Monday, December 24, 2012

Tests for coagulation disorders

Tests for coagulation disorders:

Prothombin Time (PT):
PT is the time in seconds that takes the blood to clot. This test result can vary depending on laboratory system used. A longer PT means that the patient’s blood is taking longer to clot.

Fig. BD Vacuitainer tube with Sodium citrate (Sky Blue top)

Activated Partial thromboplastin time (APTT):
APTT is a measure of how well the patient’s blood would clot. A sample of blood is “clot activated” and the time taken to clot is measured in seconds. Like the PT a longer aPTT means that clotting will generally take longer.

Sunday, December 23, 2012

Prokaryotic Transcription: step by step

Begins with the binding of DNA dependent RNAP holoenzyme to promoter in template DNA.

Fig. TATA sequence (Source : Lehninger's Biochemistry, 4th Edition)
There are variations in nucleotide sequences in these promoter sites which provide differential gene expression.

Alpha subunit binds to UP element and sigma factor covers -35 to -10 region.

Binding of RNAP to -35 forms closed complex whereas to -10 region forms open complex, due to the presence of AT region which has low Tm and also these sites have low nucleosomal density.

RNA Metabolism : Basics


RNA is the only macromolecule having role in storage and transmission of information and in catalysis (ribozyme).

Fig. Classes of Eukaryotic RNA (Source: Harper's Illustrated Biochemistry, 28th Edition)

DNA-Dependent RNA synthesis

Synthesis by RNA Polymerase (RNAP)  that adds NTPs to 3’-OH end using one strand of DNA as template, similar to DNA Polymerase (DNAP). RNA formed is similar to coding strand.

Elongated from 5’ to 3’ end with reading from 3’ to 5’ end on DNA.

Tuesday, December 18, 2012

Cross sectional studies

What is research ?
It is a scientific process of Systematic collection of data, Compilation of data, analysis of data to convert in to information for suitable interpretation to get the answer of the question

Why research is needed in health sector ?

To identify:

  • Health problems along with their magnitude to prioritize the diseases for action and then to manage  by initiating curative action
  • Risk behavior or causal factors to restrict  partially or fully for further addition by preventive action

Need of research in health sector
6 Ws are the guide for research:

  • W: who are the affected people (age, gender, race)
  • W: where are the affected people (rural/urban, geographical location)
  • W: when are affected (seasonal, cyclic)
  • W: why are affected (cause)
  • W: what action is required (intervention)
  • W: what happened after the action (evaluation of intervention)

Answer of 6 Ws are by different study designs

Cross sectional studies

  • Cross sectional studies are those carried at a point in time or over a short period of time
  • Cross sectional studiesa are useful for both describing the situation in terms of magnitude (prevalence) of disease and comparing (to measure the association), but causal association can not be established
  • While to assess causal association, the cohort study design and case control (for rare diseases) study designs are followed.

Cross-sectional studies measure:

The prevalence of not only disease but also the health related behavior or characteristics of people in a population e.g. the wearing of seat belts or participation in exercise

  • Its result can often suggest causative or risk factors associated with particular illness or behavior
  • For instance, the causal relationship between cataracts and vitamin status was originally investigated through a cross-sectional study (Coggon et al., 2003). 
  • Cross-sectional studies, need not always to investigate the whole population: a sample is usually sufficient, provided that the sample is representative and adequate in size.
  • Cross-sectional studies are useful in planning public health interventions.
Descriptive Cross-sectional survey
are used to measure the prevalence of disease in a population


  • Repetition of these surveys can be very useful in finding out whether changes are occurring over time – called monitoring or surveillance

Analytical Cross Sectional Surveys
Obtained data on prevalence of outcome and exposure are compared for differences in outcome between exposed and unexposed

  • A defined population at a particular time is considered
  • Exposure and outcomes are determined at the same point in time
  • Analytical Cross Sectional Surveys

Tuesday, December 11, 2012

What is Hypersensitivity ?


1. Basic concepts

Hypersensitivity (HS) reactions are  harmful antigen-specific immune responses  which produce tissue injury and dysfunction.

Allergen:the antigens that give rise to immediate HS.

Fig. Allergy

Familial tendency to make IgE more frequently against the common environmental substances to which most of the population is not reactive.
Fig. Types of Hypersensitivity

Type I  hypersensitivity


• Occur and resolve quickly
• Mediated by serum IgE
• Systemic and regional tissue dysfunction
• Genetic predisposition

Components and cells:

  1. Allergen :
    • pollen, dust mite, insects etc
    • selectively activate CD4+Th2 cells and B cell
  2. IgE and its production
    • IgE: mainly produced by mucosal B cells in the lamina propria 
    • IL-4 is essential to switch B cells to IgE production  
  3. High affinity receptor of the IgE on mast cell and basophi
  4. Eosinophil
Factors which stimulate Mast cells
• Cross linking of IgE by Allergen
• C3a, C4a, C5a receptor present in mast cell
• Drugs like Codeine and Morphine
• Venon (Snake bite- Mellitin), Bee sting, Ant Sting
• Physical agents like cold, heat, physical trauma
• Strong sun light

 Production of IgE Ab

  • In individuals who are prone to allergies (Atopic persons) encounter with some Ag activation of TH2 cells--->production of IgE Ab
  • IL-4 and IL-13 stimulate B lymphocytes specific for the foreign Ags to switch to IgE producing  plasma cells.
  • Strong genetic basis
Both Genetics and the Environment play a significant roles in determining who has allergies and which kind of allergy.

Certain allergies and autoimmunity can be mapped to specific gene loci.

Activation of mast cells and secretion of mediators

  1. Priming stage/Sensitizing
    • IgE Ab produced in response to an allergen binds to high affinity Fc receptors specific for the ε heavy chain on mast cell
  2. Activating stage
    • When mast cells sensitized by IgE are exposed to the allergen, the cells are activated to secrete their mediators 
  3. Effect stage:
    • Vasoactive amines and proteases—from granules, products of arachidonic acid metabolism, and cytokines. (Immediate and late phase) 
Immediate/early phase : mediated by histamine, start within seconds, last several hours.
Late phase: Mediated by new-synthesized lipid mediators, take up 8-12 hours to develop, last several days 

Sequence of events in immediate HS:

  • Intitiated by the introduction of an allergen, which stimulates Th2 reactions and IgE production. 
  • IgE binds to Fc receptors (FCERI) on mast cells, and subsequent exposure to the allergen activates the mast cells to secret the mediators that are responsible for the pathologic reactions of immediate HS.

Schematic diagrams of the high-affinity FcRI and low-affinity FcRII receptors that bind the Fc region of IgE.

