Automation in urine analysis

Urine analysis is an integral part of a clinical laboratory. automation techniques in urine biochemistry, their priniciplas and microscopy along with their advantages and disadvantages are outlined.

1. Automation in urine analysis Varun Kumar Singh

2. It all started over 2,000 years ago. Many cultures once regarded urine as a mystical fluid, and some still do. Its uses have included wound healing, stimulation of the body’s defenses , and disease diagnosis.

3. Hippocrates (approx. 400 BC) -urine characteristics ( odor / color ) - altered with different diseases. Gilles de Corbeil - related 20 different types of urine to conditions of the body (differences in sediment and color ). FRANCOIS RAYER & EUGENE N. VIGLA - 1837 “test strips” - Jules Maumene -1850 By the end of 19 the century all particles identified. AUTOMATED urine microscopy 1993

4. Introduction Many different diseases display abnormalities in urine Progression /Regression of various lesions can be monitored with minimal distress to patient Systemic d/s ,Endocrine/Metabolic detected through recognition of abnormal amount of disease-specific metabolites excreted in urine Simple, non invasive, economical investigation

5. Most labor intensive subspecialties. Least automated Least favored . Traditionally: chemistry targeted. Time: suspended for urine microscopy.

6. Why automate... Increases productivity Labor Savings Improvement in TAT Supports Lean Management principles Better use of staff Reduces errors Better response to clinician’s and administration’s concerns Improves precision and accuracy of data Better compliance to new federal Medicare regulations Ease of Use

7. Out of all of the analyses performed in the clinical laboratory the urinalysis has one very distinct advantage: - it’s a non-invasive test

8. Elements of urine analysis 2 step process Biochemical analysis Microscopy

9. Elements of urine analysis The Specimen Physical Characteristics - Color , Clarity, Specific Gravity Chemical Analysis - Glucose, Protein, Bilirubin , Urobilinogen , pH, Blood, Ketones , Nitrite, Leukocytes, and Ascorbic Acid Microscopy - Formed elements ( particles ), e.g., epithelial cells , blood cells, crystals, casts, bacteria, sperm, mucus, etc.

10. Sample collection 1st morning void. Clean catch. Midstream. Wide mouthed plastic container. Prerequisite – Wash genitalia. Avoid – During menstruation. Time lag b/w collection & analysis MINIMIZED to avoid : microbial over growth casts & crystals disintegration. Ideal - < 2hrs .

11. Options in automation ... Dip sticks Automated biochemistry Automated microscopy

12. Dipsticks Micro chemical system permits qualitative and semi quantitative analysis within a minimum time span by simple observation Clear plastic strips Reagent impregnated paper & Absorbent paper underneath are held in place on a stiff white carrier foil by fine Nylon Mesh Different reagent areas are affixed on the strip Different cellulose areas are impregnated with specific testing chemicals according to test required

13. Two external interference factors include:- Glue – interferes with the colour reading Ascorbic acid –inhibits the oxidation reaction - false negative in c/o hematuria and glycosuria Solution : - Adhesion by means of nylon mesh Iodated mesh – prevents ascorbic acid effect

14. Different dipsticks Uristix : Glucose , Protein Multistix –SG : PH, Specific gravity, Glucose, Protein, Ketone, Bilirubin, Blood, Urobilinogen Multistix -10SG : Also Nitrite & Leukocyte Combistix –SG : PH, Specific gravity, Glucose, Protein Keto –DIASTIX : Ketone, Glucose

15. Advantages of dip stick automation Enhances work flow saving labour and time Standardizes some aspects of manual urinalysis Reduces subjective errors Large number of samples in short time Performed on UNCENTRIFUGED urine

16. Chemical examination using reagent strip Requirements: Uncentrifuged, fresh, well mixed urine Reagent strips Procedure: Dip the test area in urine Remove excess of urine Compare test areas with corresponding color charts, at times specified in good light

17. pH Principle : Color of reaction area changes depending on PH ,based on double indicator principle INDICATORS : Methyl red (PH- 4.4-6.2) Orange red yellow Bromothymol blue(PH-8.0-9.6) Yellow blue

18. Influencing factors Nutrition :animal protein-acidic urine, vegetarian diet - alkaline urine. Metabolic status,diseases ,medicines Limitations • stands for too long, alkaline pH values are diagnostically meaningless - bacterial decomposition • Residues of disinfectants (quaternary ammonium compounds) -false results

