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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 33  |  Issue : 1  |  Page : 52-61

Flow cytometric analysis of lymphocyte subsets in alcoholic liver disease


1 Department of Pathology, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India
2 Department of Medicine, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India
3 Department of Microbiology, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India

Date of Web Publication14-Oct-2019

Correspondence Address:
M Banyameen Iqbal
Department of Pathology, Dr. D. Y. Patil Medical College, Hospital and Research Center, Pune - 411 018, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jms.jms_43_18

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  Abstract 


Background: The World Health Organization defines alcohol-related diseases as the third major cause of death and disability worldwide accounting for approximately 2.5 million deaths per year. Immune dysregulation has possible role for pathogenesis of alcohol liver injury. We aimed to study lymphocyte subsets in alcoholic liver disease (ALD).
Materials and Methods: A prospective cross-sectional study was done over 2 years in a tertiary care hospital in Western Maharashtra. The cases were classified into four groups: nonalcoholics (n = 18), fatty liver (n = 13), alcoholic hepatitis (n = 11), and alcoholic cirrhosis (n = 51). ALD was confirmed by abdominal ultrasonography and liver function tests. Blood sample collected in ethylenediaminetetraacetic acid was run on electronic cell counter for total leukocyte count and on flow cytometer for CD45, CD3+, CD4+, and CD8+ counts.
Results: Hemoglobin and platelet count showed a statistically significant reduction (P < 0.0001) from nonalcoholics to fatty liver to hepatitis to cirrhotic patients. There was a significant alteration in liver function tests in ALD. Total leukocyte count and lymphocytes were reduced; however, it was not statistically significant. CD3+ (1745.41 ± 917.57–1021.43 ± 500.71/cumm), CD4+ (903.41 ± 401.83–634.45 ± 309.11/cumm), and CD8+ (818.06 ± 584.31–393.9 ± 258.49/cumm) showed decreasing trend. CD4+/CD8+ ratio was progressively increased (1.39 ± 0.66–1.91 ± 0.89) from nonalcoholics to alcoholic cirrhosis.
Conclusion: In this study, we found a decrease in the concentration of lymphocytes and its subpopulation (CD3+, CD4+, and CD8+) while as CD4+/CD8+ ratio was comparatively increased in ALD patients in comparison to nonalcoholics. These findings signify an immune dysregulation in ALD.

Keywords: Alcoholic liver disease, cirrhosis, lymphocytes


How to cite this article:
Buch AC, Iqbal M B, Patil A, Haldar N, Kakrani AL, Bamanikar S, Pandit D, Kumar H. Flow cytometric analysis of lymphocyte subsets in alcoholic liver disease. J Med Soc 2019;33:52-61

How to cite this URL:
Buch AC, Iqbal M B, Patil A, Haldar N, Kakrani AL, Bamanikar S, Pandit D, Kumar H. Flow cytometric analysis of lymphocyte subsets in alcoholic liver disease. J Med Soc [serial online] 2019 [cited 2019 Nov 18];33:52-61. Available from: http://www.jmedsoc.org/text.asp?2019/33/1/52/269111




  Introduction Top


According to the World Health Organization, alcohol-related diseases are the third cause of death and disability worldwide and alcohol ingestion is responsible for approximately 2.5 million deaths/year.[1] Although several organs are injured by ethanol, alcoholic liver disease (ALD) is the most common medical consequence of excessive alcohol intake accounting for about 70% of recorded mortality.[2] Thus, understanding the mechanism responsible for alcohol liver injury has a relevant clinical and social impact.

ALD includes three major lesions: fatty liver, alcoholic hepatitis, and cirrhosis. Two or more of such lesions may coexist, fatty liver being the most common. A small percentage of heavy drinker's progress to alcoholic hepatitis leading to cirrhosis.[3]

A wide range of immunologic alterations have been reported in alcoholics, including changes in both cellular and immune parameters.[4],[5] Manifestations of humoral changes include increased immunoglobulin or functional B-cell alterations such as degree of responsiveness to T-independent antigens.[6] Both increase and decrease in T-cells or T-cell subsets have been reported in patient series and experimental animals.[7],[8]

