Impact of biological and genetic features of leukemic cells on the occurrence of “shark fins” in the WPC channel scattergrams of the Sysmex XN hematology analyzers in patients with chronic lymphocytic leukemia

Introduction

Chronic lymphocytic leukemia (CLL), a low-grade B-cell lymphoproliferative disorder, is the most common type of leukemia in the Western countries [1].

In the initial diagnostic work-up a complete automated blood count, the examination of a peripheral blood smear (PBS) and immunophenotyping are required for the diagnosis of CLL [2]. Furthermore, in addition to the laboratory results, physical examination and staging by computed tomography, prognostic information is obtained based on various biological and genetic markers that can predict the time to progression and the response to treatment [3].

In patients with CLL, the white progenitor cell (WPC) channel of the Sysmex XN series hematology analyzers typically yields a varying proportion of cells that appear in the WPC scattergram in the upper left field due to a lower side scattered light (SSC) and a high side fluorescent light (SFL), resembling the shape of shark fins [4].

WPC channel analysis, which is performed as an automatically defined reflex testing in case of abnormalities in the white blood cell differential (WDF) channel, is capable of detecting blasts and differentiating abnormal and atypical cells of the lymphocyte class [5]. Hereby, dedicated WPC reagents penetrate the cell membrane of leukocytes and stain the intranuclear deoxyribonucleic acid (DNA). The SSC and the SFL intensity of the cells are measured and expressed as 2D scattergrams [6].

To our knowledge, the underlying mechanism leading to the “shark fins” in WPC scattergrams of CLL patients has not been elucidated so far. A potential hypothesis may be the pronounced fragility of the leukemic cells, which is a known phenomenon in CLL, leading to the occurrence of the characteristic smudge cells in PBS [7].

The aim of this study was to evaluate biological and/or genetic features of CLL cells as potential influencing factors on the blood cell measurements of Sysmex XN hematology analyzers. Therefore, the effects of the lymphocyte count, the genetic background and morphological changes in the leukemic cells, such as smudge cells, on the appearance and extent of the “shark fins” in the Sysmex WPC scattergrams were investigated.

Materials and methods

Automated blood counts and scattergrams of 95 consecutive CLL patients from the Institute of Laboratory Diagnostics of the Hospital of St. John of God in Graz, Austria, and the Ordenskrankenhäuser Linz Zentrallabor in Linz, Austria in the period from June 2021 to May 2022 were analyzed in this study. The specimens were obtained during routine blood count determination. The lymphocyte counts were measured from ethylene-diamine tetraacetic acid (EDTA) anticoagulated blood using Sysmex XN 2000 hematology analyzers (Sysmex, Vienna, Austria) according to the manufacturer’s instructions. During the data collection period the Sysmex XN software 22.12 was applied.

The automated hematology analyzers were constantly checked by internal controls and external quality assurance programs (Austrian Society for Quality Assurance and Standardization of Medical Diagnostic Tests (ÖQUASTA)).

The WPC scattergrams were exported in flow cytometry standard (FCS) format and analyzed for the presence, count (10ˆ9/L) and percentage of cells in the “shark fins” using the flow cytometry software Kaluza v2.1 (Beckman Coulter, CA, USA). The scattergrams were anonymized and coded and were finally examined in a blinded fashion.

PBS were stained according to the May-Grunwald-Giemsa technique based on the standardized operating procedures of the laboratories. Smudge cells were defined as disintegrated cells lacking intact cytoplasm and having a disrupted nuclear membrane [7]. The percentage of smudge cells was determined by experienced technologists using light microscopy.

Demographic and clinical characteristics, as well as data on the diagnosis, therapy and genetic findings were collected retrospectively from the medical records and anonymized for analysis. The following prognostically significant genetic changes were included in our investigation: The chromosomal alterations del(13q), del(11q), trisomy 12 and del(17p) determined by fluorescence in situ hybridisation (FISH) panel testing. The immunoglobulin heavy chain variable region (IGHV) and the tumor suppressor protein (TP)53 mutational status assessed by next generation sequencing using the Ion Torrento S5 platform (Thermo Fisher Scientific, Waltham, MA, USA) in Graz and the NextSeq 550 instrument (Illumina, San Diego, CA, USA) in Linz.

The study was approved by the Ethics Committee of the Hospital of St. John of God in Graz in accordance with the Declaration of Helsinki.

Results

Factors influencing the occurrence of “shark fins” in the WPC scattergrams

In all 95 CLL patient samples, cells were detected in the conspicuous lymphocyte gate of the SSC/SFL scattergram of the Sysmex XN WPC channel. Examples of such “shark fins” and of a normal WPC scattergram are shown in Figure 1.

Figure 1:

Normal WPC scattergram (left) and “shark fins” in two CLL-patients (middle, right).

The lymphocyte count was identified as a highly statistically significant and strong predictor of “shark fins” (p<0.0001). After adjustment for the lymphocyte count, the absence of del(17p), unmutated TP53 and mutated IGHV as well as the percentage of smudge cells showed a statistically significant influence on the percentage of cells in the “shark fins”. No statistically significant effect on the proportion of cells in the “shark fin” was observed for age, sex, del(13q), del(11q), trisomy 12 and treatment. The statistical results are summarized in Table 2.

