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40619_PRACGUIDE_Rebrand.04.09:Layout 1 22/04/2009 09:39 Page 1 Your Practical Guide to Urine Analysis Siemens Healthcare Diagnostics s 40619_PRACGUIDE_Rebrand.04.09:Layout 1 22/04/2009 09:40 Page 2 Contents Specimen Collection 2 History of Urine Analysis 7 Clinical Significance of Test Results 3 Specimen Collection 8 Factors Affecting Urine Chemistry Tests on Siemens Reagent Test Strips 4 Correct Technique for Visual Reading of Reagent Strip Tests 10 Test Selection Made Easy 5 Quality Assurance 11 Urinary Tract Infection Testing Pathway 6 Chemical Principles of Reagent Strip Tests 12 Services You Can Rely on for Better Patient Care History of Urine Analysis 1500 B.C. Egyptian writings refer to ‘polyuria.’ 700 B.C. Hindus describe ‘honey urine.’ 400 B.C. Hippocrates stresses uroscopy 7th Century A.D. Protospharius suggests diagnosis through urine colour. 13th Century A.D. The era of the ‘pisse-prophet.’ 1674 Thomas Willis recommends evaporation, distillation and precipitation. 2 1673 Dekkers describes test to detect protein. 1827 Bright discovers that ‘dropsy’ can be signalled by a urine test. 1830 Routine urine analysis consists of: • Visual observation • Test for protein • Test for urea. Mid-19th Century • Heller develops ring test for albumin. • Bence-Jones discovers new protein. • Bird publishes description of urine crystals. • Trommer introduces test for glucose. Late 19th Century Ehrlich devises diazo reaction for bilirubin. Early 20th Century • Folin introduces colorimetric method to estimate creatinine. • 1909 – Benedict develops qualitative/ quantitative method to detect sugar. 1941 CLINITEST® Reagent Tablets introduced for the measurement of reducing sugars. This test was the forerunner of the wide range of convenience urine tests made by Siemens and used all over the world. Tablet Tests 1945 – ALBUTEST® Reagent Tablets. 1949 – HEMATEST® Reagent Tablets. 1950 – ACETEST® Reagent Tablets. 1953 – ICTOTEST® Reagent Tablets. 1956 CLINISTIX® Reagent Strips, the first dip-andread test strip. 1957 ALBUSTIX® and KETOSTIX® Reagent Strip 1960s and 1970s A range of multianalyte reagent test strips are developed. 1980s and 1990s A range of instruments for the automated and semiautomated assessment of multiple reagent test strips are developed, while continually improving the reagent tests. 2003 The launch of Clinitek Status®. A simple, easy to use automated urine testing solution. The Clinitek Status® provides a full audit trail and removes the subjectivity of visual testing. Sample Preparation • First-voided morning urine is best for routine analysis (most concentrated). • Void directly into clean, dry container/bedpan. • Sterile containers are best. • Label container where possible. • It is best to test samples immediately. If unable to do this, test within 4 hours. Use uncentrifuged urine and mix prior to testing. OR • Refrigerate specimen immediately at 4-8 °C, and allow to return to room temperature before testing. Note: • It is especially important to use fresh urine to obtain the best results with the tests for bilirubin and urobilinogen, as these compounds are relatively unstable when exposed to room temperature and light. • Prolonged exposure of unpreserved urine at room temperature may result in microbial proliferation, with resultant changes in pH. • A highly alkaline pH may cause false positive results with the protein test area. Urine containing glucose may decrease in pH as organisms metabolise the glucose. Ureasplitting organisms convert urea to ammonia, causing an alkaline shift in pH. • Bacterial growth from contaminating organisms may cause positive blood reactions from the peroxidases produced. • Red and white blood cells may be lysed upon standing. This will affect microscopy but NOT the reagent strip tests. Preservatives Thymol – a commonly-used preservative, in amounts of 0.1 gm/dl (1 g/L) or greater, may give false positive reactions for albumin determinations. Formaldehyde may give false positive results for glucose determinations using the tablet test. Hydrochloric Acid – acidification using concentrated hydrochloric acid is recommended for 24-hour urine collections. This is the most frequently-used specimen for steroid determinations, and will also preserve cellular structures. Boric acid is generally added for the preservation of bacteria in urine. However, the concentration must not be excessive as this will prevent growth of bacteria during culture. The leucocyte reagent pad may be inhibited to varying degrees. Please refer to the pack insert for further information. “A properly collected urine sample is the first stage in the generation of quality clinical information” Disclaimer This is a training guide and does not replace the package insert for each test. Prior to preparing any test, please read and operate instruments and strips according to Siemens instructions. 