Urine Analysis-lab Report Biochem 3

NURSYAZMIN BINTI KHAIRUDDIN (12273208031) MEDICAL BIOCHEMISTRY (HCB 21203)

LAB PRACTICAL: URINE ANALYSIS Objectives: 1. To learn the lab technique to do the urine analysis. 2. To observe the microscopic view of normal urine and the contents of it. 3. To observe the changes on the dipstick strip when dip into the urine. Introduction: A urinalysis (or "UA") is an array of tests performed on urine and one of the most common methods of medical diagnosis. It is performed by collecting a urine sample from the patient in a specimen cup. Usually only small amounts (10-15 ml's) may be required for urinalysis testing. Urinalysis can disclose evidence of diseases, even some that have not caused significant signs or symptoms. Therefore, a urinalysis is commonly a part of routine health screening. Urinalysis is commonly used to diagnose a urinary tract or kidney infection, to evaluate causes of kidney failure, to screen for progression of some chronic conditions such as diabetes mellitus and high blood pressure (hypertension). It also may be used in combination with other tests to diagnose some diseases. Examples of this include kidney stones, inflammation of the kidneys (glomerulonephritis), or muscle break breakdown (rhabdomyolysis). Interpretation of urinalysis is generally based on reviewing all the components of the test as well as the clinical symptoms and signs of the patient. Urinalysis is done by collecting a urine sample from a patient. The optimal sample tends to be an early morning urine sample because it is frequently the most concentrated urine produced in the day. Methods of collection are slightly different for female and male patient. 

For females, the patient is asked to clean the area around the urethra with a special cleansing wipe, by spreading the labia of the external genitals and cleaning from front to back (toward the anus).

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For men, the tip of the penis may be wiped with a cleansing pad prior to collection.

NURSYAZMIN BINTI KHAIRUDDIN (12273208031) MEDICAL BIOCHEMISTRY (HCB 21203)

The collected urine sample should be taken to the laboratory for analysis, typically within one hour of collection. If transportation to the lab could take more than one hour, then the sample may be refrigerated. A part of a urinalysis can be performed by using urine dipsticks, in which the test results can be read as colour changes. The dipstick is dipped entirely into a sample of urine before it is analyzed. The colour changes if being read too early or too long after the strip was dipped, the results may not be accurate. A proper analysis will likely provide key insight into the presence or absence of bodily abnormalities. The squares on the dipstick represent the following components in the urine: 

specific gravity (concentration of urine),

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acidity of the urine (pH),

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protein in the urine (mainly albumin),

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glucose (sugar),

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ketones (products of fat metabolism),

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blood, leukocyte esterase (suggestive of white blood cells in urine),

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nitrite (suggestive of bacteria in urine),

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bilirubin (possible liver disease or red blood cell breakdown), and

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urobilinogen (possible liver disease).

Presence or absence of each of these colour changes on the strip provides important clues for physicians to make clinical decisions based on the urinalysis results. Urinalysis cans also being done through microscopic examination. The microscopic urinalysis is the study of the urine under the microscope. It requires only a relatively inexpensive light microscope. Cells and cellular debris, bacteria, and crystals in the urine can be detected by this examination to provide further clinical clues.

NURSYAZMIN BINTI KHAIRUDDIN (12273208031) MEDICAL BIOCHEMISTRY (HCB 21203)

Materials: 1. Urine sample 2. Urine container 3. Measuring cylinder 4. Dipstick strip 5. Dropper 6. Bunsen burner 7. Alcohol and cotton 8. Slides 9. Coverslip 10. Microscope 11. Oil immersion 12. Centrifuge 13. Methylene blue 14. Tubes Methods: 1. Urine sample was collected from a volunteered student. The urine was collected using urine container.

(a) Dipstick analysis 1. A 100ml of urine was poured into a 100ml measuring cylinder from the urine container. 2. A dipstick then immediately inserted into it for a few seconds. 3. The excess of urine was drained from the tip of the stick by touching it around the mouth of the cylinder. 4. Wait for three minutes, then after three minutes, the results was compared for the change of colour with the colour code given on the bottle.

(b) Microscopic urine analysis 1. About 1.5ml of urine from the remaining urine from the urine container was dropped using dropper into a tube.

