Modified and Novel Lorrca Oxygenscan Measurement Parameters with Physiological Relevance

BACKROUND: Despite arising from a single-point hemoglobin (Hb) mutation (βGlu6 replaced by valine), patients with sickle cell disease (SCD) display wide phenotype variation. This likely arises from sickling and its resulting pathology having multiple physiologic triggers, many of which are red blood cell (RBC) related, i.e., sickle hemoglobin (HbS) and fetal hemoglobin (HbF) concentration, membrane permeability, cell hydration, etc. These triggers are inadequately captured by single measurement parameters and require a more holistic functional RBC assessment (reflecting their composite effects). To provide such, oxygen (O₂) gradient ektacytometry (LORRCA oxygenscan RR Mechatronics) was designed as a commercial, rapid, reproducible, and automated laboratory assay, which mimics in vivo physiology, quantifying RBC deformability across the physiological O₂ gradient. This assay captures the process of sickling/unsickling of a RBC population when challenged with deoxygenation and reoxygenation.

Significant variance in the oxygenscan point of sickling measurement was observed (POS - defined as the pO₂at which EI_max declined 5% - representing the initiation of sickling), despite following all guidelines for sample handling. EI fluctuation at higher O₂ partial pressure (pO₂) was noted as the cause for this. We consequently modified the POS determination, centering our analysis on EI rate of change (i.e., the first derivative of the EI trace), rather than the single point software determination. We further identified two new analyses parameters which reflect aspects of RBC physiology of potential physiologic benefit in SCD. Firstly, we used the rate of change in EI (a first derivative-based analysis) on the reoxygenation arm of the LORRCA trace, to identify the pO₂ at which EI was observed to significantly increase from EI_min, defining this as the point of unsickling (POU). Lastly, we set about quantifying the hysteresis between the O₂ unloading and O₂ loading EI curves, calling this the area between curves.

METHODS: RBCs from SCD patients were incubated with the Hb allosteric effector 5-hydroxymethylfurfural (5HMF; 20% Hct, 37C, HMF: 0-5mM, 0-3 hrs); 5HMF binds to Hb NH₂ terminal αValine1, increasing oxyHbS abundance, thereby inhibiting the mechanistic trigger for deoxygenation induced HbS polymerization.Oxygenscans were performed with elongation index (EI) determined by laser light diffraction, across the physiologic O₂ gradient (pO₂ ~150-10 mmHg; sample gassed with nitrogen ~1300 seconds followed by swift re-oxygenation in room air ~280 seconds). Samples were analyzed utilizing standard parameters offered by the LORRCA software in addition to our modified POS determination and analysis of POU and area between curves.

RESULTS: Little change in mean POS was observed in baseline measurements (minus 5HMF) between the standard LORRCA software analysis and our modified analysis (POS pO₂ = 55.1 ± 20.4 vs 56.9 ± 10.1mmHg mean±SD, respectively), however the coefficient of variance (COV) of our modified analysis was ~20% lower. The same trend was observed in samples across all doses of 5HMF. No significant difference in POS was detected with the standard analysis between any of the 5HMF doses. However, a significant reduction in POS between 0 and 5mM 5HMF final was determined with our modified analysis (POS pO₂ = 56.9 ± 10.1 vs 39.8 ±11.6mmHg, respectively). A significant difference was also observed in the POU between 0 and 5mM HMF (POU pO₂ = 38.3 ± 9.1 vs 21.3 ± 5.7mmHg, respectively) with HMF resulting in a lower pO₂ at which unsickling was observed. This has obvious patient benefit implications, with SRBCs regaining deformability at lower pO₂. Finally, a significant difference was also observed in the area between the deoxygenation and reoxygenation EI curves between 0 and 5mM HMF (area between curve = 8.9 ± 4.6 vs 4.6 ± 1.6AU, respectively). This smaller area between curves implies less change in deformability behavior between oxygenation states in the presence of 5HMF.

SUMMARY: We have developed a more robust analysis methodology for the determination of POS by oxygenscan. This appears to work well even on sub-optimal scans and can detect a change in sickling characteristics for the allosteric modifier 5HMF. In addition, we suggest two further analyses parameters for oxygenscan (POU and area between curves) which reflect aspects of RBC physiology of potential physiologic benefit in SCD.

Baer:Forma: Research Funding; Kite, a Gilead Company: Research Funding; AbbVie: Research Funding; Takeda: Research Funding; Kura Oncology: Research Funding; Ascentage: Research Funding.