2.7. Clinical protocol

A method comparison study was undertaken to compare Sight OLO with the Sysmex XN‐1000 System. The study design was based on the methods outlined in CLSI H20‐A2, ¹⁴ CLSI H26‐A2 ¹⁵ and CLSI EP09‐A3, ¹⁶ and is consistent with the approach conducted for FDA submissions for hematology devices.

Residual whole blood clinical samples (N = 679) were collected from both adult (>22 years old, N = 462) and pediatric patients (3 months to 21 years old, N = 217). The majority of samples were venous whole blood samples, while roughly 20 pediatric samples were capillary whole blood samples. All samples were collected in standard K₂EDTA collection tubes (Becton Dickinson, Franklin Lakes, NJ, USA) and processed within 8 h of collection. Processing included testing on both analyzers within 2 h of each other, as well as preparing three blood smears for each sample. The study included both normal and pathological samples in order to assess Sight OLO's performance across the analytical measuring range and around medical decision points.

A Passing‐Bablok regression analysis was performed for each CBC parameter after excluding any result which is invalidated by Sight OLO or by the comparative method. For each regression analysis, the slope and intercept and the 95% two‐sided confidence interval (CI) around the slope and intercept were calculated, as well as the correlation coefficient. The overall bias was calculated as the median of differences, where differences were taken either as absolute or relative, according to the nature of the data.

Within‐run repeatability studies were performed using residual K₂EDTA whole blood venous samples (minimum of 2 ml per sample). In order to span healthy and pathological values and the medical decision points between them, each site tested at least four samples within lab reference ranges, three samples around medical decision levels for HGB (6–10 g/dl), PLT (<50 × 10³/μl) and WBC (<2 × 10³/μl), and four samples around the upper range for RBC (>6 × 10⁶/μl), HGB (>17 g/dl), WBC (>12 × 10³/μl) and PLT (>600 × 10³/μl). In total, this requirement led to 38 samples being scanned, with each sample measured 20 consecutive times (after excluding invalidations or rejects). Standard deviation (SD) and coefficient of variation were calculated for each run. The first 20 successful runs per measurand and were analyzed. If fewer than 20 successful scans were obtained within the required time slot from phlebotomy (8 h), the sample was still analyzed so long as 17 or more replicates were scanned. For the anemic samples (HGB 6–10 g/dl) only RBC, HGB and HCT were analyzed, while for the thrombocytopenic (<50 × 10³/μl) and leukopenic (<2 × 10³/μl) samples, only PLT and only the WBC concentration and differential were analyzed, respectively. This was in accordance with the CLSI guidance H26‐A2, ¹⁵ which refers to the ICSH protocol for evaluation of blood cell counters.

Reproducibility studies were conducted using three levels of commercial control materials (low, normal and high ‐ below, within and above the reference ranges of main parameters, respectively) for all reported parameters. A total of 240 samples were included for each level of control. Controls (R&D Systems, Minneapolis, MN) were measured over five operating days at three sites (Boston Children's Hospital, Columbia University and Sight's laboratory), with two devices at each site for a total of six devices. Two runs per day, and four replicates per run were performed. At each site the testing was performed by two operators, where the first operator conducted the first run of all days and the second operator conducted the second run. Every replicate required a new test kit and, every day, a new quality control material tube per level was opened at each site. This was in accordance with the CLSI guideline EP‐05‐A3.

The flagging capabilities of Sight OLO were compared to manual microscopy for WBC distributional abnormalities and WBC morphological abnormalities, which include blasts, immature granulocytes, nucleated RBCs, and atypical lymphocytes. Other invalidating messages, such as platelet clumps and RBC agglutination were not included in the flagging study. The 108 negative and 100 positive samples with WBC count larger than 4 × 10³/μl were enrolled from the method comparison study. For each sample, the three prepared smears were sent to analysis by trained morphologists from Columbia University Irving Medical Center staff, who had no access to either clinical information or reference method results. The testing design was based on the test methods outlined in CLSI H20‐A2 ¹⁴ and CLSI H26‐A2. ¹⁵

A matrix comparison study was performed in two parts to assess the equivalence between venous and capillary samples, and between capillary and direct‐from‐finger samples. In the first part of the study, 67 samples (of 52 subjects, 15 of which repeated the test after a 6‐month interval) were collected in pairs. A venous sample was drawn, and a capillary sample was collected into 350 μl microtainers. Healthy volunteers were primarily tested, and additional samples at medical decision points and across the analytical measuring range (i.e.,: HGB 6–10 g/dl; WBC <2 × 10³/μl; PLT <50 × 10³/μl; WBC >12 × 10³/μl; PLTs >500 × 10³/μl; RBCs >6 × 10⁶/μl; HGB >17 g/dl) were enrolled from subjects in Tel Aviv Sourasky Medical Center. Each pair of samples was tested on the same Sight OLO device four times: two replicates of the venous sample and two of the capillary samples. After excluding invalidated results, a Passing‐Bablok regression analysis was performed between the pair‐averaged capillary and pair‐averaged venous samples.

For the second part of the matrix‐comparison study, 40 apparently healthy patients were enrolled, and capillary samples were collected from each of them using two different methods. First, a capillary sample was collected into a 350 μl microtainer. Then, two fingerprick samples from different fingers were collected, 27 μl from each, directly into the Sight OLO test kit's microcapillaries and immediately processed on the OLO. The two fingerprick samples and the two repeats of the capillary sample collected into the microtainer were averaged, and results were compared to each other using Passing‐Bablok analysis.