KAU-Faculty of Science- Biochemistry department Clinical biochemistry lab (Bioc 416) 2012 T.A Nouf Alshareef and T.A Bahiya Osrah considerations-for-routine-urinalysis/

Identification of Pathological Physical and Chemical Urine Constituents Abnormal (Pathological) constituents of urine: 1- Macroscopic analysis:  physical tests  chemical tests 2- Microscopic analysis: Pathological urine constituents are substances which are not usually present in urine such as glucose, protein, ketones, RBCs, Hb, bilirubin…. etc.

How to detect abnormal constituents: Urine strip: Strip is filter paper or plastic which has chemical substance (reagent) coated on it on different pads. It gives color when react with substance in urine. The produced color is compared with chart color visually or mechanically assessed.

Results are reported as: In concentration (mg/dl) As small, moderate, or large Using the plus system (1+, 2+, 3+, 4+) As positive, negative, or normal Automated Urine Testing Machine Urinalysis test strip

Urine strip: This method is rapid, easy, give early indication and qualitative. Therefore, usually there are other confirmatory tests: (chemistry, microbiology and microscopic analysis). Reaction in strip is effected by time, to reduce timing errors and to limit variations in color interpretation; automated instrument is used to read the reaction color on each test pad.

1- Proteinurea: is the presence of abnormal amount of protein in urine. Urine of healthy individual contains no protein or only traces amounts, due to: In normal physiology, protein is reabsorbed by kidney tubules (proximal tubule) large M.wt of protein so it can't pass through kidney tubule to urine. unless kidney tubule has damage. The main protein in urine is albumin therefore, proteinurea=albumin urea

Microalbumin urea: Is the presence of small amounts of albumin in urine. It is very important in detection of early stage of nephronpathy and in diagnosis of DM complication (nephropathy). High protein in urine makes urine looks foamy. Associated with face or feet abnormal edema, due to disturbance of liquid balance in body due to protein loss.

Detection: Qualitative test: using a reagent test strip. Quantitative test: depends on volume and time of urine (protein conc. in urine may vary with time and volume) Most assays are performed on urine sample of 12-24h. Reference value: Quantitative for 24-h urine: Male:1-4 mg/dl Female: 3-10 mg/dl Child: 1-10mg/dl Qualitative reference value: Normal = Negative

2- Glucoseurea: is the presence of abnormal conc. of glucose in urine. Normally, glucose is reabsorbed by active transport in proximal tubule and therefore id doesn't appear in urine. If the blood glucose level exceeds the reabsorption capacity of kidney tubules (renal threshold), glucose will appear in urine. Renal threshold of glucose: is around 160 mg/100 ml.

Glucosuria indicates that glucose concentration in blood exceeds this amount and the kidneys are unable to reabsorb it efficiently. Glucosuria occurs in DM, which characterized by: hyperglycemia, usually polyurea (increased volume of urine), high Serum Glucose urine may be light in color.

3- ketonurea: is the presence of abnormal amount of ketone bodies in urine. Body normally uses carbohydrates as source of energy. If carbohydrate source depleted or there is defect in carbohydrate metabolism, body use fat as a source of energy. Fat metabolism is occurred for certain time, at certain point, fatty acid utilization occurs incompletely results in production of intermediate substances (keton bodies). Three ketone bodies: acetone, acetoacetate,  hydroxybutayric acid Oxidation Fat Fatty AcidsH 2 O+CO 2 +energy

Normally ketone bodies are removed by liver. elevated levels of keton bodies in blood and urine cause acidosis which leads to coma and death. Ketonurea is common in uncontrolled DM (why?) because diabetic patient has high blood glucose but can't use by cells, so lipids are used as source of energy (Fat breakdown). Ketonurea present in:  Disease  Nutrition  Vomiting for long time Results effected by: diet and drugs Normal values: negative test result is normal. Small: 80 mg/dl

4- Haematourea: It is the presence of red blood cells (RBCs) in the urine. Can’t detected by the naked eye so detection by strip or microscope anucleated cells Positive result may be: normally: no pathological cause abnormally: due to stones or tumors. Need other confirmatory test.

