AUTOMATION IN URINE ANsccdvdvdvrvALYSIS.pptx

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1. AUTOMATION IN URINE ANALYSIS Presented by: Swati Mishra Moderator : Dr P. Kusaraju (Asst. Prof)

2. CONTENTS Introduction Indications Elements of urinalysis Sample collection Automation Comparison of automated analyzers Comparison of manual and automated urinalysis Summary

3. Introduction Urine analysis is one of the most commonly performed laboratory tests in clinical practice. Microscopic examination of urine is also called as the ‘ liquid biopsy of the urinary tract’.

4. Indications Suspected renal diseases - glomerulonephritis , nephrotic syndrome, pyelonephritis , and renal failure. Urinary tract infection Metabolic disorders like diabetes mellitus Differential diagnosis of jaundice Plasma cell dyscrasia . Diagnosis of pregnancy.

5. Elements of urinalysis Physical examination – Volume , specific gravity, odor, appearance/clarity, color Chemical examination – pH, proteins, glucose, ketones , bilirubin, bile salts, urobilinogen , blood, hemoglobin, myoglobin , nitrite/ leucocyte esterase Microscopy – cells, casts, crystals, organisms

6. Sample Collection First morning void, midstream, clean catch Wide mouthed plastic container Time lag between collection and analysis should be avoided

7. Automation in Urinalysis Manual microscopic urine sediment examination is labor intensive, time consuming and lacks standardization in high volume laboratories. Goal of automation – maximize productivity and testing quality, while keeping expenditures and turnaround time minimum.

8. Several automated instruments are currently available to standardize: Sample processing Biochemical test strips analysis Microscopy analysis Report results

9. Automated instruments in urinalysis include Reagent test strips/Dipsticks : performed manually, or by Semi-automated chemistry analyzers : test strip dipped in urine manually and analyzed by an instrument Fully automated chemistry analyzers : add urine to the reagent strip and process.

10. Automated urine cell analyzers : mix, aspirate, dilute, and stain urine to classify urine sediment particles. Automated urine systems : perform a complete urinalysis that includes the physical, chemical, and microscopic parts of a routine urinalysis.

11. Reagent test strips/Dipsticks Microchemical system Qualitative and semi-quantitative analysis within minimum duration Reagent impregnated paper and absorbent paper held in place by fine nylon mesh Different reagent areas (cellulose areas impregnated with specific testing chemicals) affixed on strip

12. Uses of Dipsticks

13. Different dipsticks Uristix : Glucose, Proteins Multistix -SG : pH, specific gravity, glucose, proteins, ketone , bilirubin, blood, urobilinogen Multistix-10SG : +nitrite & leucocyte Combistix –SG : pH, specific gravity, glucose, proteins Keto -DIASTIX : Ketone , glucose

14. Chemical examination using reagent strip Requirements: Uncentrifuged , fresh, well mixed urine 2.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

15. pH Principle- Test area contains polyionic nylon polymer bound to Hydrogen ions, which is released on reaction with cations in urine causing change in color of the pH-sensitive dye.

16. Proteins The reagent area of the strip is coated with an indicator and buffered to an acid pH which changes color in the presence of proteins. The principle is known as “ protein error of indicators”.

17. Glucose Based on glucose oxidase-peroxidase reaction. Reagent area of the strips is impregnated with two enzymes (glucose oxidase and peroxidase ) and a chromogen .

18. Blood Lowest detectable concentration – 5 intact RBCs

19. Ketones : Bilirubin : based on reaction of with diazo reagent; color change is proportional to the concentration of bilirubin. Urobilinogen : area is impregnated with either p- dimethylaminobenzaldehyde or 4-methoxybenzene diazonium tetrafluoroborate .

20. If tests results are questionable/ inconsistent with expected findings & clinical history, steps recommended are- 1.Confirm product is within expiry date 2. Retest with fresh sample 3.Check performance against known Negative & Positive control materials 4. Check for False positive & False negative

21. Limitations of dip sticks Differences in daylight 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

22. Automated chemistry analyzers Working principle – reflectance spectrophotometry URI PLUS 1A : 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.

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

24. UriPlus 900 Fully automatic 10 & 11 parameter strips used Based on Reflectance photometery Uses high luminosity 4 wavelength cold light source reflection determination technology.

25. Manufacturers use one of three technologies to perform automated urine microscopic analysis: Digital flow microscopy, Flow cytometry Cuvette -based digital microscopy

26. Principle Machine Digital flow microscopy Iris iQ200 microscopic analyser, URISED Flow cytometry UF-1000i, UF-100 analyser, AUTION HYBRID Cuvette-based digital microscopy 77 Elektronika

27. Iris iQ200 microscopic analyser The machine for Iris Diagnostics USA uses digital imaging and auto particle recognition to classify and quantify urine particles in uncentrifuged urine based on size and shape. Works on the principle of digital flow microscopy .

