Block B - testing for allergy

Question 1

hayfever ?

Answer 1

Hay fever which is triggered by the allergen pollens, symptoms include sneezing, mucus secretion, itchy teary eyes

Question 2

asthma ?

Answer 2

Asthma is triggered by the allergen pollens, House dust mites, animal fur which affect the lungs. Symptoms include coughing and constriction of airways

Question 3

insect bites ?

Answer 3

Insect bites are triggered by saliva. Symptoms include wheal and Flare, Itch

Question 4

food allergy ?

Answer 4

food Allergy is triggered by peanuts, shellfish. Symptoms include welling of lips, tongue larynx.

Question 5

anaphylaxis shock system?

Answer 5

Anaphylactic Shock Systemic response to food allergy occurs (ie non-localized) is a symptom along with include increased vascular permeability, swelling of lips, tongue and larynx,Smooth muscle contraction in lungs causes a fall in blood pressure and DEATH IS A POSSIBILITY.

Question 6

benefit for testing for allergy ?

Answer 6

Benefits of testing include the ability to identify allergens for a person so that they can be avoided. This may be especially important in the industrial setting to monitor desensitization treatment.

Question 7

passive cutaneous anaphylaxis ?

Answer 7

Passive Cutaneous anaphylaxis: is a test carried out by injecting rats intravenously with Evans Blue , an azo dye with a high affinity for serum albumin. Then inject rats with serum sample from patient. Any IgE present will bind to Fc receptors on the rat’s mast cells. The rat is then allergic to whatever the patient is allergic too. Inject each rat intradermally with a different allergen. If IgE specific for any of the allergens is present, they bind to the allergen and cause mast cell degranulation. Where mast cell degranulation occurs, a blue area appears next to injection site due to increased vascular permeability. Instead of a red wheal and flare reaction with a mosquito bite, a blue area arises on the rat from the Evans blue injected.

Question 8

What is a postive read out for passive cutaneous anaphylaxis ?

Answer 8

Antibodies cross link with IgE and cross linking occurs this causes increased vascular permeability and the Evans blue escapes into the skin which causes skin wheal and flare reactions. This is a positive read out.

Question 9

skin testing ?

Answer 9

This can be done in the person , not requiring a rat. The persons back marked into grids and small doses of allergens applied and skin scratched. Back is examined after 30 minutes for characteristic wheal and flare reaction.

A wheal and flare reaction is a raised area of skin that is red, itchy bump that symbolizes the presence of an allergy antibody. The larger the wheal and flare reaction the greater the sensitivity to the allergen

Question 10

radioimmunosorbant (RIST) assay

Answer 10

measures total IgE

Paper disks coated with anti-IgE is incubated with serum sample. Any IgE present binds to the disk. Disks are washed and incubated with anti-IgE-radiolabeled antibody. Disks are washed and the amount of radioactivity present recorded.

Question 11

benefits and disadvantges of RIST?

Answer 11

This is a quantifiable reaction , with the appropriate standards the amount of IgE present can be calculated. However, it doesn’t tell you what allergen the IgE binds to.

An advantage of using this test is that the dye allows both visualisation and relative quantification of the localised allergic reaction.

Question 12

radioalllergosorbant assay ( RAST )

Answer 12

Paper disks coated with known allergens and incubated with serum sample. Any IgE specific for any specific allergen binds to the allergen. Disks are washed and incubated with anti-IgE-radiolabelled antibody. Disks are washed and the amount of radioactivity present recorded.

Question 13

advantages of RAST ?

Answer 13

Here you can quantify the amount of IgE present using a standard curve

RAST can determine which allergens a person responds

Question 14

how to remeber the difference between RIST and RAST ?

Answer 14

Remember by A for allergen and I for immunoglobulin.

Question 15

future protein array - high throughput out methods ?

Answer 15

Miniaturization and testing of hundreds of different antigens on a micro array. Replacement of radiolabel by fluorescent label.

