Radioimmunoassay Measuring Substance In The Blood Biology Essay

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Radioimmunoassay (RIA) is a sensitive method for measuring very small amounts of a substance in the blood. Radioactive versions of a substance, or isotopes of the substance, are mixed with antibodies and inserted in a sample of the patient's blood. The same non-radioactive substance in the blood takes the place of the isotope in the antibodies, thus leaving the radioactive substance free.

The assay is based upon the competition of 125I-peptide and peptide (either standard or unknown) binding to the limited quantity of antibodies specific for peptide in each reaction mixture. As the quantity of standard or unknown in the reaction increases, the amount of125I-peptide able to bind to the antibody is decreased. By measuring the amount of 125I-peptide bound as a function of the concentration of peptide in standard reaction mixtures, it is possible to construct a "standard curve" from which the concentration of peptide in unknown samples can be determined.

A patient exhibits a number of symptoms as follows:-

Enlargement of left anterior neck.

Increased appetite over past month with no weight gain

More frequent bowel movements over the same period.

The heart rate is 82 and the blood pressure is 110/76.

There is an ocular stare with a slight lid lag.

The thyroid gland is asymmetric to palpation, weighing an estimated 40g (normal = 15-20g). There is a 3 x 2.5 cm firm nodule in left lobe of the thyroid.

A Total Thyroxine (T4) assay is performed using RIA

The following data is obtained


Replicate 1 (cpm)

Replicate 2 (cpm)

Total Counts added (TC)



Non specific binding (NSB)



Total Binding (0ug/dL) (TB)



Standard 1 ( 30ug/dL)



Standard 2 (15ug/dL)



Standard 3 (8ug/dL)



Standard 4 (4ug/dL)



Standard 5 (2ug/dL)



Standard 6 (1ug/dL)



Standard 7 (0.5ug/dL)



Patient sample



Using the above data calculate the concentration of T4 in the patients serum.

If, B = amount of radiolabelled T4 bound in the presence of standard

and B0 = amount of radiolabelled T4 in the absence of any unlabelled hormone

Then a standard curve can be generated by plotting B/B0 versus log[T4]

From the graph calculate the concentration of T4 in patient sample.

You should show your calculations and provide a graph of the data.

[20 marks]

A number of further tests are performed for this patient and the test results are as follows:-

Patient's value Reference range

Calcium, total (S) 10.6 mg/dl 8.4-10.2 mg/dl

Phosphorus 4.8 mg/dl 2.7-4.5 mg/dl

Alkaline phosphatase (S) 160 U/L 49-120 U/L

T3, Total (S) 311 ng/dl 100-215 ng/dl

TSH (S) <0.1 uU/ml 0.7-7 ug/dl

(S) measured in serum

What are the normal ranges for these substances?

[5 marks]

Why is Calcium and phosphorus measured for this patient?

[5 marks]

The symptoms and the ranges provided are clearly indicative of a thyroid problem. The thyroid is involved in calcatonin secretion and due to the fact that calcatonin is involved in calcium and phosphorus homeostasis. Possible rises and falls in there level could be indicative of a sporadic and unhealthy thyroid. From the data presented the patient has slightly elevated calcium and phosphorus which is in keeping with the raise in alkaline phosphates. Alkaline phosphate is normally present when calcium is in excess.

How would you interpret these results?

[5 marks]

This patient seems to quite clearly have elevated levels of both T3 and T4. This is indicative of an over-reactive thyroid gland. If we look at basic endocrinology TSH dictates the amount of T3 and T4 produced; in this instance the levels of T3 and 4 are elevated whilst levels of TSH are low. If the thyroid was acting dependently of the TSH then low levels of TSH in the system breed low levels of T3 and 4. This is simply not the case leading me to believe that the thyroid gland is secreting T3 and 4 independently of TSH. TSH is a peptide hormone which is secreted by the thyrotrope cells in the anterior pituitary gland, and is often released in relation to the amount of T3 and 4 in the system. Due to the excessive production of T3 and 4 this is leading me to believe that the patient has a tumour of the thyroid. The thyroid gland is also responsible for the production of calcatonin and this hormone is secreted by the parofollicular cells. Calcium levels are only slightly elevated which is leading me to believe the problem is with the follicular cells and not the paro. But without a count performed on calcatonin in the blood it’s hard to tell if the calcatonin hormone is in excess along with the T3 and 4 or not. One explanation of reasonable calcium levels could be that the excessive calcatonin production could be being counteracted by PTH which is produced by the parathyroid gland.

What is the likely cause of this condition and how would you confirm your diagnosis?

[5 marks]

The most obvious conclusion which can be taken from this patient is that she is suffering from hyperthyroidism; although the cause of the hyperthyroidism could be called into question. The most compelling and obvious reason for the hyperthyroidism is that the patient has a tumour of the thyroid and this is causing over production of T3 and T4. However some symptoms are leading me to believe that Graves disease could be a possibility. With Grave’s disease the most startling symptoms are increased BP and heart rate and a ocular stare with a slight lid lag. The orbital symptoms noted here are most typically associated with Grave's disease and result from inflammation and swelling of retro-orbital tissues (this effect is separate from the elevation in thryoid hormone). However, in this case the thyroid is asymmetrical and contains a nodule, whereas the thyroid gland in Grave's disease is symmetrically enlarged and homogeneous. The shape and symmetry is causing me to believe it is actually a tumour but with the ocular stare this has brought some discrepancies to my original hypothesis of the cause being a tumour.

Possible further diagnostic tools which could be used are a fine need aspiration biopsy of the thyroid to confirm whether cancer was present. Other possible tests include a CT scan, MRI which will enable the physician to detect the size of the tumour aswell as if it has spread and finally a thyroid ultrasound which can confirm if the nodule is a mass of tissue or whether the lump is infact just a cyst.

Briefly, explain the symptoms observed

[10 marks]

If a tumour of the thyroid was present this would explain the enlargement as well as its asymmetry. As i mentioned before the possibility of Graves disease is possible with the ocular stare and lid lag however unlikely due to the nature of the nodule. The Thyroid hormone is known to exacerbate the effects of noradrenalin and because noradrenalin is a neurotransmitter for the autonomic nervous system this would explain the increased heart rate and blood pressure. Thyroid hormone is used within the body to increase the metabolism of major groups such as fats carbohydrates and proteins so the food the girl eats is being metabolised at a greater rate than normal and hence not as much of the food is being stored as fat which correlates with no weight gain. The increase in bowel movements seems to be coupled with the increase in appetite meaning more food is being passed through her system and as a consequence has to be excreted out.