Each gamma-chain of the high-affinity receptor contains an ITAM, a motif also present in the Ig-/Ig- heterodimer of the B-cell receptor and in the CD3 complex of the T-cell receptor.

The low-affinity receptor is unusual because it is oriented in the membrane with its NH -terminus
directed toward the cell interior and its COOH-terminus directed toward the extracellular space.

Mast cell activation:

 Biochemical events in mast cell activation:

  • Cross linking of IgE on a mast cell by an allergen stimulates phosphorylation of ITAM (Immunoreceptor tyrosine based activation motifs) in the signalling chains of the IgE Fc receptor, which then initiates multiple signaling pathways. These signaling pathways stimulate the release of mast cell granule contents (amines, proteases). 
  • The synthesis of arachidonic acid metabolites (PG, LTs) and the synthesis of various cytokines. These mast cell mediators stimulate the various reactions of immediate HS.   

Clinical Syndrome:


  • Skin prick test
  • RAST (Radio allergo Immuno sorbent test)
    • Used to detect specific IgE against a particular Ag

DNA extraction protocol


DNA can be extracted from any blood or tissue sample. The quality and quantity of the DNA obtained will vary depending on the size, age, and cell count of the sample. As a rule, 3 ml of blood in EDTA will suffice. The DNA is extracted from all nucleated cells and is called genomic DNA.

In the nucleus, the DNA is tightly associated with many different proteins as chromatin. It is important to remove these as well as other cellular proteins to extract the DNA. This is achieved through the use of organic solvents or salt precipitation. An aqueous solution of DNA is obtained, from which the DNA is further purified by ethanol precipitation.

A number of DNA extraction kits are now commercially available. These can significantly reduce the amount of time required for DNA extraction, bypass the use of organic solvents, and provide good quality control of the reagents used. However, the use of kits in all aspects of molecular biology may inhibit the development of improvements.

Protocol: Extraction of Genomic DNA (The protocol described here is manual method)

Reagents needed:

Please note that for all the buffers and solutions,it is recommended that reagents of the highest grade available and double distilled deionised water are used throughout.

Stock Solutions 

  • NaCl 5 mol/l. Weigh 146.1 g of NaCl into a beaker and make up the volume to 500 ml with water; stir until dissolved.
  • Tris-HCl, 1 mol/l, pH 8.5. Dissolve 60.5 g of Trizma base (tris[hydroxy]methylaminomethane) in 350 ml water; add concentrated HCl until the pH falls to 8.5; make up to 500 ml with water.
  • Tris-HCl, 1 mol/l, pH 7.4. Prepare as for the above, but reduce the pH to 7.4 with HCl.
  • NaOH, 5 mol/l. Add 200 g of NaOH to 800 ml water and stir until dissolved; make up to 1 litre with water.
  • EDTA, 0.5 mol/l, pH 8.0. Weigh 93 g of EDTA disodium salt (dihydrate) and add to 400 ml of water; stir until most of it has dissolved. Add 0.5 mol/l NaOH until the pH rises to 8.0, when the rest of the solid should go into solution. Make up to 500 ml with water.
  • Phosphate buffered saline (PBS), pH 7.3.
  • Nonidet P-40 (NP40), 10%. Add 10 ml of NP40 to 90 ml water and mix well.
  • Sodium dodecyl sulphate (SDS, lauryl sulphate), 20%. Weigh 100 g of SDS and add to 350 ml of water. Stir and heat to 65°C until it is in solution and top up to 500 ml with water. 

Caution: SDS is a respiratory irritant. Wear a face mask and weigh out in a fume hood.

Working Solutions 

  • PBS + 0.1% NP40. Add 5 ml of 10% NP40 to 495 ml of PBS.
  • Ten times concentrated (×10) lysis buffer). Mix 60 ml of 5 mol/l NaCl, 20 ml of 0.5 mol/l EDTA, 10 ml of 1 mol/l Tris pH 7.4, and 10 ml of water to give 100 ml of 3 mol/l NaCl, 100 mmol/l EDTA, and 100 mmol/l Tris.
  • Lysis solution. Prepare an appropriate amount of this solution fresh every time; for 50 ml weigh 21 g of urea (7 mol/l), add 5 ml of ×10 lysis buffer, and make up to a final volume of 50 ml with water.
  • Chloroform/isoamyl alcohol (24:1). Add 20 ml of isoamyl alcohol to 480 ml of chloroform.
  • Ethanol 70%. Add 30 ml of water to 70 ml of absolute ethanol.
  • Tris EDTA (TE) (10 mmol/l Tris, 1 mmol/l EDTA). Add 5 ml of 1 mol/l Tris pH 7.4 and 1 ml of 0.5 mol/l Na2 EDTA to 494 ml water.
  • TE equilibrated phenol. The condition of the phenol is crucial to the quality of the DNA obtained. DNA is soluble in acidic water-saturated phenol, and so it is necessary to equilibrate it to a neutral pH.

  1. Take 500 ml of water-saturated phenol. Prepare 500 ml of 0.5 mol/l Tris pH 8.5, add 150 ml of this to the phenol, and mix by inversion for 2–3 min. Leave to stand until the aqueous and organic phases have separated.
  2. Remove and discard the upper aqueous layer. Add another 125 ml of 0.5 mol/l Tris, mix, stand, remove the aqueous layer as before, and then repeat.
  3. To the remaining 100 ml of 0.5 mol/l Tris, add 400 ml of water to give 500 ml of 0.1 mol/l Tris. Add 150 ml of this to the phenol, and then mix, stand, and remove. Repeat two more times.
  4. To the remaining 50 ml of 0.1 mol/l Tris add 449 ml of water and 1 ml of 0.5 mol/l EDTA to give 500 ml of TE. Add, mix, stand, and remove this in three stages as before. The phenol will have reduced in volume during this procedure but is now TE equilibrated and ready for use.


