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URINE ANALYSIS       DR.P.G.KONAPUR   PROFESSOR OF PATHOLOGY   V.M.K.V.MEDICAL COLLEGE          SALEM           www.simili...
1.Formation of Urine2.Normal constituents and composition    3.Collection of urine specimens        4.Preservation of urin...
• Urine --- most easily obtained• Examination of urine• Information--- about the functioning of  the kidney• diagnosis ---...
•   Urine is formed in the kidney.•   The functional unit of kidney is called nephrons,•    where in the ultrafilteration ...
3 NORMAL CONSTITUENTS AND COMPOSITIONOF URINEConstituent’s                                     grams/litreA.Inorganic• Chl...
COLLECTION OF URINE SPECIMENS• In. Improper collection---- may invalidate the results• Containers for collection of urine ...
Midstream urine specimen• Urine should be collected in a clean, dry and,    preferably, sterilized container.•   urethral ...
• Terminal urine sample patient voids the last portion of    urine in an open container.•   Urine specimen collection usin...
PRESERVATION OF URINE•   Urine sample < within 2 hours.•   If delay ----preservation.•   If more than 2 hours    – Keep th...
PHYSICAL EXAMINATION• Volume –     normal -- 1.2-2 L /day     the day is 3-4 times > night.     night is < 400 ml.     pol...
1.Colorless---Very dilute urine           ---Polyuria,diabetes 2.Yellow orange(high colored)--Concentrated urine          ...
• 2. Turbidity – normal urine----clear.• Turbidity in urine may be due to:         a. amorphous phosphate and carbonates ...
• b. Odour: Normal-- aromatic odour       On standing--- ammonical        there is decomposition of urea forming ammonia w...
pH•   ability to maintain normal hydrogen ion concentration• Normal kidneys are capable of producing urine the pH of which...
SPECIFIC GRAVITY• degree of concentration or dilution of the  specimen.• specific gravity measures the  concentrating and ...
PROCEDURE Urine is poured into a cylindrical or conical glass so that the vessel is    nearly full.    Froth-- removed wit...
www.similima.com   17
• Correction for temperature-• The urinometer is calibrated to read 1.000 in distilled    water at a specific temperature,...
• 1. SUGARS IN URINE:• This is a non-specific test useful for semiquantitation of marked   glucosuria• Benedict’s qualitat...
• Dissolve crystalline copper sulphate in 100 ml of  distilled water. Dissolve sodium carbonate and  sodium citrate in 700...
www.similima.com   21
Recording results The color varies from blue through green –   yellow- orange- brick red.  Negative – no change in color. ...
– COLORIMETRIC REAGENT STRIP TEST•       Principle: this test is based on a  double sequential enzyme reaction.• One enzym...
www.similima.com   24
KETONES IN URINE (ketonuria):•       fats--------carbon dioxide and water.•       inadequate carbohydrate in the diet or a...
• Rothera’s Test for Acetone and  Acetoacetic Acid:• Procedure:  – Take 5ml of urine in a test tube and saturate    it wit...
www.similima.com   27
Result: a permanganate calomel red (pink –  purple) ring forms at the junction of the  two layers------positive.• Negative...
• 3. Proteins in urine:  Normal <30 mgms / 100 ml (30 – 50  mgms/24 hours)Tests for Detection of Proteins• Semiquantitativ...
• Negative – No turbidity or cloudiness.• Trace - A faint precipitate visible against a    black background, equivalent to...
• Heat and Acetic Acid Method    Procedure :•   Take a long test tube and fill ¾ the tube with    clear urine.•   Boil the...
www.similima.com   32
• Sulphosalicylic acid method• Procedure• Take 2 ml of clear urine in a test tube.• Add an equal volume of 30 %  sulphosal...
• Nitric acid method• Procedure• Take 2 – 3 ml of concentrated nitric acid in a    test tube.•   Carefully pour 5ml of cle...
• Colorimetric reagent strip test•   PRINCIPLE: the colorimetric reagent strip test is based upon the    ability of protei...
Bence Jones Proteinuria:• Bence Jones protein is soluble at room  and body temperatures.• It precipitates upon heating bet...
OCCULT BLOOD IN URINE:    red blood cells          or    haemoglobin When hemolysis occurs    in circulation    urine ...
• Haematuria: Denotes the presence of red blood cells in urine. It is seen in various renal disorders, infectious or neopl...
• Tests for Detection of Blood:• 1. Microscopic Examination:    sediment------------RBC’S/HPF• 2. Benzidine Test    PRINCI...
Colorimetric reagent stripmethodPrinciple: the reagent area is impregnated  with tetramethylbenzidine and buffered  organi...
BILE IN THE URINE: The constituents1.          bilirubin (bile pigments),2.          bile salts,3.          urobilin and u...
