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Monday 27 August 2012

Non Alcoholic Liver Disease (NAFLD) and Non Alcoholic Steato Hepatitis(NASH)- a Review in eyes of a HistoPathologist of West Bengal, India


:Annals of Tropical Medicine and Public Health

REVIEW ARTICLE 
Year : 2013  |  Volume : 6  |  Issue : 4  |  Page : 393-400
Nonalcoholic liver disease and non-alcoholic steato hepatitis: A review in eyes of a histopathologist of West Bengal, India

1 Department of Pathology, Calcutta School of Tropical Medicine, West Bengal, India
2 7/51Purbapalli, Sodepur, 24 Parganas (north) , India
3 Department of Oncopathology, Medical College Kolkata, Kolkata, West Bengal, India
4 Swamiji Nagar, South Habra, North 24 Parganas, Medical College Kolkata, Kolkata, West Bengal, India
5  7/51 Purbapalli, Sodepur, 24 Parganas (north),  ,West Bengal, India
6 Department of Pathology, IPGME & R, Kolkata, West Bengal, India

Click here for correspondence address and email

Date of Web Publication26-Feb-2014
 
    
   Abstract 
NAFLD characterized by steatosis, when NASH by steatosis, lobular inflammation or steatosis with fibrosis. NAFLD increasingly recognized today as hepatic manifestation of systemic 'metabolic complex" also in India and in province like West Bengal with BMI greater than equal 30kg/m 2 , 67% of overweight patients in which BMI greater than or equal to 25 kg/m 2 and even in 25% normal weight population or with uncontrolled type 2- NIDDM. NAFLD/NASH today one of most common liver diseases in USA, Australia, Europe, Asian countries like china, Dallas, India, and West Bengal with emerging epidemic of obesity due to DM, consumption of fast food and rich food habits in people of industrialized cities and towns even in rural villages of West Bengal . NASH a progressive form of the disease finally leads to cryptogenic cirrhosis of liver even HepatoCellular Carcinoma requires liver transplant for cure. In most cases of NASH, Insulin resistance is found. The responsible gene is fetuin-A (FetA), ethnicity, Familial clustering in first degree relatives (20% of NASH). Natural history of NAFLD variable, although most patients experience an indolent course, some others progress to cirrhosis and liver related death. Paired liver biopsy data of predominantly NASH patients shows that over a follow up period of 2-5 years 18% - 29% patients improved with life style modification and treatment, 34% to 53% remain stable and 26% to 37% develop Cirrohosis and 9% of Cirrohosis from NASH within short time, The two hit hypothesis is widely accepted theory to explain progression of NAFLD from benign steatosis towards NASH. Diagnosis of NASH is challenging before clinicians, Radiologists by USG, with fibroscan devices, MRI, PET scan unless live needle biopsy is done and final diagnosis of NASH remains in clinical knowledge of combined Hepatologists and Hispatothologists. Liver enzymes, several biomarkers for NASH availble like TNF- alpha, Adiponectin- TNF/adiponectin ratio, serum leptin, C reactive protein.
Keywords: Diabetes mellitus, non-alcoholic liver disease, nonalcoholic steato hepatitis, obesity
How to cite this article:
Bhattacharya PK, Bhattacharya U, Bhattacharya R, Bhattacharya R, Bhattacharya S, Barman DR, Mukherjee D, Bhattacharya R, Mukherjee O, Mukherjee D, De A, Sarkar S, Haldar S. Nonalcoholic liver disease and non-alcoholic steato hepatitis: A review in eyes of a histopathologist of West Bengal, India. Ann Trop Med Public Health 2013;6:393-400

How to cite this URL:
Bhattacharya PK, Bhattacharya U, Bhattacharya R, Bhattacharya R, Bhattacharya S, Barman DR, Mukherjee D, Bhattacharya R, Mukherjee O, Mukherjee D, De A, Sarkar S, Haldar S. Nonalcoholic liver disease and non-alcoholic steato hepatitis: A review in eyes of a histopathologist of West Bengal, India. Ann Trop Med Public Health [serial online] 2013 [cited 2014 Feb 27];6:393-400. Available from: http://www.atmph.org/text.asp?2013/6/4/393/127769

   Introduction Top


Nonalcoholic liver disease (NAFLD) is characterized by hepatocytes steatosis, in the absence of history of significant any alcohol use or other commonly known the liver diseases by viruses affecting liver cells or any other drugs or AIDS and many other chemicals except drug like Tamoxifan and chemical workers with extremely high level exposer to vinyl chloride. NAFLD is an increasingly recognized today as the hepatic manifestation of the systemic complex known as "metabolic complex" also in India and provinces like West Bengal.

