Background/Case Studies: Sickle cell disease (SCD), affecting 8 million people globally, results from sickle hemoglobin (HbS) polymerization under hypoxia, causing red blood cell (RBC) sickling, vaso-occlusive crises, and life-threatening complications. DSA provides real-time sickling kinetics by integrating an enzymatic O2-scavenging system, machine learning image analysis, and multi-parameter profiling under controlled hypoxia. DSA can monitor SCD therapies including hydroxyurea, Hb-modifiers, curative and gene-editing therapies. This study aimed to evaluate the performance of DSA to ensure reliability and reproducibility and to establish its ability to distinguish among HbAA (normal), HbAS (sickle cell trait - SCT), and HbSS (SCD) genotypes. A diagnostic measuring sickling kinetics could serve as a surrogate endpoint in clinical trials, accelerating drug trials and improving patient care.
Study
Design/Methods: 54 HbSS, 20 HbAS, and 22 HbAA subjects were enrolled under IRB (FF-RBC-003v5). Blood was collected in citrate and EDTA tubes and tested using our standardized DSA procedures. Briefly, hypoxia is induced through a timed protocatechuic acid/protocatechuate-3,4-dioxygenase (PCA/PCD) enzymatic reaction. The fraction of sickled cells is determined by time-lapse imaging and reported as a function of time. Measured parameters include: mPoS@5% and mPoS@50% (time to reach 5% and 50% induced sickling, respectively in minutes), Rate@50% (highest induced sickling rate, %/min), Max Sickling (highest sickling percentage), and AUC (area under the curve during 10min, %*minute). DSA performance criteria were evaluated using blood from 10 SCD patients (6 repeats each). Patient genotypes were confirmed by capillary electrophoresis.
Results/Findings: The coefficient of variation (CV) was: 3.6% for AUC, 4.9% for mPoS@5%, 2.9% for mPoS@50%, 9.1% for Rate@50%, 2.9% for Max Sickling. DSA exhibits low variability and high reproducibility, reinforcing its reliability as a diagnostic tool for evaluating RBC sickling kinetics in SCD. DSA parameters revealed distinct genotypic differences. HbSS samples had higher AUC (453.1, vs. 87.4 HbAS, 7.8 HbAA) and max sickling (80.5 vs. 35.1 HbAS, 1.0 HbAA). HbSS samples sickled sooner than HbAS (mPoS@5% 3.2 and 7.4 min; mPoS@50% 4.4 and 9.9 min., respectively). Rate@50% was significantly higher in HbSS (45.4) compared to HbAS (8.0). All genotype pairs showed significant differences (p < 0.05).
Conclusions: These findings demonstrate the DSA’s ability to distinguish RBC sickling kinetics across genotypes, highlighting its sensitivity and specificity in identifying pathological sickling behavior among SCD and SCT subjects. The consistent performance of the DSA, regardless of anticoagulant type, further supports its potential for widespread clinical and research applications.
