4. A 41-year-old CKD 5 lady secondary to reflux nephropathy received a kidney offer from her cousin with 100 mismatch. FCXM showed negative B cell cross match, T cell was weakly positive, no DSA. She is on the waiting list for 16 years.
- What is the explanation of this crossmatch?
- Would you go ahead and transplant her?
- If you decided to go ahead, what is your immunosuppression protocol?
Dear All
The right answers are B, D and E
This is a typical cross-match picture of non-HLA antibodies. It may be a technical error, however, the tissue typing lab will repeat the test if there is a discrepancy with the patient DSA profile. This patients if you decide to transplant need aggressive induction agents and aggressive immunosuppression.
This is a real case where the kidney was transplanted with ATG induction. The graft lasted for 4 years and failed because of immunological reasons which could not be identified (the biopsy was inconclusive) but the course suggested chronic persistent/active rejection.
Winners, please contact me by email only
Dear All
I can see excellent answers. We need justification of your answers. Writing the right answer is not enough.
Dear All
I’m not impressed with most of the answers. I will put the question in a different format.
Positive T cell crossmatch with negative B cell in the absence of DSA with negative autocross match is due to
A. Low level class II antibodies due to high concentrations of class II HLA antigens in T Cells
B. Non-HLA antibodies
C. Low level class I antibodies due to high concentration of class II HLA antigens in T Cells
D. Technical erros
E. May need aggressive induction
Please justify your answers in 50 words explaining what is right and what is wrong with each of the choices given. There is a reward for the best answer.
B and d
B, D
B and D
HLA class I expressed on T cells , no class II on T cells so answer A and C are wrong
false positive T cell crossmatch can happened due to lab error or non-HLA antibodies , recent treatment with mono or poly clonal antibody, or recent vaccination, or autoantibodies which usually IgM which can be neutralized during the process of cross match to exclude it.
so B and D are the right answers
b and d
B, D
T cells are expressed in HLA class I , so A & C are wrong as there is no T cell in HLA class II.
The presence of non HLA antibodies can cause positive T cell XM in FCXM especially with the use of pronase. So B is true.
Lab error can give false positive T cell crossmatch result, so D is true
Positive T cell crossmatch when it is due to technical error will not require aggressive induction.
-B,D
-Positive T cell and negative B cell crossmatch need more work.
Maybe the patient has:
1- History of autoimmune disease (autoantibodies): so we need to retake her history with more detail.
2-Received monoclonal antibodies treatment.
3-Vaccination.
4-Technical error
B ,D ,E
Fantastic scenario Professor A. Halawa
A & C wrong because T cell expressed only class I HLA
B, D, & E are true because +T/-B FCXM may be due to lab error, non HLA Ab, & antibodies against HLA-Cw , DSA of HLA-C not tested routinely. long dialysis vintage associated with increase risk of rejection so the patient may need aggressive induction
Answer B , D
HLA class I is expressed on both B and T lymphocytes while class II are expressed only on B cells. T+B-FCXM can result from Non HLA ab against T cell, also Ab against HLA C that h as higher expression on T cell than B cells
B non-HLA antibodies ,autoantibodies, HLA-C ,and doner received rituximab ttt just transplantation and minor histocompatibility antigens H-Y antigens
Can all lead to positive T-cell Xm
D lab error which may occur up to 50 % especially with postive B cell xm
E aggressive induction is needed
Answer: ABCD&E
A&C are true
HLA-class I antigens are expressed on T cells and B cells, and some but not all studies suggest that the cell surface expression of HLA-class I antigens is higher on B than T cells{Pellegrino MA, Belvedere M, Pellegrino AG, Ferrone S. B peripheral lymphocytes express more HLA antigens than T peripheral lymphocytes. Transplantation. 1978;25(2): 93-95.} {Honger G, Krahenbuhl N, Dimeloe S, Stern M, Schaub S, Hess C. Inter-individual differences in HLA expression can impact the CDC crossmatch. Tissue Antigens. 2015;85(4): 260-266.}.
A mechanism for the T+B- FCXM test result is the differential expression of HLA-class I antigens on T cells and B cells. HLA-Cw antigen expression, as detected using mAb DT9, was found to be higher on T cells than B cells, and in this study, seven of 10 FCXM T+B- FCXM were observed with sera containing HLA-Cw DSA. Sera with anti-HLA-Cw IgG had also been shown by others to cause T+BFCXM. Our findings are consistent with the earlier findings that antibodies directed at HLA-Cw antigens may contribute to a T+B- FCXM. Importantly, we report that antibodies directed at HLA-A and antibodies directed at HLA-B are also associated with a T+B- FCXM.{T Cell Positive B Cell Negative Flow Cytometry Crossmatch (FCXM): Frequency, HLA-Locus Specificity, and Mechanisms Among 3073 Clinical FCXM Tests Prabhakar Putheti et al,: https://doi.org/10.1101/2021.05.20.21257541; }
B &D are true:
Low specificity with false positive results due to autoantibodies, immune complexes, etc.
