For Lab personnel Tuesday, 10 August 2010 Orientation on New Protein Test for Detection of Pre-eclampsia/eclampsia (PE/E): A Study at Maternity Hospital For Lab personnel Tuesday, 10 August 2010

1. Objectives of this Orientation To explain why this study is important To familiarize lab personnel throughout the hospital with the study testing protocol To explain how the study team can use their help in identifying protein-positive samples for the study (Step One)

2. Orientation Overview Objectives Purpose of the study Introduction to new point of care diagnostics Urine samples Study design Sample collection points Sample transportation Ethical aspect Procedure of the tests

3. Purpose of the Study To test the validity and specificity of this new Point of Care (PoC) test To detect any presence of protein in urine To help early detection and management of pre-eclampsia and eclampsia (PE/E) Second leading direct cause of maternal death in Nepal and other developing countries Currently, women rarely are tested for proteinuria during pregnancy in Nepal

4. Common Urine Protein Tests in Nepal Dipstick Sulphosalicylic acid Acetic acid Esbach test (occasionally)

5. New Point of Care (PoC) Diagnostics for Urine Protein Detection Developed at Johns Hopkins University Biomedical Engineering Department with Jhpiego Color-based qualitative measurement It has the reagent (tetrabromophenol blue) and acid buffer Applied to filter paper. Put urine on it and it identifies changes color if there is protein Low cost—10 times cheaper than dipstick Already proven in US lab setting

6. Type & Amount of Urine Required At least 10–15 mL urine (midstream urine) 10–15 mL urine specimen after catheterization (if in critical condition/or unable to go to lavatory)

7. Study Design Two-step study STUDY STEP SAMPLES TESTING STEP ONE 200 protein positive samples from ANC OPD, emergency lab and parasitology department Hospital dipstick result recorded Additional testing: Dipstick, PoC test, Esbach test STEP TWO 300 urine samples from ANC OPD, all pregnant women

8. Sample Collection STEP One= KEEP/COLLECT IN RED TRAY ANC OPD lab where dipstick is done Emergency laboratory Parasitology lab Wards (MICU, ANC A and ANC B) if any protein positive samples found there STEP Two=KEEP/COLLECT IN BLUE TRAY

9. Step One Sample Collection: Emergency Lab OR On duty Dr or nurse examine Emergency Patient Emergency Lab for urgent need Sample and form Gynecology Ward Inpatient Obstetric Ward provide free recycle bottle or Go to pharmacy and buy bottle or bag for urine Parasitology Lab for normal process Result + ive and -ive urine sample (-) Sample Discard Handover to Sean / Britni (+ ive) Sample Transport to new testing room Remove the label For detail see next Slide Provide sample bottle to PW Record and code the sample Collect + ive samples in red tray

9. Step One Sample Collection: Parasitology Lab collect + ive sample in red tray Emergency Inpatient Obstetric Ward Parasitology lab Sample arrives coded together with form Urine comes here for variety of test i.e, protein, sugar and other microscopic examination Sample Collection Center ANC OPD Gynecology Ward OPD Result + ive urine protein - ve urine sample Fill Result on form and register Give form to patient or family member Discard samples Handover to Sean / Britni (-) Sample (+) Sample Transport to new testing room Lab Tech/Asst tests urine using one of the Record and code the sample

9. Step One Sample Collection: Inpatient Departments Provide large sample bottle to PW (-) Sample Discard Result + ive and -ive urine sample Staff collect and test urine protein ANC A (+ ive) Sample remove the label ANC B Transport to new testing room Record and code the sample MICU Handover to Sean / Britni

9. Step One Sample Collection: ANC OPD Provide 20 mL sample bottle to PW Pregnant women Screen BP, ht, wt Registration provide free recycle bottle Collect positive samples in red tray Urine Test with Dip stick (+) Sample (-) Sample discard PW provide urine for testing Result + ive / – ive (record in ANC card ) Transport to new testing room Handover to Sean / Britni Record and code the sample

9. Step Two Sample Collection: ANC OPD Provide 20 mL sample bottle to PW Pregnant women Screen BP, ht, wt Registration provide free recycle bottle Collect all samples in blue tray Urine Test with Dip stick (+) Sample (-) Sample PW provide urine for testing Transport to new testing room Handover to Sean / Britni Record and code the sample

10. Storage of Samples Study team will collect urine within 12-24 hours Samples can be kept at room temperature

11. Transportation of Samples Lab support staff will collect samples and bring them for testing: Every morning from emergency lab and inpatient department Every hour from ANC OPD lab Close the lid of the bottle well Remove any patient identifiers from samples (lab form and ANC card) Be careful not to spill the samples Deliver samples trays to new testing room

12. Ethical Aspects Must remove any patient identifier before providing to Jhpiego/JHU researchers Study will code samples: S1–D1–1 Study step= S1 or S2 Study day=D1, D2, D3, etc Sample number Keep positive samples in red tray and other sample (unidentified) in blue tray

12. Ethical Aspects Tray labeled according to department and type of sample (e.g., positive sample from ANC OPD lab) Record in register STUDY ID # PLACE OF COLLECTION HOSPITAL RESULT LAB DIPSTICK RESULT POC RESULT ESBACH RESULT

13. Measurement of Urine Protein: Type of Tests used for the Study Dipstick test PoC diagnostics (new test) Esbach test

14. Study Procedure for PoC Collect filter paper, reagent solution, 2 mL urine and 5 mL syringe with needle Use syringe to squirt urine onto filter paper Shake excess urine off filter paper and allow 10–15 seconds for color change to settle Record the result in the register

15. Interpreting PoC Results Test result is interpreted according to color change in the filter paper (special paper used for this test) as below: Negative +1 +2 +3 Yellow Yellow–green Green Green–blue

16. Procedure for Esbach Test Pour urine up to the level mark “U” in the albuminometer Add the esbach reagent up to the mark “R” in the albuminometer Close the albuminometer with stopper Invert albuminometer slowly several times to ensure thorough mixing Keep this at room temperature for 24 hours, then measure the protein precipitate

17. Interpreting Esbach Test Result Measurement is based on the volume occupied by the precipitated protein in the bottom of the albuminometer. It is measured as grams protein per liter of urine.

18. Safety Precautions Hand washing—after handling specimens Gloving—while handling specimens

19. Waste Disposal Discard urine sample after testing Discard sample bottles Dispose all used syringes and needles Place used filter papers in bleach solution for 20 minutes and then discard