The prevalence of NAFLD has been found accelerated in the last 24 years paralleling the substantial an increase of overweight within the general population (of Median age range 51 years) with body mass index (BMI) greater than equal to 30 kg/m 2 , 67% of overweight patients in which BMI greater than or equal to 25 kg/m 2 and even in 25% normal weight population. Studies thus evaluating waist circumference noted an association between the degree of abdominal obesity and likelihood of future development of non-alcoholic steato hepatitis (NASH). The increase in incidence of the metabolic syndrome (MS) strongly supports that NAFLD is today one of most common liver diseases in USA, Australia, Asian countries, Europe paralleling also in India and in West Bengal with the emerging epidemic of obesity due to consumption of fast food and rich food habits of people marketed in shops and shopping malls of cities and towns even in rural villages set up of West Bengal. The present terminology used here by the authors as MS is a constellation of individual risk factors of diabetes mellitus (DM) type 2, of cardiovascular diseases, cryptogenic cirrhosis, and strokes. The World Health Organization was the first to publish an internationally accepted definition of MS in 1998, but the that have received the most widespread acceptance and use in the USA are established as guidelines in ATP{The US National Cholesterol Education Program Adult Treatment Panel III (2001)} III guide line. ATP III identified six components of MS like * abdominal obesity, ** atherogenic dyslipidimia*** raised blood pressure **** insulin resistance (IR) and glucose intolerance***** proinflammatory stages**** *pro-thrombotic state. For the purpose of ATP III, MS is present when ≥3 of the following criteria are present waist circumference >102 or >88 in men and women respectively; triglyceride ≥150 mg/dl; (High density lipoprotein) HDL cholesterol <40 or <50 mg/dl in men and women respectively; blood pressure ≥ 130 /

≥ 85 mmHg and fasting Blood glucose without drug or insulin ≥110 mg/dl. However, International Diabetes Federation defined MS to include central obesity with two of the following four factors: Raised triglyceride, low HDL cholesterol and raised blood pressure, raised plasma glucose. Epidemiological data from South India revealed that in the year 2003-04, 32% of all death was due to the circulatory system diseases. [1] Today within term the NAFLD a progressive form of the disease is called NASH, which finally leads to cryptogenic cirrhosis of liver and even hepatocellular carcinomas (HCC) requiring liver transplant. NASH/or NAFLD is one of the mostly seen as common liver disorders in highly industrialized countries along with type 2 diabetes, obesity, hyperlipidemia, and cardiovascular disease where sedentary lifestyle and consumption of high caloric food is very high, being the most frequently evaluated and cited risk factors for the presence of NASH and accelerated disease. Why one should like us bother with NASH,- the disease of upper or upper middle class, high income and sedentary life style people of the world who are responsible for 10/90 gap? However, surprisingly NASH is also found though hugely less amongst lean and poor people with the diabetics type 2 non-insulin dependent diabetes mellitus type 2 (NIDM) who earns on physical labor in fields and in village people. The estimated prevalence in the global general population ranges between 2.8% and 46% depending on the screening tests done for detection of NASH [2] performed within the population. Overall prevalence of NAFLD in type 2 diabetes ranges from 41% to 70% [3] Uncontrolled type 2 DM, (Maturity onset Diabetes of Young) MODY is thus also one of the leading cause of NASH in West Bengal of patients with a median BMI 29 kg/m 2 having total trigleceride mean value 2.09-3.23 mmol/L, cholesterol level 5.45-6.54 mmol/L- one provinces in India.


   IR, DM and NASH Top


In most cases of NASH, IR-+ is found in the context of obesity and NIDM. IR leads to an increased hepatic production of free fatty acids (FFAs) from glucose and to increased circulating levels of FFAs due to enhanced peripheral lipolyses. The responsible gene is of course fetuin-A (fet-A) a long known gene for entry of FFA in cells and the product of Fet A gene can act as endogenous ligand to bind up with (Toll Like Receptor 4) TLR4 receptor, and that it has a crucial role in regulating insulin sensitivity via TLR4 signaling in FFA metabolism and entry of FFA in hepatocytes and adipose tissue liopcyte resulting IR, obesity, dyslipidemia, fatty liver i.e., MS and also insulin sensitivity turned into resistance. The increased uptake of FFAs by the liver exceeds its capacity to metabolize them by mitochondrial oxidation and to remove them by secretion into the blood as (Very low density lipoprotein) VLDL. As a consequence, liver steatosis (fatty liver) develops generally first people and physicians consider them as a "benign disease" but according these authors which is the first pre-requisite of NASH. Free oxygen radicals (Reactive Oxygen Species) ROS exert their toxicity by lipid per-oxidation, which finds then a favorable environment for its perpetuation in a steatotic liver and stimulate various pro inflammatory cytokine productions, leading to inflammation and activation of hepatic stellate cells and therefore, leads fibrosis. FFA-induced proinflammatory cytokine expression in adipocytes occurs only in the presence of both fet-A-{fetuin-A} and TLR4; As a result, a relative deficiency in antioxidants may be necessary in situations promoting oxidative stress and lipid per oxidation. It should be noted that NAFLD/or NASH typically develops within a boarder range of FFA induced metabolic abnormalities that affect not only the liver but also other tissues such as skeletal muscles (one of my senior colleagues in pharmacology's spouse had long uncontrolled type 2 DM, and finally she developed NASH leading to cryptogenic cirrhosis, Gastrointestinal bleeding and she had muscle and her peripheral nerve involvement), pancreatic beta cells and vascular beds. However, it has become evident that progression from simple benign steatosis to NASH is not just a consequence of FFA derive triglyceride accumulation but rather an inadequate hepatocyte adaptation to toxic lipid derived metabolites like unsaturated fatty acids, lipid per oxidation products and others with activation of multiple inflammatory pathways, mitochondrial dysfunctions of hepatocytes, endoplasmic cytokine stress, and leading to the end to cellular (hepatocytes) apoptosis. The IR in MS and NASH is results of complex combinations of genetic, chronic illness, ageing, high Triglyceride, low Adiponectin, low HDL-c and often fasting glucose level >115 mg/dl, (Polycystic ovary syndrome) PCOs; Pregnancy, Chronic diseases like (Chronic Renal Failure) CRF, (End Stage Liver Disease) ESLD, (Non Alcoholic Fatty Liver Disease) NAFLD. So NASH is a real problem of westernized culture and society's people where obesity is playing leading and the increasing role to accelerate MS, DM and NASH and its sequel cirrhosis and HCC. Let them to go on like this! why should it bother us?- it should bother us because it is also seen that an individual who is before perfectly insulin sensitive, nonobese, poor, may become suddenly IR with just 24-72 h due to increase in plasma FFA to levels that are observed in obesity for some other cause. Thus, chronic elevation of plasma FFA appears particularly deleterious to normal metabolism in human species


