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FCXM: Flow Cytometric Crossmatch

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Pr Do

on 24 August 2015

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Transcript of FCXM: Flow Cytometric Crossmatch

Role of Flow Cytometry in Transplantation
Vital tool in transplantation work-up
Grossly underutilized
Significant information
More relevant in Indian scenario

FCXM: Flow Cytometric Crossmatch
CDC: Complement Dependant Cytotoxic Crossmatch
AMR: Antibody Mediated Rejection
PRA: Panel Reactive Antobodies


IgG FlowPRA:
1. Screening
2. Specific
3. Single antigen
C4d FlowPRA

Topics Covered
To identify antibodies against foreign HLA molecules on exposure
10-100x more sensitive than CDC (Gold standard)
CDC is not able to pick up low levels of antibodies that impair graft survival
Quantitative and Objective
However, lacks specificity

Current Protocols in Cytometry 2004; 6.16.1-6.16.2
Why do We do It???


Donor-directed HLA antibodies in the sera of recipient is a risk factor for allograft rejection or loss
Titer of these antibodies is proportional to risk of poor graft outcome
FCXM: More sensitive, but less specific

Incubation of donor cells with recipient serum
Addition of fluorescently labeled anti-human immunoglobulin reagent
Polyclonal antibody specific for Fc portion of IgG
IgM antibodies are better identified using CDC (Gold standard)


Antibodies against Class I HLA antigens bind to both B & T cells
Antibodies against Class II HLA antigens bind to only B cells
Separate fluorochromes are used to identify B and T cells
CD3 for T cells
CD19/ CD20/ CD22 for B cells


Three colour (single laser) Flow Cytometer is sufficient, with 488 nm (blue) laser:
FL1: FITC: for IgG
FL2: PE: for CD19/ CD20/ CD22 (B cells)
FL3: PerCP: for CD3 (T cells)
Results are expressed as difference in median channel value

Flow Cytometer

Positive and negative controls are used with each test
Controls can be:
Pooled sera
In our lab we use commercial controls from BAG and Invitrogen

Donor Sample: Heparin tubes (for mononuclear cells)
Patient sample: Plain vacutainer (for serum)


Mononuclear cell Separation

The separated mononuclear cells are counted using hematology counter for the percentage of mononuclear cells
~80% of the cells are lymphocytes
The cells are transferred to 15ml tubes with media
Mononuclear Cells

The 5 ml tubes are labeled as Negative Control, Positive Control & Patient
50 µl sample containing 2,50,000 cells is transferred to each tube
50 µl sera is added to respective tubes (Negative Control, Positive Control and Patient Serum)
Incubated at 4°C for 30 mins

Washed 3 times
10 µl of IgG FITC, CD3 PerCP and CD22 PE are added to each tube
All tubes are incubated in dark for 20 mins
Wash three times with media
Cells are re-suspended and acquired in Flow Cytometer
Staining contd…

At least 5000 – 15000 lymphocytes are acquired
The lymphocytes are gated on Forward scatter Side scatter analysis
These cells are then analyzed on a second plot showing CD3 and CD22 to segregate B and T cells
The B and T cells are then gated and analyzed on a histogram showing IgG FITC
Cell Acquisition

The median channel value of IgG for B and T cells is measured using the statistics of the respective histogram
The values of positive control is checked to know the validity of the test
The channel shift is measured by subtracting the median channel vale of negative from that of the patient
A positive test is reported if the value is >50 for T cells and >80 for T cells (for our lab)
CLSI Guidelines: Detection of HLA specific alloantibody by Flow Cytometry and Solid Phase Assays; Approved Guidelines
Interpretation of Results
Negative Control
Positive Control
Negative Crossmatch
Positive Crossmatch
Pre-Tx vs Post-Tx

Similarly in our cases, B cell only positivity did not impact the patient outcome on a short term
Significance of only B cell positive cross match ?
Usually results in falsely positive B cell cross match
Imperative to ask for the history from the transplant physician
Pronase pre-treatment helps to rectify this

Hum Immunol. 2004 Aug;65(8):803-9.

Cytotoxicicty using flow cytometry
Amalgamation of NIH-CDC and FCXM
Complement mediated cell death is evaluated by using
a cell viability dye like 7-AAD
More sensitive and objective

Cytometry (Part B) 2006; 70B: 82-90.

Cytotoxic FCXM


Used to identify Panel Reactive Antibodies against HLA using a panel of beads
These beads are microparticles (2-4 µ), that are coated with purified HLA antigen
Consists of 30 HLA Class I and HLA Class II beads
IgG FlowPRA®

Excludes reactions to non-HLA antibodies
Provides a percentage of PRA
Both class I and class II antibodies can be detected in one tube
Highly sensitive and reproducible

Human Immunology 1999; 60: 1293-1302

Advantages of FlowPRA®

The class I and class II beads can be separated out on the basis of positivity of the class II beads in PE channel
The positive and negative sera are part of the kit
Negative sera is used to set-up a cut-off
The shift beyond this cut-off is reported as “Percentage PRA”

Single Antigen
Types of IgG FlowPRA

This assay utilizes beads that are composed of a pool of 30 class I and 30 class II beads. The pool consists of the most of the common HLA antigens.
The class I beads are non-fluorescent, while the class II beads are fluorescent particles that are excited by 488 nm laser and detected in the FL2 channel (akin to PE).
Screening IgG FlowPRA

Negative Control

Positive Control


IgG FlowPRA Beads Class I

IgG FlowPRA Beads Class II

This assay is useful in identifying the most likely type of anti-HLA antibody that lead to positivity in the screening test. It is thus an extension of the screening test.
A panel of 32 class I and class II antigen coated beads are provided in a set of four (4), with each set containing eight (8) beads.
However, the eight bead types in each group can be resolved with varying degree of fluorescence in the FL2 (PE) channel.
Specific IgG FlowPRA

Negative Control

Positive Control


Class I:Specific Beads’ List

Class II:Specific Beads’ List

These beads very much similar to the specific beads, however one bead is coated with only a single antigen (Eg: HLA A*01 only).
Single Antigen IgG FlowPRA

Class I: Single Antigen List

Class II: Single Antigen List

Rare and underutilized test
Distinguish complement activating antibodies from non-activating ones
These antibodies are more detrimental in AMR
Instead of using FITC conjugated IgG, FITC conjugated anti-human C4d antibody
A known serum, with normal C4 levels, is used as a source of complement in the test
C4d FlowPRA
Let’s Flow In India
Dr Pranav Dorwal
Medanta The Medicity, Gurgaon
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