1. A 54-year-old potential live donor for his cousin who is 46-year-old. The result of is shown below.
- Please comment on the crossmatch result
- Will you proceed for the direct transplantation?
- If yes, what is your immunosuppression protocol?
- If no, what are the alternatives?
- What is meant by channel shift and how it influences transplantation?
Dear All
I’m impressed that you are starting thinking of the paired exchange now in spite it is not being widely practised in your countries. Yes, this is the way forward. In the UK, this programme is very successful to the extent we involve pairs with negative crossmatch to get better HLA and age matches.
Dear All
Your definition of the channel shift is not clear. You have sunk into the technique. Can you define the channel shift in a very simple term?
in a FCXM, median channel fluorescence (MCF) shift or simply channel shift (CS) is calculated by subtracting the MCF of negative control serum from the MCF of recipient serum. In the absence of a standardized method to develop CS cutoffs to classify a T-cell-positive or B-cell-positive FCXM, the cutoffs are determined by individual laboratories by evaluating a negative control serum tested against lymphocytes from 30 healthy individuals or more, resulting in a derived mean and standard deviation (SD) of the MCF.
these results from the negative individuals are then normally distributed and the labs choose at which SD will be the cutoff for channel shift.
When data are not normally distributed, highly skewed data can result in a high SD, potentially resulting in a false-negative FCXM.
So, it is practically a mechanism of determining the laboratory cut off to determine patient’s positivity or negativity for B cells or T cells on flow cytometry
Reference:
Dorwal P, Bansal SB, Chauhan R, et al. Median channel shift less than the cutoff in flow cytometric crossmatch: Not to be ignored! Asian J Transfus Sci. 2017;11(1):73-75.
Good
In flow-cytometry crossmatch, median channel fluorescence (MCF) shift is calculated by subtracting the MCF of negative control serum from the MCF of recipient serum. usually no standard form that and individual laboratories determine that by performing controls from healthy cases.
Determining Channel Shift Cutoff in T-Cell and B-Cell Flow Cytometry CrossmatchPrabhakar Putheti 1, Vijay K Sharma
Affiliations expand
intensity of fluorescence above controll, referred to as channel shifts.
Excellent
Basically the degree of the positivity. Well done
the intensity of fluorescence above the control
flow cytometry result are reported as positive or negative based upon the median channel shift. which is caused by the binding of specific antibody ,they used fluorescein -labelled goat antihuman antibody is used as reporter fluorescent dye to detect the binding of this alloantibody.
The number of channel shift positive or negative varies among laboratories and have not been standardized.
MerrillJ. PMurrayJ. EHarrisonJ. HGuildW. RSuccessful homotransplantation of the human kidney between identical twinsJAMA 19561604277282PMID: 13278189.
Extent of fluorescence above control that reveals risk of rejection. Positivity indicates incompatibility.
Please comment on the crossmatch result
positive FCXM for both T and B cell
CXM incompatible
Positive historical PRA
DSA positive with high MFI
highly sensitized patient with a high level of DSA
Will you proceed for the direct transplantation?
No, as there is a high risk of acute rejection in this case.
If yes, what is your immunosuppression protocol?
he will need desensitization with plasmapheresis with IVIG and rituximab.
If no, what are the alternatives?
better to direct him to paired kidney exchange. or wait for another suitable donor
What is meant by channel shift and how it influence transplantation?
it indicates the presence of antibodies ((DSA), when positive, it means that the degree of the fluorescence intensity is above the control level, so the DSA is clinically significant. although there is no global standardization for the degree of positivity
induction will be by r ATG and maintenance by triple immunosuppression that includes steroid plus tacrolimus plus cellcept
1-Its FCXM positive for Tcell and B cell while ,previous FCXM positive for B cell only
CXM is incompatible , positive PRA
MFI IS 37792 (DSA) so, consider patient highly sensitized
2- No I wont proceed as this patient is sensitized and +ve cross FCXM and he has high risk rejection.
3-Desnesitazation by plasmapheresis ,IVIG and rituximab
induction by rATG ; followed by triple lassical maintenance immunosuppression therapy : steroids ,MMF , TAC .
3- PKD with suitable donor has -ve cross match.
4-flow cytometry result are reported as positive or negative based upon the median channel shift. so; The channel shift : the intensity of fluorescence above the control, degree of positivity.
This a positive flow CXM for both TCells and B cells this consider contraindication for transplant because there’s high risk of AMR.
I will not proceed for direct transplant and the preferable option is paired exchange.
If l had no choice I will did desensitization by plasmapheresis and rituxmab and induction by ATG with triple immune suppression therapy of Tac,MMF and predinsilone as a maintenance
1)Please comment on the crossmatch result.
Flow cytometry positive for B and T cell.
High DSA with MFI 37792
Highly sensitize transplant recipient.
2)Will you proceed for the direct transplantation?
No in my practice.
3)If yes, what is your immunosuppression protocol?
Intensive desensitization with cycle of plasmapheresis and IV IG.
Induction : ATG
Maintenance : Tacrolimus base maintenance protocol.
4)If no, what are the alternatives?
Enroll in Pair kidney donor exchange system.
HLA / ABO compatible living donor.
5)What is meant by channel shift and how it influences transplantation?
The method to determine the intensity of B or T cell in flow cytometry. It is obtained by subtracting the median channel fluorescence of negative control serum from the median channel fluorescence of recipient serum.
positive FCXM results for both B and T cells.
High DSA level
Infer high risk of kidney rejection.
No, the risk of kidney rejection is high as infered by very high DSA levels
ATG for induction,
MMF+CNI+ steroid for maintenance
paired exchange renal transplantation
Please comment on the cross match result
o This is a flow cytometry cross match between a recipient and a potential donor.
o Current results :Positive T and B cell FCXM with high MCS>250 together with high DSA against HLA class 1(A24,B8 and C7).
o Historic results: positive for B cells (can express class 1 and 2) and negative for T cells (can be falsely negative or development
Will you proceed for the direct transplantation?
o No ,a positive T cell FCXM is a contraindication for transplantation without desensitization to achieve a negative cross match. In order to proceed for desensitization we need to check relative intensity score and MCS strength.
o if MCS>250 and RIS > 22 avoid transplantation
o MCS<250 and RIS <17 proceed for desensitization protocols
If yes, what is your immunosuppression protocol?
o Desensitization with PE, IVIG +/-Rituximab(low success rate in this case)
o Induction therapy with a depleting agent like ATG +pulse methylprednisolone
o Maintenance immunosuppression: triple therapy with TAC( maintain high trough level), MMF, steroids.
o Monitor DSAs frequently at 2,4 8 weeks then every 3 months
o Protocol biopsies
If no, what are the alternatives?
o Paired donor exchange program
o Look for a more suitable living donor
What is meant by channel shift and how it influences transplantation?
o It is the intensity of the fluorescence above the control
o It indicates the degree of the positivity of cross match
o It can influence the decision for desensitization
References:
1)Priscila de MoraesacIara, Fagun de sa, Jacqueline Moraes, et al. Accuracy of the median channel shift in the flow cytometry for predicting complement dependent cytotoxicity crossmatching in kidney transplant candidates. Transplant Immunology. 2019 (52), P 27-31.