NURSYAZMIN BINTI KHAIRUDDIN (12273208031) MEDICAL BIOCHEMISTRY (HCB 21203)

2. Then the tube was centrifuged in a bench-top centrifuge at 5000 rpm/mm for 15 minutes. 3. After 15 minutes, turn off and wait until it fully stopped. The tube was removed. 4. The urine was slowly decanted off until about 0.2ml of urine remains in the tube without disturbing the pellet at the bottom of the tube. 5. The pellet was break and a drop methylene blue was added into the tube and it was mixed. 6. A slide and a coverslip were swab using alcohol and flame under Bunsen burner, this process known as aseptic technique. 7. Then, a drop of the mixture was placed on a glass slide. It was covered using a coverslip. 8. The slide was then being observed under a microscope and the results obtained were draw. Results: (a) Dipstick analysis i. Leukocyte = Negative ii. Nitrate = Negative iii. Urobilinogen = 3.2 iv. Protein = Negative v. pH = 5.0 vi. Blood = Negative vii. Specific gravity = 1.005 viii. Ketone = Negative ix. Bilirubin = Negative x. Glucose = Negative (b) Microscopic urine analysis i. Epithelial cells Presence/absence = Structure =

NURSYAZMIN BINTI KHAIRUDDIN (12273208031) MEDICAL BIOCHEMISTRY (HCB 21203)

ii. Squamous epithelial cells Presence/absence = Structure = iii. Hyaline casts Presence/absence = Structure =

iv. Calcium carbonate crystals Presence/absence = Structure =

v. Triple phosphate crystals Presence/absence = Structure =

Discussion: From the results, the dipstick analysis shows that the presence of leukocyte, nitrate, protein, blood, ketone, bilirubin and glucose are negative. These mean that there is only a little and almost none presence of those components that can change the dipstick colour to positive. There is presence of urobilinogen, however the amount of it only 3.2 and it is normal amount. The pH of the urine is 5.0 and it is also normal pH value for normal urine sample. The specific gravity or concentration of urine is 1.005 and it also normal value for normal urine sample.

NURSYAZMIN BINTI KHAIRUDDIN (12273208031) MEDICAL BIOCHEMISTRY (HCB 21203)

The expected values to get from this dipstick urinalysis are for leukocytes, normally there are no leukocytes detectable in urine. Specific gravity for adult random urines from 1.003 to 1.040, 24-hours urines from normal adults with normal diets and normal fluid intake will have specific gravity of 1.016 to 1.022. This test permits determination of urine specific gravity between 1.000 and 1.030. For blood, hemolysis is a natural process of recycling old or damaged RBC. But when hemoglobin appears in urine, it indicates some kind of kidney disease or some kind of urinary tract disorder. The practical detection limit of this test is approximately 5 to 10 erythrocytes per microliter of urine. Blood is often, but not always, found in urine of menstruating females. This test is highly sensitive to hemoglobin and thus complements the microscopic examination. Normal urine is slightly acidic with a pH of 6 and urine pH values generally range from 5 to 8 pH units. The pH of urine is an important indicator of certain metabolic, kidney, gastrointestinal and respiratory factors. Nitrite tests in urine are to detect presence of bacteria. Any degree of pink colour after 30 seconds indicates clinically significant bacteriuria. Bacteriuria is generally due to infection of kidneys, ureters, bladder or urethra. Normal urine specimens ordinarily contain some protein; therefore, only persistent elevated levels of urine protein indicate kidney or urinary tract disease. The persistent results of trace level or over indicate significant proteinuria, and thus further clinical testing is needed to evaluate the significant results. Pathologic proteinuria generally gives values above 30mg/dl and is persistent. There is no bilirubin detectable in normal urine specimens of a healthy person by even the most sensitive methods. Elevated bilirubin in urine always indicates disease and is the earliest sign of liver cell disease and/or biliary obstruction. Even trace amounts of bilirubin are sufficiently significant to require further investigation. Ketone bodies should not be detected in normal urine specimens with this reagent. The concentrations given correlate well with the acetoacetic acid concentration in urine. The sensitivity of this test is 5mg acetoacetic acid per 100ml of urine. Detectable levels of ketone may occur with frequent vomiting, diarrhea, digestive disturbances, pregnancy, or severe physical exercise. Normally there are no glucose is detectable in urine, although a minute quantity of glucose is excreted by normal kidney. Approximately 100mg glucose /dl urine is detectable in this test strip. Concentrations of 100mg/dl may be considered as abnormal if found consistently. The normal

