Confirmation tests of Urine analysis - PowerPoint Presentation

Confirmation tests of Urine analysis

U/A: Provides data and interpretations obtained without, pain ,danger and distress to the patient

The basic (routine) urinalysis consists of four parts 1 specimen evaluation, 2-Physical Tests (Gross examination and Specific Gravity) 3- Chemical tests (Biochemical analysis) 4-Microscopic examination (Sediment Examination)

Types of Urine Collections First – morning voided urine specimen is choice Other type of urine specimen : Random Specimen First – morning or eight hours Specimen Timed Specimen (complete 24 hours collection) Midstream – voided Specimen ( either Random or first morning – used for routine analysis) Clean – catch (midstream) Specimen (for bacterial culture )

Examination of Physical Properties of URINE

Specimen for routine urinalysis Well – mixed First morning ( eight hours concentrated ) Tested at room temperature Ideally the specimen should be tested within 30 min of voiding within 2 hours of collection Specimens should not be accepted if left at room temperature for more than 2 hours

CHEMICAL SCREENING Reagent strips are the primary method used for the chemical examination of urine. Although easily used, they represent multiple complex, state-of the-art chemical reactions.

pH: The kidneys and lungs normally work in concert to maintain acid-base equilibrium Urine pH is a reflection of the ability of kidneys to maintain normal H+ concentration in plasma and extra cellular fluid Normal Adult: about 6 ( 4.6 – 8 ) diet with high meat protein = Acid urine diet with high vegetable = Alkaline urine

Reagent Strip. Indicators methyl red and bromothymol blue give a range of orange, green, and blue colors as the pH rises, permitting estimation of pH values to within half a unit within the range of 5 to 9. It should be read immediately , Measurement of urine pH and acidity must always be made on freshly voided specimens. The container should be kept cold, preferably on ice, but not frozen On standing, the pH tends to rise because of loss of carbon dioxide and because bacterial growth produces ammonia from urea.

Protein in Urine Normally , up to 150 mg of protein is excreted in the urine daily, with the average urine protein concentration varying from 2 to 10 mg/ dL , depending on urine volume About one third is albumin , and the remaining plasma proteins include small globulins, such as α-, β-, and γ-globulins . Tamm- Horsfall glycoprotein ( uromucoid ), secreted by distal tubular cells and cells of the ascending loop of Henle, constitutes one third or more of the total normal protein loss.

Functional proteinuria is usually less than 0.5 g/day and can be seen in various situations in which dehydration contributes to the level of protein measured in urine. With strenuous exercise, a mixture of high and low molecular weight proteins appears in the urine, and many casts, bothhyaline and granular, can be seen. Functional proteinuria may also accompany congestive heart failure, cold exposure, and fever.

Proteinuria in Older Adults The incidence of significant proteinuria found on urinalysis in the older adult population is substantially increased when compared with patients younger than 60 years of age older adult population in general has a threefold to fourfold greater incidence of glomerulonephritis ,. Occult malignancies in this population may also give rise to membranous glomerulonephritis , with resultant proteinuria

Heavy Proteinuria (>4 g/day). Characteristically seen with nephrotic syndrome a low serum albumin level , generalized edema, and increased serum lipids (cholesterol, triglycerides , and phosphatides) accompany this disorder

Minimal Proteinuria (<1.0 g/day). in chronic pyelonephritis

Microalbuminuria. albumin in urine above the normal level but below the detectable range of conventional urine dipstick methods. lower urine albumin levels ranging from 20 to 200 mg/L indicator of early and possibly reversible glomerular damage. In diabetic patients, microalbuminuria is associated with a fourfold to sixfold increase in cardiovascular mortality, and is an independent risk factor for renal mortality It is also more prevalent in hypertensive subjects Various methods have been introduced, including Immunologic test systems and dye-binding chemical test strips,