2) Keith DS, Vranic GM. Approach to the highly sensitized kidney transplant candidate. Clin J Am Soc Nephrol. 2016;11:684–693
Cross match result
Proceed for direct transplantation or not
I will not proceed for transplant with this donor. Will check for alternatives such as kidney paired donation. However, if there is no other available donor due to donor scarcity, then I would proceed for transplant after desensitization protocols.
Immunosuppression protocol
Alternatives
Kidney paired exchange scheme
Channel shift
Channel shift can be interpreted in the base as the extent of positivity above control. It can help to ascertain the risk of rejection in the given recipient using flow cytometry. The intensity of fluorescence is key to diagnosis of risk. The more positivity, the higher the risk of rejection post transplant or even on the table.
This patient is highly sensitized to this donor, he has a positive cross-match with positive B cell channel shift, & significant DSA high titers against this donor , the transplant will have a high risk of developing AMR , so it is not a suitable donor.
We are not proceeding with this transplant .
The alternatives are the followings:
paired exchange program
Search for another donor
cadaveric list .
Channel shift is the intensity of the fluorescence in FXCM above the control, it predicts the outcome of the transplant if positive, high risk for rejection.
High risk transplantation offer, incompatible cross match, high DSA against HLA A, B, C7.
I will not proceed for this transplant offer because it is high risk
If yes; the recipient needs to have Desensitization by Plasma exchange, IVIG till a negative crossmatch is achieved. Then immunosuppression should be formulated for high risk group:
Induction: ATG or Alemtuzumab.
Maintenance: Tacrolimus+ MMF+ prednisolone.
Regular monitoring of DSA and renal function tests.
If no–à wait for another donor, or paired exchange program
Channel shift predicts the positivity of CDC.
There is positive flow crossmatch of the current serum for both B and T cells, and the previous serum positive for B cell only. So incompatible crossmatch.
No .There is high risk of rejection.
Alternative is paired kidney donation.
If to proceed: desensitization with rituximab, plasma pheresis and IVIG.
Induction with ATG and maintenance with Tac based immunsupression.
1-pateint current positive FCXM positive for Tcell and B cell .
previous FCXM positive for B cell and negative for T cell
CXM result is incompatible and also had positive PRA on 11/21.
MFI IS 37792 (DSA) for HLA a24, B5 and C7 is very high
2- No , i will not proceed as this patient is sensitized and positive cross matching and he has high risk for acute or hyperacute rejection.
3-Desnesitazation by plasmapheresis ,IVIG and rituximab
induction by rATG or Alemtuzumab
followed by triple maintenance immunosuppression therapy
MMF1gm po bid
TAC trough level 8 to 10 ng/l
prednisolone after pluse 500 mg for 3 days tapering to 5 mg over 8 weeks.
3- alternative is PKD
4-flow cytometry result are reported as positive or negative based upon the median channel shift. which is caused by the binding of specific antibody ,they used fluorescein -labelled goat antihuman antibody is used as reporter fluorescent dye to detect the binding of this alloantibody.
The number of channel shift positive or negative varies among laboratories and have not been standardized.
MerrillJ. PMurrayJ. EHarrisonJ. HGuildW. RSuccessful homotransplantation of the human kidney between identical twinsJAMA 19561604277282PMID: 13278189.
Current serum shows positive B and T cell FCXM, DSA against A24 (22926), B8 (2931), C7 (11935)
Previous sample showed positive B cell and negative T cell FCXM
No
Desensitization with plasmapheresis, IVIG and Rituximab
crossmatch should be repeated if negative or MCS <250 can proceed with transplantation and considered as high immunological risk
Induction with ATG and maintenance with triple therapy, Tacrolimus, MMF and steroids.
avoid any reduction of maintenance immunosuppression and ensure patient adherence (4)
close monitoring of DSA post transplant
Paired exchange program
the intensity of fluorescence above control; the degree of positivity and it can predict the outcome of transplant; hence, to proceed or no for highly sensitized patient
What is meant by channel shift and how it influences transplantation?
– In a FCXM, median channel fluorescence (MCF) shift or simply channel shift (CS) is calculated by subtracting the MCF of negative control serum from the MCF of recipient serum. In the absence of a standardized method to develop CS cutoffs to classify a T-cell-positive or B-cell-positive FCXM, the cutoffs are determined by individual laboratories.
– Desensitisation is considered successful if patient has an acceptable crossmatch which is defined at our Center as a negative CDC crossmatch in equal or more than 1:2 dilution in patient sera , a negative Flowcytometry crossmatch or a positive Flowcytometry crossmatch with MCS equal or less than 250 .
If no, what are the alternatives?
– Paired kidney exchange programme.
Or
– Wait for more compatible donor.
If yes, what is your immunosuppression protocol?
– Desensitisation by plasmapheresis, low dose of IVIG and rituximab .
– Induction by ATG or Aleumtzumab.
ATG dose : 1 -1.5 mg/kg per dose for 4-6 days (total dose 6 mg/kg ) .
alemtuzumab : usually given as single dose of 30 mg intra-operatively and second dose is sometimes given .
– Triple immunosuppression:
Tacrolimus: with achieve trough level ( 8-10 ng/ml)
MMF : 1 g /12h.
Prednisolone : 500 mg IV on day of surgery , followed by tab 1 mg/kg for 3 days
then 20 mg/ day
to be tapered to 5 mg/kg over next 6-8 weeks.
POST TRANSPLANT FOLLOW UP:
– CBC , Urine examination and kidney functions.
– follow up proteinuria by 24 hour urinary protein.
– monitor DSA level for >>> 2 weeks.
>>1,3,12 month.
– Protocol biopsy once every three month to detect subclinical AMR.
– If there’s any pictures of AMR >>> should be treated.
Will you proceed for the direct transplantation?
No , unless good desensitisation done for him until CM changed to negative.
OR
* pair exchange if possible.
* Change donor
Please comment on the crossmatch result :
– Flowcytometry positive for B and T cells.
– Patient has antibodies against class I and class II.
– CXM incompatible.
– PRA positive.
– DSA against A24= 22926 , B8= 2931 and C7=11935.
– So patient has DSA against class I only with high risk for hyper-acute rejection.
The cross match is positive by FCXM cross match; T cell cross match and B cell cross match are positive with significant levels of MCS >250 in both. However historically the FCXM had only B cell MCS being significant and T cell FCXM being negative.
The patient also has positive PRA in the past signifying highly sensitized renal transplant recipient.
Proceeding for renal transplant is a contraindication as the patient has significant MCS >250 and positive T cell and B cell cross match. Patient needs to be desensitized before transplantation
Desensitization needs to be carried out with IV rituximab 375mg/m2 minimum 2 doses with alternate day plasma exchange 2.5 L plasma replacement with AB+ plasma and 5 doses of IVIG 20gm. FXCM MCS needs to be repeated. If the MCS<250, we need to do SAB DSA to determine the strength of the DSA. In general if the MFI DSA is less than 5000 Desensitization can be attempted. Relative Intensity score (RIS) is calculated by giving points to DSA based on their strengths.. no DSA – 0 points..Weak DSA (0-5000) 2 points..moderate DSA (5000-9999).5 points..strong DSA (>10000) 10 points. The sum of the scores gives the Relative Intensity score (RIS). RIS>17 is contraindication for desensitization. Those patients with RIS<17, they can be taken up for desensitization with the above protocol.
