Plasma-induced nanoparticle aggregation for stratifying COVID-19 patients according to disease severity

Abstract

Stratifying patients according to disease severity has been a major hurdle during the COVID-19 pandemic. This usually requires evaluating the levels of several biomarkers, which may be cumbersome when rapid decisions are required. In this manuscript we show that a single nanoparticle aggregation test can be used to distinguish patients that require intensive care from those that have already been discharged from the intensive care unit (ICU). It consists of diluting a platelet-free plasma sample and then adding gold nanoparticles. The nanoparticles aggregate to a larger extent when the samples are obtained from a patient in the ICU. This changes the color of the colloidal suspension, which can be evaluated by measuring the pixel intensity of a photograph. Although the exact factor or combination of factors behind the different aggregation behavior is unknown, control experiments demonstrate that the presence of proteins in the samples is crucial for the test to work. Principal component analysis demonstrates that the test result is highly correlated to biomarkers of prognosis and inflammation that are commonly used to evaluate the severity of COVID-19 patients. The results shown here pave the way to develop nanoparticle aggregation assays that classify COVID-19 patients according to disease severity, which could be useful to de-escalate care safely and make a better use of hospital resources.

Keywords: AST, aspartate aminotransferaseALT, alanine aminotransferase; Alb, albumin; C1.75, protein concentration 1.75 × 10-4 g·dL-1; CPImin, protein concentration at PImin; CRP, C-reactive protein; Colorimetric; Creat, creatinine; D-D, D-dimer; Ferr, ferritin; GGT, gamma-glutamyl transferase; Glu, glucose; Gold; Hb, hemoglobin; ICU, intensive care unit; INR, international normalized ratio (prothrombin time); LDH, lactate dehydrogenase; LSPR, localized surface plasmon resonance; MCV, mean corpuscular volume; MPV, mean platelet volume; Mono, monocytes; NIR, near-infrared; NLR, neutrophil-to-lymphocyte ratio; NTA, nanoparticle tracking analysis; PDW, platelet distribution width; PI, pixel intensity; PI1.75, pixel intensity at C1.75; PIdil, pixel intensity at plasma dilution 1:31250; PImin, minimum value of pixel intensity; PLR, platelet-to-lymphocyte ratio; Plasmonic; RBC, red blood cells; RDW, red cell distribution width; SARS-CoV-2; Sepsis; TG, triglycerides; TotProt, total protein concentration; WBC, white blood cells.

Graphical abstract

Fig. 1

Schematic representation of COVID-19 severity evaluation with plasma-induced nanoparticle aggregation. Probing a well-defined plasma dilution with gold nanoparticles generates a colorimetric signal associated to disease severity.

Fig. 2

Representative example of plasma-induced nanoparticle aggregation generated with a sample from a critical patient; A) Schematic representation of the impact of adding proteins (purple dots) at different concentrations to gold nanoparticles (yellow dots); B) Photographs of the colloidal suspensions after adding fifteen decreasing dilutions of a sample from a critical patient with an initial total protein concentration value of 8 g·dL^-1 (superscripts indicate the protein concentration in diluted samples prior to nanoparticle addition in g·dL^-1) C) Extinction spectra of tests highlighted in panel B; D) Extinction at 530 nm (Ex530); and E) Pixel intensity (PI) as a function of the protein concentration prior to nanoparticle addition in diluted samples.

Fig. 3

Pixel intensity as a function of the protein concentration after adding diluted plasma samples from ICU (red) or ward patients (black) to gold nanoparticles. Dotted lines are a guide to eye. PI_min: minimum value of pixel intensity; C_PImin: protein concentration at PI_min; C_1.75: indicates that the protein concentration is 1.75 × 10^-4 g·dL^-1.

Fig. 4

Comparison between different parameters proposed here to differentiate patient populations; (A) PI_min (minimum value of pixel intensity); (B) C_PImin (protein concentration at PI_min); (C) PI_1.75 (pixel intensity at C_1.75); and (D) PI_dil (pixel intensity at plasma dilution 1:31250) yielded by plasma samples from ICU (red) and ward (black) patients. The dotted lines represent two standard deviations above the mean of ICU patients. Data are expressed as median with percentiles 25th and 75th; *p-value was obtained with a Mann-Whitey test.

Fig. 5

Potential factors influencing nanoparticle aggregation after adding diluted plasma samples; (A) Distribution of total protein concentration values for ICU and ward patients; (B) Correlation plot comparing the total protein concentration and PI_dil; (C) Distribution of albumin levels for ICU and ward patients; (D) Correlation plot comparing the albumin concentration and PI_dil; (E) Extinction spectra of gold nanoparticles after adding 3 diluted ICU (red) and 3 ward (black) samples covering the whole dilution range shown in Fig. 3. Data in A and C are expressed as median with percentiles 25th and 75th. Mann-Whitey test proved non-significant (n.s.) statistical differences. P_dil; pixel intensity at plasma dilution 1:31250.

Fig. 6

Correlation studies between PIdil and clinical variables; (A) Principal component analysis; (B-D) Correlation plots comparing PI_dil with the neutrophil-to-lymphocyte ratio (NLR, B), the platelet-to-lymphocyte ratio (PLR, C) and the number of biomarkers out of normal range (within selected biomarkers highlighted in red and green in (A), D). PI_dil; pixel intensity at plasma dilution 1:31250.