Dr Piyush Tailor

A)Purpose of examination:

1. To find out metabolic or endocrine disturbances of the body like hepatic disease or diabetes
2. To detect intrinsic condition that may adversely affect the urinary tract.

B) Principle of examination: Physical Examination various physical characteristics of urine like Volume, color, Appearance, Sediment formation, Odor, Reaction and pH & Specific gravity are examined

Chemical Examination- By 2 parameters Reagent Strips- Protein In the presence of protein Tetra bromo Phenol Blue change the color from green to blue

Glucose In the presence of glucose in urine, Glucose Oxidase convert glucose into Glucuronic acid & Hydrogen Peroxide. Peroxidase converts Hydrogen Peroxide into water & Oxygen. Oxygen reacts with chromogen which changes the color from green to chocolate brown.

Chemical Examination by Manual method-

Protein:(Heat test) precipitation of protein due to coagulation by heat.

Glucose (Benedict’s test) When 5ml of Benedict’s qualitative reagent is heated with eight drops of urine; glucose present in the urine reduces cupric ions to cuprous ions in an alkaline medium. The original blue colour of the benedict’s reagent is changed to green, yellow, orange, or red according to the concentration of glucose in urine.

Ketones (Rothera’s test) Nitroprusside reacts with both acetone and acetic acid in an alkaline medium to produce a purple colored compound.

Bile Salts (Hay’s sulfur flower test) Bile salts when present lower the surface tension of urine. When sulfur powder is added on the surface of urine, sulfur particles sink to the bottom of the beaker. In case of a normal urine sample; sulfur particles float on the surface of urine.

Bile Pigments (Fouchet’s test) & Urobilinogen When Barium chloride reagent is added to urine, it combines with sulfate radicals in urine and precipitate of barium sulfate is formed. If bile pigments are present in urine, they will adhere to these large molecules. Ferric chloride present in Fouchet’s reagent oxidizes yellow bilirubin, in the presence of trichloroacetic acid to green biliverdin. Urobilinogen reacts with p–dimethyl aminobenzaldehyde in acidic medium to form pink colored compound.

Blood (Benzidine test) Saturated solution of benzidine in glacial acetic acid- 1 ml+ 1 ml H2O2+ 2 ml urine gives green to blue color in 5 mins.

Microscopic Examination The microscopic elements present in urine are collected in the form of deposit by centrifugation at 2500 RPM for 5 min. A small drop of the sediment is examined by making a cover slip preparation under microscope.

C) Performance specification Not applicable

D) Sample type required freshly voided midstream urine specimen

E) Preservative needed not required

F) Reagents required

a. Physical Examination- pH: pH paper

b. Chemical Examination- By 2 parameter Reagent Strips- multistix reagent strips coated with Tetra bromoPhenol Blue for protein and Glucose Oxidase for Glucose

c. Chemical Examination by Manual method

Protein (Heat test): - Glacial Acetic Acid

Glucose (Benedict’s test): Benedict’s reagent

Ketones (Rothera’s test): Rothera’s powder (sod. Nitroprusside & ammonium sulfate), Liquor Ammonia

Bile Salts (Hay’s sulfur flower test): Sulfur powder

Bile Pigments (Fouchet’s test): Barium Chloride, Fouchet’s reagent (Ferric Chloride)

Blood (Benzidine test): Benzidine powder, Glacial acetic acid, Hydrogen peroxide.

Urobilinogen: Ehrlich’s reagent (p-dimethyl amino benzaldehyde)

d. Microscopic Examination- not applicable

G) Calibration method not applicable

H) Detailed work bench instruction a. Physical Examination - see for the following physical characteristics;