Can get a lot of results so much quicker.

Read in automated machines. Possible to test against 100s of specific allergens

Question 16

future bead arrays - cytometric bead array ?

Answer 16

Miniaturization and testing of many different antigens on fluorescent beads. Replacement of radio-label by fluorescent label conjugated to an antibody

Question 17

advantages to bead array above ?

Answer 17

daptable: can be used as a capture system too, instead of antigen on the beads it can be antibodies present. (e.g. measuring cytokines in serum).

Question 18

what will flow cytometry allow ?

Answer 18

Once incubabtion occurs , flow cytometry can be used for the beads.

Standards can be run with different concentrations of analyte . Beads can be separated based on the bead colour.

Standards curves can be generated for each analyte

Concentration of samples can be determined from standard curves , easily quantified.

Question 19

antihistamines ?

Answer 19

Antihistamines block release or action of histamines

Question 20

desensitisation therapy ?

Answer 20

Desensitisation therapy uses increasing doses of antigen ( allergen) injected subcutaneously results in a shift from IgE to IgG production. The IgG will mop up any left over IgE and prevent the symptoms

Question 21

why is therapeutic monitoring important ?

Answer 21

Therapeutic monitoring is important to achieve the correct dose of drug in patients as you gain the maximum therapeutic benefit. Furthermore, you can prevent unwanted side effects.

Question 22

how is the correct dose of drug determined ?

Answer 22

The correct dose of drug was determined for patients from formulae taking account of weight, age or surface area. Doses were increased or decreased based on the clinicians interpretation of the patients response . Poorly experienced clinicians were at a disadvantage and so were their patients.

Question 23

why are empirically derived formulae of little use to unique patients ?

Answer 23

Empirically derived formulae are of little use for unique patients. For example those having other forms of confounding treatment such as other drugs or dialysis due to poor kidney function. It is also hard to accurately adjust doses if patient compliance is low i.e.. Person not taking drug or the person giving the drug to a person in care does not give drug.

Question 24

how can you measure the drug in the patients serum ?

Answer 24

Measure Drug in serum by direct method by using HPLC machines , however this requires a skilled user.

Measure drug in serum by Immunoassay e.g. FPIA.

Question 25

fluorescent polarisation immunoassay ?

Answer 25

Based on principle described by Danliker and Feigen, polarized light is used to excite a fluorescent label on a drug. It is Homogenous assay as no washing steps -competitive assay as a labelled drugs competes against a non labelled drug.

Use restricted to molecules with Molecular Weight less than 20,000 Includes Insulin, Cortisol, hCG, Drugs (Digoxin, Theophyline & Gentamycin). It requires Fluorescently labeled Drug and rrequires antibody to drug as well as requires machine (TDxFLxｨ System).

Question 26

control one - drugs with fluorescent light in cuvette ?

Answer 26

Light causes the ‘free’ drug to spin in the cuvette. Polarization is therefore lost in emitted light as the drug is small , this is detected by the detector.
Calculates how much light is conserved and lost.

Question 27

control 2 - drugs with fluorescent label antibodies that bind drugs ?

Answer 27

As drug is bound with Ab in the cuvette it is too large it is prevented from spinning. Polarization is therefore kept and not lost.

Question 28

sample one - drug with fluorescent label antibodies that bind drug sample containing high concentration ?

Answer 28

The drug in the sample is competing for the antibody, and binds which leaves the drugs free to rotate . Non-labelled drug binds Ab leaving labeled drug free to rotate. Polarization is therefore lost.

Question 29

sample 2 - drugs with fluorescent label antibodies that bind with sample containing low drug concentration ?

Answer 29

Not sufficient unlabeled drug to bind Ab therefore labeled drug also bound by Ab and does not rotate. Polarization is therefore conserved and not lost.

Question 30

why is FPIA beneficial ?

Answer 30

Is responsible for success of Therapeutic drug Monitoring and can measure drug concentrations in serum: accurately, quickly and easily