Examination of Urine_Essentials of pathology.pdf

1 Examination of Urine COMPOSITION OF NORMAL URINE COLLECTION OF URINE Urinalysis is one of the most commonly performed There are various methods for collection of...

1 Examination of Urine COMPOSITION OF NORMAL URINE COLLECTION OF URINE Urinalysis is one of the most commonly performed There are various methods for collection of urine. Method laboratory tests in clinical practice. Composition of of collection to be used depends on the nature of normal urine is shown in Table 1.1. investigation (Boxes 1.1 and 1.2). Time of Collection INDICATIONS FOR URINALYSIS 1. A single specimen: This may be a first morning 1. Suspected renal diseases like glomerulonephritis voiding, a random specimen, or a post-prandial nephrotic syndrome, pyelonephritis, and renal failure specimen. 2. Detection of urinary tract infection The first voided specimen in the morning is the most concentrated and has acidic pH in which formed 3. Detection and management of metabolic disorders elements (cells and casts) are well preserved. This like diabetes mellitus specimen is used for routine examination, fasting 4. Differential diagnosis of jaundice glucose, proteins, nitrite, microscopic analysis for 5. Detection and management of plasma cell dyscrasias cellular elements, pregnancy test, orthostatic 6. Diagnosis of pregnancy. proteinuria, and bacteriological analysis. Table 1.1: Composition of normal urine (24 hour) in adults Parameters Values 1. Volume 600-2000 ml 2. Specific gravity 1.003-1.030 3. Osmolality 300-900 mOsm/kg 4. pH 4.6-8.0 5. Glucose 3.5 gm/24 hr) vertebral column. The condition disappears in adulthood. Hypoalbuminemia (300 >200 >300 >200 >300 >25 Detection of microalbuminuria or early diabetic permeability to albumin and denotes microvascular nephropathy disease. Microalbuminuria precedes the development To follow response to therapy in renal disease of diabetic nephropathy by a few years. If blood Proteinuria >1500 mg/ 24 hours indicates glomerular glucose level and hypertension are tightly controlled disease; proteinuria >3500 mg/24 hours is called as at this stage by aggressive treatment then progression nephrotic range proteinuria; in tubular, hemodynamic to irreversible renal disease and subsequent renal and post renal diseases, proteinuria is usually < 1500 mg/ failure can be delayed or prevented. 24 hours. 2. Microalbuminuria is an independent risk factor for Grading of albuminuria is shown in Table 1.5. cardiovascular disease in diabetes mellitus. There are two methods for quantitation of proteins: (1) Estimation of proteins in a 24-hour urine sample, and Detection of microalbuminuria: Microalbuminuria cannot be detected by routine tests for proteinuria. Methods for (2) Estimation of protein/creatinine ratio in a random detection include: urine sample. Measurement of albumin-creatinine ratio in a random 1. Quantitative estimation of proteins in a 24-hour urine sample urine sample: Collection of a 24-hour sample is given Measurement of albumin in an early morning or earlier. Adequacy of sample is confirmed by random urine sample calculating expected 24-hour urine creatinine Measurement of albumin in a 24 hr sample excretion. Daily urinary creatinine excretion depends Test strips that screen for microalbuminuria are on muscle mass and remains relatively constant in available commercially. Exact quantitation can be done an individual patient. In adult males creatinine by immunologic assays like radioimmunoassay or excretion is 14-26 mg/kg/24 hours, while in women enzyme linked immunosorbent assay. it is 11-20 mg/kg/24 hours. Various methods are available for quantitative estimation of proteins: Bence Jones Proteinuria Esbach’s albuminometer method, turbidimetric methods, biuret reaction, and immunologic methods. Bence Jones proteins are monoclonal immunoglobulin 2. Estimation of protein/creatinine ratio in a random light chains (either κ or λ) that are synthesized by urine sample: Because of the problem of incomplete neoplastic plasma cells. Excess production of these light collection of a 24-hour urine sample, many labora- chains occurs in plasma cell dyscrasias like multiple tories measure protein/creatinine ratio in a random myeloma and primary amyloidosis. Because of their low urine sample. Normal protein/creatinine ratio is molecular weight and high concentration they are < 0.2. In low-grade proteinuria it is 0.2-1.0; in excreted in urine (overflow proteinuria). moderate, it is 1.0-3.5; and in nephrotic- range Bence Jones proteins have a characteristic thermal proteinuria it is > 3.5. behaviour. When heated, Bence Jones proteins precipi- tate at temperatures between 40°C to 60°C (other proteins Microalbuminuria precipitate between 60-70°C), and precipitate disappears on further heating at 85-100°C (while precipitate of other This is defined as urinary excretion of 30 to 300 mg/24 proteins does not). When cooled (60-85°C), there is hours (or 2-20 mg/dl) of albumin in urine. reappearance of precipitate of Bence Jones proteins. This Significance of microalbuminuria test, however, is not specific for Bence Jones proteins and 1. Microalbuminuria is considered as the earliest sign both false-positive and -negative results can occur. This of renal damage in diabetes mellitus (diabetic test has been replaced by protein electrophoresis of nephropathy). It indicates increase in capillary concentrated urine sample (Fig. 1.7).  12 Essentials of Clinical Pathology glucosuria or glycosuria (Box 1.7). Glycosuria results if the filtered glucose load exceeds the capacity of renal tubular reabsorption. Most common cause is hyper- glycemia from diabetes mellitus. Causes of Glycosuria 1. Glycosuria with hyperglycemia: Fig. 1.7: Urine protein electrophoresis showing heavy Bence Endocrine diseases: diabetes mellitus, acromegaly, Jones proteinuria (red arrow) along with loss of albumin and Cushing’s syndrome, hyperthyroidism, pancrea- other low molecular weight proteins in urine tic disease Non-endocrine diseases: central nervous system diseases, liver disorders Further evaluation of persistent overt proteinuria is Drugs: adrenocorticotrophic hormone, cortico- shown in Figure 1.8. steroids, thiazides Alimentary glycosuria (Lag-storage glycosuria): Glucose After a meal, there is rapid intestinal absorption of glucose leading to transient elevation of blood The main indication for testing for glucose in urine is glucose above renal threshold. This can occur in detection of unsuspected diabetes mellitus or follow-up persons with gastrectomy or gastrojejunostomy of known diabetic patients. and in hyperthyroidism. Glucose tolerance test Practically all of the glucose filtered by the