Clinical Chemistry Lab - 10 Endocrinology Methodologies

o CLINICAL CHEMISTRY LABORATORY ENDOCRINOLOGY METHODOLOGIES OUTLINE • • Introduction to ELISA • o ELISA Hormone Analysis o Thyroid Function Test o Pancreatic Hormones o Sex Hormones INTRODUCTION TO ELISA • • • Enzymology → kinetic assay (multiple absorbance of sample) Electrolytes → concentration determination (Beer’s law) Endocrinology (hormones) → ELISA (antibodies against specific analyte) o ELISA - enzyme-linked immunosorbent assay o Measure multiple absorbance from calibrator (stndrd) o Plotting values on a graph; line is usually straight slant ELISA • • • • Direct ELISA Indirect ELISA Sandwich ELISA Link o Used for detection of antigen o Found in home test kits o Uses two different abs for the same target ag o Abs bind to diff epitopes o Requires 3 components ▪ Capture ab ▪ Target ag (trapped between ab) ▪ Enzyme-linked ab o Procedure ▪ Capture abs specific for the target ag are immobilized in microtiter well • Well is rinsed to remove unbound abs ▪ Serum sample containing the Ag is added ▪ Enzyme-lined antibodies are added in the well ▪ Substrate chromogen is added • Color development = positive o Direct Sandwich ELISA o Indirect Sandwich ELISA ▪ Primary ab is binding to target ag ▪ Conjugated 2nd ab is detecting primary ab Process o Capture antibody - antibody that should be immobilized or attached to the well ▪ For each well, capture ab is immobilized ▪ Most test kits today have pre-treated abs ▪ These are specific to target • Addition of sample ▪ Contains various analytes for testing ▪ Attaches to capture ab o Addition of enzyme-linked/conjugated antibody ▪ Specific to analytes ▪ Attaches to attached abs forming sandwich ▪ The antibody has linked enzyme • Horseradish peroxidase - most common enzyme used; coupled with conjugated ab o Washing ▪ Incubation 1.5-2 hrs may be done prior to washing • Allows reagents bind with each other • Non-specific agents will float and not bind ▪ Wash 3x ▪ Removes unbound abs and other components ▪ Most crucial step ▪ When washing, there should be no visible remnants (floating abs, cpds) is present in well • Add washing buffer ▪ Washer can be used • Has pressurized buffer that be efficient in washing the wells ▪ Air dry after washing • Paper towel can be used to blot but cannot be used to wipe • Wiping may disturb the sandwhich elisa o Addition of substrate ▪ Tetramethylbenzidine (TMB) - common coupled substrate • Results in blue complex ▪ Allows color change ▪ Color changes due to prolonged standing (darken) o Addition of stop solution ▪ Strong acids are usually used • Eg. Sulfuric acid (H2SO4) ▪ Upon addition, blue → yellow complex ▪ Yellow complex will not darken in prolonged standing o ELISA Reader measure absorbance on each well Reporting o Based on placement of samples ▪ 1-5 - calibrator ▪ 6-7 - control, if required ▪ 8… - samples/batch tests o 8 wells per column reading o o • Determine concentration shift Computer programs usually compute exact conc Common Errors o Graphs with plateau ▪ Due to inadequate washing ▪ Occurs in manual washing 4. Pipette 100 µL of the conjugate working solution into each well. (We recommend using a multichannel pipette). 5. Incubate on a plate shaker (approximately 200 rpm) for 90 minutes at room temperature. 6. Wash the wells 3 times with prepared wash buffer (300 µL/well for each wash) and tap the plate firmly against absorbent paper to ensure that it is dry (The use of a washer is recommended). 7. Pipette 150 µL of TMB substrate into each well at timed intervals. 8. Incubate the plate on a plate shaker at room temperature for 15-20 minutes. (or until Calibrator A attains dark blue colour for desired OD). 9. Pipette 50 µl of stopping solution into each well at the same timed intervals as in step 7. 