   Non Classical Phenotype of NAFLD in Normal Weight Patients or NonObese People Top


Although, NAFLD and NASH are more common amongst obese affluent class patients, it is also recognized widely that a fraction of NASH patients do not meet the criteria of obesity at all at least in West Bengal is probably due to the ethnicity. They are probably metabolically obese which includes IR in spite of their normal or low BMI. Whether a separate group of lower BMI patients with primary lipoprotein abnormalities but without IR at all exists amongst them may be one hypothesis but not yet proved. Are they genetically susceptible at all? Familial association of NASH and cryptogenic cirrhosis in one or more first degree relatives has been documented. However, the studies were in association with the obesity diabetes and features of MS. A genetic predisposition and family history is found positive in 20% of NASH [4] the finding of IR and impaired muscle mitochondrial metabolism


   Is NASH Genetically Pre-Determined and Familial? Top


Familial clustering and ethnic variation in prevalence of NASH has been described that consistently have shown that fatty liver is less common among African American people and though NASH is common in USA among people of Hispanic descent. The percent of subjects with steatosis is significantly lower amongst African American (24%) compared with nonhispanic whites (33%) and hisppanic 45%. [5] NAFLD is clearly emerging in Asian population especially in regions of countries with the significant industrialization and affluence. It is found that in china, prevalence of NASH has doubled than it was in the past decade (Now 15% of the total population of china has NASH). Dallas heart study group showed that distinct similar level of IR compared with Hispanic Americans the African American had lower intra peritonial fat, liver fat and hepatic fat in all groups. [6] Hence, is clear that substantial genetic risk exist in the development of steatosis and perhaps in the development of hepatocytes injury and subsequent genetic severity and the gene is related with lipoprotein metabolism properly. Several candidate genetic polymorphisms have been described related either to lipogenetic gene or genes alternating the response to oxidation stress. (Patatin likephospholipase domain containing protein-3) PNPLA3 is a liver expressed trans-membrane phospolipase, also known as adiponutrin that is up regulating during the adipocyte differentiation. PNPLA3 polymorphism more likely to have a steatosis and evidence of cell injury. [7]


   Mitochondriopathy and Lipodystrophies Associated with NASH Top


NAFLD manifestation may be seen an associated with the primary mitochondriopathies. Several isolated elements of systemic mitochondrial diseases have been observed in NASH patients including, ophthalmoplegia, deafness, depression of gut dysmotility, lipomatosis and neurodegenerative diseases. Components of MS specifically IR, dislipidimia are found in patients affected by the maternally inherited DM type 2 and deafness syndrome(Maternally inherited diabetes and deafness) MIDD. Hepatic mitochondrial DNA mutation have been also identified in some patients with NASH and cryptogenic cirrhosis. [8] Lipodystrophy, whether acquired in diabetic or inherited are also characterized by fatty liver, DM, Hyper-triglyceridimia, panniculities, and focal or diffuse loss of subcutaneous fat and they are thought to result from failure of differentiation of pre-adipocytes, because of leptin deficiency. [9]


