Urine Calcium and Chloride Determination PDF

URINE CALCIUM DETERMINATION: SAMPLE OF CHOICE : For both urine chloride and urine calcium determination, the sample of choice is typically a **24-hour urine collection**. This type of sample provides a comprehensive measure of the levels of these ions over a full day, which helps in diagnosing and...

URINE CALCIUM DETERMINATION: SAMPLE OF CHOICE : For both urine chloride and urine calcium determination, the sample of choice is typically a **24-hour urine collection**. This type of sample provides a comprehensive measure of the levels of these ions over a full day, which helps in diagnosing and monitoring various health conditions. Why 24-Hour Urine Collection? - **Accuracy**: It accounts for fluctuations in urine output and concentration throughout the day. - **Comprehensive**: Provides a complete picture of excretion rates for these ions. There are several methods used to determine calcium levels in urine: 1. Colorimetric Method (Calcium Arsenazo III)** This method uses Arsenazo III reagent to form a blue-colored complex with calcium ions. The intensity of the color is proportional to the calcium concentration. 2. Atomic Absorption Spectroscopy (AAS) AAS measures the absorption of light by calcium atoms in a sample. It's highly accurate and can detect low levels of calcium. 3. Ion-Selective Electrode (ISE) Method This method uses an electrode that is selective to calcium ions. The potential difference measured is proportional to the calcium concentration in the sample. 4. Complexometric Titration (EDTA Titration) EDTA (ethylenediaminetetraacetic acid) forms a complex with calcium ions. The amount of EDTA used is measured to determine the calcium concentration. 5. Sulkowitch Reagent Method This method involves adding Sulkowitch reagent to the urine sample, which reacts with calcium to form a precipitate. The amount of precipitate is measured to determine calcium levels. Each method has its own advantages and is chosen based on the specific requirements of the analysis. Do you have a particular method in mind or need more details on any of these? The Sulkowitch reagent IS COMPOSED OF” - 2.5 g of oxalic acid - 2.5 g of ammonium oxalate -5 mL of glacial acetic acid - Distilled water to make 150 mL When this reagent is added to a urine sample containing calcium, a milky precipitate of calcium oxalate forms. This reaction helps in detecting the presence of calcium in the urine. URINE CHLORIDE DETERMINATION There are a few common methods for testing chloride levels in urine: ### 1. **Volhard-Harvey Method** This method involves a titration process using silver nitrate and ammonium thiocyanate. Steps: 1. Add a measured excess of silver nitrate to the urine sample. 2. Filter the solution to remove the precipitate of silver chloride. 3. Titrate the excess silver nitrate with ammonium thiocyanate using ferric ammonium sulfate as an indicator. 2. **Mohr’s Method** This method uses mercurimetric titration with silver nitrate and potassium chromate as an indicator. Steps: 1. Dissolve the urine sample in distilled water. 2. Add potassium chromate indicator to the sample. 3. Slowly add silver nitrate solution until a red-brown precipitate of silver chromate forms, indicating the endpoint. 3. Ion-Selective Electrode Method This method uses an ion-selective electrode to measure chloride concentration directly. Steps: 1. Dissolve the urine sample in distilled water. 2. Calibrate the ion-selective electrode with standard chloride solutions. 3. Measure the chloride concentration in the sample using the electrode. A common method for urine chloride determination that uses precipitation is the **Volhard-Harvey Method**. Volhard-Harvey Method: 1. Add a measured excess of silver nitrate (AgNO₃) to the urine sample. 2. The chloride ions in the urine react with silver nitrate to form a white precipitate of silver chloride (AgCl). 3. Filter the solution to remove the precipitate of silver chloride. 4. **Back Titration**: Titrate the excess silver nitrate with ammonium thiocyanate (NH₄SCN) using ferric ammonium sulfate as an indicator. The appearance of a red-brown color indicates the endpoint of the titration, and the amount of chloride in the urine sample can be calculated based on the volume of ammonium thiocyanate used. ### Procedure: 1. **Sample Preparation**: - Take a known volume of the urine sample (usually 10-20 mL). 