http://www.vivo.colostate.edu/hbooks/pathphys/digestion/liver/histo_lobule.html

http://en.wikipedia.org/wiki/Lobules_of_liver

• The basic structural unit of the liver is the lobule hexagonal arrangement of plates of hepatocytes radiating outward from a central vein (CV) in the centre. Central vein are quite prominent and makes orientation in understanding liver histology easy for beginners. The term “hepatic lobule”, without qualification, typically refers to the classical lobule or anatomical lobule. It is hexagonal; divided into concentric centrilobular, midzonal, periportal parts

• Normal hepatocytes are arranged in narrow plates, which are usually no more than 2 cells thick, with intervening sinusoids radiating out from central vein to periphery

• At the vertices of the lobule are regularly distributed portal triads (also known as portal tracts) – containing an artery, vein and bile duct. Due to plane of section, one can often observe more than one of each of these structures in a given portal tract or absence of one or more structures.

• On the other hand from a metabolic perspective, the functional unit is the hepatic acinus elliptical or diamond-shaped; divided into zone I (periportal), zone II (transition zone), and zone III (centrilobular). each of which is centred on the line connecting two portal triads and extends outwards to the two adjacent central veins. The periportal zone I is nearest to the entering vascular supply and receives the most oxygenated blood. Conversely, the centrilobular zone III has the poorest oxygenation and is relatively more sensitive to ischemic injury.

• Delicate bands of collagen invest hepatocytes– reticulin stains highlight the 2 cell thick trabeculae.

• Starting point is an adequate biopsy  characterised by 20-25mm in length, 1.4mm in diameter, and contains at least 11 Portal tracts

• Attempt an  initial assessment ‘blind’ to the clinical information and then review in light of clinical information which should include the following depending on the circumstances- BMI, alcohol intake, hepatitis screen results, US findings and clinical presentation and any particular questions from the treating clinicians. Special informations like vascultitic screen (hepatic vasculitis), presence of lymph nodes  or organomegaly ( lymphoma or malignancies) are needed in special situations. Remember better the information , better the interpretation

• Special stains:

1.     Reticulin – stains Type III collagen fibres- to assess architecture

2. Trichrome/ Haematoxylin Van Gieson (HVG)- stains Type I collagen fibres-to assess fibrosis

3.     Orcein – stains elastic fibres- to assess HBsAg, copper associated protein and elastic fibres- found in long standing fibrosis

4.     PAS p to identify glycogen (also useful to look for small foci of necrosis or granulomas)

5.     d-PAS (PAS diastase) to identify ceroid pigment within kupffer cells, mucin in bile ducts, some hepatic neoplasms and ?1antitrypsin granules

6.     Perl’s stain to assess iron

7.     Immunohistochemical stains (where appropriate)

§  Viral antigen markers (HBsAg, HBcAg, HDV, CMV, EBV)

§  Hepatocyte inclusions (ubiquitin for MH, ?1antitrypsin)

§  Biliary cks (CK7, 19, AE1/AE3) to assess bile duct loss and ductular reaction in and around pts

§  Liver tumour markers (PCEA, CD10, heppar-1, ?fp etc) to distinguish between primary and metastatic neoplasms

§  Proliferation marker Ki67 and CD34 used to distinguish between regenerative, dysplastic and neoplastic nodules

• The order of analysis of a liver biopsy:

1.     Liver Architecture: connective tissue stains are required for accurate assessment of liver architecture

• Are the normal vascular relationships maintained?
• Is there any evidence of fibrosis/cirrhosis?
• Any subtle architectural abnormalities eg. Atrophy, NRH

2. Portal Tracts And Periportal Regions

• Bile ducts – present in normal numbers, any bile duct lesions (eg. Granulomas in pbc, fibrosing cholangitis in psc)?
• Hepatic arteries – look for inflammation (eg. Pan, drug reactions), amyloid
• Portal veins – look for pv lesions (eg. Obliteration and/or dilatation in non-cirrhotic portal ht, hepatoportal sclerosis, inflammation in liver allograft rejection
• Then assess for other abnormal features:

§  Inflammatory cells (type, density, distribution)

§  Interface hepatitis

§  Ductular reaction (common reaction to many forms of liver injury, especially those with cholestasis)

3. Liver Parenchyma

• Hepatocytes – degenerative changes (eg. steatosis, ballooning, bilirubinostasis), liver cell death (apoptosis or necrosis), and severity thereof (spotty, confluent, bridging, panacinar, multiacinar), nuclear or cytoplasmic inclusions (Mallory hyaline, eosinophilic globules) and whether these are zonal (eg. zone 1 MH in cholate stasis, and zone 3 MH in ALD)
• Sinusoidal cells – Kupffer cell inclusions may be seen in some metabolic storage diseases and parasitic infections, Kupffer cell enlargement with ceroid pigment accumulation is non-specific reaction to previous hepatocellular injury
• Then look for other abnormal features:

§  Inflammatory cells (type, density and distribution)

§  Sinusoidal dilatation, sickle cells, intravascular malignancy

§  Deposits eg. Amyloid

§  Note the presence/absence of liver cell dysplasia or neoplasia

4. Hepatic Veins

• Is there evidence of endothelial inflammation?
• Are veno-occlusive lesions present?
• Is there inflammation within the vein wall?
• Are there any lesions in the tissue surrounding the hepatic veins (inflammation, necrosis, fibrosis)?

• Try to  identify the main patterns of injury/damage present. eg. steatosis, steatohepatitis, acute hepatitis, chronic hepatitis, chronic biliary disease with bile duct loss
• Beware of the possibility of sampling error due to patchy distribution of changes
• In some cases , might need to use of semi-quantitative scoring systems eg Ishak’s score , Metavir score etc.
• Electron microscopy might need to be considered  in some cases of metabolic disease (glycogen storage, Niemann-Pick), certain viral infection (paramyxovirus in giant cell hepatitis)
• Non-histological tests eg. biochemical measurements of liver iron or copper, detection of low levels of viral RNA or DNA by PCR is also sometime needed

2.     Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC)

