Clinical Chemistry

CHM 60304

Assignment 2

Group Member:
Yeap Tze Huay 0331462
Dinie Aqilah binti Ahmad Fariz 0330846
Nur Aliah Husna Bt Mohamed Azmi 0312923
Topic 1: 13C Urea breath test

1. Indicate the tests for their diagnosis.

13C Urea breath test is diagnostic procedure used to identify infection by Helicobacter pylori
(H. pylori). H. pylori is a common type of bacteria that grow and infect the stomach lining. H.
pylori infections are usually harmless, but they are responsible for the majority of ulcers in the
stomach and duodenum (Kusters et al., 2006). H. pylori has the ability to survive and invade
the mucosal lining of stomach and duodenum due to the production of high level of urease
which can help in rising the local pH of the site of infection. The urease produced can be used
as one the parameter for confirmation of diagnosis of H. pylori infection (Kusters et al., 2006).

A few different tests can be carried out to determine and confirm an H. pylori infection.

Blood test

Blood test can be carried out by determining the antibodies to H. pylori in the blood
specimen of the patient. The antibodies are proteins that are produced by immune system of
body when adaptive immune system take place after H. pylori is detected. However, blood test
is not recommended for routine diagnosis or monitoring of H. pylori infection as the positive
indication of antibody present in blood will no distinguish between a present or previous
infection. If the ordered test shows positive result, stool antigen or breath test should be carried
out to confirm the ongoing infection.

Stool test

Other than blood test, stool test can be carried out. A stool sample will be obtained from
the patient and the presence of H. pylori is tested on the faeces. Stool test for confirmation of
H. pylori infection involves the detection of H. pylori antigen in the stool sample (Shimoyama,
2013). This test can be carried out to diagnose the infection and monitoring of the infection
after treatment of antibiotics.
Biopsy

A biopsy from the antrum of the stomach can be taken during endosclooopic procedure
(Malik et al., 1999) and this is the most accurate way to diagnose the infection of H. pylori, but
it is an invasive procedure. A few test can be carried out on the biopsy sample obtained.

The biopsy specimen can be sent for histology to be examined under a microscope for
any abnormalities of the stomach or duodenum lining.

A culture can also be carried out on the biopsy specimen to detect growth of H. pylori
in/on a nutrient agar (Mégraud et al., 2007). As stomach is an acidic environment, only H.
pylori will have the ability to invade and colonize, hence the positive growth on growth
medium has a high possibility to be H. pylori. The growth on the medium will undergo further
investigation thru biochemistry test such as differential agar and also urease test to confirm the
presence of H. pylori of a patient which confirms the infection of H. pylori.
On the other hand, rapid urease testing can be carried out by the detection of enzymatic
activity of urease (Uotani et al., 2015). The rapid urease test is carried out by placing the biopsy
specimen onto a substrate containing urea and pH indicator (phenol red). In a positive urease
test, H. pylori will produce urease which will hydrolyse urea into ammonia and increase the
pH of the medium causing the changes in the colour of the specimen from yellow (negative) to
red (positive) (Uotani et al., 2015).

13C Urea Breath Test

The urea breath test (UBT) was a commonly used non-invasive method and frequently
required in a clinical practice in order to diagnose Helicobacter pylori (H.pylori) infection and
to monitor its abolition.
 2. Discuss the mechanism of the test.

In 13C urea breath test, labelling urea with 13C will be prescribed to the patient to be
taken, the breath sample will be collected before ingestion of isotopically labelled urea and
after ingestion to provide reliable diagnostic information. The advantage of this test was that it
can be carried out as a diagnostic and monitoring tool of H. pylori infection and this is a non-
invasive procedure.

Typically, urease act as a cytosolic enzyme which catalyses the hydrolysis of urea into
ammonia and carbon dioxide and H.pylori highly produce these respective enzymes
(Sciencedirect.com, 2015). Once urea has been hydrolysed into its respective substances, they
will then diffuse into the blood and excreted by the lungs (Ferwana et al., 2015). The excreted
material by the lungs was CO2 and it can be detected in breath by isotopically labelled CO2 by
using mass spectrometry or by mass correlation spectrometry. Two UBTs was made available
and approved by Food and Drug Administration (FDA) which was 13C and 14C tests. Both test
was said to be reasonable and have the capabilities to provide real-time results. However, 13C
presents as a non-radioactive compound which resembled a preferred isotope especially in
young children and women of childbearing age with a minimal dose of approximately one
microCi (Savarino et al., 2017).

