DETERMINATION OF HORMONES

MOHAMED A.ALI BSc.HLS, ADMLS, DMLS

1.INTRODUCTION
Hormones are chemicals that carry messages from glands to cells within tissues or organs in the body. They are protein or lipid by nature. They also maintain chemical levels in the bloodstream to help achieve homeostasis, which is a state of stability or balance within the body.

Classification of hormones
Hormones can be classified in different ways such as basing on chemical structures, site of production, the way they are produced and acting and other ways. Therefore we can have such as endocrine hormones or exocrine hormones, or autocrine hormone acts on the cells of the secreting gland, and a paracrine hormone acts on nearby but unrelated cells.

How They Work

These chemicals circulate freely in the bloodstream, waiting to be recognized by a target cell, which is their intended destination. The target cell has a receptor that can be activated only by a specific type of hormone, after which the cell knows to start a certain function within its walls. Genes might get activated, for example, or energy production resumed

2. DISCUSSION ON HORMONES. A. THYROTROPIN (TSH)

Thyroid-stimulating hormone (also known as TSH or thyrotropin) is a hormone that stimulates the thyroid to produce thyroxine (T4), and then triiodothyronine (T3) which stimulates the metabolism of almost every tissue in the body. It is a glycoprotein hormone synthesized and secreted by thyrotrope cells in the anteriorpituitary gland, which regulates the endocrine function of the thyroid gland.

THYROTROPIN (TSH)
TSH stimulates the thyroid gland to secrete the hormone thyroxine (T4), which has only a slight effect on metabolism. T4 is converted to triiodothyronine (T3), which is the active hormone that stimulates metabolism. About 80% of this conversion is in the liver and other organs, and 20% in the thyroid itself Normal range 0.47-5.01uIU/L

B. THYROID HORMONES
The thyroid hormones, triiodothyronine (T3) and thyroxine (T4), are tyrosinebased hormones produced by the thyroid gland that are primarily responsible for regulation of metabolism. Tyrosine (abbreviated as Tyr or Y) or 4hydroxyphenylalanine, is one of the 22 amino acids that are used by cells to synthesize proteins. Iodine is necessary for the production of T3 and T4.

THYROID HORMONES
The major form of thyroid hormone in the blood is thyroxine (T4), which has a longer half-life than T3. The ratio of T4 to T3 released into the blood is roughly 20 to 1.

Production
Thyroid hormones (T4 and T3) are produced by the follicular cells of the thyroid gland and are regulated by TSH made by the thyrotrophs of the anterior pituitary gland. The main atom used which used to the formation of these two hormones is Iodine. Iodide is actively absorbed from the bloodstream by a process called iodide trapping

Production.
Via a reaction with the enzyme thyroperoxidase, iodine is bound to tyrosine residues in the thyroglobulin molecules, forming monoiodotyrosine (MIT) and diiodotyrosine (DIT). Linking two moieties of DIT produces thyroxine. Combining one particle of MIT and one particle of DIT produces triiodothyronine. But also T4 is converted to the active T3within cells by deiodinases. Proteases digest iodinated thyroglobulin, releasing the hormones T4 and T3, the biologically active agents central to metabolic regulation.

Effect of Thyroid hormone synthesis In relation to TSH

If there is a deficiency of dietary iodine, the thyroid will not be able to make thyroid hormone. The lack of thyroid hormone will lead to increased production of thyroid-stimulating hormone, which causes the thyroid to enlarge (goiter).

Diagnosis of disease
TSH concentrations are measured as part of a thyroid function test in patients suspected of having an excess (hyperthyroidism) or deficiency (hypothyroidism) of thyroid hormones. Interpretation of the results depends on both the TSH and (T4 and T3)concentrations. Normal range T3 0.8-2ng/ml, T4 4.5-12Ug/L

SOURCE OF PATHOLOGY

TSH LEVEL

THYROID HORMONES LEVEL HIGH LOW HIGH LOW

DISEASE CAUSING CONDITIONS

Hypothalamus/pitui HIGH tary Hypothalamus/pitui LOW tary Thyroid Thyroid LOW HIGH

Begining tumor of the pituitary. Hypopituitarism Hyperthyroidism or Graves' disease Congenital hypothyroidism (cretinism), hypo thyroidism or Hashimoto's thyroiditis

C.PROSTATE SPECIFIC ANTIGEN

PSA-Prostate-specific antigen-secreted by epithelial cells of the prostate gland which lies beneath the bladder.
PSA is a glycoprotein (serine protease) with a molecular weight of 28.4kD, it liquefies semen, and allows sperm to swim freely. It is believed in dissolving cervical mucus, allowing the entry of sperm into the uterus. PSA is present in small quantities in the serum of men with healthy prostates, but is often elevated in the presence of prostate cancer or other prostate disorders. PSA is not only detected in men but also in women with mamma carcinoma.