E is true
if a truly positive T; either no transplant as T cell positive crossmatch is a contraindication
or aggressive induction should be attempted.
B, D, and E
Dear Dr Ahmed,
I believe the right answer will be (D).
A- is wrong as T cell has class I HLA.
B- is wrong as isolated positive T cell crossmatch in absence of DSA and with Negative auto crossmatch will make IgM autoantibodies and Non-HLA antibodies unlikely (1).
C- is wrong as B cell crossmatch will be positive also if the recipient has class I anti HLA antibodies.
E- is wrong as even if the patient has non-HLA antibodies it will not clinically affect the allograft because non-HLA antigen is expressed on lymphocytes but not on allograft tissue (1).
References:
1) Melissa Y Yeung. Kidney transplantation in adults: Overview of HLA sensitization and crossmatch testing. © 2021 UpToDate. (Accessed on 7 December 2021).
”As we all know a positive T cell crossmatch with CX as done with Flowcytometry which is sensitive to low titers of antibodies reflects anti HLA due to class I antigens. but because B FCX is negative this is not accepted and we need further workup. It could be due to technical error but speaking in general, it may be true due to autoantibodies (so we need the history of autoimmune disease). The patient may be treated before with monoclonal antibodies lite rituximab etc. History of vaccination. as in history, it is reflux nephropathy and nothing evident or no clue for autoimmune disease etc. recent vaccination could be a reason.
this is an excellent crossmatch with just one mismatch so it is low risk. many centers may prefer 1 low dose rATG because the history of 16 years carries many possibilities of being transfused sensitized etc. we may need auto flow to crossmatch ( by mixing recipients lymphocyte and serum to clarify this phenomenon). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4961613/
,,, I will prefer 1 dose of rTG may be 1.5 mg/kg as this is a low risk transplant for ABMR”
Back to question and answers:
A and D should have positive B FCX (as suggested by high class II)
E is not necessary as this is anyhow a low-risk transplant
son I suggest B and D be the correct answer
HLA class I expressed on T cells , no class II on T cells so answer A and C are wrong
false positive T cell crossmatch can happened due to lab error or non-HLA antibodies , recent treatment with mono or poly clonal antibody, or recent vaccination, or autoantibodies which usually IgM which can be neutralized during the process of cross match to exclude it.
so B and D are the right answers
A and C were wrong because T cell express class 1
B it is right answer,non HLA antibodies can cause positive T cell cross match like MICA and angiotensin 2 receptors antigen
D technical error is possible in any test so it is right answer
E definitely right due to any positive T cell cross match need aggressive induction.
Other causes of positive T cell cross match is : recent treatment with rituximab, recent vaccination, antibodies to HLA-cw
The correct answer is B & D because these are the possible explanation compared to other stems in the question. A is wrong because B cell XM will be positive if class II is present and T cell XM is negative for class II . C is also wrong because B cell XM cross will be positive as well which is not the case in the scenario. E is incorrect because this not high risk patient by definition to justify aggressive induction protocol and we have been told DSA is negative.
B and D
as far as i learnt
T cell positive B cell negative flow cytometry cross match(T+B- FCXM)
1- Donor HLA specific IgG antibodies directed HLA class 1 antigen HLA-A,B and or C locus
2- summed MFI of class 1 DSA and the Median Channel fluorescence (MCF) T cells has significant association with T+B- FCXM
3- HLA-Cw antigen expression (detected as mAb DT9) found to be higher on T cell than B cell – so antibody directed toward HLA-Cw (IgG HLA-Cw)- T+B-FCXM
4- Autoantibodies directed towards T cells could result in T+B- cells
5- HLA 1 Expressions lower on B cells isolated from deceased donor blood compared to B cells isolated for spleen, lymph node or B cells
6- Previously used Zenopax (Daclizumab) – anti IL2R/CD25 (if activated T cells) will give rise to Positive T cell —if T cells are activated and negative B cells on FCXM
Back to the question
Positive T cell crossmatch with negative B cell in the absence of DSA with negative autocross match is due to
A. Low level class II antibodies due to high concentrations of class II HLA antigens in T Cells
-wrong answer as T cell expresses HLA class 1 only
B. Non-HLA antibodies
-correct answer as Non -HLA reactivity will give rise to T+B- FCXM
C. Low level class I antibodies due to high concentration of class II HLA antigens in T Cells
-wrong answer as T cell expresses only HLA class 1
D. Technical erros
-correct answer- wrong Channel shift (CS) used to test the T and B cell positivity might give rise to error
E. May need aggressive induction
-correct answer- HLA-Cw antigen expression will lead to T+B- FCXM, as we know mismatch in HLA -c showed emergent evidences in poor graft function. So, i might choose more potent induction
so, B,D,E are my answer
A -wrong -T cell expresses HLA class 1 only
B -correct – Non -HLA reactivity will give rise to T+B- FCXM
C -wrong – T cell expresses only HLA class 1
D-correct – wrong Channel shift used to test the T and B cell positivity might give rise to error
E-correct answer- HLA-Cw antigen expression results in T+B- FCXM.mismatch in HLA -c showed poor graft survival
Positive T-cell cross match with negative B cell in the absence of DSA with negative autocross match;
Possible causes for the above scenario are ;
B- none HLA antibodies
D- technical
Cross match for this patient is done by flow cytometory ,which is more specific than CDC_XM ,it detect IgG and does not detect IgM .At the same time it cannot differentiate between HLA and non-HLA antibodies. Absence of DSA and auto antibodies in the setting of positive T-cell ,raise the possibility of none HLA antibodies as a cause .