• certain liver diseases --unable to  conjugate all the bilirubin-----an increase  in both conjugated and unconjugated  ...
Tests for detection of bile salts:Hay TestProcedure:flowers of sulphur --sprinkled on the surface   of the urine,Results:i...
Tests for detection of bile pigments    1. Foam Test:        Shake urine in a test tube. If the foam on         top is yel...
3. Fouchets Test:FOUCHETS REAGENT             Trichloroacetic acid – 25 gms             Distilled water - 100 ml          ...
Colorimetric Strip Reagent Test• Principle: this test is base on the coupling of    bilirubin with diazotized 2, 4-•      ...
Tests for detection ofurobilinogen:1. Qualitative Ehrlich’s Test EHRLICH’S REAGENT           Paradimethylaminobenzaldehyde...
Results:  pink color  observable when viewed from the top of the test tube. Against a white background placed beneath the ...
MICROSCOPIC EXAMINATIONQualitative technique:the urine must be freshly voidedexamined without excessive delay in order to ...
10-15 ml of urine ----from freshly mixed urine     specimen and centrifuged at a standard speed,    usually 1500 to 2000 ...
low power-  Casts tend to congregate at the edges of the  cover slip.  A minimum of 10 – 15 high power fields should  be ...
• NORMAL SEDIMENTNormal sediment contains a limited number of formed elements. it can be divided into two classes.• unorga...
A. Unorganized sediment - these are the crystals of various    substances present in the urine and they vary with the    p...
B.Calcium Oxalate:They are commonly found in diets rich intomatoes, spinach etc. they are typicallyenvelope shaped crystal...
• e.Tyrosine:–    fine needles arranged in concentric sheaves, constriction at the  middle.   liver disorders.• f.Sulpha c...
Crystals in acid urine Amorphous urates-Brick – Red-  Granules Uric Acid -Yellow – Brown--  Polymorphous – Whetstones,...
Cholestrol (rare) -ColorlessBroken window panes with notched corners ,Flat platesLeucine (rare)-1. Yellow or Brown2.High...
Acid, Neutral or SlightlyAlkaline UrineCalcium oxalate--Colorless Octahedral Dumbbells,Often smallHippuric acid Colorle...
Alkaline UrineCalcium Carbonate-Colorless --Needles,Spheres,DumbbellsAmmonium biurate-Yellow Opaque BrownThorn apple,Sp...
A- represents the residue of normal human urine, as seen under the   microscope.B is represented oxalate of urea. An exces...
A and B--highly magnified urinary deposits, which indicate different   degrees of impairment of the digestive functions ar...
epithelial cellsIn division A is represented pus and mucus, the presence of which  indicates suppuration of the kidneys (B...
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Uric acid• In division A are represented the mixed urates as they appear  during idiopathic fevers, as intermittent, remit...
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varieties of cancer.           www.similima.com   67
• Organized Sediment: the components of organized    sediment include casts, red blood cells, white blood cells,    epithe...
1. Hyaline casts:   are colorless,  semi-transparent and  occasionally refractile cylinders and are soluble in acetic acid...
4. Red Blood Cell Casts:   are casts with red blood cells embedded  in the coagulated protein in the tubule.  Their presen...
• CELLS:• a. Red blood cells: Normally 1-2 red blood cell    are found per high power field•   .they appear pale , light r...
• Quantitative Evaluation of the urine  sediment – Addis count• The Addis count is a quantitative  measurement of the excr...
DETECTION OF BACTERIA• Microscopic Examination:   sediment - >20 or more bacteria per high  power field may indicate a ur...
Results:uniform pink color --positive resultthe presence of 100,000 or more organisms per ml,A negative result should nev...
