Dr Piyush Tailor

PURPOSE OF EXAMINATION It is Quick, simple and Cost effective test to diagnose and monitor various diseases of Urinary tract as well as systemic disorders like Diabetes.

PRINCIPLE OF EXAMINATION

Glucose- This test is based on double sequential enzyme reaction. 1st enzyme glucose oxidase catalyzes formation of gluconic acid and hydrogen peroxide from glucose. Second enzyme peroxidase catalyzes reaction of H2O2 with KI chromogen to oxidize the chromogen to colours ranging from green to brown.

Bilirubin – This test is based on coupling of bilirubin with diazotized dichloroaniline in a strongly acidic medium. The colour ranges through various shades of tan.

Ketone – This test is based on reaction of acetoacetic acid with sodium nitroprusside producing colours ranging from buff-pink, for a negative reading, to purple when acetoacetic acid reacts with nitroprusside.

Specific gravity – This test is based on the apparent pKa change of certain pretreated polyelectrolytes in relation to ionic concentration. In the presence of an indicator, colour range from deep blue-green in urine of low ionic concentration through green and yellow-green in urines of increasing ionic concentration.

Blood – This test is based on the peroxide like activity of hemoglobin, which catalyzes the reaction of diisopropylbenzene dihydroperoxide and 3, 3, 5, 5-tetramethyl benzidine.The resulting colour ranges from orange through green; very high levels of blood may cause the colour development to continue to blue.

pH- This test is based on the double indicator principle that gives a broad range of colours covering the entire urinary pH range. Colour range from orange through yellow and green to blue.

Protein - This test is based on the protein-error-of-indicators principle. At a constant pH, development of any green colour is due to the presence of protein. Colours range from yellow for negative through yellow-green and green to green –blue for positive reactions.

Urobilinogen - This test is based on a modified Ehrlich reaction, in which p-diethylaminobenzaldehyde in conjunction with a colour enhancer reacts with urobilinogen in a strongly acid medium to produce a pink-red colour.

Nitrite - This test is based upon the conversion of nitrate (derived from the diet) to nitrite by the action of gram negative bacteria in the urine. At the acid pH of the reagent area , nitrite in the urine reacts with p-arsanilic acid to form a diazonium compound. This diazonium compound inturn couples with 1, 2, 3, 4, tetrahydrobenzoquinoline-3-ol to produce a pink colour.

Leucocytes – Granulocytic leucocytes contain esterases that catalyse. The hydrolysis of the derivatised pyrolle amino acid ester to liberate 3- hydroxy 5- phenol pyrolle .This pyrolle then reacts with the diazonium salt to produce a purple product.

PERFORMANCE SPECIFICATION

• Glucose 5.5-111 mmol/L(100-2000 mg/dL) glucose
• Bilirubin > 0.4 mg/dl
• Ketone 0.5-16 mmol/L(5-160 mg dL) acetoacetic acid
• Blood 10-200 cells/ul
• Protein 30-2000 mg/dL
• Nitrite > 0.1mg/dLnitrite ion
• Leucocytes 15-500 cells/µL
• Specific gravity – 1.000 and 1.030
• Ph - 5 - 8.5
• Urobilinogen - 3.2-131 µmol/L (0.2- 8 mg/dL)

SAMPLE TYPE REQUIRED Clean voided Mid stream random urine sample.

PRESERVATIVE Not required.

REAGENT Siemens reagent strip

CALIBRATION Calibration and preventive maintenance is not required for small clinitek urine chemistry analyser. DETAIL WORK BENCH PROCEDURE: • Completely immerse all the reagents area into fresh, well mixed, uncentrifuged urine .Dip briefly and remove immediately to avoid dissolving out reagents. • While removing the strip run the edge against the rim of the urine container to remove excess urine. • Switch Power button on the stabilizer and wait for the GREEN light to appear on the indicator. • Press the Power button provided on Analyzer and wait until the Instrument shows SYSTEM OK. • Enter Operator ID. • Enter Patient ID. • Blot by touching the edge of Strip to Tissue paper to remove excess urine. • Insert the Reagent Strip in the Space provided for the Strip with reagent side facing upwards. • Press START and Select Type of Urine specimen according to its colour, appearance and then instrument analyses the Strip. • Wait for Results to appear on Screen and to have results in printed format.