10. Read the plate on a microwell plate reader at 450 nm within 20 minutes after addition of the stopping solution. HORMONE ANALYSIS • Utilizes ELISA as method of determination THYROID FUNCTION TEST • • • • • • The thyroid gland is a butterfly-shaped endocrine gland that is normally located in the lower front of the neck. The major thyroid hormone secreted by the thyroid gland is thyroxine, also called T4 because it contains four iodine atoms. To exert its effects, T4 is converted to triiodothyronine (T3) by the removal of an iodine atom. This occurs mainly in the liver and in certain tissues where T3 acts, such as in the brain. The amount of T4 produced by the thyroid gland is controlled by another hormone, which is made in the pituitary gland located at the base of the brain, called thyroid stimulating hormone (abbreviated TSH). The amount of TSH that the pituitary sends into the bloodstream depends on the amount of T4 that the pituitary sees. If the pituitary sees very little T4, then it produces more TSH to tell the thyroid gland to produce more T4. Once the T4 in the bloodstream goes above a certain level, the pituitary’s production of TSH is shut off. Principle of the test: o The principle of the following enzyme immunoassay test follows a typical one-step capture or ‘sandwich’ type assay. The assay makes use of two highly specific monoclonal antibodies: A monoclonal antibody specific for TSH is immobilized onto the microwell plate and another monoclonal antibody specific for a different region of TSH is conjugated to horse radish peroxidase (HRP). TSH from the sample and standards are allowed to bind simultaneously to the plate and to the HRP conjugate. The washing and decanting steps remove any unbound HRP conjugate. After the washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stopping solution. The absorbance is measured on a microtiter plate reader. The intensity of the colour formed by the enzymatic reaction is directly proportional to the concentration of TSH in the sample. A set of standards is used to plot a standard curve from which the amount of TSH in patient samples and controls can be directly read. Materials: o Manufacturer: Labor Diagnostika Nord GmbH o TSH ELISA o Precision pipettes to dispense 50, 100, 150 and 300 µl o Disposable pipette tips o Distilled or deionized water o Plate shaker o Microwell plate reader with a filter set at 450nm and an upper OD limit of 3.0 or greater* Procedure: 1. Prepare working solutions of the anti-TSH-HRP conjugate and wash buffer. 2. Remove the required number of microwell strips. Reseal the bag and return any unused strips to the refrigerator. 3. Pipette 50 µl of each calibrator, control and specimen sample into correspondingly labelled wells in duplicate. • • • • TSH test o A high TSH level most often means you have hypothyroidism, or an underactive thyroid. o This means that your thyroid isn’t making enough hormone. As a result, the pituitary keeps making and releasing TSH into your blood. A low TSH level usually means you have hyperthyroidism, or an overactive thyroid. o This means that your thyroid is making too much hormone, so the pituitary stops making and releasing TSH into your blood. T4 tests o A high blood level of T4may mean you have hyperthyroidism. o A low level of T4may mean you have hypothyroidism. o changes in binding protein levels don’t affect free T4levels, many healthcare professionals prefer to measure free T4. T3 test o If your health care professional thinks you may have hyperthyroidism even though your T4 level is normal, you may have a T3 test to confirm the diagnosis. o Sometimes T4 is normal yet T3 is high, so measuring both T4 and T3 levels can be useful in diagnosing hyperthyroidism. Thyroid antibody tests o Measuring levels of thyroid antibody (Links to an external site.)may help diagnose an autoimmune thyroid disorder such as Grave's disease—the most common cause of hyperthyroidism—and Hashimoto's disease the most common cause of hypothyroidism. o Thyroid antibodies are made when your immune system attacks the thyroid gland by mistake. o Grave’s disease - autoantibody produced mimics TSH ▪ TSH receptor antibody (TSHRAb) - mimics TSH ▪ Promotes increased production of T3/T4 due to stimulation of thyroid by TSH-like ab o Hashimoto's thyroiditis - destroys TPO & TG ▪ TPO & TG - important components for the formation of T3 & T4 • Axis/Feedback loop for Thyroid Hormones o Hypothalamus → TRH → Anterior pituitary gland → TSH → thyroid → T3/T4 o Increased T3/T4 is detected by hypothalamus, counts as successful stimulation ▪ Hypothalamus halts TRH production after success • • • o o o o Primary hypothyroidism ▪ Thyroid is not responding to TSH ▪ High TRH & TSH, lower T3/T4 Second hypothyroidism ▪ Issues in anterior pituitary ▪ ↑ TRH, ↓TSH, ↓ T3/T4 Tertiary hypothyroidism ▪ Issues in hypothalamus Hyperthyroidism ▪ Caused by increased stimulation, cancer PANCREATIC HORMONES • • • Pancreas is both exocrine and endocrine gland. The exocrinal part secretes pancreatic fluid into the duodenum after a meal. The endocrinal part secretes various types of hormones. These are produced by a specialized tissue in the pancreas and then released to the capillary system and reached the liver by the portal venous circulation. The specialized tissue is called islets of langerhans. Islets of Langerhans represent approximately 1-2 % of the pancreas. Types of cells are regonized in these islets. o A cells – producing glucagon (25% of all islet cells). ▪ Promote glucose increase o B cells – producing insulin (60% of all islet cells). ▪ Control glucose levels o D cells – producing somatostatin (10% of all islet cells). o F cells – producing panceratic polypeptide (5% of all islet cells). Islets of Langerhans play a crucial role in carbohydrate metabolism and so in a plasma glucose concentration. It involves: o Glycolysis– the anaerobic conversion of glucose to lactate. Occurs in the red blood cells, renal medulla and skeletal muscles. o Glycogenesis– the synthesis of glycogen from glucose. Glucose is stored ( in liver, muscle) in the form of glycogen and this serves to maintain a constant plasma glucose concentration. o Glycogenolysis– the breakdown of glycogen to glucose. Gluconeogenesis – the production of glucose from non-sugar molecules (amino acids, lactate, glycerol) o Lipolysis– the breakdown of triacylglycerols into glycerol and free fatty acids. o Lipogenesis– the synthesis of triacylglycerols. Function of Pancreas o Pancreatic hormones are responsible for storage of fat and glucose, as glycogen, after meal. o Enables the mobilisation of energy reserves as a result of food deprivation, stress, physical activity. o Maintain the constant plasma glucose concentration. o Promote growth. Pancreatic Hormones: o Insulin o Glucagon o Somatostatin Principle of the test: o The human insulin assay used is a sandwich ELISA. The solid phase microplate is coated with a blend of monoclonal antibodies directed against distinct epitopes of the human insulin peptide. Samples, standards and controls are pipetted into these wells, along with a conjugate monoclonal antibody against human insulin, which has horseradish peroxidase (HRP) covalently linked to it. During the incubation the insulin antigen binds to the immobilized capture antibodies. At the same time the conjugate antibody is binding to the insulin present in the well forming the sandwich. After washing to remove unbound materials, a chromogenic-substrate for HRP, consisting of tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) is added. The substrate is converted to a colored compound by the action of HRP. The reaction is halted by the addition of H2SO4 and the microplate is read spectrophotometrically. The absorbance of the solution is proportional to the amount of insulin present in the original sample. Materials o BioTek (Machine and commercially prepared reagent) o Serum sample o Pipettes Procedure: 1. Samples and standards were prepared off line and 50 µL of each was pipetted manually into the assay plate. 2. After loading samples and standards, 50 µL of assay conjugate was added to all wells. The plates were incubated at room temperature for 30 minutes on a microplate rotary shaker. 3. After incubation the plates were transferred to ELx405 Automated Microplate Washer and washed three times with 400 µL of washer buffer with a 15 second soak period between wash cycles. After washing, 100 µL of substrate solution was added. 4. Plates were returned to the microplate shaker and the color development allowed for 7.5 minutes. 