Clin. Lab. 2/2024 305 Clin. Lab. 2024;70:305-310 ©Copyright ORIGINAL ARTICLE Verification of a Benchtop Hematology Analyzer with a 5-Part Differential Count: There is Nothing Wrong with Being Small Anima Baiden, Elise Schapkaitz, Maynolia Naidoo, Susan Louw Department of Molecular Medicine and Hematology, School of Pathology, Faculty of Health Sciences, University of The Witwatersrand (Wits) and National Health Laboratory Service (NHLS), Johannesburg, South Africa SUMMARY Background: The benchtop ADVIA 560 AL hematology analyzer (Siemens Healthineers Tarrytown, NY, USA) offers a small footprint and ease of operation making it suitable for satellite laboratories and intensive care units. A verification study of this analyzer was performed. Methods: Between- and intra-run precision, carry-over, linearity, and throughput were evaluated on the ADVIA 560 AL . Accuracy was assessed on 94 patient samples by comparing the results obtained on the ADVIA 560 AL to the results on the reference Sysmex XN1000 analyzer (Sysmex Corporation, Kobe, Japan). Results: The ADVIA 560 AL showed acceptable imprecision on control material and minimal bias in comparison to the XN 1000 on patient samples with a throughput of 60 samples per hour. The percentage carryover was not significant and the linearity was within acceptable limits. Conclusions: The ADVIA 560 AL bench-top analyzer is suitable for acute care centers and satellite laboratories owing to its small footprint, ease of use, and reproducible and accurate results. (Clin. Lab. 2024;70:305-310. DOI: 10.7754/Clin.Lab.2023.230630) Correspondence: Dr. Susan Louw P.O. Box 94 Cresta, Gauteng, 2118 South Africa Phone: +27 (82) 606 3487 Fax: +27 866 99 99 01 Email: Susan.louw@nhls.ac.za ORCID https://orcid.org/0000-0002-4315-1496 Anima Baiden ORCID https://orcid.org/0000-0001-6912-8617 Elise Schapkaitz ORCID https://orcid.org/0000-0002-1534-2930 Maynolia Naidoo ORCID https://orcid.org/0000-0002-1195-9992 _______________________________________________________ Manuscript accepted August 6, 2023 KEYWORDS benchtop hematology analyzer, 5-part differential count, verification, satellite laboratories INTRODUCTION During the Coronavirus disease 2019 (COVID-19) pan- demic, the utility of hematological, coagulation and bio- chemical parameters as predictors of the severity of in- fection were established [1]. Components of the full blood and differential counts (FBC and DIFF) including low platelets [2], low hemoglobin [3], and changes in white blood cell subtypes, namely lymphopenia and neutrophilia, were predictive of the outcomes of patients with COVID-19 [4]. It was against this background that the National Health Laboratory Service (NHLS) in South Africa validated testing platforms for acute care centers and satellite lab- oratories [5]. The automated ADVIA 560 AL (Sie- mens Healthineers, Tarrytown, NY, USA) is a small (height, 52 cm, width, 41 cm, and depth, 49 cm) bench- A. Baiden et al. Clin. Lab. 2/2024 306 top analyzer that offers improved turnaround times ow- ing to the suitability for near-patient-testing to assist with emergency patient management. The analyzer has a throughput of 60 samples per hour, and a result can be generated within 60 seconds on emergency samples which can be processed without completing the analysis of samples in the auto-loader tray [6]. Furthermore, it can be connected to a printer, interfaced with a laborato- ry information system and a handheld bar-code reader is available. A volume of only 110 μL, excluding dead volume, is required for open and closed mode analyses. The analyzer stores up to 100,000 patient results as well as quality control (QC) and calibration results including graphical scatter diagrams and histograms. The ADVIA 560® analyzer is compatible with ethylenediaminetetra- acetic acid (EDTA) sample tubes, such as Becton Dick- inson (BD)® vacutainers, and pediatric samples in Sarst- edt