Methods positive screening test may have serious implications, it is important to be able to confirm results by a second, different method Common screening tests include the qualitative/ semiquantitative colorimetric reagent strip test and precipitation-based testing Accurate results are obtained with reagent strips only when albumin is increased. Because of the lack of sensitivity of the reagent strip to globulins,

Reagent Strip. This method takes advantage of the protein error of pH indicators. Because proteins carry a charge at physiologic pH, The reagent strip is tetrabromphenol blue buffered to an acid pH of 3, or tetrachlorophenoltetrabromosulfophthalein . In the absence of protein, the strip is yellow; 30 to 60 seconds following urine application, variable shades of green develop , depending on the type and concentration of protein present. be read in a “plus” system as negative, trace, and 1+ to 4+. Most methods will detect 5 to 20 mg/dl of albumin.

1-Reagent Strip: Cut off 5 -20 mg/dl Albumin False Pos : Highly Alkaline Urine (PH>9) Phenazopyridine False Neg : Highly diluted urine

2-Sulfpsalicylic Acid 3%: cut off 5 – 10 mg/dl Albumin,Globulin‘s , Glycoproteins,Bens – Jones protein False Pos : Tulbutamid,Penicilin,Sulfnamide False Neg : ↑↑ diluted & ↑↑ alkaline urine

Procedure. Specimens should be centrifuged, and a clear supernatant used . To approximately 3 mL of supernatant urine in a clean test tube , aliquot an equal amount of 3% SSA. Invert to mix. Let stand exactly 10 minutes . Invert again twice. Using ordinary room light (not a lamp ), observe the degree of turbidity and/or precipitation, and grade the results according to the following descriptions:

Negative—no turbidity (≈5 mg/ dL or less) Trace—perceptible turbidity (≈20 mg/ dL ) 1+ —Distinct turbidity, but no discrete granulation (≈50 mg/ dL ) 2+ —Turbidity with granulation, but no flocculation (≈200 mg/ dL ) 3+ —Turbidity with granulation and flocculation (≈500 mg/ dL ) 4+ —Clumps of precipitated protein, or solid precipitate (≈1.0 g/ dL or more )

Glucose : Glucosuria usually occurs when the blood level is more than 180 – 200 mg/dl Method: Reagent strip: Pos in: D M and also Normal Neonates during the first 10 to 14 days of life owing to glucose , galactose , fructose ,lactose Normal Pregnant and Postpartum woman due to lactose

Methods Reagent Strip. This method is based on a specific glucose oxidase and peroxidase method, a double sequential enzyme reaction; reagent strips differ only in the chromogen used False-positive readings may be produced by strongly oxidizing cleaning agents in the urine container. Low specific gravity may falsely elevate results . Sodium fluoride used as a preservative will cause false-negative readings , as can high specific gravity and occasionally ascorbic acid. Glycolytic enzymes from cells and bacteria will reduce glucose levels in urine on standing; prompt refrigeration or testing is essential

Copper Reduction Tests. As a screening test, the glucose oxidase method will not detect increased levels of galactose or other sugars in urine. It is therefore important that a copper reduction method be used, especially for young pediatric patients Of the copper reduction methods used for screening purposes, the qualitative Benedict method is more sensitive to reducing substances in urine than is the single-tablet ( Clinitest ) copper reduction method Strong reducing substances such as ascorbic acid, gentisic acid, or homogentisic acid may inhibit the

Keton's: Incomplete fat metabolism (Acetoacetic,acetone,3 hydroxy butyric acid) Pos in: Diabetic Ketosis Febrile Dis and Toxic states with Vomiting or Diarrhea (Infants & Children) Vomiting of Pregnancy Cachexia,Starvation , After Anesthesia ,Exposure to Cold Sever Exercise

Depending on the methods used, total ketone bodies (as acetone) can range as high as 17 to 42 mg/ dL . up to 2 mg/dl acetoacetic acid is normal Reagent Strip. This method is based on a nitroprusside ( sodium nitroferricyanide ) reaction for ketones . Different methods measure acetoacetic acid alone, or both acetone and acetoacetic acid. Ferric chloride (Gerhardt’s test) detects acetoacetic acid. These methods do not measure 3-hydroxybutyrate, the predominant ketone body . In urine and plasma, reagent strips and tablets react to 10 mg/dl of acetoacetic acid and are less sensitive to acetone .