3 40619_PRACGUIDE_Rebrand.04.09:Layout 1 22/04/2009 09:41 Page 4 Correct Technique for Visual Reading of Reagent Strip Tests Quality Assurance Procedure Use Chek-STIX for checking reactivity of Siemens test strips procedure Chek-STIX strips offer a convenient method of generating an artificial urine sample which will give a positive or negative result to all Siemens multiple strip tests. Always check the appearance of the urine sample as it may give useful information. Siemens tests are scientifically designed to react progressively and produce colour changes in the case of positive reactions at the times specified. Accurate timing is essential for reliable quantitative results.N Chek-STIX has been developed specifically so the user can be assured of the reactivity of the reagent areas of Siemens multiple reagent strips. The product may also be used as a check of user technique as well as an aid to teaching. Example Test The control solution generated by Chek-STIX Positive solution gives results on Siemens strips in the same manner as urine specimens. If the strips are reacting properly a positive result will be seen on each test area, with expected values as shown in the pack insert. 1. Completely immerse all reagent areas into fresh, wellmixed, uncentrifuged urine. Dip briefly and remove immediately to avoid dissolving out reagents. “Don’t forget to read the package insert carefully” 2. While removing the strip, run the edge against the rim of the urine container to remove excess urine. Hold the strip in a horizontal position to prevent possible mixing of chemicals from adjacent reagent areas or soiling of hands with urine. 3. After the appropriate time, compare test areas closely with the corresponding colour chart on the bottle label or bench reader at the time specified. Hold strip close to colour blocks and match carefully. Always record the results. 4. For enhanced convenience and standardisation, use a CLINITEK analyser to read the reagent strip and print the results. 1. Place 12 ml of distilled water in test tube provided, by filling to graduation line. Immerse totally one Chek-STIX strip. 3. Invert tube once more. Remove and discard Chek-STIX strip. Solution is now ready to test reactivity of Siemens strips or user technique. Glucose 5.5 mmol/L Bilirubin Positive Ketones Small-Moderate Specific Gravity 1.000 to 1.015 Blood Moderate-Large pH 7.0-8.0 Protein 0.3-1.0 g/L Urobilinogen Positive Colours may be atypical Nitrite Positive Leucocytes Positive “Expected results may change from the above. Always check the pack insert” Precautions It is important to make sure the tests are being used and stored properly. A few precautions will ensure meaningful results for patient management. Do not Do Do read package insert carefully before testing 4 2. Cap tube tightly. Invert the tube gently back and forth for 2 minutes. Allow to stand for 30 minutes. Expected Results Do replace cap immediately and close tightly Do check expiry date on bottle before use Do not remove desiccant from bottle Do not touch test areas of the strip Do not take out more strips than are required for immediate use 5 40619_PRACGUIDE_Rebrand.04.09:Layout 1 22/04/2009 09:41 Page 6 Chemical Principles Reagent Strip Tests Glucose Reagents glucose oxidase; peroxidase; potassium iodide; buffer; non-reactive ingredients. Reagents 2, 4-dichloroaniline diazonium salt; buffer; non-reactive ingredients. Chemical Principles The test is based on a double sequential enzyme reaction utilising glucose oxidase and peroxidase with a potassium iodide Chromagen. Colours range from green to brown Chemical Principles Sensitivity / Specificity The test is specific for glucose; no substance excreted in urine other than glucose is known to give a positive result. Approximately 5.5 mmol/L of glucose is detectable. The test is more sensitive than the copper reduction test, CLINITEST, which will detect 13.9 mmol/L (1/4%) glucose as a trace. 