www.similima.com   75
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  1. 1. URINE ANALYSIS DR.P.G.KONAPUR PROFESSOR OF PATHOLOGY V.M.K.V.MEDICAL COLLEGE SALEM www.similima.com 1
  2. 2. 1.Formation of Urine2.Normal constituents and composition 3.Collection of urine specimens 4.Preservation of urine 5.Physical examination of urine 6.Chemical Examination of Urine 7. Microscopic Examination Of Urine Deposits
  3. 3. • Urine --- most easily obtained• Examination of urine• Information--- about the functioning of the kidney• diagnosis ---of urinary tract diseases• diagnosis -----of certain metabolic and systemic diseases. www.similima.com 3
  4. 4. • Urine is formed in the kidney.• The functional unit of kidney is called nephrons,• where in the ultrafilteration of plasma takes place,• followed by absorption of most of the water and some of the solutes.• The kidneys through the nephrons ---excrete many of the waste products of the body www.similima.com 4
  5. 5. 3 NORMAL CONSTITUENTS AND COMPOSITIONOF URINEConstituent’s grams/litreA.Inorganic• Chloride 9.0• Phosphorous 2.0• Sulfur 1.5• Sodium 4.0• Potassium 2.0• Calcium 0.2• Magnesium 0.2• Iron 003B.Organic• Urea 25• Uric acid 0.6• Creatinine 1.5• Ammonia 0.6• Sugar not detected by benedicts test trace• Ketone bodies trace• Carbonates, bicarbonates & free carbonic acid trace• Mucin & mucin like substances Diastase www.similima.com 5
  6. 6. COLLECTION OF URINE SPECIMENS• In. Improper collection---- may invalidate the results• Containers for collection of urine should be wide mouthed, clean and dry• First morning sample –concentrated urine --- chemical constituents casts and crystals• Random specimen chemical screening microscopic examinations• 24 hours urine sample quantitative estimation of proteins, sugars, electrolytes, and hormones. Urine specimen is collected in a clean 2 liters bottle with a stopper. The first morning sample is not collected. All the urine passed during the rest of the day and night and next day I st sample is collected. Volume is measured and immediately sent to the laboratory. www.similima.com 6
  7. 7. Midstream urine specimen• Urine should be collected in a clean, dry and, preferably, sterilized container.• urethral opening is cleaned with a moist cotton swab.• first 10 – 25 ml of urine is not collected (discarded)• since it contains urethral and prostatic secretions which may be required if investigating urethra and prostrate.• First morning samples ---- these are the concentrated samples and casts, crystals etc .if present www.similima.com 7
  8. 8. • Terminal urine sample patient voids the last portion of urine in an open container.• Urine specimen collection using a catheter. This procedure is used for certain bacteriological tests• Urine specimens from infants• Urine can be collected into a plastic bag with an adhesive mouth.• The bag is fixed around the genitalia and left in place for 1- 3 hours, depending on the examination requested.• Colostomy bags can be used www.similima.com 8
  9. 9. PRESERVATION OF URINE• Urine sample < within 2 hours.• If delay ----preservation.• If more than 2 hours – Keep the sample in the refrigerator without any preservative. – Toluene – add a few drops till it forms a thin layer on the surface of urine. – Conc.HCL – 1 ml of conc.HCL for 125-150mlof urine. – Formaldehyde -1 drop for 15 ml of urine. Cells and casts are well preserved. www.similima.com 9
  10. 10. PHYSICAL EXAMINATION• Volume – normal -- 1.2-2 L /day the day is 3-4 times > night. night is < 400 ml. polyuria >2000ml / day. Oliguria <500ml / day. Anuria is total suppression of urine <100 ml per day.• Appearance – color & turbidity• 1. Color - normal ---- amber yellow ( to the presence of urobilin,uroerythrin,and urochromes) www.similima.com 10
  11. 11. 1.Colorless---Very dilute urine ---Polyuria,diabetes 2.Yellow orange(high colored)--Concentrated urine -----------Excess urobilin ------------Bile pigments ------------Intake of carrots (Yellow foam ++) 3.Red/ smoky------Hemoglobin/ ---- RBC ----MyoglobinBeetroot / ---------- anilinedyes -------Menstrual contamination -------porphyrins 4.Cloudy--------Phosphates & carbonates, Urates & uric acid Pus cells bacteria,yeast,spermatozoa 5.Milky-------Pyuria,Fat ,chyluria 6. Brown black----Methemoglobin,,Homogenesticacid(alkaptonuria),Melanin7. orange--------Bile pigments,Drugs – pyridium,rifampicin www.similima.com 11
  12. 12. • 2. Turbidity – normal urine----clear.• Turbidity in urine may be due to: a. amorphous phosphate and carbonates – alkaline or neutral urine disappears on addition of dilute acetic acid b. crystals, cellular exudates, bacteria and fungus c. chyle and fat d. pus e. amorphous urates in acidic urine www.similima.com 12
  13. 13. • b. Odour: Normal-- aromatic odour On standing--- ammonical there is decomposition of urea forming ammonia which gives a strong ammonical smell..• Abnormal odour of urine Fruity---- Ketonuria Mousy---- Phenylketonuria Rancid------ Tyrosinaemia www.similima.com 13