If the patient has very levels of DSA MFI or MCS, desensitization is not carried out and the patient has to wait for a long time till the MFI in repeat DSA are negative. The patient can get listed in the paired kidney exchange program.
The results from FCXM are expressed as median channel shift (MCS), the relative median fluorescence of a particular sample is calculated by dividing the median fluorescence of that sample by the median fluorescence of the negative control. The relative median fluorescence is then compared to a predetermined cut-off value. The MCS > 250 implies high chance of AMR if transplanted. They need to be desensitized depending on the DSA MFI strengths on single antigen bead
Crossmatch result shows positive flow cytometry crossmatch to B & T cells. This patient has DSA against HLA- A24, B8 and C7 with high MFI.
Will you proceed for the direct transplantation?
There is very high risk for hyper acute rejection with positive T and B cell cross match. So ,we can proceed for either desensitization or paired exchange program.
immunosuppression protocol if proceeding for transplantation:
Desensitization (Plasmapheresis + IVIG + rituximab).
*Induction therapy (ATG + pule steroids)
*Maintenance therapy (Tac, MMF, Steroid)
What is meant by channel shift and how it influence transplantation?
It is the intensity of the fluorescence exceeding the control , and it indicates degree of the positivity of crossmatch and the influence influence the decision for desensitization. Calculation of the median channel shift (MCS) of median fluorescence intensity (MFI) of the test serum is done in relation to negative control .Some labs use the 10% as a cutoff by considering the fluorescence index (FI), which is the percentage shift in the test serum as compared to the positive control ;test serum MFI − negative control MFI/[positive control MFI − negative control MFI] × 100.
FCXM current serum positive for T and B- cells. with channel shift
FCXM previous serum negative for T cells but positive for B cells
CXM incompatible
Positive DSA class 1 for HLA (A24, B8, C7) with high MFI ,so this patient consider as high immunological risk for renal transplantion.
* will not proceed with Tx
As This pt is highly sensitized with positive T-cell CXM
☆immunosuppression protocol :
Desensitization (Plasmapheresis + IVIG + rituximab). When CXM became negative:
*Induction therapy (ATG + pluse steroids)
*Maintenace therapy (Tacrolimus, MMF, Steroid)
*It is important to follow this pt by regular DSA level.
Alternative
Kidney paired donation program
☆What is meant by channel shift :
*The channel shift is a fluorescent value. The median channel fluorescence (MCF) shift is calculated by subtracting the MCF of negative control serum from the MCF of recipient serum
* It can predict the CDC assay results and is used to help decide if the renal transplant recipient can be desensitized.
Here we have:
– Flow previus with a technique error because T was postive and B negative.
– Later, in Flow current serum this was overcome and So, with both positive we have DSAs to HLA class I or mixture of DSAs to class HLA I and II
Both have low MFIs. (cut point 5000)
– CXM : incompatible. Confirming the flowmetry result.
– Positive PRA – means that the panel is at risk for rejection, although the percentage of incompatibility was not quantified.
Identified high DSA MFI
No. I´m not. There is a high risk of hyperacute ande acute rejection due to the presence of DSA.
I would opt for prior desensitization
Channel shift is a type of analysis (similar to MFI), where the ratio of fluorescence intensity of antibodies present in incubations between donor lymphocytes with negative and positive controls is calculated. It is used to assess the risk in terms of non-detection of low-titer anti-HLA antibodies.
Please comment on the crossmatch result
Will you proceed for the direct transplantation?
No, because there is very high risk for hyperacute rejection with positive T and B cell cross match.
If no, what are the alternatives?
– Desensitization.
– Paired exchange program.
What is meant by channel shift and how it influences transplantation?
– FCXM is a test that detects both complement and non-complement-fixing antibodies and has advantages of increased sensitivity, automation, rapid results and lower cost.
– Median channel fluorescence (MCF) is calculated by subtracting the MCF of negative control serum from the MCF of recipient serum.
☆Comment on the crossmatch result
▪︎ FCXM current serum positive for T and B- cells. with channel shift
▪︎FCXM previous serum negative for T cells but positive for B cells
▪︎ CXM incompatible
▪︎Positive DSA class 1 for HLA (A24, B8, C7) with high MFI ,so this patient consider as high immunological risk for renal transplantion.
☆Will you proceed for the direct transplantation?
___________________________
NO,
This pt is highly sensitized with positive T-cell CXM
☆If yes, what is your immunosuppression protocol?
____________
▪︎Desensitization (Plasmapheresis & IVIG+ rituximab). When CXM became negative:
▪︎Induction therapy (ATG + methylpred)
▪︎Maintenace therapy (Tacrolimus, MMF, Steroid)
▪︎ It is important to follow this pt by regular DSA level.
☆ If no, what are the alternatives?
______________________________
Enrolled in Kidney paired donation program
☆What is meant by channel shift and how it influences transplantation?
______________________________
▪︎The channel shift is a fluorescent value. The median channel fluorescence (MCF) shift is calculated by subtracting the MCF of negative control serum from the MCF of recipient serum
▪︎ It can predict the CDC assay results and is used to help decide if the renal transplant recipient can be desensitized.
Please comment on the crossmatch result
Will you proceed for the direct transplantation?
If no, what are the alternatives?
What is meant by channel shift and how it influence transplantation?
comment on cross matching result?
postive crosmatching for both T&B cells
DSA graft HLA class1 postive
FCXM postive
past HX of high PRA
SO the patient , senstiized
will you preceed for transplantion?
no unless pateint desenstaization .
immunosupression protocol?
plasmopharsis 6 session low or high dose followed by IVIG
Rituxmab.
Induction ATG.
follow up by DSA and protocol biopsy.
alternative?
either wating till have other match donor or paired exchange.
meant of channel shifit?
IN FCXM some result shift less than or equal to negative control that means lies on grey area and immunologist call them negative and further evaluation revealed weak DSA by luminex SAB in some of the cases.so it influence transplant that the risk still existe/
# Please comment on the crossmatch result
FCXM previous serum negative for T- cell , but positive for B- cell
FCXM current serum positive for both T,B cells (incompatible CXM)
Positive DSA class 1 for HLA(A24, B8, C7) with high MFI ,so this patient consider as high immunological risk for renal transplantion.
# Will you proceed for the direct transplantation?
NO, becuse according to our national protocol current positive T-cell CXM is contraindication for transplantion.
# If yes, what is your immunosuppression protocol?
– Desensitization with Plasmapheresis & IVIG
,when CXM become negative induction therapy ATG & methylprednisolone
Maintenace therapy (Tacrolimus, MMF, Steroid)
Regular followup for DSA
# If no, what are the alternatives?
Kidney paired donation from living donors.
# What is meant by channel shift and how it influences transplantation?
– Indicate the degree of positivity of cross match
– Influence the decision for desensitization.
Please comment on the crossmatch result
FCXM positive for T cell and B cell
Positive crossmatching
History of positive PRA
DSA against class I HLA
Highly sensitized patient
Will you proceed for the direct transplantation?