28. Automatically analyzes and classifies urine particles into 12 categories. The sample is mixed and aspirated to a planar flow cell where 500 digital photomicroscopic images are taken per sample. This system uses Auto particle recognition (APR) software.

29. Flow cytometry – Sysmex UF-1000i Particles labeled with fluorophores / fluorochromes ( Polymethine dye). Flow cytometric analyzers identify and categorize particles in urine based on : forward scatter fluorescence staining characteristics impedance signals adaptive cluster analysis side scatter (specific for bacteria).

30. Identification of particles based on – size, shape, volume and staining characteristics Separate channel for bacterial analysis (improves bacterial detection) Sample volume : 4-5ml (1ml in manual mode) Results ( scattergrams & histograms) – in cells/ microL

31. UF-1000i Particle detection categories Particles enumerated Flagged particles RBCs Non hyaline (pathologic) casts * WBCs Crystals * Epithelial casts Small round cells * Hyaline casts Yeast Bacteria Mucus Sperm *Manual microscopic examination required to specifically identify and categorize

32. Manual microscopic examination to: Classify Non hyaline casts (granular, cellular, RBC, WBC, Crystalline) Identify crystals Identify yeast Categorize particles identified as small round cells as transitional cells, renal cells or another small particle.

33. A. Forward scatter v/s fluorescent light intensity – high sensitivity B. Forward scatter v/s fluorescent light intensity – low sensitivity

34. Results from UF-1000i can be electronically linked to urine chemistry analyzer  Integrated urinalysis report AUTION HYBRID : integrated analyzer (chemistry and microscopy analyzers within single unit) User defined criteria adjusted to reduce review rate of specimens and increase productivity.

35. Cuvette -based Particle analysis Recent innovation Urine specimen centrifuged in a cuvatte multiple images captured by high resolution digital camera coupled to a microscope Sedimax / UriSed & Cobas U 701 are the only cuvette based urine analyzers

36. The UriSed 2 microscopic urine sediment analyzer is a stand-alone instrument which can be connected to the LabUMat 2 urine strip reader. Together, the two instruments make a Complete Urine Laboratory System .

37. Technical specifications of the 3 machines iQ 200 Sprint UF 1000i UriSed Technologies Flow cell digital imaging with automatic particle recognition software Fluorescence flow cytometry with diode laser and hydrodynamic focussing conductometry Microscopic urine sediment analysis, digital imaging, automatic particle recognition. Samples/hour 101 Normal mode – 100; special mode - 80 80 Sample volume 2ml 4ml (1ml in manual mode) 0.2 ml Data storage 10000 pt results with images 10000 samples graphics, 5000 patie nt info, 1000 selective test orders. 50000 sample results and images Size (mm) and weight (kg) 559x610x530 ; 46kg 615x710x580; 75.5kg 600x600x600; 58kg

38. Comparisons between different automation machines for urinalysis SYSMEX UF 1000i Iris iQ 200 UriSed Advantages Improved bacteria counting and flags for UTI Uses uncentrifuged sample Manual operation for urgent testing. uncentrifuged samples. Accurately quantifies bacteria. Results may be viewed from archived images No reagents required Results viewed from digital images. Images same as manual microscopy so minimal retraining needed Disadvantages Pathologic casts not differentiated from benign casts Flagged results require manual microscopy Technical training to view images. Mistakes crystals for RBC and cannot classify dysmorphic RBCs Ultracentrifugation causes particle loss Mistakes yeast for rbc Mistakes EC for WBC and not FDA approved

39. Comparison with Manual SN Variable Manual Automation 1 Bias ++ Nil 2 Standardisation + Absent 3 Precision +/- ++ 4 Reproducability +/- ++ 5 Variance ++ Nil 6 Crystal, cast, microbial subcategorization Excellent Absent 7 Quantitation of RBCs & WBCs Estimate Exact no 8 Time More Less 9 Cost Effective Expensive

40. Summary So a complete urinalysis using macroscopic examination, reagent strips , automation and manual microscopy can be used in conjunction for standard and accurate results. Automation has more benefit in setups with high sample load. Automation helps to screen samples for UTI or kidney disease.

41. REFERENCES Henry’s clinical diagnosis and management by laboratory methods, 24th edition Nancy A. Brunzel - Fundamentals of urine and body fluid analysis, 4 th edition - chapter 16 Shirish M. Kawthalkar -Essentials of clinical pathology

42. THANK YOU