
==== Front
J Cytol
J Cytol
JCytol
J Cytol
Journal of Cytology
0970-9371
0974-5165
Wolters Kluwer - Medknow India

JCytol-41-176
10.4103/joc.joc_122_23
Original Article
Diagnostic Utility of High Fluorescence Cells in Detecting Malignant Effusions
Mishra Shashank 1
Parikh Biren P. 1
Singh Jayvardhan 2
1 Department of Oncopathology, Gujarat Cancer and Research Institute, Ahmedabad, Gujarat, India
2 Department of Physiology, GSVM Medical College, Kanpur, Uttar Pradesh, India
Address for correspondence: Dr. Biren P. Parikh, Department of Oncopathology, Gujarat Cancer and Research Institute, Ahmedabad, Gujarat, India. E-mail: biren.parikh@gcriindia.org
Jul-Sep 2024
18 7 2024
41 3 176180
14 7 2023
03 6 2024
21 6 2024
Copyright: © 2024 Journal of Cytology | Indian Academy of Cytologists
2024
https://creativecommons.org/licenses/by-nc-sa/4.0/ This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
Background:

Most modern haematology analysers have a dedicated body fluid mode for cell counts of body fluids. Many analysers also count the number of high fluorescence cells (HF cells). HF cells have a large nuclear size and emit high fluorescence when stained with fluorescent dyes. Due to their large nuclear size, Malignant cells are counted as HF cells.

Aims and Objectives:

We aim to determine the diagnostic utility of HF cells in predicting the presence of malignant cells in serous effusions.

Materials and Methods:

HF cell counts were done on 209 serous fluid samples using the body fluid mode of Mindray BC-6800 plus haematology analyser. Papanicilaou-stained smears of all samples were examined for the presence of malignant cells by a panel of cytopathologists. ROC curve analysis was done to determine the sensitivity and specificity of HF cells in malignant effusions.

Results:

Out of 209 samples, malignant cells were found by microscopy in 97 cases (46.4%). The absolute number and percentage of HF cells were significantly higher (P < 0.001) in malignant effusions (HF# = 24.9 cells/ul, HF% = 10.4%) when compared to non-malignant samples (HF# = 4.95 cells/ul, HF% = 5.76%). ROC curve analysis determined an optimal cut-off of ≥30 HF cells/ul (sensitivity = 73.91, specificity = 55.66%) for the prediction of malignant cells.

Conclusion:

HF cells in serous effusions can be a helpful tool to aid the pathologist, but it is not an ideal screening test due to its low sensitivity (67.74%) and negative likelihood ratio (0.5) at a cut-off of ≥30 HF cells/ul. However, due to high specificity of 83.18% at a cut-off of ≥72 HF cells/ul, a meticulous search for malignant cells should be done on microscopy.

BC-6800 plus
body fluids
cytopathology
high fluorescence cells
malignant effusions
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pmcINTRODUCTION

Ascitic and pleural fluids of patients having varied underlying pathology are frequently received in a clinical laboratory. Routine analysis of serous fluids includes biochemical analysis for glucose, proteins, lactate dehydrogenase, and cell counts. In addition, microscopic examination of stained slides for the presence or absence of malignant cells is vital in cases where malignancy is suspected.[1] However, due to the manual nature of microscopic examination for malignant cells, the technique is time-consuming and has significant inter-observer variability.[2] Most of the automated analysers used in modern laboratories have a body fluid mode for total and differential counts of body fluids. The BC-6800 plus (Mindray Medical International Ltd., Shenzhen, China) is a type of automated haematology analyser commonly used for the complete blood count (CBC) and differential count of blood cells. The analyser also has a body fluid (BF) mode that can be used for the analysis of body fluids such as pleural, peritoneal, and cerebrospinal fluid. The BC-6800 uses laser scattering to generate forward-scatter (FS), side-scatter (SS), and lateral fluorescence (FL) signals. The intensity of forward scatter indicates the size of the cell, side scatter is proportional to cytoplasmic granules, and lateral fluorescence reflects the nuclear size and complexity.[3] These three signals are used for the calculation of total white blood cells in BF (WBC-BF), polymorphonuclear (PMN) cells, mononuclear (MN) cells, and high-fluorescence body fluid (HF-BF) cells.[4] Malignant cells have larger nuclear size than leucocytes and therefore emit high levels of fluorescence when exposed to certain dyes or probes. In body fluids, high fluorescence (HF) cells indicate the presence of atypical or malignant cells. If HF cells are detected in the body fluid sample, cytological examination of stained slide is required for confirmation. The BC-6800 analyser’s body fluid mode is a useful tool in the initial rapid screening of malignant cells in body fluids. However, mesothelial cells and macrophages also have larger nuclear size and thus are counted as HF cells in body fluids. This contributes to the low specificity of this technique in detection of malignant cells in body fluids.[5] In this study, we aim to compare the sensitivity and specificity of the detection of HF cells in pleural and peritoneal fluids by BC-6800 haematology analyser with the detection of malignant cells using microscopic cytological analysis of stained slides.