  14. 14. pH• ability to maintain normal hydrogen ion concentration• Normal kidneys are capable of producing urine the pH of which varies from 4.5 to slightly higher than 8.0.• A pH below 7 indicates acid urine and a pH above7 alkaline urine• PROCEDURE• Dip the litumus paper strips in the urine, remove and read the color change immediately.• Blue litmus turns red - acid• Red litmus turns blue - alkaline• Blue and red litmus turns purple - neutral• nitrazine paper method :The Ph---- nitrazine paper which are sensitive and specific in the pH range of 4.5 – 8.0 range. www.similima.com 14
  15. 15. SPECIFIC GRAVITY• degree of concentration or dilution of the specimen.• specific gravity measures the concentrating and diluting abilities of the kidney.• The normal --------------1.015 and 1.025 (in a 24 hours specimen).ESTIMATION OF SPECIFIC GRAVITY BY URINOMETER• urinometer: This is a weighted bulb- shaped a scale calibrated----- from 1.000 to 1.060 www.similima.com 15
  16. 16. PROCEDURE Urine is poured into a cylindrical or conical glass so that the vessel is nearly full. Froth-- removed with a filter • The instrument is floated in the urine and care should be taken to see that it does not touch the slides. • The depth to which it sinks in urine indicates the specific gravity of urine • read on the urinometer scale at the junction of the urine with the air. • The reading is taken at eye level, the lowest part of the meniscus being taken. • In case the urine is insufficient, it may be diluted with an equal volume of distilled water and the last two figures of the reading are then multiplied by 2. www.similima.com 16
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  18. 18. • Correction for temperature-• The urinometer is calibrated to read 1.000 in distilled water at a specific temperature, indicated on each instrument e.g. 15 C or 20 C.• There is a change in the specific gravity of 0.001 for each 3 C above and below this temperature. Therefore add 0.001 to the reading for each 3 C above the temperature for which the urinometer is calibrated,• substract 0.001 for each 3 C, the temperature below the standard temperature.• For example - urinometer calibrated for 20 C• Specific gravity of urine at 32 C is 1.001• Corrected specific gravity {(32 – 20)/ 3 X 0.001} + 1.001 = 1.005• Correction is also recommended when glucose or protein are present. It is recommended that .003 be subtracted from the urinometer reading for each 1000 mg / dl of glucose or protein. www.similima.com 18
  19. 19. • 1. SUGARS IN URINE:• This is a non-specific test useful for semiquantitation of marked glucosuria• Benedict’s qualitative test:• This test is not specific for sugars and is affected by most of the reducing substances.• Composition of Benedict’s reagent: Copper sulphate - 17.3 gms Sodium carbonate (anhydrous) – 100 gms Or Crystalline sodium carbonate - 200 Gms Sodium citrate - 175 gms Or Potassium citrate www.similima.com 19
  20. 20. • Dissolve crystalline copper sulphate in 100 ml of distilled water. Dissolve sodium carbonate and sodium citrate in 700 cc. of distilled water. Slowly add the latter to the former solution with constant stirring. When complete, make up the volume to 1000ml with distilled water. Procedure• Take 5ml of benedicts reagent• Boil for 3 – 5 minutes• add to it 0.5ml (8 drops)of protein free urine.• Cool and note the color. www.similima.com 20
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  22. 22. Recording results The color varies from blue through green – yellow- orange- brick red. Negative – no change in color. Trace - solution appears pale green to slightly cloudy. 1+ - Definite cloudy green (0.5% sugar) 2+ - Yellow to orange precipitate, supernatant fluid pale blue (1% sugar) 3+ - Orange to red precipitate, supernatant fluid pale blue (1.5% sugars) 4+ - Brick red precipitate, supernatant fluid decolorizes (2 % sugar) www.similima.com 22
  23. 23. – COLORIMETRIC REAGENT STRIP TEST• Principle: this test is based on a double sequential enzyme reaction.• One enzyme, glucose oxidase, catalyzes the formation of gluconic acid and hydrogen peroxide from the oxidation of glucose.• A second enzyme, peroxides catalyzes the reaction of hydrogen peroxide with potassium iodide chromogen to oxidize the chromogen to colors ranging from green to brown. www.similima.com 23
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  25. 25. KETONES IN URINE (ketonuria):• fats--------carbon dioxide and water.• inadequate carbohydrate in the diet or a defect in carbohydrate metabolism or absorption,• fatty acids------------metabolized.• When the fatty acid utilization is incomplete------- the intermediary products of fat metabolism appear in the blood and the urine.• These products -------- acetone,• diacetic acid (acetoacetic acid) betahydroxybutyric acid.• Diabetes mellitus,• Other Causes of Ketonuria: – Fever – Anorexia – Gastrointestinal disturbances – Fasting – Starvation – Severe vomiting www.similima.com 25