Monovette® tubes. The instrument uses impedance variation to measure the red blood cell and platelet count and optical flow-cytometry for the white blood cell and DIFF counts. Many small benchtop FBC ana- lyzers offer a 3-part DIFF but the ADVIA 560® ana- lyzer performs a 5-part DIFF consisting of lymphocytes, monocytes, neutrophils, eosinophils, and basophils. The hemoglobin is measured by spectrophotometry. MATERIALS AND METHODS Ethics Ethics approval for the study was obtained from the Hu- man Research Ethics Committee (HREC) of the Univer- sity of the Witwatersrand (Protocol M1911201). Analyzer The ADVIA 560® analyzer was installed and calibrated by the manufacturer and laboratory staff received opera- tor training. This study was performed in accordance with the ISO 15189 International Standard for Medical Laboratories, the International Committee on Standard- isation in Hematology (ICSH 2014), and the Clinical and Laboratory Standards Institute (CLSI 2010) method comparison guidelines [5,7]. Samples Commercial controls and residual blood specimens from 94 samples with normal and abnormal hematology profiles submitted for testing at Charlotte Maxeke Jo- hannesburg (CMJAH) and Chris Hani Baragwanath (CHBAH) Academic Hospitals in Johannesburg, South Africa, were included in the study. Patient samples with possible interfering substances, such as lipids and biliru- bin as well as hemolyzed samples were included. Fifty- one samples were from patients who were investigated for COVID-19, and of these, 35 (69%) were positive on real-time polymerase chain reaction (PCR) for this viral infection. Patient samples were collected in K2EDTA tubes (Vacutainer®, Becton Dickinson, Plymouth, UK) and stored at room temperature until analysis by a dedi- cated technologist within 8 hours of collection at the CMJAH, NHLS laboratory [8]. Pediatric samples were excluded from the study since the ADVIA 560 AL® an- alyzer is not compatible with microtainers, which are the collection tubes utilized for pediatric patients at CMJAH and CHBAH. Precision Between-run precision was assessed with commercial controls (high, low, and normal) processed daily for 10 days. Within-run precision was assessed with commer- cial controls processed 20 times in both the closed auto- mated and open manual mode. Different lots of control material to the internal quality control were used. The mean, standard deviation (SD), and coefficient of varia- tion (CV) were collated on an Excel® spreadsheet and compared to state of the art (SOTA) and manufacturer precision limits [7,9]. Comparison study Ninety-four samples covering the range of FBC param- eters as per ICSH [7] recommendation, were analyzed on both the ADVIA 560 AL® and the already validated Sysmex XN 1000® (Sysmex® Corporation, Kobe, Ja- pan) automated hematology analyzers. The ADVIA 560 AL® DIFF and morphological flags were compared with manual smear evaluation by 2 morphologists on 40 samples as per the ICSH criteria for grading of periph- eral blood morphology [10]. Bland-Altman method comparison and regression analyses, with a statistical significance of p < 0.05, were performed. Linearity Linearity was assessed with serial normal saline dilu- tions (1:2; 1:4; 1:8, and 1:16) of a patient’s samples fol- lowed by duplicate analysis. Carryover Carryover from a patient sample with high counts to a sample with low counts was assessed by analyzing the high count sample 3 times (H1, H2, and H3) followed by 3 consecutive analyses of the low count sample (L1, L2 and L3). Carry-over was calculated with the follow- ing formula: Carryover (%) = (L1 - L3)/(H3 - L3) x 100. Throughput Throughput of the auto-sampler for FBC and DIFF was assessed with 60 patient samples analyzed as a batch. The samples were loaded on sample racks which hold 10 samples each. Throughput was defined as the time from bar code reading of the first sample to the last sample reaching the output