NURSYAZMIN BINTI KHAIRUDDIN (12273208031) MEDICAL BIOCHEMISTRY (HCB 21203)

urobilinogen range is 0.1 to 1.0mg/dl. If results exceed the concentration of 2.0mg/dl, the patient and/or the urine specimen should be evaluated further. From the results of microscopic urine analysis it shows the presence of epithelial cells, squamous epithelial cells, hyaline casts, calcium carbonate crystals and triple phosphate crystals. Epithelial cells may originate from any site in the genitourinary tract from the proximal convoluted tubule to the urethra or from vagina. Normally few cells (3 to 5) per h.p.f. from these sites can be found in the urine due to sloughing off of old cells. There are three main types of epithelial cells may be recognized: (a) tubular (b) transitional and (c) squamous. Squamous epithelial cells are the largest cells seen in normal urine specimens. They are thin, flat cells, usually with an angular or irregular outline and a small round nucleus. They may be present as single cells or as variably-sized clusters. It is common in low numbers in voided specimens and generally represents contamination from the genital tract. Their main significance is as an indicator of such contamination. Hyaline casts are formed in the absence of cells in the tubular lumen. They consist of Tamm-Horsfall protein and have a smooth texture and a refractive index very close to that of the surrounding fluid. They are very difficult to see in wet preparations of urine and must be distinguished from mucus strands. Generally, hyaline casts have parallel sides with clear margins and blunted ends, whereas mucus strands are irregular in size with irregular margins. Hyaline casts are far easier to visualize using phase contrast microscopy but this is not available on standard microscopes. Hyaline casts can be present in low numbers (0-1/LPF) in concentrated urine of otherwise normal patients and are not always associated with renal disease. Greater numbers of hyaline casts may be seen in association with proteinuria of renal (e.g., glomerular disease) or extra-renal (e.g., overflow proteinuria as in myeloma) origin. In such cases it has been proposed that the presence of excessive serum protein in the tubular lumen promotes precipitation of the Tamm-Horsefall mucoprotein. Triple phosphate crystals, resemble prisms or "coffin lids". They are found normally in alkaline or neutral urine. They are colourless. Healthy people often have only a few crystals in their urine. A large number of crystals, or certain types of crystals, may mean kidney stones are present or there is a problem with how the body is using food.

NURSYAZMIN BINTI KHAIRUDDIN (12273208031) MEDICAL BIOCHEMISTRY (HCB 21203)

However not all components that are normally seen in urine are manage to be observe. This is due to some error during the procedure of the experiments. The volume of urine that remains inside the tube after the centrifuge and decanted was still high, so the pellet at the bottom of the tube dissolve back and when a drop of methylene blue was added into the tube, the mixed inside the tube become too diluted. This cause the pellet unable to be observes under the microscope because of the diluted staining. Other than that, other error that occurs during this experiment is the urine concentration is too diluted due to the high amount of water intake or drank by the students before the experiments. This will also affect the pellets obtained from the centrifuge and cause the components inside urine difficult to be observed under the microscope. Conclusion: As a conclusion, in a normal urine sample, it is common to have the results of dipstick test negative for leukocyte, nitrate, protein, blood, ketone, bilirubin and glucose. The amount of urobilinogen presence is only 3.2. The urine pH around 5.0. And the specific gravity or concentration of urine is 1.005. In microscopic urine analysis, the presence of red blood cells, white blood cells, bacteria of normal flora, casts, epithelial cells and crystals in a small amount are considered common in normal urine sample. References: 1. http://www.patient.co.uk/doctor/Urine-Dipstick-Analysis.htm 2. http://www.vet.uga.edu/VPP/clerk/Sine/index.php 3. http://www.ehow.com/about_5465199_urine-dipstick-analysis.html 4. http://www.ranvet.com.au/urine_analysis.htm 5. http://www.medicinenet.com/urinalysis/page2.htm