2. **Addition of Silver Nitrate**: - Add a measured excess of silver nitrate solution to the urine sample. This will react with chloride ions to form a white precipitate of silver chloride (AgCl). \[ \text{AgNO}_3 + \text{Cl}^- \rightarrow \text{AgCl} \, (s) + \ text{NO}_3^- \] 3. **Precipitation and Filtration**: - Allow the precipitate to form completely and filter the solution to remove the silver chloride precipitate. References - **"Quantitative Chemical Analysis" by Daniel C. Harris**: A comprehensive resource on analytical chemistry techniques, including precipitation titrations. - **"Vogel's Textbook of Quantitative Chemical Analysis"**: An excellent reference for detailed procedures and explanations of various quantitative analysis methods. - CALCIUM AND CHLORIDE DETERMINATION AUBF Laboratory CALCIUM Calcium is the most abundant mineral in the body. Human body is composed of 1-2 kg Ca (99% in bones). 3 types: ionized, protein bound, complexed Reference range: 8.8 – 10.4 mg/dL (2.2 – 2.6 mmol/L) Factors affecting Ca absorption: acidic pH, Vitamin D and PTH, high protein diet CALCIUM Ionized / free – 50% of circulating calcium; biologically active; diffusible Complexed – 10% of circulating calcium; bound to phosphate, bicarbonate, and citrate (low MW anions); diffusible Protein bound – 40% of circulating calcium; biologically inactive; not excreted / not diffusible CALCIUM HYPOCALCEMIA HYPERCALCEMIA decrease in total plasma Ca increase in total plasma Ca concentration (i.e. renal tubular concentration (i.e. early disease) malignancy) causes hyper-excitability of the can lead to renal calculi, kidney neuromuscular junction; damage, constipation, symptoms include pins and dehydration, tiredness, and needles, tetany, paralysis, and depression (remembered by the convulsions phrase “stones, moans, and groans”) SULKOWITCH TEST This test is used to detect calcium in urine samples. The reagent is composed of oxalic acid, ammonium oxalate, glacial acetic acid and distilled water. Positive result is indicated by the presence of turbidity with precipitate. SULKOWITCH TEST Principle: Precipitation of Ca ions upon reaction with reagents The turbidity indicates the level of calcium in the sample: a. Clear or low turbidity – low calcium b. Normal turbidity – normal calcium c. Heavy or milky turbidity – high calcium CHLORIDE Normal chloride excreted in the urine is 8 – 15g/24 hours (110 – 250 mEq/day). It is excreted as NaCl. Measuring urine chloride level is used to diagnose metabolic alkalosis (chloride-responsive alkalosis or chloride-resistant alkalosis). Metabolic alkalosis occurs when the blood becomes too alkaline which may be due to: CHLORIDE Increased Levels Decreased Levels (Chloride-Resistant MA) (Chloride-Responsive MA) Polydipsia Excessive sweating, fasting, diarrhea, vomiting Use of diuretics Diabetes insipidus Addison’s Disease Infections Cushing’s Syndrome URINE CHLORIDE TEST In this test, chloride is precipitated as silver chloride with silver nitrate in the presence of nitric acid. Specimen: 1st morning urine sample 2mL urine + 0.5mL nitric acid + 1mL silver nitrate Positive result is indicated by the presence of curdy white precipitate.  FECAL OCCULT BLOOD TEST AND PREGNANCY TEST AUBF Laboratory Fecal Occult Blood Test (FOBT) FOBT is a non-invasive test which detect hidden blood in the stool. It can be used as a screening test to check for colorectal disease (i.e. colon cancer). Types of FOBT: Guaiac smear test and flushable reagent pads Fecal Occult Blood Test (FOBT) Advantages: non-invasive, low cost Disadvantages: detects blood, but not its cause; false positives and negatives Positive result may indicate the following: colon polyp / colon cancer, esophagitis, gastritis, GI trauma and tumor, hemmorhoids, fissures, peptic ulcer, complications in recent GI surgery Fecal Occult Blood Test (FOBT) Collect stool sample from three consecutive bowel movements. Smear the stool sample on a paper card. Add a drop or two of the testing solution on the opposite side of the card. A blue color change indicates a positive reaction (presence of hidden blood). Pregnancy Test Pregnancy tests are based on the detection of human chorionic gonadotropin (hCG). Human chorionic gonadotropin (hCG) is produced by the placenta shortly after fertilization and reaches detectable levels in urine and serum about one week after implantation attachment of the early embryo to the uterine lining. Levels of hCG continue to rise during the first trimester of pregnancy, making it an excellent marker for pregnancy. Pregnancy Test Pregnancy tests can be done either in urine or in blood samples. Both tests detect the presence of hCG. URINE TEST: Urine specimen must be collected without preservatives in a clean, dry container. 