glomeruli reveals a peak at 1 hour above renal threshold is reabsorbed by the proximal renal tubules and returned (which causes glycosuria); the fasting and 2-hour to circulation. Normally a very small amount of glucose glucose values are normal. is excreted in urine (< 500 mg/24 hours or 2.0 grams/dl): Brick- red precipitate. Fig. 1.9: Principle of Benedict’s qualitative test for sugar in urine. Sensitivity is 200 mg of glucose/dl  14 Essentials of Clinical Pathology Sensitivity of the test is about 100 mg glucose/dl of urine. False positive test occurs in the presence of oxidizing agent (bleach or hypochlorite used to clean urine containers), which oxidizes the chromogen directly. False-negative test occurs in the presence of large amounts of ketones, salicylates, ascorbic acid, and severe Escherichia coli infection (catalase produced by organisms in urine inactivates hydrogen peroxide). Ketones Excretion of ketone bodies (acetoacetic acid, β-hydroxy- butyric acid, and acetone) in urine is called as ketonuria. Fig. 1.10: Grading of Benedict’s test (above) and reagent Ketones are breakdown products of fatty acids and their strip test (below) for glucose presence in urine is indicative of excessive fatty acid metabolism to provide energy. B. Clinitest tablet method (Copper reduction tablet test): This Causes of Ketonuria is a modified form of Benedict’s test in which the reagents Normally ketone bodies are not detectable in the urine are present in a tablet form (copper sulphate, citric acid, of healthy persons. If energy requirements cannot be met sodium carbonate, and anhydrous sodium hydroxide). by metabolism of glucose (due to defective carbohydrate Sensitivity is 200 mgs/dl of glucose. metabolism, low carbohydrate intake, or increased 2. Reagent strip method This test is specific for glucose metabolic needs), then energy is derived from break- and is therefore preferred over Benedict’s and Clinitest down of fats. This leads to the formation of ketone bodies methods. It is based on glucose oxidase-peroxidase (Fig. 1.12). reaction. Reagent area of the strips is impregnated with two enzymes (glucose oxidase and peroxidase) and a 1. Decreased utilization of carbohydrates chromogen. Glucose is oxidized by glucose oxidase with a. Uncontrolled diabetes mellitus with ketoacidosis: In the resultant formation of hydrogen peroxide and diabetes, because of poor glucose utilization, there is gluconic acid. Oxidation of chromogen occurs in the compensatory increased lipolysis. This causes presence of hydrogen peroxide and the enzyme peroxi- increase in the level of free fatty acids in plasma. dase with resultant color change (Fig. 1.11). Nature of Degradation of free fatty acids in the liver leads to chromogen and buffer system differ in different strips. the formation of acetoacetyl CoA which then forms The strip is dipped into the urine sample and color is ketone bodies. Ketone bodies are strong acids and observed after a specified time and compared with the produce H+ ions, which are neutralized by bicar- color chart provided (Fig. 1.10). bonate ions; fall in bicarbonate (i.e. alkali) level This test is more sensitive than Benedict’s qualitative produces ketoacidosis. Ketone bodies also increase test and specific only for glucose. Other reducing agents the plasma osmolality and cause cellular dehydration. give negative reaction. Children and young adults with type 1 diabetes are Fig. 1.11: Principle of reagent strip test for glucose in urine. Each mole of glucose produces one mole of peroxide, and each mole of peroxide reduces one mole of oxygen. Sensitivity is 100 mg glucose/100 ml  Examination of Urine 15 No method for detection of ketonuria reacts with all the three ketone bodies. Rothera’s nitroprusside method and methods based on it detect acetoacetic acid and acetone (the test is 10-20 times more sensitive to acetoacetic acid than acetone). Ferric chloride test detects acetoacetic acid only. β-hydroxybutyric acid is not detected by any of the screening tests. Methods for detection of ketone bodies in urine are Rothera’s test, Acetest tablet method, ferric chloride test, Fig. 1.12: Formation of ketone bodies. A small part of and reagent strip test. acetoacetate is spontaneously and irreversibly converted to 1. Rothera’s’ test (Classic nitroprusside reaction) Acetoacetic acetone. Most is converted reversibly to β-hydroxybutyrate acid or acetone reacts with nitroprusside in alkaline solution to form a purple-colored complex (Fig. 1.13). Rothera’s test is sensitive to 1-5 mg/dl of acetoacetate especially prone to ketoacidosis during acute illness and to 10-25 mg/dl of acetone. and stress. If glycosuria is present, then test for ketone bodies must be done. If both glucose and ketone Method bodies are present in urine, then it indicates presence of diabetes mellitus with ketoacidosis (Box 1.8). 1. Take 5 ml of urine in a test tube and saturate it with In some cases of diabetes, ketone bodies are increased ammonium sulphate. in blood but do not appear in urine. 2. Add a small crystal of sodium nitroprusside. Mix Presence of ketone bodies in urine may be a warning well. of impending ketoacidotic coma. 3. Slowly run along the side of the test tube liquor b. Glycogen storage disease (von Gierke’s disease) ammonia to form a layer. 2. Decreased availability of carbohydrates in the diet: 4. Immediate formation of a purple permanganate a. Starvation colored ring at the junction of the two fluids indicates b. Persistent vomiting in children a positive test (Fig. 1.14). c. Weight reduction program (severe carbohydrate False-positive test can occur in the presence of L-dopa restriction with normal fat intake) in urine and in phenylketonuria. 