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Title: The investigation and diagnosis of 2 cases of liver disease.

Introduction:
Liver is the largest internal organ in the body, performing more than 5,000 separate bodily functions, from cleansing the blood of toxins to converting food into nutrients to controlling hormone levels. Usually Liver disease is reflected by biochemical abnormalities of 1 of 2 different hepatic systems or of liver function. Even though tests that measure the level of serum liver enzymes are commonly referred to as liver function tests (LFTs), they generally reflect hepatocyte integrity or cholestasis rather than liver function. Biochemical markers of liver include:
Alanine Aminotransferase (ALT) and Aspartate aminotransferase (AST) are enzymes found in the heart, liver and muscle. They are sensitive biomarker of hepatocellular damage. However, ALT is considered more specific to liver damage as it has less activity in extra-hepatic tissue than AST.

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Alkaline Phosphatase (ALP) is an enzyme found in the bone, bile duct, placenta and liver. In liver, It is a canicular enzyme that plays a role in bile production. Therefore, increased levels of ALP may indicate liver inflammation and/or blockage of the bile duct (cholestasis).

Gamma glutamyl transferase (GGT) is an enzyme found in the cell membranes of liver, pancreas, intestine and kidneys. Its function is to catalyze transfer of glutamyl group from peptides to other amino acids. Its correlated levels with ALP indicate hepatobiliary origin problems (Cholestasis).

Bilirubin is a breakdown product of heamolysis taken up by liver cells and conjugated to water-soluble product excreted in bile. Elevations may indicate hepatic or extra-hepatic disorders. It also helps in diagnosing Jaundice and its fractionation aid in distinguishing conjugated from unconjugated hyperbilirubinaemia.
Clinical Details:
Patient A:
A 26 years old female presented to Tallaght University Hospital (TUH) with symptoms of abdominal pain, itch, arthralgia (x2/52), new facial rash and a sore throat in July 2018. After admission, liver function tests (LFTs) were performed over the course of several months and results showed consistent increase in liver enzymes. She had no past medical history. She was not on regular medication but received penicillin following the onset of sore throat, after other symptoms.

Patient B:
A 59 years old female was presented to oncology unit in TUH with symptoms of intra-hepatic infection. The patient was already attending TUH.

Laboratory Investigation
Patient A:
Over the course of several months, Liver function tests (LFTs) were performed in the clinical chemistry lab in Tallaght Hospital. After registration, samples were centrifuged. The aim of centrifugation is to separate blood contents (cells & platelets) from plasma. Then, LFTs were performed on these samples using automated system Roche C8000.
The LFTs in TUH include:
-Total protein
-Albumin
-Bilirubin
-Alkaline Phosphatase (ALP)
-Alanine Aminotransferase (ALT)
– Gamma glutamyl transferase (GGT)
The LFTs showed extremely elevated liver enzymes levels (with ALT level exceeding 1000 IU/L), which is an indication of acute liver damage.
Differential diagnosis of Hepatitis
Acute or chronic hepatitis refers to an inflammatory condition of the liver. Hepatitis’s most common cause is viral infection. However, there are other possible causes of hepatitis such as autoimmune hepatitis which occurs when the body makes antibodies against liver tissue. Hepatitis can also be caused as a secondary result of medications, drugs, toxins and alcohol (3).
With hepatitis being caused by the virus A, B, C, and E diagnosis is a lot more complicated as all the isoforms show similar symptoms. Thus, laboratory testing is important to identify the exact causative virus so that the appropriate treatment can be initiated appropriately (4).
Approach for Differential diagnosis of Hepatitis
Diagnosis is a critical decision point at which sufficient evidence has to be collected to get a clear clinical picture about the patient before beginning of the treatment. Laboratory tests for hepatitis are critical for the clinician to confirm ?ndings about the clinical condition of the patient because of various causative agents of the disease (3).

Detection of the specific virus that causes viral hepatitis is only possible by detection of serological markers of respective viruses through
immunological tests. The speci?c causative virus can be detected using these diagnostic tests which can help aid in initiating the right treatment (4).