Pathology
Causes ;The main cause of PC is unknown but change of DNA of the normal prostate cells which can be inherited(5-10%) or non inherited. Risk factors; 1. age. 2.family history.3.Others Diet,Race,obesity,smoking ,infection and inflammation of the prostate. An enlarged prostate is often called benign prostatic hyperplasia (BPH) The symptoms of prostate enlargement are called lower urinary tract symptoms (LUTS). As the prostate enlarges it may cause narrowing of the first part of the urethra.

Method
Many PSA immunoassays are based on the sandwich principle, which uses antibodies to PSA labeled with an enzyme or a conventional or luminescent dye. Normal range of PSA in serum is <4ng/ml.

D.HCG
human chorionic gonadotropin (hCG) is a hormone produced during pregnancy that is made by the developing placenta after conception, and later by the placenta component syncytiotrophoblast.

hCG
Some cancerous tumors produce this hormone; therefore, elevated levels measured when the patient is not pregnant can lead to a cancer diagnosis. However, it is not known whether this production is a contributing cause or an effect of tumorigenesis.

Functions
Human chorionic gonadotropin promotes the maintenance of the corpus luteum during the beginning of pregnancy, causing it to secrete the hormone progesterone. It has also been hypothesized that hCG may be a placental link for the development of local maternal immunotolerance.

Functions
It has also been suggested that hCG levels are linked to the severity of morning sickness in pregnant women. Because of its similarity to LH, hCG can also be used clinically to induce ovulation in the ovaries as well as testosterone production in the testes. Human chorionic gonadotropin also plays a role in cellular differentiation/proliferation and may activate apoptosis.

As a tumor marker
Human chorionic gonadotropin can be used as a tumor marker, as its subunit is secreted by some cancers including seminoma, choriocarcinoma, germ cell tumors, hydatidiform mole formation, teratoma with elements of choriocarcinoma, and islet cell tumor. For this reason a positive result in males can be a test for testicular cancer. The normal range for men is between 0-5 mIU/mL. Combined with alphafetoprotein, -HCG is an excellent tumor marker for the monitoring of germ cell tumors.

Methodology
Most tests employ a monoclonal antibody, which is specific to the -subunit of hCG (-hCG). This procedure is employed to ensure that tests do not make false positives by confusing hCG with LH and FSH. (The latter two are always present at varying levels in the body, whereas the presence of hCG almost always indicates pregnancy.) Many hCG immunoassays are based on the sandwich principle, which uses antibodies to hCG labeled with an enzyme or a conventional or luminescent dye. Pregnancy urine dipstick tests are based on the lateral flow technique. (chromatographic immunoassay )

Methodology
The serum test, using 2-4 mL of venous blood, is typically by immunoassay that can detect hCG levels as low as 5 mIU/ml and allows quantification of the hCG concentration. The ability to quantitate the hCG level is useful in the monitoring germ cell and trophoblastic tumors, followup care after miscarriage, and in diagnosis of and follow-up care after treatment of ectopic pregnancy. The lack of a visible fetus on vaginal ultrasound after the hCG levels have reached 150,000 mIU/ml is strongly indicative of an ectopic pregnancy.

Methodology
NB: The international unit of hCG was originally established in 1938 and has been redefined in 1964 and in 1980. At the present time, 1 international unit is equal to approximately 2.3510-12 moles, or about 610-8 grams.

E.AFP-ALPHA FETOPROTEIN
AFP is a large glycoprotein molecule which is closely relate to human albumin both genetically and structurally. It is produced initially by the fetal yolk sac in small quantities and then in large quantities by the fetal liver as the yolk sac degenerates.

AFP-Alpha fetoprotein
In adults, high blood levels (over 500 nanograms/milliliter) of AFP are seen in only three situations: HCC Germ cell tumors (cancer of the testes and ovaries) Metastatic cancer in the liver (originating in other organs)

The sensitivity of AFP for diagnosing liver cancer.

The sensitivity of AFP for liver cancer is about 60%. In other words, an elevated AFP blood test is seen in about 60% of liver cancer patients. That leaves 40% of patients with liver cancer who have normal AFP levels. Thus, any patient with cirrhosis and an elevated AFP, particularly with steadily rising blood levels, will either most likely develop liver cancer or actually already have an undiscovered liver cancer.

