Flow Cytometry

1. Flowcytometry & Transplantation

2. Every rose has it’s thorn !

3. And if u want to enjoy the roses u need to learn to
handle the thorns !

4. •Detection
•Identification
•Role in transplants
HLAAntibodies

5. Transplant Management
 PreTransplant Monitoring
 PostTransplant Monitoring

6. Pre Transplant Monitoring

7. Challenges ??
Monitoring of Sensitized PatientsAwaitingTransplantation
• Circulating antibodies must be periodically monitored in patients awaiting
transplantation
• Variable response to desensitization therapies
• Antibodies can rebound following completion of a course of treatment
• Further sensitizing events may take place

8. Sensitised Patient ??
A sentitised patient is one who is having HLA antibodies circulating
in the body.
HLA antibodies are not naturally occurring but arise from the
following :
PREGNANCY
TRANSFUSION
PREVIOUS TRANSPLANTATION

9. Post Transplant Monitoring
 Pre-transplant desensitization may reduce the alloantibody burden sufficiently to
allow transplantation to proceed with a negative cytotoxic crossmatch
 Concern for a post-transplant rebound in antibody levels and subsequent risk of
delayed acute rejection

10. Goals of Flowcytometric Assays in
Transplantation
• Identify Presence Of HLA antibodies
• Determine the titre of Antibodies
• Identify the DSA (Donor Specific Antibodies)
• Planning appropriate transfusion support strategies
• Elucidates the specificities (i.e. HLA antigens) to which they react

11. Cell Based
FlowcytometricAssays
Very Sensitive
Lacks Specificity
Uses Cell as base element
to study
E.g. Flowcytometric
Crossmatch
Solid phase
FlowcytometricAssays
Uses Microparticles coated with
purified HLA molecules
Very Specific/ PreciseAssay
Widely Used
E.g. Flow PRA

12. Role of Flowcytometry
Elementary flow cytometric investigations have been employed to;
 Quantify cell numbers eg.Absolute CD3+T cells counts
 Population Ratios eg. CD4+:CD8+ ratios
 Activation status of different cellular subsets eg. Expression of CD69/HLADR on CD3+ or
CD8+T cells

13. Role of Flowcytometry
 Elevated frequencies of CD4+Tregs in patients post transplant have been reliably
correlated with reduced risk of graft rejection
 Other regulatoryT cells subsets have been identified by flowcytometry like
CD8+/CD28 –
 Less is known about these regulatory cells role in inducing tolerance

14. Advantages Of Flowcytometry In Organ
Transplantation
 Enhanced sensitivity offered by indirect fluorescence effectively compensates for low titre
antibodies
 Use of subset specific monoclonal antibodies permits the electronic dissection of cell
subpopulations for the identification of both class I and class II antibodies
 Ability of flow cytometer to detect antibody independently of complement fixation
 Wider assay of clinically relevant immunoglobulins
 Improved specificity of assay