3. Increased metabolic needs: a. Fever in children 2. Acetest tablet test This is Rothera’s test in the form of a b. Severe thyrotoxicosis tablet. The Acetest tablet consists of sodium nitro- c. Pregnancy prusside, glycine, and an alkaline buffer. A purple- d. Protein calorie malnutrition lavender discoloration of the tablet indicates the presence of acetoacetate or acetone (≥ 5 mg/dl). A rough estimate Tests for Detection of Ketones in Urine of the amount of ketone bodies can be obtained by The proportion of ketone bodies in urine in ketosis is comparison with the color chart provided by the variable: β-hydroxybutyric acid 78%, acetoacetic acid manufacturer.The test is more sensitive than reagent strip 20%, and acetone 2%. test for ketones. Box 1.8: Urine ketones in diabetes Indications for testing At diagnosis of diabetes mellitus At regular intervals in all known cases of diabetes, and in gestational diabetes In known diabetic patients during acute illness, persistent Fig. 1.13: Principles of Rothera’s test and reagent strip test hyperglycemia (>300 mg/dl), pregnancy, clinical evidence for ketone bodies in urine. Ketones are detected as acetoacetic of diabetic acidosis (nausea, vomiting, abdominal pain) acid and acetone but not β-hydroxybutyric acid  16 Essentials of Clinical Pathology Table 1.6: Urine bilirubin and urobilinogen in jaundice Urine test Hemolytic Hepatocellular Obstructive jaundice jaundice jaundice 1. Bilirubin Absent Present Present 2. Urobilinogen Increased Increased Absent In acute viral hepatitis, bilirubin appears in urine even before jaundice is clinically apparent. In a fever of unknown origin bilirubinuria suggests hepatitis. Presence of bilirubin in urine indicates conjugated hyperbilirubinemia (obstructive or hepatocellular jaundice). This is because only conjugated bilirubin is water-soluble. Bilirubin in urine is absent in hemolytic jaundice; this is because unconjugated bilirubin is Fig. 1.14: Rothera’s tube test and reagent strip test for water-insoluble. ketone bodies in urine Tests for Detection of Bilirubin in Urine 3. Ferric chloride test (Gerhardt’s): Addition of 10% ferric Bilirubin is converted to non-reactive biliverdin on chloride solution to urine causes solution to become exposure to light (daylight or fluorescent light) and on reddish or purplish if acetoacetic acid is present. The test standing at room temperature. Biliverdin cannot be is not specific since certain drugs (salicylate and L-dopa) detected by tests that detect bilirubin. Therefore fresh give similar reaction. Sensitivity of the test is 25-50 mg/ dl. sample that is kept protected from light is required. Findings associated with bilirubinuria are shown in 4. Reagent strip test: Reagent strips tests are modifications Box 1.9. of nitroprusside test (Figs 1.13 and 1.14). Their sensitivity Methods for detection of bilirubin in urine are foam is 5-10 mg/dl of acetoacetate. If exposed to moisture, test, Gmelin’s test, Lugol iodine test, Fouchet’s test, reagent strips often give false-negative result. Ketone pad Ictotest tablet test, and reagent strip test. on the strip test is especially vulnerable to improper 1. Foam test: About 5 ml of urine in a test tube is shaken storage and easily gets damaged. and observed for development of yellowish foam. Similar result is also obtained with proteins and Bile Pigment (Bilirubin) highly concentrated urine. In normal urine, foam is Bilirubin (a breakdown product of hemoglobin) is white. undetectable in the urine of normal persons. Presence of 2. Gmelin’s test: Take 3 ml of concentrated nitric acid bilirubin in urine is called as bilirubinuria. in a test tube and slowly place equal quantity of urine There are two forms of bilirubin: conjugated and over it. The tube is shaken gently; play of colors unconjugated. After its formation from hemoglobin in (yellow, red, violet, blue, and green) indicates positive reticuloendothelial system, bilirubin circulates in blood test (Fig. 1.15). bound to albumin. This is called as unconjugated 3. Lugol iodine test: Take 4 ml of Lugol iodine solution bilirubin. Unconjugated bilirubin is not water-soluble, (Iodine 1 gm, potassium iodide 2 gm, and distilled is bound to albumin, and cannot pass through the water to make 100 ml) in a test tube and add 4 drops glomeruli; therefore it does not appear in urine. The liver of urine. Mix by shaking. Development of green color takes up unconjugated bilirubin where it combines with indicates positive test. glucuronic acid to form bilirubin diglucuronide (conjugated bilirubiun). Conjugated bilirubin is water- soluble, is filtered by the glomeruli, and therefore appears Box 1.9: Clinical and laboratory findings in bilirubinuria in urine. Jaundice Detection of bilirubin in urine (along with urobili- Urine color: Dark yellow with yellow foam nogen) is helpful in the differential diagnosis of Elevated serum conjugated bilirubin jaundice (Table 1.6).  Examination of Urine 17 Fig. 1.16: Positive Fouchet’s test for bilirubin in urine bile salts and conjugated bilirubin regurgitate into blood from biliary canaliculi (due to increased intrabiliary pressure) and are excreted in urine. The test used for their Fig. 1.15: Positive Gmelin’s test for bilirubin showing detection is Hay’s surface tension test. The property of play of colors bile salts to lower the surface tension is utilized in this test. 4. Fouchet’s test: This is a simple and sensitive test. Take some fresh urine in a conical glass tube. Urine i. Take 5 ml of fresh urine in a test tube, add 2.5 should be at the room temperature. Sprinkle on the ml of 10% of barium chloride, and mix well. A surface particles of sulphur. If bile salts are present, precipitate of sulphates appears to which bilirubin sulphur particles sink to the bottom because of lowering is bound (barium sulphate-bilirubin complex). of surface tension by bile salts. If sulphur particles remain ii. Filter to obtain the precipitate on a filter paper. on the surface of urine, bile salts are absent. iii. To the precipitate on the filter paper, add 1drop Thymol (used as a preservative) gives false positive of Fouchet’s reagent. (Fouchet’s reagent consists test. of 25 grams of trichloroacetic acid, 10 ml of 10% ferric chloride, and distilled water 100 ml). Urobilinogen iv. Immediate development of blue-green color Conjugated bilirubin excreted into the duodenum around the drop indicates presence of bilirubin through bile is converted by bacterial action to urobilino- (Fig. 1.16). 5. Reagent strips or tablets impregnated with diazo gen in the intestine. Major part is eliminated in the feces. reagent: These tests are based on reaction of bilirubin A portion of urobilinogen is absorbed in blood, which with diazo reagent; color change is proportional to undergoes recycling (enterohepatic circulation); a small the concentration of bilirubin. Tablets (Ictotest) detect amount, which is not taken up by the liver, is excreted in 0.05-0.1 mg of bilirubin/dl of urine; reagent strip tests urine. Urobilinogen is colorless; upon oxidation it is are less sensitive (0.5 mg/dl). converted to urobilin, which is orange-yellow in color. Normally about 0.5-4 mg of urobilinogen is excreted in Bile Salts urine in 24 hours. Therefore, a small amount of urobili- Bile salts are salts of four different types of bile acids: nogen is normally detectable in urine. cholic, deoxycholic, chenodeoxycholic, and lithocholic. Urinary excretion of urobilinogen shows diurnal These bile acids combine with glycine or taurine to form variation with highest levels in afternoon. Therefore, a complex salts or acids. Bile salts enter the small intestine 2-hour post-meal sample is preferred. through the bile and act as detergents to emulsify fat and reduce the surface tension on fat droplets so that enzymes Causes of Increased Urobilinogen in Urine (lipases) can breakdown the fat. In the terminal ileum, bile salts are absorbed and enter in the blood stream from 1. Hemolysis: Excessive destruction of red cells leads where they are taken up by the liver and re-excreted in to hyperbilirubinemia and therefore increased bile (enterohepatic circulation). formation of urobilinogen in the gut. Bilirubin, being Bile salts along with bilirubin can be detected in urine of unconjugated type, does not appear in urine. in cases of obstructive jaundice. In obstructive jaundice, Increased urobilinogen in urine without bilirubin is  18 Essentials of Clinical Pathology typical of hemolytic anemia. This also occurs in megaloblastic anemia due to premature destruction of erythroid precursors in bone marrow (ineffective erythropoiesis). 