6 Bilirubin This test is based on the coupling of bilirubin with diazonium dichloroaniline in a strongly acid medium. The colour ranges through various shades of tan. Sensitivity / Specificity Test has sensitivity of 7 to 14 µmol/L bilirubin. Ketones Reagents sodium nitroprusside; buffer Chemical Principles This test is based on the development of a pink or maroon colour by the reaction of acetoacetic acid with nitroprusside. Sensitivity / Specificity The test reacts with acetoacetic acid in urine, but does not react with acetone or beta-hydroxybutyric acid. The test detects as little as 0.5 mmol/L acetoacetic acid. Specific Gravity Reagents bromthymol blue; poly (methyl vinyl ether/maleic anhydride); sodium hydroxide. Chemical Principles This test is based on the pKa change of certain pre-treated polyelectrolytes in relation to ionic concentration. In the presence of an indicator, colours range from deep blue-green through green and yellowgreen in urines of increasing ionic concentration. Sensitivity / Specificity The test determines the specific gravity of urines in the range 1.000 to 1.030. In general, the result correlates within 0.005 with refractive index values. For increased accuracy add 0.005 to readings obtained from urines with pH values equal to or greater than 6.5. Blood Reagents disopropylbenzene dihydroperoxide; 3,3,’ 5.5,’ tetramethylbenizidine ; buffer; non-reactive ingredients. Chemical Principles This test is based on the peroxidase-likeactivity of haemoglobin, which catalyses the reaction of the reagent with the chromogen. The resulting colour ranges from orange through green to dark blue. Sensitivity / Specificity The test is capable of detecting 150 to 620 µg/L free haemoglobin or 5 to 20 intact red blood cells per microlitre in urines with an S.G. of 1.005. The test is less sensitive in urines with a high S.G. and is more sensitive to free haemoglobin than intact red blood cells. The appearance of green spots on the reacted reagent pad indicates the presence of intact erythrocytes in urine. Clinical Significance of Test Results pH Protein Urobilinogen Reagents methyl red: bromthymol blue: non-reactive ingredients. Reagents tetrabromphenol blue; buffer; non-reactive ingredients. Reagents paradiethyl-aminobenzaldehyde; non-reactive ingredients. Chemical Principles Chemical Principles Chemical Principles The test is based on the double indicator principle, which gives a broad range of colours covering the pH range 5 to 8.5. Colours change from orange through yellow and green to blue. The test is based on the protein-error-ofindicators principle. The pKa of some indicators is changed when they bind to protein, changing colour when protein is added at a constant buffered pH. Colour ranges from yellow for “negative” through yellowgreen, green to green-blue for “positive” reactions. The test is based on the Ehrlich reaction, in which paradimethyl aminobenzaldehyde reacts with urobilinogen in a strongly acid medium to produce a brownorange colour. Sensitivity / Specificity The test provides quantitative differentiation of urine pH in the range 5 to 8.5. Readings are not affected by urinary buffer variations, but the pH may be changed by certain medications. Sensitivity / Specificity Quantitative test, with “trace” result indicating albumin concentration of between 0.15 to 0.30 g/L. Test is less sensitive to globulin, Bence-Jones protein and muco-protein, so a “negative” result does not rule out the presence of these proteins. Sensitivity / Specificity Quantitative test that detects urobilinogen concentrations as low as 3 µmol/L in urine. Absence of urobilinogen cannot be determined by this method. Nitrite Reagents para-arsanilic acid; 1, 2, 3, 4-tetrahydrobenzo (h)-quinolin, 3-ol; buffer; non-reactive ingredients. Chemical Principles This test is based on the conversion of nitrate (derived from dietary metabolites) to nitrite by the action of certain species of bacteria in the urine. Any degree of pink colour is considered ‘positive.’ Sensitivity / Specificity The test is specific for nitrite, with a sensitivity of 13 to 22 µmol/L nitrite in urines of normal specific gravity. Leucocytes Reagents Derivatised pyrrole amino acid ester; diazonium salt; buffer; non-reactive ingredients. Chemical Principles This test is based on the principle that esterases found in granulocytic leucocytes catalyse the hydrolysis of a derivatised pyrrole amino acid ester to liberate 3-hydroxy-5phenyl pyrrole. This pyrrole then reacts with a diazonium salt to produce a purple complex. Sensitivity / Specificity This test provides a semi-quantitative result for the presence of leucocytes, ‘trace’ indicating approximately 5 to 15 cells/µL, + + + indicating approximately 500 cells/µL. The test for leucocytes is a specific enzymatic reaction which is able to detect lysed cells in addition to undamaged cells. In clinical studies involving over 1000 specimens, the sensitivity was 91.4% when compared with the chamber count of numbers of 10 and more cells/µL. Glucose Significance of Positive Results Glucose is found in urine when its concentration in plasma exceeds the renal threshold. Commonest Causes of Positive Results In patients with raised blood glucose concentration: •Diabetes mellitus •Glucose infusion In patients without raised blood glucose concentration: •Pregnancy •Renal glycosuria. Bilirubin Significance of Positive Results Bilirubin in urine indicates an excess of conjugated bilirubin in plasma. Commonest Causes of Positive Results Liver cell injury, as in viral or drug-induced hepatitis; paracetamol overdose; late-stage cirrhosis. Biliary tract obstruction, e.g. by gall-stones; carcinoma of the head of pancreas; biliary atresia in infants; late-stage primary biliary cirrhosis; druginduced intrahepatic cholestasis. Ketones Specific Gravity Significance of Positive Results Indicates accumulation of acetoacetate secondary to excessive breakdown of body fat. Significance of Positive Results A measure of total solute concentration health varies widely according to the need to excrete water and solutes. Acetone and ß-hydroxybutyrate are released at the same time. Commonest Causes of Positive Results Characteristic findings High values are found in dehydration, except when due to in urine. In impaired kidney function as in chronic renal failure. Low values are found in individuals with intact renal function and high fluid intake; diabetes insipidus; chronic renal failure; hypercalcaemia, hypokalaemia. Commonest Causes of Positive Results Fasting, particularly with fever and/or vomiting; most often seen in children. Diabetic ketoacidosis (grossly uncontrolled insulin-dependent diabetes). Ketotic hypoglycaemia in young children. Blood Significance of Positive Results Intact red cells in urine are found with diseases of the kidneys or urinary tract. Free haemoglobin has either been excreted from plasma or liberated in-vivo or in-vitro from red cells in the urine. Commonest Causes of Positive Results Haematuria (intact red cells) Due to kidney disorders, including glomerulonephritis; polycystic kidneys; tumours. Due to urinary tract disorders, including stones; tumours; infection; benign prostatic enlargement. Haemoglobinuria (free haemoglobin) Severe haemolysis, e.g. crises in sickle cell disease or red cell glucose-6-phosphate dehydrogenase deficiency; haemolytic breakdown of red cells in urine (especially when dilute and when testing is delayed after voiding). N.B. Myoglobin, released from muscle, gives a diffuse reaction indistinguishable from that given by free haemoglobin. pH Significance of Positive Results Urinary pH usually reflects the pH of body fluids, but may also be affected by the ability of the kidneys to eliminate hydrogen ions and by infection of the urinary tract by organisms containing an enzyme which promotes production of ammonia from urea. In health the pH of uncontaminated urine varies between 4.5 and 8.0 depending on the rate of elimination of hydrogen ions. (pH < 7.0 = Acid, pH > 7.0 = Alkaline). Commonest Causes of Positive Results Low values are found in acidaemia, e.g. diabetic ketoacidosis and lactic acidaemia; starvation; potassium depletion. High values are found in alkalaemia (except when due to potassium depletion), e.g. due to vomiting and consumption of large amounts of antacids; renal tubular acidosis; urinary tract infection with ammoniaforming organisms. The commonest cause of high values is the use of stale urine: such specimens should not be used. Protein Significance of Positive Results Strip tests will detect a range of proteins but are most sensitive to albumin. Excess