tray. Verification of a Benchtop Hematology Analyser Clin. Lab. 2/2024 307 Table 1. Full blood count and differential count precision results. Parameter (units) Observed precision (%) Manufacturer claim (%) SOTA (%) Criteria met Within-run Between-run Within-run Between-run Within-run Between-run SD CV SD CV SD CV SD CV CV CV Full blood count (FBC) WBC (109/L) 0.21 1.81 0.29 2.20 < 0.18 < 2.7 < 0.20 < 3.4 2.5 2.5 manufacturer; SOTA RBC (1012/L) 0.05 1.44 0.05 1.59 < 0.11 < 1.7 < 0.13 < 2 1.1 1.1 manufacturer only HGB (g/L) 0.17 1.58 0.18 1.47 < 2.0 < 2.0 < 0.22 < 2.4 0.9 1.0 manufacturer only MCV (fL) 0.37 0.41 0.56 0.60 < 1.0 < 1.7 < 1.2 < 2 0.6 0.8 manufacturer; SOTA RDW (%) 0.26 1.62 0.36 2.26 < 0.4 < 2.5 < 0.45 < 3 2.0 2.0 manufacturer; SOTA PLT (109/L) 8.21 4.24 7.97 3.67 < 23 < 6 < 27 < 7 3 3 manufacturer only MPV (fL) 0.31 3.16 0.21 2.18 < 0.45 < 8.7 < 0.50 < 10 2.5 2.5 manufacturer; SOTA Differential count (Diff) Percentage (%) of total white blood cell count NEU 1.24 2.23 1.74 3.20 < 3.5 - < 3.5 - - - manufacturer LYM 0.95 3.08 1.28 4.00 < 3.1 - < 3.1 - - - manufacturer MON 0.71 14.72 1.11 21.30 < 2.0 - < 2.0 - - - manufacturer EO 0.32 7.29 0.47 10.10 < 2.0 - < 2.0 - - - manufacturer BAS 0.01 7.23 0.00 0.00 < 0.5 - < 0.5 - - - manufacturer Absolute counts (109/L) Total WBC 0.15 1.82 0.12 1.49 < 0.16 < 2.7 < 0.16 < 2.7 - 2.5 manufacturer; SOTA NEU 0.12 2.14 0,17 3.22 - - - - - 2.5 did not meet SOTA LYM 0.10 3.77 0.14 5.25 - - - - - 3.5 did not meet SOTA MON 0.07 16.08 0.12 20.86 - - - - - 8.5 did not meet SOTA EO 0.95 52.48 0.05 9.94 - - - - - 10 SOTA BAS 0.00 0.00 0.00 3.90 - - - - - 20 SOTA SD - standard deviation, CV - coefficient of variation, SOTA - state of the art, WBC - white blood cell, RBC - red blood cell, HGB - hemoglobin, MCV - mean cell volume, RDW - red cell distribution width, PLT - platelets, MPV - mean platelet volume, NEU - neutrophils, LYM - lymphocytes, MON - monocytes, EO - eosinophils, BAS - basophils, - - parameter target not available. RESULTS Precision Results of the precision study of the FBC and DIFF are presented in Table 1. The ADVIA 560 AL® showed ac- ceptable imprecision for FBC and % DIFF parameters according to the manufacturer’s criteria. The absolute counts of neutrophils, lymphocytes, and monocytes did however not meet SOTA criteria [9], and the manufac- turer imprecision limits for these parameters were also not available. The results obtained on open vs. closed mode were comparable without statistically significant differences. Comparison study The results of the comparison study on 94 patient sam- ples analyzed on the ADVIA 560 AL® and the Sysmex XN 1000® analyzers are depicted in Table 2. The corre- lations between the ADVIA 560 AL® and the XN 1000® analyzers, with the exception of the mean platelet volume (MPV), were excellent (Table 2 and Figure 1). Mean differences represented by the Bland‐Altman sta- A. Baiden et al. Clin. Lab. 2/2024 308 Table 2. Full blood count and differential count accuracy results. Parameter (units) SD a R value Bias % TEobs EFLM TEa Full blood count WBC (109/L) 0.21 > 0.99 1.94 2.36 ± 13.1 RBC (1012/L) 0.05 > 0.99 -3.27 -3.17 ± 3.8 HGB (g/L) 0.17 0.99 3.30 3.64 ± 3.9 HCT (L/L) 0.52 0.94 -4.01 -2.97 ± 3.0 MCV (fL) 0.37 0.94 -1.03 -0.29 ± 1.6 PLT (109/L) 8.21 0.99 -7.80 8.62 ± 10.3 MPV (fL) 0.31 0.84 -29.52 -28.9 ± 3.6 Differential count NEU (109/L) 0.16 0.96 -5.76 -5.44 ± 18.6 LYM (109/L) 0.10 0.95 -9.55 -9.35 ± 14.1 MON (109/L) 0.07 > 0.99 -8.16 -8.02 ± 17.3 EO (109/L) 0.95 0.76 3.53 5.43 ± 27.6 BAS (109/L) 0.00 0.35 -62.61 -62.91 ± 16.9 SD a - within-run standard deviation, TEobs - total error observed, EFLM Tea - European Federation of Clinical Chemistry and Laboratory Medicine Total allowable error, WBC - white blood cell, RBC - red blood cell, HGB - hemoglobin, HCT - hematocrit, MCV - mean cell volume, PLT - platelets, MPV - mean platelet volume, NEU - neutrophils, LYM - lymphocytes, MON - monocytes, EO - eosinophils, BAS - basophils. tistics were small for white blood cell (WBC) and he- moglobin (HGB) with few outliers. For platelets (PLT), however, a mean difference of -21.37 (95% CI, -65.54 to 22.80) was found. The correlation between the manu- al morphological and automated differential counts was excellent with the exception of the basophil count. The following 5 morphology flags were triggered on the ADVIA 560 AL® analyzer during the analysis of 40 samples: White blood cell high linearity range exceed- ed; Monocyte-Neutrophil differentiation; Platelet-red blood cell fragment differentiation; abnormal differen- tial counts and Monocyte-Lymphocyte differentiation. These samples were assessed with manual morphologi- cal examination and the false negative rate of the flags was < 5%. Carryover The percentage carryover for white blood cells was 0.26%, 0% for hemoglobin and 0.31% for platelets. These results were not significant and within the manu- facturer's limit of 1%. Linearity The linearity for hemoglobin (44 - 223 g/L), white blood cells (0.29 - 112.92 x 109/L), and platelets (3 - 1,006 x 109/L) were within acceptable limits for high and low ranges. Throughput The throughput for 60 samples was 62 minutes for FBC-DIFF analysis. DISCUSSION The ADVIA 560 AL® hematology analyzer is suitable for sample analysis in clinical settings such as emergen- cy departments, critical care units, and field hospitals since this analyzer has a small footprint, requires mini- mal operator expertise and maintenance while produc- ing accurate and reproducible results. In this study, the performance of the ADVIA 560 AL® automated analyzer was compared with the reference large benchtop Sysmex XN 1000® analyzer and manual slide morphological examination. The ADVIA 560 AL® showed acceptable accuracy in comparison with the Sysmex XN 1000® for FBC and DIFF parameters which is consistent with previously published studies investi- gating large bench-top Sysmex® and ADVIA analyzers [11]. The exception to this was the PLT and mean plate- let volume (MPV) which were lower on the ADVIA 560 AL® versus the XN 1000®. This discrepancy has al- so been documented in a previous validation study in- volving multiple analyzers [12]. Impedance technology potentially underestimates platelet numbers and mean size, particularly in the presence of large platelets [13]. This recognized limitation has been addressed on the ADVIA 560 AL® with the platelet-red blood cell frag- ment differentiation morphology flag signaling the need for morphological examination. The outliers noted on the HGB correlation (Figure 1) comprised samples with very high white cell counts of ˃ 70 x 109/L. The poten- tial for increased turbidity in such samples resulting in a disturbance of the HGB measurement has been previ- Verification of a Benchtop Hematology Analyser Clin. Lab. 2/2024 309 Hemoglobin (HGB) (g/dL) 5 1 0 1 5 2 0 2 5 - 1 0 1 2 3 4 A v e r a g e U p p e r 9 5 % l i m i t o f a g r e e m e n t = 1 . 3 5 4 L o w e r 9 5 % l i m i t o f a g r e e m e n t = - 0 . 4 7 B i a s 0 . 4 3 7 B l a n d - A l t m a n D i f f e r e n c e v s . A v e r a g e H a e m o g l o b i n ( H b ) ( g / d L ) A d v i a - S y s m e x D if f e r e n c e ( A d v ia - S y s m e x ) Test parameter Correlation coefficient (r) Bias Slope (95% CI) Intercept (95% CI) Mean SD HB 0.9724 0.4372 0.46 0.920 (0.898 - 0.941) 1.341 (1.079 - 1.602) White blood cells (WBC) (x 109/L) 0 5 0 1 0 0 1 5 0 - 3 0 - 2 0 - 1 0 0 1 0 A v e r a g e L o w e r 9 5 % l i m i t o f a g r e e m e n t = - 6 . 0 8 D if f e r e n c e ( A d v ia - S y s m e x ) B i a s - 0 . 1 5 6 U p p e r 9 5 % l i m i t o f a g r e e m e n t = 5 . 