• Classically middle-aged female , anti-mitochondrial Ab (AMA) in 95% and may be associated with may be associated with Sjögrens, progressive systemic sclerosis, coeliac disease etc
• PBC is characterised histologically by progressive destruction of intrahepatic (interlobular) bile ducts by lympho-plasma cellular infiltrate and granulomatous inflammation (~30%).
• Sequence of events are- portal inflammation (by T lymphocytes, plasma cells, histiocytes, neutrophils and eosinophils) leading to interface hepatitis. Bile stasis causes ductular reaction (cytoplasmic eosinophilia, cell loss, nuclear pseudo-stratification, lymphocyte infiltration) and bile duct damage causing ductopenia- ultimately leading to periportal fibrosis with P-P linkage with preservation of normal vascular relationships.
• Epithelioid granulomas (usually without giant cells) centred on damaged bile ducts is nearly always due to primary biliary cirrhosis (Fig. 8a to 8e). Sarcoidosis granulomas for comparison are-better-formed, more numerous and randomly distributed .
• Biliary pattern of cirrhosis results in a ‘jigsaw puzzle’ arrangement of nodules and peripheral pallor around nodules due to cholate stasis.
• Remember there are other diseases of bile ducts associated with vanishing bile duct syndrome in developmental anomaly ( extrahepatic biliary atresia,?1antitrypsin deficiency), Immune mediated diseases (autoimmune sclerosing cholangitis, sarcoidosis, liver allograft rejection, GVHD) , Vascular causes (ischaemic cholangitis, portal vein thrombosis), Infective causes ( cryptosporidia, CMV, recurrent pyogenic cholangitis) , Neoplastic (Hodgkin’s lymphoma)

• PSC differs from PBC in that it affects not just small interlobular bile ducts, but also medium (septal), large (hilar) and extrahepatic bile ducts (but this distinction is not usually made on liver biopsies)
• The patients in PSC show a lymphocytic infiltrate associated with atrophy of bile duct epithelium, luminal obliteration and concentric ‘onion-skin’ fibrosis (Fig. 9a to 9e). Similar lesions can occur in 2° causes of sclerosing cholangitis
• Cholestasis is marked in both PSC and PBC , reflected in the deposition of copper binding protein (Orcein positive) (Fig. 9g and 9h).

3.     Liver infiltrates and deposits

• Steatosis refers to the accumulation of lipid within hepatocytes and is typically described as macrovesicular (single large cytoplasmic vacuole filling the cell, Fig. 6a) or microvesicular (multiple small vacuoles imparting a bubbly appearance to the cytoplasm of hepatocytes).
• Iron

1.     Iron is  one of the more common deposits seen in the liver (after fat) and occurs in the setting of hereditary haemochromatosis and acquired forms of siderosis (eg. Transfusion-related).

2.     The iron is deposited as haemosiderin, which has a fine, golden-yellow, refractile appearance on H&E and is not to be confused with lipofuscin (which is deposited in ‘aging cells’) Fig. 5a, 5b and 5c.

3.     The amount of haemosiderin deposition can be graded (1-4), depending on how much of the lobule is involved – initially the iron is deposited within the central portions of the lobule (in haemochromatosis) and as this progresses it is deposited across the entire lobule (a Perls histochemical stain confirms the presence of iron which is stained blue) (grade 4, Fig. 5d).

• Amyloid is an abnormal protein which may be deposited in the liver as part of systemic amyloidosis (Fig. 6b and 6c) and typically has an amorphous (‘structure-less’), eosinophilic appearance, confirmed by performing Congo red or Thioflavin-T histochemical stains (‘apple-green’ birefringence under polarised light noted with the former).
• ?-1 antitrypsin deficiency results in the accumulation of AAT globules in hepatocytes, which have a pink colour with the diastase-Periodic acid Schiff stain and can be highlighted with the aid of a specific immunohistochemical marker (Fig. 10a to 10c).

4.     Alcoholic and non-alcoholic fatty liver disease

• fatty liver disease (whether alcoholic or non-alcoholic) comprises a spectrum of changes ranging from simple steatosis (fatty change) to steatohepatitis and fibrosis, to cirrhosis.
• mainly macrovesicular steatosis is seen (Fig. 6a), but minor microvesicular change may be seen (Fig. 11a).
• the inflammatory infiltrate consists of neutrophil polymorphs and hepatocyte damage is manifested by ballooning of hepatocytes and deposition of Mallory’s hyaline (Fig. 11b).
• with continued hepatocyte damage, increasing amounts of pericellular and portal fibrosis are seen (Fig. 11c and 11d) eventually culminating in micronodular cirrhosis.

NAFLD

•          Microvesicular steatosis

•          Macrovesicular steatosis

•          Mallory bodies- eosinophilic accumulations of intracellular material

•          Ballooning degeneration

•          Perisinusoidal zone 3 fibrosis

•          Scattered, predominantly lobular, neutrophilic or mixed inflammation

•          In children, portal inflammation may be more prominent than in adults

5.     Metastases to the liver

• The liver is a common site of metastatic disease, the commonest of which is metastatic adenocarcinoma from a variety of sites, but in particular the gastrointestinal tract and pancreas.
• Metastatic adenocarcinoma varies from well-differentiated forms demonstrating well-formed glandular structures, to poorly differentiated tumours showing little morphological evidence of their glandular origin (fig. 4a and 4b).
• Metastatic small cell carcinoma has a distinctive appearance (but may be confused with lymphoma and other ‘small round blue cell tumours’) , fig. 4c and 4d.
• Immunohistochemistry is usually employed to confirm the site of origin of metastatic tumour, but may not necessarily be diagnostic (final diagnosis requiring correlation with clinical and/or radiological findings).

6.     Cirrhosis

• With early cirrhosis, increased amounts of collagen are noted within, and later, between portal tracts (Fig. 2a shows a vaguely nodular outline, which is better seen on the HVG and reticulin stains – the fibrous septa do not completely link the portal tracts, therefore established cirrhosis cannot be confirmed histologically).
• Fig. 2f reveals linking fibrosis and a micronodular architecture, indicating established cirrhosis is present (these biopsies are often fragmented).
• Focal nodular hyperplasia is not to be confused with cirrhosis (usually easily distinguished due to the focal nature of the architectural abnormality on imaging and the lack of fibrosis)

7.     FNH (Focal nodular hyperplasia)

• Benign, non-neoplastic hepatocellular lesion usually seen in young subjects (30-50 yrs)
• Usually presents with pain or as incidental finding, very low risk of haemorrhage
• Solitary (multiple in 20-30%) in a background of normal liver
• Related to ?change in blood flow around arterial malformation with resulting hyperplastic growth response
• Macroscopically – nodular appearance, often near capsule ,unencapsulated but well-demarcated because of nodularity, small (usually <5cm), but varies and may involve entire lobe and normally there is a central fibrous scar
• Microscopically -normal-appearing hepatocytes arranged in incomplete nodules partially separated by fibrous tissue, intact reticulin framework (similar to normal liver) .key features include  variable numbers of bile ductular structures within the fibrous stroma at the edge of the nodules and  presence of medium to large, thick-walled muscular vessels .normal pts are not found in the lesion