19. Principle : Based on Pka change of pretreated polyelectrolytes in relation to ionic concentration of urine. Indicator substance changes color relative to ionic concentration ,this is translated to specific gravity values Deep blue green Yellow orange Specific Gravity

20. • Explains differences between microscopy and test strip results: WBC and RBC - lysed in low concentrated urine • Interpretation of borderline results of test strip parameters: dilution or concentration of the urine can confirm or invalidate the pathological significance

21. Limitations • does not indicate the contribution of non-ionic urinary constituents- urea, creatinine or glucose • pH >7.0, specific gravity test strip reading may be too low and has therefore to be increased by 0.005 g/ mL • protein -100 to 500 mg/ dL or ketoacidosis , - elevated • glucose concentrations >1,000 mg/ dL (>56 mmol /L) is not determined Influencing factors Fluid intake Heavy sweating Increased urine output - diuretics

22. Glucose Principle: Specific GLUCOSE-OXIDASE and PEROXIDASE, a double sequential enzyme reaction. Glucose + O2 Gluconic acid +H2O2 H2O2 + Chromogen H 2 0 + Oxidized chromogen changes REAGENT CLINISTIX MULTI STIX CHEM STRIP CHROMOGEN O- toluidine KI chromogen Amino propylcarbazol Color change Pink blue Blue brown Yellow Orange brown Time --- 30 sec 60 sec

23. Limitations • The urine glucose concentration -glucose excretion - does not necessarily correlate with the actual blood glucose value Influencing factors Low or false-negative glucose • Metabolic products and drug metabolites which have a reducing action False-positive glucose • Presence of residues of peroxide-containing or other strongly oxidizing cleaning agents

24. Bilirubin Principle: Based on coupling reaction of bilirubin with a diazonium reagent. Reagent strip Chemstix Multistix Reagent 2,6 dichlorobenzene diazonium tetrafluoroborate 2,4-dichloroaniline Time 30—60 sec 20 sec Result Pink Violet Creambuff Tan

25. Limitations • High ascorbic acid concentrations lower the sensitivity of the bilirubin test Influencing factors False-negative bilirubin • Prolonged standing - direct sunlight, -oxidation False-positive bilirubin • Medicines that color the urine red or that are themselves red in an acid medium, e.g. phenazopyridine • Yellow or green reaction color of the UBG test in the presence of high bilirubin concentrations

26. Urobilinogen Principle : MODIFIED EHRLICH ALDEHYDE reaction : Acidic medium Urobilinogen + chromogen Red colour REAGENT STRIP MULTISTIX CHEMSTIX Reagent P-dimethyl amino benzaldehyde & Acid buffer 4-methoxy benzene- diazonium tetrafluoroborate Time ---- 10-30 sec Result yellow -red brown Red azo dye Advantage specific 0.2-1mg/dl Specific 0.4 mg/dl

27. Limitations • specific for urobilinogen - not react with other diazo -positive substances ( porphobilinogen , indican , p – aminosalicylic acid, sulfonamides , sulfonylureas ) Influencing factors False-negative urobilinogen • Oxidation - in direct sunlight. • Formaldehyde > 200 mg/ dL - preservative False-positive urobilinogen • Drugs or metabolites which turn red in an acid medium ( e.g.phenazopyridine )

28. Principle: liberation of oxygen in the reagent strip by peroxidase like activity of heme from free Hb , lysed Red cell or Myoglobin leading to oxidation of Chromogen and change in color Blood Dipsticks capable of detecting intact RBC, Free Hb and Myoglobin Lowest detectable concentration is 5 INTACT RBC/UL or Free Hb to 10RBC/UL

29. REAGENT STRIP CHEMSTIX MULTISTIX Chromogen & peroxidase 2,5 dimethyl –2,5 dihydro peroxyhexane & tetramethyl benzidine peroxidase 3,3’,5,5’tetramethyl benzidine & cumene hydroperoxide Time 60 sec 40 sec Results Yellow green Orange green dark blue Method of estimation

30. Discrepancy between test and microscopy • Old specimens- RBCs lyzed in urine upon sitting-not detected under microscope • Urine not swirled, RBCs - bottom, pad at the end of strip being dipped in a concentrated area • Over-centrifugation can cause destruction of RBCs

31. Influencing factors False-positive blood • Expired, contaminated or improperly stored strips. Residues from strong oxidizing reagents in urine containers or cleansing tissues • Menstrual contamination, not collecting clean catch midstream False-negative blood • Formalin (used as a preservative) • Nitrite (in excess of 10mg/ dL ) delays the reaction

