Chemical constituents: Dipstick testing

Glucose:

For glucose measurement reagent pad is impregnated with glucose oxidase, peroxidase, potassium iodide, and blue dye. The reaction employs glucose oxidase and peroxidase to produce hydrogen peroxide, which is subsequently reduced with concurrent oxidation of potassium iodide to release iodine. This free iodine blends with the background color to produce a variety of colors from green to dark brown. The lower detection limit is 70-130 mg/dl with an upper limit of 2018 mg/dl. The color change should be read exactly 30s after sample addition.

Glycosuria – Normal excretion is <20 mg/24 hr. Glycosuria occurs due to increased blood glucose (hyperglycemic glycosuria) or decreased renal threshold, impaired proximal absorption (renal glycosuria). TmG (Tubular maximum for glucose) is 350mg/min but when blood glucose rises above the renal threshold of 180 mg/dl of urine, then excess is passed in urine.

Hyperglycaemic glycosuria:

       Alimentary glycosuria: When a large carbohydrate diet is taken, blood sugar rises and cross renal threshold.

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   Nervous or emotional glycosuria: Stimulation of sympathetic nerves during emotion, stress that lead breakdown of liver glycogen producing hyperglycemia and glycosuria.

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 Glycosuria due to endocrine disorder: E.g. diabetes mellitus, hyperthyroidism, epinephrine, hyperpituitarism due to increased secretion of GH, glucagonoma, etc.

Renal glycosuria:

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   Hereditary: There may be defect in carrier protein e.g. in Fanconi syndrome.

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 Acquired: This may be due to damaged renal tubules, heavy metal poisoning like lead, cadmium, mercury, aminoacidurias, renal tubular acidosis etc.

·         Lowered renal threshold: This is seen during pregnancy.

Protein:

The dipstick test for total protein includes a cellulose test pad impregnated with tetrabromphenol blue and citrate pH 3 buffer. The reaction is based on the protein error of indicators phenomenon in which certain chemical indicators demonstrate one color in the presence of protein and another in its absence. Thus tetrabromphenol blue is green in the presence of protein at pH3 but yellow in its absence. The color is read exactly 60 s after and test has lower detection limit of 150-300 mg/L. The reagent is most sensitive to albumin and less to others.

Proteinuria - Normal excretion is <150 mg/24 hr and not detectable. Proteinuria occurs due to

Secreted or nephrogenic (derived from epithelium of urinary tract)

Glomerular (increase permeability)

Overflow (due to raised plasma concentration of low molecular weight proteins)

Tubular (decreased tubular reabsorption or saturated reabsorption)

The positive dipstick test should be confirmed in the lab by measuring either the protein/creatinine or albumin/creatinine ratio on an early morning or random urine sample. Dipstick for albumin measurement which includes dyes to which albumin binds producing color.

For positive dipstick result; the result should be confirmed using protein/creatinine ratio. Patients with two or more positive (>30mg total protein or albumin/mmol creatinine) test on early morning samples 1 to 2 weeks apart should be diagnosed having persistent proteinuria and referred to CKD management program.

Hemoglobin

Presence of blood in urine can be done by doing microscopy to see red blood cells or dipstick can be used. The chemical detection of hemoglobin in urine depends on the peroxidase activity of hemoglobin employing peroxidase substrate an oxygen acceptor. Here the oxidizable substrate is tetramethyl benzidine (TMB) and organic peroxide. The color change varies from orange through pale to dark green. Reading should be done after 60s.

The presence of Hb or red cells in urine indicates renal or bladder disease, glomerulonephritis, sickle cell disease, vasculitis, etc.

Specific gravity

It consists of absorbent cellulose pad impregnated with indicators which change color due to change in pK brought about by solutes in urine; the hydrogen ions released are detected by the pH indicator. The color changes from dark blue at low specific gravity to yellow green at specific gravity of 1.030. Test is read 45s after sample addition. Diabetic patients with uncontrolled hyperglycemia and glycosuria may have high urine specific gravity even when normal renal concentrating function is impaired. It can also be measured by refractometer. Here refractive index of urine specimen will vary directly with total amount of dissolved solids in sample. This instrument measures RI of urine as compared with water on a scale.

pH

To measure the pH of a sample, the test pad is impregnated with indicators-one example being a mixture of methyl red and bromthymol blue. Methyl red in a diluted form is red at pH below 4.2 and yellow at above 6.2. Bromothymol blue is yellow at pH<6.0 and blue at values above 7.6. At pH within these values the indicators give shads of orange and green, respectively. Thus the reagent blocks are evaluated at exactly 60s and compared with color chart where the lowest pH block at 5.0 is orange and highest at 8.5 is blue.

Bilirubin/Urobilinogen

The test includes fouchetes test and Ehrlichs test.

Ketone bodies

    

     This is done by Rothras test where only acetone and acetoacetate will give positive result with nitroprusside. The nitroprusside based strips are also used which gives different shades of color.