• Volume
• Color
• Appearance
• Sediment formation- allow the urine to stand for some times and see at the bottom for sediment formation
• Odor
• Reaction and pH – place a drop of urine on a blue litmus paper and not down the reaction (Red colour/ no colour change)
• Specific gravity- 1)Fill the container ¾ full of urine, 2) Float the urinometer in the urine & rotate it carefully and note the specific gravity reading from the scale

b. Chemical Examination By Multistix Reagent Strips

• Take fresh, well mixed and uncentrifuged urine.
• Dip the test areas of the strip in the urine.
• Remove excess of the urine by tapping the edge of the strip against the container.
• Compare the test areas closely with corresponding colour charts on the bottle at the time specified (60 Sec) in good light.
• Note the result.

c. Chemical Examination by Manual method- Protein (Heat test):

• Take 10ml of urine in a test tube
• Boil the upper portion of the urine
• If turbidity develops, add 2 drops of glacial acetic acid if the turbidity is due to phosphate, it will be cleared
• Reboil the specimen.

Glucose (Benedict’s qualitative Test):

• Pipette out 5ml of Benedict’s reagent in a test tube.
• Add 8 drops of urine
• Heat on the flame till boiling
• Cool under tap water

Ketones (Rothera’s test)

• Take 5ml of urine in a test tube
• Add 1gm of Rothera’s powder mixture and mix well.
• Layer 2ml of conc. ammonium hydroxide on urine gently by allowing it to flow down the side of the inclined test tube.
• Observe for pink-purple ring at the interface.

Bile salts (Hay’s sulfur flower test)

• Take about 10 ml urine in a beaker.
• Sprinkle dry sulfur powder on the surface of urine.
• Observe the sulfurs particles, whether it sinks to bottom or it remains floating on the surface.

Bile pigments and Urobilinogen

• Take 3-4 ml urine in centrifuged test tube by pipette.
• Add equal amount of 10gm/dl barium chloride, mix well.
• Centrifuge at 1,500 RPM for 10 minutes (Or filter by using Whatman no. 1 filter paper.)
• Place supernatant in another test tube for urobilinogen test.
• Add 1-2 drops of Fochet’s reagent to the sediment (or to the precipitate on a filter paper).
• Add about 0.5 ml of Ehrlich reagent to the supernatant.

Blood (Benzidine test)

• Prepare saturated solution of benzidine in glacial acetic acid
• Take 1 ml of this solution
• Add 1 ml of Hydrogen peroxide to this solution
• Add 2 ml of urine sample in this 2 ml of solution

d. Microscopic Examination-

• Mixed the urine & pour into a test tubes.
• Centrifuge it for 5 mins at 2500 RPM.
• Discard the supernatant completely & resuspend the deposit by shaking the tubes.
• Place 1 drop of deposit on glass slide & cover it with cover slip.
• Mark the slide with identification number & observe it 1st under low power in subdued light (partially closing iris diaphragm & condenser is downward.
• Note the contents of various fields.

I) Quality control procedure INTERNAL QUALITY CONTROL

• Daily one quality control sample (level 1/ level 2) is run on urine auto analyzer.
• Inter kit comparison : Daily one patient sample is run by Two/Three parameter strips, multistix & on automated urine analyzer. Every 6 months analysis by manual method is also done.
• Weekly one random urine sample is tested technically and microscopically by two different persons (two different technician and two different pathologists).
• Yearly twice precision check is done on one random sample of urine by auto analyzer.
• Reports of quality control tests are filed in CP: C\record\file\9\internal quality control records

EXTERNAL QUALITY CONTROL

As an alternative approach to EQA, participation split sample testing is done by two different technicians once in 3 months.

The reports are filed in - CP: C\record\file\2\Split sampling.

J) Interference contaminated urine containers or centrifuge tubes if used it may interfere with the results

K) Calculation of results and uncertainty:

Chemical examination-

By Multistix reagent strip method:

Protein: With increasing intensity of green color (see label on strip bottle) grade the result: negative, Trace, + (up to 30mg/dl), ++ (30- 100mg/dl), +++ (100-300mg/dl), ++++ (>2000mg/dl) Glucose: according to colour change from green to brown (see label of strip bottle) grade the result as: negative, trace (up to 100mg/dl), + (100-250mg/dl), ++ (250-500mg/dl), +++ (500-1000mg/dl), ++++ (>1000mg/dl)

Protein (Heat test)

No Turbidity- Protein absent

Presence of turbidity-Protein is present

Grade the result according to the degree of Turbidity as +, ++, +++, ++++.