Regarding kidney transplantation in patient with positive none HLA anti bodies ;
The presence of Non-HLA abs are not an absolute contraindication to transplantation . Immunologic risk stratification before transplantation, by comprehensive diagnostic assessment strategies focusing on both HLA-DSA and Non HLA abs responses, could help to better define sub phenotypes of antibody-mediated rejection, or delayed graft function, and lead to timely initiation of targeted therapies . Accordingly, early treatment of patients with increased immunologic risk factors and with circulating Non HLA abs is required . The risk factors include re-transplanted, male gender, young age, and those with FSGS at time of transplantation were positive for AT1RAbs and AECAs prior to transplantation . Furthermore, testing for non-HLA antibodies is often performed when histological evidence suggests an antibody mediated process in the absence of HLA-DSA .
As the cross matching of this patient is positive ,her transplant immunosuppressant protocol is similar to those with HLA antibodies and positive cross match including desensitization ,induction and triple TAC. Angiotensin receptor blockers such as losartan have also been used to block the activity of angiotensin receptor in patients with AT1R-Ab mediated rejection . However, a more recent study shows that chronic use of losartan can upregulate AT1R expression resulting in worse outcomes.
The explanation of the other wrong answers is;
A-low level of class 2antibodies due to high concentration of class 2 HLA antigens t-cells.
B- low level class 1 antibodies due to high concentration of class 2 antigen in T-cells
T-cell cross match cannot be affected by MHC II polymorphisms , because MHC class II molecules do not dissociate at the plasma membrane. The mechanisms that control MHC class II degradation have not been established yet, but MHC class II molecules can be ubiquitinised and then internalized in an endocytic pathway. While MHC class I polymorphisms and the high diversity of peptides that can bind to MHC class I in different individuals make it virtually impossible to have a perfect tissue match between donor and recipient, and thus are responsible for graft rejection .
.
Referance;
Vaidya S, Cooper TY, Avandsalehi J, et al. (2001) Improved
ÀoZ c\tometric detection of HL$ alloantiEodies using pronase: Potential implications in renal transplantation. Transplantation 71: 422-428.
Philogene MC, Jackson AM (2016) Non-HLA antibodies in transplantation:
when do they matter? Curr Opin Organ Transplant 21: 427-432
Jordan P, Kübler D (1996) Autoimmune diseases: nuclear autoantigens can be
found at the cell-surface. Mol Biol Rep 22: 63-66.
Cardinal H, Dieudé M, Hébert MJ (2017) The Emerging Importance of NonHLA Autoantibodies in Kidney Transplant Complications. J Am Soc Nephrol.
28: 400-406.
Djamali A, Kaufman DB, Ellis TM, Zhong W, Matas A, et al. (2014) Diagnosis
and management of antibody-mediated rejection: current status and novel
approaches. Am J Transplant 14: 255-271.
Lukitsch I, Kehr J, Chaykovska L, Wallukat G, Nieminen-Kelhä M, et al.
Renal ischemia and transplantation predispose to vascular constriction
mediated by angiotensin II type 1 receptor-activating antibodies. Transplantation
94: 8-13.
Song MA, Dasgupta C, Zhang L (2015) Chronic Losartan Treatment UpRegulates AT1R and Increases the Heart Vulnerability to Acute Onset of
Ischemia and Reperfusion Injury in Male Rats. PLoS One 10: e0132712.
Dieudé M, Bell C, Turgeon J, Beillevaire D, Pomerleau L (2015)
The 20S proteasome core, active within apoptotic exosome-like vesicles, induces
autoantibody production and accelerates rejection. Sci Transl Med 7: 318ra200.
