Do NEUT-RI, NEUT-GI, RE-LYMP, and AS-LYMP provide diagnostic value in differentiating acute cholecystitis from biliary colic?

Abstract

OBJECTIVE

In the differential diagnosis of biliary colic and acute cholecystitis, physical examination, non-specific biochemical tests, and imaging methods are used. The aim of this study was to investigate the differential diagnostic power of NEUT-RI, NEUT-GI, RE-LYMP, and AS-LYMP parameters, which show neutrophil and lymphocyte activation, and are obtained with fluorescence flow cytometry during full blood count.

METHODS

In this prospective cohort study, patients presenting with right upper quadrant pain were separated into two groups according to the diagnoses of acute cholecystitis and biliary colic. The conventional inflammatory markers and the NEUT-RI, NEUT-GI, RE-LYMP, and AS-LYMP values were compared. Receiver Operating Characteristics (ROC) Curve analysis was performed to investigate the diagnostic power of the inflammatory markers.

RESULTS

In the acute cholecystitis group, C-reactive protein, white blood cells, neutrophil, monocyte, and immature granulocyte counts were higher, and the lymphocyte count was lower. The NEUT-RI and NEUT-GI values were significantly higher, and the RE-LYMP value was significantly lower in the acute cholecystitis group. The AS-LYMP values were similar in both groups. In the ROC analyses, the AUC values were 0.914 for CRP, 0.842 for WBC, 0.873 for neutrophils, and 0.823 for immature granulocyte count. A poor diagnostic performance was determined for NEUT-RI and NEUT-GI with AUC values of 0.662 and 0.612, respectively. The AUC for RE-LYMP and lymphocyte count was non-significant at <0.6.

CONCLUSION

The results showed that NEUT-RI and NEUT-GI have limited diagnostic power in the differential diagnosis of acute cholecystitis from biliary colic, and RE-LYMP and AS-LYMP are insufficient diagnostically.

Highlight key points

• Conventional inflammatory markers (CRP, WBC, neutrophils, and immature granulocytes) showed the highest diagnostic accuracy in differentiating acute cholecystitis from biliary colic.

• NEUT-RI and NEUT-GI levels were significantly higher in acute cholecystitis, but their diagnostic performance was inferior to that of conventional inflammatory parameters.

• RE-LYMP and AS-LYMP did not demonstrate sufficient diagnostic value for distinguishing acute cholecystitis from biliary colic.

Stones and sludge form in the gallbladder with disruption to cholesterol and lipid metabolism [1]. When gallbladder stones temporarily obstruct the cystic duct, temporary pain occurs in the right upper quadrant [2]. The pain resolves when the stone is displaced and the cystic duct is reopened for drainage. This condition often occurs after the intake of fatty foods, is named biliary colic, and is treated symptomatically with non-steroidal anti-inflammatory drugs (NSAIDs) [3, 4]. Sometimes, the gallbladder stone becomes lodged in the cystic duct, prolonging the process. Bacterial infection develops over time in the gallbladder when drainage cannot be performed [5]. In this situation, the right upper quadrant pain is not relieved by waiting or NSAIDs. This condition is called acute cholecystitis, which usually requires hospitalization, and cholecystectomy is the currently accepted treatment [6]. These two clinical conditions both present with right upper quadrant pain, so they can be confused with each other [7, 8]. This confusion can result in unnecessary use of antibiotics, detailed tests, and long follow-up periods, and sometimes a necessary surgical intervention can be delayed.

Neutrophils are the first line of defense in immunity, clearing pathogens through phagocytosis and the oxidative burst pathway. They present antigens, synthesize various proinflammatory cytokines, and produce surface molecules for T-cell activation [9, 10]. B and T lymphocytes are the primary cells controlling the acquired immune response. T lymphocytes function in cell-mediated immunity, while B lymphocytes are involved in antibody-mediated immunity [11, 12]. Morphological changes (size, shape, and structure) and activity changes (chemotaxis and migration) occur in neutrophils and lymphocytes during inflammation [13]. Activated cells have a different membrane lipid composition, and their activity increases due to cytokine production in the cytoplasm [14]. Full blood count analyzers using the fluorescence flow cytometry method allow differentiation of activated neutrophils and lymphocytes from resting cells [13]. For each white blood cell passing in front of the laser beam, forward scattered light (FSC), side scattered light (SSC), and fluorescence intensity (SFL) signals are recorded and presented graphically [15].

