1. Chemical Examination of Urine Part III: Ketones, Blood, Bilirubin & Urobilinogen Ricki Otten MT(ASCP)SC uotten@unmc.edu

2. 2 Ketones: Purpose Ketones are intermediary products of fat metabolism

3. 3 Ketones Three ketone bodies –Acetone2% –Acetoacetic acid20% –Beta-hydroxybutyric acid78% Characteristic ‘fruity breath’ ~ acetone

4. 4 Ketones: Normal Normal: negative Abnormal: –Inability to utilize carbohydrates –Excessive loss of carbohydrates –Inadequate intake of carbohydrates

5. 5 Ketones: Methods Reagent strip Acetest: tablet test

6. 6 Ketones: Method Glycine: also measures acetone –Reagent strip: check package insert –Acetest tablets: contain glycine

7. 7 Ketones Reagent strip –Sensitivity: 5-10 mg/dl –Specificity: acetoacetic acid and/or acetone False positive: highly pigmented urine False negative: improper specimen handling Acetest –Specificity: acetoacetic acid and acetone False positive: highly pigmented urien False negative: improper specimen handling

8. 8 Blood: Purpose Blood in urine indicates pathology Two forms found in urine –Intact RBC –Hemolyzed RBC

9. 9 Blood: Terms Hematuria Hemoglobinuria Myoglobinuria All will give a positive blood reaction

10. 10 Blood: Reagent strip Test can detect hemolyzed RBC Heme moiety imparts peroxidase activity and catalyzes the reaction

11. 11 Blood Sensitivity Specificity –Intact RBC –Hemolyzed RBC (hemoglobin) –Myoglobin –False positives: myoglobin, oxidizing agents –False negatives: ascorbic acid

12. 12 Blood: Correlate reagent strip results –Microscopic findings –Color and clarity

13. 13 Bilirubin and Urobilinogen Bilirubin in urine is always pathologic: liver disease Urobilinogen in urine: normal to have a small amount: 0.2 – 1.0 mg/dl

14. 14 Three mechanisms Pre-hepatic: liver is healthy Hepatic: liver disease Post-hepatic: liver is healthy, obstruction indicated

15. 15 Bilirubin: Methods Reagent strip Ictotest: tablet test Foam test

16. 16 Bilirubin: Methods Reagent strip Ictotest: tablet test Same reaction Same specificity: conjugated bilirubin –False positive: urine color –False negative: low concentration, ascorbic acid, improper specimen handling

17. 17 Bilirubin: Methods Reagent strip Ictotest: tablet test Sensitivity differs Reagent strip: ~0.5 mg/dl Ictotest: 0.05 – 0.1 mg/dl

18. 18 Bilirubin: Methods Possible to have a negative reagent strip test and positive ictotest –Difference in sensitivity levels Always perform Ictotest when –Urine bilirubin test specifically ordered –Urine appearance is amber: even if bilirubin reagent strip test is negative –Positive reagent strip test

19. 19 Bilirubin: Foam Test Shake urine and observe resulting foam Yellow foam = bilirubin

20. 20 Urobilinogen: Methods Reagent strip test –Two reactions dependent upon manufacturer Para-dimethylaminobenzaldehyde Diazonium salt –Cannot determine absence of UBG Watson-Schwartz assay

21. 21 Urobilinogen: Methods Para-dimethylaminobenzaldehyde –Sensitivity: 0.2 mg/dl –Specificity: False positive: any ‘Ehrlich reactive compound’; color masking; urine at body temp False negative: improper specimen handling Diazonium salt –Sensitivity: 0.4 mg/dl –Specificity: reacts only with UBG False positive: color masking False negative: improper specimen handling

22. 22 Urobilinogen: Watson Schwartz Classic method used to differentiate urobilinogen from porphobilinogen using a differential extraction method Para-dimethylaminobenzaldehyde