State Medical University,
Volgograd, Russia
State Medical University,
Volgograd, Russia
Positive T cell crossmatch with negative B cell in the absence of DSA with negative autocross match is due to
A. Low level class II antibodies due to high concentrations of class II HLA antigens in T Cells…wrong t cell express only class 1
B. Non-HLA antibodies….right as solid phase crossmatch use beads coated with HLA only
C. Low level class I antibodies due to high concentration of class II HLA antigens in T Cells…. wrong t cells don’t express class 2
D. Technical erros… most probably
E. May need aggressive induction….no and still considered low immunological risk and if still persistent positive after repeating the test(assuming that living related donation and can be postponed) non depleting basilixmab plus the triple conventional therapy will be enough.
Would you go ahead and transplant her
No i will repeat the test as i mentioned assuming the donor is living related.
But i should focus the light on the important to role out UTI in such patient with history of reflux nephropathy which sometimes needs bilateral nephrectomy.
If you decided to go ahead, what is your immunosuppression protocol
Non depleting basilixmab induction and triple conventional immune suppression medications tac/MMF/Steroids
In completing my comments that have been posted, I think that right answers are B, D, and E.
Positive T cell crossmatch with negative B cell in the absence of DSA with negative autocross match is due to
A. Low level class II antibodies due to high concentrations of class II HLA antigens in T Cells
False: B cells express class I and class II HLA-antigens, while T cells only express class I HLA antigens.
B. Non-HLA antibodies
True: Pronase treatment is also prone to give false-positive reactions in T-FCXM test, probably due to the participation of non-HLA antibodies including autoantibodies.
C. Low level class I antibodies due to high concentration of class II HLA antigens in T Cells
False: B cells express class I and class II HLA-antigens, while T cells only express class I HLA antigens.
D. Technical errors
True: A positive T cell crossmatch alone is usually due to technical errors. Such as false positive T cell FCXM in pronase treated FCXM due to disclosing hidden epitopes or non-HLA antibodies including autoantibodies.
E. May need aggressive induction
True: There is the probability of anti HLA class I particularly anti HLA-Cw. Thus, with regard to positive T cell crossmatch, recipient is considered to be at high risk for acute allograft rejection and induction therapy with rATG and intravenous methylprednisolone will be suggested
D and E
Explanation:
T cell expresses class I only while B cell expresses both class I and class II. If T cell positive and B cell negative either technical error which is common to occur or more work is needed for other differential diagnosis :
– Monoclonal antibodies target T cell not B cell.
– Autoimmune disease binds only to T cell.
– Covid 19 vaccine which express surface antigen which make antibodies bind non specific.
-Auto-immune disease with autoantibodies bind only to T cell.
So here we need aggressive induction.
B and D are correct
Positive T cells cross match alone most probably represent technical error or may be non HLA Abs
Tcells carry class I antigens, B cells carries Class I and II antigens
DSAs against class I would cause both B and T cell positive CM
DSAs against class II would cause only a positive B CM
Positive T cell crossmatch with negative B cell in the absence of DSA with negative autocross match is due to
B, D, E: True
A, C: False
A. Low level class II antibodies due to high concentrations of class II HLA antigens in T Cells: Wrong (as Class II HLA not seen on T cells)
B. Non-HLA antibodies: True (A positive T cell crossmatch maybe due to non-HLA antigens)
C. Low level class I antibodies due to high concentration of class II HLA antigens in T Cells: Wrong (as Class II HLA not seen on T cells)
D. Technical errors: True (A false negative B cell cross match due to viability issues of B cells can be seen)
E. May need aggressive induction: True {Non-HLA antibodies (e.g. MICA, AT1R) may lead to AMR, hence need aggressive induction}
B ,D
HLA Class l expressed on Tcell,No class ll expression on T cell ,class l and class ll present on B cell
False positive result occurs because
FCXM cannot differentiate between HLA and non-HLA antibodies. One method of further increasing its sensitivity and specificity is with the addition of enzyme pronase to digest the Fc receptors on donor lymphocytes . Pronase treatment minimizes the binding of non-specif- ic antibodies, thereby improving overall specificity. This becomes useful in interpretation of results where the CDC-XM was negative with a positive FCXM
Other causes of false positive results are treatment with monoclonal or polyclonal IS, recent vaccination, presence of IgM AB or may be lab errors.
Would you go ahead and transplant her?
yes I will transplant
If you decided to go ahead, what is your immunosuppression protocol?
I will use Basiliximab for induction and triple IS for maintenance
The correct answer are B & D.