The protocol for the urea breath test was to be performed with relatively low doses
(<100mg) of urea or 75mg or even 50mg was considered sufficient to perform UBT. Preferably,
the UBT should be carried out ten to fifteen minutes after urea ingestion. A positive urea breath
test (UBT) shows an active H.pylori infection which requires treatment or further analysis with
invasive procedures for an accurate confirmation (Gisbert and Pajares, 2004).
 3. Indicate the possible test outcome compared to that of normal patient.

Blood test for H. pylori antibody testing will show positive in individual with previous or
present infection of H. pylori. In a normal patient, the blood test will indicate negative result
of absence of H. pylori antibody. However, the result might come back as positive if the patient
had H. pylori previously. This indicates that the blood test for antibody detection of H. pylori
is not confirmative and additional test have to be carried out.

Stool test for H. pylori antigen in a patient with H. pylori infection will show positive result
as antigen of H. pylori will be detected. The test of H. pylori antigen is accurate which can be
used for diagnostic of H. pylori infection and monitoring tool after antibiotic treatment.
However, in a normal patient H. pylori antigen will show as negative.

Microscopic examination of the biopsy of a patient with chronic (long term) H. pylori
infection will show inflammatory lesions with infiltrates of polymorphonuclear cells and
plasmocytes (plasma cell of lymphocyte B cell) and lymphocytes aggregates. In a normal
patient without H. pylori infection, the biopsy specimen will show normal history of gastric
mucosa.

Rapid urease test of the biopsy specimen will show colour change of the medium from
yellow to red which indicates positive result of H. pylori infection. However, in a normal
patient, the colour of the medium will remain as yellow.

The culture of the biopsy specimen will show positive growth of H. pylori in a patient with
H. pylori infection. In normal patient, no growth will be observed on the growth medium.
Topic 2: Mast and Rast Allergy Tests

1. Indicate the tests for their diagnosis.

Diagnostic investigations (‘Allergy testing’) serves only to confirm an allergic trigger

suspected on the basis of the patient’s clinical history. The different allergy tests are based on
the mechanism of the allergic reaction, which is generally divided into two groups, which are
IgE-mediated and non IgE-mediated allergy tests IgE-mediated (Type I or immediate
hypersensitivity) allergy tests in particular, includes tests such as total IgE, allergen specific-
IgE, skin prick tests and component tests. Type I immunoglobulin E (IgE)-mediated allergy
testing is evaluated by measuring allergen-specific IgE. This can be done through:

i. In-Vitro Testing: RAST and MAST Allergy Tests

The RAST (radioallergosorbent test) and MAST (multiple allergosorbent test) allergy
tests detect antigen-specific IgE antibodies in the patient’s serum. RAST testing uses a
radioimmunoassay of the blood serum used to detect specific allergens. MAST can
examine multiple antigens simultaneously in a short period of time and it is a RAST-
type test that uses an enzyme rather than a radioactive marker (Ross, 2018). The levels
of allergy antibody (IgE) produced when blood is mixed with a series of allergens in a
laboratory is measured. IgE antibodies are present in the blood only if there is a true
allergic reaction. It is noted that RAST is a method of demonstrating allergic reactions
and should only be performed on patients who cannot undergo skin testing or when skin
test results are uncertain (Australasia, 2018).

ii. In-Vivo Testing: Skin Prick Tests (SPTs)

SPTs are used to identify immediate IgE-mediated hypersensitivity against a group of
allergens by measuring the wheal reaction of an allergen against a positive histamine
control. It is an indirect measure of cutaneous mast cell reactivity due to the presence
of specific IgE. Mast cells reside in the subepithelial layer of the skin and the respiratory,
nasolacrimal, and gastrointestinal tracts, and the skin happens to be the most accessible
organ to test.
Skin testing detects allergen-specific IgE bound to mast cells. The allergen cross-links
specific IgE bound on the mast cell. This causes degranulation of preformed mediators,
including histamine and tryptase. Histamine release is the major mediator that results
in a hive at the prick site and surrounding erythema, called a wheal and flare.
SPTs are done by placing the purified allergen extract on the skin. The skin is then
pricked with a special lancet, in a standardised manner. The test site is then evaluated
after 20 minutes for a wheal-and-flare reaction (secondary to histamine release from
mast cells).
 2. Discuss the mechanism of the tests.

Laboratory assays for allergen specific-IgE (sIgE) are most often used to confirm the
suspected clinical diagnosis of allergic disease. MAST and RAST allergy tests are used to
diagnose Type I allergic reactions.