  1. Freeze an anticoagulated blood sample at -20°C. EDTA is the preferred anticoagulant. It is convenient to collect the blood in a tube that can be centrifuged, such as a disposable plastic 25 ml universal container. Any sample size from 2 to 20 ml will be satisfactory. Blood can be shipped at room temperature to a reference laboratory, preferably within a few days of taking it. The sample can be stored at -20°C for several weeks; storage for longer periods is better at -80°C.
  2. Thaw the blood and centrifuge at 700 g for 15 min. Carefully pour off the supernatant. The pellet is hard to see at this stage and may be quite loose.
  3. Resuspend the pellet in 1–2 ml of PBS + 0.1% NP40 by mixing up and down in a wide-bore standard plastic transfer pipette. Top up the suspension to the original volume with PBS + 0.1% NP40.
  4. Centrifuge again at 700 g for 15 min and pour off the supernatant. If necessary, repeat until the pellet has lost most of its red colour.
  5. Add 2–3 drops of lysis solution. Break up the pellet into this solution using a nonwettable sterile stick (e.g., a plastic disposable bacterial inoculating loop) or a clean siliconised glass rod. The solution will become viscous. Make it as homogeneous as possible.
  6. Add successive 0.5 ml volumes of the lysis solution, mixing each time, until the viscosity is such that the solution can be pipetted up and down without difficulty. The final volume will depend on the size, nature, and quality of the blood sample. For 10 ml of freshly frozen normal blood, use 2–3 ml of lysis solution.
  7. Add 1/10 volume of 20% SDS. Mix gently with a transfer pipette and incubate at 37°C for a minimum of 15 min. The samples can be left overnight at this stage.
  8. Transfer the sample to a capped polypropylene tube. Add an equal volume of chloroform/isoamyl alcohol and an equal volume of phenol. Mix gently by inversion for 5 min. Centrifuge at 1300 g for 15 min.
  9. Transfer the upper aqueous phase to a new polypropylene tube. Leave behind the white protein interface and the organic phase. This may be difficult if the solution is too viscous, in which case further dilution with lysis solution is necessary.
  10. Repeat steps 8 and 9 at least once more and continue until the interface is clear. Add an equal volume of chloroform/isoamyl alcohol and mix gently by inversion for 5 min. Centrifuge as before and again transfer the aqueous phase to a universal or capped 10 ml tube.
  11. Add 2.5 volumes of absolute ethanol. Mix the solution by inverting the tube several times. The DNA should precipitate as a “cotton wool” ball. Using a micropipette tip, transfer the DNA to a microcentrifuge tube containing 1 ml of 70% ethanol.
  12. Centrifuge in a microcentrifuge at 12,000 g for 5 min. Pour off the residual ethanol, and remove all of this with a micropipette.
  13. Leave to dry on the bench for 10 min.
  14. Add 50–500 μl of TE depending on the size of the pellet. Aim to have a DNA concentration of approximately 0.5 mg/ml. Leave to resuspend for at least one night. Mix gently by flicking the tube; never vortex. The DNA can be stored for long periods at 4°C or frozen at -20° C.

Extraction of DNA from Other Sources 
For the analysis of Ig and TCR gene rearrangements, it is necessary to enrich a peripheral blood sample for lymphocytes prior to DNA extraction. This is achieved through the separation of mononuclear cells on Ficoll/Hypaque (or Lymphoprep). After washing the cell pellet in PBS, lysis and DNA extraction can proceed as from step 5.

DNA is extracted from bone marrow aspirates in the same way as from peripheral blood, except that before freezing they are diluted in at least 5 volumes of PBS.

Tissue biopsies vary greatly in nature, size, and cell content and, as a result, so does the quality and quantity of DNA obtained from them. To obtain sufficient quantities of high molecular weight DNA for Southern blot analysis, the biopsy must be several mm3 in size and fresh frozen. If such a biopsy is available, sufficient cells can be obtained by mechanically disrupting it into PBS first by chopping it finely with a clean blade and then breaking it up with the blunt end of a 5 ml syringe plunger. The suspension is centrifuged to obtain a pellet, from which DNA is extracted as before (from step 5). For smaller biopsies, it is better to treat the sample with proteinase K prior to extraction, as follows:

  1. Place the tissue in 700 μl of 50 mmol/l Tris-HCl, pH 8.0, 100 mmol/l NaCl, 1% (w/v) SDS containing 100 μg/ml proteinase K.
  2. Cut up the tissue with a fine pair of scissors, and incubate at 50°C overnight.
  3. Proceed with phenol/chloroform extraction and DNA precipitation as from step 8 earlier.

Determining the DNA Concentration 
Take 5 μl of the DNA solution and dilute into 245 μl of water. Mix well by vortexing. (This DNA is to be discarded and can therefore be treated in this way.) Read the absorbance (A) in a spectrometer at 260 nm against a water blank. An A of 1.0 is obtained from a solution of DNA at a concentration of 50 μg/ml. Therefore, multiply the A reading obtained by 2500 to get the concentration of the original DNA solution in μg/ml. The ratio of the A260 to the A280 gives an indication as to the purity of the DNA solution. This ratio should be in the range of 1.7–2.0.

(Source: Dacie and Lewis Practical Haematology, 10th Edition)

Visit to AIIMS, New Delhi , India

Me at AIIMS, New Delhi, India...

We (including my friends Dr. Seraj and Niraj)  visited All India Institute of Medical Sciences (AIIMS), New Delhi in last year at Christmas. The visit was so fruitful and helpful for us.  We are thankful for all the faculties, PG and PhD students.



Dry skin, slow speech, constipation, decrease GI motility, weight gain despite of reduced appetite, dry hair and fallout, facial puffiness, angina, hyperlipidemia, etc.

Pale, dry skin, goiter, cool peripheries, alopecia, bradycardia, ascites, hydrocele, cretinism in children, etc.

This is the most common disease occurring in 5-15% women after 65 years of age. Myxedema is a severe form of hypothyroidism in which there is accumulation of mucopolysaccharides in the skin and other tissues, leading to a thickening of facial features and a doughy induration of the skin. Cretinism is the term used to describe severe hypothyroidism that develops in the newborn.

Fig. Hypothyroidism examination
Primary hypothyroidism: 
This occurs due to extrinsic factors that affect thyroid gland or due to disease of the thyroid gland. As a compensatory mechanism there is release of TRH and TSH which cause thyroid hyperplasia causing enlargement (goiter). Non goiterous hypothyroidism occurs due to functional loss of thyroid gland despite of increased production of TSH and TRH. Primary hypothyroidism is frequently associated with circulating Antithyroid antibodies (autoantibodies).

