Tuesday, January 15, 2013

Dipstick method for Urinalysis (MULTISTIX METHOD)


DIPSTICK METHOD OF URINALYSIS

Multistix reagent strip method

Multistix reagents are clear plastic strips. Seven different reagent areas are affixed on the strip. These different cellulose areas are impregnated with specific testing chemicals according to the test which reacts with specific substances present in urine by changing the color. Color change chart is observed and compared to the color chart for the presence of abnormal levels of substances .Special care in their use is required to prevent inaccurate results and confirmation of quantitative test is appropriate if the results from dipstick testing reveal abnormalities.


The various determinations done by multistix are
pH, Specific Gravity, Glucose, Protein, Ketones, Urobilinogen, Blood, Bilirubin


Fig. Multiple strip for Urinalysis and their interpretaiton
The reagent strips are also available for only one or two tests such as glucose, glucose and protein, glucose and ketones.

Procedure
  1. Collect fresh urine specimen in a clean dry container with labeling
  2. Mix well immediately before testing and enter in routine record book and give lab number too.
  3. Open the cover and remove the one strip from strip bottle and replace cap.
  4. Completely immerse reagent areas of strip in fresh urine and remove immediately to avoid dissolving out the reagent
  5. While removing, run the edge of the entire length if strip against the rim of urine container to remove the excess urine.
  6. Hold the strip in a horizontal to prevent possible mixing of chemicals from adjacent reagent or contaminating the hands with urine.
  7. If reading visually compare the reagent areas to corresponding color chart on the bottle labeled at times specified.
  8. Hold the strip close to color blocks and match carefully.
  9. Avoid the layering of the strip directly on color chart as this will result  on urine flow in the chart
  10. If reading instrumentally, carefully follow the direction given in the appropriate instrument operating manual.
Determination of pH

The test is based on the double indicator principle that gives a broad range of colors covering the entire urinary pH ranges i.e. orange (6.0) through yellow and green to blue (9.6).

The indicators impregnated in strip are

Methyl red: pH: 4.4-6.2, changes color from red to yellow

Bromothymol blue: pH: 8.0-9.6, changes color from yellow to blue.

Note
  1. Measurement of urine ph should be always made on freshly voided urine. On standing pH tends to rise because of loss of carbon dioxide and production of ammonia from urea by bacterial growth
  2. Care should be taken not to have  excessively wet strip where acid buffer from the protein  patch  runs  into pH patch causing it to become orange
Determination of Specific Gravity

This is the indirect method for measuring specific gravity. The reagent area has three main ingredients present – polyelectrolyte, indicator substances and buffer. The principle of methodology is based on Pka change of pretreated electrolytes in relation to ionic concentration of urine. The polyelectrolytes in the reagent area contain acid groups which dissociated according to ionic concentration of specimen and the release of hydrogen ions from reagent area cause change in PH thus changes the color of indicator bromothymol blue from deep blue green to yellow green.

If there is low ionic concentration color remains blue green of low specific gravity and if high ionic concentration color remains yellow green of high specific gravity. The value must be read in 45 secs and it measures specific gravity from 1.000 to 1.030 with increment of 0.005

Note
  • This method is not affected by high amount of glucose, protein or radiographic contrast material all of which tends to elevate the specific gravity readings obtained from refractometers and urinometers.
Determination of Glucose

The test is based on a specific glucose oxidase peroxidase method a double sequential enzyme reaction. The reagent strips contain chromogen KI also along with glucose oxidase and peroxidase enzymes. The method is specific for glucose

Reaction
                glucose oxidase
Glucose ----------------------> Gluconic acid + hydrogen peroxide

                    peroxidase
H2O2 ---------------------------> H2O + O2 

O2  + K ------------------> color changes from sky blue to blue to green to chocolate brown depending on concentration of glucose

Note
  1. The strip  reacts with lactose , galactose , fructose or reducing metabolites of drugs
  2. It may be used for semi quantitative results by reporting  as approximately gm/dl
  3. Combination  of glucose and ketone strips are available which helps  to detect ketonuria  and suppression of glucose reaction by ketone  seen with other some reagent strip
  4. False positive result is seen if there is strongly oxidizing cleaning agents in urine container or low specific gravity of urine.
  5. False negative result is seen if sodium fluoride is used as preservative. Urine has high specific gravity and pH.
  6. Ascorbic acid occasionally also gives false negative results.
  7. Prolonged exposure of urine sample to room temperature  may lower glucose  results because of glycolysis due to microbial contamination
  8. Acidification of urine cause strip to turn black
  9. The detectable range is 75-125 mg/dl
Determination of Protein

The test area is impregnated with tetrabromophenol blue buffered o tans acid ph-3 .The area is yellow in absence of protein but at same ph changes to green to blue in presence of protein depending on type and concentration of protein present. The result is read in plus system as negative trace, and 1+ to 4+.