Table 2:

Influence of potential factors on the presence of “shark fin” cells determined by Spearman correlation.

Parameter	p-Value
Unmutated TP53	0.01
Absence of del(17p)	0.02
Mutated IGHV	0.03
Smudge cells	0.04
Trisomy 12	0.19
Del(11q)	0.30
Del(13q)	0.32
Sex	0.28
Age	0.30
Treatment	0.48

1. TP53, tumor suppressor protein 53; IGHV, immunoglobulin heavy chain variable region.

Discussion

In all 95 analyzed blood samples of CLL patients, cells were located in the conspicuous lymphocyte gate of the SSC/SFL scattergram of the Sysmex XN 2000 WPC channel, which is consistent with previous data showing that the Sysmex XN hematology analyzers equipped with a WPC channel perform very well the differentiation between neoplastic and reactive lymphocytes [4]. Furthermore, the number of lymphocytes in the peripheral blood of the CLL patients was identified as a statistically highly significant and strong predictor for the occurrence of cells in this shark fin-shaped lymphocyte gate (p<0.0001).

The reason for the leukemic cells showing such a high intensity of SFL in the WPC scattergram could be due to the fact that malignant lymphocytes have more apparent DNA compared to normal leukocytes, based on an enhanced proliferation kinetics [6].

In the current study the proportion of smudge cells on PBS did exert a statistically significant influence on the occurrence of “shark fin” cells after adjustment for the lymphocyte count (p=0.04). This observation is in accordance with the fact that the differentiation of lymphocytes in the XN-WPC channel is based on the different membrane properties of the cells, especially the lipid composition, after application of specific detergents [9]. Smudge cells are thought to be caused by an increased vulnerability of CLL cells, and probably they are thus located in the “shark fin” gate in the SSC/SFL scattergram not only because of the increased amount of nucleic acids in leukemic cells mentioned above, but also due to increased permeability of the abnormal cell membrane [4], 7]. It has been previously shown that smudge cell formation in CLL is associated with an altered concentration of vimentin in lymphoid cells, a protein that is crucial for the stiffness and integrity of lymphocytes, and further, from a clinical point of view, it was demonstrated that a higher smudge cell percentage predicts prolonged overall survival [10], 11].

In addition to this constitutional feature of the leukemic cells, a statistically significant influence on the presence of “shark fins” was detected in study participants with unmutated TP53 (p=0.01), absence of del(17p) (p=0.02) and mutated IGHV (p=0.03).

The prognostically favourable mutated IGHV status was observed in 49 % (n=47) of the CLL patients investigated, which is lower compared to a previously published rate of 60–65 % [12], 13]. In this context, however, it has to be taken into account that in 13 % (n=11) of our study subjects a mutational status of IGHV was not available, which may have lowered the rate of IGHV mutations.

In our CLL study population del(17p) was detected in 3 % and a TP53 mutation in 10 %. This is in line with prior study groups that found deletions in the short arm of chromosome 17 in 4–8 % of untreated CLL patients and thereby in most cases of band 17p13, where the tumor suppressor gene TP53 is located [14]. In that study mutations of TP53 were detectable in 4–37 % of CLL patients and were associated with a very poor outcome [14].

In 88 % of our study patients in whom the TP53 mutational status was available, no TP53 mutation and also no del(17p) was present, which is regarded as prognostically favourable [2], 3]. Furthermore, a significant correlation for the occurrence and extent of the “shark fin” in the WPC-channel was found for the unmutated TP53 (p=0.01) and the lack of del(17p) (p=0.02).

No significant correlation with the number of “shark fin” cells was observed for del(13q), which is the most frequently described chromosomal alteration in CLL [15] and was also detectable in 55 % of the CLL patients included in our study. No significant association was identified for the genetic aberrations del(11q) and trisomy 12 as well.

Thus, with the absence of the TP53 mutation, the lack of del (17p), the mutated IGHV together with the increased percentage of smudge cells on the PBS, all statistically significant factors influencing the occurrence and number of cells in the “shark fins” were found to be prognostically favourable [3], 8], 11], 15]. Whether our observations may contribute to the assessment of prognosis cannot be answered based on our study protocol. Further studies incorporating prospective data are needed to answer that question.

As “shark fin” cells were also detectable even if none of the chromosomal alterations analyzed were found, it can be assumed that at least none of the genetic aberrations examined has to be present for the occurrence of “shark fins” in the Sysmex WPC channel.

Whether other mutations that have not been investigated or are not known so far may influence the “shark fin” phenomenon, cannot be ruled out.

Conclusions

In summary, the occurrence and extent of the “shark fins” in the Sysmex XN series WPC channel were highly significantly correlated with the number of lymphocytes in the peripheral blood of the investigated CLL patients. Furthermore, the absence of TP53 mutations and del(17p), the presence of mutated IGHV as well as the proportion of smudge cells in the PBS did exert a significant influence on the appearance of cells in the “shark fins”. Our observations clearly indicate an impact of biological and genetic properties of the leukemic cells on the formation of “shark fins” in the Sysmex XN series WPC scattergrams.