8.     NRH (Nodular regenerative hyperplasia)

• Innumerable nodules throughout the liver (sometime focal) varying from 1mm to 1cm in size, hyperaemic rim around larger nodules, nodules of uniform, bland hepatocytes without fibrous septa (distinguishes from cirrhosis),
• associated with PV obstruction, polycythaemia vera, rheumatoid arthritis, Budd-Chiari, lymphoma and myeloproliferative disorders etc
• often mistaken macroscopically for cirrhosis or metastatic tumour

9.     HCC (Hepatocelluar carcinoma)

• The deranged liver architecture of hepatocellular carcinoma (HCC) is evident in Fig. 3a – solid sheets of atypical hepatocytes with occasional acinar (ring-like) structures are seen (the presence of bile within these acinar structures is a helpful clue to the presence of HCC, Fig. 3b).
• Immunohistochemical staining with monoclonal CEA is another helpful indicator of HCC, highlighting the canalicular architecture of these tumours.
• The fibrolamellar variant of hepatocellular carcinoma is characterised by increased amounts of collagen which is arranged in parallel bands around and between malignant cells showing focal bile production and occasional eosinophilic cytoplasmic bodies (Fig. 3f and 3g).

10.  Acute hepatitis

• Acute hepatitis = <6 months and chronic hepatitis= >6 months
• distinguishing between the two may be difficult, but useful differentiating features include: Acute hepatitis – the pattern of inflammation is usually mixed portal and parenchymal whereas in chronic (Fig. 7a -7d) it is usually portal/periportal. Cholestatic features are common in acute and rare in chronic (except in endstage disease). Fibrosis is mild and reversible in acute and more prominent in chronic hepatitis.
• liver biopsy is performed in setting of hepatitis when the clinical presentation is atypical or the cause of the acute hepatitis is uncertain from the clinical, biochemical or serological findings. The three questions facing the pathologists are – Is this acute or chronic damage? How severe is the damage? What is the aetiology?

MICROSCOPICALLY

è  Inflammatory infiltrates within the liver are described according to the location of the infiltrate (portal or lobular) and the nature of the inflammatory cells

è  Inflammatory infiltrate à mainly lymphocytes (t more than b), but plasma cells may be seen in aih, neutrophils in alcoholic hepatitis and eosinophils in drug reactions (in chronic hepatitis, the inflammation is mainly mononuclear and there may be lymphoid aggregates eg. HCV, AIH, PBC ± interface hepatitis)

è  Exception = acute hav infection with plasma cell-rich portal/periportal infiltrate mimicking aih

è  Hepatocellular damage often most marked in the perivenular regions -is indicated by ballooning, bile pigment accumulation (bilirubinostasis), lobular disarray and cell death (either necrosis or apoptosis)

è  Bilirubinostasis is rarely a feature of chronic hepatitis except in decompensated end stage disease

è  Different degrees of severity of cell death may be identified:

a.     Spotty necrosis à apoptosis of individual hepatocytes resulting in  formation of intensely eosinophilic ‘councilman’ bodies which represent necrotic hepatocytes (fig. 7d).

b.    Confluent necrosis (zone 3) à loss of groups of adjacent liver cells

c.     Bridging necrosis à confluent necrosis linking vascular structures (c-c or c-p bridging)

d.    Panacinar necrosis à loss of hepatocytes within an entire acinus

e.     Multiacinar necrosis à panacinar necrosis involving several adjacent acini

è  Ductular reaction is common (associated with severity of cholestasis) and neutrophils may be present (‘cholangiolitis’) à ductular reaction is less common in chronic hepatitis and is associated with the severity of the fibrosis

è  Fibrosis (if present) is mild and may be reversible

a) Clues to aetiology:

1.     Viral à A, B, C, D, E and other (CMV, EBV, HSV)

2.     Drugs

3.     Autoimmune hepatitis (AIH)

4.     Idiopathic (‘seronegative hepatitis)

a.     Viral

i.    HAV infection à perivenular cholestasis with little inflammation, plasma cell-rich portal/periportal inflammation

ii.    Certain viral infections are associated with characteristic (but non-specific) patterns of inflammation (eg. formation of lymphoid nodules with steatosis is suggestive of Hepatitis C virus infection, Fig.7a) and with certain cytopathic effects (eg. ground glass cytoplasmic inclusions (Orcein positive) in Hepatitis B virus infection, Fig. 7e and 7f).

iii.    HEV infection suggested by à prominent cholestasis, microvesicular steatosis and prominent cholangiolitis

iv.    EBV and CMV may be associated with erythrophagocytosis by sinusoidal KCs and portal histiocytes (mistaken occasionally for sinus histiocytosis with massive lymphadenopathy or for malignant histiocytic neoplasm), EBV also  associated with atypical lymphocytes in sinusoids/PTs and occasionally epithelioid granulomas, CMV, HSV, VZV and adenovirus infections in the immunocompromised host are associated with typical nuclear inclusions

b.    Drugs

Paracetamol toxicity à discrete, centrilobular coagulative necrosis with little inflammation

c.     Autoimmune hepatitis (AIH)

1.     Plasma cell-rich portal infiltrate (sometimes parenchymal) with prominent interface hepatitis (need to exclude HAV)

2.     Centrilobular ballooning and hepatocyte necrosis, often with bridging necrosis

3.     Rosetting, fibrous entrapment of periportal hepatocytes and giant cell transformation of hepatocytes is sometime seen

4.     Differential includes AMA negative PBC, but in AIH the inflammation is more uniform and intense, shows lobular activity and lacks the typical bile duct damage

11.  Chronic hepatitis:

1.     The common differential diagnoses of chronic hepatitis include  chronic viral hepatitis B, C or D, autoimmune hepatitis (AIH), steatohepatitis, PBC, PSC, ?₁antitrypsin deficiency and Wilson disease and drug reaction.

2.     The inflammatory infiltrate is mainly mononuclear (± lymphoid follicles) and is predominantly portal/periportal ± interface hepatitis, much less lobular inflammation is seen (Fig. 7a -7d).

