Hematology instruments don’t speak the same language: a comparison study between flagging messages of sysmex XN-1000 and alinity H

Main findings

Previously was reported that, despite using different technologies, there is a good agreement regarding cell differentiation and counting between the Alinity hq hematology analyzer and the Sysmex platforms [7]. This study aimed to further explore the comparability of these two platforms by investigating the accuracy and agreement of WBC-related flagging messages. In the present study, the agreement between the two instruments ranged from poor (for the Abnormal Lymphocytes) to good agreement (for IG flags). The high degree of agreement for IG between the instruments is also demonstrated by the results of the Bland Altman test and Passing-Bablok regression analysis.

Left shift

According to the ISLH guidelines, the laboratory should follow its own SOP when samples are flagged as “Left shift”. For this flag, a Se of 32.3 % was obtained for the Sysmex analyzer, and in 10/11 flagged samples, there was also an “Immature granulocyte” (IG) flag. A study investigating the flagging performance of the Sysmex XN series instruments reported that 7/15 samples flagged as “Left shift” also presented a secondary flag indicating atypical lymphocytes or blasts. However, the authors did not provide information regarding the number of samples with increased IG counts or IG flags [8]. In another study, the Se reported was 59.7 % with a Sp of 95.1 % for the Sysmex instrument but the population included were mostly patients undergoing chemotherapy or with known haematological malignancies [9].

For the Alinity analyzer, the Se was 68.8 %. In our study, a much higher PPV was found compared with the initial evaluation of the Alinity prototype (77.5 versus 61.4 %) [10]. This result may be explained by the different software version used by our Alinity hq.

Only a fair agreement was obtained between the two analyzers. Consequently, in cases where samples flagged positively on the Sysmex system, repeating the analysis on the Alinity platform may reduce the necessity for PBS needed. However, it is essential to note that when data concerning “Left shift” are concomitant with IG flags or values, the SOP should follow the recommendations for IG from ISLH.

Immature granulocytes

A PBS positive finding for immature granulocytes is defined by the ISLH guidelines as either >2 % metamyelocytes or >1 % (pro)myelocytes. In the present study, Sysmex XN-1000 showed a good Sp of 87.6 %. A study where Sysmex XN series instruments were compared with the Advia hematology analyzer platform, reported a specificity that is comparable to the one observed in the actual study (82.0 %) [11]. In a comparison between Sysmex analyzer and PBS, a Sp of 98.9 % was reported [9].

Alinity hq showed a similar Sp (97.7 %). An 81.5 % Se for the IG count with a flagging threshold >1 % was found independently of left shift flags. In a previous evaluation, Alinity hq prototype showed an identical left shift Se of 58.2 % and a slightly higher left shift Sp (92.7 vs. 97.7 %) compared to the CELL-DYN Sapphire automated hematology system [10]. However, in that study, the left shift and IG count >1 % were evaluated together [10].

It is important to understand that, while Alinity hq also counts IGs, the two analyzers have different flagging criteria: IG >0.1 × 10⁹/L for Sysmex and IG >1 % for Alinity. Thus, at least in theory, flagging differences between the two analyzers may be a result of just that: different approaches to flagging thresholds (absolute count vs. percentages).

In six samples from group B (IG>0.1 × 10⁹/L), the difference of the IG count between the two instruments was outside the confidence interval of the mean difference. However, all six samples were from patients with hematological disorders (Acute Myeloid Leukemia or Myelofibrosis), three samples with >20 % monocytic blast cells and three samples with erythroblasts.

In other study, a small difference (−0.6 %) was found between results of the IG obtained with Alinity hq in comparison with flowcytometry and manual differential count [12]. Similarly, for the Sysmex instrument and the manual review comparison, small and clinically insignificant differences (less than 3 %) was found in samples with IG ≤6 % [13]. In samples with IG >6 % it is expected to have supplementary flags triggered which should prompt a PBS evaluation. Group’s A deviation from linearity may be explained by a high number of samples with “0” values (52 samples) on Alinity, such small values could not be successfully correlated by a regression equation, the coefficient of determination of the regression being small (0.2). In the study [13] that compared the Sysmex IG with PBS findings, the regression equation was y=1.011 + 0.2039*x; the difference may be explained that in our group only samples with IG<0.1 × 10⁹/L were included while in the referenced study samples with a high range of values were included. Also, the regression was performed with PBS not between two instruments. The group B regression equation is more suited for absolute value comparation, with no significant deviation from linearity. Although the intercept can be considered statistically significant (CI: 0.085–0.101), it is reasonable to accept this difference as not medically significant.