Note
  1. This test takes the advantages of protein error of pH in indicators because protein carries a charge at physiological pH.
  2. Indicators apart form tetra bromophenol blue are tetra chlorophenol, tetrabromo sulphthalein.
  3. More sensitive to albumin than  globulin, bences jones protein, or mucoprotein
  4. High salt level will lower the result
  5. A false positive result is seen on alkaline /highly buffered urine samples, quaternary ammonium compounds. Amidoamines in fabric softeners, chlorohexidine and with excessive leaking of acid buffer of test strip by excessive wetting.
Determination of Ketones

The method is based on nitroprusside reaction for ketone .the multistix strip contain buffers and sodium nitroprusside which reacts with acetoacetic acid producing a pink maroon color in 15 secs. It doesn’t react with acetone

Note
  1. The chemo strip reagent strip contain sodium nitroprusside buffer and glycine which reacts with acetoacetic acid in presence of alkaline medium to form violet dye
  2. Positive result is indicated by change in color from beige to violet within 60 secs. It is not sensitive to gamma hydro butyric acid.
  3. This method detects 10 mg/dl of acetoacetic acid and 70 mg/dl of acetone.
  4. Reagent strip without alkali react to acetoacetate not acetone.
  5. With large results like 3+, urine may be diluted and remeasured.
  6. False positive is seen  after the use of phthaleins or in presence of extremely large amount of phenyl ketones , l dopa metabolites aerosol , Methyl dopa and catophil
  7. False negative results occur because of loss of reagent. Reactivity or may also occur  with improper specimen handling which allows acetone  to volatile or bring consumed by bacterial action
Determination of Bilirubin

The test is based on coupling reaction of bilirubin with diazonium salt in strongly acidic medium .If bilirubin is present color changes from cream buff to tan which is read at 20 secs .This uses the diazonium salt – diazotized 2,4-dichloroaniline.

Note
  1. The multistix reagent strip detects 0.8 mg/dl of bilirubin
  2. Chemo strip reagent contains 6-dichlorobenzene diazonium tetraflouroborate as salt which changes color from pink to violet at 30 -60 secs
  3. Urine must be fresh because bilirubin glucorinide in urine quickly hydrolyses to less reactive free bilirubin.
  4. False negative result is seen when specimen stand too long due to oxidation if bilirubin.
  5. Large amount of ascorbic acid and nitrite can also lower bilirubin results
  6. Rifampicin and large  amount of chlorpromazine metabolites may cause false positive results
Urobilinogen
  • The test is based on modified Ehrlich’s Aldehyde reaction .The test area is impregnated with p-dimethyl aminobenzaldehyde in conjunction with color ethanol which reacts with urobilinogen in strongly acidic media to form yellow shades of red brown.
  • This is not specific for urobilinogen .Other porphobilinogen, indole, sterol can also give same color development.
Note
  1. The chemo strip reagent test area is impregnated with 4-methoxybenezene tetra flourate .This is specific for urobilinogen
  2. Freshly voided urine is best because urobilinogen is quite labile and will potentially form nonreactive urobilin in acidic urine.
  3. Test can also be altered by p-aminosalicyclic acid, sulphonamide and p-aminobenzoic acid.
  4. If formalin is present it gives false negative result
  5. Bilirubin if present gives green color.
Determination of Blood
  1. This method is based on the liberation of oxygen from peroxide in reagent strip by peroxidase in reagent strip  by peroxidase like activity of haem in free hemoglobin lysed RBC or myoglobin .Intact RBC are lysed on strip causing hemoglobin to react . The reagent area is impregnated with buffered mixture of organic peroxide and chromogen tetra methyl benzidine
  2. Hemoglobin catalyses the oxidation of chromogen to produce green color which is read at 60 secs. 
  3. If green spot is present then intact RBCs are present
  4. If uniform green color is present then it indicates the presence of hemoglobin or myoglobin
Note
  1. Sensitivity is up to 0.05-3 Hb/dl of urine
  2. False positive result is seen in presence of hypochlorite bacterial content ,microbial peroxidase associated with UTI
  3. Presence of ascorbic acid and formalin gives false negative results
  4. Presence of nitrites in large amount will delay the reaction.
Determination of Nitrite

This is based on conversion of nitrate in urine to nitrite by the action of bacteria like Proteus species, E.coli, Enterobacter, and Klebsiella.
                                           Acidic pH
Nitrite+ p-arsinilic acid ----------------> Diazonium complex

This diazonium compound then couple with 1,2,3,4, tetra hydrobenzoquinolin-3- ol to produce pink color. This indicates 105. Or more organism per ml,

Note
  1. This method detects the presence of 0.075 gm of nitrite per dl.
  2. False positive is seen in stored sample due to contaminants and post collection, bacterial proliferation, and medication (phenazopyridine)
  3. False negative result is seen due to ascorbic acid, urobilinogen or low pH, lack of dietary nitrites.
Leucocytes esterase

Esterase of granulocytes catalyses the conversion of indoxyl carboxylic acid ester to form indoxyl which reacts with diazinium salt to give beige purple. This tests detects the lower concentration of 10- 15 mg/dl

Note
  • Alteration of test is seen in urine with  having high specific gravity ,high glucose concentration, drugs like gentamycin and high protein content
Precaution
  1. Read after correct time indicated
  2. Don’t leave strip outside for a long time
  3. Ensure expiry date is over or not
  4. Container should be clear, dry and detergent free.
  5. Hold strip horizontally.
Advantage of Multistix Strip
  1. Quick screening of urine chemistry
  2. Reliable ,specific and sensitive
  3. Avoid the use of various corrosive reagents , different type of glass wares and other laboratory material required for wet chemical testing of urine
It can be performed in uncentrifuged urine and doesn’t require acidification
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