3.     Ductular reactions are less common and fibrosis to some degree is usually seen (the most reliable criteria for diagnosing chronic hepatitis)

4.     Role of liver Bx in chronic hepatitis:

a)     Establish histological diagnosis

b)    Identify or confirm aetiology

c)     Assess necro-inflammatory activity (grade) and extent of fibrosis (stage)

d)    Identify additional lesions

a)     Hepatitis B (HBV) infection

• HBV and HCV infections are diagnosed serologically, but the liver is Bx for prognostic and/or therapeutic reasons
• A characteristic and diagnostic feature of chronic HBV infection is the expression of HBsAg or HBcAg in hepatocytes
• Cytoplasmic HBsAg expression corresponds to ground-glass hepatocytes seen on H&E (Orcein+ inclusions) Fig. 7e and 7f
• HBcAg expression may be cytoplasmic, membranous or nuclear and infers active replication of virus

b) Hepatitis C (HCV) infection

• METAVIR and modified Ishak staging systems are the most widely used, but suffer problems with inter-observer variation and sampling variation. Agreement is generally good for fibrosis, but less so for inflammation. Histological concordance (ie. Sampling) is good for inflammation but less so for fibrosis (the opposite to above)

• Metabolic and HCV-related (genotype 3) pathways lead to steatosis and the severity of the steatosis affects the response to antiviral Rx

• Minor degrees of siderosis are common in HCV infection (hepatocytes and kcs) but more severe siderosis in an hepatocellular distribution raises the possibility of genetic haemochromatosis. Siderosis also has an adverse effect on antiviral Rx

• Microscopically the biopsy typically shows

a)     Mononuclear portal inflammation

b)    Lymphoid aggregates are typically prominent, but may also be seen in AIH, PBC (Fig 7a)

c)     Interface hepatitis is usually mild

d)    Lobular inflammation is often mild and confluent necrosis is not seen with HCV alone, except in the setting of immunocompromised patients (eg. Post-Tx) or if coexistent AIH present

e)     Fatty change is another feature, but is usually mild and mainly macrovesicular

Metavir score:

The fibrosis is graded on a 5-point scale from 0 to 4.

Fibrosis score:
F0 = no fibrosis
F1 = portal fibrosis without septa
F2 = portal fibrosis with few septa
F3 = numerous septa without cirrhosis
F4 = cirrhosis

The activity, which is the amount of inflammation (specifically, the intensity of necro-inflammatory lesions), is graded on a 4-point scale from A0 to A3.

Activity score:
A0 = no activity
A1 = mild activity
A2 = moderate activity
A3 = severe activity

Knodell Scores

The Knodell scoring system, also called the Histologic Activity Index (HAI), classifies liver biopsy specimens according to scores into four categories of histologic features:

• Periportal and/or bridging necrosis (scores from 0 to 10)

• Intralobular degeneration and focal necrosis (scores from 0 to 4)

• Portal inflammation (scores from 0 to 4)

• Fibrosis (scores from 0 to 4)

The Knodell Necroinflammatory Score is the sum of scores from parts I-III, hence a range of 0 to 18, and measures the degree of acute necroinflammatory activity in the liver.

The Knodell Fibrosis Score (part IV, above) measures the degree of scarring in the liver. Scarring builds up over time due to chronic necroinflammatory activity, ultimately leading to cirrhosis.

Recently, the Ishak fibrosis score has become the preferred method for evaluating liver fibrosis because it rates fibrosis according to seven categories on a continuous integer scale, as opposed to the discontinuous 4-point Knodell fibrosis score

Ishak’s Fibrosis Score

A scoring system that measures the degree of fibrosis (scarring) of the liver, which is caused by chronic necroinflammation.

A score of 0 represents no fibrosis,

Score of 1 and 2 indicate degrees of portal fibrosis;

Score  3 and 4 indicate bridging fibrosis.

A score of 5 indicates nodular formation and incomplete cirrhosis.

Score of  6 is established cirrhosis.

• Azathioprine (AZA) is a pro drug which undergoes conversion to 6- mercaptopurine (6-MP).  The conversion is done non-enzymatically by glutathione present in red blood cells and other tissues.
• 6-MP is then metabolized in the liver and gut by one of three enzymes; 6-thiopurine methyl transferase (TPMT), Xanthine oxidase, and Hypoxanthine-guanine-phosphoribosyl transferase (HGPRT).
• HGPRT converts 6-MP to active metabolite 6-thioguanine (6-TG) nucleotides whereas the other two enzymes metabolises 6-MP to inactive metabolites. Deficiency of TPMT may lead to increased active metabolite i.e. 6-TG and thus increased risk of toxicity
• Toxicity of AZA or 6-MP is largely related to the activity of TPMT. Deficiency of TPMT leads to preferential metabolisation of 6-MP and AZA to thioguanine nucleotides responsible for much of the drug toxicity.
• Low TPMT activity has been observed in up to 10 percent (heterozygous) of the population, with 0.3 percent (homozygous) having negligible activity.
• Either TPMT genotype or TPMT enzyme activity can be measured. TPMT enzyme activity is often measured in clinical practice in UK
• Low TPMT enzyme activity may lead to increased risk of myelosuppression.  However, the majority of patients who develop myelosuppression while taking AZA do not have detectable TPMT gene mutations. Thus a normal TPMT screening test does not preclude bone marrow and/or liver toxicity.  Thus, even when TPMT testing is performed, regular FBC and liver function tests must still be obtained. Experts vary in their use of TPMT.
• However, in current clinical practice TPMT levels are often measured before initiating treatment. Those with absent TPMT enzyme activity should not receive AZA or 6-MP. Patients with normal TPMT enzyme activity can be treated either by beginning with a low dose and increasing incrementally to the target dose or by beginning with the target dose at the outset.
• In non responding patients 6-TG levels may be obtained to check compliance.

5-10ml of whole blood (EDTA) is needed for the test

Precaution

• Recent blood transfusions will confuse the enzyme phenotype.
• TPMT is inducible so the analysis should be done before commencing therapy with thiopurines

http://acbroi.org.uk/science/documents/tumourmarkerbooklet3rdedn2005_001.pdf (archived copy at WebCite)

An excellent and concise reference resource.

• Patients with symptoms clinically suggestive of GORD, who fail to respond during a high dose therapeutic trial of a PPI
• Patients with symptoms clinically suggestive of GORD without oesophagitis or with an unsatisfactory response to a high dose PPI in whom anti-reflux surgery is contemplated
• Patients with persistent GORD symptoms despite anti-reflux surgery

Discuss whether PPI treatment needs to be stopped prior to pH study?

This depends on the purpose of pH study. So PPI should be continued if the pH study is being done to analyse correlation between symptoms and acid exposure. However, if the purpose is to exclude excess acid exposure, PPI should be stopped.
Mostly pH studies are done without stopping PPI.

Discuss the technical details of the test?

