A paper-based multiplexed transaminase test for low-cost, point-of-care liver function testing

Abstract

In developed nations, monitoring for drug-induced liver injury through serial measurements of serum transaminases [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)] in at-risk individuals is the standard of care. Despite the need, monitoring for drug-related hepatotoxicity in resource-limited settings is often limited by expense and logistics, even for patients at highest risk. This article describes the development and clinical testing of a paper-based, multiplexed microfluidic assay designed for rapid, semiquantitative measurement of AST and ALT in a fingerstick specimen. Using 223 clinical specimens obtained by venipuncture and 10 fingerstick specimens from healthy volunteers, we have shown that our assay can, in 15 min, provide visual measurements of AST and ALT in whole blood or serum, which allow the user to place those values into one of three readout "bins" [<3× upper limit of normal (ULN), 3 to 5× ULN, and >5× ULN, corresponding to tuberculosis/HIV treatment guidelines] with >90% accuracy. These data suggest that the ultimate point-of-care fingerstick device will have high impact on patient care in low-resource settings.

Fig. 1

Schematic of the paper-based AST/ALT test design and protocol. (A) The device consists of two layers of similarly patterned paper, a plasma separation membrane, and a laminated cover of polyester film. (B and C) A drop of whole blood (either a fingerstick specimen or 30 μl of a specimen obtained by venipuncture) is applied to the back of the device. Red and white blood cells are filtered out by the plasma separation membrane, while plasma wicks to the five detection zones via patterned hydrophobic channels in the paper (B). After 15 min, the AST and ALT test zones are matched to a color “read guide” (C) to obtain a concentration value. Results are interpreted as being within one of three “bins” of values: <3x ULN (ULN defined as 40 U/l); 3-5x ULN; or >5x ULN. (D) Detailed schema of the paper-based transaminase test and possible colorimetric readouts for the 5 zones (i to v). A schematic of test and control zones (prior to receiving a sample) is shown in the center of the figure. (i) AST test zone: low/normal AST values (<80 U/l) result in a dark blue color, whereas high AST values (>200 U/l) result in a bright pink color. (ii) ALT test zone: low/normal ALT values (<60 U/l) result in a yellow color, whereas high ALT values (>200 U/l) result in a deep red color. (iii) ALT negative control zone: a change from white to yellow indicates appropriate device activation; in the event of sample hemolysis, the zone becomes orange/red and the device is read as “invalid.” (iv) AST negative control zone: the baseline blue color remains unchanged if dye chemistry is functioning properly, whereas the zone becomes bright pink in the event of nonspecific dye reaction and the device is read as “invalid.” (v) AST positive control zone: the zone changes from blue to pink if AST reagents are functioning properly, but remains dark blue if either the reagents are not functioning or the zone is not activated, in which case the device is read as “invalid.”

Fig. 2

Paper device performance with ALT and AST spiked into fresh whole human blood. Thirty μl were pipetted onto each device. (A and B) Device readout linearity for color intensity versus concentration of ALT (A) or AST (B). Each datum is an average of 3 independent measurements from 3 separate devices. Dashed lines represent the upper and lower 95% prediction intervals. ALT values are normalized by subtracting each point from 255 so that higher color intensity values correspond to higher ALT concentrations (Supplementary Methods). (C and D) LOD curves for ALT (C) and AST (D). Data are normalized such that the lowest intensity measured yields a value of 0 color intensity on the y-axis. Solid lines represent a nonlinear regression of the Hill Equation. The data are averages of 7 measurements ± SD.

Fig. 3

Direct comparison of transaminase measurements made with the paper-based device to those made with the standard automated method. (A to D) ALT values in paired serum (A) and whole blood (B) clinical specimens and AST values in paired serum (C) and whole blood (D) clinical specimens are plotted against values (in U/l) measured in the serum specimen using a gold-standard automated method (Roche Modular Analytics System). Results for each whole blood specimen (n = 1 per reader) were averaged from 3 blinded readers to generate each datum. All serum specimens with sufficient volume remaining after initial testing were stored at 4°C for up to 2.5 weeks and blindly retested in triplicate (three readers blinded to automated results read each of up to three assays performed per specimen) and then averaged. In each plot, the blue boxes represent AST or ALT “bins” within which values for both the paper-based device and the automated method are within the same range: <3× ULN (0-119 U/l), 3-5× ULN (120-200 U/l), or >5× ULN (>200 U/l). The red line corresponds to the line of equality, and the green and purple lines correspond to ±40 U/l from the line of equality, respectively. Points circled in red are samples that were neither in the correct bin nor within 40 U/l of the value measured by the gold-standard automated method. For scaling purposes, x-axis values measured by the Roche system that exceeded 400 U/l were plotted between 400 and 500 U/l.

Fig. 4

Bland-Altman plots of visual transaminase measurements in clinical specimens. (A) ALT values in serum. (B) AST values in serum. (C) ALT values in serum (after log transformation). (D) AST values in serum (after log transformation). (E) ALT values in whole blood. (F) AST values in whole blood. The purple and blue lines represent the 95% limits of agreement. The red line is the line of equality and the green line is the average difference of the methods. Values are derived from the difference between the gold-standard method (Roche) and the paper test value (average of all recorded values per sample) (supplementary methods).