Glucose (Benedict’s test)

Ketones (Rothera’s test)

No Purple ring:-Ketones bodies absent

Appearance of Purple Ring:-Ketones bodies present.

Bile salts (Hay’s sulphur flower test)

Sulfur particles sink to bottom: Bile salts present.

Sulfur particles remain floating: Bile salts absent.

Bile pigments and Urobilinogen: 1)Sediments:

• No change in colour : Bile pigments absent.
• Colour change to green: Bile pigments present.

(2) Supernatant:

• Development of pale pink color: Urobilinogen present: Normal
• Development of cherry red color: Urobilinogen present: Increased.

Blood

L) Biological reference interval:

Physical Examination

• Volume: 50-200 ml
• Color: pale yellow
• Appearance: clear
• Sediment formation:no sediment
• Reaction and pH:slightly acidic 4.8-7.5
• Odor:aromatic
• Specific gravity: 1.003-1.030

Chemical Examination

      Protein:Absent
      Glucose:Absent
      Ketones:Absent
      Bilirubin:Absent
      Bile Salts:Absent
      Urobilinogen:Present (very low concentration- 0.2-1 EU/dl)
      Blood: Absent
      
      

Microscopic Examination

• Pus cells- 2 to 3 per HPF
• Epithelial cells- (M- 2-3 per HPF, F-2-5 per HPF)
• Cast- Absent
• Amorphous Material- Amorphous urates of sodium, potassium or calcium,Amorphous phosphates of calcium & magnesium
• Crystals-
  • Acidic urine: uric acid, calcium sulfat,Acid/ Neutral/ slightly alkaline urine: Calcium Oxalate, Hippuric acid Alkaline/ Neutral/ slightly acidic urine: Triple phosphates
  • Alkaline urine: Calcium carbonate, ammonium biurates, calcium phosphate
• Bacteria- Absent
• Yeast cells- Absent
• Parasites- Absent

M) Reportable interval for examination results: Physical Examination

Volume: 20-500 ml.

Color: pale yellow, dark yellow,red,brown, green

Appearance: clear, turbid, hazy, smokey, milky

Sediment formation- sediment-nt/ +nt

Reaction and pH –4.8-7.5

Odor: - aromatic, fruity, ammonical, foul smelling

Specific gravity- 1.003-1.060

Chemical Examination

Protein: trace to++++ (30-2000mg/dl)

Glucose: trace to ++++ (100- 2000mg/dl)

Ketones: Absent/ +nt

Bile Pigments: Absent/ +nt

Bile Salts : Absent/ +nt

Urobilinogen: Present (very low concentration)/ increased

Blood: Absent- ++++

N) Critical values:

• Strongly positive test for glucose(3+ or more) ,albumin (3+ or more) and strongly positive ketone.
• Presence of field full of pus cells and/or RBCs.

O) Interpretation by the laboratory Physical Examination-

Volume: normal: 50-200 ml

            
            > 500ml    Polyuria, Diabetes mellitus, Diabetes insipidus
            
            < 20ml: Oliguria, anuria, renal condition, post renal condition

Color: normal: pale yellow

        
        Dark yellow:   Jaundice
        Reddish: Hematuria,hemoglobinuria ,myoglobinuria, acute  febrile disease
        White- chyluria, pus
        Brownish black- alkeptonuria, malignant melanoma
         

Appearance: normal: clear

           Turbid:  abnormal no of Leukocytes, epithelial cells, bacteria
           Smokey:  RBC
           Hazy: mucus
           Milky: chyle

Sediment formation- normal: no sediment

Sediment present: pus cells, epithelial cells, RBC, Cast, Presence of amorphous phosphates or urates