2. Hemorrhage in tissues: There is increased formation of bilirubin from destruction of red cells. Causes of Reduced Urobilinogen in Urine 1. Obstructive jaundice: In biliary tract obstruction, delivery of bilirubin to the intestine is restricted and very little or no urobilinogen is formed. This causes stools to become clay-colored. 2. Reduction of intestinal bacterial flora: This prevents conversion of bilirubin to urobilinogen in the intestine. It is observed in neonates and following antibiotic treatment. Testing of urine for both bilirubin and urobilinogen can provide helpful information in a case of jaundice (Table 1.6). Fig. 1.17: Ehrlich’s aldehyde test for urobilinogen Tests for Detection of Urobilinogen in Urine test is used. Add 1-2 ml of chloroform, shake for 2 Fresh urine sample should be used because on standing minutes and allow to stand. Pink color in the chloroform urobilinogen is converted to urobilin, which cannot be layer indicates presence of urobilinogen, while pink detected by routine tests. A timed (2-hour postprandial) coloration of aqueous portion indicates presence of sample can also be used for testing urobilinogen. porphobilinogen. Pink layer is then decanted and shaken Methods for detection of increased amounts of urobili- with butanol. A pink color in the aqueous layer indicates nogen in urine are Ehrlich’s aldehyde test and reagent porphobilinogen (Fig. 1.18). strip test. False-negative reaction can occur in the presence of 1. Ehrlich’s aldehyde test: Ehrlich’s reagent (p- (i) urinary tract infection (nitrites oxidize urobilinogen dimethylaminobenzaldehyde) reacts with urobili- to urobilin), and (ii) antibiotic therapy (gut bacteria which nogen in urine to produce a pink color. Intensity of produce urobilinogen are destroyed). color developed depends on the amount of urobili- 2. Reagent strip method: This method is specific for nogen present. Presence of bilirubin interferes with urobilinogen. Test area is impregnated with either the reaction, and therefore if present, should be p-dimethylaminobenzaldehyde or 4-methoxy- removed. For this, equal volumes of urine and 10% benzene diazonium tetrafluoroborate. barium chloride are mixed and then filtered. Test for urobilinogen is carried out on the filtrate. However, Blood similar reaction is produced by porphobilinogen (a The presence of abnormal number of intact red blood substance excreted in urine in patients of porphyria). cells in urine is called as hematuria. It implies presence Method: Take 5 ml of fresh urine in a test tube. Add 0.5 of a bleeding lesion in the urinary tract. Bleeding in urine ml of Ehrlich’s aldehyde reagent (which consists of may be noted macroscopically or with naked eye (gross hydrochloric acid 20 ml, distilled water 80 ml, and para- hematuria). If bleeding is noted only by microscopic dimethylaminobenzaldehyde 2 gm). Allow to stand at examination or by chemical tests, then it is called as room temperature for 5 minutes. Development of pink occult, microscopic or hidden hematuria. color indicates normal amount of urobilinogen. Dark Causes of Hematuria red color means increased amount of urobilinogen (Fig. 1.17). 1. Diseases of urinary tract Since both urobilinogen and porphobilinogen Glomerular diseases: Glomerulonephritis, Berger’s produce similar reaction, further testing is required to disease, lupus nephritis, Henoch-Schonlein distinguish between the two. For this, Watson-Schwartz purpura  Examination of Urine 19 Fig. 1.18: Interpretation of Watson-Schwartz test Nonglomerular diseases: Calculus, tumor, infec- cells) as well as myoglobin. Heme proteins in tion, tuberculosis, pyelonephritis, hydronephrosis, hemoglobin act as peroxidase, which reduces polycystic kidney disease, trauma, after strenuous hydrogen peroxide to water. This process needs a physical exercise, diseases of prostate (benign hydrogen donor (benzidine, orthotoluidine, or hyperplasia of prostate, carcinoma of prostate). guaiac). Oxidation of hydrogen donor leads to 2. Hematological conditions: Coagulation disorders, sickle development of a color (Fig. 1.19). Intensity of color cell disease produced is proportional to the amount of hemo- Presence of red cell casts and proteinuria along with globin present. hematuria suggests glomerular cause of hematuria. Chemical tests are positive in hematuria, hemo- Tests for Detection of Blood in Urine globinuria, and myoglobinuria. 1. Microscopic examination of urinary sediment: Benzidine test: Make saturated solution of benzidine Definition of microscopic hematuria is presence of 3 in glacial acetic acid. Mix 1 ml of this solution with 1 or more number of red blood cells per high power ml of hydrogen peroxide in a test tube. Add 2 ml of field on microscopic examination of urinary sediment urine. If green or blue color develops within 5 in two out of three properly collected samples. A minutes, the test is positive. small number of red blood cells in urine of low specific Orthotoluidine test: In this test, instead of benzidine, gravity may undergo lysis, and therefore hematuria may be missed if only microscopic examination is orthotoluidine is used. It is more sensitive than done. Therefore, microscopic examination of urine benzidine test. should be combined with a chemical test. Reagent strip test: Various reagent strips are 2. Chemical tests: These detect both intracellular and commercially available which use different extracellular hemoglobin (i.e. intact and lysed red chromogens (o-toluidine, tetramethylbenzidine). Fig. 1.19: Principle of chemical test for red cells, hemoglobin, or myoglobin in urine  20 Essentials of Clinical Pathology Fig. 1.20: Evaluation of positive chemical test for blood in urine Causes of false-positive tests: glucose-6-phosphate dehydrogenase deficiency