CMV-PP65

CMV-PP65 

Introduction: 

Cytomegalovirus (CMV) lower matrix protein (phosphoprotein) pp65 by indirect immunofloresence using microscopy in isolated peripheral blood leukocytes (WBC) obtained from erthyen diamine tetra acetic acid (EDTA) or heparin anti-coagulated human peripheral blood. The detection of CMV pp65 in human peripheral blood cells aids in the diagnosis of acute or reactivated CMV infection. This product is not FDA cleared (approved) for use in testing of blood or plasma donors.

Requirement:  

1. EDTA blood Sample
2. P65 Reagent A, B, C, D, E   
3. Control Slide   
4. Centrifuge Machine
5. Pipette or Micropipette
6. Hemocytometer
7. Cytocentrifuge 
8. Cytofunal
9. Coplin jars 
10. Glass Slide
11. Mounting media
12. Stop watch
13. Cover Slip
14. PBS or PU Solution
15. Universal Container
16. Pasture Pipette
17. Immunofloresence Microscope   

Procedure:    

Step – 1: - Preparation of Sample                              
Collection of Venous Blood Sample 3 – 4 ml in EDTA Tube or Purple topNote: Processing should be performed within 6 – 8 hours of sample collection  

Step – 2Data inter in Computer:  – Patient ID Number – PP65 – RT –   U - Change Date & Time, Write LV No. & Age/ Sex in Requisition Form
 

Step – 3:         Preparation of working solution A, B & C   

1. Solution (A) :  Lazing solution: - Take a universal container and add 1 ml of Solution (A), 9 ml of Cool PU Solution and mix well than put in 2 – 8 ⁰C 
2. Solution (B) : Take a Coplin jars  – add 7 ml of Solution (B) and 27 ml of PBS than mix well
3. Solution (C) : Take a Coplin jars  – add 7 ml of Solution (C) and 27 ml of PBS than mix well

Step – 4         Preparation of Slide

1. Take universal container from step - 3 (A) and add 2 ml of EDTA Blood sample than mix well
2. Incubate at 2 – 8 ⁰C for 10 minutes
3. After that Centrifuge at 2500 RPM for 5 minutes
4. Removed Supernatant Part and wash with PBS 4 – 5 times
5. Put in little amount of PBS and mix well with pasture pipette
6. Take glass slide and write LV No. or insert the slide in cytofunal
7. Transfer 6 drops of mixing amount (WBCs) in cytofunnel
8. Centrifuge in Cytocentrifuge for 5 minutes at 60 RPM       
9. Ari dry slide at room temperature   
10. After air dray Round Circle with diamond pencil   

Step – 5       Fixation and permeabilization

1. Put slide in Solution (B){ from step – 3} and wait for 5 minutes
2. Transfer slide in PBS Solution and wait for 5 minutes
3. Transfer slide in Solution (C) { from step – 3} and wait for 5 minutes
4. Transfer slide in PBS Solution and wait for 5 minutes

Step – 6

1. Wish Outside of Round Circle and put 35 µL Solution (D) and incubate for 20 minutes at 37 ⁰C in a humid chamber
2. Put slide in fresh PBS Solution and wait for 5 minutes
3. Wish Outside of Round Circle and put 35 µL Solution (E-conjugate) and incubate for 20 minutes at 37 ⁰C in a humid chamber
4. Put slide in fresh PBS Solution and wait for 5 minutes
5. Transfer slide in Distilled Water  and wait for 5 minutes
6. Wish Outside of Round Circle and  Mount with Mounting Media and Cover with Cover Slip

Step - 7

1. See the slide Immunofloresence (IFA) Microscope under 40X  object  
2. Result :           

a)      Positive result = Yellow Green

 b)      Negative Result  = No Yellow Green 

Clinical Signification: 

Clinical significance of cytomegalovirus (CMV) antigenemia in the prediction and diagnosis of CMV gastrointestinal disease after allogeneic hematopoietic stem cell transplantation. 

*********************************************************************