificity of PNH testing assays reflects the choice and validation of all antibodies/reagents and corresponding fluorochromes (**Tables 1**–**3**).

#### *4.2.2. Clinical specificity*

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

by assay of a series of abnormal samples and scoring for abnormality in comparison to a suitable reference method, such as clinical findings [27]. The values for clinical sensitivity of

Accurate and High Sensitivity Identification of PNH Clones by Flow Cytometry

http://dx.doi.org/10.5772/intechopen.71286

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The validation of assay performance characteristics comprises the determination of repeatability (intra-assay imprecision) and reproducibility (inter-assay imprecision). It is generally recommended to assay a few replicates from at least five samples within a single analytical run for repeatability and a few replicates from at least five samples in separate analytical runs for reproducibility [29]. For confirmation of good performance characteristics, CV% below 10% should be obtained for samples with more than 1% target PNH cells and below 20% for

The validation of specimen, processed specimen and reagent stability has been reviewed in

Flow cytometry is a highly versatile and complex technology, which is routinely applied in clinical diagnostic laboratories. The vast majority of assays are laboratory developed tests based on publications, without any gold standard reference [30] mostly with poor validation. For the purpose the ICSH/ICCS workgroup published in 2013 the practice guidelines for validation of cell-based fluorescence assays [31], subsequently several relevant publications addressed various aspects of the validation of PNH testing by flow cytometry [12, 13, 32]. Each laboratory applying for ISO 15189 accreditation should confirm optimal validation of instrument setup, assay performance characteristics, laboratory information system and

Highly sensitive and specific PNH testing of all three lineages (RBC, neutrophils and monocytes) has become the standard of care for patients with suspected PNH. This is a rare disease and therefore often overlooked as a diagnostic possibility. It is important for the ordering physician to test the high-risk patients for PNH [12] and also to receive informational reports as an important part for best patient management. The laboratories are challenged with the validation of multiple steps, including instrument optimization, selection of best antibody clones/conjugates, panel design and targeted acquisition and interpretation of data. Developing competency in PNH testing and reporting is critical for laboratories and is directly related to awareness of best practices, following guidelines which are developed by experts based on extensive evaluation of

well-established PNH assays is usually >99%.

**4.4. Repeatability and reproducibility**

samples with minor clones (<1%).

**4.5. Stability**

Section 2 [13, 16, 23].

**5. Accreditation**

result reporting [33].

**6. Conclusion**

#### **4.3. Sensitivity of PNH assays**

### *4.3.1. Analytical sensitivity*

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, lower levels should be validated in each laboratory.

#### *4.3.2. Functional sensitivity*

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.

#### *4.3.3. Clinical sensitivity*

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 by assay of a series of abnormal samples and scoring for abnormality in comparison to a suitable reference method, such as clinical findings [27]. The values for clinical sensitivity of well-established PNH assays is usually >99%.