  1. Hashimoto thyroiditis – This is characterized by autoimmune destruction of thyroid gland. Autoantibodies are directed against TPO, Tg and other tissues. Initially there is hyperthyroidism due to Overactivity of thyrocytes where stored thyroid hormones are released about for 6-8 weeks, gland becomes enlarged, palpable and tender i.e. goiterous.  But further gland damage leads to permanent hypothyroidism and goiter regresses.
  2. Riedel’s syndrome – Gland become fibrosed
  3. Iodine deficiency – Lowered gland iodine; impaired hormone synthesis. This is the most common cause of goiterous hypothyroidism.
  4. Acute iodine excess – Transient inhibition of hormone synthesis may become permanent in the presence of coexisting thyroid destructive autoimmune activity
Fig. Growth retardation : effect of Hypothyroidism (All the men showing are of same age) 
 Congenital hypothyroidism

    • Caused by structural abnormalities like absent gland, ectopic site, enzymes defects like iodide transport, Organification, peroxidase, deiodinase, mutation in T3, transporter, maternal antibodies. Screening of this condition is done in almost all countries of the world. Here T4 and TSH are measured.
  • Drug induced defect – Lithium, glucocorticoid, propranolol, iodine 

Secondary hypothyroidism: 
Due to extrathyroidal disease. Here TSH is low or normal and T3 and T4 are low due to inadequate tropic hormones.
Anterior pituitary failure – Loss of TSH stimulation of thyroid
Hypothalamic dysfunction – Loss of TRH stimulation of anterior pituitary

In hypothyroidism serum TSH is elevated (due to lack of feedback regulation) whereas thyroid hormone level is suppressed in case of primary hypothyroidism. In secondary hypothyroidism due to defect in pituitary or hypothalamus, TSH is undersecreted which causes reduced synthesis of thyroid hormones.
In many patients of primary hyperthyroidism no goiter or history of goiter is found.

Subclinical hypothyroidism
In this condition TSH level is raised (but <10 mU/L) but thyroid hormone level are normal.

ATA Guidelines for hypothyroid screening

Measurement of TSH
At age 35
Every 5 years after 35 yrs.
More frequently with risk factor or symptoms: goiter, family history, lithium use, amiodarone use.

After giving thyroxine as medication to treat hypothyroidism it is important to wait at least five half-lives (7x5 = 35 days, since levothyroxine has half life of 7 days) before rechecking thyroid function tests in order to achieve a new steady state.


Hyperthyroidism is a hypermetabolic condition caused by excessive production of thyroid hormones. This is also called thyrotoxicosis. The prevalence is low 0.3 to 0.6% in population.

Symptoms: Increase irritability, sweating, palpitation, SOB, loss of weight despite increase appetite (classical feature), increase bowel movement, malabsorption, loss of appetite, etc.

Signs: Tachycardia, tremor, warm and moist peripheries due to increased cutaneous blood flow and sweating, arrhythmias, etc.

Causes of thyrotoxicosis are divided into two types: (I) those associated with frank hyperthyroidism and increased production and secretion of thyroid hormones from the gland,
and (2) those that are not.

TSH measurement shows suppressed level with highly increased thyroid hormones in all cases (except in pituitary adenomas where TSH is inappropriately secreted). For follow up of treatment measurement of free T4 and T3 should be done with TSH until TSH returns to normal.

Graves ‘disease
There is presence of TSH mimicking autoantibodies to TSH receptor. There is diffused goiter due to thyroid hyperplasia, Opthalmopathy, myxedema and hyperthyroidism. It predominately affects female. There is staring eyes with forward protrusion of eyeball and lid lagging behind globe. During this condition there is long period of hyperthyroidism and again hypothyroidism ensues due to excess damage of thyroid cells. Iodine-131 can be used therapeutically to treat hyperthyroidism due to Graves’ and other thyroid disorders, this radioiodine interferes with Organification of iodine, inhibits thyrocytes replication by inducing radiation damage thus controlling thyroid Overactivity. Male to female ratio is 5:1 in having this disease. Laboratory test shows very high level of T3, T4 with low or undetectable TSH except in those rare conditions where there is TSH secreting pituitary adenoma or pituitary resistance to thyroid hormones. TSH within the euthyroid reference range eliminates the diagnosis of hyperthyroidism. When TSH is low and T4 within normal range T3 should be measured as it is highly increased during Graves’s disease and in multinodular toxic goiter (T3-toxicosis).

Toxic Nodular Goiter (Plummer’s disease)
This occurs by autonomously functioning thyroid tissue without requiring TSH. Here multiple sites within thyroid gland autonomously produce thyroid hormones. The biochemical diagnosis of hyperthyroidism is suppression of TSH but thyroid hormones lies at URL. Radioiodine is the treatment of choice. MNG usually results from a low-grade, probably intermittent stimulus to the thyroid gland from iodine deficiency, goitrogens (foods that induce hypothyroidism and goiter in the diet such as cabbage, broccoli, cauliflower, and brussels sprouts), decreased thyroid hormone production, or an autoimmune disease, which causes multiplication and growth of small groups of thyroid cells.

TSH-secreting pituitary tumour
Rarely adenomas of pituitary gland secreting TSH (TSHomas) may produce hyperthyroidism. There is persistence of TSH secretion despite overproduction of thyroid hormones. There is thyroid gland Overactivity and hyperplasia leading to goiter.

Iodine Induced
In individual with goiter due to previous iodine deficiency, chronic administration of excess iodine in the diet can induce a hyperthyroid state. This phenomenon (sometimes called the Jod-Basedow phenomenon) and occurs in patients who already have pre-existing thyroid autonomy, expression of which may have been masked by lack of iodine.

hCG secreting Trophoblastic tumor
Here hCG functions as TSH since it has common alpha subunit and its receptor and TSH receptor have similar ligand binding domain.

[Amiodarone is anti-arrhythmic drug that contain 1-12 mg iodine, this prevent peripheral conversion of T4 to T3 with increased production of rT3 and it also inhibits both iodine uptake by thyroid and entry of T4 into cells and can cause both iodine induced hypothyroidism and hyperthyroidism]. This is also a wolf chaikof effect. Some develop hyperthyroidism if the medication leads to inflammation of the thyroid gland (subacute thyroiditis) and subsequent leakage of stored thyroid hormone into the circulation.

Subclinical hyperthyroidism
Here TSH is suppressed but normal concentration of T3 and T4, usually in URL.

Hyperthyroidism or NTI
Suppressed TSH but elevated fT4 is common picture but it’s difficult to differentiate whether this is due to NTI or hyperthyroidism. A raised T4 is uncommon in NTI. The typical signs and symptoms of hyperthyroidism are absent in NTI. Laboratory pattern shows low T4 and TSH. Here illness decreases 5’ monodeiodinase activity, less T4 is converted to T3. This leads to decreased T3 but high rT3.

How to identify Thyroid dysfunction causes ?

Test to identify cause of thyroid dysfunction

a. Antibodies to thyroid peroxidase
These antibodies are found in almost 95% patients with autoimmune hypothyroidism secondary to Hashimoto’s thyroiditis early in the course of the disease and in some patients with other autoimmune thyroid disease. Their target antigen is thyroid peroxidase enzyme. These antibodies can fix the complement and play a major pathogenic role in autoimmune thyroiditis leading to impaired formation of T3 and T4.

b. Antibodies to thyroglobulin
This occurs with lower frequency and do not fix complement and are not known to play a direct pathogenic role in aetiology of autoimmune thyroid disease in man. These antibodies can interfere in Tg measurement giving falsely decreased concentration in IMA. The main reason to measure them to indicate possible assay interference in Tg assay.

c. Antibodies to TSH receptor
This is called Grave’s disease where autoantibodies (IgG) against TSH receptor are formed and these antibodies mimic the function of TSH leading to overt hyperthyroidism, in this condition the TSH level is reduced. There is thyroid gland Hyperfunction. Other TSH receptor antibodies (blocking antibodies) although infrequently encountered can inhibit gland function and lead to hypothyroidism.