1st morning urine usually contains the highest concentration of hCG. BLOOD TEST: Quantitative blood test or qualitative hCG blood test may be done. Pregnancy Test No. Method Time Consumed 1 Inhibition Agglutination Method 2 minutes 2 Strip or Cassette by 10 minutes Immunochromatographic Assay 3 Digital Midstream Device 3 minutes 4 Beta hCG Test by 40 minutes Electrochemiluminescence (ECL) technology 5 Beta hCG Test by Enzyme-Linked 45 – 120 minutes Immunosorbent Assay (ELISA)  Pregnancy Test by Strips or Cassette It is a rapid chromatographic immunoassay for the qualitative detection of hCG in urine or serum to aid in the early detection of pregnancy. This test has a specificity of 98%. The test uses two lines to indicate results. This test is conducted by immersing the test strip in a urine or serum specimen and observing the formation of colored lines.  Pregnancy Test by Strips or Cassette 1. Remove the testing device from the foil pouch by tearing at the notch. Hold the strip by the colored end. 2. Immerse the strip into the urine with the arrow end pointing towards the specimen. Do not immerse past the MAX line. 3. Take the strip out after a minimum of 10 seconds and lay the strip (MAX side facing up) flat on a clean, dry, non-absorbent surface. 4. Read the test result in 10 minutes. Ensure that the background of the test area is white before interpreting the result.  Pregnancy Test by Strips or Cassette 1. Collect 1st morning urine specimen. 2. Take the urine sample with dropper and add about 2-3 drops into the sample wells. 3. Do not add urine sample in the window marked as C and T. 4. Wait for the colored lines to appear. Read the results within 5 minutes. Name: _______________________________________________ Section: ________________ ANALYSIS OF URINE AND OTHER BODY FLUIDS Laboratory Activity No. 13 Calcium Determination Diet plays a vital role in the detection of calcium in the urine. In normal adults with regular diet, the daily output in urine is 100 to 300 mg/day or 2.5 to 7.5 mmol/24 hours. Other factors aside from diet which cause appearance of calcium n urine include endocrine factors and weight. Increased calcium in urine maybe seen in conditions such as bone diseases, hyperparathyroidism, and osteoporosis. Decreased, however, is seen in renal tubular acidosis. Learning Objectives: Upon completion of this activity, the student should be able to: a. Perform various tests for the detection of urine calcium b. Know the different renal diseases associated with urine calcium SULKOWITCH TEST Reagent Composition: oxalic acid, ammonium oxalate, glacial acetic acid, distilled water Procedure: 1. Place 3mL of urine sample in a test tube. 2. Add an equal volume of Sulkowitch reagent in the sample. 3. Mix and observe. 4. Record your results. Positive result: production of turbidity with precipitate Manner of reporting: - Negative +/- Trace 1+ Presence of fine cloudiness without precipitate 2+ Moderately turbid with little, fine precipitate 3+ Heavy turbidity with fine precipitate 4+ Milky with granular precipitate Results: A. Sulkowitch Test: ___________________________ Name: _______________________________________________ Section: ________________ ANALYSIS OF URINE AND OTHER BODY FLUIDS Laboratory Activity No. 14 Chloride Determination A chloride test measures the level of chloride in your blood or urine. Chloride is one of the most important electrolytes in the blood. It helps keep the amount of fluid inside and outside of your cells in balance. It also helps maintain proper blood volume, blood pressure, and pH of your body fluids. Tests for sodium, potassium, and bicarbonate are usually done at the same time as a blood and urine test for chloride. Most of the chloride in your body comes from the salt (sodium chloride) you eat. Chloride is absorbed by your intestines when you digest food. Extra chloride leaves your body in your urine. Sometimes a test for chloride can be done on a one-time random sample of urine or on a sample of all your urine collected over a 24-hour period. High chloride levels detected may be caused by dehydration, such as from diarrhea or vomiting, eating a lot of salt, kidney disease or parathyroidism. On the other hand, low chloride levels may be caused by conditions that cause too much water to build up in the body, such as with syndrome of inappropriate antidiuretic hormone secretion (SIADH) , Addison’s disease, a condition that raises the pH of the blood above the normal range (metabolic alkalosis), heart failure, and ongoing vomiting. Learning Objectives: Upon completion of this activity, the student should be able to: a. Perform various tests for the detection of urine chloride b. Know the different renal diseases associated with urine hloride URINE CHLORIDE TEST Materials and reagents: first morning urine specimen, 6N nitric acid, silver nitrate Principle: Chloride is precipitated as silver chloride from the urine using silver nitrate and nitric acid. Procedure: 1. Place 2mL of urine sample in a test tube. 2. Add 0.5mL of nitric acid and 1mL of silver nitrate in the solution. 