• The basic equipment requirements for ambulatory oesophageal pH studies are a portable data logger for data storage, a pH electrode and software (with computer) for analysis of pH data.
• The pH electrode is passed through a nostril and positioned 5 cm above the superior margin of the lower oesophageal sphincter (LOS). The LOS is identified by manometry. Alternative methods of pH electrode placement like pH step-up, fluoroscopy, endoscopic measurements and body height formulas are inferior to manometry.
• The rationale behind the 5 cm spacing is to avoid possible electrode displacement into the stomach, especially during swallow-induced oesophageal shortening.
• Once the pH electrode is placed and connected to the data logger, the patient carries on his usual day to day activity. He returns the data logger the next day.
• Data loggers sample intra oesophageal pH eight times every minute.  It also has an event marker that can be activated by the patient during the study to indicate the timing of symptoms.
• Thus, the raw data obtained from an ambulatory pH study are the number of reflux events, the oesophageal acid exposure time associated with each event, and the timing and nature of symptoms.

What are the pitfalls of pH study?

• pH recordings in asymptomatic subjects reveal that acid reflux is a normal, physiological occurrence.
• Up to a quarter of patients with oesophagitis had a normal pH study, emphasising that oesophageal pH monitoring is associated with a significant false negative rate.
• Thus, pH monitoring has clear limitations in defining pathological acid reflux. However, it is the only investigation that can provide information on whether patients’ symptoms are related to episodes of acid reflux (symptom index)
• In the absence of oesophagitis, there is no gold standard for the definition of GERD, making it impossible to establish the accuracy of any diagnostic test.

Discuss the analysis of pH study?

• A fall below pH 4 in oesophageal pH has been conventionally taken to indicate acid reflux.
• 24 hours pH study generates 14,400 data points besides event marker data.
  A number of variables have been described that have value in discriminating patients with acid reflux symptoms from asymptomatic controls: percentage total time oesophageal pH<4; percentage time upright oesophageal pH<4, percentage time supine oesophageal pH<4; number of episodes oesophageal pH<4; number of episodes oesophageal pH<4 for more than 5 minutes; and the longest single episode oesophageal pH<4.  A composite score like Demeester score was developed to express them. However, the composite score has no advantage over the simpler percentage total time oesophageal pH<4 in discriminating patients with GORD from asymptomatic controls.
• Physiological acid reflux is defined as (from the largest published series): percentage total time oesophageal pH<4 <5%; percentage upright time oesophageal pH<4 <8%; percentage supine time oesophageal pH<4 <3%; number of episodes pH<4 for >5 minutes <3.
• The symptom index is the number of symptom episodes associated with acid reflux as a percentage of the total number of symptom episodes. However, few symptoms and frequent acid reflux episodes may lead to a positive symptom index due to a chance association.
• The symptom sensitivity index was developed in an attempt to account for the limitations of the symptom index. It is defined as the number of acid reflux episodes associated with symptoms as a percentage of the total number of acid reflux episodes.  A positive symptom sensitivity index was arbitrarily defined as at least 10%.

Discuss wireless pH study?

• A catheterless pH monitoring system comprises a plastic capsule that houses a pH sensor and a transmitter. The capsule is fixed on the oesophageal wall at endoscopy. The capsule continuously monitors oesophageal pH and transmits the data every few seconds to a small receiver worn by the patient. After a few days, the capsule detaches from the oesophageal wall and passes through the digestive tract.
• Catheterless oesophageal pH monitoring is suitable for patients who do not tolerate nasal intubation.
• The wireless system has the potential advantage of extended recording up to 48 hours but is also considerably more expensive than catheter-based monitoring.
• Catherless pH system is NICE approved in UK (http://www.nice.org.uk/nicemedia/pdf/IPG%20187%20A4v2.pdf)

Discuss impedance monitoring?

This is a new technique designed to detect intraluminal bolus movement. When combined with pH it allows for detection of gastroesophageal reflux independent of pH (i.e., both acid and non-acid reflux).  Clinical value of it is uncertain.

Oesophageal manometry

Discuss the indications for oesophageal manometry?

• To diagnose suspected primary oesophageal motility disorders (e.g. achalasia and diffuse oesophageal spasm)
• To diagnose suspected secondary oesophageal motility disorders occurring in association with systemic diseases (e.g. systemic sclerosis)
• To guide the accurate placement of pH electrodes for ambulatory pH monitoring studies
• As part of the pre-operative assessment of some patients undergoing anti-reflux procedures (patients with atypical symptoms)
• To reassess oesophageal function in patients who have been treated for a primary oesophageal disorder (eg. sub-optimal clinical response to pneumatic balloon dilatation) or undergone anti-reflux surgery (e.g. dysphagia following fundoplication)

Discuss the technical details of manometry?

• The basic hardware required for manometry comprises a pressure sensing apparatus that detects changes in luminal pressure and converts this to an electrical signal, and a recording device that amplifies and stores this information for subsequent analysis.
• The position of the catheter is verified by asking the patient to take a deep breath. Intra-abdominal pressure readings go up with inspiration and down on expiration. Conversely, pressure readings taken within the thoracic cavity go down on inspiration and up on expiration.
• The station pull-through technique allows identification of the location and length of the sphincter high pressure zone (HPZ). The lower oesophageal sphincter (LOS) resting pressure (pressure of HPZ minus intra-gastric pressure) is then estimated.
• A series of wet swallows (5mL of water) are used to examine LOS relaxation.
• Oesophageal body motility is subsequently assessed using a further series of wet swallows

Discuss the contraindications to oesophageal manometry and pH monitoring?

• Suspected or confirmed pharyngeal or upper oesophageal obstruction
• severe coagulopathy (but not anticoagulation within the therapeutic range)
• Bullous disorders of the oesophageal mucosa
• Cardiac conditions in which vagal stimulation is poorly tolerated.

Patients with peptic strictures, oesophageal ulcers, oesophageal or junctional tumours, varices or large diverticulae are at increased risk of complications from blind oesophageal intubation and such conditions are a relative contra-indication to performing manometry and pH monitoring. In such cases, endoscopic or radiologic guidance may be considered.

Ref

1. British Society of Gastroenterology: Guidelines for oesophageal manometry and pH monitoring.

Faecal elastase (FE)

• FE is increasingly being used as a non-invasive first line test to diagnose exocrine pancreatic insufficiency. It is essentially the only test for exocrine pancreatic insufficiency available in most centres
• It is comparable to pancreatic function tests such as the pancreolauryl test and the gold standard secretin test and has higher sensitivity and specificity for pancreatic insufficiency than other pancreatic enzymes such as Faecal Chymotrypsin.
• Faecal Elastase is a proteolytic enzyme secreted by the acinar cells of the pancreas. Unlike other pancreatic enzymes such as Chymotyrpsin, Elastase-1 is not degraded during intestinal transit, and so the stool concentration reflects exocrine pancreatic function.
• Digestive enzyme substitution therapy has no influence on the determination of faecal elastase. The monoclonal antibodies used in the test do not cross-react with elastases of animal origin, which are contained in enzyme substitution preparations.
• Values above 200 µg elastase/g stool indicate normal exocrine pancreatic function.
• Values below 200 µg elastase/g stool indicate exocrine pancreatic insufficiency.
• A single spot stool sample is sufficient.
• Pancreatic elastase is detected using ELISA with two monoclonal antibodies highly specific for human pancreatic elastase 1.