Contamination of urine by menstrual blood in following exposure to oxidant drugs, immune females hemolysis (mismatched blood transfusion, paroxy- Contamination of urine by oxidizing agent (e.g. smal cold hemoglobinuria), paroxysmal nocturnal hypochlorite or bleach used to clean urine containers), hemoglobinuria, hemolytic uremic syndrome, and or microbial peroxidase in urinary tract infection. disseminated intravascular coagulation. Causes of false-negative tests: Presence of a reducing agent like ascorbic acid in high Tests for Detection of Hemoglobinuria concentration: Microscopic examination for red cells is positive but chemical test is negative. Tests for detection of hemoglobinuria are benzidine test, Use of formalin as a preservative for urine orthotoluidine test, and reagent strip test. Evaluation of positive chemical test for blood is shown in Figure 1.20. Hemosiderin Hemosiderin in urine (hemosiderinuria) indicates Hemoglobin presence of free hemoglobin in plasma. Hemosiderin Presence of free hemoglobin in urine is called as appears as blue granules when urine sediment is stained hemoglobinuria. with Prussian blue stain (Fig. 1.21). Granules are located inside tubular epithelial cells or may be free if cells have Causes of Hemoglobinuria disintegrated. Hemosiderinuria is seen in intravascular 1. Hematuria with subsequent lysis of red blood cells hemolysis. in urine of low specific gravity. 2. Intravascular hemolysis: Hemoglobin will appear in Myoglobin urine when haptoglobin (to which hemoglobin binds in plasma) is completely saturated with hemoglobin. Myoglobin is a protein present in striated muscle (skeletal Intravascular hemolysis occurs in infections (severe and cardiac) which binds oxygen. Causes of myoglo- falciparum malaria, clostridial infection, E. coli binuria include injury to skeletal or cardiac muscle, e.g. septicemia), trauma to red cells (march hemo- crush injury, myocardial infarction, dermatomyositis, globinuria, extensive burns, prosthetic heart valves), severe electric shock, and thermal burns.  Examination of Urine 21 reagent strip format that can detect significant bacteriuria: nitrite test and leucocyte esterase test. These tests are helpful at places where urine microscopy is not available. If these tests are positive, urine culture is indicated. 1. Nitrite test: Nitrites are not present in normal urine; ingested nitrites are converted to nitrate and excreted in urine. If gram-negative bacteria (e.g. E.coli, Salmonella, Proteus, Klebsiella, etc.) are present in urine, they will reduce the nitrates to nitrites through the action of bacterial enzyme nitrate reductase. Nitrites are then detected in urine by reagent strip tests. As E. coli is the commonest organism causing urinary tract infection, this test is helpful as a screening test for urinary tract infection. Some organisms like Staphylococci or Pseudomonas do not reduce nitrate to nitrite and therefore in such infections nitrite test is negative. Also, urine must be retained in the bladder for minimum of 4 hours for Fig. 1.21: Staining of urine sediment with Prussian blue stain to demonstrate hemosiderin granules (blue) conversion of nitrate to nitrite to occur; therefore, fresh early morning specimen is preferred. Sufficient dietary intake of nitrate is necessary. Therefore a negative nitrite Chemical tests used for detection of blood or test does not necessarily indicate absence of urinary hemoglobin also give positive reaction with myoglobin tract infection. (as both hemoglobin and myoglobin have peroxidase The test detects about 70% cases of urinary tract activity). Ammonium sulfate solubility test is used as a infections. screening test for myoglobinuria (Myoglobin is soluble 2. Leucocyte esterase test: It detects esterase enzyme in 80% saturated solution of ammonium sulfate, while released in urine from granules of leucocytes. Thus hemoglobin is insoluble and is precipitated. A positive the test is positive in pyuria. If this test is positive, chemical test for blood done on supernatant indicates urine culture should be done. The test is not sensitive myoglobinuria). to leucocytes < 5/HPF. Distinction between hematuria, hemoglobinuria, and myoglobinuria is shown in Table 1.7. MICROSCOPIC EXAMINATION Chemical Tests for Significant Bacteriuria Microscopic examination of urine is also called as the (Indirect Tests for Urinary Tract Infection) “liquid biopsy of the urinary tract”. In addition to direct microscopic examination of urine Urine consists of various microscopic, insoluble, solid sample, chemical tests are commercially available in a elements in suspension. These elements are classified as Table 1.7: Differentiation between hematuria, hemoglobinuria, and myoglobinuria Parameter Hematuria Hemoglobinuria Myoglobinuria 1. Urine color Normal, smoky, red, Pink, red, or Red or brown or brown brown 2. Plasma color Normal Pink Normal 3. Urine test based on Positive Positive Positive peroxidase activity 4. Urine microscopy Many red cells Occasional red cell Occasional red cell 5. Serum haptoglobin Normal Low Normal 6. Serum creatine kinase Normal Normal Markedly increased  22 Essentials of Clinical Pathology organized or unorganized. Organized substances examined within 2 hours of voiding because cells and include red blood cells, white blood cells, epithelial cells, casts degenerate upon standing at room temperature. If casts, bacteria, and parasites. The unorganized sub- preservative is required, then 1 crystal of thymol or 1 stances are crystalline and amorphous material. These drop of formalin (40%) is added to about 10 ml of urine. elements are suspended in urine and on standing they settle down and sediment at the bottom of the container; Method: A well-mixed sample of urine (12 ml) is therefore they are known as urinary deposits or urinary centrifuged in a centrifuge tube for 5 minutes at 1500 sediments. Examination of urinary deposit is helpful in rpm and supernatant is poured off. The tube is tapped diagnosis of urinary tract diseases as shown in Table 1.8. at the bottom to resuspend the sediment (in 0.5 ml of Different types of urinary sediments are shown in urine). A drop of this is placed on a glass slide and Figure 1.22. The major aim of microscopic examination covered with a cover slip (Fig. 1.23). The slide is examined of urine is to identify different types of cellular elements immediately under the microscope using first the low and casts. Most crystals have little clinical significance. power and then the high power objective. The condenser Specimen: The cellular elements are best preserved in should be lowered to better visualize the elements by acid, hypertonic urine; they deteriorate rapidly