MATERIALS AND METHODS

The study received approval from the institutional ethics committee has been taken on 04 July 2023. The study was conducted at a tertiary care cancer hospital over 6 months between July 2022 and December 2022. The laboratory is a NABL accredited lab and performs daily internal quality control of an automated hematology analyser. A total of 209 serous body fluid samples were included in the study; these included 112 pleural effusion samples, 96 ascitic fluid samples, and 1 drainage fluid. Patients having confirmed or suspected carcionomas, sarcomas, and hematolymphoid malignancies are included in the study. A minimum of 3 ml of serous fluid was collected in lavender top vacutainers, which have K2- EDTA as an anticoagulant. Samples with insufficient volume, inappropriate containers, and mismatch between requisition form and sample were excluded from the study.

Cell counts on Mindray BC-6800 plus analyser

Body fluid samples were gently mixed by inversion, and any visible clot was manually removed before aspiration. Total and differential counts of these body fluids were done using the body fluid (BF) mode of Mindray BC-6800 plus a haematology analyser, which employs labxpert software. The analyser is based on the principle of fluorescence flow cytometry to differentiate cells as per their size (forward scatter) and cytoplasmic granularity (side scatter), and requires a minimum volume of 500 ul for cell counts. In body fluid mode, the analyser gives the absolute number of total white blood cells (WBC-BF), number, and percentage of polymorphonuclear cells (PMN#/%), mononuclear cells (MN/MN%), and total nucleated cells (TC-BF#). HF cell number and percentage (HF#/HF%) are included as research parameters. HF cells have large nuclei, which emit higher fluorescence intensity and are found above the WBC region in the scatter plot. The total nucleated cell count (TN#) is the sum of the number of WBC and HF cells. A Wright Giemsa stained slide was also prepared from the same sample in the haematology laboratory for initial screening for the presence of malignant cells and for correlating the results of the automated analyser.

Cytological examination

Each body fluid sample was also sent to the cytopathology laboratory simultaneously, and a microscopic examination was performed on Papanicolaou-stained slides prepared using centrifuged samples. A cell block was also prepared from the remaining fluid, when feasible, and stained with haematoxylin and eosin. Immunohistochemistry was also performed on cell block for confirmation of diagnosis, whenever necessary. The slides were examined by a panel of experienced cytopathologists and were classified into either positive for malignant cells (n = 97) or negative for malignant cells (n = 112). Interpretation of cytological examination of serous fluids by microscopic examination of PAP stained smears and H and E stained cell block were taken as true positive/negative for malignant cells.

Data analysis

Statistical analysis was performed using ‘R’ software.[6] The difference in HF cell count (HF#) and percentage (HF%) was assessed between serous fluids, which were positive and negative for malignant cells using the Mann–Whitney U test. A P value of <0.05 was considered statistically significant. The sensitivity and specificity were calculated using the area under the receiver operating characteristics (ROC) curve. Optimal cutoffs were defined as cutoff values with the highest sum of sensitivity and specificity, based on ROC curve analysis.

RESULTS AND DISCUSSION

Out of a total of 209 patient samples, 129 were from female patients (61.7%) and 80 were from male patients (38.3%). The age of patients who tested positive for presence of malignant cells ranged between 1 and 84 years, with a median age of 44.7 years. Patients who tested negative for presence of malignant cells ranged between 6 and 82 years, with a median age of 48.5 years. A total of 97 samples were positive and 112 were found negative for malignant cells after microscopic examination by a panel of experienced cytopathologists. Amongst serous effusions, which were positive for malignant cells, ovary was found to be the most common primary site (n = 36, 37.9%), followed by haematolymphoid malignancies (n = 18, 18.9%), breast (n = 15, 15.8%), and lung (n = 11, 11.6%). The distribution of site of primary tumour in malignant effusions is summarised as Table 1.