Opthalmopathy in Grave’s disease is poorly understood. Orbital muscle, connective tissue and adipose tissue become infiltrated with lymphocytes and macrophages. The extracellular compartment of extraocular muscle and orbital fibro-adipose tissue becomes oedematous owing to water deposition caused by production of glycosaminoglycans by orbital fibroblasts. TSH receptor is also expressed in orbital connective tissue, orbital fat and extraocular muscle fibers which are also the target of these autoantibodies.

99mTc given intravenously as pertechneate, is concentrated within the gland but not organified into thyroid hormones and therefore diffuses out of the gland with time, and the functioning thyroid tissue can be obtained by imaging technique and determining the total dose taken per time, activity of gland can be determined.

Use of 123I being best isotope to use for imaging thyroid tissue, this provides the idea on Organification process. Here radioactive isotope of iodine is given and proportion of total administered dose of isotoe concentrated within the thyroid gland during a given time period is quantified to estimate the activity of gland.

Thyroid profile testing or Thyroid function test (TFT)

Thyroid blood tests:

The blood tests that may be done as part of a thyroid diagnosis include the following:
  1. Thyroid Stimulating Hormone (TSH) Test
  2. Total T4/ Total Thyroxine
  3. Free T4 / Free Thyroxine
  4. Total T3 / Total Triiodothyronine
  5. Free T3 / Free Triiodothyronine
  6. Thyroglobulin/Thyroid Binding Globulin/TBG
  7. T3 Resin Uptake (T3RU)
  8. Reverse T3
  9. Thyroid Peroxidase Antibodies (TPOAb) / Antithyroid Peroxidase Antibodies
  10. Antithyroid Microsomal Antibodies / Antimicrosomal Antibodies
  11. Thyroglobulin Antibodies / Antithyroglobulin Antibodies
  12. Thyroid Receptor Antibodies (TRAb)
  13. Thyroid-Stimulating Immunoglobulins (TSI)

Test for thyroid dysfunction

a. Measurement of TSH
TSH is measured by sandwich ELISA method by using anti TSH antibodies (Ab against β subunit of TSH) as primary and HRP enzyme conjugated secondary antibody, here TSH is sandwiched between these two antibodies and color is produced when chromogenic substrate like TMB (Tetramethyl Benzidine) is added. Substrate A containing TMB and substrate B containing H2O2 is mixed and added. HRP will release [O] by hydrolysis of H2O2 which will oxidize TMB producing color.

Reference range for TSH is 0.39 – 6.16 µIU/L.

b. Measurement of free T4 and T3
The plasma concentration of free thyroid hormones are extremely small and as most of them are protein bound and especially in NTI or under medication, there is alteration in protein level or hormone itself making their measurement less informative. So, free hormone estimate is used for quantification. The binding of T4 to TBP is overcome by using barbital buffer which will selectively inhibit the binding. Similarly Anilino naphthalene sulfonic acid (ANS) is also used for this purpose. These agents displace T4 from TBG. This is in case of measurement of total T3 and T4.



This is done by evaluating sign and symptoms of thyroid disorders or by palpation. Normal thyroid is rubbery feel; in Graves’s disease and diffused colloidal goiter it has soft consistency. In Hashimoto’s disease it is firm, in thyroid carcinoma and Riedel’s thyroiditis it is rock hard and irregular outline.

Doctors performing ultrasound of thyroid

Symptoms: Increase irritability, sweating, palpitation, SOB, loss of weight, increase bowel movement, loss of appetite, etc.

Signs: Tachycardia, tremor, warm and moist peripheries, arrhythmias, etc.

Symptoms: Dry skin, slow speech, constipation, weight gain, dry hair and fallout, facial puffiness, angina, etc.

Signs: Pale, dry skin, goiter, cool peripheries, alopecia, bradycardia, ascites, hydrocele, etc.

(Courtesy:, Thyroid disease)


The tests used to investigate thyroid dysfunction can be grouped into:

1. Test for thyroid dysfunction: TSH, T4, T3 measurement in serum
2. Test to identify cause of thyroid dysfunction: E.g. autoantibodies and serum TBG, thyroid enzyme activities, biopsy, etc.
3. Test to monitor treatment and detect recurrences of follicular carcinoma: TBG measurement

Factors affecting thyroid function


a. Age
The level of TSH and thyroid hormones are higher in neonates and children which is required for growth and development. In old age slight decrease is seen.

b .Pregnancy
In pregnancy due to effect of estrogen and diminished clearance there is increase in TBG. Also there is increase in deiodination of thyroid hormones in developing placenta. So, during pregnancy there is increase in requirement for iodine (200µg/day) and more T4 and T3 is produced to compensate for overutilization. In early pregnancy due to thyroid stimulating action of hCG there is slight rise in fT3 and fT4, and this suppresses TSH but as pregnancy progress this pattern subsides since hCG also falls thus TSH rises.

c. Non-thyroidal illness
Patients in hospital with NTI have abnormalities in thyroid function tests. A low T3 may be found even though patients are clinically euthyroid; this has been termed as sick euthyroid syndrome. Several mechanisms are involved, including:

  1. Hypothalamic-pituitary-thyroidal malfunction leading to decrease store of TRH and  suppression of TSH due to increased concentration of dopamine, cytokine, cortisol, etc. 
  2. Alteration in plasma concentration and affinity of binding proteins (usually reduced concentration and affinity). High concentration FFA can compete with thyroid hormone binding to plasma proteins so there may be slight rise in T4.
  3. Impaired uptake of thyroid hormones in tissue
  4. Decreased conversion of T4 to T3 in peripheral tissue and receptor dysfunction. This cause marked decline in T3 and slight increase in T4. Excess T4 is converted to rT3 which is metabolically inactive and there is marked increase in rT3.

TSH is the most reliable test of thyroid function in hospitalized patients. Normalization of thyroid parameters occurs during recovery from NTI or refeeding after starvation. These changes in TSH helps in differential diagnosis of thyroid disorder which lead to hyper and hypothyroidism. In NTI plasma proteins are altered, since thyroid hormones are bound by proteins, so measurement of T3, T4 would not indicate exact picture.

In non-thyroidal illness 5’-mono-deiodination (D1) is impaired leading to a decreased production of T3 but increased rT3 due to impaired clearance; however total T4 remains unchanged.