Chemstrip reagent strips contain sodium nitroferricyanide and glycine, which react with acetoacetic acid and acetone in an alkaline medium to form a violet dye. A positive result is indicated by a color change from beige( رنگ بژ ) to viole t, which is read at 60 seconds. The method detects about 10 mg/ dL of acetoacetic acid and 70 mg/ dL of acetone,

Blood: Detection limit: 5 – 15 RBC/ml 0.06 – 0.15 mg/dl Hb Pos in : Hematuria Hemoglobinuria Myoglobinuria ( destruction of muscle fibers (rhabdomyolysis) occurs )

Reagent Strip for Heme Compounds (Hemoglobin, Myoglobin 0.05 to 0.3 mg/dl of hemoglobin urine . 0.3 mg/dl of hemoglobin = 10 lysed erythrocytes per microliter. Normal erythrocytes contain approximately 30 pg of hemoglobin per cell.

common problem with the method is the inhibition of the hemoglobin reagent strip by interfering ubstances , commonly ascorbic acid , and this problem emphasizes the need for a routine microscopic examination to screen for hematuria and confirm the presence of hematuria in patients with a positive reagent strip hemoglobin test A positive test for hemoglobin with a normal urinary sediment suggests that a fresh urine sample should be examined for erythrocytes, because an alkaline pH or urine specific gravity of less than 1.010 may cause lysis of erythrocytes

Lab Finding in Myoglobinuria ( rhabdomyolysis) Urine : Red ,Brown -occasional Proteinuria // Supernatant not clear in ammonium Sulfate test Plasma color : Normal marked increase of C K increase Aldolase Normal Haptoglobin

Qualitative Test for Myoglobin Use a fresh urine specimen Mix 1 mL of urine and 3 mL of 3% sulfosalicylic acid to assay for protein. If the pigment is precipitated, it is a protein. Filter . If the filtrate is a normal color, no abnormal nonprotein pigment is present . ( Note: The heat and acetic acid test does not precipitate myoglobin or hemoglobin.)

To 5 mL of urine in a test tube, add 2.8 g of ammonium sulfate Dissolve by mixing. This is optimal for precipitation of hemoglobin. Filter or centrifuge. If the supernatant shows a normal color, the precipitated pigment is hemoglobin . If the supernatant fluid is colored, this is presumptive evidence of myoglobin .

Nitrite : Bacteria reduce urinary Nitrite to nitrate Pos Nitrite indicate significant number of bacteria (> 10 6 Gm - ,Escherichia Coli) False Pos : Poorly Collected and stored specimen

Leukocyte Esterase: Esterase of Azurophilic Granules of PMN and other cells are labile in urine Useful in detecting the Enzyme remnants of cells that are not visible microscopic Cut off: 10 – 30 Leukocyte /ml urine

Reagent strip. The nitrite testing area of Multistix is impregnated with p - arsanilic acid, which forms a diazonium salt when it reacts with nitrite present in the urine. This compound is then able to couple with benzoquinoline to form a pink azo dye. This method detects 0.075 mg/dl of nitrite in solution and is read at 40 seconds False-positive with poorly collected/stored specimens as the result of contaminants and postcollection bacterial proliferation . False-negative nitrite results may be due to ascorbic acid, urobilinogen , or low pH (<6).