Positive T cell XM and negative B cell XM with no DSA may be related to non- HLA antibodies or autoantibodies. Auto antibodies were ruled out by auto crossmatch so this is not the answer .Due to high sensitivity of Luminex for detecting DSA, there is no class I or ІІ HLA antibodies in this case. Technical errors especially long incubation period due to delays in washing is the other answer. E is false because due to standard risk and low mismatch you don’t need aggressive induction and an IL2-receptor Ab induction or even no induction is preferred.
. A and c are wrong because Class 11 are present in B cell , FCXM can detect all transplant-related antibodies, including non-HLA antibodies. Positive cross match may be duto Lab error ,rituximab and vaccination. So B and D are right .
A is false , T cells had no class II HLA antigens on it’s surface.
B is true , non HLA antigens can lead to positive flow crossmatch with negative DSA.
C is false , T cells had no class II HLA antigens on it’s surface.
D is true , lab error like denature antigens or incomplete HLA typing can lead to positive FC-XM with negative DSA.
E is true , patient had long time on dialysis , so she had increased risk of DGF and rejection .
Negative B and positive T FXCM may be caused by:Non-HLA antibodies, Technical errors
Better to be repeated to Rule out technical errors if the same result we should have desensitization
Also we should rule out UTI due to reflux nephropathy
Immunosuppressive protocol :It would be ATG and methylepred ,maintenance will be tacrolimus , MMF and prednisone
The possible causes here for positive t cell crossmatch and negative b cell crossmatch
Non HLA ab
Technical errors .
A . Is a wrong answer because t cell positive expressed class I only .
E. Is correct as the patient need induction immunosuppression.
B, D, E: True
A. Low level class II antibodies due to high concentrations of class II HLA antigens in T Cells false. as Class II HLA not seen on T cells
B. Non-HLA antibodies: True. positive T cell cross match may be due to non-HLA antigens, also the differential expression of HLA-class I antigens on T cells(higher HLA-Cw) than B cells. Auto AB will be excluded by autocross match
C. Low level class I antibodies due to high concentration of class II HLA antigens in T Cells: false as Class II HLA not seen on T cells
D. Technical errors True A false negative B cell cross match due to B cells viability can be encountered
E. May need aggressive induction True Non-HLA antibodies can lead to AMR so need aggressive induction therapy.
T cells express HLA class l while B cells express both class l & ll so if B cell crossmatch is positive, T cell crossmatch should be positive but if T cell is positive with negative B cell crossmatch , it may occur in some situations like autoimmune diseases, technical error, non HLA antibodies…, so my answer is B and D
T-cells has HLA class one ,B cells has HLA class 2
so i proceed with B&D answers
to exclude non HLA AB virtual SPxm should be done
yes I’ll go ahead for procedure
with precautions (recommend double nephrectomy before transplantation of living donor kidney )
no need for heavy induction
may use corticosteroid only in induction & recommend use of cortisol free protocol with tacrolimus & MMF
A &C are wrong as HLA class II antigens are not expressed on T cells.
B right : non HLA antibodies against T cells .Non -HLA AB response leads to38 % of allograft loss vs 18% n case of HLA mismatches. FCXM revealing T positive and B negative suggests non specificity of antibodies against HLA antigens .Examples of non HLA antigens :
Antibodies directed against MICA &B antigens which are expressed on endothelial cells.
Anti ETAR
Antibodies against HY antigen
Antibodies against AT1R and others like vimentin ,tubulin, myosin and collagen : not a possibility in our case as they are autoantibodies and there is negative autocross matching
AECA
Differential expression of HLA onT cells &B cells and /or differential binding of ABs to T cells and B cells could explain
D right L-SAB is needed to address the status of class A,B & Cw ABs. Cut off points of channel shift used for qualitatively determination of FCXM positivity or negativity are to be considered.
B right in spite absence of DSA with match of 100,live related donor and no evidence of sensitization, possibility of non-HLA antibodies make induction including Bsiliximab a rational protocol.
REFERENCES:
BTS guidelines,2014
Putheti,P et al : T cell positive Bcell negative FCXM :frequency,HLA locus specificity and mechanisms among 3073 clinical FCXM tests, Med Rexv ,Reprint,May,2021.
Rmirez,CBand McCaulty, editors, Contemporary kidney transplantation,2018
The result should be interpreted with caution.
This can be due to autoantibodies or due to non HLA antibodies .
Or due to incomplete typing of the HLA of the donor HLA C ,
or HLA antibody to rare HLA antigen that not expressed in the Luminex SAB panel
So we need to go the lab and ascertain if the auto cross match had been done or not , pronase digestion is done or not , and check for complete typing od the HLA of the donor and recipient and checking for non HLA antibodies if possible .
If we comfirm that this poaitivity is false , we can proceed with transplantation.