Assessment of thyroid illness in ill patient should be postponed until the illness resolves.

Hypothyroidism in euthyroid sick syndrome shows a reduced total T4 and a slightly subnormal FT4. Serum TSH is probably the best single test to distinguish between euthyroid sick syndrome and hypothyroidism (in the absence of suspected pituitary or hypothalamic disease or medications, such as dopamine or glucocorticoids). A clear elevation of the TSH concentration (>20 mIU/L) would indicate hypothyroidism. Lesser TSH elevations may be seen transiently in euthyroid sick syndrome patients during recovery. If the question of hypothyroidism in acutely ill patients cannot be resolved with TSH and FT4 testing, measurement of rT3 may help (rT3 being low in hypothyroidism and normal or high in euthyroid subjects). Documentation of a normal serum cortisol may help-distinguish euthyroid sick syndrome patients from those with hypothalamic or pituitary hypothyroidism.

Hyperthyroidism in euthyroid sick syndrome shows subnormal TSH values often associated with the acute phase of illness or with glucocorticoid or dopamine therapy. In these ill patients TSH level is mildly suppression in 0.05 to 0.1 mIU/L range as compared to hyperthyroid patients where TSH is highly suppressed.

d. Drugs
Dopamine, glucocorticoid, cytokine decreases TSH secretion. Lithium, iodide decrease or increase thyroid hormone secretion; Propylthiouracil, carbimazole decrease thyroidal synthesis; Oestrogens increases TBG whereas androgen, glucocorticoid decreases TBG. NSAIDs, Phenytoin, carbamazepine, furosemide and salicylate compete with thyroid hormone binding to plasma binding proteins and may increase plasma fT4 concentration.

How Thyroid hormone synthesis and secretion are controlled ?


The most important regulator is TSH. This dimeric peptide hormone comprises a specific beta subunit, that bind to receptor and alpha receptor which is common to gonadotrophins; both subunits along with associated carbohydrate moieties are required for bioactivity. TSH secretion has circadian rhythm, plasma concentration being highest between midnight and 4.00h and lowest at midday.  The classic hypothalamo-pituitary-thyroidal axis for regulation of thyroid hormone synthesis is shown below.

Fig. Regulation of Thyroid hormones(Source:Bishop's clinical chemistry) 

TSH itself auto regulates its release from hypothalamus and pituitary. Pituitary have TSH receptors, on binding of TSH its secretion is inhibited. Interaction of TSH receptor antibodies with TSH receptor in pituitary explains why patients with Grave’s disease may continue to have suppressed TSH weeks or months after normal thyroid hormone concentration achieved after therapy.

Other mechanisms like release of cortisol under stress condition, release of cytokines like IL-1, TNF during illness, somatostatin released during malnutrition all these inhibits TSH release. Thus these factors are important during non thyroidal illness (NTI).

TSH acts via cell surface receptor which is coupled to G protein. This receptor has extracellular N-terminal domain for hormone binding, 7 transmembrane domains and short intracellular C-terminal domain involved in activation of G protein modulators of adenylate cyclase-protein kinase A system.  These characteristics are shared with receptors for gonadotropins which has 40% homology in extracellular domain. This may explain the weak thyroid stimulating activity of hCG. Binding of TSH results in activation of AC and accumulation of cAMP. The calcium and phosphoinositol signaling pathways may also be activated by TSH.



This fraction of free hormone (fT3) is the active and can cross plasma membrane (due to low size) and directly bind to nuclear receptor (TR-RXR heterodimer) which are already bound to Thyroid hormone response cis-element in DNA (TRE) along with repressor, in the promoter region of target gene. Binding of hormone to receptor cause the release of repressor and subsequent recruitment of activators which will activate gene transcription. 

TR have high affinity and high specificity for T3. T3 binds to TR in target tissue with 10 times the affinity of T4. T3 and glucocorticoids enhance transcription of GH gene and so more GH is produced, the anabolic effect of T3 may be due to GH.

Fate of T4 and T3 hormone


 In circulation most of T3 and T4 are carried by plasma carrier proteins. They are,

1.Thyroxine binding globulin (TBG) – bind 70% of circulating thyroid hormones. It binds 80% T3 and 68% T4.
2.Thyroxine binding prealbumin or transthyretin (TBPA) – binds 20% of circulating thyroid hormones. It binds 9% T3 and 11% T4.
3.Albumin – binds 10% of circulating hormones. It binds 20% T3 and 11% T4.

Thus about 99.97% of T4 and 99.80% of T3 is protein bound. Low-density lipoprotein (LDL) specifically binds and transports <1% of total circulating T4-LDL facilitates entry of T4 into cells by forming a T4-LDL complex that is recognized by the LDL receptor

T4 is more tightly bound than T3 to all these proteins. Approximately 0.2% of T3 and 0.03% of T4 are in free form and are active. So although total T4 is 40 times more than T3 but in free form T4 is only 3 times that of fT3. The half life of T3 is 1-2 days and that of T4 is 5-7 days. In circulation normal T4:T3 ratio is 7:1. This protein bound T4 is the reservoir of T4 and accounts for constant supply of free T4 as well as free T3.

Specific transporters are involved in transport of thyroid hormones into cells either in peripheral tissue or to brain and neurons.

Tg is a preprohormone, T4 is prohormone while T3 performs all the biological actions.  In periphery 45% of T4 is deiodinated to T3 and 45% to rT3 by deiodinases (D1, 2, 3). All these deiodinase are selenoenzymes and requires adequate dietary intake of selenium for their expression. So, normal T4, production of about 100 nmol daily, approximately 40 nmol of T3 and 45 nmol of rT3 are produced by peripheral deiodination. In euthyroid state at least 85% of T3 production and all of rT3 are produced by peripheral deiodination of T4. Peripheral 5’ deiodination of T4 to T3 and 5-deiodination to rT3 is catalyzed by D1 and D2.

D1 carries out either 5’-deiodination giving T3 or 5-deiodination giving inactive rT3 and this enzyme is most abundant found mostly in liver and kidney and is responsible for largest contribution to circulating pool of T3, especially in hypothyroid condition. During hyperthyroidism when T4 is high, T3 production is primarily derived from D1. D2 provides important source of T3 in pituitary, brain and brown adipose tissue as well as in other tissues. This maintains the constant level of T3 in CNS. D3 converts T4 to rT3 and is thought to be important extrathyroidal control mechanism or regulate T3 action. Propylthiouracil and propranolol inhibits the conversion of T4 to T3.

Sulphation, glucuronidation, deamination, oxidative decarboxylation, ether cleavage and deiodination are the main routes of inactivation and degradation which are excreted via bile or urine.