5. After color development, 100 µL of Sulfuric acid stop solution was added and the absorbance of each well at 450 (650 nm reference wavelength) was determined using Synergy™ Mx Multi-Mode Microplate Reader (BioTek Instruments). o • • • • • • Results of the test: o Normal values for Insulin Determination ▪ Optimal: 3-8 uIU/mL (18-48 pmol/L) ▪ Low: <3 uIU/mL (<18 pmol/L) ▪ High: >8 uIU/mL (>48 pmol/L) Low Insulin o Many labs cannot detect insulin lower than 2 uIU/mL. In such cases the result is reported as “below detectable limits.” If there is no detectable fasting insulin ( <2 uIU/mL, or <12 pmol/L) and blood sugar is significantly elevated (ex HgbA1C >6.4%, or >46 mmol/L), the patient could have type 1 diabetes or Latent Autoimmune Diabetes of Adulthood (LADA) and should be immediately assessed. High Insulin o High insulin on a fasting blood test indicates a chronically anabolic state – effectively storing sugars while building fat and muscle. Practically, high insulin results in central obesity and high blood pressure. High fasting insulin is rare in a thin or frail person. Future risk of type 2 diabetes begins to go up in those with fasting insulin above 8 uIU/mL (48 pmol/L). ELISA is not usually done due to expensive procedures and procedures for insulin determination can be done with FBS procedures. • • MATERIALS • • • Sex hormone, a chemical substance produced by a sex gland or other organs that has an effect on the sexual features of an organism. Progesterone, testosterone, androgens, estrone, and estradiol are steroid hormones synthesized either in the testes, ovaries, or adrenal glands. Each of these steroid hormones has cholesterol as its precursor. • • Morning blood sample for men reagents for immunoassays PROCEDURE SEX HORMONES • Total testosterone o Generally draw blood for testosterone lab tests between 7 a.m. and 10 a.m. Total testosterone measures free, albumin bound and SHBG bound testosterone in the blood and is the most commonly used measure of testosterone levels. It is the test usually used to assess testosterone levels in men with suspected hypogonadism or testosterone deficiency. There are several methods available to measure total testosterone. Free Testosterone o A test for free testosterone measures only the 2% of testosterone which remains unbound to proteins in blood. Free testosterone levels may be measured in men whose total testosterone test results showed testosterone levels which are on the borderline of levels considered to indicate testosterone deficiency. Total testosterone o Automated immunoassay systems ▪ Automated platform immunoassay systems are testosterone tests in which most steps (e.g. adding the blood sample, adding reagents) are performed automatically by a machine, rather by people in the laboratory. These tests are the fastest method of assessing testosterone levels. They are considered accurate enough for assessing testosterone levels in men but not in women. Free Testosterone o Direct immunoassay test kits ▪ Direct immunoassay test kits are the easiest and fastest available methods of assessing free testosterone. They are test kits in which reagents containing testosterone antibodies and tracer particles (either radioactive or non-radioactive) which bind to free testosterone in the sample. The tracer particles are easily identifiable and can be counted to measure the concentration of free testosterone in the sample. The results are less accurate than other methods though and lower results are typically obtained compared to equilibrium dialysis. RESULTS OF THE TEST PRINCIPLE OF THE TEST • • Testosterone level range: o MEN: 300 to 1,000 nanograms per deciliter (ng/dL). o WOMEN: 15 and 70 ng/dL. Testosterone levels can decrease naturally due to your age or other health conditions. After the age of 40, men’s testosterone levels decrease by an average of at least 1 percent every year. Some symptoms of low testosterone, particularly erectile dysfunction, are commonly seen in men over 40. Low testosterone levels have often been observed in people with obesity, no matter their age. The most common testosterone-related problem in men is hypogonadism, also called low testosterone.