   What Is Then Natural History of NAFLD/NASH ? Top


What is then mechanism of progression of NAFLD to NASH to Cirrhosis if the condition of fatty liver is considered to be benign? Benign fatty liver appears to have remarkably little likely how of development of additional fibrosis! For the development of fibrosis there needs inflammation and if the inflammation is of lowgrade then usually it should not progress to advanced fibrosis (grade F3 and F4). Crosssectional and longitudinal biopsy studies so constitute the best available evidences for establishing the developing of progressive fibrosis and NASH. It requires longitudinal but not cross-sectional studies. Crosssectional studies may be necessary to understand the severity of NAFLD at a time of its presentation. There are many cohort studies to understand geographic locations with a longer periods followup and shows fibrosis in NASH is consistent. NASH is so defined as steatosis with lobular inflammation or steatosis with the fibrosis [10] These cohorts studies reflect a population that is predominantly female 961%) obese (63%) IR (38% frankly type 2 DM and mean age 47 yearsfifth decade. Approximately 1/3 rd patients had advanced fibrosis (stage F3 or stage F4). Progression of fibrosis is an associated with age, presence of degree of inflammation, apoptosis of hepatocyte on the initial presenting biopsy. The natural history of NAFLD is highly variable, Although most patients experience an indolent course some others progress to cirrhosis and liver related death paired liver biopsy data of predominantly NASH patients shows that over a follow-up period of 2-5 years, 18-29% patients improved with life style modification and treatment 34-53% remained stable and 26-37% developed cirrhosis and 9% of cirrhosis from NASH within a short time. [11]


   Two/or Three Hit Hypothesis? Top


The two hit hypothesis is today widely accepted theory to explain the progression of NAFLD from benign steatosis (fatty liver) toward NASH. The first hit involves dys regulated hepatic lipid accumulation (steatosis) and second hit include, oxidative, metabolic and cytokine stress that leads to hepatocyte cell death by the process of apoptosis. Indeed NASH differs from benign simple steatosis mainly in the degree of hepatocyte injury and hepatocyte apoptosis. Hepatocyte apoptosis should trigger also regenerative mechanism to replace dead hepatocytes. However, in NASH the mechanism somehow fails, and aberrant repair response occur in some individuals resulting activation of hepatic stellate cells to convert in myo-fibroblasts and pro fibrogenic immune cells. Hepatocyte apoptosis thus indirectly promotes inflammation and release of neutrophil chemo attractant. There is liberation of pro inflammatory chemokines such as (They are all pro inflammatory cytokines & apoptosis related factors and named as above TNFR1 = Tumor Necrosis Factor Receptor-1, TRAIL R1and R2= TNF related apoptosis inducing ligand receptors 1 and 2; NF-KB = Nuclear factor kappa beta kinase) CXCL1, MIP2, TRAIL-R1 and R2 TNF-R1/CD120a, NF-kB activation and many other pro inflammatory adhesion molecules. Apoptosis initiates fibrosis. The phagocytes of apoptotic hepatocytes by macrophage kupffer cells stimulates production of (Transforming growth factor Beta) TGFB - a pro fibrogenic cytokine. Engulfment of apoptotic bodies by the stellate cells trigger their own activation and promote fibrosis. NASH is more advanced lesion than simple steatosis is characterized by increased hepatocyte injury by apoptosis. As it is like alcoholic steato hepatitis. Immunohistology when carried out with liver true cut biopsy of patients with NASH shows up regulated expression of NF-kB, and (TNF related apoptosis inducing ligands) TRAIL.

The second hit is believed to mediate the progression of NASH. At low-level ROS activate NFkB to induce synthesis of pro-inflammatory cytokines and death receptor (FasL={Apoptosis cell suicidal Fas Ligand0}) expression. Jun-N terminal kinase (JNK) and BAX={A cell Cycle Regulatory intra nuclear protein} protein regulate cellular apoptosis through regulation of Bcl2. JNK1 protein has been shown to express high in NASH hepatocytes by inmmunohistological stain. The TNF alpha, TRAIL receptors level is high in NASH. Treatment of mice with Jo2 (anti-Fas antibody can enhance hepatocyte apoptosis, hepatic injury, chemokine production (CXCL1) and infiltration of neutrophils Diagnosis of NASH = diagnosis of NASH is a real challenging job before the clinicians and radiologists even with fibroscan devices and magnetic resonance imaging (MRI) (where it is present), unless a liver true cut needle biopsy is carried out (today needle liver biopsy has been almost becoming obsolete due to market available sophisticated radiological imaging techniques in cities and Industrial towns like Doppler (Ultra Sono graphy) USG; computed tomography (CT) (contrast/non contrast), MRI, Fibroscan device for liver and (positron Emission Tomography) PET technology and due to some complications and limitation in doing liver biopsies and today histopathologists in Kolkata, West Bengal can rarely find and report a true cut needle liver biopsy specimen for last two decades or either when abdomen is opened for occult diagnosis and liver is operated or a wedge biopsy is taken in suspected metastasis in liver after dissecting one involved lobe of liver, or some other diseases like gall bladder diseases or NASH or a USG or CT or MRI guided (Fine Needle Aspiration Cytology) FNAC or after a very rarely performed postmortem carried out in provinces of West Bengal, India) and final diagnosis of NASH remains in the clinical knowledge of combining Hepatologists and Hispatothologist. Before one performs liver biopsy, a hepatologist must do the following steps as per authors of this article