albumin in urine is usually due to increased permeability of the basement membrane of glomeruli. Commonest Causes of Positive Results Clinically important albuminuria, as in acute and chronic glomerulonephritis; glomerular involvement in systemic lupus erythematosus; nephrotic syndrome; pre-eclampsia (toxaemia of pregnancy). Clinically unimportant albuminuria, as in postural (orthostatic) proteinuria; fever; heart failure. Urobilinogen Significance of Positive Results Urinary excretion of urobilinogen reflects the combined effects of production of bilirubin, conversion of bilirubin to urobilinogen in the gut, reabsorption into the blood stream. Commonest Causes of Positive Results Increased excretion, increased production, e.g. inborn red cell disorders including sickle cell disease, glucose-6-phosphate dehydrogenase deficiency and final elimination by the kidney; hereditary spherocytosis; acquired immune-and drug-induced haemolysis; ineffective erythropoiesis, e.g. in thalassaemias Nitrite Significance of Positive Results Most of the organisms which infect the urinary tract contain an enzyme system which catalyses the conversion of nitrate, which is normally present in urine, to nitrite, which is not found in urine in the absence of a urinary tract infection. Commonest Causes of Posoiive Results Urinary tract infection due to nitrite-producing organisms. Leucocytes Significance of Positive Results Some of the leucocytes which have entered inflamed tissue from the blood are shed into the urine. Commonest Causes of Positive Results Urinary tract infection; especially when acute inflammation of urinary tract. N.B. Infection is by far the commonest cause of urinary tract inflammation. N.B. A negative result does not exclude infection, because some organisms are unable to convert nitrate to nitrite. Decreased uptake by liver, e.g. in viral hepatitis and cirrhosis. Decreased excretion Biliary tract obstruction, as with gallstones, carcinoma of pancreas. (Prevention/restrictio n of bilirubin entering the gut). Sterilisation of colon by administration of unabsorbable antibiotics, e.g. neomycin. (Prevents/reduces bacterial conversion of bilirubin to urobilinogen). 7 40619_PRACGUIDE_Rebrand.04.09:Layout 1 22/04/2009 09:42 Page 8 Factors Affecting Urine Chemistry Tests on Siemens Reagent Test Strips Glucose Bilirubin DIASTIX Reducing sugars other than glucose CLINITEST CLINISTIX (Multiple Strips) Galactose + no effect no effect Lactose Fructose Maltose + + + no effect no effect no effect no effect no effect no effect Pentose + no effect no effect High concentrations of L-Dopa may cause false + false – false – Ketones no effect no effect high concentrations May mask a small reaction (May give red and other colours) Phenothiazines High doses can cause hepato-cellular damage giving true + Chlorpromazine metabolites development. Can also cause false + by reacting with reagent area. Urine not freshly voided may depress colour Ascorbic acid Azo dye metabolites and other highly coloured compounds, e.g. rifampicin and phenazopyridine high concentrations moderate concentrations high concentrations may cause false + may cause false – may slightly depress colour development. May give false – Lodine (Etodolac) Hypochlorite or chlorine no effect false + false + Peroxide no effect false + false + Nalidixic false + no effect no effect Cephalosporins give black colours no effect no effect Probenecid false + no effect no effect High specific gravity no effect decreases sensitivity no effect Low specific gravity no effect intensifies colour no effect May give false + Positive or atypical colour. Ketones Mesna Red/Violet colour L-Dopa Atypical colours PKU (large quantities of phenylketones) + BSP Test (Bromosulphthalein) False + Phenazopyridine Atypical colour Captopril May give false + Specific Gravity Blood pH Protein Elevated readings may be obtained in the presence of 1-7.5 g/L protein Ferrous sulphate peroxidase Acid High protein diet – like activity gives + reaction Glucose Urea Urines containing glucose or urea greater than 1% may cause a low specific gravity reading relative to other methods Myoglobin Gives a + reaction Prolonged exposure of unpreserved urine at room temperature pH For increased accuracy, 0.005 may be added to the readings from urines with pH equal to or greater than 6.5. Ascorbic acid High concentrations reduce