The parameters of Neutrophil Reactivity Intensity (NEUT-RI) and Neutrophil Granularity Intensity (NEUT-GI) provide information related to the activation stage of neutrophils [16]. This occurs by obtaining data related to metabolic activity, internal structure, and cell size using the flow cytometry method [13]. Reactive lymphocytes (RE-LYMP) refer to all lymphocytes with a higher fluorescent signal within the lymphocyte population, in other words, activated B and T lymphocytes [13]. Antibody-synthesized lymphocytes (AS-LYMP) help differentiate antibody-synthesized B lymphocytes from within the activated lymphocyte population. AS-LYMP is the subgroup with the highest fluorescent signal within all the lymphocytes [17].

It is thought that new inflammation parameters can bring a fresh perspective to inflammation, in addition to the information provided by commonly used parameters such as white blood cells (WBC), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) [18]. These new hematological parameters, which measure neutrophil and lymphocyte activation, have been previously studied in conditions such as some autoimmune skin diseases, autoimmune liver diseases, differentiation of bacterial-viral infections in childhood diseases, and mostly in the differential diagnosis, prognosis follow-up, and risk of mortality in sepsis and COVID-19 infection [17, 19–24]. The aim of this study was to investigate the diagnostic value of these new inflammation parameters, which can be obtained from full blood count values, in the differentiation of biliary colic and acute cholecystitis, both of which present with right upper quadrant pain.

MATERIALS AND METHODS

This prospective cohort study included patients who presented at the Emergency Department of a tertiary-level healthcare center with complaints of right upper quadrant pain between 1 September – 28 October 2024, 1 December – 16 December 2024, and 1 March – 24 April 2025, and were diagnosed with acute cholecystitis or biliary colic by the General Surgery consultant. Patients were excluded from the study if another abdominal pathology was determined during the evaluation, if they had another concomitant acute inflammatory disease, malignancy, or any other gastrointestinal, chronic inflammatory, hepatobiliary, cardiac, pulmonary, or hematological disease other than gallbladder pathology, if they were pregnant, had recently received a blood transfusion, or were using steroids.

Upon presentation at the ED, venous blood samples were routinely taken from the patients. The white blood cells (WBC), red blood cells (RBC), hemoglobin value, neutrophil, lymphocyte, monocyte, and platelet counts, red cell distribution width (RDW) count and percentage, platelet distribution width (PDW), immature granulocyte (IG) count and percentage, and C-reactive protein (CRP) value were recorded. A Sysmex XN-1000 analyzer (Sysmex, Kobe, Japan) was used for the full blood count, and a Cobas 8000 analyzer (Roche Diagnostics, Switzerland) for measurement of the CRP value. During the routine blood count, the NEUT-RI, NEUT-GI, RE-LYMP, and AS-LYMP values, which are markers of neutrophil and lymphocyte activation, were obtained with the flow cytometry method used by the Sysmex hematological analyzer and recorded. No other blood samples were taken for this study, and the samples were not stored. The new inflammatory parameter results recorded in this study were not disclosed to the clinicians managing the diagnosis and treatment of the patients.

The patients presenting at the ED with right upper quadrant pain underwent a physical examination (sensitivity, rigidity, Murphy sign) and were separated into two groups according to the diagnosis of acute cholecystitis or biliary colic made by the General Surgery consultant from the examination findings, laboratory test results (full blood count, acute phase reactants), and imaging findings (increased gallbladder wall thickness on ultrasonography, pericholecystic fluid). The routinely used conventional inflammatory parameters and the newly researched inflammatory parameters were compared between the two groups.

Approval for the study was granted by the Ethics Committee of Ankara Etlik City Hospital (Date: 16.10.2024, No: AEŞH-BADEK-2024-940). The need for informed consent was waived as no additional blood was taken from the patients, there was no intervention, no randomization, and no recording of personal information. All the study procedures were in compliance with the World Health Organization Helsinki Declaration, and national and international ethical values.

Statistical Analysis

Data obtained in the study were analyzed statistically using SPSS v. 26.0 software (SPSS Inc., Chicago, IL, USA) and MedCalc v. 22.023 (MedCalc Software, Ostend, Belgium).