32. Principle : Based PH INDICATORS i.e. proteins carry a charge at physiologic pH, their presence will elicit a pH change Reagent strip : Tetra bromophenol blue Time : 30—60 sec Protein Interpretation: Yellow Blue 5-20 mg/dl Albumin can be detected Reagent strip more sensitive to albumin than to Globulin, Bence Jones proteins & mucoproteins

33. Limitations • Microalbuminuria cannot be detected - first positive result -15–30 mg/ dL • The sensitivity to other proteins (e.g. globulins, proteases, peptones, mucoproteins ) is lower Influencing factors Low or false-negative protein • Proteinuria is mainly consisting out of other proteins than albumin False-positive protein • During or after infusion of poly vinyl pyrrolidone (blood substitute) • Strongly basic urine (pH > 9) during therapy with phenazopyridine • Residues of disinfectants based on quaternary ammonium compounds or chlorhexidine

34. Principle : Nitroprusside reaction for ketones Sodium Nitroferricyanide & Glycine+Acetone / Acetoacetic acid Ketones Alkaline Medium Violet colour REAGENT STRIP CHEMSTIX MULTISTIX Reagent Sod nitroferricyanide & Glycine Sodium nitroferricyanide & Buffers Reaction Acetoacetic acid & Acetone Acetoacetic acid Color Time Beige Violet 60 sec Pink Maroon 15 sec Results Acetoacetic acid 10mg/dl,acetone70mg/dl Acetoacetic acid-5-10mg/dl No reaction

35. Limitations • Phenylketone or phthaleine compounds - (red-orange ) Influencing factors False-positive ketones • Captopril , MESNA (2-mercapto-ethanesulfonic- acid sodium salt) and other substances containing sulfhydryl

36. NITRITE Test principle: - The aromatic amine sulfanilamide reacts with nitrite in the presence of an acid buffer to form a diazonium compound, which is coupled with 3-hydroxy-1,2,3,4- tetrahydrobenzo -(h)- quinoline to form an azo dye. Nitrate that is present in the urine is converted by bacterial reduction into nitrite. Sulfanilamide + Nitrite  Diazonium salt Diazonium salt + Coupling component  Azo dye (red)

37. Limitations • The intensity of the red color is a measure of the nitrite concentration but cannot be correlated to the severity of the infection Influencing factors False-positive nitrites • Expired, contaminated or improperly stored Strips • Drugs that color the urine red e.g. phenazopyridine • Bacterial contamination from sample collection - nitrate to nitrite in specimens >4 hours old False-negative nitrites • Bacteria causing UTIs may not be able to convert nitrate to nitrite • Antibiotic therapy • Insufficient nitrate intake or too short retention of urine in the bladder

38. Leukocytes Test principle :- The leukocytes excreted in the urine are almost exclusively granulocytes, whose esterase activity is detected in the test strip reaction. The test zone contains an indoxyl ester, which is cleaved by the granulocyte esterase. The free indoxyl reacts with a diazonium salt to form a violet dye.

39. Limitations • The test does not react to pathogenic bacteria and trichomonads in urine • Protein excretion in excess of 500 mg/ dL and glucose excretion of over 2 g/ dL could - weaker color development Influencing factors False-positive leukocytes: • contamination by vaginal secretion • Expired, contaminated or improperly stored strips • Nitrofurantoin , imipenem , meropenem , clavulanic acid (antibiotics) False-negative leukocytes: • Specimen not mixed well or at a low temperature • Proteinuria > 500 mg/ dL • Glucosuria > 2,000 mg/ dL • Cephalexin , gentamycin • Boric acid, sodium azide , mercury salts, hydrochloric acid

40. Limitations of dip sticks Differences in lightning conditions Difference in individual skill, failure to keep specified time Loss of reagent reactivity due to improper storage Discoloration of strips by bilirubin, blood or other constituents

41. Role of Quality control in Dip sticks If tests results are questionable/ inconsistent with expected findings & clinical history, steps recommended Confirm product is within expiry date Retest with fresh sample Check performance against known Negative & Positive control materials Check for False positive & False negative

42. REFLECTANCE SPECTROPHOTOMETERY

43. Instruments intended for single use Semi automated urine analysis systems Fully automated urine analysis systems

44. Principle : REFLECTANCE SPECTROPHOTOMETERY Analyses color and intensity of light reflected from reagent area and reports results in clinically meaningful units NO calculations required Automatic calibration : Runs a self test each time before each strip is read or power is switched on. URI PLUS 1A

45. Method of Operation Strips laid on the instrument Sensor detects strip presence and activates strip movement ,reading cycle Has an optional Bar code reader. QC done once in morning

46. UriPlus 900 Fully automatic 10 & 11 parameter Strips used Based on Reflectance photometery { colorimetry } Uses high lumonosity 4 wavelength cold light source reflection determination technology.

