Design of testing device
The Sickle SCAN™ test was designed as a rapid, highly sensitive test. It was created using advanced, qualitative lateral flow technology to identify sickle cell disorders of hemoglobin A, S, and C allowing for detection of results with the naked eye in the POC setting. The test was specifically developed to allow for the confirmation of sickle cell trait (HbAS) and SCD in persons with HbSS, HbSC, HbSβ0, and HbSβ+ genotypes.
The test requires 5 μL of blood added to a provided buffer-loaded module designed to release hemoglobin by lysing erythrocytes. The resulting hemolyzed solution is dropped onto the sample inlet of the Sickle SCAN™ cartridge. The treated sample flows through the test cartridge in order to interact with antibody-conjugated colorimetric detector nanoparticles and travel to the capture zones (identified by lines on the device). A total of four detection lines are possible, including hemoglobin variants A, S, C, and a control line (which confirms the test is functioning). Samples containing two hemoglobin variants (such as compound heterozygotes) will have both hemoglobin variants detected (Fig. 1).
Test principle
The Sickle SCAN™ assay employs the sandwich format chromatographic immunoassay approach for the qualitative measurement of human HbA, HbS, and HbC in whole blood samples. A mouse monoclonal antibody (MsxHb-15001, BioMedomics, Inc., Durham, NC, USA) against the C-terminus of human hemoglobin α-chain is used as the detection antibody. This detection antibody is conjugated to blue colored nanoparticles (BMBB-32-14001, 300 nm, BioMedomics, Inc.). Three polyclonal antibodies against the initial N-terminal amino acid sequence (BioMedomics, Inc.) of human sickle cell hemoglobin (HbS), human hemoglobin C (HbC), and adult normal hemoglobin (HbA) are used as capture antibodies on test lines. A separate goat anti-mouse IgG (H&L) antibody (GtxMu-003-E, ImmunoReagents, Inc., Raleigh, NC, USA) is used as the capture antibody to form the control line.
As the test sample diffuses through the absorbent test strip, the antibody-conjugated colorimetric detector nanoparticles bind to the hemoglobin in the specimen, forming an antibody-antigen complex. The specimen then migrates across a membrane toward three test lines containing HbA, HbS, and HbC antibodies to selectively detect the presence of each Hb. The specific complex with each Hb is captured at the test line with the corresponding antibody and produces a blue colored band. Excess conjugate will flow past the test lines and be captured on the control line. Therefore, to serve as a procedural control, a colored band will always appear at the control line region if the proper volume of sample has been added and membrane wicking has occurred. Once the test has run and the control line appears, the presence of any of those test lines indicates that the respective hemoglobin is present in the blood. This allows us to identify persons with hemoglobin A, sickle trait (AS), HbSS, and HbSC disease. However, the LoD of hemoglobin A (40 %) is higher than that of hemoglobin S (2 %). This was an intentional design in order to ensure persons with HbSβ+ were identified with sickle cell disease (although they are seen with this test as HbSS) and still differentiate sickle cell trait (HbAS). As a result of this distinction the intensity of the test lines does not correlate in this initial Sickle SCAN™ test to the quantity of the specific hemoglobin. In accordance, the test is not designed to identify less common hemoglobin variants (such as hemoglobin Constant Spring, hemoglobin O-Arab, or others). Thus, results indicating hemoglobin A alone is not diagnostic and further testing should be considered in the context of an individual’s ethnic background.
Design of testing device
In an effort to improve rapid test specificities and sensitivities, this test was developed using advanced, qualitative lateral flow technology and detection requiring only the naked eye. This Sickle SCAN™ test was created to identify sickle cell disorders of hemoglobin A, S, and C.
Sickle SCAN™ is a qualitative lateral flow immunoassay that tests for the presence of hemoglobin A, S, and C (Fig. 2). The kit includes the immunoassay, capillary sampler, and pretreatment buffer. Five microliters of sample is taken using the capillary sampler (Fig. 3a) and then diluted in the pretreatment buffer (Fig. 3b). Once diluted, five drops of the sample are dispensed onto the immunoassay and the test is read after 2 minutes (Fig. 3c).
Ex vivo laboratory testing methods
Patient samples were obtained from venipuncture performed at the Medical University of South Carolina (Charleston, SC, USA), Duke University (Durham, NC, USA), and Children’s Hospital Oakland (Oakland, CA, USA). The collection and use of these samples for test development were approved by the local institutional review boards (IRBs) in each institution above. Patients were recruited from the regular SCD clinic populations. Samples were collected in EDTA and kept at room temperature for shipment to BioMedomics, Inc. Testing occurred within 4 weeks of sample receipt. Those who had received a blood transfusion within the last 60 days were excluded from analysis.