The ideal approach for detection of specific causative agent is demonstrated below.

Immunological Tests Panel:
Hepatitis A IgM Antibody (Anti HAV – lgM)
Hepatitis E IgM Antibody (Anti HEV- lgM)
surface antigen of the hepatitis B virus (HBsAg)
Anti-Hepatitis B core total antibodies (Anti HBc)
Anti-Hepatitis C virus (Anti HCV)
Human immunodeficiency virus (HIV)
Autoimmune Hepatitis diagnostic panels (4)
Following initial findings, differential diagnosis of hepatitis was made. However, the serology results were negative for hepatitis.

The patient didn’t show any signs of suspicious lesions upon Magnetic resonance imaging (MRI) test results either. MRI is a noninvasive spectroscopic imaging technique used in medical settings to produce detailed pictures of the inside of the human body. The MRI test is based on the principles of nuclear magnetic resonance (NMR), which is a spectroscopic technique used to obtain microscopic chemical and physical data about molecules. this magnetic resonance imaging is accomplished through the absorption and emission of energy of the radio frequency (RF) range of the electromagnetic spectrum. It can be used to help diagnose or monitor treatment for several of conditions within the chest, abdomen and pelvis (5).

Patient B:
A blood sample was received into the clinical chemistry main lab in TUH. It was registered and centrifuged. Then, LFTs were performed on this sample using automated system Roche C8000. LFTs results showed elevated liver enzymes levels (with GGT & ALP levels exceeding 800 IU/L) which is indication of obstructive pattern liver damage (Cholestasis).
Ultrasound (US) of the liver was performed but no obvious biliary duct dilation was detected. However, upon magnetic resonance cholepancreatography (MRCP) test the results showed moderate intrahepatic biliary ductal dilatation secondary to large central necrotic nodal mass at the porta hepatis.

Results:
Table SEQ Table * ARABIC 1: Results of Liver profile test (LFTs) for patient A
Test Result Reference range
Total protein 82 g/L 65-85
Albumin 38 g/L 35-50
Total bilirubin 12 umol/L <17
ALT 1235 IU/L <35
ALP 235 IU/L 35-105
GGT 260 IU/L <40
Table SEQ Table * ARABIC 2: Results of differential diagnosis of hepatitis for patient A
Test Result
Hep B antigens Negative
Hep B antibodies >1000
Hep C antigens Negative
Hep C antibodies Negative
Autoimmune antibodies Negative
HIV Negative
The results from the initial LFTs showed normal levels of total protein, albumin and total bilirubin. However, increased ALP and GGT levels (Table 1) were detected, and elevated ALT value exceeding 1000 IU/L (Table 1) which is an indication of acute hepatocellular disease such as acute hepatitis. Though, no signs of hepatitis were found when the serology testing was performed (Table 2). The Hep B antibodies result was exceeding 1000 which indicates that the patient was fully immunized against Hep B. MRI test did not show any suspicious lesions either.

Table SEQ Table * ARABIC 3: Results of Liver profile test (LFTs) for patient B
Test Result Reference range
Total protein 66 g/L 65-85
Albumin 35 g/L 35-50
Total bilirubin 57 umol/L <17
ALT 147 IU/L <35
ALP 827 IU/L 35-105
GGT 843 IU/L <40
Results from initial LFTs showed normal levels of total protein and albumin and a slight increase in total bilirubin value (Table 2). However, liver enzymes were elevated, specifically ALP and GGT, with values exceeding 800 IU/L, which indicates obstruction of the liver (Intra-hepatic cholestasis). Ultrasound results showed no obvious extrahepatic biliary duct dilatation. However, MRCP results showed moderate intrahepatic biliary ductal dilatation secondary to large central necrotic nodal mass at the porta hepatis.