  26. 26. • Rothera’s Test for Acetone and Acetoacetic Acid:• Procedure: – Take 5ml of urine in a test tube and saturate it with ammonium sulphate. – Add 1 crystal of sodium nitroprusside.Run liquor ammonia carefully at the side of the tube so as to form a layer on top of the saturated urine. www.similima.com 26
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  28. 28. Result: a permanganate calomel red (pink – purple) ring forms at the junction of the two layers------positive.• Negative result shows no ring or a brown ring.• Ferric Chloride Test for Diacetic Acid – Gerhardt’s Test• Harts Test for Beta-Hydroxybutyric Acid• Colorimetric Reagent Strip Test www.similima.com 28
  29. 29. • 3. Proteins in urine: Normal <30 mgms / 100 ml (30 – 50 mgms/24 hours)Tests for Detection of Proteins• Semiquantitative precipitation tests: 1.The heat and acetic acid method, 2.sulfosalicyclic acid method and 3.the concentrated nitric acid protein precipitation method precipitation. www.similima.com 29
  30. 30. • Negative – No turbidity or cloudiness.• Trace - A faint precipitate visible against a black background, equivalent to about 5 mg / dl protein.• 1+ - Definite cloud without flocculation equivalent to 10 – 30 mg / dl.• 2+ - Heavy and granular cloud without flocculation equivalent to 40 – 100 mg / dl.• 3+ - dense cloud with marked flocculation equivalent to 200 – 500 mg / dl .• 4+ - cloudiness with precipitation equivalent to 500 mg / dl or more. www.similima.com 30
  31. 31. • Heat and Acetic Acid Method Procedure :• Take a long test tube and fill ¾ the tube with clear urine.• Boil the upper portion over a flame .the lower portion serves as the control.• If proteins, phosphates or carbonates are present in the urine a white cloud develops.• Add 1-3 drops of glacial acetic acid. Any turbidity due to phosphate precipitation will clear or if it is due to carbonates they disappear with effervescence.• If it persists, it is due to albumin .• precipitates due to mucin or nucleoproteins will disappear on addition of 2 drops of nitric acid. www.similima.com 31
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  33. 33. • Sulphosalicylic acid method• Procedure• Take 2 ml of clear urine in a test tube.• Add an equal volume of 30 % sulphosalicylic acid.• Mix thoroughly, allow it to stand for 10 minutes and estimate the amount of turbidity. www.similima.com 33
  34. 34. • Nitric acid method• Procedure• Take 2 – 3 ml of concentrated nitric acid in a test tube.• Carefully pour 5ml of clear urine down the inner side of the inclined test• tube so that the urine forms a layer over the nitric acid.• A ring of white precipitated protein will form at the interface. Estimates the amount of precipitate. www.similima.com 34
  35. 35. • Colorimetric reagent strip test• PRINCIPLE: the colorimetric reagent strip test is based upon the ability of proteins to alter the color of some acid-base indicators without altering the pH. When an indicator such as tetrabromphenol blue is buffered at pH 3.it is yellow in solutions without protein. But in the presence of protein the color will change to green and then to blue with increasing protein concentration.• PROCEDURE: protein is determined by dipping the strip into well mixed un centrifuged urine and immediately comparing the resultant color with the chart provided on the reagent strip bottle.• RESULTS: the results are reported as negative (yellow color), trace, 1+ to 4+.trace readings may detect 5 to 20 mg of protein/ dl. Albumin reacts more strongly than the other proteins. Highly buffered alkaline urine and contamination of the urine specimen with quaternary ammonium compounds or skin cleansers containing chlorohexidine may produce false results. www.similima.com 35
  36. 36. Bence Jones Proteinuria:• Bence Jones protein is soluble at room and body temperatures.• It precipitates upon heating between 45 C and 60 C• redissolves when the urine is further heated to the boiling point.• Reappear on cooling www.similima.com 36
  37. 37. OCCULT BLOOD IN URINE: red blood cells or haemoglobin When hemolysis occurs in circulation urine Normally an occasional red cell may be found on microscopic examination of the urine sediment. In women during menstruation, the urine may get contaminated with menstrual blood and hence examination of urine should not be done during that period. www.similima.com 37
  38. 38. • Haematuria: Denotes the presence of red blood cells in urine. It is seen in various renal disorders, infectious or neoplastic or trauma related to any part of urinary tract.• Hemoglobinuria: is the presence of blood pigments in the urine without the presence of red blood cells. It is associated with certain hemolytic anemia’s that cause hemolytic anemia, transfusion reactions, malaria, and paroxysmal nocturnal hemoglobinuria www.similima.com 38
  39. 39. • Tests for Detection of Blood:• 1. Microscopic Examination: sediment------------RBC’S/HPF• 2. Benzidine Test PRINCIPLE Heme acts as a catalyst when hydrogen peroxide is mixed with benzidine. REAGENTS A: Saturated solution of benzidine in glacial acetic acid B: Hydrogen peroxide PROCEDURE Mix equal parts of A and B in a test tube and an equal amount of the mixed reagent.RESULTS• A blue color indicates the presence of hemoglobin. www.similima.com 39