the sensitivity N.B. The commonest cause of a so-called false-positive reaction for blood is that the laboratory method, microscopy, only measures red cells and, since these readily lyse, a positive result for haemoglobin with the blood test can be obtained without any cells being present when the urine is ultimately examined under the microscope Ammonium chloride medication Alkaline Diet high in vegetables, citrus fruits, milk and other dairy products Prolonged exposure of unpreserved urine at room temperature Antacid buffers. Protein Highly buffered alkaline urines May cause false + Quaternary ammonium salts, e.g. Benzalkonium chloride, Chlorhexidine May cause false + Phenazopyridine May give atypical colour. Urobilinogen Nitrite Leucocytes PAS (p-aminosalicylic acid) metabolites Atypical colours Ascorbic acid Large quantities may cause false – reaction Decreased Test Results Elevated glucose concentration Azo dye metabolites Mask reaction (phenazo-pyridine gives an orange/red colour) High Specific Gravity Sensitivity is reduced High specific gravity Urine not freshly voided False – Lodine (Etodolac) May give false+ Azo dye metabolites (phenazopyridine) Could mask or mimic + reaction. Presence of Cephalexin, Cephalothin, oxalic acid or Tetracycline Masked colour reaction Nitrofurantoin (brown urine) Any substance that causes abnormal urine colour Boric Acid Inhibits reaction to varying degrees. Captopril May cause decreased reactivity. development High doses of salicylates 8 false + false – false – “For technical and product support information or queries, please contact our Technical Solutions Centre on: 0845 600 1955” 9 40619_PRACGUIDE_Rebrand.04.09:Layout 1 22/04/2009 09:42 Page 10 Test Selection Made Easy Urinary Tract Infection Testing Pathway If ALL of the following are Creatinine Albumin NEGATIVE DISCARD Nitrite Leucocytes Blood or Protein Report as no evidence of infection Leucocytes Nitrite Urobilinogen Urine Specimen Protein Visual Appearance Clear Test with Multistix* 8SG/10SG If ANY of the following are pH POSITIVE Blood Nitrite Leucocytes Blood or Protein Specific Gravity Ketones Culture + Microscopy Culture + Microscopy Bilirubin 10 100 100 25 100 100 100 100 100 100 50 50 50 50 25 25 25 2304 Multistix* 8SG 2283 Multistix* GP 2740 N-Multistix* SG 2741 Multistix* SG 2743 Labstix* SG 2815 N-Labstix* 2814 Bili-Labstix* 2810 Labstix* 2877 Hema-Combistix* 2857 Uristix* 2872 Albustix* 2816 Hemastix* Microalbustix Clinitek Microalbumin Microalbumin 9 Obviously infected or blood stained 2300 Multistix* 10SG Code No Product Pack size Glucose This approach gives the power to confidently avoid sending noninfected urine samples for microscopy and culture, and enables appropriate, timely and cost-effective patient management in the clinic, ward or surgery. 11 40619_PRACGUIDE_Rebrand.04.09:Layout 1 22/04/2009 09:42 Page 12 Siemens Healthcare Diagnostics, the leading clinical diagnostics company, is committed to providing clinicians with the vital information they need for the accurate diagnosis, treatment and monitoring of patients. Our comprehensive portfolio of performance-driven systems, unmatched menu offering and IT solutions, in conjunction with highly responsive service, is designed to streamline workflow, enhance operational efficiency and support improved patient care. Multistix and all associated marks are trademarks of Siemens Healthcare Diagnostics Inc. All other trademarks and brands are the property of their respective owners. Product and service availability may vary from country to country and is subject to varying regulatory requirements. Please contact your local representative for availability. Siemens Global Headquarters Siemens AG Wittelsbacherplatz 2 80333 Muenchen Germany Local Contact Information Siemens Healthcare Diagnostics Ltd Sir William Siemens Square, Frimley, Camberley, Surrey GU16 8QD UK Telephone 01276 696000 E-mail: [email protected] www.siemens.co.uk/diagnostics Global Siemens Healthcare Headquarters Siemens AG Healthcare Sector Henkestrasse 127 91052 Erlangen Germany Telephone: +49 9131 84-0 www.siemens.com/healthcare www.siemens.com/diagnostics Practical guide to urine analysis leaflet I Printed in UK I © 04/2009 Siemens Healthcare Diagnostics. Global Division Siemens Healthcare Diagnostics Inc 1717 Deerfield Road Deerfield, IL 60015 0778 USA www.siemens.com/diagnostics