As a marker
An AFP greater than 500 ng/ml is very suggestive of liver cancer. In fact, the blood level of AFP loosely relates to (correlates with) the size of the liver cancer. Finally, in patients with liver cancer and abnormal AFP levels, the AFP may be used as a marker of response to treatment. For example, an elevated AFP is expected to fall to normal in a patient whose liver cancer is successfully removed surgically (resected).

Other significance of the test.

Check the developing baby (fetus) of a pregnant woman for brain or spinal problems (called neural tube defects). Check the developing baby (fetus) of a pregnant woman for Down syndrome.

Methodology
Many AFP immunoassays are based on the sandwich principle, which uses antibodies to AFP labeled with an enzyme or a conventional or luminescent dye.

F.Carcinoembryonic antigen(CEA)
CEA is normally produced during the development of a fetus. The production of CEA stops before birth, and it usually is not present in the blood of healthy adults May be elevated in the serum of some patients with colon and certain other cancers(rectal ,pancreases, breast, ovary or lung)

Diagnostic significance.
The carcinoembryonic antigen (CEA) test measures the amount of this protein that may appear in the blood of some people who have certain kinds of cancers, especially large intestine (colon and rectal) cancer. It may also be present in people with cancer of the pancreas, breast, ovary, or lung.

Find how widespread cancer is for some types of the disease, especially colon cancer.

Diagnostic significance
Check the success of treatment for colon cancer. CEA levels may be measured during treatment with medicines to destroy cancer cells (chemotherapy). This provides information about how well the treatment is working. CEA levels may be measured both before and after surgery to evaluate both the success of the surgery and the person's chances of recovery. Check to see if cancer has returned after treatment.

Methodology
Many CEA immunoassays are based on the sandwich principle, which uses antibodies to CEA labeled with an enzyme or a conventional or luminescent dye. Normal range <10ng/ml Less than 2.5 or 5.0ng/ml

3.HORMONES ANALYSIS
Basing on measuring of hormones main methods can be used, which are 1. Automated by using the machine directly 2. Manually by using ELISA instruments On our site we use the second method to analyze hormones

Manually by using ELISA instruments

This is the quantitative determination of the hormones. By using the ELISA it can be competitive, sandwitch e.t.c

Definitions in the test

Standards- solution/ substance with known concentrations. Antibody coated wells-is 1plate wells coated with monoclonal anti human antibody. Assay buffer-solution containing phosphate buffer with protein as stablizer. Enzyme conjuagates-Non protein moety attached to enzymes Chromogen substrate reagent-solution contains tetramethyl benzidine and hydrogen peroxide. Wash solution- contains phosphate buffer salt solution with 0.05%Tween 20 as detergent, pH=6. Stop solution-1molar Hcl soln. pH <1 Cardboard sealer- to cover the plate during incubation. ELISA- Enzyme linked Immunosorbent Assay.

Specimen collection and preparation

Serum/ plasma are specimens of choice and should be prepared from a whole blood obtained by approved aseptic technique. Separate and refrigerate the specimen at 2-8C 48hrs if testing cannot be done within an hour and let it return to RT before testing. If prolonged storage is required , samples should be stored at -20C for 30days. Freeze-thaw once the specimen during storage.

WORKING WITH REAGENTS AND SPECIMENS

All reagents should be allowed to reach RT before use Do not mix kit reagents from different lot numbers. To prevent risk of contamination, use personal protective equipments like gloves, lab coats,etc. and avoid direct contact with reagents. Do not use expired date reagents Kit should be stored at 2-8C upon receipt and when it is not in use Keep un-used wells in their sealed bag.

Cont
There are different companies which provide reagent kits for the determinations of hormones. Each kit contain antibody-coated microtiter wells, enzyme conjugate, chromogen substrate, wash solution, stop solution, cardboard sealer, standards and controls.

Cont
The standards are there for the preparation of the calibration graphs and then chats used for reading the patients and controls results. They are prepared when the new kit is opened and the controls are run to verify the graph. The standard operating procedures should be followed clearly basing on the specific kit so make sure you read the SOP in a kit before starting procedure.

Cont
After following the SOPs lastly Calculate mean absorbance value of standards during the preparation of the curve at 450nm. Construct a point to std curve by ploting the mean absorbance for each stds against its concentration in in ng/ml or mIU/L on linear graph paper.

References
Fundamental of Clinical chemitry 5th Edition byTietz Natonal Committee for Clinical Laboratory Standards, Evaluation of Precision perfomance of Clinical Chemistry Devices. Approved Guidelines EP5-A(1999) The Merck Manual 18th Edition.