  40. 40. Colorimetric reagent stripmethodPrinciple: the reagent area is impregnated with tetramethylbenzidine and buffered organic peroxide. This forms a green to dark blue compound when hemoglobin catalyzes the oxidation reaction of tetramethylbenzidine with a peroxide. www.similima.com 40
  41. 41. BILE IN THE URINE: The constituents1. bilirubin (bile pigments),2. bile salts,3. urobilin and urobilinogen. Bilirubin in appears IN JAUNDICE Bilirubin in Urine: bilirubin in the urine indicates the presence of hepatocellular disease or intra or extrahepatic biliary obstruction in the reticuloendothelial cells breakdown of hemoglobin. linked to albumin---------- liver. This albumin-bound form, which is also known as unconjugated bilirubin( indirect bilirubin) insoluble in water and does not appear in the urine. In the liver cells,--conjugated with glucuronic and sulfuric acids to form water soluble conjugated bilirubin (direct bilirubin). It is secreted into the bile and then excreted into the intestinal tract through the bile duct. This conjugated bilirubin -------------to urobilinogen. www.similima.com 41
  42. 42. • certain liver diseases --unable to conjugate all the bilirubin-----an increase in both conjugated and unconjugated bilirubin --bilirubinuria.• obstructive biliary tract disease-- bilirubinuria.• hemolytic anemia’s unconjugated bilirubin-------- urine free from bilirubin(Acholuric) www.similima.com 42
  43. 43. Tests for detection of bile salts:Hay TestProcedure:flowers of sulphur --sprinkled on the surface of the urine,Results:if bile salts are present they sink to the bottom.Otherwise they float on the surface.This is due to the property of bile salts to lower surface tension www.similima.com 43
  44. 44. Tests for detection of bile pigments 1. Foam Test: Shake urine in a test tube. If the foam on top is yellow, bile pigments are present. 2. Gmelins test: PROCEDURE: 1. Place ½ inch column of yellow nitric acid in a test tube. 2. Overlay with equal amounts of urine RESULT: A play of colored rings, the most distinct being green indicates the presence of bile pigments. www.similima.com 44
  45. 45. 3. Fouchets Test:FOUCHETS REAGENT Trichloroacetic acid – 25 gms Distilled water - 100 ml 10% Ferric chloride solution – 10 ml.PROCEDURE 1. Place 10 ml of acidified urine in a test tube. 2. Add 2.5ml of 10 % barium chloride. 3. Mix and filter. 4. Unfold the filter paper and spread it on a dry filter. 5. Add 1 drop of Fouchets reagent to the residual precipitate.RESULT: A green or blue color indicates the presence of bilirubin. www.similima.com 45
  46. 46. Colorimetric Strip Reagent Test• Principle: this test is base on the coupling of bilirubin with diazotized 2, 4-• dichloroaniline in a strong acid medium to form a brown purple azobilirubin• Compound. The color ranges through various shades of tan.• Procedure: the reagent strip is dipped into fresh, uncentrifuged urine tapped to• remove excess urine and after 20 seconds, compared to the color chart• on the reagent strip bottle• Result: the results are interpreted as negative, small (+), moderate (++), and large (+++) amounts of bilirubin. www.similima.com 46
  47. 47. Tests for detection ofurobilinogen:1. Qualitative Ehrlich’s Test EHRLICH’S REAGENT Paradimethylaminobenzaldehyde – 2 gms 20%5 HCL - 100 ml PROCEDURE a. Place 10 ml of urine in a test tube. b. Add 2.5 ml of barium chloride (to remove bilirubin). c. Mix well and filter. d. Add 0.5 ml of Ehrlich’s reagent e. Allow it stand for 3 – 5 minutes. www.similima.com 47
  48. 48. Results: pink color observable when viewed from the top of the test tube. Against a white background placed beneath the bottom of the test tube. cherry red color Abnormally high amounts of This test must be done with fresh urine or else urobilinogen is oxidized on exposure to air urobilin. Excessively cold water should not be used in diluting the urine. www.similima.com 48
  49. 49. MICROSCOPIC EXAMINATIONQualitative technique:the urine must be freshly voidedexamined without excessive delay in order to prevent cellular deterioration.Cellular debris from the urethral meatus and secretions from the vagina may contaminate the urine specimen. www.similima.com 49
  50. 50. 10-15 ml of urine ----from freshly mixed urine specimen and centrifuged at a standard speed, usually 1500 to 2000 rpm for 5 minutes. This is sufficient to bring to the bottom casts, pus cells, blood and crystals. For bacteria however a higher speed of 3,000 rpm is required. the sediment resuspended in 1 ml.of the same fluid. A drop of resuspended sediment is placed directly on a microscope slide and covered with a cover slip.. www.similima.com 50
  51. 51. low power- Casts tend to congregate at the edges of the cover slip. A minimum of 10 – 15 high power fields should be scanned for this examination.• Red blood cells, leucocytes,epithelial cells--- per high power field(/hpf);• casts --- per low power fields(/lpf).Other elements such as bacteria, parasites, crystals and spermatozoa are reported as well www.similima.com 51