in reducing the illumination. alkaline, hypotonic solution. A mid-stream, freshly voided, first morning specimen is preferred since it is Cells the most concentrated. The specimen should be Cellular elements in urine are shown in Figure 1.24. Table 1.8: Urinary findings in renal diseases Condition Albumin RBCs/HPF WBCs/HPF Casts/LPF Others 1. Normal 0-trace 0-2 0-2 Occasional – (Hyaline) 2. Acute 1-2+ Numerous; 0-few Red cell, Smoky urine or glomerulonephritis dysmorphic granular hematuria 3. Nephrotic syndrome >4+ 0-few 0-few Fatty, hyaline, Oval fat bodies, Waxy, epithelial lipiduria 4. Acute pyelonephritis 0-1+ 0-few Numerous WBC, granular WBC clumps, bacteria, nitrite test HPF: High power field; LPF: Low power field; RBCs: Red blood cells; WBCs: White blood cells. Fig. 1.22: Different types of urinary sediment tahir99 - UnitedVRG vip.persianss.ir  Examination of Urine 23 Red Blood Cells Normally there are no or an occasional red blood cell in urine. In a fresh urine sample, red cells appear as small, smooth, yellowish, anucleate biconcave disks about 7 μ in diameter (called as isomorphic red cells). However, red cells may appear swollen (thin discs of greater diameter, 9-10 μ) in dilute or hypotonic urine, or may appear crenated (smaller diameter with spikey surface) in hypertonic urine. In glomerulonephritis, red cells are typically described as being dysmorphic (i.e. markedly variable in size and shape). They result from passage of red cells through the damaged glomeruli. Presence of > 80% of dysmorphic red cells is strongly suggestive of glomerular pathology. The quantity of red cells can be reported as number Fig. 1.23: Preparation of urine sediment for of red cells per high power field. microscopic examination Causes of hematuria have been listed earlier. Fig. 1.24: Cells in urine (1) Isomorphic red blood cells, (2) Crenated red cells, (3) Swollen red cells, (4) Dysmorphic red cells, (5) White blood cells (pus cells), (6) Squamous epithelial cell, (7) Transitional epithelial cells, (8) Renal tubular epithelial cells, (9) Oval fat bodies, (10) Maltese cross pattern of oval fat bodies, and (11) spermatozoa tahir99 - UnitedVRG vip.persianss.ir  24 Essentials of Clinical Pathology White Blood Cells (Pus Cells) diamond- or pear-shaped (caudate cells). Large numbers or sheets of these cells in urine occur after catheterization White blood cells are spherical, 10-15 μ in size, granular and in transitional cell carcinoma. in appearance in which nuclei may be visible. Degene- rated white cells are distorted, smaller, and have fewer Oval Fat Bodies granules. Clumps of numerous white cells are seen in These are degenerated renal tubular epithelial cells filled infections. Presence of many white cells in urine is called with highly refractile lipid (cholesterol) droplets. Under as pyuria. In hypotonic urine white cells are swollen and polarized light, they show a characteristic “Maltese the granules are highly refractile and show Brownian cross” pattern. They can be stained with a fat stain such movement; such cells are called as glitter cells; large as Sudan III or Oil Red O. They are seen in nephrotic numbers are indicative of injury to urinary tract. syndrome in which there is lipiduria. Normally 0-2 white cells may be seen per high power field. Pus cells greater than 10/HPF or presence of Spermatozoa clumps is suggestive of urinary tract infection. They may sometimes be seen in urine of men. Increased numbers of white cells occur in fever, pyelonephritis, lower urinary tract infection, tubulo- Telescoped urinary sediment: This refers to urinary interstitial nephritis, and renal transplant rejection. sediment consisting of red blood cells, white blood cells, In urinary tract infection, following are usually seen oval fat bodies, and all types of casts in roughly equal in combination: proportion. It occurs in lupus nephritis, malignant Clumps of pus cells or pus cells >10/HPF hypertension, rapidly proliferative glomerulonephritis, and diabetic glomerulosclerosis. Bacteria Albuminuria Organisms Positive nitrite test Organisms detectable in urine are shown in Figure 1.25. Simultaneous presence of white cells and white cell casts indicates presence of renal infection (pyelo- Bacteria nephritis). Bacteria in urine can be detected by microscopic Eosinophils (>1% of urinary leucocytes) are a examination, reagent strip tests for significant bacteriuria characteristic feature of acute interstitial nephritis due to (nitrite test, leucocyte esterase test), and culture. drug reaction (better appreciated with a Wright’s stain). Method of collection for bacteriologic examination is given earlier in Box 1.2. Renal Tubular Epithelial Cells Significant bacteriuria exists when there are >105 Presence of renal tubular epithelial cells is a significant bacterial colony forming units/ml of urine in a clean- finding. Increased numbers are found in conditions catch midstream sample, >104 colony forming units/ml causing tubular damage like acute tubular necrosis, of urine in catheterized sample, and >10 3 colony- pyelonephritis, viral infection of kidney, allograft forming units/ml of urine in a suprapubic aspiration rejection, and salicylate or heavy metal poisoning. sample. These cells are small (about the same size or slightly larger than white blood cell), polyhedral, columnar, or oval, and have granular cytoplasm. A single, large, refractile, eccentric nucleus is often seen. Renal tubular epithelial cells are difficult to distin- guish from pus cells in unstained preparations. Squamous Epithelial Cells Squamous epithelial cells line the lower urethra and vagina. They are best seen under low power objective (×10). Presence of large numbers of squamous cells in urine indicates contamination of urine with vaginal fluid. These are large cells, rectangular in shape, flat with abundant cytoplasm and a small, central nucleus. Transitional Epithelial Cells Transitional cells line renal pelvis, ureters, urinary Fig. 1.25: Organisms in urine: (A) Bacteria, (B) Yeasts, bladder, and upper urethra. These cells are large, and (C) Trichomonas, and (D) Egg of Schistosoma haematobium tahir99 - UnitedVRG vip.persianss.ir  Examination of Urine 25 1. Microscopic examination: In a wet preparation, mellitus. Usually pyuria is present if there is infection presence of bacteria should be reported only when by Candida. Candida may also be a contaminant in the urine is fresh. Bacteria occur in combination with pus sample and therefore urine sample must be examined in cells. Gram’s-stained smear of uncentrifuged urine a fresh state. showing 1 or more bacteria per oil-immersion field Trichomonas vaginalis suggests presence of > 105 bacterial colony forming units/ml of urine. If many squamous cells are present, These are motile organisms with pear shape, undulating then urine is probably contaminated with vaginal membrane on one side, and four flagellae. They cause flora. Also, presence of only bacteria without pus cells vaginitis in females and are thus contaminants in urine. indicates contamination with vaginal or skin flora. They are easily detected in fresh urine due to their 2. Chemical or reagent strip tests for significant motility. bacteriuria: These are given earlier. 