Conformity of numerical variables to normal distribution was examined with visual (histogram and probability graphs) and analytical methods (Kolmogorov-Smirnov/Shapiro-Wilk tests). Descriptive statistics were stated as mean±standard deviation (SD) values for continuous variables with normal distribution and as median (minimum-maximum) values when the distribution was not normal. Categorical variables were stated as number (n) and percentage (%). In the comparisons of numerical data, the Student’s t-test was used when the distribution was normal, and the Mann-Whitney U-test when data were not normally distributed. Pearson’s chi-square test was applied to categorical data. To determine the diagnostic power of the inflammatory markers in the differentiation of acute cholecystitis and biliary colic, Receiver Operating Characteristics (ROC) curve analysis was performed, the Area under the Curve (AUC), sensitivity, and specificity values were calculated, and cutoff values were determined. A value of p<0.05 was accepted as the level of statistical significance.

results

Evaluations were made of a total of 236 patients: 145 (61.4%) diagnosed with biliary colic, and 91 (38.6%) diagnosed with acute cholecystitis. The demographic characteristics and inflammatory parameter values are presented in Table 1. There was no significant difference between the groups in respect of gender distribution. The mean age of the patients was 53.34±17.00 years in the acute cholecystitis group and 45.58±14.81 years in the biliary colic group, and the difference was statistically significant (p<0.001). In the acute cholecystitis group, the CRP, WBC, neutrophil, monocyte, and immature granulocyte counts and percentages were statistically significantly higher, and the lymphocyte count was statistically significantly lower. The hemoglobin, platelet, RDW, and PDW values were similar in both groups (Table 1).

Table 1.

Demographic characterictics and inflammatory parameters

	Biliary colic n=145 (61.4%)	Acute cholecystitis n=91 (38.6%)	p	95% confidence interval of the differerence
Age*	45.58±14.81	53.34±17.00	<0.001	-13.89 to -5.62
Gender (male)***	81 (55.9%)	43 (47.3%)	0.197
Neut-RI (FI)**	41.90 (36.60–49.70)	43.30 (36.80–56.50)	<0.001
Neut-GI (SI)*	150.54±3.88	152.01±3.85	0.005	-2.48 to -0.44
Re-Lymph# (103/μL)**	0.03 (0.00–0.20)	0.02 (0.00–0.15)	0.386
Re-Lymph%**	0.30 (0.00–2.40)	0.20 (0.00–1.40)	<0.001
As-Lymph# (103/μL)**	0.00 (0.00–0.08)	0.00 (0.00–0.07)	0.746
As-Lymph%**	0.00 (0.00–1.10)	0.00 (0.00–0.70)	0.300
CRP (mg/L)**	4.18 (0.31–74.00)	116.24 (0.68–447.90)	<0.001
WBC (103/μL)**	8.92 (4.51–15.50)	14.99 (3.51–34.56)	<0.001
Hemoglobin (g/dL)*	13.75±1.67	13.95±1.98	0.420	-0.66 to 0.27
Neutrophyl (103/μL)**	5.75 (2.36–14.04)	12.12 (1.45–29.76)	<0.001
Lymphocyte (103/μL)**	2.23 (0.57–5.96)	1.64 (0.21–3.45)	<0.001
Monocyte (103/μL)**	0.63 (0.15–1.27)	0.95 (0.05–3.37)	<0.001
Platelet (103/μL)**	284.00 (48.00–583.00)	262.00 (131.00–625.00)	0.494
RDW# (fL)**	41.90 (35.80–56.70)	42.90 (36.20–58.00)	0.058
RDW%**	13.10 (11.80–18.80)	13.30 (11.40–17.40)	0.234
Ig# (103/μL)**	0.03 (0.00–0.10)	0.07 (0.01–0.72)	<0.001
Ig%**	0.30 (0.00–0.90)	0.40 (0.10–4.00)	<0.001
PDW (fL)**	11.40 (8.00–18.70)	11.80 (8.40–18.00)	0.515

^*: Mean±standard deviation, Student’s t test, **: Median (min – max), Mann-Whitney U test, ***: Pearson’s Chi-square test. Neut-RI: Neutrophil reactivity intensity; FI: Fluorescence intensity; Neut-GI: Neutrophil granularity intensity; SI: Scatter intensity; Re-Lymph#: Reactive lymphocytes count; Re-Lymph%: Reactive lymphocytes percentage; As-Lymph#: Antibody-synthesizing lymphocytes count; As-Lymph%: Antibody-synthesizing lymphocytes percentage; CRP: C-reactive protein; WBC: White blood cells; RDW#: Red cell distribution width count; RDW%: Red cell distribution width percentage; Ig#: Immature granulociytes count; Ig%: Immature granulocytes percentage; PDW: Platelet distribution width.