Table 1 The distribution of the site of the primary tumour in malignant serous effusions

Primary site	Counts	% of Total	Cumulative %	
Bone	2	2.1%	2.1%	
Breast	15	15.8%	17.9%	
Buccal mucosa	2	2.1%	20%	
Cervix	1	1.1%	21.1%	
Esophagus	1	1.1%	22.1%	
Hematolymphoid	18	18.9%	41.1%	
Lung	11	11.6%	52.6%	
Lymph node	1	1.1%	53.7%	
MUO	1	1.1%	54.7%	
Oral cavity	1	1.1%	55.8%	
Ovary	36	37.9%	93.7%	
Periampullary	1	1.1%	94.7%	
Stomach	3	3.2%	97.9%	
Thyroid	1	1.1%	98.9%	
Uterus	1	1.1%	100%	

Cell counts on BC-6800 plus haematology analyser

Complete cell counts were done for all serous fluid samples, and a comparison was made between malignant effusions and non-malignant effusions using the Mann–Whitney U test and student t-test. The mean absolute HF count (HF#) in malignant effusions was 24.9 HF cells/ul, which was significantly higher than non-malignant effusions, having a mean HF# of 4.95 HF cells/ul (P value <0.001). The mean HF% was 10.4% in malignant samples versus 5.76% in non-malignant samples (P value <0.001).

Receiver operator curve analysis for HF cells was performed. The area under the ROC curve was 0.712 for HF# and 0.676 for HF%. Figure 1 shows the ROC curve for HF cells in malignant effusions. An HF% cut-off of 2.7% (metric score 1.30 and Youden’s index 0.296) had a sensitivity of 73.91% and specificity of 55.66% for the detection of malignant cells in serous fluids. Similarly, at an HF# cut-off of 30 HF cells/ul (metric score 1.32 and Youden’s index 0.322), the sensitivity was 67.74% and the specificity was 64.49%. HF# proved to be more specific than HF%.

Figure 1 Figure showing the ROC curve for high fluorescence cells in malignant effusions. (1-specificity) on the x-axis is plotted against sensitivity on the y-axis. The area under the curve for absolute HF cell count (HF#) is 0.712 and for HF cell percentage (HF%) is 0.676

Using a HF# cut-off of >30 HF cells/ul, a total of 101 samples were screened as suspicious for malignant cells. On microscopic examination in the cytology laboratory, amongst these 101 samples, 63 were found to be positive for malignant cells (true positives) and 38 were found to be negative for malignant cells (false positives).

The remaining 99 samples had an HF number <30/μL. In 69 cases (69.7%), no malignant cells were found (true negative). Whereas 30 samples had HF# <30/ul but were positive for malignant cells in cytology (false negative). At a higher HF# cut-off of 72 HF cells/ul, the specificity increased to 83.18% at a sensitivity of 48.39%.

DISCUSSION

HF cells have large nuclear sizes and show increased fluorescence intensity using flowcytometry. In serous effusions, in addition to malignant cells, mesothelial cells, plasma cells, and macrophages also fall in the HF region.[7]

In our study, we have compared the number and percentage of HF cells detected using Mindray BC-6800 plus haematology analyser in body fluid mode in malignant versus non-malignant serous effusions. We have found that there is a significant difference (P value <0.001) in the absolute number and percentage of HF cells between malignant and non-malignant effusions.

ROC curve analysis in our study showed that at a HF% cut-off of 2.7%, the sensitivity for detection of malignant cells in serous effusions was 73.91% but specificity was 55.56%. A total of 24 cases were false negative at a HF% cut-off of 2.7/ul. These included 7 cases of lung carcinoma, 3 cases of carcinoma breast, 2 cases of carcinoma stomach, and 12 cases of haematolymphoid malignancies (ALL–05, AML-02, NHL-05). Therefore, serous effusions with haematolymphoid malignancies had a relatively higher false negative rate when compared to other tumours. For HF#, a cutoff of >30 HF cells/ul had a sensitivity of 67.74%, specificity of 64.49%, positive predictive value of 62.38%, and negative predictive value of 69.7%. This implies that absolute HF cell count (>30 HF cells/ul) has better specificity than HF% >2.7%. A higher cut-off of 72 HF cells/ul increased the specificity to 82.29%, but the sensitivity came down to 48.39%.

Labaere D et al.[8] also did a study on the presence of HF cells in serous fluids using a Sysmex XN 2000 haematology analyser and found out that the absolute numbers and percentage of HF cells were significantly higher in malignant effusions. In this study, the investigators got a sensitivity of 88% and specificity of 61% at a lower HF# cutoff of 17 HF cells/ul.

