BACKGROUND and AIM

Measurement of Factor IX is required for quantification of residual plasma FIX levels with a diagnostic purpose (Haemophilia B) or for performing recovery studies during treatment by replacement therapies. The one-stage clotting assay is the most used in routine laboratories; there are only two chromogenic assays commercially available at present. We aim to evaluate the analytical performance of the Biophen FIX chromogenic assay on the Sysmex CS-2400 analyzer.

METHODS

PATIENTS

• 40 with haemophilia B (HB severe, moderate and mild),
• 120 healthy Italian subjects (controls aged 18-69 years).

The clotting tests were performed on citrate platelet poor plasmas stored at -80°C.

The results obtained with the chromogenic assay BIOPHEN Factor IX (HYPHEN BioMed, France) were compared with the one-stage assays (using Actin FS and FIX deficient plasma; Siemens, Germany). All the tests were performed using Sysmex CS 2400 analyzer (Sysmex, Kobe, Japan).

Analytical performance and method comparison analysis were performed according to the Clinical & Laboratory Standards Institute (CLSI) guidelines.

RESULTS

Imprecision analysis results are reported in Tables 1.
Linearity was good up to 1/128 (r=0.99);
mean recovery was 97.28%.
Analytical sensitivity was 0.2 IU/dL.

The reference range calculated on the 120 healthy subjects was 60 to 138% (95% Reference interval, Double-sided). Biophen FIX:C assay showed a good correlation with the one-stage assay (Spearmen’s Rank correlation 0.94) as shown also by the Bland and Altman Plot (Figure 1).

The diagnostic agreement between Biophen FIX:C and one-stage assay was satisfactory (K Cohen coefficient = 0.83); The K coefficient was 0.89 when Biophen FIX:C was compared with the historical classification of the patients, demonstrating an optimal diagnostic accuracy in HB patients.

CONCLUSIONS

Our results showed a good analytical performance of Biophen FIX:C as a suitable Chromogenic FIX functional assay. The introduction of chromogenic FIX test in addition to standard one-stage assay is important for the diagnosis and monitoring of therapeutic effects particularly in haemophilic patients treated with extended half-life products.