Bilirubin: Normal Adulate contains 0.02 mg/dl in urine (not detected by usual tests) Should be performed on fresh urine Pos in : Jaundice( obstruction to bile outflow ) & Cong hyperbilirubinemia (Dub in – Johnson, Rotor ) False Pos : Chloropromazopine,Phenazopyridine False Neg :↑ Vita C ,↑Nitrite

Bilirubinuria is associated with yellow-brown to greenish brown urine that may have a yellow foam , elevated serum bilirubin (conjugated ),A positive test for urinary bilirubin with a negative test for urobilinogen in urine is indicative of intrahepatic or extrahepatic biliary obstruction . This test is valuable in the differential diagnosis of jaundice, because bilirubinuria is not found with hemolytic jaundice .

Reagent Strip coupling reaction of bilirubin with a diazonium salt in acid medium. When this method is used , normal urine contains no detectable bilirubin Chemstrip uses 2,6-dichlorobenzene-diazonium tetrafluoroborate , and the color changes from pink to violet at 30 to 60 seconds . This test detects 0.5 mg/DL urine. Urine must be fresh because bilirubin glucuronide in urine quickly hydrolyzes to less reactive free bilirubin .

Urobilinogen in Urine Conjugated bilirubin from the liver eventually reaches the duodenum , complexed with cholesterol, bile salts, and phospholipids within the bile The free bilirubin is then reduced to urobilinogen , mesobilirubinogen , and stercobilinogen . Up to 50% of the Urobilinogen is reabsorbed into the portal circulation and is reexcreted , unconjugated , into the bile .

Normal output of urobilinogen in the urine is 0.5 to 2.5 mg or units/24 hours These substances are colorless and labile, as opposed to the urobilins , the oxidation products of urobilinogen that impart a yellow-orange color to normal urine. Output of urobilinogen is increased in alkaline urine; the level is decreased in acid urine . Increased excreted in the urine hepatocellular damage due to viral hepatitis, drugs, or toxic substances, or in some cases of cirrhosis

Reagent Strip. Testing is based on the Ehrlich aldehyde reaction or on the formation of a red azo dye from a diazonium compound . Multistix method ; its test area is impregnated with an acid buffer solution and p - dimethylaminobenzaldehyde , which produces a reddish brown color with Urobilinogen Color varies from light yellow to shades of red-brown, and values from 0.2 to 1 mg /dl are considered normal . This test method is not specific for Urobilinogen and react with porphobilinogen , p - aminosalicylic acid metabolites, sulfonamides, procaine , 5-hydroxyindoleacetic acid, indole, and methyldopa

ASCORBIC ACID Or Vitamin C is neither a normal nor pathologic constitute of urine The presence of ascorbic acid may interfere with the measurement of other chemical tests like BLOOD and GLUCOSE Detection of these analyte depend on the release of hydrogen peroxide Ascorbic acid causing reduce or false negative reaction

COLOR Normal Color: The Normal color of urine varies considerably even between specimen from the same person in a given day The color of normal urine results from the presence of three pigments: Urochrome , Uroerythrin , Urobilin

Abnormal Color Highly colored urine specimen are often the result of various medications chemicals, dyes, vitamins, fruits, or vegetables . Although these substances have little clinical significance, they are a problem for their interfere with or mask the various chemical reaction on reagent strips that indicate abnormal constituents in urine

Abnormal Color Amber ( Dark yellow or Orange – red) indicate a very concentrated specimen, usually with low volume It is often seen in condition such as fever or dehydration The color may be similar to specimen containing the pigments bilirubin or urobilin

Abnormal Color Brown (yellow – brown or Green – brown) Indicates the presence of bilirubin , related to Jaundice , on standing bilirubin oxidize to biliverdin a green pigment resulting in a green – brown specimen Urine specimen containing bilirubin will Foam when shaken and the foam is yellow Orange (Orange – red or Orange – Brown) Is very similar when urine contain bilirubin and results of Urobilin an oxidant product of urobilinogen

Abnormal Color Red or Pink Caused by the presence of RBC, Hemoglobin, and Myoglobin A concentrated specimen such as dehydration or fever may show a characteristic precipitate and color caused by the presence of amorrh urates or uric acid . This precipitate is PINK to red Many drugs and foodstuffs may cause a red or pink urine e.g. laxatives, levodopa, methydopa