No I will not unless good desensitization done for him.
If yes, what is your immunosuppression protocol?
Desensitization by plasmapheresis,IVIG and Retuximab
Induction by ATG
Maintenance by triple IS
Follow up DSA and by protocol biopsy
If no, what are the alternatives?
Paired kidney exchange or just wait for more compatable donor.
What is meant by channel shift and how it influences transplantation?
It mean the intensity of fluorescence above controll, and it indicate the degree of positivity of the crossmatch.
Q1. The current cross match report shows: +ve Cross match for both B and T-cells, mostly due to anti HLA- class I.
Q2. Will you proceed for the direct transplantation?
Q3. In case of transplantation: we need all the following:
ü desensitization protocol as PEX + IVIG or Rituximab.
ü Induction therapy with ATG.
ü Triple maintenance immunotherapy (tacrolimus, MMF and prednisolone).
Q4. Alternatives to desensitization?
Q5. What is meant by channel shift and how it influences transplantation?
Please comment on the crossmatch result
FCXM positive for B and T cells
, so the patient is highly sensitized with a high risk for acute AMR
Will you proceed for the direct transplantation?
No. I will not proceed positive FCXM ,high risk increase risk of rejection .
If yes, what is your immunosuppression protocol?
Desensitization : Plasma Exchange + rituximab + IVIg
Induction with ATG
Manutention with Tacrolimus + MMF + Prednisone
with follow up of proteinuria , DSA , protocol biopsy
If no, what are the alternatives?
Kidney paired donation (from living donors)
What is meant by channel shift and how it influences transplantation?
It is a measure of immunological incompatibility; higher the value, higher the risk.
q1. cross match of MFI between donor and recipient are positive for T & B cell.
Previous sample positive for B cell igG
incompatible by flow cytometry
positive PRA and positive DSA
it’s positive cross match; so donor not suitable for recipient transplant because high risk of AMR.
q2. No i will not proceed transplant.
better avoid transplant by Other donor compatible
If there’s no option patient undergoing desensitisation protocol or
deceased allocation system or Paired donor exchange program
q4.
Flow cytometry is sensitive, quantitative and objective but not specific
Any shift in flow cytometry mean indicated further evaluation to detect low titer of HLA Ab. it’s may indicates positive antibodies and chance of desensitisation protocol.
Median channel fluorescence shift is calculated by subtracting mean channel shift of negative control serum from mean channel shift of recipient serum.
References:
Pranav Dorwal et al; Median channel shift less than the cut off in flow cytometric cross match: not to be ignored! Asian journal of transplant Science 2017 jan-july 11(1):73-75.
Please comment on the crossmatch result
It showed positive B and T cell in current cross match ,negative T cell and positive B cell in previous test done by FCXM
Will you proceed for the direct transplantation?
I will not proceed positive FCXM ,high risk increase risk of rejection .
If yes, what is your immunosuppression protocol?
I can proceed with desensitization protocol with Plasma Exchange + rituximab + IVIg
Induction with ATG
maintenance with Tacrolimus + MMF + Prednisone
If no, what are the alternatives?
To look for anther donor or pair exchange .
What is meant by channel shift and how it influences transplantation? In a FCXM, median channel fluorescence (MCF) shift or simply channel shift (CS) is calculated by subtracting the MCF of negative control serum from the MCF of recipient serum.Its highly sensitive may lead to positive results affect transplant plan .
·
Dear All
The Channel Shift also indicate the degree of positivity of the crossmatch. Also may influence the decision for desensitization.
Current serum shows positive B and T cell FCXM, DSA against A24 (22926), B8 (2931), C7 (11935)
Previous sample showed positive B cell and negative T cell FCXM
No, as it has high immunological risk, MCS >250 and relative intensity score (RIS) is 22 so this donor better to be excluded (The RIS give 0 points for no specific antibody, 2 points for DSA with MFI <5000, 5 points for DSA with MFI between 5000 and 10,000, and 10 points for DSA with MFI >10,000) (1)
DSA relative intensity score >17 was shown to have high predictive value for AMR. (2)
HLA incompatible transplants with desensitization have high rate of AMR up to 50% (3)
It will need desensitization to proceed to transplant which is expensive and carries high risk of morbidity due to potent immunosuppression
Desensitization with plasmapheresis, IVIG and Rituximab
crossmatch should be repeated if negative or MCS <250 can proceed with transplantation and considered as high immunological risk
Induction with ATG and maintenance with triple therapy, Tacrolimus, MMF and steroids.
avoid any reduction of maintenance immunosuppression and ensure patient adherence (4)
close monitoring of DSA post transplant
Paired exchange program, it is considered the best option for patients with HLA incompatible live donors to avoid the cost and risk associated with desensitization therapy. (5)
Results from FCXM are expressed as median channel shift, the relative median fluorescence of a particular sample is calculated by dividing the median fluorescence
of that sample by the median fluorescence of the negative control. The relative median fluorescence is then compared to a predetermined cut-off value. (6)
Anti-HLA DSA strength is commonly assessed by MCS
(1) Sinangil A, Ucar ZA, Koc Y, Barlas S, Abouzahir S, Ecder ST, Akin EB. Outcome of Desensitization Therapy in Immunologically High-Risk Kidney Transplantation: Single-Center Experience. In Transplantation proceedings 2019 Sep 1 (Vol. 51, No. 7, pp. 2268-2273). Elsevier.
(2) Vo AA, Sinha A, Haas M, et al. Factors predicting risk for antibody mediated rejection and graft loss in highly human leukocyte antigen
sensitized patients transplanted after desensitization. Transplantation.
2015;99:1423–1430.
(3) Keith DS, Vranic GM. Approach to the highly sensitized kidney transplant candidate. Clin J Am Soc Nephrol. 2016;11:684–693
(4) Garces JC, Giusti S, Staffeld-Coit C, Bohorquez H, Cohen AJ, Loss GE. Antibody-mediated rejection: a review. Ochsner Journal. 2017 Mar 20;17(1):46-55.
(5) Cooper JE. Desensitization in Kidney Transplant: A Risky (but Necessary?) Endeavor for Those With Limited Options. Transplantation. 2019 Dec 1;103(12):2460-1.
(6) Callus R, Buttigieg J, Anastasi AA, Halawa A. Basic concepts in kidney transplant immunology. British Journal of Hospital Medicine. 2017 Jan 2;78(1):32-7.
Thanks Heba
Please comment on the crossmatch result
FCXM positive for B and T cells
This patient is highly sensitized with a high risk for acute AMR
Will you proceed for the direct transplantation?
No. In our service, we do not carry out desensitization protocols. Unfortunately, wait longer in search of a more viable body with fewer CREGs involved.
If yes, what is your immunosuppression protocol?
Desensitization with Plasma Exchange + rituximab + IVIg
Induction with ATG
Manutention with Tacrolimus + MMF + Prednisone
If no, what are the alternatives?
Kidney paired donation (from living donors)
What is meant by channel shift and how it influences transplantation?
In, FCXM, median channel fluorescence (MCF) shift or simply channel shift (CS) is calculated by subtracting the MCF of negative control serum from the MCF of recipient serum. It prevents false-negative FCXM results, avoiding hyperacute rejection.