Furthermore, emerging evidence suggests that chronic inflammation represents the driving force in the evaluation of alcohol liver injury. Early studies have shown that liver inflammatory infiltrate in alcoholic hepatitis and active alcoholic cirrhosis contains both CD8+ and CD4+ T lymphocytes.[9] In either chronic alcohol-treated mice or alcohol abusers liver infiltrating T-cells express an activation/memory phenotype and respond to T-cell receptor stimulation by producing Th-1 cytokines such as tumor necrosis factor (TNF)-γ and TNF-α.[10],[11] A significantly increased spontaneous production of interleukin (IL)-6, IL-1β, IL-10, and IL-12 was also observed on peripheral blood monocytes among alcoholic individuals.[12]

With this background, this study was undertaken to see the alteration in cell-mediated immunity in various clinical stages of ALD and to compare the lymphocyte subsets between ALD patients and nonalcoholic controls.


  Materials and Methods Top


A prospective cross-sectional study was carried over 2 years extending from July 2015 to June 2017 in a tertiary care hospital in Western Maharashtra. The Institutional Ethical Committee clearance was obtained before the onset of the study. Informed consent was obtained from all the participants. Sample size was decided according to the feasibility and availability of the funds as the reagents are costly. A total of 93 cases were enrolled in the study. Seventy-five patients diagnosed with ALD clinically, who were admitted in our hospital, were further grouped into fatty liver, alcoholic hepatitis, and alcoholic cirrhosis and were included in the study along with 18 healthy nonalcoholic participants as control in the separate group. History of average daily intake of 40–80 ml of alcohol for minimum of 10 years along with clinical signs and symptoms ranging from upper abdominal pain, nausea, malaise, anorexia, jaundice, ascites, variceal bleeding, or encephalopathy was considered as case of ALD. This was further confirmed by abdominal ultrasonography and liver function tests. Patients suffering from HIV, tuberculosis, diabetes mellitus, cancer, or on any immunosuppressant drug were excluded from the study, as these conditions may alter the lymphocyte subset ratio, and thus confound the results.

About 5 ml of blood from the patients was collected by venepuncture into a sterile ethylenediaminetetraacetic acid vacutainers. Total and differential leukocyte count was done using Benesphera 5-part Differential Hematology Analyzer. 2 ml of blood was collected in plain vacutainer and was run on semi-automated biochemistry analyzer for liver function tests.

Flow cytometric analysis was done using FACS Jazz (Beckton Dickinson, San Jose, CA, USA). Calibration was done using BD Calibrite TM 3 beads (Beckton Dickinson, San Jose, CA, USA). The subsets of lymphocytes that were measured were CD45, CD3+, CD4+, and CD8+. Fluorescent-tagged antibodies against these were utilized for the same. Anti-CD3 FITC (BD multitest CD3 fluorescein isothiocyanate), anti-CD4 PE (phycoerythrin), anti-CD8 APC (allophycocyanin), and anti-CD45 V450 were added with appropriate dilution to the collected blood and then processed in the three-color BD flow cytometer.

A 100 μL sample of blood was taken in a test tube to which were added 10 units of FITC, 10 units of APC, 10 units of PE, and 5 units of V 450. They were then mixed by tapping and subsequently incubated at room temperature for 30 min. Meanwhile, the FACS lysing solution was prepared by mixing 18 ml of distilled water per 2 ml of lysing solution. 2 ml of the resulting solution was added to each tube. Each tube was then subjected to vortex for 10 s, followed by incubation in dark for 10 min and centrifugation at 1500 rpm for 5 min. The supernatant was then discarded. 2 ml of sheath fluid was added to the cell pellet. This was again subjected to vortex, followed by centrifugation and discarding of supernatant. 500 μL of sheath fluid was added and the resulting fluid was stored in a refrigerator at 4°C. This was then run in the flow cytometer within 24 h.

Hematological parameters, liver function tests, and the lymphocyte subsets were measured across nonalcoholics controls and different categories of ALD using one-way analysis of variance test. The results were statistically analyzed using Statistical Package for the Social Science (SPSS) Statistics data editor 20 software Version 7.0 and Microsoft Office. Groups were characterized using descriptive statistics, means, standard deviations, and percentages. These tests were considered to be statistically significant when their P < 0.05 and extremely statistically significant for values <0.01.