Immediate (Type I) hypersensitivity refers to a specific type of immune response in which

Immunoglobulin E (IgE) plays a central role. Clinical manifestations of immediate
hypersensitivity most commonly involve the skin (urticaria), eyes (conjunctivitis), respiratory
tract (rhinitis and asthma) and cardiovascular system (hypotension). IgE is produced after first
exposure to an allergen (an environmental molecule that induces an allergic response). This
IgE subsequently binds to mast cells in tissues, such as those mentioned above. Subsequent
exposure to allergen causes activation of the mast cells, to which the allergen-specific IgE is
bound, causing release of chemicals that cause the clinical manifestations of allergy.

Inflammatory chemicals including histamine, serotonin, proteases, bradykinin generating

factor, chemotactic factors (molecules that stimulate the immune response), leukotrienes,
prostaglandins and thromboxanes are released. These chemical mediators cause symptoms
associated with allergies such as hives, runny nose, watery eyes, sneezing, wheezing or itching.
Type I allergic reactions are often triggered by food (like peanuts, shellfish and milk), drugs
(like penicillin), inhaled allergens (like dust, pollen and pet dander), latex and insect venom
(bees, wasps, etc.).

Figure 1: Schematic representation of an allergen specific–IgE assay principle. The

allergen/allergen components are covalently bound to the solid phase antibody. The patient’s
serum containing IgE antibodies against the target allergen is added. The non-bound Ig-E
antibodies are washed away, and the bound IgE antibodies are detected by a signal (either
radiolabel or enzyme-colorimetric label).
https://www.pathcare.co.za/webfiles/files/Allergy_Investigations_June2014.pdf

In RAST and MAST allergy tests, immunoassays measure interactions between

antigens and antigen-specific antibodies. Immunoassays are often referred to as
radioallergosorbent (RAST) testing, though the term is outdated as radiation is rarely used
nowadays.
Multiple Antigen Simultaneous Test (MAST) was the common tool to identify a
specific allergen. MAST was then upgraded into two test which MAST-CLA
(chemiluminescent assay) and MAST-immunoblot assay. MAST-CLA has been widely
utilized. Reason being, it does not use any radioactive agent and it was not expensive which
enabled a simultaneous multiple allergens examination. However, MAST-immunoblot was the
upgraded test after MAST-CLA was introduced. MAST-immunoblot was rather simpler and
provide results at a faster rate of about two and a half hours and lesser sample serum volume
approximately 12.5 Litre (l) per test. However, its diagnostic values remained to be further
investigate (Park et al., 2004).
Similarly, radioallergosorbent test (RAST) was used to detect allergen specific IgE in the
blood by utilizing a solid phase radioimmunoassay method. In particular, this test enables the
detection of allergic diseases such as asthma and allergic rhinitis. This test was normally
performed secondarily to skin prick test. The serum sample of a patient would be incubated
with an allergen bound to a solid material. The Allergen-specific IgE will then be detected by
using antibodies specific for hum IgE labelled with either enzyme or a fluorescent compound
(Australasian Society of Clinical Immunology and Allergy (ASCIA), 2010)
 3. Indicate the possible test outcome compared to that of normal patient.

MAST and RAST Allergy Tests

An elevated allergen-specific IgE result indicates that the person tested likely has an allergy.
However, the amount of specific IgE present does not necessarily predict the potential severity
of a reaction.

If the specific IgE test is positive, a person may or may not ever have an actual physical
allergic reaction when exposed to that substance (Labtestsonline.org, 2018). The person may
be sensitive to that particular allergen, which means that the person have the ability to mount
an allergic response when they are exposed to that allergen. It does not mean that they will
necessarily have a clinical allergic reaction to that substance, however, nor does it mean that
any reactions will be severe. Patients who have allergies may be advised to avoid the allergen
or they may be prescribed allergy medications, such as antihistamines (like diphenhydramine)
or anti-inflammatories (Livingnaturally.com, 2018).
Negative results indicate that a person probably does not have a "true allergy," an IgE-
mediated response to the specific allergens tested. It is likely that they do not have the ability
to mount an allergic reaction to that allergen and any symptoms experienced are probably not
due to an allergic reaction to that substance.
Results of allergy blood testing must be interpreted with care. False negatives and false
positives can occur. Even if an IgE test is negative, there is still a small chance that a person
does have an allergy (MyVMC, 2018). The results of the RAST and MAST allergy tests need
to be interpreted in conjunction with one’s clinical history and pattern of allergic reactions.
Hence, a person's clinical history and additional medically-supervised allergy tests may be
necessary to confirm an allergy diagnosis.
Figure 2: Summary of interpretation of results from RAST and MAST allergy tests for type 1
allergy.