5- Hemoglobinuria: Presence of heamoglobin in urine due to rupturing of RBCs This may occur in malaria, typhoid, yellow fever, hemolytic jaundice and other diseases.

6- Bilirubin (Bile): Result from hemoglobin breakdown Elevated in hepatitis and jaundice.

7- Nitrite: used for screening for bacteria. Normal urine doesn’t contain nitrate but not contain nitrites. In the presence of bacteria, the normally present nitrate in the urine is reduced to nitrite. Positive test indicates presence of more than 10 organisms/ml. reduction nitrate nitrite "pink"

8- Urine leucocytes: This test detects any microbial infection in the body. Depends on esterase method: +ve result: means more than 5 leucocytes/hpf. (high power field) If urine stand long time leucocytes lysis and more intense reaction occur. False positives: occurs with vaginal contamination, presence of glucose, albumin, ascorbic acid large amounts of oxalic acid can inhibit the reaction. Esterase + Ester 3-0H-5-phenyl pyrrole diazonium salt pink -purple color neutrophils reagent strip

Microscopic Examination: \ Urine sedimentation may contain cells, casts and crystals and is examined microscopically after centrifugation of a urine sample. A very small amount of all of the above sediments is normal. Concern begins when any of these components is significantly elevated.

Procedure: 1.centrifuged well-mixed urine 10 ml at 3000 r.p.m for 10 min until precipitate. 2.supernatant is decanted and ml is left inside the tube. 3.The sediment is resuspended in the remaining supernatant. 4.drop of sediment is poured in slide and cover-slipped. 5.The sediment is first examined under low power to identify most crystals, casts, squamous cells, and other large objects. 6.Next, examination is carried out at high power to identify crystals, cells, and bacteria.

Microscopic Findings Cells Crystals Casts RBCs WBCs Epithelial cells Bacteria Yeast Tiny-tube shaped made up of RBC, WBC, kidney cells, held up together by a protein

1- Crystals Crystals are common findings in urine especially if urine is allowed to stand long before examination. Type of crystal depends on: pH and constituents of urine. Kidney stones is the most common cause. Most common crystals: oxalate, phosphate, uric acid, cystine.

1- Oxalate crystals occur in urine at any pH. Calcium oxalate crystals are square shape with a characteristic “X” mark

2- Phosphate crystals found if urine pH > 6.5 Its formation depends on ammonia conc. usually associated with bacterial growth. can indicate urinary tract infection.

3- Uric acid crystals uric acid crystals: needle shaped gut Uric acid crystal commonly appear in gut patient.

4- Cystine crystals cystine crystals are hexagonal in shape

5- amorphous crystals aggregates of finely granular material without any defining shape. 1- Amorphous urates: (Na, K, Mg, or Ca salts) urate form in acidic urine (pH: 5-6) 2- Amorphous phosphates contains calcium and phosphate formed in alkaline urine (pH=7-9) why? (alkaline pH decreases solubility of calcium phosphate) alkaline pH of urine may be due to: 1- caused by diet (vegetarian, rich in phosphates..) 2- Pathology Usually, the presence of these crystals is non significant.

Differentiation between amorphous urates and amorphous phosphates : Urine pH Centrifugation: the precipitate of calcium phosphate is white, amorphous urate is pink.

Microscopic analysis RBCs: RBCsWBCs 2- Cells

1- Epithelial cells Little amount normally appear in female due to reproductive period.

2- RBCs: RBCs up to 5/HPF (high power field) are commonly accepted as normal. High RBCs in urine is called hematuria, may be due to: - hemorrhage (bleeding), - inflammation, nephrtitis (inflammation in nephrons) - necrosis (death of body tissues), - trauma (wound), or neoplasia(tumor) in urinary tract.

3- WBCs presence of WBCs suggests infection. WBC up to 5/HPF are commonly accepted as normal. Higher numbers (pyuria) indicate presence of inflammation somewhere in urinary tract.

4- Bacteria Bacteria are the commonest organism seen commonly due to contaminants.

Others Sperm Worm Schistosoma & Hematobium (causes liver disease and bladder) Tricochomonas vaginal

Exercises:

Oxalate

Phosphate

Phosphate crystals