  Results Top


The mean age in the study cohort suffering from fatty liver, alcoholic hepatitis, and alcoholic cirrhosis was 45.69, 40.49, and 45.53 years, respectively. The mean age in nonalcoholic control group was 38.56 years. It was seen that patients with ALD were slightly older and were predominantly males (97%). The results of laboratory parameters in all four groups are shown in [Table 1]. Despite the difference in sex distribution, hemoglobin values were higher in nonalcoholics than in the patients with ALD, and the difference was statistically significant (P < 0.0001). All the hematological indices (mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration) although statistically not significant, were on the higher side in ALD cases as compared to nonalcoholic group. Platelet count was higher in nonalcoholic group, and the association was statistically significant (P < 0.0001). There was no statistically significant difference in total leukocyte counts among all the groups; however, it was slightly higher in ALD patients as compared to nonalcoholics. ALD patients had lymphopenia; however, it was not statistically significant. The lymphocyte subsets (CD3+, CD4+, and CD8+) were showing a decreasing trend as the severity of ALD increase from fatty liver to cirrhosis. However, we found a relative increase in these lymphocytes in alcoholic hepatitis which then revealed decreasing trend in cirrhosis. CD4+/CD8+ ratio shows a steady increase as the disease progresses from fatty liver to cirrhosis. Among the liver function tests, albumin, globulin, serum glutamic-oxaloacetic transaminase (SGOT), Alkaline phosphatase (ALK), and international normalized ratio (INR) were showing a statistically significant relationship with the severity of the disease (P < 0.0001). Bilirubin, on the other hand, does not show any significant relationship with the disease [Figure 1], [Figure 2], [Figure 3], [Figure 4].
Table 1: Laboratory parameters in different study groups

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Figure 1: Shows CD4 and CD8 Cell counts in control group

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Figure 2: Scatter plot Showing CD4 and CD8 Cell counts in patients suffering from fatty liver

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Figure 3: Scatter plot showing CD4 and CD8 Cell counts in hepatitis patients

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Figure 4: Figure Showing CD4 and CD8 Cell counts in cirrhosis patients

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  Discussion Top


Liver is a major organ of the body with extensive range of functions including metabolic, biochemical, and immunologic. In the recent past, immunologic mechanisms have been implored to explain liver damage in chronic alcoholics leading to chronic failure of the liver. Not only hemoglobin but platelets as well as lymphocytes are also reduced in cases of ALD. In this study, we found a steady decrease in the hemoglobin as well as platelet concentration as the disease progressed from fatty liver to cirrhosis and the association was statistically significant (P < 0.001). We also found variation in red cell indices. Romo et al. in his study also found similar results.[13] Progressive macrocytosis is seen from nonalcoholics to different groups of ALD, however, not statistically significant.[14] Anemia of chronic liver disease can be because of acute or chronic gastrointestinal hemorrhage, hypersplenism secondary to portal hypertension, and hemorrhage because of impaired blood coagulation. Alcohol ingestion may lead to Vitamin B12 and folic acid deficiency which occurs secondary to malabsorption and/or inadequate dietary intake.[15]

Biochemical parameters were all deranged in patients suffering from ALD. Their association was statistically significant in SGOT, ALK, and INR (P < 0.001). Similarly, albumin and globulin values were also significantly deranged. Studies done by Torkadi et al., Ray et al., and Das and Vasudevan also found similar results.[16],[17],[18] Chronic liver disease causes hypoalbuminemia and increase in gamma globulins. The increase in the latter is due to increased synthesis of antibodies which causes altered albumin/globulin (A/G) ratio. Our study revealed significant decrease in A/G ratio in patients with hepatitis and cirrhosis in comparison to fatty liver and nonalcoholic control group.

While the role of lymphocytes in ALD pathogenesis has been the subject of different lines of research, results, and ultimate interpretations are discrepant. Couzigou et al. were among the first researchers to describe lymphopenia and increased CD4+/CD8+ ratio in patients with liver failure due to alcoholic cirrhosis.[19] In this study, we also found a decreasing trend in the lymphocyte subsets as the disease progresses from fatty liver to cirrhosis with a spike in lymphocyte subsets in alcoholic hepatitis, as this phase of the disease involves liver inflammation with lobular inflammatory infiltrate composed of lymphocytes, neutrophils, macrophages, and plasma cells in alcoholic steatohepatitis.[10] It is widely accepted that immune system activation is relevant for alcoholic steatohepatitis pathogenesis and ALD progression.