The mean and p values of the new inflammatory parameters are shown in Table 1.

The NEUT-RI and NEUT-GI values were determined to be statistically significantly high in the acute cholecystitis group (p<0.001, p=0.005, respectively). The RE-LYMP count and percentage values were statistically significantly low in the acute cholecystitis group (p=0.386, p<0.001, respectively). The AS-LYMP count and percentage values, which provide information about antibody-synthesized B lymphocytes, were similar in both groups.

ROC analysis was performed for the inflammatory parameters with significant results in the comparisons between the groups. The AUC values, cutoff values, sensitivity, and specificity rates are shown in Table 2. The AUC values were 0.914 for CRP, 0.842 for WBC, 0.873 for neutrophils, and 0.823 for immature granulocyte count. In contrast to the strong diagnostic performance of the conventional inflammatory markers listed above, poor diagnostic performance was determined for NEUT-RI and NEUT-GI with AUC values of 0.662 and 0.612, respectively (Table 1). The ROC-AUC graph of the conventional and new inflammatory parameters is shown in Figure 1.

Table 2.

ROC analysis results of conventional and new inflammatory parameters

	ROC-AUC	95% confidence interval	Cut-off value	Sensitivity %	Specifity %	p
Neut-RI	0.662	0.598 to 0.722	>41.9	73.6	50.3	<0.001
Neut-GI	0.612	0.547 to 0.675	>152.1	49.5	69.0	0.003
CRP	0.914	0.870 to 0.948	>30.8	77.5	95.5	<0.001
WBC	0.842	0.789 to 0.886	>12.0	72.5	84.1	<0.001
Neutrophyl	0.873	0.823 to 0.912	>6.8	90.1	68.3	<0.001
Ig#	0.823	0.768 to 0.869	>0.04	69.2	79.3	<0.001
Ig%	0.722	0.660 to 0.778	>0.3	71.4	63.4	<0.001

ROC: Receiver operating characteristics; AUC: Area under the curve; Neut-RI: Neutrophil reactivity intensity; Neut-GI: Neutrophil granularity intensity; CRP: C-reactive protein; WBC: White blood cells; Ig#: Immature granulociytes count; Ig%: Immature granulocytes percentage.

Figure 1.

ROC-AUC of inflammatory parameters.

ROC-AUC: Receiver operating characteristics-area under the curve.

The ROC-AUC graph for the acute cholecystitis diagnostic power of RE-LYMP count and percentage, and the lymphocyte count, which were lower in the acute cholecystitis group in the inter-group comparisons, is shown in Figure 2. For RE-LYMP count, the AUC value (95% confidence interval) was 0.466 (0.388–0.544), for RE-LYMP percentage, the AUC was 0.340 (0.267–0.413), and for lymphocyte count, the AUC was 0.335 (0.263–0.403) (p=0.388, p<0.001, p<0.001, respectively).

Figure 2.

ROC-AUC of Re-Lymph count, percentile and Lymphocite count.

DISCUSSION

The differential diagnosis of biliary colic and acute cholecystitis requires tests and evaluations such as detailed biochemical tests, at least 6 hours of patient monitoring with frequent physical examinations, evaluation of gallbladder wall thickness and pericholecystic fluid on ultrasonography, and ultrasonographic Murphy’s sign [2, 4]. Biochemical tests such as CRP, ESR, and WBC have been mentioned in the literature in the differential diagnosis of biliary colic and acute cholecystitis. Although most studies accept changes in the above-mentioned conventional inflammatory parameters as significant, there is also a limited number of studies reporting a contrasting view [25, 26]. CRP and procalcitonin do not increase in the early stage and require a separate blood sample to be examined with a biochemical analysis device and kit [27]. Therefore, the focus of this study was on the new inflammatory parameters of NEUT-RI, NEUT-GI, RE-LYMP, and AS-LYMP, indicating neutrophil and lymphocyte reactivity and antibody synthesis power, which can be studied together with other full blood count parameters in the same sample in the routinely used hematology analyzer [28].