3. Culture: On culture, a colony count of >105/ml is Eggs of Schistosoma haematobium strongly suggestive of urinary tract infection, even Infection by this organism is prevalent in Egypt. in asymptomatic females. Positive culture is followed by sensitivity test. Most infections are due to Gram- Microfilariae negative enteric bacteria, particularly Escherichia coli. They may be seen in urine in chyluria due to rupture of If three or more species of bacteria are identified on a urogenital lymphatic vessel. culture, it almost always indicates contamination by vaginal flora. Casts Negative culture in the presence of pyuria (‘sterile’ Urinary casts are cylindrical, cigar-shaped microscopic pyuria) occurs with prior antibiotic therapy, renal structures that form in distal renal tubules and collecting tuberculosis, prostatitis, renal calculi, catheterization, ducts. They take the shape and diameter of the lumina fever in children (irrespective of cause), female genital (molds or ‘casts’) of the renal tubules. They have parallel tract infection, and non-specific urethritis in males. sides and rounded ends. Their length and width may be variable. Casts are basically composed of a precipitate of Yeast Cells (Candida) a protein that is secreted by tubules (Tamm-Horsfall These are round or oval structures of approximately the protein). Since casts form only in renal tubules their same size as red blood cells. In contrast to red cells, they presence is indicative of disease of the renal parenchyma. show budding, are oval and more refractile, and are not Although there are several types of casts, all urine casts soluble in 2% acetic acid. are basically hyaline; various types of casts are formed Presence of Candida in urine may suggest immuno- when different elements get deposited on the hyaline compromised state, vaginal candidiasis, or diabetes material (Fig. 1.26). Casts are best seen under low power Fig. 1.26: Genesis of casts in urine. All cellular casts degenerate to granular and waxy casts tahir99 - UnitedVRG vip.persianss.ir  26 Essentials of Clinical Pathology objective (×10) with condenser lowered down to reduce muscle exercise in healthy persons and during fever. the illumination. Increased numbers are found in conditions causing Casts are the only elements in the urinary sediment glomerular proteinuria. that are specifically of renal origin. Granular casts: Presence of degenerated cellular debris in Casts (Fig. 1.27) are of two main types: a cast makes it granular in appearance. These are Noncellular: Hyaline, granular, waxy, fatty cylindrical structures with coarse or fine granules (which Cellular: Red blood cell, white blood cell, renal represent degenerated renal tubular epithelial cells) tubular epithelial cell. embedded in Tamm-Horsfall protein matrix. They are Hyaline and granular casts may appear in normal or seen after strenuous muscle exercise and in fever, acute diseased states. All other casts are found in kidney glomerulonephritis, and pyelonephritis. diseases. Waxy cast: These are the most easily recognized of all Non-cellular Casts casts. They form when hyaline casts remain in renal tubules for long time (prolonged stasis). They have Hyaline casts: These are the most common type of casts homogenous, smooth glassy appearance, cracked or in urine and are homogenous, colorless, transparent, and serrated margins and irregular broken-off ends. The ends refractile. They are cylindrical with parallel sides and are straight and sharp and not rounded as in other casts. blunt, rounded ends and low refractive index. Presence They are light yellow in color. They are most commonly of occasional hyaline cast is considered as normal. Their seen in end-stage renal failure. presence in increased numbers (“cylinduria”) is abnormal. They are composed primarily of Tamm- Fatty casts: These are cylindrical structures filled with Horsfall protein. They occur transiently after strenuous highly refractile fat globules (triglycerides and cholesterol Fig. 1.27: Urinary casts: (A) Hyaline cast, (B) Granular cast, (C) Waxy cast, (D) Fatty cast, (E) Red cell cast, (F) White cell cast, and (G) Epithelial cast tahir99 - UnitedVRG vip.persianss.ir  Examination of Urine 27 esters) in Tamm-Horsfall protein matrix. They are seen Normal Crystals in nephrotic syndrome. Crystals present in acid urine Broad casts: Broad casts form in dilated distal tubules and a. Uric acid crystals: These are variable in shape are seen in chronic renal failure and severe renal tubular (diamond, rosette, plates), and yellow or red-brown obstruction. Both waxy and broad casts are associated in color (due to urinary pigment). They are soluble in with poor prognosis. alkali, and insoluble in acid. Increased numbers are found in gout and leukemia. Flat hexagonal uric acid Cellular Casts crystals may be mistaken for cysteine crystals that also form in acid urine. To be called as cellular, casts should contain at least three b. Calcium oxalate crystals: These are colorless, refractile, cells in the matrix. Cellular casts are named according to and envelope-shaped. Sometimes dumbbell-shaped the type of cells entrapped in the matrix. or peanut-like forms are seen. They are soluble in Red cell casts: These are cylindrical structures with red dilute hydrochloric acid. Ingestion of certain foods cells in Tamm-Horsfall protein matrix. They may appear like tomatoes, spinach, cabbage, asparagus, and brown in color due to hemoglobin pigmentation. These rhubarb causes increase in their numbers. Their have greater diagnostic importance than any other cast. increased number in fresh urine (oxaluria) may also If present, they help to differentiate hematuria due to suggest oxalate stones. A large number are seen in glomerular disease from hematuria due to other causes. ethylene glycol poisoning. RBC casts usually denote glomerular pathology e.g. acute c. Amorphous urates: These are urate salts of potassium, glomerulonephritis. magnesium, or calcium in acid urine. They are usually yellow, fine granules in compact masses. They are White cell casts: These are cylindrical structures with white soluble in alkali or saline at 60°C. blood cells