Five microliters of venous sample (taken from the EDTA-stored samples) were mixed in 1 ml of hemoglobin solubility buffer (used to lyse erythrocytes) designed specifically for this device. The sample was mixed by inversion for 20 seconds and then five drops (using the designated dropper) of hemolyzed solution were dropped onto the inlet to the Sickle SCAN™ testing platform. Ten minutes elapsed prior to quantification of the test line color intensity. Data collection and reference standards were planned as per the test principle prior to the index test.
The initial quantification of the Sickle SCAN™ test line color intensity was accomplished by removing the assay strip from the device cartridge and scanning it using a portable flatbed scanner (CanoScan LiDE210, Canon, Melville, NY, USA). The image was then analyzed using a custom-coded algorithm (MATLAB, MathWorks, Cambridge, UK) to determine the color intensity of the test lines. The quantitative analysis of the test line intensities was determined by the RGB color model values of the image at the test line positions. The software automatically determined the test line positions by searching for the control line, present on all tests, and measuring a set distance to the next test line position. An intensity cutoff was determined to distinguish between positive and negative results for each line. Sickle SCAN™ was compared to either hemoglobin electrophoresis (HYDRASYS acid assay, Sebia, Norcross, GA, USA) or high performance liquid chromatography (HPLC) for each sample using standard guidelines. Confirmatory testing using the above techniques was performed individually at the designated institutions. The results of these confirmatory tests are thus described interchangeably as the gold standard diagnostics. Of note, a scanner is not required for the POC test but was used here for confirmation during analysis.
Limit of detection (LoD)
The LoD is the minimum percent of a specific Hb, that is, HbA, in which the indicator can be read. The percent Hb is the percentage of the specific hemoglobin out of the total Hb concentration. The LoD for the HbA indicator was tested in samples ranging from 0–60 % HbA in increments of 10 %. The LoD for the HbS was tested with samples ranging from 0–40 % HbA in varying increments, and the LoD for the HbC indicator was tested with samples containing 0–10 % HbC in increments of 2 %. The samples for the HbA, HbS, and HbC LoD studies were prepared, respectively, by mixing HbA, HbS, and HbA + C standards with either HbS or HbA standards to create the desired specific Hb percentages. Each sample was loaded onto the Sickle SCAN™ assay and allowed to run for 10 minutes.
Interfering factors
Whole blood samples with BSA at concentrations of 0–100 mg/mL, penicillin at concentrations of 0–500 μg/mL, hydroxyurea at concentrations of 0–75 μg/mL, bilirubin at concentrations of 0–2.5 μg/mL, and cholesterol at concentrations of 0–4 mg/mL were tested in this interference study.
Capillary testing methods
All patients signed written consent to participate in testing by finger stick at the POC. For pediatric patients under 18 years of age, informed consent to participate in the study was obtained from their parent or guardian. IRB approval was obtained from the Medical University of South Carolina (MUSC) to conduct a pilot trial using this POC test device in patients with SCD and their family members followed at the MUSC SCD clinic. All SCD providers in the clinic were trained in reading the testing device prior to the initiation of testing.
Subjects of all ages were included in the trial. According to the approved IRB protocol, all included patients with SCD had to have a current HPLC hemoglobin variant result. In most infants between 6–12 months of age, HPLC is not assessed routinely after the initial confirmatory assessment. Thus, although the LoD for both HbS and HbC (1 % and 2 %, respectively) discussed above in the ex vivo study demonstrates that the test will likely be useful in newborn children, they were not included in this validation study and a follow-up study in neonates will be undertaken. Those who had received a blood transfusion within the last 60 days or whose hemoglobin genotype was not previously known were excluded. Patients receiving hydroxyurea therapy were included. At the time of consent, 71 patients met inclusion and exclusion criteria based on their history and were consented to undergo testing. Blood was obtained from the finger pad using a standard lancet for capillary testing. Five microliters of blood was collected using the capillary sampler provided in the testing kit and added to the buffered loaded module. After inverting the module × 3, five drops of hemolyzed sample were dropped onto the inlet of the Sickle SCAN™ cartridge. Results were read within 120 seconds. The Sickle SCAN™ test result was determined after visual inspection sequentially by two independent providers who determined the results separately without reference to one another. Due to timing, the initial provider read the test at 2 minutes and the second provider read the test between 4–10 minutes after sample had been added. In affected patients, the first provider had knowledge of the patient’s genotype prior to testing. However, the second provider was always blind to the sample being tested. On average, all testers reviewed each sample within 5 minutes of testing.