Ref- http://www.schebo.co.uk (archived copy at WebCite)

Secretin stimulation test (Gold standard)

• The secretin stimulation test is the most sensitive and specific testing available for the diagnosis of chronic pancreatitis. However it is not available widely.
• Secretin is a hormone secreted by the small intestine. Secretin stimulates the pancreas to release bicarbonate to neutralize gastric acid and aids in digestion. The secretin stimulation test measures the ability of the pancreas to respond to secretin.
• A collection tube is placed in the 3rd part of duodenum under fluoroscopic guidance. After a test dose (0.2 mcg) of synthetic secretin, a full dose (0.2 mcg/kg) is administered as an intravenous bolus at time 0.
• Duodenal fluid is continuously collected in 15 minute aliquots for one hour. A bicarbonate concentration less than 80 mEq/L in all of the four aliquots represents exocrine insufficiency.

Pancreolauryl test

• A tablet containing fluorescein dilaurate is taken on day 1 and urine is collected for 10 hours. On day two, a tablet containing fluorescein alone is given and urine collected again for 10 hours. This allows for correction in individual variations in intestinal, hepatic and renal function.
• Fluorescein dilaurate is hydrolysed by cholesterol ester hydrolase, an enzyme normally present in pancreatic juice. Fluorescein is absorbed by the intestine, conjugated in the liver, and excreted in the urine where its fluorescence can be measured.
• Results are expressed as the ratio of fluorescein excreted after fluorescein dilaurate and after free fluorescein. A ratio of less than 20% is considered abnormal.
• Sensitivity for detecting severe pancreatic insufficiency is at least 85%.
• Pancreolauryl test is no longer available in UK. It was withdrawn by Pfizer in 2005

Fat malabsorption

• Stool tests
  • 72 hour stool collection for fat estimation- this was the standard test for malabsorption for decades. However, it is poorly reproducible and unpleasant and thus its use is discouraged. Normal fat excretion is < 6gm/day. Steatorrhea is present, if fat excretion is more than 14 g/day. More than 6gm/day fat is pathologic; however fecal fat excretion can be increased up to 14g/day in diarrheal diseases even without fat malabsorption
  • Single stool analyses- such as faecal fat concentration (g faecal fat/100 g wet stool weight) and semi-quantitative methods such as acid steatocrit or Sudan III staining correlate moderately well with three day faecal fat quantification and offer an alternative method of assessing fat malabsorption but are not readily available in most centres. The stool steatocrit involves separating a faecal homogenate by centrifugation into a lipid, water, and solid phase. Faecal acidification much improves this method.
  • Faecal elastase- is the test most often used. It helps in differentiating between pancreatic and intestinal causes of steatorrhoea. See details on pancreatic function tests.
• Breath test
  • 14 C- triolein breath test- measures 14CO2 in breath after ingestion of a triglyceride that has been labeled with 14C. Fat malabsorption results in decreased pulmonary excretion of 14CO2. It has been reported to have 100% sensitivity and 96% specificity however limitations such as the confounding effects of co-morbidities altering fat metabolism or respiratory function have also been documented. The test assesses both lipolysis and absorption. The test is inappropriate in patients with diabetes, liver disease, or obesity.

Carbohydrate (CH) malabsorption

• D-xylose test- Limited clinical value today. The patient ingests a 25 g dose of D-xylose, and urine is collected for the next five hours. A venous blood sample is also collected after one hour. Normal excretion of D-xylose is 6.0 +/- 1.5 g. Excretion of lesser amounts of D-xylose or a serum D-xylose concentration less than 20 mg/dL suggests abnormal absorption.
  An abnormal D-xylose test suggests mucosal disease. Absorption is usually normal in pancreatic insufficiency. Renal failure and impaired gastric emptying can lead to false positive results.
• Hydrogen breath (lactose tolerance) tests- H2 is produced by bacterial action on unabsorbed CH in the colon. Thus in CH malabsorption, a breath H2 peak will occur 90 minutes after its ingestion when it first arrives in the colon and is broken down by the bacteria. False positive results are possible particularly with small bowel bacterial overgrowth. It can be used to diagnose specific forms of carbohydrate malabsorption (e.g., lactose, fructose, sucrose isomaltase and others).
  Concurrent antibiotic administration will alter the results of H2 breadth test as it relies on bacterial fermentation of carbohydrate.
• Stool pH less than 5.5 is a qualitative indicator of CH malabsorption. (SCFA and lactic acids are the products of CH metabolism)

Protein malabsorption

• Not generally tested. The tests are technically difficult and intestinal protein loss is generally due to either bacterial overgrowth or protein losing gastroenteropathy. Enteral protein loss is demonstrable by measurement of the alpha-1 antitrypsin clearance. In massive enteral protein loss, the exact site of protein leakage may be localized by the infusion of 99mTc-albumin and gamma camera scintigraphy.
• Alpha-I antitrypsin (AIAT) is excreted intact in stool because it is resistant to degradation in the gut lumen. Further it is not secreted or absorbed. Faecal excretion of AIAT is thus used as an indirect measure of enteric albumin loss.
  Limitations:
  • There is poor correlation between random stool concentrations of AIAT and clearance measurements. A 24 hour stool specimen is thus needed.
  • Diarrhoea can increase AIAT clearance. A1AT clearance greater than 24ml/day (4.8mg/g of stool) in patients without diarrhoea and greater than 56ml/day (11.2mg/g of stool) in pts with diarrhoea is considered abnormal.
  • There is an inverse correlation between AIAT plasma clearance and the serum albumin concentration; as serum albumin levels fall below 3 g/dl, the clearance of AIAT exceeds 180 ml/day.
  • AIAT is degraded at a gastric pH below 3 and thus cannot be used to measure gastric protein loss. PPI can be used to avoid this.
  • Intestinal bleeding can also significantly increase the clearance rates. Thus positive OBT make the reading unreliable.
• Technetium 99m-labelled human serum albumin (Tc HSA) scintigraphy is a less sensitive test than AIAT to detect and monitor enteric protein loss. However, it can localise the specific site of gastric or enteric protein loss. It can also be used like AIAT to monitor response to therapy.