Thanks Filipe
Please comment on the crossmatch result
Will you proceed for the direct transplantation?
If yes, what is your immunosuppression protocol?
If no, what are the alternatives?
What is meant by channel shift and how it influences transplantation?
Well done
This reflects a better understanding
●comment on the crossmatch result:
Results for current serum:
Flow cytometry is positive for B and T .
So he has antibodies against class-I and class-II HLA antiges
CXM is incompatible .
Previous FCXM was postive only for B cells with High DSA ppp MFI 37792.
DSA are Specific for A24, B6, and C7 so he had antibodies to class 1 HLA antigens only.
This patient is highly sensitized with high risk for acute AMR .
●Will you proceed for the direct transplantation?
no i cant go for direct transplantation as there is great risk for hyperacute and acute rejection.
I wil recommend paired kidney exchange if possible,or the second option is to go for desensitization until the positive cross-match become negative.
●If yes, what is your immunosuppression protocol?
First desensitization by Plasmapheresis, low dose IVIg and rituximab .
As this recipient is high risk patient i will give him ATG for induction followed by triple immunosuppression with Tacrolimus, MMF and steroid
Frequent monitor od DSA at 2 werks then 1,3,and 12 months post transplantation ,protocol biopsies also will detect subclinical AMR.
●If no, what are the alternatives?
I will recommend paired kidney exchange programme
●What is meant by channel shift and how it influences transplantation?
Flow cytometric crossmatch (FCXM) is a sensitive, quantitative, and objective, yet cost-effective method for detection of donor-specific antibodies. Although it lacks the specificity that would be offered by a Luminex single antigen bead (SAB) assay, it is a useful screening method.
In a FCXM, median channel fluorescence (MCF) shift or simply channel shift (CS) is calculated by subtracting the MCF of negative control serum from the MCF of recipient serum. In the absence of a standardized method to develop CS cutoffs to classify a T-cell-positive or B-cell-positive FCXM, the cutoffs are determined by individual laboratories by evaluating a negative control serum tested against lymphocytes from 30 healthy individuals or more, resulting in a derived mean and standard deviation (SD) of the MCF.
these results from the negative individuals are then normally distributed and the labs choose at which SD will be the cutoff for channel shift.
When data are normally distributed, 68.3%, 95.4%, and 99.7% of the distribution falls within 1SD, 2SD, and 3SD of the mean.Laboratories choose 2SD or 3SD as the CS cutoff.
So MCF is a cutoff to dermine postive FCXM
Prabhakar Putheti,Vijay K. Sharma ,Determining Channel Shift Cutoff in T-Cell and B-Cell Flow Cytometry Crossmatch
Thanks
The Kidney transplant recipient candidate has high positive FCMX (according to provided MCF cutoffs) recently against T and B cells, previously against only B cells. so he is sensitized and has both class I and class II antibodies. this is supported by positive PRA on 15-11-2021 and a very high level of donor-specific antigens with high MFI (against a24, b8, and c7). In Turkey, there is a wide paired donation program in addition to cadaveric transplantation so I would prefer paired kidney transplantation rather than desensitizing the patient. The patient is 54 years old so if no other choice and in case he is on the waiting list for a long time desensitization I may prefer desensitization with rituximab plus plasmapheresis and perform transplantation after negative crossmatch with high dose of ATG induction, followed by triple maintenance (TAC/MMF/DC) with ver close monitoring of DSA)
Capital letters are needed here A27, etc
Why not paired exchange programme?
Please provide feedback on the crossmatch result:
FXC for both B and T cells is positive, with high MFI for both and positive CXC; please provide feedback.
Initially, DSA against HLA class II was detected in the first sample; however, since the current sample revealed positive FXC for both B and T cells, it is possible that de-novo DSA against HLA class I is being developed. This is supported by the presence of positive PRA and DSA 37762 in the first sample, and the development of de-novo DSA against HLA class II in the second sample (HLAA 24, B 8, C7 )
The receiver is extremely sensitized and at high risk of rejection, indicating that this is an incompatible cross-match and that the recipient is very sensitive.
Are you planning to go forward with the direct transplantation?
No, I’d prefer not to go along with it.
If this is the case, what are the alternatives?
– Donation of a kidney in exchange for another
– Desensitization until negative cross-match findings are obtained.
• If you have immunosuppression, what is your regimen?
– Plasmapheresis and IVIg are used to desensitize the patient.
– ATG is used for induction and ATG is used for maintenance ( Tac, MMF and steroid)
– Maintain tacrolimus trough levels on the higher side.
– Check the DSA level on a regular basis.
Negative CDC crossmatch and positive flow cytometry crossmatch – In patients with a negative CDC crossmatch but a positive flow cytometry crossmatch, our approach depends upon the strength of the flow crossmatch, as determined by the median channel shift (MCS):
•
If the MCS is ≤250, we offer HLA desensitization depending upon the overall specificity and strength of DSAs. To assess this, we calculate a relative intensity score (RIS) for the patient, assigning 10 points for each DSA in the strong-binding range (MFI ≥10,000), 5 points for each moderate-intensity DSA (MFI 5000 to 9999), and 2 points for each weak DSA (MFI <5000). If the RIS is less than 17 points, we proceed with our standard desensitization protocol and transplantation. In rare cases in which a patient has an MCS ≤250 but a RIS greater than 17 points (most patients with a RIS >17 have an MCS >250), we would not attempt desensitization and would pursue other potential donors or KPD.
•
If the MCS is >250, we typically do not perform desensitization and prefer to pursue other potential donors or KPD.
Excellent
Excellent Weam
Yes, also it indicates if the desensitization would be successful or not.
Please comment on the cross match result
This patient has positive FCXM for B and T cells indicating antibodies against HLA class1 and 11 antigens. High DSA of 37792. MFI- A24-22926 B8-2931 C7-11935
Will you proceed for the direct transplantation?
This patient is at very high risk for rejection. I will not proceed with Renal transplantation.
If yes, what is your immunosuppression protocol?
This patient is high risk and will need desensitization protocols with Plasmaphresis, IVIG and Rituximab. Induction therapy with ATG and maintenance immunosuppression with Tacrolimus, MMF and corticostroids
If no, what are the alternatives?
Continue of renal replacement therapy, wait for suitable donor or enrol in paired kidney exchange
What is meant by channel shift and how it influences transplantation?
