PLASMA TRANSFUSIONS AND CROSSMATCHING BLOOD IN ELEPHANTS

Transcription

1 PLASMA TRANSFUSIONS AND CROSSMATCHING BLOOD IN ELEPHANTS Christine Molter Houston Zoo Maryanne Tocidlowski Houston Zoo Fieke Molenaar ZSL Whipsnade Zoo 11th International EEHV Workshop 17 May 2017

2 CROSS MATCHING IN ELEPHANTS Role of plasma in EEHV treatment Plasma collection and storage Minor and major crossmatching instructions Q&A

3 ROLE OF PLASMA INEEHV TREATMENT EEHV haemorrhagic disease Loss of circulating volume, coagulation factors and platelets, electrolytes Plasma Colloid Coagulation factors Platelets (if not stored frozen) Antibodies

4 PLASMAADMINISTRATION Place IV catheter(s) 1-2 plasma units/treatment event Start slow, then increase rate

5 PLASMA COLLECTION AND STORAGE Materials: Citrate blood collection bags (450 ml) Large bore needles Separation: Centrifuge /Overnight (5 C) Manual / Press Storage: Fresh 5 C 48h Frozen -20 C 2 months -80 C 5 years Collect from donors on a rotating schedule

6 PLASMA COLLECTION AND STORAGE

7 PLASMA COLLECTION AND STORAGE

8 PLASMA COLLECTION AND STORAGE

9 PLASMA COLLECTION AND STORAGE

10 PLASMA COLLECTION AND STORAGE Prior to donation blood should be tested CBC Chemistry PCR

11 CROSSMATCHING Minor Crossmatch -Plasma transfusion Major Crossmatch -Whole blood transfusion -Packedred cell transfusion Recipient Donor

12 CROSS MATCHING STEP BY STEP Materials EDTA (preferred) or serum tube without separator Centrifuge Small clear tubes (3 tubes/animal) Saline 0.9% Pipettes Incubator (35-37 C) Marker pen Paper

13 CROSS MATCHING STEP BY STEP Step 1: Prepare a 3-5% RBC suspension 1. Collect blood from recipient and donor EDTA preferred Serum separator tubes acceptable, but gel plug might form

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15 CROSS MATCHING STEP BY STEP 2. Centrifuge tube and separate plasma from RBCs Save both

16 CROSS MATCHING STEP BY STEP 3. Place 1 drop of recipient RBCs into a small (2-5 ml) clean, labelled test tube

17 CROSS MATCHING STEP BY STEP 4. Add 1-2 ml saline to the tube with RBCs 1 RBC drop:40 saline drops These steps wash antigens from RBC surface

18 CROSS MATCHING STEP BY STEP 5. Centrifuge at 2500 RPM for 20 seconds

19 CROSS MATCHING STEP BY STEP 6. Remove supernatant, leaving the red cell button on the bottom

20 CROSS MATCHING STEP BY STEP 7. Repeat steps 4-6 three times Total of 4 washes

21 CROSS MATCHING STEP BY STEP 8. Add 1 drop of newly washed recipient red cells to a new test tube.

22 CROSS MATCHING STEP BY STEP 9. Add drops of saline and mix to suspend the red cells This should be a ~3-5% red cell suspension

23 CROSS MATCHING STEP BY STEP Check suspension % with capillary tube Centrifuge and measure Adjust saline drops based on measurements

24 CROSS MATCHING STEP BY STEP Step 2: Minor crossmatch 1. Add 1 drop of the recipient s 3-5% red cell suspension to a labeled test tube Add 1 drop of the recipient s 3-5% red cell suspension to another labeled test tube to be used as a control

25 CROSS MATCHING STEP BY STEP 2. Add 2 drops of donor plasma or serum to the test tube

26 CROSS MATCHING STEP BY STEP 3. Add 2 drops of saline to the control tube

27 CROSS MATCHING STEP BY STEP 4. Incubate these tubes at 37 o C for 15 minutes (optional) Enhances reaction

28 CROSS MATCHING STEP BY STEP 5. Centrifuge the tubes at 2500 RPM for 20 seconds

29 CROSS MATCHING STEP BY STEP 6. Observe supernatant for signs of hemolysis If present in the test tube and not the control tube, the match is not compatible If present in both, start again with a new cell suspension

30 CROSS MATCHING STEP BY STEP 7. If no hemolysis, then gently rock the test tube back and forth to re-suspend the cell button

31 CROSS MATCHING STEP BY STEP 7. Observe the cell button while rocking the tube and grade for the presence of agglutination. Grade on a 0-4 scale where 0 is no agglutination and 4 is heavy clumping.

32 CROSS MATCHING STEP BY STEP

33 CROSS MATCHING STEP BY STEP Microagglutination(weak postive)

34 CROSS MATCHING STEP BY STEP Microagglutination(weak postive)

35 CROSS MATCHING STEP BY STEP Microagglutination (strong positive)

36 CROSS MATCHING STEP BY STEP SRouleaux formation (acceptable)

37 CROSS MATCHING STEP BY STEP 8. Record results

38 CROSS MATCHING STEP BY STEP Step 3: Major Crossmatch Same steps as minor crossmatch Use recipient plasma and donor RBC

39 QUESTIONS AND POTENTIAL ANSWERS