What is Wolff Chaikoff effect ?

Wolff-Chaikoff effect:

Iodine deficiency leads to severe mental deterioration especially in children. The recommended dietary intake of iodine is 150µg/day for adults but increases during pregnancy and lactation. If iodine intake drops below 50µg/day then hypothyroidism results. High iodine intake (e.g. in amiodarone therapy) can induce hypothyroidism, goiter and sometimes hyperthyroidism. A large excess of iodide, when given acutely, inhibits the adenylate cyclase response to TSH and iodination of thyroglobulin also inhibit thyroglobulin hydrolysis. This is termed the Wolff-Chaikoff effect.

After few day of exposure to high iodide concentration, thyroidal uptake is very low, the intrathyroidal iodide concentration falls and the synthesis of iodinated Tg recommences. This effect can be used to prepare a thyrotoxic patient for thyroidectomy by giving excess KI.

Thyroid Hormone: Synthesis, Storage, Release and Biological action

Thyroid gland lies in front of trachea, just below the larynx and is the largest gland and butterfly shaped. The gland is bilobed with central isthmus and weighs 10-20g in adult. Behind thyroid gland there are 4 parathyroid glands.

Thyroid gland consists of thousands of follicles filled with colloid inside and cuboidal epithelial follicular cells called thyrocytes. Colloid is composed of thyroglobulin. The interfollicular stroma contains C cells (parafollicular cells) which secrete calcitonin involved in calcium homeostasis. The primary function of thyroid is to synthesize and secrete thyroid hormones.

Fig. Hypothalamus Pituitary Axis

Fig. a) Location of thyroid gland b) Histological structure of Thryoid follicle (Source: Bishop's Clinical chemistry, 6th edition)

Under normal circumstances 10 nmol of tri-iodothyronine (T3) and 110 nmol of thyroxine (T4) are formed per day. The actions of thyroid hormone are exerted through modulation of gene expression. Thyroid hormones promote differentiation and growth; they are essential for normal fetal and neonatal development. Thyroid hormones increases mitochondrial oxidative phosphorylation, increase calorigenesis and oxygen consumption in tissue, except brain. They stimulate protein synthesis, gluconeogenesis, liver glycogenolysis, enhance carbohydrate absorption from GI, lipolysis and accelerate insulin degradation.

Hypothyroidism cause the increase in plasma cholesterol, CK-MM, TBG, creatinine, decreased sodium. T3 also stimulate urea cycle enzyme (CPS) so that urea is excreted rather than ammonia.


Synthesis of T4 and T3 occurs on thyroglobulin (Tg), a glycoprotein of molecular weight 660 kDa with 5000 amino acids, 115 tyrosine residues. Thyroglobulin is synthesized by the thyrocytes and exported to be stored within the colloid of follicular lumen. Other enzyme TPO is also synthesized in thyrocytes and present in apical membrane and involved in oxidation of Iodide to Iodine using NADP+ and H2O2. Use of radioactive iodine uptake (RIU) method has helped in elucidation of the pathway of synthesis of thyroid hormones. About 70% of iodide is present as MIT and DIT in Tg and 30% as in T3 and T4.

Trapping of iodide: 
Iodide from plasma is actively transported by sodium-iodine symporter (2Na+ and 1I- are symported inside cell) situated in basal membrane of thyrocytes. This is an ATP dependent process since iodide is taken against concentration gradient. This is also a rate limiting step in thyroid hormone synthesis. In the apical membrane a anion transporter or iodide transporter protein pendrin mediates iodide efflux into colloid. Pertechnetate (which is used for radioactive imaging of gland since it inhibits the pump for iodine but itself gets transported inside), Perchlorate, thiocyanates (found in certain foods) competitively inhibit iodide pump but are not taken up. The ratio of iodide in thyroid to in serum (T:S) is about 25:1 in normal iodine diet. Small amount of iodide also enters by diffusion so any extra iodide not incorporated in MIT, DIT (about <10%) leaves by this mechanism.

Oxidation of iodide to iodine by thyroid peroxidase: 
Oxidation of iodide only occurs in thyroid gland. This occurs in the luminal side of apical membrane and requires heme containing TPO and H2O2, which is generated by calcium dependent flavoprotein enzyme system situated at apical membrane. Antithyroid drugs like propylthiouracil, thiourea, methimazole, carbimazole inhibits the oxidation of iodide by TPO.

Iodination or Organification:
This occurs in tyrosyl residues present in Tg. MID and DIT are formed through the action of TPO. At first 3rd position of tyrosine is iodinated followed by 5th position. Free tyrosine can also be iodinated but it will not be incorporated into protein since there is no tRNA for iodotyrosine.

Fig. Structure of Thyroid hormones (Source: Tietz Textbook of clinical chemistry, 4th Edition)
Coupling of iodine into tyrosyl residue on thyroglobulin: 
This is also catalyzed by TPO and produces T3 (Tri-iodotyrosine) and T4 (Tetra-iodotyrosine) that remain linked to Tg. Here DIT + DIT forms T4 and MIT + DIT forms T3. Iodinated Tg is stored in follicular lumen. When iodine supply is limited proportion of T3 in Tg is increased.

Internalization of Tg and release of T4 and T3:
Thyroglobulin is internalized by pinocytosis and appears as colloid droplets that fuse with lysosome and undergo proteolytic degradation to release T3 and T4. Some uncoupled MIT and DIT are again deiodinated to Iodide and Tyrosine which are recycled. While in thyrocytes some amount of T4 is converted to T3 by deiodinase D1 and D2. Release into circulation occurs by fusion of endocytic vesicle containing thyroid hormones with plasma membrane.

Iodide released by deiodinase (of MIT, DIT) in thyroid cell constitute the important pool within thyroid in contrast to iodide that enters the blood.

TSH stimulates each of the above process by cAMP mediated Ca++/PI pathway by binding to cell surface G-protein coupled receptor. Prolonged TSH action cause hypertrophy and increase vascularity of thyroid gland leading to thyroid enlargement (Goiter).