We found highest total leukocyte count, T cell, and CD4+ and CD8+ lymphocyte cells in alcoholic hepatitis. However, CD4+/CD8+ ratio increased steadily as the disease progressed. In this study, alteration in the subsets of lymphocytes was not very significant in patients with fatty change as compared to nonalcoholic healthy controls. Mild fatty change has no effect on cellular functions while severe fatty change may transiently impair cellular functions. This could explain our findings. Spinozzi et al. reported no alterations in the total lymphocyte count, but did find an increased CD4+/CD8+ ratio in ALD patients.[20] Other studies have identified significant lymphopenia with decreased numbers of CD4+ and CD8+ populations, in both alcohol abusers without liver disease and in patients with alcoholic cirrhosis.[21],[22] Lombardo et al. also found a reduction in CD3+ and CD4+ T lymphocytes subsets with the severity of liver cirrhosis, which correlates with our study as well.[23] In alcoholic patients, there is overgrowth of lipopolysaccharide (LPS)-secreting Gram-negative bacteria in the intestines. Alcoholic consumption is a known cause of increased intestinal permeability. This increase in gut permeability facilitates the transfer of gut pathogens into the circulation which, in turn, leads to increased liver exposure to LPS. These LPS endotoxins are responsible for pathogenesis and progression of ALD. These endotoxins along with the toxic metabolites of alcohol are supposed to decrease the lymphoid cell numbers by Fas-mediated apoptosis of lymphoid cells which is responsible for the lymphocytopenia.[24] Several events which include bacterial translocation (transfer of bacteria and its products from intestine to extraintestinal organs), toll-like receptors, and intestinal bacterial overgrowth and dysbiosis are responsible for events leading to ALD.[25]

The present study shows the impact of alcoholism in our country, as the majority of ALD patients are young or middle aged (average age of cirrhosis being 45.53 years), mostly male, and with a high daily alcohol intake often since childhood.

Our sample size was small which could be the reason for insignificant P value, despite the correlating trends of lymphocyte subsets. We realized that there are many variables which affect the CD4+ and CD8+ counts such as age, sex, diurnal variations, history of smoking, body mass index, and associated undiagnosed viral infections which may give rise to variabilities in the results.[23]

The reagents and the equipment used in the study are costly, require good infrastructure, and trained staff which may not be feasible to utilize this test as a routine practice.

We could not correlate our results with liver biopsy, which was the limitation of our study. In the future, a larger study with CD4+, CD8+, and natural killer cell counts on liver biopsy tissue can be undertaken to study the immune base of the pathogenesis of ALD.


  Conclusion Top


Our study found the statistically significant association between biochemical parameters (albumin, globulin, SGOT, ALK, and INR) and stages of ALD. These tests are easy, cheap, and form the first line of investigation for diagnosing ALD. However, to ascertain the relationship of immune dysregulation in causation of ALD, flow cytometric analysis of lymphocyte subsets has an important role to play. In this study, we found a decrease in lymphocyte counts and its subsets (CD3+, CD4+, and CD8+) and increase in CD4+/CD8+ ratio in groups of ALD cases as compared to nonalcoholic healthy controls. Among the cases of ALD, we found raised T-lymphocytes in alcoholic hepatitis as compared to fatty liver and cirrhosis explaining the active inflammatory activity in alcoholic hepatitis.

Acknowledgment

Mr. Dhananjay Lende, Technical supervisor in Flow cytometry for assisting in sample processing.

Financial support and sponsorship

Financial support and funding was provided by Dr D.Y Patil Vidyapeeth.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Couzigou P, Vincendeau P, Fleury B, Richard-Molard B, Pierron A, Bergeron JL, et al. Changes in circulating lymphocyte subsets in alcoholic hepatopathies. Respective role of alcohol, hepatocellular insufficiency and malnutrition. Gastroenterol Clin Biol 1984;8:915-9.  Back to cited text no. 19
    
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