Mean channel shift- CS shows the strength of cross match or immunological incompatibility. In FCXM donor lymphocytes are mixed with recipient serum. Fluorescence on antibodies scan be seen due to presence of antibodies. Median channel fluorescence -MCF on lymphocytes exposed to recipient serum- MCF rs and on lymphocytes exposed to negative control serum MCFncs are required to calculate channel shift. CS= MCF rs – MCFncs
Priscila de Moraes. et al. Accuracy of the median channel shift in the flow cytometry for predicting complement dependent cytotoxicity crossmatching in kidney transplant candidates. Transpl immunol. 2019:52:27-31
Thanks Well done
1-cross match Comment
Current flow cytometry cross match is positive for both Tand B cell
previous flow cytometry cross match was positive only for B cell
high titer of DSA against HLA A24,B8, C7
high sensitized recipient considered high risk group of transplant patient with high risk of hyperacute and acute rejection
2-will not proceed for this transplantion because carry high risk of AMR
So the patient will stay on waiting list until more compatible donor is available
Or paired kidney transplant done for him
3-if yes, Desensitization regime should be done before transplant If MCS less than 250 and RIS less than 17
Then flow cytometry and CDC should be repeated after transplant if negative so we can do transplant
Induction with ATG
Maintenance with TAC,steroid,MMF
4- The MCS a semi-quantitative approximation of the amount of antibody attached to the cells and the determination of positivity an arbitrary decision.
median channel shift MCS, is used to help decide if the renal transplant recipient can be desensitized.
flow cytometry semiquantitative assay of antibody strength correlates with the titer of the antibody or antibodies in the recipient’s serum and its numerical value can aid in evaluating whether or not a renal transplant should be performed and what immunosuppression should be used, particularly when the MCS is in the range. These values can be more helpful than whether the crossmatch is positive or negative.
REFERENCE
1- Up to date
2- Histocompatibility Laboratory, Gift of Life Michigan, Ann Arbor, MI. A Critical Analysis of Quantization of the Flow Cytometry Crossmatch: Its Changing Role Since 1969.
Abstract# C1872. Transplantation: July 15, 2014 – Volume 98 – Issue – p 600-601.
Can you explain the range of MCS you consider and its correlation withRIS to decide to proceed with the transplantation.
criteria for an acceptable cross match affer desensitization may vary by transplant centers
acceptable cross match define as negative CDC cross match in a 1:2 dilution of patient sera, negative flow cytometry ,or positive flow cytometry with MCS < 250
reference
Up to date
Patient is positive FCXM for both B and T cell. Means antibodies against both HLA
class one and HLA class two or HLA class 1 alone.
DSA 37792 AND mfi.
This patient IS high risk.
NO high risk of rejection
If yes, what is your immunosuppression protocol?
Desensitization PP and IVIg as per protocol, the patient received a live donor renal
transplantation.
Immunosuppressive therapy:
Induction with Thymoglobulin given over five days for a total dosage of 6 mg/kg.
Corticosteroids were also initiated intraoperatively (Solu-medrol 500 mg every 12
hours for 3 days with a prednisone taper).
Mycophenolate mofetil 1.0 g twice a day was also started on the first postoperative
day
. TAC at 0.1 to 0.2 mg/kg twice a day with dosages adjusted to keep serum trough
levels at 8–15 ng/mL
Wait for suitable donors.
Paired donation.
MCF is calculated by subtracting the MCF of negative control serum from the MCF
of recipient serum.
The median channel shift MCS, is fluorescence value which is used to help decide
if the renal transplant recipient can be desensitized.
If YES what is the range to accept
How can you judge if your desensitization procedure reached acceptable levels.
positive fcxm for both t and b cells with a 482,480 respectivly on current crossmatch.
DSA against HLA- A24, B8 and C7 with very high MFI (37792).
highly sensitized patient with incompatible crossmatch.
no as patient higly sensetized with incompatible cxm makes him susceptible for ABMR .
if some tell yes according to my weak knowledge i suggest not so much decrease in dsa level to zero only suppressing by plasmapharesis plus ivig first then induction by atg plus maintenance with tac mmf ccs.
continue on either RRT till desensetization and finding another donor.
Great you are trying for correct information kindly read your colleagues answers above .
Better preceded by reading the immunology chapter in Donovitch offered to all by Professor Halawa.
Your follow up plan is fine.
Try to revise the MCS and relation to RIS to help you make the decision of accepting or declining the desensitization plan.
Read your college’s comments they are sufficient.
Thnx prof , noted
Please comment on the cross match result:
Previous positive B cells FCXM indicate either false positive due to previous treatment with monoclonal ABS like rituximab, autoimmune disease as primary disease. like SLE , or false negative T cell due to low titer of class1 DSA, or due to Non HLA abs. B cells can express both class1, 11 .while T cells express class1 , so the repeated recent wet FCXM was also positive for both T and B cells with the historic B cell positive FCXM this indicate true positive XM due to the presence of performed HLA CALSS 1, CLASS11 ,no previous CDCXM as its outdated practice in west but still in our center still common practice to do auto or allo CDCXM with PRA % IF CDCXM also positive for T and B then this patient at high risk for hyper acute rejection and decline the transplant.
Historic positive PRA, DSA positive for class 1 HLA (A24, B8, C7), this confirm he is highly sensitized with performed anti HLA class1
Will you proceed for the direct transplantation?
better to change the donor and offer him paired kidney donation if I will accept this offer this patient should go for desensitization to convert the FCXM to negative and reduce the DSA level
Desensitization by use the combination of plasma pheresis low dose or high dose IVIG, with or without rituximab based on local center resources ,Induction therapy with r ATG or almeutuzumab with triple IS including tacrolimus , MMF, steroid followed by close monitoring of DSA by lumenix SAB at 2weeks ,4 ,8 then 3months ,12 months then annual ,graft biopsy per indication, urine p/c ratio
If no, what are the alternatives?
Ask for another donor or paired kidney donation
What is meant by channel shift and how it influences transplantation?
FCXM is more sensitive , semi quantitative but less specific technique , usually need the donor lymphocytes incubated with recipient sera in addition of fluorescence labelle Ig , with positive and negative control , MCF refer to the calculation of the CS from the negative control and cutoff value setup by 2-3SD , no standardized cutoff value each lab set its own . The cutoff was calculated to be 50 for T‑cells and 80 for B‑cells, using the 3SD rule (1).
Any MCS below the negative control directed for additional work by doing lumenix SAB
References:
1-Dorwal P, Bansal SB, Chauhan R, Jain D, Raina V, Kher V. Median channel shift less than the cutoff in flow cytometric crossmatch: Not to be ignored!. Asian J Transfus Sci. 2017; 11(1):73-75.
Thankyou for the follow up plan.
The flowcytometry crossmatch result for the recipient-donor pair reveals a positive result for both T cell and B cell with current serum, and a B cell positive result with historical serum. A positive T and positive B cell crossmatch denotes DSA to HLA class I and II antigen. A negative T with positive B cell crossmatch denotes DSA to HLA class II or low level DSA to HLA class I antigen. (1)
This report also gives details of DSA in the recipient with high MFI against HLA-A24, B8 and C7. Hence it is a high immunological risk donor-recipient pair.
Considering a positive crossmatch with high MFI DSA, I will not proceed with the transplant directly.
The MCS (Median Channel Shift) for this patient is more than 50.
The DSA-RIS score for this patient is 22 (10+10+2), which is more than 17. (2)
This patient should not be taken up for desensitization.
If we take up this patient for transplant then the patient will require desensitization with Plasmapheresis and IVIG and rituximab to achieve a negative crossmatch before proceeding with the transplant.
Since this patient is a high immunological risk category patient, the patient will require induction immunosuppression and tacrolimus based triple drug maintenance immunosuppression.(3)
1) Induction therapy: Injection Anti Thymocyte Globulin (ATG) in dose of 1-1.5 mg/kg per day for 4-6 days (total dose 6 mg/kg).