Monday, December 3, 2012


To detect
  • Malabsorption
  • Pancreatitis
  • Cystic fibrosis    
  • Enzyme levels
The pancreas is a vital organ in the human body. It is a gland which secretes hormones that control various functions in the body. It is also responsible for pancreatic juice which is a digestive aid that is passed into the digestive tract to help with the breaking down of proteins, fats and carbohydrates. The pancreas is situated in the abdominal cavity and sits below the stomach. Because it is vital in digestion, injury or infection that occurs to the pancreas is considered to be extremely dangerous and must be attended to immediately.
Pancreatic cancer is one of the most dangerous forms of cancer with an extremely low rate of patient survival. The pancreas is responsible for the production of insulin, somatostatin and glucagon which are important hormones in the human body.
The pancreas is an organ that plays a significant role in the performance of the digestive tract within the body. It is responsible for the breakdown of proteins, carbohydrates, and fats and does this with the help of digestive juices that are present within the organ as well as a certain proportion of juices that are created by the intestines. As with any cancer, the development of abnormal cells within the organ will start to have an adverse effect on the overall functionality of the organ. This would cause a lot of internal damage making tests for pancreatic cancer a necessity.
This is because just like with any medical complication, performing a medical test will play a vital role in the diagnosis of the condition as well as helping the medical staff and the patient have a better understanding of how serious the condition is. Performing tests for pancreatic cancer is a very important part of the diagnostic process and is really the only mode of confirmation that the condition has developed. Blood tests for pancreas are usually performed when some of the more common symptoms of the condition are seen. Affected individuals will usually show symptoms of pain, nausea, vomiting, jaundice or weakness in addition to almost debilitating abdominal pain.

There are a number of different tests for pancreatic cancer that will be chosen from on the basis of preference of the doctor as well as variant of the condition. 
Tests For Detecting Pancreatic Cancer
Secretin Pancreatic Function Test
Secretin pancreatic function test is a test that is primarily focused on the hormone 'secretin' that triggers the secretion of a fluid that neutralizes stomach acids and assists in digestion. The test is carried out by the doctor or nurse guiding a tube down the throat and till it reaches the stomach before it is guided into the upper part of the small intestine. A small amount of secretin is then administered before any secretions from the duodenal region are removed through a suction process and then examined for over 2 hours.
Endoscopic Pancreatic Function Test
In the endoscopic pancreatic function test, a probe with an attached light on the head is guided down the throat and into the stomach. Sound waves are emitted from the probe in order to generate images on a monitor in front of the doctor in the operating theater. This method of detecting pancreatitis can also help reveal the presence of gallstones in an effort to prevent invasive methods such as ERCP from making the condition worse. The probe used for this test is also capable of retrieving samples of tissue from the organ thereby allowing for a biopsy method of further diagnosis as well.
Pancreas Blood Test Levels and Results
A patient may suffer from many symptoms associated either with the hormones produced in the pancreas or with the functioning of the digestive system. In either case a pancreas blood test may be ordered to diagnose the problem. In such a situation the blood test for pancreas will check the levels of various hormones as well as the digestive enzymes that are produced by the pancreas. A deviation of these levels from the normal range can indicate the presence of a problem related to the pancreas.
Some of the other methods of pancreas blood tests for diabetes as well as other causing factors will all be governed by their own pancreas blood test levels as well as pancreas blood test results. Before undergoing any of these pancreas tests, it is important to make it a point to consult your doctor and identify if any medications of even lifestyle choices are likely to manipulate the test results.
When it comes to pancreatic cancer most people are affected by a cancer is known as adenocarcinoma of the pancreas. This variety of cancer is known to be one of the most aggressive of all cancers. Recent studies have shown that about thirty thousand individuals are diagnosed with the condition on a yearly basis and more than 90% of these people eventually succumb to the disease. While a family history of the condition definitely puts some individuals at a greater risk than others, certain lifestyle factors also contribute to the development of the condition such as an advancing age, smoking and diabetes mellitus. In depth research on the subject has also shown that males tend to be slightly more prone to the condition than women at a ratio of about 1.3:1. It is necessary to begin treatment for the condition as soon as the condition is detected.
Test For Checking The Functionality of Pancreas
There are many tests that could check the functioning of the pancreas apart from a simple blood test. Fecal matter can be tested for a digestive enzyme known as elastase. One may also use the secretin stimulation test as a pancreas function test. Secretin is found in the upper part of the small intestine and this substance stimulates the release of bicarbonates from the pancreas.

During such a test, an endoscope will be inserted till it reaches the small intestine and secretin will be introduced to the area known as the duodenum. This should stimulate the pancreatic secretions. Within a period of about two hours, the area is drained of its fluid by aspiration. This type of testing for pancreas irregularities will show whether the pancreas is responding properly in terms of its digestive function. Other testing could be performed in order to visually check the pancreas. An ultrasound test can show if there is any inflammation or change in the size of the pancreas that is unexpected. It could also reveal a problem of fluid accumulation around the pancreas. When pancreatic cancer is suspected, a biopsy of the pancreas may be performed to confirm this.

(Courtesy: )




Diagnosis of:

  • Gastric-ulcer/ Peptic-ulcer
  • Pernicious anaemia
  • Zollinger Ellison Syndrome
  • Completion of surgical vagotomy


Examination of
  • resting juice content.
  • fractional gastric juice.
  • gastric juice after stimulation.

alcohol stimulation
insulin/ pantagastrin

  • Tubeless gastric analysis (Azure-A-resin)/ cation exchange resin - excreted into urine (H+ conc) 

Sample collection: Ryle’s Tube


Increased Gastric HCl: 

  1. Zollinger – Ellison Syndrome (a tumor of gastrin secreting cells of the pancreas)
  2. Chronic duodenal ulcer
  3. Excessive histamine production 

Decreased Gastric HCl:  Gastritis, gastric carcinoma, pernicious anaemia.


Lactose Tolerance Test –
  • 50 gm lactose in 200 ml of water
  • Blood specimen at Fasting, 30, 60 and 120 minutes.
  • Normal : More than 30mg/dL.
  • 20-30mg/dL borderline and <20mg/dL - lactase deficiency

D-Xylose absorption test –
  • 25 gm (0.5 gm/kg body weight in children) in 250ml  water
  • blood level after 30’, 1 hr and 2 hrs.      (<25 mg/dL abnormal).
  • urine for 5 hrs (normally 4 gm excretion) (> 20%)
  • normal blood concentration with decreased urinary excretion ---> renal impairment/ incomplete urine collection

To differentiate pancreatic steatorrhoea and malabsorption steatorrhoea

Serum carotenoids –
e.g. lycopene, xanthophyll and carotene malabsorption  
carotenoids  (normal ranges 50 - 250mg/dL)

Fecal fat analysis –

  • normally 1 - 4gm/ 24 hrs (lipid free diet)
  • < 7gm / (lipid rich diet)
  • excretion in biliary obstruction
  • exocrine pancreatic insufficiency
  • diseases of small intestine

Qualitative screening test –
  • Sudan staining for fecal fat
  • Normally 40 to 50 small neutral lipid droplets/ high power

Quantitative fecal fat analysis –
  • 72 hrs stool collection
  • lipid rich diet for 2 days prior to test
  • 50 – 100 gm of lipid each day
  • reference range – 1 to 7gm/ 24 hrs 

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