  52. 52. • NORMAL SEDIMENTNormal sediment contains a limited number of formed elements. it can be divided into two classes.• unorganized sediment• Organized sediment www.similima.com 52
  53. 53. A. Unorganized sediment - these are the crystals of various substances present in the urine and they vary with the pH of the urine .crystals of normal urine is formed as the specimen cools.1. Crystals in acidic urine:a. Uric acid and Urates; – crystals are seen when the urine is allowed to stand for sometime and are not seen in freshly passed urine. Amorphous urates appear as red granules and are dissolved by heat and sodium hydroxide but not acetic acid. Uric acid crystals vary in shape and are yellow brown in colour and are not dissolved by heat, acetic acid or HCL but are soluble when heated with sodium hydroxide. disturbances of uric acid metabolism fevers where the urine is concentrated. www.similima.com 53
  54. 54. B.Calcium Oxalate:They are commonly found in diets rich intomatoes, spinach etc. they are typicallyenvelope shaped crystals but occasionallyappear dumb- bell shaped .they are insoluble instrong Hcl. c. Cystine Crystals: highly refractile, hexagonal plates and aresoluble in Hcl but insoluble in acetic acid. Theyare seen cystinosis which is an inborn error ofmetabolism in which cystine crystals are found inthe urine, reticuloendothelial system and eyes. d.Leucine: slightly yellow, oily looking spheres with radialand concentric striations .they are not soluble inHcl or ether .they are found in liver disorders. www.similima.com 54
  55. 55. • e.Tyrosine:– fine needles arranged in concentric sheaves, constriction at the middle. liver disorders.• f.Sulpha crystals :- patients taking sulphonamides. 2. Crystals in alkaline urine:• a. Ammonium Magnesium Phosphates: ( triple phosphate) coffin lid, feathery or leaf like forms. In freshly passed urine they indicate stones in the bladder or kidney. Phosphates may occur as amorphous deposits in alkaline urine and are dissolved in acetic acid.• b. Dicalcium Phosphates: hey are also seen in slightly acid or neutral urine. They are colorless prisms arranged in stars and rosettes They are soluble in acetic acid. c. Calcium carbonate: amorphous granules or colorless spheres 55 www.similima.com and dumb-bells which are soluble in acetic acid with gas formation.
  56. 56. Crystals in acid urine Amorphous urates-Brick – Red- Granules Uric Acid -Yellow – Brown-- Polymorphous – Whetstones,Rosettes of prisms,Rhombohedral prisms, hexagonal plateSodium urateColorless to YellowFan of slender prismsCystine (rare)1. Colorless2.Highly refractileFlat hexagonal plates with well – defined edges singly or in clusters www.similima.com 56
  57. 57. Cholestrol (rare) -ColorlessBroken window panes with notched corners ,Flat platesLeucine (rare)-1. Yellow or Brown2.Highly refractile-Spheroids with striations pure form hexagonalTyrosine (rare) Colorless or YellowFine silky needles in sheaves or rosettesBilirubin-Reddish Brown--Cubes, Rhombic plates, Amorphous needles www.similima.com 57
  58. 58. Acid, Neutral or SlightlyAlkaline UrineCalcium oxalate--Colorless Octahedral Dumbbells,Often smallHippuric acid Colorless- Rhombic plates Four sided prisms www.similima.com 58
  59. 59. Alkaline UrineCalcium Carbonate-Colorless --Needles,Spheres,DumbbellsAmmonium biurate-Yellow Opaque BrownThorn apple,Spheres,Dumbbells,Sheaves of needlesCalcium Phosphate-Colorless- Prisms, Plates, Needles - - - - www.similima.com 59
  60. 60. A- represents the residue of normal human urine, as seen under the microscope.B is represented oxalate of urea. An excess of this element indicates indigestionNitrate of urea is represented in division C. www.similima.com 60
  61. 61. A and B--highly magnified urinary deposits, which indicate different degrees of impairment of the digestive functions are represented. www.similima.com 61
  62. 62. epithelial cellsIn division A is represented pus and mucus, the presence of which indicates suppuration of the kidneys (Brights disease). In B pus globules are alone represented. In the division marked C are shown blood corpuscles as they are arranged in blood drawn from a vein or artery. D represents the same separated, as they always are when present in the urine. In E highly magnified oil globules are represented. If present in the urine, they indicate disease of the kidneys. In the division marked F are represented epithelial cells, the presence of which in large numbers is indicative of disease of the mucous lining of the urinary organs. www.similima.com 62
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  64. 64. Uric acid• In division A are represented the mixed urates as they appear during idiopathic fevers, as intermittent, remittent, etc. When appearing as seen in division B, a less violent affection of the same character is indicated. Division C represents urate of ammonia, occasionally observed when there is a tendency towards albuminuria, In division D which is present in the urine of persons suffering from gout. The crystals shown in division E consist of the same salt. www.similima.com 64