Zimmerman et al.[9] studied the correlation between CSF samples that were positive for malignant cells and the presence of HF cells detected by the Sysmex XE 5000 haematology analyser. They concluded that 78.5% of CSF samples (51 of 65) that were positive for malignant cells using microscopy had HF cells. However, 33 of 126 CSF samples that were negative for malignant cells also had HF cells. This was explained by the presence of macrophages and plasma cells on microscopy, which also falls in the HFC region of the haematology analyser. As the sensitivity of detecting HF cells in samples with low tumour cell count is not good enough, therefore they have advised against the use of HF cell count as a screening tool for the detection of malignant cells and have advocated the use of microscopy on cytospin CSF smears due to high sensitivity of manual microscopy in detecting malignant cells.[9]

Cho et al.[10] compared the automated cell counts done using Sysmex XN 2000 haematology analyser in body fluid mode with manual microscopy and found that malignant samples had significantly higher HF cells (P value <0.001) than benign samples (17.8 vs 4.15/100 WBC). They have advocated a cut-off of 6.9 HF cells/100 WBC for the detection of malignant cells using ROC curve analysis with the area under the curve of 0.791. This is comparable to our results, which also show a significant difference between the number of HF cells in malignant and non-malignant effusions (P value <0.001).

The diagnostic accuracy of a test is its ability to identify the target condition of interest. For a diagnostic test to be used for patient care, it should have appropriate sensitivity, specificity, predictive value, and likelihood ratio. Unlike predictive value, likelihood ratios are not affected by disease prevalence. A positive likelihood ratio (LR+) of a diagnostic test is the probability that a patient having the disease will test positive divided by the probability of testing positive by a person without the disease. Similarly, a negative likelihood ratio (LR−) is the probability of testing negative for a patient who has the disease divided by the probability of testing negative for a person who does not have the disease.[11] A positive likelihood ratio is a very good indicator for ruling in a disease. A higher LR+ for a diagnostic test indicates a higher probability of having a disease. An LR+ of >10 is considered significant for a diagnostic test. A negative likelihood ratio is a good indicator to rule out a disease. The lower the LR-value, the better the diagnostic test is at ruling out a diagnosis. A diagnostic test with an LR-value of <0.1 is considered good.[12]

The ROC curve is another estimate of the discriminative power of a test. To make a ROC curve, at each cut-off, a pair of sensitivity and specificity values are plotted. The value of 1-specificity is plotted on the x-axis, and the corresponding sensitivity is on the y-axis. The higher the area under the curve (AUC) and the closer the curve is to the upper left-hand corner, the better the test is at discriminating between diseased and non-diseased. An ideal diagnostic test should have an AUC of 1, whereas a non-discriminating test has an area of 0.5.[12]

For a test to be used as a screening test, it should have high sensitivity and a low negative likelihood ratio (<0.1). In our study at HF# and HF% cut-off of 30 HF cells/ul and 2.7% the sensitivity for prediction of malignant cells in serous effusions is 67.74% and 73.8% respectively. The positive and negative likelihood ratios for HF# are 1.90 and 0.5 and for HF% are 1.67 and 0.47, respectively. The AUC of the ROC curve for HF# and HF% is 0.712 and 0.676, respectively. From the results of the above tests of diagnostic accuracy, it is clear that due to low sensitivity and negative likelihood ratio, the detection of HF cells is not a reliable screening test for predicting the presence of malignant cells in serous effusions. However, at a higher cut-off of 72 HF cells/ul, the sensitivity and specificity are 48.39% and 83.18%, respectively. This implies that if the absolute number is > 72/ul, then the probability of finding malignant cells in the effusion is high. Therefore, if the number of HF cells is > 72/ul, extreme caution must be exercised during manual microscopy before reporting a case as negative for malignant cells.

CONCLUSION

Malignant effusions have a significantly higher HF cell count (Mean HF# = 24.9 HF cells/ul, mean HF% = 10.4%) in comparison to non-malignant effusions (Mean HF# = 4.95 HF cells/ul, HF% = 5.76%). The sensitivity and negative likelihood ratio of HF cell detection are not good enough to be used as a screening test for the detection of malignant cells in serous fluids.

Reactive mesothelial cells, macrophages, and plasma cells, which are commonly found in serous effusions, are also counted as HF cells due to their large nuclear size. This leads to reduced specificity and positive predictive value of HF cell count as a test to determine the presence of malignant cells in serous fluids. Therefore, manual microscopy by an experienced cytopathologist using centrifuged smears and cell block preparation continues to be the gold standard in diagnosing malignant effusions. However, despite its low sensitivity in comparison to microscopy, the detection of HF cells is a useful ancillary technique that provides the pathologist a helpful clue of the probability of finding malignant cells, especially in high-volume cancer centres that have a high cytopathology workload.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.
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