Clear Red : Indicates presence of Hemoglobin Hb may result from intravascular hemolysis or lysis of RBC in urine specimen or urinary tract The specimen may be pink, red, red – brown or even black ,which is a result of conversion of hemoglobin to met hemoglobin Cloudy Red : Suggests th e presence of red blood cells and its intensity will depend on the number of RBC The possibility of menstrual contamination should be considered in female patient

Phenazopyridin a drug commonly used for UTI results an intense orange – colored specimen that looks very much like bilirubin and interferes with several reagent strip tests Pale suggests diluted urine, clinically pale urine may be associated with diabetes mellitus or diabetes insipidis . Pale urine also seen in kidney and urinary tract disease where there is a loss of the ability to concentrate urine

Dark Red – Brown A color similar to cola is characteristic of MYOGLOBIN Its associated with extensive muscle injury from trauma or extreme exercise Dark Red or Red – Purple Color of port wine is characteristic for PORPHYRINS the urine may be colorless when voided but darkens upon standing

Brown or Black Urine containing MELANINE or HOMOGENTISIC ACID , may be normally colored when voided but become brown or black upon standing PHENOL poising may also result an olive – green to black urine BLUE or GREEN Most likely caused by presence of dyes , drugs or ingested substance like CHLOROPHYLL in mount deodorants Pathogenic finding include infection of the small intestine and PSEDOMONAS infection

T RASPARENCY Urine is normally clear when voided but often becomes Cloudy when it stands due to precipitation of amorphous crystals or mucus Growth of bacteria in an improperly stored urine Standard terms are Clear, Hazy, Cloudy, Turbid Clear No visible particular matter present Hazy Some visible particular matter present

amorphous. for cloudy urine is broad and includes several nonpathologic entities. Turbidity may simply be due to the precipitation of crystals or nonpathologic salts referred to as amorphous. Phosphate, ammonium urate, and carbonate can precipitate in alkaline urine these redissolve when acetic acid is added. Uric acid and urates cause a white , pink, or orange cloud in acid urine and redissolve on warming to 60° C.

2-Leukocytes ( may form a white cloudy appear) 3- Bacterial growth (causes a uniform opalescence's ) 4- Gross hematuria (Smokiness) 5- ↑ Epithelial cell 6- Spermatozoa and prostatic fluid Turbidity may be due to:

MUCUS: from the urinary passages may cause a BULKY DEPOSITE that increased in inflammatory states of the lower urinary tract as genital tract

Pseudochyluria : ( use of paraffin based vaginal creams for the treatment of candida infections)

Lipiduria : Oily contaminations, Cholesterol and triglycerides (in nephrotic syndrome) Major Skeletal Trauma with one or more Fractures

Specific Gravity (S .G) Urea (20%), sodium chloride (25%), sulfate, and phosphate contribute most of the specific gravity of normal urine reflect the ability of kidney to concentrate or dilute the urine Normal range = 1.005 – 1.033 less than 1.007 Hyposthenuria more than 1.033 either contamination ( high level glucose , Iv radiopaque dye) Methods: Reagent Strip Refractometer urinometer , and ( Picnometer ).

Chemical Examination pH Albumin or protein Glucose Bile Acetone Calcium Bilirubin Urobilinogen Blood Hb

Osmolality The normal adult with a normal fluid intake will produce urine of about 500 to 850 mOsm /kg water. The normal kidney is able to produce a urine osmolality in the range of 800 to 1400 mOsm /kg water in dehydration, and a minimal osmolality of 40 to 80 mOsm /kg water during water diuresis. After a period of dehydration, the osmolality of the urine should be three to four times that of the plasma (e.g., with a plasma osmolality of 285 mOsm /kg water, the urine osmolality should be at least 855 mOsm /kg water). Methods. The freezing-point depression method is commonly employed . A solution containing 1osmol or 1000 mOsm /kg water depresses the freezing point -1.86 ° C below the freezing point of water.