Discussion
The liver has to perform different kinds of biochemical, synthetic and excretory functions, so no single biochemical test can detect the overall functions of liver. All laboratories usually employ a liver function profile. These tests are helpful to recognize the pattern of liver disease. For example, they are helpful in differentiating between acute viral hepatitis and various cholestatic disorders and chronic liver disease (6).
The results of LFTs from the 2 patients showed extremely elevated liver enzymes but the pattern of elevation was different between the 2. For patient A, LFTs results showed extremely elevated ALT with level exceeding 1000 IU/L which indicates acute liver damage. Whereas, for patient B, the results of LFTs showed significant increase in ALP & GGT with levels exceeding 800 IU/L which indicates cholestasis. These were the outcomes of initial investigation for both patients.
Patient A
Initial investigation results indicated acute liver damage, which is most likely to be caused by hepatitis. Therefore, a differential diagnosis of hepatitis was carried out but no signs of hepatitis were found. The patient did not show any sign of suspicious lesions upon MRI test. As a result of laboratory investigation and clinical details, it was suggested that the patient has Adult onset Still’s disease.

Still’s disease or as commonly known as adult-onset Still’s disease (AOSD) is a rare rheumatic condition. It was first described by Bywaters in 1971 as a distinct clinical entity in adults which is quite similar to the one observed in children known as systemic juvenile idiopathic arthritis (sJIA) (7). It has an approximate prevalence of 1.5 cases per 100,000–1000,000 people (8). The exact etiology of AOSD is unknown (7). There are several factors such as genetics, infectious (bacterial and viral) agents, and environmental factors that have been suggested to play a causative role. It is mainly associated with several symptoms, such as fever higher than 39°C, cutaneous rash during fever peaks, joint or muscle pain, lymph node hypertrophy, increase of white blood cells (especially polymorphonuclear neutrophils) and abnormalities of liver metabolism. Other symptoms may also be present. However, these signs are insufficient to establish the diagnosis. Also, several other diseases (specifically infectious or neoplastic diseases) may produce similar symptoms. Therefore, adult onset Still’s disease usually corresponds to a diagnosis of exclusion, in that the diagnosis is retained when the other possible diagnoses have been completely ruled out (7). The evolution of the disease is hard to predict, it may be limited to only one occurrence or may recur over a period of several months or years. On some occasions, joint erosions may happen (roughly 1 patient out of 3). Treatments can either limit the intensity of the symptoms of the disease, through the use of Aspirin and NSAIDs, or to control the disease evolution, through the use of corticosteroids, methotrexate, ciclosporine A, and sometimes intravenous immunoglobulins or tumour necrosis factor (TNF) alpha inhibitor as Etanercept or Infliximab (9).
Patient B
Initial investigation results indicated Intra-hepatic cholestasis based on liver enzyme elevation pattern. Ultrasound was performed but no obvious biliary duct dilatation was detected. Upon MRCP test However, the patient showed Moderate intrahepatic biliary ductal dilatation secondary to large central necrotic nodal mass at the porta hepatis, which correlates with the liver enzymes elevation pattern. Moreover, multiple liver metastasis and large gastro-hepatic ligament nodal mass were detected when the patient was examined by interventional radiology. In July 2017, The patient was diagnosed with oesophageal cancer stage 4 at the esophagogastric junction which seems to be spread to the liver.

Esophageal cancer is an aggressive malignancy and a leading cause of cancer-related mortality worldwide. Squamous cell carcinoma and adenocarcinoma are the two predominant histological subtypes with varying geographical and racial distribution. Globally, squamous cell carcinoma remains the most common histological type. In Western countries, however, adenocarcinoma has become the leading histological subtype, corresponding to a rise in the incidence of obesity, gastro-esophageal reflux disease and Barrett’s esophagus (10). The risk of esophageal adenocarcinoma conferred by Barrett’s esophagus depends on factors such as genomic instability, race and gender of the patient. Treatment requires a multidisciplinary team approach and optimal therapy is still debated. Endoscopic therapies, including radiofrequency ablation, endoscopic mucosal resection and endoscopic sub mucosal dissection, have become the standard treatment modality for Barrett’s esophagus and early carcinoma. Multimodal treatment, which includes chemotherapy, radiation therapy followed by surgical resection or without surgical resection, in varying orders remains the main mode of treatment for most patients (11).