Bile Acid Malabsorption
This can cause symptoms of chronic diarrhoea. Three types of bile acid malabsorption are recognised:
Type 1: following ileal disease or resection or bypass surgery.
Type 2: primary idiopathic malabsorption.
Type 3: associated with cholecystectomy, peptic ulcer surgery, chronic pancreatitis, coeliac disease and diabetes mellitus.
Bile acids are cathartic to colonic mucosa, and impair sodium and water absorption. Selenium-75 labeled Homotaurocholic Acid Test (⁷⁵SeHCAT) involves the administration of a selenium75 labeled synthetic bile acid (homotaurocholic acid) orally, followed by measurement of retention of the bile acid by whole body scan or gamma camera at seven days (abnormal is less than 5 percent, normal is >12%, equivocal is between 5 and 12%).

• IDA is diagnosed by haemoglobin less than the normal limit for the lab, MCV <76fl and ferritin <15ug/dl. Both microcytosis and hypochromia (MCH) are sensitive indicators of IDA. However they are also present in thalassaemia, sideroblastic anaemia, lead poisoning and anaemia of chronic disease.
• Ferritin is an acute phase reactant may be elevated, if concurrent inflammation is present. In such cases iron deficiency can be diagnosed by low serum iron and high TIBC or low transferrin saturation. In contrast anaemia of chronic disease has low serum iron and low TIBC with normal transferrin saturation.
• A bone marrow aspirate stained for iron (Perls stain) is diagnostic of iron deficiency. While this largely has been displaced in the diagnosis of iron deficiency by performance of serum iron, TIBC, and serum ferritin, the absence of stainable iron in a bone marrow aspirate is the criterion standard for the diagnosis of iron deficiency. It is diagnostic in identifying the sideroblastic anaemia’s by showing ringed sideroblasts in the aspirate stained with Perls stain.

Discuss iron replacement treatment?

• Oral replacement- This is achieved most simply and cheaply with ferrous sulphate 200 mg twice daily although ferrous gluconate and ferrous fumarate are as effective and may be better tolerated. A liquid preparation may be tolerated when tablets are not. Ascorbic acid enhances iron absorption and should be considered when response is poor.
• Parenteral replacement- should be used when there is intolerance to oral preparations or non-compliance.
• Parenteral preparations:
  • Intravenous iron sucrose is well tolerated Bolus IV iron sucrose (200mg iron) over 10minutes is licensed and more convenient than a two hour infusion.
  • IV iron dextran can replenish iron and Hb levels in a single infusion, but serious reactions can occur (0.6-0.7%) and there have been fatalities associated with infusion. However, it can be given via IM route when venous access is problematic.
  • Sodium ferric gluconate
  • IV iron sucrose and sodium ferric gluconate are rarely associated with severe allergic reactions and deaths, and are better tolerated than iron dextran even at high doses and thus are preferred.

Discuss the calculation of the parenteral iron dose?
Dose in mg of iron= body weight in kg X (12-present Hb. X10) X 0.24
Add to this a depot of 500mg.

Link to calculate parenteral iron dose

IV iron sucrose can be administered at 200mg weekly for five weeks. Alternatively, iron sucrose can be given at 500mg IV (in 250cc of NS) over four hours once weekly for two weeks.

• The diagnostic accuracy of the UBT is more than 95% in studies.
• UBT is based upon the hydrolysis of urea by H. pylori to produce CO2 and ammonia. A labeled carbon isotope is given by mouth; H. pylorus liberates tagged CO2, which can be detected in breath samples.
• Two UBTs are FDA approved: the non-radioactive 13C test and the radioactive 14C test. Both tests can be performed in 15 to 20 minutes and have similar cost and accuracy. 13C is preferred by some as it does not use a radioactive isotope; however the dose of radiation in the 14C test is minimal.
• Test- A baseline breath sample is collected before the patient ingests 13C-urea, i.e., urea labeled with a naturally occurring, non-radioactive carbon isotope. A second sample is collected shortly after the ingestion. H pylori-associated urease degrades the urea, producing ammonia and CO2. The resultant CO2 is absorbed in the blood and then exhaled. An increase in the ratio of 13CO2 to 12CO2 between the pre- and post-ingestion samples indicates the presence of H pylori-associated urease.
• Before conducting the UBT, the patient should be off antibiotics, PPI and bismuth for at least 2 weeks. Patients can remain on H2 blocker therapy.

Stool antigen tests

• Sensitivity and specificity I than 90%.
• The test is based on a sandwich EIA with antigen detection. This is a qualitative test with an animal anti-H.pylori antibody adsorbed to microwells as capture antibody.
• The patient simply collects a stool sample in a clean, dry container.
• PPI, antibiotic and bismuth suppresses H. pylori and thus reduces faecal antigen values, resulting in a decreased accuracy.

Serology

• ELISA technology is used to detect IgG or IgA antibodies
• The diagnostic accuracy is low (80–84%) with high sensitivity (90 to 100 percent), but variable specificity (76 to 96 percent).
• Positive serology indicates current or past infection with H.pylori. A positive test does not confirm active disease.
• A negative serological test provides >95 % assurance that there is no H.pylori infection.
• Serology should not be used in the evaluation of treatment of H.pylori because the antibody titre takes many months (or even years) to decline.

Rapid urease test (RUT test)

• The sensitivity and specificity is more than 90%.
• Obtaining tissue samples from the antrum and the fundus may increase the sensitivity of the test in these patients.
• The CLO test is a qualitative RUT assay based on the detection of urease, produced by H. pylori.
• The test system consists of a test well filled with a urea containing gel where the suspected tissue is inoculated and allowed to incubate. If H. pylori is present in the patient’s sample, urease will hydrolyze the urea in the gel leading to an accumulation of ammonium ions (NH4 +). This causes a rise in pH which is detected by a pH indicator in the test system changing from yellow to magenta. Other shades of red such as pink or orange are also considered positive. Yellow is considered a negative screen.
• Gastric biopsy specimen should be taken preferably from an area that is not as eroded or denuded; H. pylori is present in smaller numbers in these areas than in otherwise normal looking tissue.
• A CLO is reported as negative if there is no change in colour until the end of the 24 hour period.
• CLO test may be falsely negative  in patients with recent gastrointestinal bleeding or with the use of PPIs, H2 antagonists, antibiotics, or bismuth-containing compounds as these agents suppress H. Pylori

Histology
The density of HP may vary at different sites and this may lead to sampling error. There may also be interobserver variability with histology. The sensitivity of histology may be decreased in patients taking anti secretory therapy, but is still higher in this setting than rapid urease testing. The sensitivity and specificity of histology is more than 90%
HP culture

Biopsies for culture should be obtained before the forceps are contaminated with formalin. The tissue should be placed into a container with a few drops of saline. Cultures have 80-90% sensitivity and 100% specificity

Which test to use?

