Microparticle - PowerPoint Presentation

Slide1

Microparticle

Enzyme Immunoassay

MEIA)

)Slide2

Microparticle

Enzyme Immunoassay (MEIA)

is an immunoassay method that utilizes the isolation of antibody/antigen complexes on a solid phase surface of small beads called

microparticles

.

MEIA

has been widely adapted to automate the measurement of large molecules such as markers associated with cardiac, fertility, cancer, metabolic, hepatitis, and thyroid testing.Slide3

Chemiluminescent

Magnetic Immunoassay (CMIA):

Chemiluminescent

label conjugated to the antibody or antigen, and it produces light when combined with it’s substrate. This method is similar to MEIA, thought the

chemiluminescent

reaction offers high sensitivity and ease of measurement. Slide4

CMIA

used for

quantitative determination of human thyroid stimulating hormone in serum and plasma

.

In ARCHITECT instrument, TSH assay is a two-step immunoassay to determine the presence of TSH in serum or plasma

by CMIA

technology with flexible assay protocols referred to as

chemiflex

.Slide5

CMIA principle

In the first step, sample,

anti

TSH antibody coated

paramagnetic micro particles

and TSH assay

diluent

are combined. TSH present in the sample binds to the anti TSH antibody coated micro particles. After washing, anti- alpha TSH

acridinium

labeled conjugate is added in the second step

.

Pre-trigger and trigger solutions are then added to the reaction mixture: the resulting

chemiluminescent

reaction is measured as

relative light units(RLUs

).Slide6

A direct relationship exists between the amount of TSH in the sample and the RLUs detected by the instrument optical system.Slide7
Slide8

Detection technology

Label

Separation step

Solid phase

Techno-logy

Fluorscence

detector

Alkaline

phosphatase

enzyme

Glass fiber matrix

Latex

microparticle

MEIA

Chemiluninescence

photomultiplier tube

Chemiluminescent

compound

magnet

Magnetic

micriparticle

CMIASlide9

Assay components

The components of

MEIA

include the following, all suspended in a specific buffer optimized for the assay:

Microparticle

-Antibody Solid Phase:

Latex

microparticles

that are coated with antibody to bind the specific analyte being measured;

Antibody-Enzyme

Conjugate:

Alkaline

Phosphatase

enzyme conjugated to antibody;Enzyme Substrate: Fluorescent 4-Methyl Umbelliferyl Phosphate (MUP) in solution that is available for a reaction with the enzyme on the antibody.Slide10

Assay principle

MEIA:

technology uses a solution of suspended, submicron sized latex particles to measure analytes.

The

particles are coated with capture molecule specific for the analyte being measured.

The

effective surface area

of micro particles

decreases

assay incubation time. This permits MEIA assays to be

completed

in less time than other immunoassay.Slide11

In the sampling center, reactants and sample for one assay are transferred to a reaction vessel. The reaction vessel is transferred to the processing center where reagents and sample are incubated to allow them to come to reaction temperature.

The reagents and sample are combined and the reaction mixture is transferred to an inert

glass fiber matrix.

Irreversible binding of the

microparticles

causes the immune complex to be retained by glass fibers while the reaction mixture flows rapidly through the large pores in the matrix.Slide12

An alkaline phosphate- labeled conjugate is added to the glass fiber matrix

prior

to the addition of 4-

methylumbelliferyl

phosphate MUP. The conjugate catalyzes the hydrolysis of MUP to

methylumbelliferone(MU).

Measurement

of the fluorescent

MU

as it is generated on the matrix is proportional to the concentration of the analyte in the test sample.Slide13
Slide14

MEIA reaction

sequences

There are two types of reaction sequences or formats for MEIA assays

:

One step:

Sample,

microparticles

and conjugate are combined in the incubation well of the reaction vessel

.

Two step:

Sample and

microparticles

are combined in the incubation well of the reaction vessel and the conjugate reaction takes place on the matrix cell.Slide15

Procedure

Analytes bind to micro particles:

Samples and micro particles are combined and incubated at reaction temperature. During the incubation period analytes bind to the micro particles creating an immune complex.

Immune complex binds to glass fiber matrix.

The processing probe aspirates the reaction vessel and dispenses it onto the matrix cell. The immune complex binds irreversibly to glass fiber matrix. A matrix cell wash removes unbound materials. The immune complex is retained by the glass fibers while the excess reaction mixture flows rapidly through the large ores in the matrix.Slide16

Thyroid

hormones Slide17

The thyroid gland

The thyroid gland is a small butterfly-shaped gland at the base of the neck. It weighs only about 20 grams. However, the hormones it secretes are essential to all growth and metabolism. The gland is a regulator of all body functions. Thyroid disorders are found in 0.8-5% of the population and are 4 to 7 times more common in women.

Types of thyroid disease

There are many types of thyroid disease. However, the main conditions present in most thyroid illnesses are

hypothyroidism

(thyroid under activity) and

hyperthyroidism

(thyroid over activity).Slide18

The amino acid tyrosine is the starting point in the synthesisSlide19

Thyroid hormones releaseSlide20
Slide21

Radioactive iodine tests, testing uptake by the thyroid gland

Hormones concentration T3,T4

Thyroid Binding

Globulin

Concentration of TSH

Urinary excretion of thyroid hormones

Tests influenced by the actions of thyroid hormones.(glucose tolerance test, calcium,

cholestrol

)

Thyroid antibody tests for cases autoimmune

.

Commonly

used thyroid

testsSlide22

The main contribution of chemical measurements to the investigation of thyroid disease is to help sub-divide patients into the categories of effects of treatment.

