ABSTRACT

A common problem in clinical laboratories is maintaining the stability of analytes during sample storage, destined for batch analysis. Factors such as equipment breakdown, lack of reagents, electricity outages can cause losses/deterioration in specimens collected and thus the samples will need storage. This study was designed to assess the stability in blood of hepatobiliary disease biomarkers (alanine amino transferase (ALT), aspartate amino transferase (AST), gamma glutamyl transferase (γ-GT), alkaline phosphatase (ALP) and albumin (ALB)) under varying storage conditions. Thirty study participants (five males and twenty-five females) were recruited from the Buea Regional Hospital and the Muea Sub-divisional Hospital. Serum samples from these participants was used to measure the biomarker levels using spectrophotometric commercial assay kits. Each biomarker’s activity/concentration was measured over the course of thirty days in samples collected and preserved at 25, 4, -20 and -86 °C with/without the use of glycerol. The concentration/activities of AST, ALP and ALB measured remained stable at -20 °C for up to 14 days as well as at -86 °C for up to 30 days in samples that were stored without glycerol. AST was stable at 4 °C for up to 3 days in the samples stored with glycerol. Albumin equally was stable at -86 °C for up to 30 days in the samples stored with glycerol. Based on this study’s findings, it is recommended that: Same day analysis be done for the measurement of ALT, γ-GT. ALP can be stored at 4 °C for short periods such as 3 days. Albumin and AST can be stored for up to 14 and 30 days when stored at -20 °C and -86 °C respectively, and is suitable for research purposes.

Keywords: Assessment, Stability, Hepatobiliary Disease Biomarkers

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1) INTRODUCTION

Hepatobiliary diseases are a group of diseases involving the liver, gall bladder and biliary tract caused by viral, bacterial, parasitic infections, neoplasia, toxic chemicals, alcohol consumption, poor nutrition, metabolic disorders, and cardiac failure amongst others (Nicholas et al., 2010).

In 2015, hepatitis B virus infection resulted in an estimated 887,000 deaths, mostly from cirrhosis and hepatocellular carcinoma (i.e. primary liver cell cancer), with the hepatitis B prevalence being the highest in the WHO Western Pacific Region and the WHO African Region, where 6.2% and 6.1% of the adult population were infected respectively (WHO, 2019). In 2015, hepatitis C virus infection resulted in an estimated 500,000 deaths mostly from cirrhosis and hepatocellular carcinoma (GBD, 2016)

Hepatobiliary diseases are primarily diagnosed using liver function tests (LFTs). Enzymes such as alanine amino transferase (ALT), aspartate amino transferase (AST), gamma glutamyl transferase (γ-GT) and alkaline phosphatase (ALP) and serum proteins such as albumin form the major analytes measured in liver function tests (Allan et al., 2008). Liver function tests (LFTs) reflect the liver’s diverse functions and they include true tests of hepatic synthetic function (such as, serum albumin levels), tests of excretory function (including, serum bilirubin levels), and tests that reflect hepatic necro-inflammatory activity (serum aminotransferase levels) or cholestasis (such as, alkaline phosphatase levels), (Friedman et al., 2018).

Most hospitals in low- and medium-income countries usually have large daily requests for laboratory investigations which cannot be executed immediately as expected due to limited human and material resources. As a solution, they resort to batch analysis which requires the samples to be stored at specific temperatures to guarantee the stability of the analyte under measurement.

There exists therefore the huge problem of sample storage in hot, humid climates to ensure the analyte being measured remain stable. In most developing countries, these laboratories are usually understaffed, experience frequent equipment breakdown due to a poor maintenance culture, lack of reagents, as well as, persistent electricity outages. These factors can cause losses in patient history and specimens collected resulting in delays in issuing results of laboratory measurements which in some cases maybe incorrect due to poor storage conditions

Samples are usually stored in a refrigerator (4–8 °C) for samples to be analysed immediately and in a freezer (−20 °C to −80 °C) for samples to be analysed after a longer period. Serum samples could also be stored at physiological pH and in the presence of a stabilizer such as ethylene glycol and glycerol (Basten, 2010).