The patient had long time on dialysis , so she had high risk of DGF , and higher risk of rejection , cPRA also should be taken into account and previos history of sensitization, induction with ATG , triple immunosuppression ( TAC, MMF , steroid ).
T-lymphocytes express HLA class I and B lymphocytes HLA class I and II
So A ,C are false
A mechanism for the T+B- FCXM test result is the differential expression of HLA-class I antigens on T cells and B cells. HLA-Cw antigen expression which is detected by using mAb DT9, found to be higher on T cells than B cells.
Autoantibodies directed at T cells could result in a T+B- result. Non -HLA and technical error
may cause +T,-B
So B,D are correct
E : Induction with basiliximap and maintenance with ( steroids,MMF,CNI).
Reference
1- Prabhakar Putheti, Vijay K Sharma, Rex Friedlander, Arvind Menon, Darshana Dadhania, Thangamani Muthukumar, Manikkam Suthanthiran
doi: https://doi.org/10.1101/2021.05.20.21257541
T Cell Positive B Cell Negative Flow Cytometry Crossmatch (FCXM): Frequency, HLA-Locus Specificity, and Mechanisms Among 3073 Clinical FCXM Tests
2- Nicholas Torpey, Nadeem E Moghal, Evelyn Watson, and David Talbot. OSH Renal Transplantation. Published online: Oct 2011
T cells Express only HLA class I while B cells express both HLA I and II so for this scenario 100 mismatch means HLA -A mismatch while while weak positive T cell indicate either lab. error need to be repeated , presence of auto antibodies and in this situation auto crossmatch must done by mixing recipient serum with own lymphocytes ,non HLA antibodies like IgG and IgM and history of vaccination and recent treatment with ATG also give T cell positive alone .
The patient can proceed the transplantation as DSA was negative and we can put her on triple immunosuppressants.
. Positive crossmatch with negative DSA can be explained by presence of nonHLA antibodies.
The most reported non-HLA antibodies include those directed against angiotensin II type 1 receptor (AT1R-Ab), endothelin receptors, MHC class I chain-related antigen A (MICA-Ab), vimentin (AVA), tubulin (anti-Kα1 tubulin), collagens (anti-Col) and antiendothelial cell antibodies (AECA).
Transplantation is better for her rather than HD so Iwill go ahead for transplantation. Desensitization treatment to reduce levels of Non HLA abs is similar to what is commonly used for HLA antibodies (intravenous immunoglobulin, plasmapheresis, rituximab, and bortezomib) .However, Combination therapies with Plasmapheresis (pre- and/or posttransplant), intravenous immunoglobulin (100mg/kg) and rituximab may lead to more durable antibody elimination.
Angiotensin receptor blockers such as losartan have also been used to block the activity of angiotensin receptor in patients with AT1R-Ab mediated rejection. However, a more recent study shows that chronic use of losartan can upregulate AT1R expression resulting in worse outcome .
Negative B cell FCXM with weakly positive T cell FCXM but no DSA is related to non-HLA antibody or auto antibodies and may be technical errors such as delay washing and prolonged incubation. As there is no DSA and only one mismatch, this transplantation will be of standard risk and it’s ok to transplant her. Because of considering primary kidney disease which is reflux nephropathy and not immune-mediated GNs there is no need for potent immunosuppression. We can do TX with an induction with Simulect or even methylprednisolone and Tacrolimus and cellcept. Then maintenance immunosuppression could be either triple therapy or steroid-withdrawal protocol
The cross match will probably need to be repeated , as valid results include either positive B or positive both .
That’s because B cells carries both class I and II antigens , T cells carries only class I .
Or this may represent non HLA antibodies
I would repeat it at 1st then
I would ask for a virtual Cross match, and decide desensitization accordingly
I would proceed with induction therapy using thymoglobulin
*What is the explanation of this crossmatch?
In such patient with negative B cell crossmatch and weekly positive T cell crossmatch may be technically error as T cell expresses class I only while B cell expresses both class I and class II.
*Would you go ahead and transplant her?
I will wait for further assessment and clear evaluation for possible complications as patient with history of reflux nephropathy .
*If you decided to go ahead, what is your immunosuppression protocol?
Must done with aggressive induction therapy
ATG OR BASILIXIMAB and triple therapy steroid , MMF and tacrolimus .
FLCX detects both anti HLA and non anti HLA antibodies, non-complement fixing antibodies, and only IgG antibodies.
B cells express class I and class II HLA-antigens, while T cells only express class I HLA antigens.
B cells express class I HLA-antigens much more, and it is one reason for B cell crossmatch positive and T cell cross match negative flow cytometry, which, of course, is not the case in our scenario.
A positive T cell crossmatch alone is usually due to technical errors.