Tests for evaluate thyroid glandSlide23

It must be emphasized that

a single

thyroid function test

is

NOT

absolute in diagnostic accuracy and thus, a careful selection

of tests, so that their combination

can give

comprehensive data, would enhance

the diagnostic

accuracy.

Thyroid hormones Slide24

Type of sample: blood,

urine or saliva

M

ost

blood tests involving hormones measure the bound protein.  However, saliva-based testing measures the free level of hormone. 

Conditions for test: you should ask the patient if he/she under treatment and receive drug or made

surgery thyroidectomy

recently.

Preparation the patient: be kind when you deal with patient.

You should read the diagnosis status on request.

Sample collection Slide25

Concentrations are usually lowered in

thyroxicosis

, due feedback inhibition of hypothalamus and pituitary

Conversely, plasma TSH is increased in hypothyroidism, except for the rare cases of hypothyroidism secondary to pituitary disease.

TSH Slide26

TSH

is the most appropriate test when monitoring patients receiving thyroxine for the treatment of hypothyroidism.

It should be measured no sooner than

6-8 weeks

after the start of treatment.

In the unusual situation where thyroid function needs to be assessed before this time, FT4 should be used, as the TSH will not have plateaued at this stage. Slide27

TSH:

In

most situations TSH

analysed

using a high sensitivity assay is now accepted as the first line test for assessment of thyroid function. A TSH between 0.4 and 4.0

mIU/L gives 99% exclusion of hypo- or hyperthyroidism,12 while the TSH is considered more sensitive than FT4 to alterations of thyroid status in patients with primary thyroid disease.

Thyroglobulin

:

Levels

are increased in all types of thyrotoxicosis, except

thyrotoxicosis

factitia

caused by self-administration of thyroid hormone. The main role for thyroglobulin is in the follow-up of thyroid cancer patients. After total thyroidectomy and radioablation, thyroglobulin levels should be undetectable; measurable levels (>1 to 2ng/mL) suggest incomplete ablation or recurrent cancer. Slide28

Thyrotropin (TSH)-secreting adenomas

These rare tumors make too much thyroid-stimulating hormone (TSH), which then causes the thyroid gland to make too much thyroid hormone. This can cause symptoms of hyperthyroidism (overactive thyroid),Slide29

Is more biologically active than T4 but both hormones have similar actions in the body.

T3 is not usually used in confirming the diagnosis of suspected hypothyroidism because other tests can demonstrate hypofunction of the thyroid gland.

T3 tests are often useful to diagnosis hyperthyroidism or

todetermine

its severity

Sometimes, however, a patient may have clinical signs of thyrotoxicosis with a normal T4. Measurement of the T3 is then needed, because T3 may be elevated in thyrotoxicosis while other thyroid tests are still the normal range.

T3 testing rarely is helpful in the hypothyroid

patient,since

it is the last test to become abnormal.

T3Slide30

FT3

- FT3 has little specificity or sensitivity for diagnosing hypothyroidism and adds little diagnostic information. The main value of FT3 is in the evaluation of the 2 to 5% of patients who are clinically hyperthyroid, but have normal FT4. In this situation, an elevated FT3 would be suggestive of T3

toxicosis

, in which the thyroid secretes increased amount of T3 or there is excessive conversion of T4 to T3.Slide31

The interpretation of results for serum T4 needs to take into account alterations in the thyroid-binding proteins.

The free thyroxine index (FTI) is determined by the following calculation:

FTI = Thyroxine (T4)/Thyroid Binding

Capacity

The FTI is a normalized determination that remains relatively constant in healthy individuals and compensates for abnormal levels of binding proteins

.

Hyperthyroidism causes increased FTI and hypothyroidism causes decreased

value.

T4Slide32

FT4

: This

test measures the metabolically active, unbound portion of T4. Measurement of FT4 eliminates the majority of protein binding errors associated with measurement of the outdated total T4, in particular the effects of oestrogen.Slide33

FreeT4

ng\dl

{.8 – 2.2}

TotalT4

Ug

\ml

{4.5 – 12.5}

Free T3

Pg\ml

{1.95- 5.85}

Total T3

ng\ml

{.6 – 1.85}

TSH

MlU\ml

{.5-5 }

Parathyroid hormone(PTH)

Pg\ml

{9-52}

Reference rangesSlide34

There are marked variations in the thyroid function with age, evident in all of the vitro hormone and proteins measurements.

In general thyroid function parameters in healthy subjects show little or no clinical significant gender or racial variation.

Clinical interpretation Slide35
Slide36
Slide37

Some persons produce antibodies against their thyroid that either stimulate or damage the

gland.The

two major anti bodies that interfere with thyroid function are

antithyroid

peroxidase (anti-TPO) and

antithyroglobulin.

Both

anti bodies are readily detected in the serum. The presence of anti-TPO and/or

antithyroglobulin

antibodies in a patient with clinical hypothyroidism is diagnostic for Hashimoto’s

thyroiditis

.When these same antibodies are detected in a patient with clinical hyperthyroidism, suspect autoimmune thyroid disease.Slide38
Slide39

Quality control, the best step for all analytical tests to have a reliable result and a voiding the errors.

Pre analytical

Analytical process

Post analytical process

Clinical errors Slide40

A 63-year-old woman has Hashimoto’s disease. Her thyroid laboratory values today include the following

:

She feels consistently run down and has dry skin that does not respond to the use of hand creams.

The hormones levels:

TSH

10.6 mIU/L (normal (0.5–4.5 mIU

/L

)

A

free T4 concentration of 0.5 ng/

dL

(normal 0.8–1.9 ng/

dL

).Case studySlide41

That’s all