3. Observe and record your results. Positive result: a white cloudy precipitate indicates the presence of chloride Results: A. Urine Chloride Test: ___________________________ Name: _______________________________________________ Section: ________________ ANALYSIS OF URINE AND OTHER BODY FLUIDS Laboratory Activity No. 15 Microscopic Examination of Urine The last part of routine urinalysis is the microscopic examination, which also happens to be the most time consuming and least standardized among all the parameters in urinalysis. The main reason in performing this is to detect the insoluble substances in urine that maybe clinically significant. Identification and quantitation of these insoluble substances is important in detecting renal disorders. Correlation of the three parameters namely physical, chemical, and microscopic examination is critical to produce accurate and valid results. The microscopic examination has been subjected to a wide variety of procedural variations and laboratory protocols. Whatever procedural variation is introduced; the following parameters should be taken into consideration such as: a. Specimen preparation b. Specimen volume c. Centrifugation Learning Objectives: Upon completion of this activity, the student should be able to: a. Perform microscopic examination of urine properly b. Know the different urinary sediments which may be seen under the microscope c. Correlate the different urinary sediments to renal diseases MICROSCOPIC URINALYSIS Sample: freshly collected urine sample Procedure: 1. Place at least 8mL of urine sample in a test tube. 2. Perform urinary reagent strip testing for four parameters (pH, SG, protein, and glucose). 3. Centrifuge the sample for 5 minutes. 4. After centrifugation, decant the solution from the test tube. At least 0.5mL of fluid must remain in the test tube after decantation. 5. The sediment is re-suspended by agitation or by tapping to allow complete resuspension of urinary sediment. This is important to ensure equal distribution of the cells. 6. Pipet the resuspended urine on to a glass slide to allow uniform volume concentration. 7. Cover the glass slide and examine under the microscope under LPO and HPO. Results: Name of Patient: _________________________ Age and Sex: ____________________ Date: __________________________________ LABORATORY RESULT Physical Examination Result a. Color __________________________ b. Clarity __________________________ Chemical Examination a. pH __________________________ b. Specific Gravity __________________________ c. Protein __________________________ d. Glucose __________________________ Microscopic Examination a. Red Blood Cells __________________________ b. White Blood Cells __________________________ c. Epithelial Cells __________________________ d. Bacteria __________________________ e. Casts __________________________ f. Crystals __________________________ g. Others __________________________ Name: _______________________________________________ Section: ________________ ANALYSIS OF URINE AND OTHER BODY FLUIDS Laboratory Activity No. 16 Pregnancy Testing Human chorionic gonadotropic (HCG) is a hormone produced by viable placental tissues with a molecular weight of approximately 50, 000. This can be detected in various body fluids such as urine, colostrum, milk, blood, and amniotic fluids of pregnant individuals. Ita presence in the urine generally indicates a positive result for pregnancy. It is quantitated in international units (IU) of gonadotropic. Importance of Pregnancy Testing a. Confirm pregnancy cases b. Investigate complete and incomplete cases of abortion c. Differentiate pregnancy from other conditions such as hydatid form mole Classifications of Pregnancy Test A. Biological Tests 1. Aschheim-Zondek Test (immature female white mice) 2. Friedmann’s Test (female rabbit) 3. Hoffman’s Test (female rabbit) 4. Rat Ovarian Hyperemia Test 5. Galli Maonini Test (male frog) B. Immunological Tests 1. Hemagglutination Inhibition Test 2. Latex Agglutination Inhibition Test 3. Direct Latex Agglutination Test LATEX AGGLUTINATION INHIBITION TEST Principle: The test consists