2) Maintenance immunosuppression: Triple drug therapy including
a. Tacrolimus: Target trough level of 8-10 ng/ml
b. Mycophenolate mofetil (MMF): 1000 mg twice a day
c. Corticosteroids: Injection methylprednisolone 500 mg intravenous on the day of surgery, followed by tablet prednisolone 1mg/kg/day for 3 days and then 20 mg/day, to be tapered to 5 mg/day over next 6 to 8 weeks.
Post-transplant, patient requires close follow-up with clinical and laboratory evaluation.(3)
i) Complete blood count, urine examination and serum creatinine.
ii) To look for proteinuria: Spot urine protein-to-creatinine ratio.
iii) DSA testing: Once in first three months, then annually or whenever there is any graft dysfunction, alteration in immunosuppression or suspicion of non-adherence.(4,5)
iv) Protocol biopsy: Once in first three months.(4) If the biopsy shows features of AMR, it should be treated.
In a patient with DSA and positive flowcytometry crossmatch, it is better to change the donor – by either enrolling in a paired-exchange program, or asking for another donor in the family.
In a flowcytometry crossmatch, donor lymphocytes are incubated with recipient serum. In presence of antibodies, fluorescence on the lymphocytes can be visualized. The median channel fluorescence (MCF) on the lymphocytes can be measured. To get a value of channel shift, we need MCF on lymphocytes exposed to recipient serum (MCFrs)and MCF on lymphocytes exposed to a negative control serum (MCFncs). The channel shift is the difference between the 2 values.
Channel shift = MCFrs – MCFncs
It is a measure of immunological incompatibility; higher the value, higher the risk.
Different laboratories choose different cut-offs, usually in the range of 2-3 standard deviation (SD). (6,7)
References:
1) Kumar A, Mohiuddin A, Sharma A, El Kosi M, Halawa. An Update on Crossmatch Techniques in Transplantation. J Kidney 2017,3:4.
2) Sethi S, Choi J, Toyoda M, Vo A, Peng A, Jordan SC. Desensitization: Overcoming the Immunologic Barriers to Transplantation. J Immunol Res. 2017;2017:6804678. doi: 10.1155/2017/6804678. Epub 2017 Jan 3. PMID: 28127571; PMCID: PMC5239985.
3) Kidney Disease: Improving Global Outcomes (KDIGO) Transplant Work Group. KDIGO clinical practice guideline for the care of kidney transplant recipients. Am J Transplant. 2009 Nov;9 Suppl 3:S1-155. doi: 10.1111/j.1600-6143.2009.02834.x. PMID: 19845597.
4) Tait BD, Süsal C, Gebel HM, Nickerson PW, Zachary AA, Claas FH, et al. Consensus guidelines on the testing and clinical management issues associated with HLA and non-HLA antibodies in transplantation. Transplantation. 2013 Jan 15;95(1):19-47. doi: 10.1097/TP.0b013e31827a19cc. PMID: 23238534.
5) Crespo M, Zárraga S, Alonso Á, Beneyto I, Díaz Corte C, Fernandez Rodriguez AM, et al; GREAT Study Group and Spanish Network for Research in Renal Diseases (REDINREN, RED16/0009). Monitoring of Donor-specific Anti-HLA Antibodies and Management of Immunosuppression in Kidney Transplant Recipients: An Evidence-based Expert Paper. Transplantation. 2020 Aug;104(8 Suppl 2):S1-S12. doi: 10.1097/TP.0000000000003270. PMID: 32658025.
6) Dorwal P, Bansal SB, Chauhan R, Jain D, Raina V, Kher V. Median channel shift less than the cutoff in flow cytometric crossmatch: Not to be ignored! Asian J Transfus Sci. 2017 Jan-Jun;11(1):73-75. doi: 10.4103/0973-6247.200773. PMID: 28316449; PMCID: PMC5345290.
7) Putheti P, Sharma VK. Determining Channel Shift Cutoff in T-Cell and B-Cell Flow Cytometry Crossmatch. Exp Clin Transplant. 2020 Jun 2. doi: 10.6002/ect.2020.0021. Epub ahead of print. PMID: 32490759.
With level of DSA you need closer follow up at2,4,8 then every 3 months.
Otherwise very good as usual.
Crossmatch result:
FCXM with positive result for both T(482 CS) and B lymphocyte (480CS) /
anti HLA it is DSA =37792MFI(22926,2931,11935)for A24,B8,C7 resectively .
mean anti HLA class 1 which explain positive against both T and B lymphocyte since HLA gene present in both .
Will you proceed for the direct transplantation?
NO , not proceed for transplantation high risk patient ( anti HLA ,DSA ,high MFI, against T ,B lymphocyte) high risk of hyper acute or accelerated rejection .
if low MFI titer we can ask for CDC if it is negative can proceed for transplantation with desensitization protocol but such high MFI unlikely to be negative CDC.
If yes, what is your immunosuppression protocol?
after desensitization protocol immunosuppressive regimen include ATG induction ,TAC ,steroid and MMF.
If no, what are the alternatives?
alternative approach is paired exchange donation but we should keep in our mind that this not solve all problem but minimize the risk because it is change the DSA to non DSA and anti HLA non DSA high MFI still carry risk of rejection .
In Flow XM median channel fluorescence (MCF) or simply channel shift (CS)is calculated by substracting the MCF of negative control from serum from the MCF of recipient serum ,in the absence of standardization method cutoff for CF to classifying T,B positive FCMX, negative cutoff obtained by evaluation of negative serum of at least 30 patient .
Limaye S, O’Kelly P, Harmon G, et al. Improved graft survival in highly sensitized patients undergoing renal transplantation after the introduction of a clinically validated flow cytometry crossmatch. Transplantation. 2009;87(7):1052-1056.
CrossRef – PubMed
CDC will be off course needed to accept this decision
There are earlier guiding calculations as MFI , RIS you can get figures which clearly contraindicated transplantation.
This patient has a positive flow cytometry crossmatch to both B and T cells. It also appears that this patient has DSA against HLA- A24, B8 and C7 with very high MFI.
My answer is No. I would not proceed with the transplantation.
Based on this result it looks difficult to proceed with this transplant. I would have been more comfortable if I knew the donor HLA to make sure that the donor has unacceptable antigens.
I would also like to know the results of the single antigen bead (Luminex) analysis.
Being a living donor transplant, I would recommend a paired exchange transplant.
My answer was No
Being a living donor transplant, I would recommend a paired exchange transplant.
The patient could also be desensitized and then transplanted.
Channel shift is a measure of the strength of the crossmatch when using flow cytometry.
It is determined as the difference between the mean fluorescence negative control serum from the recipient.
This is what is used as a cut-off to decide if a T cell or B cell result is positive.
The channel shift data can be used to predict the likelihood of a positive CDC crossmatch and hence the likelihood of hyperacute rejection.
Thanks, Innocent
Well done
Please comment on the crossmatch result
Flow cytometry crossmatch positive for both T cells and B cells, Abs aagaist HLA class I and Class II (evidenced by previous flow cytometry which was positive for B Cells)
positive DSA testing with MFI of 37792, A24 with MFI of 22926, B8 with MFI of 2931, C7 with MFI of 11935
incompatible cross match results
Will you proceed for the direct transplantation?