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  67. 67. varieties of cancer. www.similima.com 67
  68. 68. • Organized Sediment: the components of organized sediment include casts, red blood cells, white blood cells, epithelial cells, bacteria, yeast, parasites, spermatozoa and artifacts.• a. Casts:• Casts are formed in the tubules and is composed of proteinaceous material. They are washed out by the glomerular secretion into the collecting tubules and the bladder. They are cylindrical in shape with round or broken ends with uniform diameter but varying in length. They require acidic conditions, high salt concentration, reduced urine flow and protein to be formed. Practically all casts have a hyaline matrix, which may or may not contain inclusions such as desquamated cells.• The casts are named according to the matrix of the inclusions contained in them e.g. red blood cell casts. www.similima.com 68
  69. 69. 1. Hyaline casts: are colorless, semi-transparent and occasionally refractile cylinders and are soluble in acetic acid. They are seen when there is damage to the glomerular capillary membrane, permitting leakage of proteins through the glomerular filtrate. They are seen in fever, orthostatic proteinuria, and emotional stress or strenuous exercise. 2. Granular casts: are casts containing large or fine granules embedded in coagulated protein. They are not found in normal urine and their presences indicate pyelonephritis. They are also seen in chronic lead poisoning.3. Epithelial casts: are formed of fused desquamated tubular cells. They are coagulated protein in which are embedded desquamated epithelial cells from the renal tubules .they are seen in diseases where there is damage to the tubular epithelium as in nephrosis, eclampsia, amyloidosis and heavy metal poisoning. www.similima.com 69
  70. 70. 4. Red Blood Cell Casts: are casts with red blood cells embedded in the coagulated protein in the tubule. Their presences indicate acute inflammation or vascular disorder in the glomerulus causing hematuria. They are seen in pathological conditions such as acute glomerulonephritis, renal infarction and collagen vascular disorder.5. White Blood Cell Casts (Pus cell)6. Fatty Casts7. Waxy Casts www.similima.com 70
  71. 71. • CELLS:• a. Red blood cells: Normally 1-2 red blood cell are found per high power field• .they appear pale , light refractive, biconcave discs when viewed under high power magnification .they have no nuclei. Red blood cells in fresh, unstained sediment appear pale in color; in urine that is not fresh, they are pale or colorless shadow cells .in concentrated urine, they may be small and crenated; and in dilute urine, they are large and swollen and sometimes rupture to produce ghost cells.• b. White cells• c. Epithelial Cells: Normally a few epithelial cells occur in the urine .A marked increase• is these cells in the urine is seen destruction of the tissues in the urinary tract. www.similima.com 71
  72. 72. • Quantitative Evaluation of the urine sediment – Addis count• The Addis count is a quantitative measurement of the excretion of red cells, leucocytes and casts in the urine during a 12 hour period.• e. Bacteria:Bacteruria is considered significant when there is the presence of 100,000 or more bacteria per ml of urine specimen. www.similima.com 72
  73. 73. DETECTION OF BACTERIA• Microscopic Examination: sediment - >20 or more bacteria per high power field may indicate a urinary tract infection Reagent strips: PRINCIPLE: This test depends upon the conversion of nitrate to nitrite by the action of gram negative bacteria in urine. At the acid pH of the reagent area, nitrite in the urine reacts with p-arsanilic acid to form diazonium compound. This compound in turn couples with 1, 2, 3, 4-tetra hydrobenzoquinolin-3-ol to produce pink color.• Procedure: The strip is dipped in the urine specimen for 5 seconds. www.similima.com 73
  74. 74. Results:uniform pink color --positive resultthe presence of 100,000 or more organisms per ml,A negative result should never be interpreted as indicating absence of bacteruria.There are several reasons for this. – First morning urine or urine that has remained in the bladder for several hours is more likely to yield a positive nitrite test result in the presence of significant bacteruria than a random urine sample that may have been in the bladder for short time. in the latter type of specimen there may have been insufficient time(less than 4 hours) for conversion of nitrate to nitrite to have occurred. – When dietary nitrate is absent, even if organisms containing reductase are present and bladder incubation is ample. – Ascorbic acid concentrations of more than 25 mg / dl or greater may cause false negative results with specimens containing small amounts of nitrate. www.similima.com 74
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