Liver enzymes- the uses of serum liver enzymes are used routinely to screen up chronic liver disease only. Hence, it is also justified to do in NAFLD/NASH. Elevated serum transaminases level is commonly found in NAFLD/NASH as per hepatologist and biochemist friends of principal authors. Usually (Alanine amino transferase) ALT and (Aspartate amino transferase) AST tends to be higher in NASH compared when with benign NAFLD (mean ALT is usually 1.72 ± 0.92 and mean AST is 1.39 ± 1.40 ± 0.75). Thus, the sensitivity of elevated Alanine transferase for diagnosis of NASH is 40-59% and specificity is also 50%, and predictive value is <30% in West Bengal according these authors. However, what should be cut-off value for AST and ALT in NASH in diabetic and obese patients? In diabetic AST and ALT remains a little elevated. Some people say AST/ALT ratio (AAR) can predict probable NASH versus simple steatosis of NAFLD in diabetes. Usually AAR greater than 0.8 may be a good predictor and this AAR may be used for prediction of advanced fibrosis as per these authors. Before AAR to be taken as a predictor of NASH, heptologists must exclude hepatites B and C., (Gumma-glutamyl transpeptides) GGT (for Alcoholic) and for AIDS in the set-up of Kolkata population of West Bengal, and if patients belongs to have affluent middle class and both the histopathologists and the clinicians want to seize a sizable amount of money from patients pocket, there remains some kinds of justification of performing other serological tests also such as alpha-1 anti-trypsin deficiency, Anti-nuclear antibody, anti-smooth muscle antibody and anti-mitochondrial antibody, serum leptin, TNF, insulin level, tunnel test, mandolic acid (MDA) level etc. The problem occurs when AST, ALT GGT remains within normal range (range varies from 19% to 37%) of NAFLD. The crucial question remains to pathologists like me is there any real bio markers for NASH?


   The Biomarkers Related with NASH May Be Top


Biomarkers of inflammation-though there may be several biomarkers for NASH such as TNF-alpha, adiponectin-TNF/adiponectin ratio, serum leptin, C reactive protein, IL 6, TGFBeta, insulin like growth factor; however, these authors consider high sensitive CRP is enough to distinguish from benign NAFLD with NASH. Performing other biomarkers may be just of academic or research interest and to drain patients hard money into heptologists and pathologists pocket. The authors have seen that many clinicians/gastroenterologist asked the patient party/patients to perform the bio markers for oxidative stress, and the hepatocyte apoptosis the poor class party of some diabetic patients with the differential diagnosis of the NAFLD has been some time advised in a hospital or chamber prescriptions to do MDA/serum thioredixine/tunnel tests etc). There is, in fact, no need to perform serum thioredixine/or MDA which are marker for oxidative stress because final diagnosis of NASH remains in hands histopathology only.

However, one may perform basic (Low density Lipoprotein) LDL and if possible Oxidative LDL (P < 0.1) in NASH, which is increased rather one biomarker for hepatocyte for apoptosis like caspase-3 cytokeratine-18 may be done whose cut off level is 400 UlL and positive predictive value for NASH is 99.9% and negative predictive value 85.7% [12] rather to do tunnel tests. All these are however, academic research avenues. Why patients and his/her parity will be harassed and paid for these tests in the private laboratory set-up a crucial question to me.


   Imaging Technology and NASH Top


Current imaging technology can detect steatosis with increasing accuracy by high resolution USG and Doppler USG. However, USG cannot detect NASH as it cannot detect inflammation or fibrosis or pre-cirrhosis changes or remodeling of liver parenchyma. In clinical practice, however, USG is the first time for assessment of liver abnormalities. In this setting of hepatic steatosis, liver is typically hyperechotic or bright caused by reflection and alteration of the sound beam by fat. Additionally there may be decreased visualization of hepatic vasculature, loss of diaphragmatic definition, sparing of gall bladder fosse. Standard USG however, provide no information to the clinician regarding the presence or absence of NASH or steatohepatitis, though hepatic fibrosis, which is a component of NASH may be detected; however, the sensitivity is extremely poor when there is mild fibrosis. Doppler USG can enhance standard ultrasound by assessing blood flow in the hepatic and portal vasculature. Hepatic blood flow is impaired as a consequence of hepatic vein obliteration (Rt hepatic vein blood flow). Steatosis alone is also associated with impaired microcirculation in liver probably as a result of sinusoidal compression by hepatocytes engorged with the triglycerides. Another Imaging technology is dynamic elastography for detection of hepatic fibrosis (called fibroscan). This facility is available only in gastroenterology department of IPGME & R SSKM Hospital in Kolkata capital metropolis city of province West Bengal. This technique has adequate sensitivity (>80% as per hepatologists and gastroenterologists friends of mine Prof. Jayanta Dasgupta) for detecting cirrhosis in a variety of liver disease including hepatities B and C causative cirrhosis, primary biliary cirrhosis, primary slerosing cholangities. Alcoholic fibrosis (it is highly needed to differentiate NASH from Alcoholic steato heptities and alcoholic lipo-fibro-granulomas). Fibroscan can detect more subtle form of fibrosis (F1-F3). Failure of this technique is nearly 5% in subjects who are however, very obese. In fact, it replaced liver biopsy for fibrosis detection. Other Imaging technology may be used CT, but contrast CT is never used to diagnose NASH as it does not distinguish between benign steatosis from steatohepatities.


