Performance evaluation of the prototype Abbott Alinity hq hematology analyzer

The analytical and clinical performance as well as the workflow efficiency of the novel, prototype Alinity hq hematology analyzer was evaluated in the clinical laboratory of Universitair Ziekenhuis Brussel, Department of Hematology, Brussels, Belgium.


| INTRODUC TI ON
Modern hematology analyzers provide complete blood cell counts (CBC), white blood cell (WBC) differential, reticulocyte (RETIC), and related measurand concentrations with high sample throughput and reduced turnaround time, using electrical particle counting (impedance), optical and fluorescence flow cytometry, and most recently, digital imaging-based principles. 1 Despite the increasing accuracy and precision of the results, manual microscopic review of blood smears remains necessary for certain blood samples, to confirm morphological abnormalities for WBC, red blood cells (RBC), and platelets (PLT), or to confirm abnormal numerical results. 2,3 It is still considered the reference method for evaluating RBC, and PLT morphology and for WBC differentiation. In addition to reporting numerical results, analyzers provide various morphological flags to indicate the presence of certain immature or pathological cell types and other morphological findings. Accuracy of these flags may have significant impact on slide review rate.
The aim of this study was to evaluate the performance characteristics of a prototype Alinity hq hematology analyzer (Abbott Laboratories, Diagnostics Division, Hematology, Santa Clara, CA, USA) in a routine laboratory setting. A complete performance evaluation was conducted in accordance with professional recommendations 4 including both analytical (within-run and within-laboratory imprecision, linearity, and carryover) and clinical performance characteristics. Results were compared to those obtained with CELL-DYN Sapphire. 5 In addition, Alinity hq WBC differential was evaluated in comparison with the manual microscopic differential as the reference method, and flagging performance was analyzed in comparison with manual smear review. Workflow efficiency was assessed by comparing re-run and reflex testing rates on the two analyzers.

| Alinity hq
Alinity hq is a novel, high throughput hematology analyzer from Abbott Laboratories. Alinity hq uses optical principles for all measurements. Hemoglobin (HGB) is determined using spectrophotometry. Nucleated cells are measured using a proprietary, fluorescent nuclear dye-containing reagent that stains nuclei for subsequent multi-angle light scatter and fluorescence analysis. Nucleated cells are reported as a 6-part WBC differential (including immature granulocytes [

| CELL-DYN Sapphire
CELL-DYN Sapphire (Abbott) uses MAPSS™ technology for WBC differential and PLT measurements, and impedance for RBC. It reports a standard 5-part WBC differential. 11 Other differences between CELL-DYN Sapphire and Alinity hq include the lack of the RBC fragments flag on CELL-DYN Sapphire, and the presence of an IG flag, instead of reporting the concentration and %IG. In addition, CELL-DYN Sapphire features a band alert that warns the user about the potential presence of band NEUs/IGs. It also features a CD61 monoclonal antibody-based ImmunoPLT method, which has been reported to be equivalent with the CD41/CD61 reference method. 12,13

| Within-run
Thirty-five clinical samples were used to determine within-run imprecision for all primary CBC parameters and WBC differential, ensuring that each measurand is represented in normal, low, and high concentration ranges in at least two samples. Samples were tested in 10 replicates in the same run. Mean, standard deviation (SD), and coefficient of variation (%CV) were calculated for each measurand.
%CV obtained on samples were compared to Ricos' desirable specifications 15 and to imprecision results published in the operator's manual of CELL-DYN Sapphire.

| Sample carryover
Carryover was determined for WBC, RBC, HGB, and PLT, on each incubation block separately, per ICSH guidelines, 14 by running a whole blood sample with elevated parameters in triplicate, immediately followed by one with low parameters in triplicate. The sequence was performed three times, with three different high/low sample pairs for each measurand. Some samples were concentrated to obtain the desirable concentration.
Alinity hq features a data flag (alert), which warns the user for the presence of potential carryover, and invalidates the affected analyte (WBC or PLT). This flag is triggered if a sample with less than 0.5 × 10 9 /L WBC is preceded by one with more than 40 × 10 9 /L WBC, or when a sample with less than 50 × 10 9 /L PLT is preceded by one with more than 1000 × 10 9 /L PLT. If the flag is triggered, the run needs to be repeated, which eliminates the opportunity for carryover impacting the result.

| Linearity
Linearity of the analytical measuring ranges (AMR) for WBC, RBC, HGB, and PLT was tested using the commercially available CBC-LINE CL011 kit containing separate full range and low range kits.
Testing was performed according to the relevant package inserts.

| Interinstrument comparison
Alinity hq absolute and percent NEU and IG results were added up, and the sum of the results was compared to Sapphire NEU concentration and %NEU, as CELL-DYN Sapphire does not report IG concentration separately, instead, includes that in the NEU count. In addition to the whole AMR, regression and correlation analysis was performed separately for NEU, HGB, and PLT data on a subset of clinical samples in the low range, to assess performance at medical decision points. Measurands with invalidating data flags either on CD-Sapphire or Alinity hq or both analyzers were excluded from the calculations on a measurand-by-measurand basis; therefore, the final number of samples included in the comparison is different for each parameter and is shown in the Results section ( Table 2). In two samples with hypereosinophilic syndrome (with over 70% EOs), EOs were misclassified as NEUs by Alinity hq. Manual smear review revealed hypogranular EO granulocytes. These two samples were excluded from the WBC differential comparison calculations.

| Comparison of PLT count with ImmunoPLT results
Platelets counts on 98 samples (included in the 417 samples used for method comparison) were additionally compared with the results of the CD61 ImmunoPLT method on CELL-DYN Sapphire. 12,13 Samples were selected to cover a wide range, however, enriched in samples with low PLT count (below the reference range and around medical decision levels). Statistical analysis was performed for all 98 samples and separately for the samples with PLT results of <100 × 10 9 /L.

