Proposed Use of the Microfluidic Impedance Red Cell Assay (MIRCA) to Identify Individuals with Sickle Cell Disease in Need of Second Line Therapies

Background

Individuals with sickle cell disease (SCD) have red blood cell functional abnormalities which contribute to acute complications. Hydroxyurea (HU), a fetal hemoglobin (HbF) inducer, is a mainstay therapy that benefits up to 50% of adult individuals with SCD. The patients who experience an increase in HbF levels in response to HU are considered HU responders. Although high HbF is protective on a population level, %HbF does not always correlate with clinical severity on an individual level. Typically, second-line drugs like voxelotor are added to HU after clinical worsening is noted. We hypothesize that worsening RBC function may indicate need for a second-line therapy initiation before clinical decline is apparent.

One way to measure RBC deformability is with the MIRCA (Microfluidic Impedance Red Cell Assay) a microfluidics device that mechanically measures the ability of a red blood cell (RBC) to squeeze between pillars. Trapped, poorly deformable cells are measured by an impedance analyzer. The device readouts are an occlusion index (OI) under normoxia (NOI) or with sodium metabisulfite (MBS)-induced hypoxia (HOI).

Here we present a clinical validation of MIRCA for use in monitoring the need for second-line agents in individuals with SCD on HU.

Methods

One hundred and nineteen peripheral blood samples from 66 adults and 35 pediatric individuals (87 HbSS/Sβ⁰, 14 HbSC/Sβ⁺/SE) were collected in EDTA under an Emory University IRB approved protocol.

Whole blood was run on an ADVIA 2120i hematology analyzer to obtain complete blood counts and percent dense red blood cells (%DRBC). Washed RBCs were suspended to 20% (v/v) in 1X PBS or 1.5% (w/v) MBS in 1X PBS to run on MIRCA. Vaso-occlusive events (VOE), acute chest syndrome (ACS), avascular necrosis (AVN), and SCD complications (retinopathy, nephropathy, priapism, splenic sequestration, chronic pain) were determined by chart review. VOE and ACS in the past year were summed to define an acute events (AE) variable. A composite variable was used for disease complications (DC).

The association of MIRCA readouts with laboratory data, AE, AVN, and DC were analyzed with linear mixed model, negative binomial model, or Kruskal-Wallis test using StataNow 18.5 (College Station, TX); a p < 0.05 was considered significant. Age, genotype, and maximum tolerated HU dose (MTD - HU ≥ 35 mg/kg/day or absolute neutrophil count < 4000/µl). were added as covariates while analyzing the laboratory data. Genotype, age, %HbF, %DRBC, absolute reticulocyte count (ARC), and absolute neutrophil count (ANC) were added as covariates while analyzing AE and DC.

Results

Out of 101 patients, 32 were on HU, and 39 were on HU MTD.

Higher NOI was associated with higher DC (p = 0.02) after adjusting for genotypes and DRBC. Age, %HbF, ARC, and ANC were not retained in the model due to their p values >0.05. Higher NOI was associated with higher AE (p = 0.02) in the univariate regression, but the association was not seen when adjusted for HbF and age. HOI was not associated with AE or DC. HOI and NOI were not associated with AVN.

NOI and HOI values were 2.64% (p = 0.04) and 10.75% (p = 0.004) higher in adults than in children, respectively. NOI and HOI were lower by 6% (p = 0.002) and 18.2% (p = 0.001) in HbSC, HbSβ⁺, and HbSE individuals compared to HbSS and HbSβ⁰ individuals, respectively. NOI and HOI were not associated with sex and ARC.

Individuals on HU at MTD had 6.9% lower HOI than those not at MTD (p = 0.04). Higher HOI was associated with higher ANC (p < 0.001), higher DRBC (p = 0.04), and lower Hb (p = 0.049).

Higher NOI was associated with higher DRBC (p < 0.001) and lower Hb (p = 0.001). NOI was not associated with MTD and ANC.

Discussion

Higher NOI values were associated with higher DC after adjusting for covariates, suggesting that rising NOI may be predictive of increased risk of SCD complications. Our cohort included 71 (70.3%) subjects on HU (39 at MTD), indicating that NOI values can be used to monitor patients on HU. NOI may be used to identify subsets of high-risk patients after HU optimization that may benefit from second-line therapies.

We plan to use HOI and NOI in combination with traditional clinical laboratory tests to predict the need for additional therapeutic intervention. MIRCA can be used to monitor RBC deformability at routine clinic visits; worsening of deformability and poor laboratory results will trigger initiation of second-line therapies before patients experience clinical decline.