   Biopsy is the Final Step for Diagnosis and Evaluation of Treatment and Progression of NASH and Differentiation from Benign NAFLD Top


The best available evidence of establishing the progressive fibrosis in NAFLD is based on needle biopsy of liver and then follows up biopsies. Problems we histo pathologists in Kolkata, West Bengal based tertiary level even teaching hospital settings face are extremely often/or almost regular inconsistent clinical data supplied with biopsy tissues from the clinicians, sample variation and personal expertise variability from one histopathlogist to others and limitation on liver biopsy interpretations. Yet, I must say the liver biopsy still remains the gold standard diagnostic criteria of NASH and progression of the diseases, not the imaging. There are of course several risk factors for the liver needle biopsy to be done particularly in patients over the age of 55 years, highly obese patients (BMI say over 32 kg/m 2 ), abnormal (Liver Function Tests) LFT specially abnormal Serum Prothombin Time. Liver biopsy is useful not only for diagnosis and grading of fibrosis but also for exclusion of NASH. One should remember while interpretation of liver biopsy that not all obese or obese and diabetic individuals may have fatty liver or fatty liver disease from previous. Liver biopsy interpretation may vary in regard to the degree of fibrosis The authors consider them as (1) the lobes from which the biopsy is being taken (2) the length of needle employed (3) surgical versus non-surgical attainment of biopsy tissues sent (4) Histo pathologists expertise to diagnose NASH (5) adequacy of biopsy specimen (6) nature of sections (thick, thin, ultra-thin) and what stains are used to diagnose and quality of stain (7) inter observer variation in interpretation between lesions of NAFLD and NASH.

The choice of lobe: Capsule/parenchyma ratio-needle biopsy from left lobe results more sub-capsular parenchyma what authors experience is and thus include a greater amount of connective tissues element associated with capsule and larger portal tract is also authors experience.

Whether the liver biopsy is taken during a bariatric or any other surgical procedure-then aggregation of ploy-morho-nuclear leukocytes (PMN) may be more due to surgical procedure itself or handling liver during operation or due to anesthetic agents used, and it is then referred by the author as surgical hepatitis.

Whether the patient has suffered previously from hepaties B or C ever and has an association with NASH- and PMN and fibrosis, may not be then for NASH but due to hepatitis B or Hep C infection. In Hep C infection, PMN infiltrate may be also component of lobular inflammation of NASH. Finally, cirrhosis will lead to challenge in interpretation of NASH. In fact in cirrhosis due to NASH may or may not retain the feature of steatosis.


   Histological Findings in NASH Authors Experiences Top


The histological finding of NASH as per principal author of the article is almost similar to alcoholic steato hepatitis and extremely difficult to diagnose it as NAFLD, more difficult to diagnose it as NASH, but if one take minute history patients are non-alcoholic, overweight, commonly women and diabetic and they often receiving metformin as Oral hypoglycemic agent They if give a history of previous jaundice the diagnosis become extremely difficult because none preserve the necessary documents what caused them jaundice Hep B or Hep C. In alcoholic hepatitis, there are associated cholangities, Mallory hyaline bodies more common than in NASH. Apoptotic body hepatocyte (councilman bodies) is rather more seen in NASH. Steatosis, lobular and portal inflammation, hepatocyte ballooning and fibrosis are characteristic in NASH. NASH histology can be divided again in two types. In adult NASH, liver biopsy these changes are more commonly seen in zone 3 which shows predominant macro vascular steatosis, ballooning and peri-sinusiodal fibrosis when present. This fibrosis is known as chiken wire fibrosis. This type of fibrosis resembles a piece of mesh and collagen fibers when demonstrated by (Van Gesion or Elastic van Gesion) VG or EVG stain is extremely delicate (type 1a collagen in NASH). Additional fibrosis is found in periportal, and portal region with progression of the disease Lobular inflammation is not necessarily concentrated in zone 3 but may be found.

In pediatric group NASH liver biopsy, there may be pan acener steatosis or steatosis concentrated in zone-1 (peri-portal) hepatocytes, ballooning degeneration may be absent and lobular inflammation may be absent or extremely mild if present. The most common form of steatosis in NASH in adult and in child type is droplet of fat that is known as Macro vesicular steatosis. This may be composed of a single droplet that displaces the cytoplasm and nucleus peripherally or may be composed of a mixture of small and large droplets of fat. One should remember Micro vesicular steatosis is usually not seen in NASH. One should also remember in mind that in Adult NASH liver biopsy, the zone-3 steatosis may also occur in alcoholic steatosis or steato-hepatitis, with many drugs or lipodystrophy in diabetic patients. Zone-1 steatosis has to be differentiated from NAFLD, hepatitis C, (Protein Calorie Malnutrition) PCM, AIDs, total parental nutrition, cystic fibrosis, use of corticosteroids, and amiodarone drugs [13] Inflammation in NASH is either lobular and portal or in one of them. The lobular infiltrates of NASH are mixed cells with PMN and chronic inflammatory cells like mononuclear cells, lymphocyte, monocytes, plasma cells and eosinophils. Portal chronic inflammation is most notable in setting of NASH pediatric liver biopsy with NASH, resolution of NAFLD with treatment and in case of severe steato hepatitis of adults. There is an increase of Kupffer cells aggregate and loss of zone-1 predominance of this cell type. In NASH Kupffer cells aggregate mostly in zone-3. Lipogranulomas like in alcoholic liver disease spectrum may occur also in NASH, but they are seen in peri veinular, lobular and in portal areas. A lipogranuloma in NASH is very small in comparison to that is usually seen in alcoholic Lipogranuloma. It may be as small as a single droplet of Macro vesicular steatosis surrounded by aggregate of Kupffer cells. Some times lipofuschin granules found in zone-3 hepatocytes and is probably related to oxidative stress in these heptocyets.