Left shift and immature granulocytes

For Sysmex combining the two flags resulted in an improved Se and lower Sp compared to Left shift alone, while for Alinity improvement was noted in Se compared to Left shift alone. The performance of the combined flags overcame the insufficiencies of each flag individually, the only exception was for NPV (87.3 %) where on Alinity both flags had higher NPVs independently.

Blasts and abnormal lymphocytes

On the Sysmex instrument, 96 samples were flagged as “Blast/Abn Lympho”. Of these, 22 also had an “Atypical lympho” flag, 27 showed positive IG findings on PBS, 47 had lymphocytosis and/or monocytosis, and only 9 samples were confirmed with blast cells after PBS review, resulting in a Sp of 53.4 %. A similar result was reported by another study evaluating the Sysmex XN series instruments by PBS reviews and reflex WPC channel testing, where only 4/55 flagged samples presented blast cells, while 30 exhibited abnormal or atypical lymphocytes [8]. Other studies that assessed the performance of other instruments in XN series showed high specificities of 84.9 and 97.0 % [9], 14]. In the present study, the Se of Sysmex vs. manual review was 100 %, aligning with the reported Se of Sysmex XN series (100 %) when compared to the Advia platform [14].

Alinity hq flagged 39 samples as “Blast?” Among the 32 false-positive samples, 20 displayed a “VAR LYM” flag, 10 had lymphocytosis and/or monocytosis, and 2 were from patients with malignant pathologies. In a previous evaluation of Alinity hq’s prototype, the highest Se achieved was 70 %, focusing on samples with >5 % blasts [10]. In the current study, even when assessing a more diverse sample set, a slightly higher Se (77.7 %) was recorded, which could also be explained by the differences in the software version used.

Regarding the comparison between analyzers, out of the 96 samples flagged by Sysmex, 60 were negative on Alinity hq and only a fair agreement was obtained. Therefore, a sample flagged as “Blast/Abn Lympho” by Sysmex may be retested on Alinity in order to reduce the need for manual PBS reviews. This divergence in results between the two analyzers is attributed to their distinct technologies and the wide morphological variety of abnormal/atypical lymphocytes.

Atypical lymphocytes

“Atypical Lympho” and “VAR LYM” are similar flags for both analyzers. Sysmex’s “Atypical Lymphocytes” flag showed a low Se (40.0 %). Out of 37 samples with a positive manual review finding (>5 % atypical/reactive lymphocytes [1]), 14 were flagged with “Atypical Lymphocytes” and 21 with “Blast/Abn Lympho”. While Sysmex effectively identified atypical/abnormal lymphocytes, the presence of a flag expressed as “Blast?Abn Lympho” can be confusing, despite ISLH guidelines mandating slide reviews in both cases.

The Alinity analyzer’s “VAR LYM ” flag showed a Sp of 72.1 %, but its Se may be impacted by the presence of lymphocytosis or monocytosis in nearly half of the samples. Even if the two instruments have different technologies, it seems that when atypical lymphocytes are present, much like in manual review, an agreement is not easily reached. The agreement between the two instruments was moderate.

Blasts, abnormal lymphocytes and atypical lymphocytes

Considering the morphology of blasts (myeloblasts or lymphoblasts), reactive lymphocytes and the technology challenges in identifying them, an analysis combining the flags regarding these cells seemed mandatory. As well known, the Sysmex instrument already has a flag “Blast?Abn Lympho” which may include the blast cells or abnormal lymphocytes. Combining this flag with the “Atypical lymph” did not bring an overall improvement. The Alinity instrument has designated flags for blasts and atypical lymphocytes. Combining the “Blast” and “VAR LYM” resulted in a better performance regarding PPV (37.5 %), but lower Sp (65.9 %) than each flag individually.

Using two CBC platforms may not apply to all laboratories, it is common in large hospitals with multi-site labs. Based on our experience in an emergency laboratory, reanalyzing samples flagged as Blast/Abnormal Lymphocytes by the Sysmex instrument, on the Alinity platform may be beneficial. Despite ISLH recommendations to perform PBS for initial samples and not ignore any flags, performing PBS tests for this flag is inefficient compared to rerunning the sample on another platform.

Study limitations

The interpretation of Atypical Lymphocytes was not strictly guided by ISLH standards, given the subjectivity of these terms among laboratory personnel and the difficulty in reaching a consensus. Another study limitation is the low number of blast cells that were confirmed by PBS although we had a high proportion of patients with hematological pathology. The Q value (probability of presence of abnormal cells on an arbitrary scale) for the Sysmex instrument was not included in the evaluation since the Alinity Instrument does not provide a Q value and the aim of the study was to compare the performance of the instruments in laboratories that use simultaneously the two platforms. Although samples included in the study referred to multiple morphological abnormalities, regardless of cell lineage, the sample selection based on flags triggered by the Sysmex instrument is an important limitation.