As a marker of neutrophil activation, NEUT-RI and NEUT-GI have been previously shown to be early diagnostic markers in sepsis [29, 30]. These two neutrophil activation markers are useful in providing an idea about disease severity and prognosis in sepsis and in determining the risk of mortality [31, 32]. When neutrophil activation markers are evaluated together with RE-LYMP and AS-LYMP, which provide information about lymphocyte activation, significant changes are seen in the neutrophil activation markers because of bacterial causes and in the lymphocyte activation markers because of viral causes, thus providing information about the infection etiology [17]. In a previous study, NEUT-RI was seen to be useful in sepsis diagnosis and mortality evaluation in pediatric cases [33]. In 2020, when the whole world was affected by the COVID-19 pandemic, new inflammatory parameters were investigated in the differential diagnosis of COVID-19 infection from other viral agents and in the evaluation of disease severity and mortality risk. While AS-LYMP gave an idea about the severity of COVID-19 infection, RE-LYMP was found to be useful in the differentiation of coronavirus with a diagnosis of COVID-19 from other viruses [24, 34, 35]. When these encouraging results emerged, it was then investigated in autoimmune skin diseases such as Pemphigus and psoriasis, autoimmune hepatitis, and diseases related to cirrhosis, and significant results were obtained [20]. A study in Spain researched the severity of appendicitis, but no significant result was reached [36]. Most recently, a study in France reported that the diagnostic power of the new inflammatory parameters was weak in the diagnosis of sepsis compared to the conventional parameters. In that study, the control group was extremely homogeneous, formed of patients who presented at the ED and were infected but did not have sepsis [23]. In another study, which included neutrophil reactivity parameters in patients with lung cancer, sarcoidosis, and COVID-19, no significant results were found in sarcoidosis and lung cancer, but a decrease was observed in the NEUT-RI value in COVID-19 [37].

In the current study, which focused on the differential diagnosis of biliary colic and acute cholecystitis, the conventional inflammatory parameters of CRP, WBC, neutrophils, and IG showed higher AUC values in the ROC analysis (0.914, 0.842, 0.873, 0.823, respectively). The RE-LYMP and AS-LYMP parameters were not significant in the comparisons between the groups. Although NEUT-RI and NEUT-GI were significant in the differential diagnosis of acute cholecystitis from biliary colic, the diagnostic power was low compared to the conventional inflammatory parameters (ROC-AUC 0.662, 0.612, respectively). In previous papers, significance has been assigned to conventional parameters such as CRP, WBC, and Murphy’s sign in the differential diagnosis of acute cholecystitis from biliary colic [25, 26]. The current study is the first to have examined the new inflammatory parameters in terms of gallbladder pathology. Even though NEUT-RI and NEUT-GI do not have sufficient diagnostic power compared to conventional inflammatory parameters, they should be taken into consideration for deeper research and opening new research horizons. The importance of new inflammatory parameters in respect of choledocolithiasis-cholangitis differential diagnosis, and the diagnosis and prognosis of acute biliary pancreatitis, can be considered an area open to future research.

There were some limitations to this study. The clinician differentiating acute cholecystitis and biliary colic was blind to the new inflammatory parameters. However, the conventional inflammatory parameters were seen as routine practice. This could affect the clinical approach, and there could have been bias towards the CRP, WBC, and neutrophil values, which should be taken into consideration when evaluating the results. The clinician who determined the groups by making the diagnoses of acute cholecystitis and biliary colic was the General Surgery consultant on duty that day, so patients were evaluated by different clinicians on different days. This may have prevented a standard clinical approach, although the study was performed in a single center. Another limitation of this study is the statistically significant age difference between the acute cholecystitis and biliary colic groups. As age may influence baseline inflammatory status, this difference could have had a modest effect on both conventional and novel inflammatory parameters. Therefore, the results should be interpreted with caution in this respect.

Conclusion

NEUT-RI and NEUT-GI are valuable in the differentiation of acute cholecystitis from biliary colic but have lower diagnostic power than conventional inflammatory parameters. RE-LYMP and AS-LYMP are insufficient diagnostically. There is a need for further research of these new inflammatory parameters in multicenter studies with large cohorts, and it would also be useful to investigate these in respect of bile duct pathologies and pancreas inflammation.

Ethics Committee Approval

The Ankara Etlik City Hospital Ethics Committee granted approval for this study (date: 16.10.2024, number: AESH-BADEK-2024-940).

Informed Consent

Written informed consent was obtained.

Conflict of Interest

None declared.

Financial Disclosure

The author declared that this study has received no financial support.

Use of AI for Writing Assistance

None declared.

Authorship Contributions

Concept – UO, SY; Design – UO, SY; Supervision – UO, SY; Fundings – UO; Materials – UO; Data collection and/or processing – UO, SY; Analysis and/or interpretation – UO; Literature review – UO; Writing – UO; Critical review – UO.

Peer-review

Externally peer-reviewed.