Skin Prick Tests

The wheal and erythema surrounding each reaction is evaluated after 15-20 minutes. A
positive reaction is interpreted as a wheal 3 mm or larger than the ‘negative control’. The
presence of a reaction at the ‘positive (histamine) control’ is necessary to confirm the absence
of antihistamines in the subject’s system.
Reference

Australasia, L. (2018). Liver function tests. [online] Labtestsonline.org.au. Available at:

https://www.labtestsonline.org.au/learning/test-index/liver-function#sample [Accessed 22
May 2018].

Australasian Society of Clinical Immunology and Allergy (ASCIA). (2010). Laboratory Tests
in the Diagnosis of Allergic Diseases. [online] viewed 23 May 2018
<https://www.allergy.org.au/health-professionals/papers/tests-in-the-diagnosis-of-allergic-
diseases>

Erwana, M., AbdulMajeed, I., Alhajiahmed, A., Madani, W., Ferwana, B., Hasan, R., Altayar,
O., J Limburg, P., Murad, M. and Knawy, B. (2015). Accuracy of urea breath test in
Helicobacter pyloriinfection: Meta-analysis. [online] World Journal of Gastroenterology.
Viewed 23 May 2018 <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4306177/>

Gisbert, J. and Pajares, J. (2004). Review article: 13C-urea breath test in the diagnosis of
Helicobacter pylori infection - a critical review. Alimentary Pharmacology and Therapeutics,
[online] 20(10), pp.1001-1017. Viewed 23 May 2018
<https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2036.2004.02203.x>

Koreamed.org. viewed 23 May 2018

<https://www.koreamed.org/SearchBasic.php?RID=0039KJLM/2004.24.2.131&DT=1>

Kusters, J. G., van Vliet, A. H. M., & Kuipers, E. J. (2006). Pathogenesis of Helicobacter
pylori Infection. Clinical Microbiology Reviews, 19(3), 449–490.
http://doi.org/10.1128/CMR.00054-05

Labtestsonline.org. (2018). Allergy Blood Testing. [online] Available at:

https://labtestsonline.org/tests/allergy-blood-testing [Accessed 27 May 2018].

Livingnaturally.com. (2018). Allergen-specific IgE antibody test - nutrition house. [online]

Available at:
http://www.livingnaturally.com/ns/DisplayMonograph.asp?StoreID=3D9D155236034A5897
378F7C5A033221&DocID=allergy-rast [Accessed 27 May 2018].

Malik, G. M., Mubarik, M., & Kadla, S. A. (1999). Helicobacter pylori Infection in Endoscopic
Biopsy Specimens of Gastric Antrum: Laboratory Diagnosis and Comparative Efficacy of
Three Diagnostic Tests. Diagnostic and Therapeutic Endoscopy, 6(1), 25–29.
http://doi.org/10.1155/DTE.6.25

MyVMC. (2018). RAST (serum specific IgE) allergy testing information | myVMC. [online]
Available at: https://www.myvmc.com/investigations/rast-radioallergosorbent-test-serum-
specific-ige/#c1 [Accessed 27 May 2018].

Park, D., Cho, J., Lee, K., Ko, S., Kim, H., Choi, S. and Lee, Y. (2004). Detection Rate of
Allergen-Specific IgE by Multiple Antigen Simultaneous Test-Immunoblot Assay. [online]

Pathcare.co.za. (2018). [online] Available at:

https://www.pathcare.co.za/webfiles/files/Allergy_Investigations_June2014.pdf [Accessed 27
May 2018].

Ross, C. (2018). Interpretation of Liver Function Tests (LFTs) | Geeky Medics. [online] Geeky
Medics. Available at: https://geekymedics.com/interpretation-of-liver-function-tests-lfts/
[Accessed 22 May 2018].

Savarino, V., Vigneri, S. and Celle, G. (2017). The 13C urea breath test in the diagnosis of
Helicobacter pylori infection. [online] Pdfs.semanticscholar.org. viewed 23 May 2018
<https://pdfs.semanticscholar.org/6142/c4222f9e802d4c9f9757e3a59b1c8e33eefa.pdf>

Sciencedirect.com. (2015). Urease - an overview | ScienceDirect Topics. [online] Available at:

https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/urease
[Accessed 23 May 2018].

Shimoyama, T. (2013). Stool antigen tests for the management of Helicobacter

pylori infection. World Journal of Gastroenterology : WJG, 19(45), 8188–8191.
http://doi.org/10.3748/wjg.v19.i45.8188

Uotani, T., & Graham, D. Y. (2015). Diagnosis of Helicobacter pylori using the rapid urease
test. Annals of Translational Medicine, 3(1), 9. http://doi.org/10.3978/j.issn.2305-
5839.2014.12.04