• The American College of Gastroenterology guidelines recommend the UBT as “the best non endoscopic test for documenting H. pylori infection,” although stool antigen testing is equally accurate and is more widely available.
• Rapid urease test is the easiest and cheapest endoscopic test in the absence of GI bleed or current use of PPI or antibiotics
• The accuracy of various methods to detect HP may be affected in the presence of recent bleeding. Sensitivity is low but specificity is high for biopsy-based methods (RUT, histology and culture) in the presence of GI bleed. With respect to noninvasive tests in presence of GI bleed, a metanalyses showed that sensitivity and specificity were 93 and 92 percent for the (13)C urea breath test, 87 and 70 percent for the stool antigen test, and 88 and 69 percent for serology. A biopsy should be done, if possible at the time of endoscopy. If this is negative for HP, UBT should be preferentially used, although stool antigen test and serology are alternatives.
• Serology is also useful as a diagnostic test when others could be false negative, such as in patients with a low bacterial density like gastric atrophy, MALT lymphoma and with recent or current use of PPIs and antibiotics.

Discuss use of helicobacter tests to confirm eradication?

• Urea breath testing performed at least four weeks after treatment has been promoted as the test of choice to confirm eradication of infection.
• Stool antigen testing is a more widely available alternative, but it may be less accurate. Serologic testing is not useful for follow-up since many patients continue to have antibodies for months or even years after eradication therapy
• PPI, Antibiotics and bismuth should be discontinued for at least two weeks prior to testing done to confirm H. pylori cure (with urea breath test, stool antigen testing, or endoscopic testing) to reduce the chance of false-negative results.

• The best available tests are the IgA antihuman tissue transglutaminase (TTG) and IgA endomysial antibody immunofluorescence (EMA) tests. They have equivalent diagnostic accuracy with sensitivity and specificity of these two tests exceeding 95%.
• These tests may not be as accurate in the clinical setting.  Thus, in symptomatic individuals in whom there is strong clinical evidence for CD, a D2 biopsy should be considered even if the serologic test is negative.
• The serologic tests for CD are IgA based and hence will not be able to identify individuals who have both CD and selective IgA deficiency. Approximately 5-10% of celiac patients are IgA deficient.  Here, IgG-based tests may be used; however, their accuracy remains to be determined. Thus, D2 biopsies may be best in symptomatic IgA deficient patients.
• Serologic testing for celiac disease in children younger than 5 years of age may be less reliable.
• There are no data to show that a combination of tests is better than a single test using either EMA IgA or TTG IgA.
• Antigliadin antibody (AGA) tests are no longer routinely recommended because of their lower sensitivity and specificity.

Discuss the pathogenesis for coeliac antibodies?

The enzyme tissue transglutaminase (tTG) has an important role in the breakdown of proteins in the small intestine. Dietary gliadin triggers the formation of a gliadin peptide–tTG complex in the small intestine. In CD, antibodies to this peptide-tTG complex (anti-tTG antibodies) are formed, leading to mucosal inflammation.

Discuss the TTG and EMA tests?

• IgA class EMA is measured qualitatively by subjective assessment of direct immunofluorescence with monkey oesophagus or human umbilical cord as the antigenic substrate. This needs experienced microscopists and the assay can only be semi-quantitative at best, using serial dilution titers.
• In contrast, IgA anti-tTG antibody is measured by solid phase ELISA, which is fully quantitative, automated and more reproducible between laboratories. Human recombinant anti tTG is used as the antigenic substrate. These factors allow for reliable, simple, serial measurements of antibody concentration.

Discuss the role of HLA typing in coeliac disease?

Almost 100% of pts with CD have HLA DQ2 or DQ8. However, approximately 30% of the general population is also positive for the DQ2 haplotype. Thus HLA typing would not be very specific, but a negative test for both HLA DQ2 and DQ8 virtually excludes CD.
This may be clinically useful, if duodenal histology is not possible in a patient with positive coeliac serology.

Discuss coeliac serology as a marker of dietary compliance?

• Coeliac serology can be used as an approximate marker of dietary compliance, although a decrease in titer does not correlate with histopathologic improvement.
• In those whose antibody levels do not decrease within 12 months, dietary compliance should be checked and repeat biopsy examination should be performed as necessary by mutual consent.
• Because of the false positivity of tTG and EMA with other autoimmune diseases, such as type 1 diabetes and autoimmune hepatitis, these antibodies may remain elevated in a certain subset of patients despite strict adherence to a GFD.

• The C. difficile bacteria produce two principal toxins – A and B – which cause diarrhoea and colitis. Toxin A & B are both pathogenic and strains produce both.
• Enzyme immuno assays (EIA) to detect C. Difficile toxin is the most common test used.
• Cytotoxin testing- detects C. difficile toxins, using an in-vitro cell sheet assay.
• Culture – this is relatively slow and requires supplementary testing to confirm that an isolate is a toxin producer.
• Detection of an antigen (e.g. the enzyme glutamate dehydrogenase) or a gene (e.g. the toxin B gene).

Discuss the limitation of EIA kits used to detect C. Difficile infection?
All currently available EIA kits have a high negative predictive value. However, caution is required (due to false positive rates) in the interpretation of toxin-positive results to ensure that these are consistent with the clinical presentation.
The use of a confirmatory or second test (like cytotoxin test) will increase the accuracy of toxin positive results. However, the need for it is unclear at present.
Should multiple repeat samples be submitted for testing?
A repeat test (so 2 tests in all) may be considered if there remains a high clinical suspicion of C. Difficile infection. However, multiple tests should be avoided as, with tests that have a sub-optimal positive predictive value, this increases the chance of obtaining false positive results.
What is the best single test for the detection of C. difficile toxins?

Well-performed cell-culture cytotoxicity assays are still regarded as the “gold standard” for diagnostic testing. However, these tests have a disadvantage in terms of speed.

Are further tests required to demonstrate clearance of the organism?
Tests of cure (i.e. clearance testing) are not clinically helpful as toxin excretion may continue even when symptoms have stopped. Thus

• Re testing is not recommended if the person is still symptomatic within a period of 28 days
• Only re-test to confirm recurrent C. difficile infection if the symptoms resolve and then recur.

Ref
http://www.hpa.org.uk/web/HPAwebFile/HPAweb_C/1238055363795