of an antigen suspension of HCG bound to latex particles and an antibody reagent containing antibodies to HGC-B subunit. HCG bound latex particles will react with the antibody and cause agglutination of the latex. When the antibody is blocked, the latex will not agglutinate, thus HCG is present in the sample and is interpreted as a positive reaction. Since HCG is absent in non-pregnant women, the antibody is not blocked, and the latex will agglutinate interpreted as a negative reaction. Reagents: HCG bound latex (preservatives: phenol and 0.25% sodium azide), anti-HCG antibody reagent, positive and negative control Procedure: 1. Place one drop of urine specimen onto the test plate using a disposable dropper. (Positive and negative controls can also be tested onto a different test plate to observe both positive and negative reactions.) 2. Add one drop of anti-HCG reagent directly to the test sample. 3. Add one drop of HCG bound latex suspension. 4. Stir the mixture until it is spread evenly onto the test plate. 5. Observe for the presence or absence of agglutination. Interpretation: Positive – no agglutination occurs within 2 minutes Negative – agglutination occurs within 2 minutes Result: Name of Patient: __________________________ Age / Sex: ______________ Method of Testing: ________________________ Test Result: _____________ Name: _______________________________________________ Section: ________________ ANALYSIS OF URINE AND OTHER BODY FLUIDS Laboratory Activity No. 17 Fecal Occult Blood Test Fecal occult blood test is a test that checks for occult (hidden) blood in the stool. Small samples of stool are placed on special cards coated with a chemical substance called guaiac and sent to a doctor or laboratory for testing. A testing solution is put on the cards and the guaiac causes the stool sample to change color. If there is blood in the stool, the color changes very quickly. Blood in the stool may be a sign of colorectal cancer or other problems, such as polyps, ulcers, or hemorrhoids. Procedure: 1. Place a pea size amount of stool sample onto the specimen card. 2. Close the flap. 3. Wait for 3 – 5 minutes. 4. Open the back flap and add two drops of Guaiac’s reagent and wait for 1 minute. 5. Observe for color change and record your result. Interpretation: A positive result is indicated by the presence of blue color and means that hidden blood is present in the stool sample. Result: Name of Patient: _________________________________ Age / Sex: ___________________ Fecal Occult Blood Test Result: ______________________ Name: _______________________________________________ Section: ________________ ANALYSIS OF URINE AND OTHER BODY FLUIDS Laboratory Activity No. 18 Seminal Fluid Analysis Seminal fluid is a viscous fluid produced by make in the process of ejaculation during sexual contact, urge, or climax. It is produced in four main fractions that contribute to the production of a normal seminal fluid. Each fraction is different in terms of composition and the mixing of these fraction is critical in the production of a viable seminal fluid. These fractions include urethral and bulbourethral, testes and epididymis, prostate gland, and seminal vesicle. Semen analysis is performed for the following purposes: a. Investigation of infertility cases b. Conformation of the effectivity of vasectomy cases c. Forensic medicine for the confirmation of semen Methods of collecting semen include: a. Self-production b. Condom method (not advisable because of spermicidal action) c. Coitus interruptus (withdrawal method) d. Vaginal vault aspiration (not recommended) Specimen Collection and Handling a. Instruct the patient to abstain from sexual activities or self-production for 3 – 5 days prior to specimen collection. b. Specimen should be placed in a dry, sterile container. Glass containers are recommended as sperm motility is affected in plastic containers. c. The specimen should be delivered in the laboratory within one hour after collection. d. The time of collection should be noted and not the time of receipt as fresh samples are highly viscous. Samples are processed only after 30 minutes to one hour of liquefaction. Normal Values for Various Semen Analysis Volume 2 – 5 mL Viscosity Pour in droplets (thin threads) pH 7.2 – 8.0 Sperm count >40 million / ejaculate Sperm concentration >20 -250 million / mL Motility >50-60 motile (1 hour) – slow forward progression with lateral movement Morphology 40 million / ejaculate Sperm concentration >20 -250 million / mL Motility >50-60 motile (1 hour) – slow forward progression with lateral movement Morphology