Enhancing the Performance of a Blood‐Based Diagnostic Screening Tool for Dysferlinopathy: Optimising an Immunoassay Across Continents

Figures

• 

  FIGURE 1. Dysferlin immunolabelling of PBFs. Representative image of dysferlin labelling in neutrophils. C: healthy control; P: genetically confirmed dysferlinopathy patient. Staining was absent (−) or very reduced (+/−) in patients, present (+) or artifactually very reduced (+/−) in controls. Scale bar 25 μm.

• 

  FIGURE 2. Poor sample collection technique affects the test result. (A and B) Representative incorrect peripheral blood film (PBF) slide. The top arrow indicates the thick region of the smear, where excessive cell overlap limits morphological assessment. The bottom arrow highlights the central portion of the PBF, which is artifactually devoid of white blood cells, rendering it unsuitable for neutrophil evaluation. (C) Magnified view of the boxed region in (B), highlighting neutrophil clustering and morphological distortion, associated with loss of dysferlin signal and increased nonspecific background staining. (D) Representative example of reduced dysferlin labelling associated with poor smear quality. Scale bar 25 μm.

• 

  FIGURE 3. Dysferlin labelling is affected by storage conditions. (A) Graphical scheme of storage conditions of PBF slides collected in India from healthy individuals. Slides were divided into two batches: batch 1 was kept for 1 week at India RT (40°C) and then shipped to the United Kingdom, where it was processed immediately or kept at UK RT (~20°C) for 20 weeks until analysis; batch 2 was kept at India RT for 18 weeks before shipping to the United Kingdom for staining. (B) IHC labelling of dysferlin of the two batches of slides (Batch 1: row 1; Batch 2: row 2) compared to slides kept for 20 weeks at UK RT (~20°C) for 20 weeks. Dysferlin staining was absent in batch 2. Scale bar 25 μm.

• 

  FIGURE 4. Long‐term storage at 4°C does not affect dysferlin persistence in neutrophils. (A) PBF stored at 4°C up to 20 weeks (w) shows no reduction of dysferlin staining. (B) Slides stored at 4°C were thawed at UK RT or at 37°C for various lengths of time. Dysferlin was preserved when the slides spent a limited amount of time at temperatures higher than 20°C. NoAb: negative control. Scale bars 25 μm.