embedded in Tamm-Horsfall protein matrix. Leucocytes usually enter into tubules from the inter- Crystals present in alkaline urine: stitium and therefore presence of leucocyte casts indicates a. Calcium carbonate crystals: These are small, colorless, tubulointerstitial disease like pyelonephritis. and grouped in pairs. They are soluble in acetic acid and give off bubbles of gas when they dissolve. Renal tubular epithelial cell casts: These are composed of b. Phosphates: Phosphates may occur as crystals (triple renal tubular epithelial cells that have been sloughed off. phosphates, calcium hydrogen phosphate), or as They are seen in acute tubular necrosis, viral renal amorphous deposits. disease, heavy metal poisoning, and acute allograft Phosphate crystals rejection. Even an occasional renal tubular cast is a Triple phosphates (ammonium magnesium significant finding. phosphate): They are colorless, shiny, 3-6 sided prisms with oblique surfaces at the ends (“coffin- Crystals lids”), or may have a feathery fern-like appearance. Crystals are refractile structures with a definite geometric Calcium hydrogen phosphate (stellar phosphate): shape due to orderly 3-dimensional arrangement of its These are colorless, and of variable shape (star- atoms and molecules. Amorphous material (or deposit) shaped, plates or prisms). has no definite shape and is commonly seen in the form Amorphous phosphates: These occur as colorless of granular aggregates or clumps. small granules, often dispersed. Crystals in urine (Fig. 1.28) can be divided into two All phosphates are soluble in dilute acetic acid. main types: (1) Normal (seen in normal urinary sediment), and (2) Abnormal (seen in diseased states). c. Ammonium urate crystals: These occur as cactus-like However, crystals found in normal urine can also be seen (covered with spines) and called as ‘thornapple’ in some diseases in increased numbers. crystals. They are yellow-brown and soluble in acetic Most crystals have no clinical importance acid at 60°C. (particularly phosphates, urates, and oxalates). Crystals can be identified in urine by their morphology. However, Abnormal Crystals before reporting presence of any abnormal crystals, it is They are rare, but result from a pathological process. necessary to confirm them by chemical tests. These occur in acid pH, often in large amounts. Abnormal tahir99 - UnitedVRG vip.persianss.ir  28 Essentials of Clinical Pathology Fig. 1.28: Crystals in urine. (A) Normal crystals: (1) Calcium oxalate, (2) Triple phosphates, (3) Uric acid, (4) Amorphous phosphates, (5) Amorphous urates, (6) Ammonium urate. (B) Abnormal crystals: (1) Cysteine, (2) Cholesterol, (3) Bilirubin, (4) Tyrosine, (5) Sulfonamide, and (6) Leucine crystals should not be reported on microscopy alone; and appear stacked in a stair-step arrangement. They additional chemical tests are done for confirmation. are soluble in ether, chloroform, or alcohol. They are 1. Cysteine crystals: These are colorless, clear, hexagonal seen in lipiduria e.g. nephrotic syndrome and hyper- (having 6 sides), very refractile plates in acid urine. cholesterolemia. They can be positively identified by polarizing microscope. They often occur in layers. They are soluble in 30% 3. Bilirubin crystals: These are small (5 μ), brown crystals hydrochloric acid. They are seen in cysteinuria, an of variable shape (square, bead-like, or fine needles). inborn error of metabolism. Cysteine crystals are often Their presence can be confirmed by doing reagent associated with formation of cysteine stones. strip or chemical test for bilirubin. These crystals are 2. Cholesterol crystals: These are colorless, refractile, flat soluble in strong acid or alkali. They are seen in severe rectangular plates with notched (missing) corners, obstructive liver disease. tahir99 - UnitedVRG vip.persianss.ir  Examination of Urine 29 4. Leucine crystals: These are refractile, yellow or brown, CRITICAL FINDINGS spheres with radial or concentric striations. They are soluble in alkali. They are usually found in urine along Strongly positive test for glucose and ketone bodies with tyrosine in severe liver disease (cirrhosis). Positive test for reducing sugar in an infant 5. Tyrosine crystals: They appear as clusters of fine, Hemoglobinuria delicate, colorless or yellow needles and are seen in Red cell casts or >50% dysmorphic red cells on liver disease and tyrosinemia (an inborn error of microscopic examination metabolism). They dissolve in alkali. Abnormal crystals like cysteine, leucine, or tyrosine. 6. Sulfonamide crystals: They are variably shaped crystals, but usually appear as sheaves of needles. BIBLIOGRAPHY They occur following sulfonamide therapy. They are soluble in acetone. 1. Burtis CA, Ashwood ER (Eds). Tietz fundamentals of clinical chemistry (5th Ed). Philadelphia; WB Saunders REFERENCE RANGES Company, 2001. 2. Carroll MF, Temte JL. Proteinuria in adults: A diagnostic Volume in 24 hours: Adults: 600-2000 ml approach. Am Fam Physician 2000;62:1333-40. Color: Pale yellow to colorless 3. Cheesbrough M. District laboratory practice in tropical Appearance: Clear countries. Part 1 and Part 2. Cambridge; Cambridge Odor: Aromatic University Press, 1998. Specific gravity: 1.003-1.030 4. Grossfeld GD, Wolf JS, Litwin MS, et al. Asymptomatic Osmolality: 300-900 mOsm/kg of water microscopic hematuria in adults: Summary of the AUA pH: 4.6-8.0 (Average: 6.0) best policy recommendations. Am Fam Physician 2001; Proteins: Qualitative test: Negative 63:1145-54. Quantitative test: < 150 mg/24 hours 5. Henry JB (Ed): Clinical diagnosis and management by Albumin: < 30 mg/24 hours laboratory methods. (20th Ed). Philadelphia; WB Saunders Glucose: Qualitative test: Negative Company, 2001. Quantitative test: < 500 mg/24 hours (< 15 mg/dl) 6. King M. A medical laboratory for developing countries. Ketones: Qualitative test: Negative London. Oxford University Press, 1973. 7. Mathieson PW. The cellular basis of albuminuria. Clinical Bilirubin: Negative Science 2004;107:533-8. Bile salts: Negative 8. Simerville JA, Maxted WC, Pahira JJ. Urinalysis: A Occult blood: Negative comprehensive review. Am Fam Physician 2005;71: Urobilinogen: 0.5-4.0 mg/24 hours 1153-62. Myoglobin (Ammonium sulphate solubility test): 9. Wallach J. Interpretation of diagnostic tests. (7th Ed). Negative Philadelphia. Lippincott Williams and Wilkins, 2000. Microscopy: 1-2 red cells, pus cells, or epithelial cells/ 10. World Health Organization. Manual of basic techniques HPF; occasional hyaline cast/LPF; Phosphate, oxalate, for a health laboratory (2nd Ed). Geneva; World Health or urate crystals depending on urine pH. Organization, 2003. tahir99 - UnitedVRG vip.persianss.ir