Previous studies (Cuhadar et al., 2012; Kachhawa et al., 2017) have indicated that biochemical analytes (including serum amino transferases) are stable in some samples for a specific period. Cuhadar et al., 2012 reported that, the biochemical analytes in serum remain stable for a longer period than those present in plasma.

Research carried out in this area, have been done mostly on animals (Divya and Jayavardhanan, 2010). Those that have been carried out in humans (Kachhawa et al., 2017), did not analyse the kinetic stability of these analytes (the rate of deterioration) in serum and the factors that affect this deterioration.

The study reported here was designed to investigate the stability (and deterioration) of alanine amino transferase, aspartate amino transferase, gamma glutamyl transferase, alkaline phosphatase and albumin in serum stored at room temperature, 4 °C, -20 °C, -86 °C and in the presence/absence of a stabilizer (glycerol).

1.2) LITERATURE REVIEW

1.2.1 Hepatobiliary Diseases

 Hepatobiliary diseases are a group of diseases involving the liver, gall bladder and biliary tract caused by viral, bacterial, and parasitic infections, neoplasia, toxic chemicals, alcohol consumption, poor nutrition, metabolic disorders, and cardiac failure (Nicholas et al., 2010). There exist eleven different types of hepatobiliary diseases, with several of them overlapping between two or more categories.

1.2.1.1 Infectious Hepatobiliary Diseases

 Infectious hepatobiliary diseases (IHDs): IHDs are hepatobiliary diseases caused by pathogens. They are subdivided into five types namely, viral IHDs, bacterial IHDs, parasitic IHDs, fungal IHDs and IHDs resulting from worms (Dancygier, 2010).

Viral IHDs, as the name indicates are hepatobiliary diseases caused by viruses. These viruses could either be DNA or RNA viruses. A common example of a viral IHD is viral hepatitis which consists of five types, A, B, C, D and E as well as Herpes Simplex virus, Influenza virus. Viral IHDs are the leading cause of hepatobiliary diseases in Sub-Saharan Africa (GBD, 2016).

IHDs are also caused by bacteria such as Escherichia coli, Pseudomonas aeruginosa and salmonella, all of which cause liver abscess, which is characterised by the destruction of liver tissue due to bacterial invasion via the bloodstream (portal vein, hepatic artery), the biliary system or (rarely) by direct penetration (post-traumatic) of bacteria (Dancygier, 2010).

Parasitic hepatobiliary diseases are caused by parasites such as Entamoeba histolytica which causes amebiasis which primarily manifests as amoebic liver abscess. An amoebic liver abscess is a liquefaction of the liver’s parenchymal tissues by the invasive E. histolytica (Dancygier, 2010).

 Infectious hepatobiliary diseases are also caused by worms (trematodes) or helminths such as Schistosoma masoni, Schistosoma japonicum, Schistosoma mekongi, Schistosoma intercalatum (Friedman et al., 2018). These helminths cause schistosomiasis, which is the second most common human parasitic disease after malaria (GBD, 2016). Schistosomiasis manifests as liver fibrosis.

Fungal (infectious) hepatobiliary diseases are caused by fungi such as Candida albicans, which causes candidiasis. Hepatic candiadisis is mostly due to direct fungal invasion with the formation of granulomas and, most importantly, liver abscesses (Mauss et al., 2018).

1.2.1.2 Tumorous (Neoplastic) Hepatobiliary Diseases

Tumorous hepatobiliary diseases are diseases developed through a pathological process that results in the formation and growth of a tumour (neoplasm). Tumorous hepatobiliary diseases are growths formed in the parenchymal and mesenchymal tissues of the liver as well as the biliary tract and the gall bladder (Nicholas et al., 2010). Hepatic neoplasms could be sarcomas, carcinomas, lymphomas as well as benign tumours. An example of a tumorous hepatobiliary disease is hepatocellular carcinoma.