47. Calibration

48. Quality control Once a day in morning

49. How tests are done in our lab?

50. Cobas 6000 c501

51. Electrochemiluminiscence

52. Can analyse urine : Mg+ , Na+ ,K+ , Ca++, P- Protein Microalbumin Uric acid Urea Creatinine Amylase Micro total protein Light emitting substance like ruthium labelled Ab are added to the sample. Emission of light is electrically stimulated Amount of light produced is directly proportional to amount of substance to be detected is present Cobas 6000 c501 Based on Electrochemiluminiscence (ECL) technology:

53. Urine microscopy Bright light microscopy Phase contrast microscopy epithelial cells , blood cells, crystals, casts, bacteria, sperm, mucus, etc.

54. Automation in microscopy Flowcytometry Auto particle recognition SYSMEX UF-50, 100, 1000i IRIS Q 200 URI SED AVE-763Analyzer

55. Flowcytometry Particles - labeled with fluorophores measured in a laser beam classified fluorescence size impedance forward scattered light. results - scattergrams and histograms. cells per microliter or cells per field of view

56. Auto particle recognition Particle images - planar flow cell in the object plane of a microscope. Stroboscopic illumination freezes the motion - blur-free images - charge-coupled device camera sensor. Individual particle images isolated from each of the 500 captured frames - 12 categories their size, shape, contrast and structure The images - verification and manual editing Results - particles per field of view (per high-power field; HPF) or per microliter .

57. Kova systems Semi automated Kova tube (12ml), kova cap Centrifuge 1500rpm x 5 min Kova petter - decant, resuspend , charge Kova slide - counts

58. Closed slide system Censlide Fast centrifuge <2 min, 1350rpm Standarised sediment volume Transfer to scope

59. Particle count algorithm/ volume calibration = particle concentration User defined criterion met yes no LIS Flags for manual confirmation Manual microscopy

60. ADVANTAGES DISADVANTAGES Important screening tool; Reduces work load in a busy laboratory - reducing the samples to be examined manually. Not affected by Preanalytic confounding factors like improper centrifugation Excellent walk away efficiency. Fast results on large no.of samples. Accurate no. given for most particles. Review of sample possible. DOES NOT SUBCLASSIFY the particle . NO INTERNATIONAL STANDERDISATION or recognized REFERANCE MEASUREMENT procedure drawback in result validation. Insufficient mixing leads to wrong analysis. Cells like Dysmorphic WBC mistaken as artefact

61. COMPARISION WITH MANUAL SN Variable Manual Automation 1. Bias ++ Nil 2. Standardisation + Absent 3. Precision + / - ++ 4. Reproducability + / - ++ 5. Variance ++ Nil 6. Crystal,cast ,& microbial sub categorization Excellent Absent 7. Quantitation rbc wbc Estimate Exact No. 8. Time More Less 9. Cost Effective Expensive

62. Summary precise and improves the work flow in a routine laboratory Rapid biochemical analysis sediment analysis – helps in identifying pathological samples - missed in the two-step procedure. combined with dipstick testing- reduce the number of specimens submitted to microscopy. Visual microscopy - dysmorphic erythrocytes, yeasts, Trichomonas , oval fat bodies differentiation of casts and certain crystals.

63. References Henry’s clinical diagnosis and management by laboratory methods, 22 nd edition Jeff A. et al, Urinalysis: A Comprehensive Review, American Family Physician, 71,6 Compendium of urinalysis: Urine test strips and microscopy, Cobas Nousin et al, Automated urinalysis: first experiences and a comparison between the Iris iQ200 urine microscopy system, the Sysmex UF-100 flow cytometer and manual microscopic particle counting, Clin Chem Lab Med 2007;45(9):1251–1256 Automating urinalysis , Iris diagnostics Langlouis et al, Automated Flow Cytometry Compared with an Automated Dipstick Reader for Urinalysis, Clinical Chemistry 45:1,118–122 (1999)