6. Chapter 6 HCG test.pdf

Serology of HCG tests Menber (BSc, MSc) 1  Pregnancy is the period during which a woman carries a baby with in her body before giving birth.  Pregnancy begins with conception-that is, the fertilization of an egg by a sperm.  The fertilized egg is called zygote....

Serology of HCG tests Menber (BSc, MSc) 1  Pregnancy is the period during which a woman carries a baby with in her body before giving birth.  Pregnancy begins with conception-that is, the fertilization of an egg by a sperm.  The fertilized egg is called zygote. Menber (BSc, MSc) 2  During pregnancy, the chorionic membrane of the placenta produces a hormone called human chorionic gonadotrophin (hCG), which stimulates the secretion of progesterone, by the ovary. Progesterone maintains the uterus during the pregnancy and prevents any further release of eggs from the ovary. Menber (BSc, MSc) 3  Structure of the hCG  HCG is a glycoprotein composed of two non- covalently linked polypeptides:  alpha () sub units (92 aa)  beta () sub units(145 aa)  The individual sub units lack biological activity but become active when linked to form the intact complex. Menber (BSc, MSc) 4  hCG is synthesized in large amounts by the placenta, and it appears in urine, blood, amniotic fluid and serum relatively soon after implantation of the developing embryo.  The rapid rise in concentration of this hormone serves as basis for pregnancy testing(good pregnancy marker). Menber (BSc, MSc) 5  The urine and serum of pregnant women contain high concentrations of human chorionic gonadotrophin (hCG) produced by trophoblast, which provide the basis of tests for the diagnosis of pregnancy.  Specific and sensitive analytical methods for the - chain sub unit of hCG permit the detection of pregnancy as early as 8 days after ovulation (1 day after implantation).  hCG concentrations climb early in pregnancy, reaching a maximum by 8 to 10 weeks of gestation. Menber (BSc, MSc) 6  60 55 50 45 40 35 hCG U/ml 30 25 20 15 10 5 0 0 4 8 12 16 20 24 28 32 36 40 Ovulation period Menber (BSc, MSc) 7  Application of pregnancy tests  Pregnancy tests are important :  To detect early pregnancy.  To determine the adequacy of hormone production in high risk pregnancies (for example, habitual abortion)  To diagnose and monitor patients with germ cell tumours.  Ectiopic disease: the implantation of the fertilized egg in the extra uterine space. Example in the fallopian tube  In threatened abortion: to conform the abortion is complete or not. In this case the quantity of hGC is decreases gradually.  In the infection of hydated mole: to give treatments in the way not harm the fetus to check before mechanical treatment. Example X-ray. Menber (BSc, MSc) 8  Detection of hCG in urine  A number of serologic tests have been used in pregnancy testing; each designed to detect minute amounts of hCG when it appears in the urine during the first few weeks of pregnancy. Menber (BSc, MSc) 9  Specimen: Urine The amount of hormone excreted in the urine is almost the same as that found in the blood. An early morning specimen is preferable because this is the most concentrated and will therefore contain the highest level of hCG  The urine must be collected in  a clean container, which is free from the all traces of detergent.  If the specimen cannot be tested immediately it should be refrigerated at 4c, but for not longer than 48 hr’s.  Specimens preserved with boric acid are also suitable for testing. Menber (BSc, MSc) 10  If the urine is cloudy it should be flittered or centrifuged and the supernatant fluid used.  Specimens that are heavily contaminated, or contain large amounts of protein or blood, are not usually suitable for testing.  There are different types of commercially produced kits for pregnancy tests and each of this is with the technique and precaution with instructions. Menber (BSc, MSc) 11  Types of urine testing kits 1.Rapid latex slide test I. Direct agglutination test II. Indirect or inhibition test 2. Immunochromatographic test Menber (BSc, MSc) 12 Direct agglutination test Principle In the direct slide test, the latex reagent consists of particles coated with anti hCG antibodies. This reagent is mixed directly with the urine there will be agglutination, which is visible to the naked eye if the urine contains the hormone hCG. If not there is no visible agglutination. Menber (BSc, MSc) 13 Procedure  Prepare clean, dry, detergent free slides.  Take one drop of early morning urine in Pasteur pipettes and drop on slide.  Take 1 drop of reagent (anti-hCG antibody).  Mix gently; and see for agglutination.  If hCG is present in the urine---- visible agglutination--- positive tests.  If no hCG is present in the urine--- no agglutination----- negative test. Menber (BSc, MSc) 14  Indirect latex slide test (inhibition latex slide test) Principle:  Antibodies to hCG and urine are mixed and Colored latex or other visible particles coated with hCG are added.  Negative urine results visible agglutination; and positive urine results, presence of hCG in urine inhibits agglutination (or protein flocculation). Menber (BSc, MSc) 15 Reagents:  Antiserum containing anti-hCG-antibody  Latex reagent containing polystyrene particles coated (sensitized) with the hCG antigen. Menber (BSc, MSc) 16 Procedure:  Take clean slides which is free from detergent  Mix a drop of urine with one drop of antiserum on slide.  Add latex reagent  See for positive or negative test.  Agglutination ……negative  No agglutination ….positive Menber (BSc, MSc) 17  If hCG is present in the urine it will combine with the anti-hCG antibody. This will leave no antibody free to combine with the latex hCG and therefore there will be no agglutination of the latex particles. Menber (BSc, MSc) 18  If there is no hCG in the urine, the antibody will be free to combine with the latex hCG and cause agglutination of the latex particles. Menber (BSc, MSc) 19  Immunochromatographic test Most IC card and strip tests are able to detect 50 mIU/ml or even 25 mIU/ml of hCG. Latex slide tests, however, are generally less sensitive, usually becoming positive only when the hCG level is over 500 mIU/ml. Menber (BSc, MSc) 20  Principle The test band region is pre-coated with anti-alpha hCG antibody to trap hCG as it moves through the membrane caused by capillary action. When the patient specimen is added, it reconstitutes an anti beta hCG antibody(reaction zone), which is complexed to colloidal gold particles. This complex is trapped by the anti-alpha hCG and forms a colored complex in the test region in the form of a straight line Menber (BSc, MSc) 21 Interpretation Not Pregnant Pregnant Menber (BSc, MSc) 22 Limitation of the test Urine False Positives – Blood or protein in the urine – Human error in result interpretation – Ectopic production of hCG – Exogenous hCG – Drugs (aspirin, carbamazepine, methadone, high urinary pH and seminal fluid) Urine False Negatives – Early measurement after conception – Dilute urine specimen Menber (BSc, MSc) 23  Quantitative HCG assay There are several types of technique that aids to measure the levels of hCG in serum or urine quantitatively or semi quantitatively. These are: Radio immunoassay ELISA Radio receptor assay (RRA). Menber (BSc, MSc) 24  QUIZ 1. ________is serum marker indicates active HBV viral replication and a higher risk of transmissibility 2. High level of CRP in the blood test indicates ________________ 3. Write the current HIV test algorithm with its interpretation ___________ ____________ ____________ Menber (BSc, MSc) 25 Thank You !!! Menber (BSc, MSc) 26