The liver is one of the most common sites for metastatic disease, accounting for 25% of all metastases to solid organs. In the United States and Europe, secondary liver neoplasms are far more common than primary hepatic neoplasms (12). In particular, lung, breast, and gastrointestinal cancers frequently give rise to liver metastases, and for some patients, the liver may be the only site of disease. In general, systemic therapy is the preferred therapy for liver metastases. However, selected patients with limited involvement of the liver may be suitable for surgical resection, minimally invasive focal ablation, or radiotherapy (RT), delivered with the goal of improving the time to progression and overall survival. In contrast, low-dose whole-liver radiotherapy (WLRT) may be delivered to patients with symptoms from diffuse liver metastases refractory to systemic therapy, with the primary goal of reducing symptoms and improving quality of life (QL) (13).

Conclusion
Patient A
The biochemistry and microbiology contributed to the diagnosis of the patient’s condition, Adult onset Still’s disease. This was achieved by ruling out all the possible causes that have similar signs and symptoms such as hepatitis, which was negative. MRI test showed no suspicious lesions either. The patient is currently being treated by Methylprednisolone and Anakinra (recombinant human interleukin-1 receptor antagonist).

Patient B
The patient is diagnosed by multiple liver metastasis and large upper epigastric mass. This was as a result of metastatic spread from primary cancer site in the oesophagus, which was diagnosed in July 2017. The patient is currently undergoing a regime of radiotherapy and chemotherapy. The 5-year survival rate for patients with esophageal cancer is 19% (11). And for liver metastasis, the 5-year survival rate is about 11%. However, The median 5-year overall survival rate after resection of liver metastasis is 30% with a range of 12% to 41% (13).

References:
Czaja A. Autoimmune Hepatitis – Approach to Diagnosis Internet. PubMed Central (PMC). 2018 cited 23 October 2018. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1785222/
Shetty K, Wu G. Chronic Viral Hepatitis. Totowa, NJ: Humana Press; 2010 (2):115-132.

(ACR) R. Body MRI – magnetic resonance imaging of the chest, abdomen and pelvis Internet. Radiologyinfo.org. 2018 cited 24 October 2018. Available from: https://www.radiologyinfo.org/en/info.cfm?pg=bodymr
Johnston D. Special Considerations in Interpreting Liver Function Tests Internet. Aafp.org. 2017 cited 24 October 2018. Available from: http://www.aafp.org/afp/1999/0415/p2223.html
Adult-onset Still’s disease | Genetic and Rare Diseases Information Center (GARD) – an NCATS Program Internet. Rarediseases.info.nih.gov. 2018 cited 30 October 2018. Available from: https://rarediseases.info.nih.gov/diseases/436/adult-onset-stills-disease
Wakai K, Ohta A, Tamakoshi A, et al. Estimated prevalence and incidence of adult Still’s disease: Findings by a nationwide epidemiological survey in Japan. J Epidemiol. 2011;7(4):20-25. 
Gerfaud-Valentin M, Maucort-Boulch D, Hot A, et al. Adult-onset Still disease: manifestations, treatments, outcome, and prognostic factors. Medicine (Baltimore) 2014;93(2):91–99.

Charles Patrick Davis P. Esophageal Cancer Symptoms, Signs, Staging ; Treatment Internet. eMedicineHealth. 2018 cited 24 October 2018. Available from: https://www.emedicinehealth.com/cancer_of_the_esophagus/article_em.htm#what_is_the_prognosis_for_esophageal_cancer_what_is_the_survival_rate_for_esophageal_cancer
Esophageal Cancer – Statistics Internet. Cancer.Net. 2018 cited 22 October 2018. Available from: https://www.cancer.net/cancer-types/esophageal-cancer/statistic
Poston G, Audisio R, Hoff P, Vauthey J. Liver Metastases. London: Springer-Verlag London; 2009; (2):55–69.

Gazzaniga GM, Cogolo LA, Ciferri E, et al. Liver surgery for metastases: clinical results. J Surg Oncol Suppl. 2016;2:59–62.

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