Odor: fruity smell- : ketone bodies

    Pungent smell- Bacteria  
    Musty odor - Phynyleketonuria

Reaction and pH – normal: slightly acidic

                acidic: Metabolic acidosis
                Alkaline- Metabolic alklosis 

Specific gravity- nomal: 1.003-1.030

                 High: Diabetes mellitus, fever, acute nephritis
                 Low: Diabetes insipidus, chronic nephritis

Chemical Examination

• Protein: normal- absent

Present- pre-renal condition like dehydration, heart D’se, severe diarrhoea; renal condition; post renal condition like lesions of renal pelvis, bladder, prostate

• Glucose: Normal- absent

Present- Diabetes mellitus, hyperactivity of endocrine glands like thyroxine, pituitary, adrenal

• Ketones: Normal- Absent

Present- severe diabetes mellitus, fever, nervous disorder, prolonged diarrhoea & vomitting

• Bile salts: Normal- Absent

Present- hepatic & post hepatic condition

• Bile pigments: Normal- Absent

Present- hepatic & post hepatic condition

• Urobilinogen: Normal- Present (very low conc.)

Increased- hepatic & post hepatic condition Very high- Prehepatic condition

• Blood : Normal- absent

Present- Urological, nephrological and bleeding disorder

Microscopic Examination

• Pus cells- normal: 2 to 3 per HPF

Abnormal: >5 per HPF (UTI, Pyuria, acute glomerulonephritis, renal tubular acidosis, dehydration, stress, fever)

• Epithelial cells- normal- (M- 2-3 per HPF, F-2-5 per HPF)

Abnormal: increased no of tubular epithelial cells: pyelonephritis, ATN, Kidney transplant rejection

• Cast- normal:Absent

Abnormal: Hyaline cast- mild renal disease

Red cell cast- glomerular disease, acute glomerulonephritis, sub acute bacterial endocarditis, severe pyelonephritis

WBC cast- renal infection & non infectious inflammation, acute pyelonephritis, interstitial nephritis, glomerular disease

Granular cast- significant renal disease

Waxy cast- severe chronic renal failure, malignant hypertension, and tubular inflammation

Fatty cast- nephrotic syndrome, chronic glomerulonephritis, toxic renal poisoning

• Crystals- Acidic urine: uric acid, calcium sulfate +nt in fresh urine in high proportion may suggest renal calculi

Acid/ Neutral/ slightly alkaline urine: Calcium Oxalate (renal calculi), Hippuric acid (non significant)

Alkaline/ Neutral/ slightly acidic urine: Triple phosphates (+nt in fresh urine in high proportion may suggest renal calculi)

Alkaline urine: Calcium carbonate, ammonium biurates, calcium phosphate (not significant) Crystals found in acidic urine indicating abnormal metabolism: cystine, cholesterol, leucine, tyrosine, bilirubin, hematoidin, Sulfonamides

• Bacteria- normal: Absent; abnormal: +nt

• Yeast cells-normal- Absent; abnormal: +nt in acidic urine containing sugar

• Parasites- normal- Absent; abnormal: Trichomonas vaginalis, microfilaria, schistostoma Hematobium,, Enterobius Vermicularis

P) Potential sources of variability

Volume: first voided morning urine volume may not give idea of urinary output

Colour: may be affected by intake of the food colour, beets, vitB complex, serotonin, concentrated urine, drug intake

Appearance: Precipitation of amorphous phosphates in alkaline urine & amorphous urates in acid urine

Reaction- protein reach diet give acidic pH, citrus fruits give acidic pH

Odor- decomposition of urea to ammonia by bacterial action may give ammoniacal odor

Protein: excessive muscular exercise, excessive protein ingestion, prolonged cold bath & pus may give positive test

Glucose: Renal glycosuria may give positive test

Ketones: starvation may give positive test

Urobilinogen: constipation may give positive test

Pus cells& epithelial cells: few leukocytes and epithelial cells normally secreted by male & female genital tracts

Casts: Hyaline cast may be +nt due to physical exercise, dehydration

Crystals: calcium oxalate & Hippuric acid may be derived from various drugs and foods

Bacteria: after storage at room temperature bacterial growth may take place