The Pronase that is used for improving the specificity and sensitivity of B cell FCXM by reducing high background fluorescence and by preventing the binding of non-specific anti-IgG antibodies to immunoglobulin Fc-receptors on B lymphocytes (digest Fc receptors on donor’s lymphocytes), can cause false positive crossmatch results by disclosing hidden epitopes.
Pronase treatment of lymphocytes for flow cytometry crossmatching is used to reduce non-specific B cell reactivity due to the binding of immune complexes to Fc receptors. However, pronase has been shown to produce false-positive T-cell FCXM.
Pronase treatment is also prone to give false-positive reactions in T-FCXM test, probably due to the participation of non-HLA antibodies including autoantibodies. Patients might be inappropriately excluded from receiving organs. In laboratories using pronase treated single tube T/B FCXM, caution is needed to avoid false-positive reporting of results.
Pronase treated false-positive reactions in T-FCXM test was also reported in the context of HIV infection.
There are some reports about the effect of pronase on inducing false positive T cell crossmatch.
There is also a case report of false positive T cell FCXM caused by blood type antigen expressed on lymphocytes in ABO incompatible kidney transplantation.
HLA antibodies directed at HLA-A, B or Cw locus are associated with a T+B- result.
In one study, 2.3% of FCXM were T+B-.
A mechanism for the T+B- FCXM test result is the differential expression of HLA-class I antigens on T cells and B cells. HLA-Cw antigen expression, as detected using mAb DT9, was found to be higher on T cells than B cells.
Antibodies directed at HLA-Cw antigens may contribute to a T+B- FCXM. Antibodies directed at HLA-A and antibodies directed at HLA-B are also associated with a T+B- FCXM.
Aberrant expression of HLA class I on the B cells associated with a T+B- FCXM and impaired binding of anti-HLA antibodies.
In this scenario, first, technical errors should be ruled out. For example, in the case of pronase treated T and B cells flow cytometry, pronase treatment should be performed only for B cell crossmatch in different tubes.
If technical errors are not in doubt, the probability of anti HLA class I, particularly anti HLA-Cw is raised. Consequently, the next step is detection of DSA by more precise technique such as SAB.
If DSAs aren’t detected, with regard to positive T cell crossmatch, recipient is considered to be at high risk for acute allograft rejection and induction therapy with rATG and intravenous methylprednisolone will be suggested. But if DSAs are detected, given that the patient is on the waiting list for a long time, desensitization protocol (plasmapheresis, IVIG, rituximab) may be taken into account.
For maintenance therapy, triple therapy with MMF, tacrolimus and prednisolone are suggested.
Holly Sanders 1, Valia Bravo-Egana 2. 1AU Medical Center, Augusta, GA, United States; 2 Medical College of Georgia, Augusta, GA, United States. PRONASE TREATMENT CAN CAUSE FALSE POSITIVE T-CELL FLOW CYTOMETRY CROSSMATCHES IN PATIENTS WITH AUTOIMMUNE DISEASES: TWO CLINICAL CASES. Human Immunology 79 (2018) 58–187.
H. Park, Y.M. Lim, B.Y. Han, J. Hyun, E.Y. Song, and M.H. Park. Frequent False-Positive Reactions in Pronase-Treated T-Cell Flow Cytometric Cross-match Tests. Transplantation Proceedings, 44, 87–90 (2012)
Prabhakar Putheti,1,2 Vijay K Sharma,1,2 Rex Friedlander,1 Arvind Menon,1 Darshana Dadhania,1,2,3 Thangamani Muthukumar,1,2,3 and Manikkam Suthanthiran, T Cell Positive B Cell Negative Flow Cytometry Crossmatch (FCXM): Frequency, HLA-Locus Specificity, and Mechanisms Among 3073 Clinical FCXM Tests, medRxiv preprint, May 24, 2021
Daiki Iwami1, Makoto Ito2, Kiyohiko Hotta1, Haruka Higuchi1, Nobuo Shinohara1. False-Positive Flow-Cytometric TCellCrossmatch caused by Blood Type Antigen Expressed on Lymphocytes in ABO Incompatible Kidney Transplantation. 2018 Wolters Kluwer Health.
Positive T cell crossmatch with negative B cell in the absence of DSA with negative autocross match is due to
A. Low level class II antibodies due to high concentrations of class II HLA antigens in T Cells…wrong t cell express only class 1
B. Non-HLA antibodies….right as solid phase crossmatch use beads coated with HLA only
C. Low level class I antibodies due to high concentration of class II HLA antigens in T Cells…. wrong t cells don’t express class 2
D. Technical erros… most probably
E. May need aggressive induction….no and still considered low immunological risk and if still persistent positive after repeating the test(assuming that living related donation and can be postponed) non depleting basilixmab plus the triple conventional therapy will be enough.