7 7 B l a n d - A l t m a n D i f f e r e n c e v s . A v e r a g e W h i t e b l o o d c e l l s ( W B C s ) ( 1 0 9 / L ) A d v i a - S y s m e x 0 5 0 1 0 0 1 5 0 0 5 0 1 0 0 1 5 0 L i n e a r r e g r e s s i o n : W h i t e b l o o d c e l l ( W B C ) ( x 1 0 9 / L ) S y s m e x X N A d v ia 5 6 0 L i n e o f e q u a l i t y Test parameter Correlation coefficient (r) Bias Slope (95% CI) Intercept (95% CI) Mean SD WBC 0.9761 -0.1566 3.02 1.08 (1.062 - 1.106) -0.45 (-0.679 - -0.214) Platelets (PLTs) (x 109/L) 2 0 0 4 0 0 6 0 0 8 0 0 - 1 0 0 - 5 0 0 5 0 A v e r a g e U p p e r 9 5 % l i m i t o f a g r e e m e n t 2 2 . 8 L o w e r 9 5 % l i m i t o f a g r e e m e n t = - 6 5 . 5 4 B i a s - 2 1 . 3 7 D if f e r e n c e ( A d v ia - S y s m e x ) B l a n d - A l t m a n D i f f e r e n c e v s . a v e r a g e P l a t e l e t s ( P L T s ) ( 1 0 9 / L ) A d v i a - S y s m e x 0 2 0 0 4 0 0 6 0 0 8 0 0 0 2 0 0 4 0 0 6 0 0 8 0 0 L i n e a r r e g r e s s i o n : P l a t e l e t s ( P L T s ) ( x 1 0 9 / L ) S y s m e x X N A d v ia 5 6 0 L i n e o f e q u a l i t y Test parameter Correlation coefficient (r) Bias Slope (95% CI) Intercept (95% CI) Mean SD PLT 0.9857 -21.37 22.54 0.91 (0.888 - 0.932) 2.9 (-3.4 - 9.3) Figure 1. Bland Altman and linear regression analyses of white blood cells, hemoglobin, and platelets. A. Baiden et al. Clin. Lab. 2/2024 310 ously documented [14]. The ADVIA 5-part DIFF show- ed good correlation with the gold standard manual mor- phological slide examination with the exception of ba- sophils, a finding which has also been documented in other evaluations of hematology analyzers [15]. The ADVIA 560 AL® showed acceptable imprecision for FBC and % DIFF. Manufacturer’s imprecision lim- its were not available for absolute DIFF counts, and neutrophil, lymphocyte, and monocyte absolute counts did not meet SOTA criteria [9]. The false negative rate of morphology flags was < 5% when compared with manual morphology. Additionally, the ADVIA 560 AL® showed good linearity for high and low ranges and < 1% carryover which is comparable with other small benchtop hematology analyzers [16]. The study has a major limitation in that the ADVIA 560 AL® analyzer is incompatible with microtainer samples and verification of the pediatric Sarstedt Monovette® tubes was not performed. Additional evaluation of the accuracy of the automated flagging of samples which require morphologic examination is also required. The time to analysis of the samples was within 8 hours, in accordance with ICSH recommendations [5]. A shorter time to analysis was not possible as some samples were collected at a neighboring hospital. In conclusion, the ADVIA 560 AL® analyzer is a small benchtop hematology analyzer suitable for acute care centers and satellite laboratories owing to its small foot- print, high throughput, and reproducible and accurate analytical results. This analyzer can potentially assist with the triaging and appropriate escalation of the level of care of patients presenting for emergency care. Data Availability Statement: All study data will be made available on request. Ethics Approval Statement: The Human Research Ethics Committee (HREC) of the University of the Witwatersrand approved the study (Protocol M1911201). Individual patient consent was waived. Source of Support: The analyzer and reagents were sponsored by Siemens Healthineers . Declaration of Interest: The authors declare no conflict of interest with regard to this verification study. References: 1. Soraya GV, Ulhaq ZS. Crucial laboratory parameters in COVID- 19 diagnosis and prognosis: An updated meta-analysis. Med Clin (Engl Ed) 2020;155(4):143-51. (PMID: 32864456) 2. Yang X, Yang Q, Wang Y, et al. Thrombocytopenia and its asso- ciation with mortality in patients with COVID-19. J Thromb Haemost 2020;18(6):1469-72. (PMID: 32302435) 3. Bergamaschi G, Borrelli de Andreis F, Aronico N, et al. Anemia in patients with Covid-19: pathogenesis and clinical significance. Clin Exp Med 2021;21(2):239-46. (PMID: 33417082) 4. Fathi N, Rezaei N. Lymphopenia in COVID-19: Therapeutic op- portunities. 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