QUALITY CONTROL PROCEDURE

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

o Internal QC: 1. This is done by running the BIO RAD quality control samples (level 1 & Level 2) daily once on auto analyzer. 2. Daily one random patient sample is analyzed by Two/Three parameter strips and multistix strips (8 parameters/10 parameters) and by automated analyzer. Every 6 month one random urine sample is also checked by manual method. 3. Precision check is performed twice a year.

INTERFERENCE

• Glucose- Ascorbic acid concentration may cause false negative result for specimen containing small amount of glucose.

Ketone bodies may reduce the sensitivity of the test and may cause false negative result.

• Bilirubin – Indican can produce a yellow –orange to red colour response that may interfere with the interpretation of a negative or a positive bilirubin reading.

Metabolite of Iodine may cause false positive result.

Ascorbic acid concentration if greater may cause false negative results.

• Ketones – False positive result may occur with highly pigmented urine specimen.

Large amounts of levodopa metabolites may cause false positive result.

Compound such as mesna may cause false positive results.

Specific gravity –Highly buffered alkaline urine may cause low readings relative to other readings.

Protein if present in urine may cause falsely high specific gravity.

• Blood – Elevated specific gravity, Captopril and certain Oxidising compound may reduce the reactivity of the blood test.

Microbial peroxidase associated with UTI may cause false positive results.

• Protein – False positive results may obtain with highly buffered or alkaline urine.

Contamination of the urine specimen with quaternary ammonium compounds or with skin cleaners containing chlorhexidine may also produce false positive results.

• Urobilinogen – The reagent area may react with p-amionosalicylic acid and sulfonamides.

False negative results may obtain if Formalin is present.

• Nitrite – Negative result may occur when Urinary tract infection are caused by organism that do not contain reductase to convert nitrate to nitrite , when urine has not been retained in the bladder long enough for reduction of nitrate to nitrite.

Sensitivity of the Nitrite test is reduced for urines with high specific gravity.

Ascorbic acid may cause false negative results.

• Leucocytes – Elevated glucose concentration or high specific gravity may cause decreased test results.

The presence of Cephalexin or Cephalothin or high concentration of Oxalic acid may cause decreased test results. CALCULATION OF RESULT: Not Applicable.

BIOLOGICAL REFERENCE INTERVAL

• Glucose: Absent.
• Protein: Absent to Trace.
• Bilirubin: Absent.
• Ketone bodies: Absent.
• Specific gravity : 1.003 to 1.030
• Blood: Absent.
• pH : 5 to 7.5
• Urobilinogen : 0.2 to 1 mg/dl
• Nitrite: Absent.
• Leucocytes: 0 to 5 per HPF.

REPORTABLE INTERVAL FOR EXAMINATION RESULT

• Glucose: Trace - ++++ (100-2000mg/dl).
• Protein: Trace - ++++ (30-2000mg/dl).
• Bilirubin : + -+++(small-large)
• Ketone bodies : Trace- large(5-160mg/dl)
• Specific gravity : 1.000 to 1.030
• Blood : Trace-large (10-200cells/ul)
• pH : 5 to 8.5
• Urobilinogen : 2 to 8 units/dl
• Nitrite: Absent/+nt.Leucocytes : trace-+++(15 to 500 cells/ul)

CRITICAL VALUE

• 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.

INTERPRETATION BY THE LABORATORY

• Glucose: Normal- absent

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

          
*Protein: normal- absent

Present- pre-renal condition like dehydration, heart Disease, severe diarrhea; renal condition; post renal condition like lesions of renal pelvis, bladder, prostate

• Bilirubin: Normal- Absent

Present- hepatic & post hepatic condition

• Ketones: Normal- Absent

Present- severe diabetes mellitus, fever, nervous disorder, prolonged diarrhea & vomiting

• Specific gravity- Normal: 1.015-1.030

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

• BLOOD: Normal- Absent

Present- hematuria, hemoglobinuria

• pH – normal: slightly acidic

acidic: Metabolic acidosis Alkaline- Metabolic alklosis

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

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

• Nitrite: normal- absent

Present in urinary tract infection

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

POTENTIAL SOURCE OF VARIABILITY

Glucose: Renal glycosuria may give positive test

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

Ketones: starvation may give positive test

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

Urobilinogen: constipation may give positive test

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