| Comparison of Alinity hq WBC differential with manual microscopic differential
A smear was prepared from each sample that was run on the analyzers. Eventually, 398 samples were included in this evaluation, due to sample exclusions related to inappropriate test selection on Alinity hq (n = 4), WBC concentration of less than 0.20 × 10 9 /L (n = 12), or inadequate smear quality (n = 3). Measurands with invalidating data flags by Alinity hq were excluded from the calculations on a measurand-by-measurand basis; therefore, the final number of samples included in the comparison is different for each parameter and is shown in the Results section (Table 3). Two experienced morphologists performed a 200-cell microscopic WBC differential on each sample, and when results disagreed, a third morphologist was used to resolve discrepancies, as advised by the ICSH guidelines. 14

| Flagging performance
The same samples that were included in the Alinity hq comparison with manual microscopic differential were used to assess the performance of the BLAST flag and IG and NRBC detection. Failure of the analyzer to provide results for one or more WBC differential cell types was regarded as a morphological flag, according to the recommendations of CLSI H20-A2. 18

| Workflow efficiency
Workflow efficiency was assessed by comparing re-run and reflex testing rates for the same samples that were included in the interinstrument comparison on both analyzers.

| Within-laboratory
Results for the normal-level control were within Ricos' desirable specifications, except for MCV, which had slightly higher imprecision (1.2% vs 0.7%; Table 1). Imprecision for the low-and high-level controls was also within the desirable imprecision for most parameters.
TA B L E 3 Comparison of Alinity hq WBC differential with manual WBC differential The RBC (and consequently, HCT) %CV results, however, were somewhat higher than the desirable specifications in the high-level control. Imprecision for the low-level EO concentration also exceeded the desirable specification (14.7% vs 10.5%). BASO concentrations showed a high level of imprecision due to the low concentrations; however, SD values were small at all three levels (0.03-0.08 × 10 9 /L).

| Interinstrument comparison
Very strong correlation was observed between results obtained with Alinity hq and CELL-DYN Sapphire (Table 2) (Table 2).

| Comparison of Alinity hq PLT count with ImmunoPLT results
The comparison of Alinity hq PLT results to those of the CD61-based ImmunoPLT results in the range of 9.64-927 × 10 9 /L showed a correla-

| Comparison of Alinity hq WBC differential with manual differential
Results showed strong or very strong correlation for all parameters, except for %BASO (Table 3). Bland-Altman analysis revealed an average negative bias of −7.67% for %NEU, and a positive bias of 4.13% and 3.15% for %LYM and %MONO. Comparison of manual and automated %IG and NR/W showed an overall low bias of 0.19% and −1.06%, respectively (Table 3).

| Flagging performance
When using 1% as threshold, the combined presence of BLAST flag and WBC differential invalidating data flags resulted in a blast detection sensitivity of 75% on Alinity hq and 94% on CELL-DYN Sapphire (P = 0.0007), with the same specificity (96%). At 5% blast level, both analyzers identified 100% of samples with blasts (Table 4).
There were two samples in the study cohort with >5% AL. None The sensitivity of Alinity hq for IG detection was compared to that of the CELL-DYN Sapphire IG flag at a threshold that has provided the best discrimination between pathological and nonpathological samples based on manual differential results (as described in the Materials And Methods section). Alinity hq was found to be more sensitive (81% vs 76%, P = 0.0862), although not statistically significant, and specific (88% vs 78%, P = 0.0002) than CELL-DYN Sapphire.

| Workflow efficiency
A significantly lower re-run rate was observed on

| D ISCUSS I ON
The analytical and clinical performance evaluation of the prototype and was also found to be slightly more sensitive (81% vs 76%, not significant) and more specific (88% vs 78%, P = 0.0002) than CELL-DYN Sapphire for detecting IG. In addition, Alinity hq accurately reports the absolute concentration of IG, as confirmed by the strong correlation and low bias between manual and Alinity hq %IG results.
The lower re-run rate on Alinity hq can be attributed to the different technologies. When RBC resistant to lysis are detected by CELL-DYN Sapphire, the run needs to be repeated in an extended lyse mode, while no repeat is necessary with Alinity hq. Moreover, samples with WBC concentrations between 0.20 and 2.00 × 10 9 /L are repeated with extended differential counts with CELL-DYN Sapphire, but not with Alinity hq. On the other hand, WBC or PLT carryover alerts were the main reason for re-running samples with Alinity hq, which, however, can be configured as an automatic process.
The percentage of samples that required manual differential due to morphological and numerical abnormalities was high with both instruments, although somewhat lower with Alinity hq. This is attributed to the composition of the sample cohort, the majority of which was selected to be pathological.
Limitations of the study include the use of a prototype instrument and software. The most significant difference between the launch software and the prototype software was the lack of the leftshift flag, which indicates the presence of increased number of band NEUs. In addition, RETIC results were not evaluated due to technical issues. Further evaluation is needed for assessing accuracy of results around medical decision limits, such as samples with PLT concentration of <20 × 10 9 /L. 23 In conclusion, good analytical and clinical results were obtained during this performance evaluation of the prototype Alinity hq hematology analyzer. The ability of Alinity hq to report IG count and %, the higher sensitivity of NRBC and IG detection along with the lower number of samples requiring re-run or reflex testing are important steps toward the reduction of manual differentials.

ACK N OWLED G EM ENTS
We would like to thank the staff of the Hematology Laboratory for their participation in this study.

CO N FLI C T O F I NTE R E S T
Dr Hoffmann is former employee of Abbott Diagnostics. Dr Lakos is an employee of Abbott Diagnostics.

AUTH O R CO NTR I B UTI O N S
All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.