   Immuno Histopathology for NASH? Top


This is not at all normally required but may be of only research interests if one ask me the two immunohistological stain I do prefer are with monoclonal antibody K18/18 for demonstration of loss of keratin filaments to loss in NASH and it will also high light Mallory hyaline bodies. The other most important stain is M30-which is also a fragment of K18 are found to be normal steatotic hepatocytes of NAFLD, and alcoholic hepatic steatosis and steatohepatitis but M30 positivity is lost or less in NASH. However, please note carefully that prescribing or advising the immuno stain to confirm NASH is to drain money of patients and party very purposefully if done with patients own pocket expense in the name of research. It may be MD/PhD thesis work.


   NASH Leads to Cirrhosis Top


It is generally believed that inflammation and apoptosis lead to drive the disease progressing in NASH, and acute pro-fibrogenic pathway leads more fibrosis and eventually the diffuse architectural disruption, regenerating nodule which are the hall mark of cirrhosis. Cirhhosis is the final stage of fibrosis (F4 fibrosis). In adult biopsies, perisinusiodal fibrosis continues to F2 stage and may or may not persist in F3 (bridging fibrosis) or F4 (Cirrhosis). The fibrosis is here mixed with elastic fibers in stage F3 and F4.


   NASH Scoring System Top


The NAFLD/NASH are now focused on different methods extant methods for evaluation of biopsies lesions. The original proposed scoring system was done by Brunt et al. (1999), [14] which was developed for semi quantitative assessment of new inflammatory activities as grading of lesions and fibrosis as stage of lesions, specifically like chronic hepatitis. The method proposed for grading included collective assessment of steatosis, amount of lobular and portal inflammations and ballooning degeneration of hepatocytes. The feature of ballooning and inflammation is divining forces for progression of the diseases but not the steatosis. The NIDDK NASH CRN system by Kleiner 16 et al. in 2005 ={Kleiner DE, Brunt EM , Van Natta M Design and validation of a histological Scoring System in Non alcoholoic liver disease Hepatology 2005;41;(6): 1313- 21} was developed to assign a score of evaluating pre- and postliver biopsies in studies that the author of this article follows in interpretation of NAFLD biopsies. As such the entire spectrum of NAFLD including, the different pattern for children was also included by the author. His = {His} scoring system is based on steatosis amount lobular inflammation and ballooning of hepatocytes, for grading and NAFLD score and fibrosis for the purpose of staging. The fibrosis in the earliest stage resembles him like a piece of mesh and the collagen fibers in it may be delicate (1a in NASHCRN system) or when dense (1b in NASH CRN) because this is a lesion not predictable seen in children, The earliest stage of pediatric biopsies is portal expansion (1c in NASH CRN). Additional fibrosis may occur in the peri-portal region (stage 2) with progression, bridging fibrosis between central portal areas, between central and portal areas or between portal areas (stage-3. Cirrhosis becomes the final stage (stage-4).


   NASH and HCC Top


HCC (primary liver cancer) is the predominant cause of primary liver cancer, and viral hepatitis with Hep B and hepC and alcohol was commonest etiology in most cases. However, though NASH follows a benign course it can progress to cirrhosis and result in complication including HCC. A significant number of cases of HCC (15-50%) remain cryptogenic without known underlying liver diseases. But in the setup of obesity, DM with NASH it is increasingly recognized that NASH is likely accountable for a substantial portion of HCC. Most cases of HCC occur in highly endemic areas of eastern Asia, sub-Saharan Africa where incidence is over 20 cases per one lac population. Sothern European countries are considered at intermediate risk between 5 and 10 cases per one lack when north America, South America and Northern Europe have been considered of low risk areas for HCC. [15] Many areas of high prevalence are now reporting fewer cases of HCC following efforts to vaccinate newborns yet for (Hepatitis B Virus) HBV. However, incidence of NASH and HCC doubled over the past two decades in USA. The increase is most notable among African Americans and among Asians in USA.

The primary risk factors for HCC are cirrhosis which is present in 70-90% cases. Although both HBV and Hepc autoimmune hepatitis, primary biliary cirrhosis are the common causes of HCC worldwide many studies demonstrated HCC associated with HBV is stable, and HepC is a major risk factor even in USA up to 70% of HCC but a significant amount 15-50% of HCC cases occur without known causes of liver disease. There is also a relationship between DM type 2 and HCC.