I won’t proceed, this is high risk transplantation with B and T cells and high DSA which will results in hyperacute rejection!
If no, what are the alternatives?
A highly sensitized patient like our, may be offered desensitization with combination of Plasmapheresis with IVIG and Rituximab or the use of immunoadsorption.
Also, paired exchange programme will give this patient and other patients of the same condition a higher chance of negative cross match transplant.
What is meant by channel shift and how it influences transplantation?
n a FCXM, median channel fluorescence (MCF) shift or simply channel shift (CS) is calculated by subtracting the MCF of negative control serum from the MCF of recipient serum. In the absence of a standardized method to develop CS cutoffs to classify a T-cell-positive or B-cell-positive FCXM, the cutoffs are determined by individual laboratories by evaluating a negative control serum tested against lymphocytes from 30 healthy individuals or more, resulting in a derived mean and standard deviation (SD) of the MCF. So, it is practically a mechanism of determining the laboratory cut off to determine patient’s positivity or negativity for B cells or T cells on flow cytometry
Dorwal P, Bansal SB, Chauhan R, et al. Median channel shift less than the cutoff in flow cytometric crossmatch: Not to be ignored! Asian J Transfus Sci. 2017;11(1):73-75.
FXC for B and T cells are positive with high MFI for both and positive CXC
First sample showed Positive B cell and negative T cell FCXM denoting DSA against HLA class II then possibly denovo DSA are detected since current sample revealed positive FXC for both B and T cells indicating developement of denovo DSA against HLA class I
With positive PRA and DSA 37762 (HLAA 24 ,B 8 ,C7 )
Indicating that this is incompatible cross match and that the recipient is highly sensitized with high risk of rejection
No I would rather not proceed
But if the transplantation would proceed desensitization will be needed and induction with r ATG then maintenance with Tac ,MMF ,and steroids
With frequent monitoring of DSA and protocol biopsy can be needed as well
Could be paired kidney exchange program or
desensitization with IVIG , Plasma pheresis and rituximab or
awaiting for matching donor
In a FCXM, median channel fluorescence (MCF) shift or channel shift (CS) is calculated by subtracting the MCF of negative control serum from the MCF of recipient serum. In the absence of a standardized method to develop CS cutoffs to classify a T-cell-positive or B-cell-positive FCXM, the cutoffs are determined by individual laboratories .(1)
A test is only reported when positive control works enough for a specific sample. All quality control measures are taken in keeping with the accreditation guidelines as this test is accredited by the National Accreditation Board for Laboratories. Some laboratories tend to use the 10% cutoff by considering the fluorescence index (FI), which is the percentage shift in the test serum as compared to the positive control ([test serum MFI − negative control MFI]/[positive control MFI − negative control MFI] × 100).(2)
Reference
1-Putheti P, Sharma V K. Determining Channel Shift Cutoff in T-Cell and B-Cell Flow Cytometry Crossmatch .Experimental and Clinical Transplantation (2020)
2-Dorwal P, Bansal S B etal . Median channel shift less than the cutoff in flow cytometric crossmatch: Not to be ignored! Asian J Transfus Sci. 2017 Jan-Jun; 11(1): 73–75.
Good
1-Please comment on the crossmatch result
This flow cytometry showed
Previous cytometry – B cell positive with MCS 369
The cut off was to be calculated to be 50 for T cell and 80 for B cells, using 3SD rule.
I would like enquire any treatment with rituximab, anti-CD20 monoclonal antibody, binding of Rituximab will lead to high levels of binding of Fluorescein isothiocyanate (FITC).Using pronase will cleave the anti-CD20 and hence remove Rituximab binding.
Current cytometry – positive B and T cell- indicating strong class 1 antibody or combination both class 1 and 2 antibody
Patient had previous PRA and DSA 37792 ( HLA A24, HLA B8, HLA C )
2-Will you proceed for the direct transplantation?
The strength of the reactions was graded:
The DSA-RIS:
Patients with a DSA score of >17 has ∼91% chance of developing ABMR. This patient has DSA-RIS score -22, high risk for transplantation.
3-If yes, what is your immunosuppression protocol?
If its long waited transplant/ dialysis vintage, I would like to initiate desensitization protocol until desensitization was considered successful. I would like to use IVIG 2g/kg alternating with PLEX with Rituximab 375mg/m2.
Desensitization is considered successful if post therapy donor-specific CMX was acceptable, as determined by negative CDC in 1 : 2 or higher dilution, or FCMX with a shift of < 225 MCSs and DSA scores ≤17
4- If No, would like to enroll into paired kidney exchange program.
5-What is meant by channel shift and how it influences transplantation?
Patient sera is incubated with donor T and B cells. HLA antibodies in the patient sera bind to HLA antigens on the surface of the lymphocytes and are detected flow cytometrically. The results are reported as the median channel shift (MCS) over the negative control sera.
The normal sera were obtained from AB-positive, nonsensitized males, which were also tested on Luminex to confirm the lack of any human leukocyte antigen (HLA) antibody. Using these sera, the cutoff was calculated to be 50 for T-cells and 80 for B-cells, using the 3SD rule. A test is only reported when positive control works satisfactorily for that particular sample. All quality control measures are taken in keeping with the accreditation guidelines vide ISO 15189:2012 as this test is accredited by the National Accreditation Board for Laboratories.
Some laboratories also tend to use the 10% cutoff by considering the fluorescence index (FI), which is the percentage shift in the test serum as compared to the positive control ([test serum MFI − negative control MFI]/[positive control MFI − negative control MFI] × 100).
References:
Determining Channel Shift Cutoff in T-Cell and B-Cell Flow Cytometry CrossmatchPrabhakar Putheti, Vijay K. SharmaExperimental and Clinical Transplantation. 2020
S. C. Jordan et al., 2015, Vol. 114Kidney transplantation in highly sensitized patients
How did you get this scoring scheme for question 2, add a reference
An easier definition of MCS would be better as you here describing the technique
· Comment on this report :
Current Flow cytomtery CX show positive against both B and T lymphocytes due to presence of Ab against either HLA I alone or against both HLA class I and II, SAB show Anti HLA class I Ab .
· Will you proceed for the direct transplantation
No , I will not proceed as this is a very high risk patient as positive FCXM although not contraindication for transplantation , is associated with increased risk of early acute rejection and poor 1 year graft survival . Also this patient has multiple DSA against HLA class I with significant MFI value .
1- Paired kidney donation
2- Desensitization till get negative cross match results
· If yes, what is your immunosuppression protocol
1- Desensitization with plasmapharesis , IVIg
2- Induction with ATG , maintenance with ( Tac , MMF and steroid)
3- Maintain IS trough levels on high therabutic sides
4- Follow up DSA level regularly .
What is meant by channel shift and how it influences transplantation
In FCXM , the recipient serum is mixed with the donor lymphocyte then flurescent labeled antibodies specific to B and T lymphocytese are added as well as AHG congiugated with fleurescin , then a flow cytometer is used to detect concentration of anti T and anti B Ab that’s present in the serum and expressed as positive or negative results . for quantifying the positive resulrs , mean channel shift is used and it refers to number of binding fluresent units above the background number