   Acknowledgement Top


The authors acknowledge contributions of Prof. Jayanta Kumar Das Gupta DM (Gastro) (Calcutta Univ.), Prof. Pradip Kumar Dutta MD (Calcutta Univ.), Dr. Pijush Kanti Roy MS (Cal. Univ.), Dr. Hriday Ranjan Das MD Trop. Med (Calcutta Univ.) DTM&H (Calcuuta Univ.), Dr. Sumana Mukherjee MD (Calcutta Univ.), Dr. Shovon Das and Dr. Soma Das.[16]

 
   References Top

1.Joshi R, Cardona M, Iyengar S, Sukumar A, Raju CR, Raju KR, et al. Chronic diseases now a leading cause of death in rural India - Mortality data from the Andhra Pradesh Rural Health Initiative. Int J Epidemiol 2006;35:1522-9.  Back to cited text no. 1
    
2.Lazo M, Clark JM. The epidemiology of nonalcoholic fatty liver disease: A global perspective. Semin Liver Dis 2008;28:339-50.  Back to cited text no. 2
    
3.Tolman KG, Fonseca V, Tan MH, Dalpiaz A. Narrative review: Hepatobiliary disease in type 2 diabetes mellitus. Ann Intern Med 2004;141:946-56.  Back to cited text no. 3
    
4.Schwimmer JB, Celedon MA, Lavine JE, Salem R, Campbell N, Schork NJ, et al. Heritability of nonalcoholic fatty liver disease. Gastroenterology 2009;136:1585-92.  Back to cited text no. 4
    
5.Weston SR, Leyden W, Murphy R, Bass NM, Bell BP, Manos MM, et al. Racial and ethnic distribution of nonalcoholic fatty liver in persons with newly diagnosed chronic liver disease. Hepatology 2005;41:372-9.  Back to cited text no. 5
    
6.Guerrero R, Vega GL, Grundy SM, Browning JD. Ethnic differences in hepatic steatosis: An insulin resistance paradox? Hepatology 2009;49:791-801.  Back to cited text no. 6
    
7.Weiskirchen R, Wasmuth HE. The genes that underlie fatty liver disease: The harvest has begun. Hepatology 2009;49:692-4.  Back to cited text no. 7
    
8.Bohan A, Droogan.O, Nolan N. Mitochondrial DNA abnormalities without significant deficiency of intramitochondrial fatty acid beta oxidation enzymes in a well defined subgroup of patients with non alcholoic steatohepatities (NASH). Hepatology 2000;32:A387.  Back to cited text no. 8
    
9.Garg A. Acquired and inherited lipodystrophies. N Engl J Med 2004;350:1220-34.  Back to cited text no. 9
    
10.Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, McCullough AJ. Non alcoholic fatty liver disease: A spectrum of clinical and pathological severity. Gastroenterology 1999;116:1413-9.  Back to cited text no. 10
    
11.Adams LA, Sanderson S, Lindor KD, Angulo P. The histological course of nonalcoholic fatty liver disease: A longitudinal study of 103 patients with sequential liver biopsies. J Hepatol 2005;42:132-8.  Back to cited text no. 11
    
12.Wieckowska A, Zein NN, Yerian LM, Lopez AR, McCullough AJ, Feldstein AE. In vivo assessment of liver cell apoptosis as a novel biomarker of disease severity in nonalcoholic fatty liver disease. Hepatology 2006;44:27-33.  Back to cited text no. 12
    
13.Brunt EM, Tniakos DG, editors. Surgical Pathology of GI Tract, Liver, Billiary Tract and Pancreaus. 2 nd ed. Philadelpheia: Elsevier; 2009. p. 1007-14.  Back to cited text no. 13
    
14.Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA, Bacon BR. Nonalcoholic steatohepatitis: A proposal for grading and staging the histological lesions. Am J Gastroenterol 1999;94:2467-74.  Back to cited text no. 14
    
15.Gomaa AI, Khan SA, Toledano MB, Waked I, Taylor-Robinson SD. Hepatocellular carcinoma: Epidemiology, risk factors and pathogenesis. World J Gastroenterol 2008;14:4300-8.  Back to cited text no. 15
    
16.Kleiner DE, Brunt EM , Van Natta M Etal Design and validation of a histological Scoring System in Non alcoholoic liver disease. Hepatology 2005;41;1313-21.  Back to cited text no. 16
    

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Correspondence Address:
Pranab Kumar Bhattacharya
Department of Pathology, School of Tropical Medicine, Kolkata, Room No 10[c], 108 C.R Avenue, Kolkata - 700 073, West Bengal
India
Rupsa Bhattacharya
Purbapalli, Sodepur, 24 Parganas (north), IPGME & R ,West Bengal
India
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DOI: 10.4103/1755-6783.127769
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    Abstract
   Introduction
   IR, DM and NASH
    Non Classical Ph...
    Is NASH Genetica...
    Mitochondriopath...
    What Is Then Nat...
    Two/or Three Hit...
    The Biomarkers R...
    Imaging Technolo...
    Biopsy is the Fi...
    Histological Fin...
    Immuno Histopath...
    NASH Leads to Ci...
   NASH Scoring System
   NASH and HCC
   Acknowledgement
    References

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