Would you go ahead and transplant her
No i will repeat the test as i mentioned assuming the donor is living related.
But i should focus the light on the important to role out UTI in such patient with history of reflux nephropathy which sometimes needs bilateral nephrectomy.
If you decided to go ahead, what is your immunosuppression protocol
Non depleting basilixmab induction and triple conventional immune suppression medications tac/MMF/Steroids
Positive T-cell cross match with negative B cell in the absence of DSA with negative autocross match;
Possible causes for the above scenario are ;
B- none HLA antibodies
D- technical
The cross matching of this patient is done by flow cytometory ,which is more specific than CDC_XM ,it detect IgG and does not detect IgM .At the same time it cannot differentiate between HLA and non-HLA antibodies. Absence of DSA and auto antibodies in the setting of positive T-cell ,raise the possibility of none HLA antibodies as a cause. .
Regarding kidney transplantation in patient with positive none HLA anti bodies ;
The presence of Non-HLA abs are not an absolute contraindication to transplantation . Immunologic risk stratification before transplantation, by comprehensive diagnostic assessment strategies focusing on both HLA-DSA and Non HLA abs responses, could help to better define sub phenotypes of antibody-mediated rejection, or delayed graft function, and lead to timely initiation of targeted therapies . Accordingly, early treatment of patients with increased immunologic risk factors and with circulating Non HLA abs is required . The risk factors include re-transplanted, male gender, young age, and those with FSGS at time of transplantation were positive for AT1RAbs and AECAs prior to transplantation . Furthermore, testing for non-HLA antibodies is often performed when histological evidence suggests an antibody mediated process in the absence of HLA-DSA .
As the cross matching of this patient is positive ,her transplant immunosuppressant protocol is similar to those with HLA antibodies and positive cross match including desensitization ,induction and triple TAC. Angiotensin receptor blockers such as losartan have also been used to block the activity of angiotensin receptor in patients with AT1R-Ab mediated rejection . However, a more recent study shows that chronic use of losartan can upregulate AT1R expression resulting in worse outcomes.
The explanation of the other wrong answers is;
A-low level of class 2antibodies due to high concentration of class 2 HLA antigens t-cells.
B- low level class 1 antibodies due to high concentration of class 2 antigen in T-cells
T-cell cross matching cannot be affected by MHC II polymorphisms , because MHC class II molecules do not dissociate at the plasma membrane. The mechanisms that control MHC class II degradation have not been established yet, but MHC class II molecules can be ubiquitinised and then internalized in an endocytic pathway. While MHC class I polymorphisms and the high diversity of peptides that can bind to MHC class I in different individuals make it virtually impossible to have a perfect tissue match between donor and recipient, and thus are responsible for graft rejection .
.
Referance;
Vaidya S, Cooper TY, Avandsalehi J, et al. (2001) Improved
ÀoZ c\tometric detection of HL$ alloantiEodies using pronase: Potential implications in renal transplantation. Transplantation 71: 422-428.
Philogene MC, Jackson AM (2016) Non-HLA antibodies in transplantation:
when do they matter? Curr Opin Organ Transplant 21: 427-432
Jordan P, Kübler D (1996) Autoimmune diseases: nuclear autoantigens can be
found at the cell-surface. Mol Biol Rep 22: 63-66.
Cardinal H, Dieudé M, Hébert MJ (2017) The Emerging Importance of NonHLA Autoantibodies in Kidney Transplant Complications. J Am Soc Nephrol.
28: 400-406.
Djamali A, Kaufman DB, Ellis TM, Zhong W, Matas A, et al. (2014) Diagnosis
and management of antibody-mediated rejection: current status and novel
approaches. Am J Transplant 14: 255-271.
Lukitsch I, Kehr J, Chaykovska L, Wallukat G, Nieminen-Kelhä M, et al.
Renal ischemia and transplantation predispose to vascular constriction
mediated by angiotensin II type 1 receptor-activating antibodies. Transplantation
94: 8-13.
Song MA, Dasgupta C, Zhang L (2015) Chronic Losartan Treatment UpRegulates AT1R and Increases the Heart Vulnerability to Acute Onset of
Ischemia and Reperfusion Injury in Male Rats. PLoS One 10: e0132712.
Dieudé M, Bell C, Turgeon J, Beillevaire D, Pomerleau L (2015)
The 20S proteasome core, active within apoptotic exosome-like vesicles, induces
autoantibody production and accelerates rejection. Sci Transl Med 7: 318ra200.
State Medical University,
Volgograd, Russia
State Medical University,
Volgograd, Russia
Positive T cell crossmatch with negative B cell in the absence of DSA with negative autocross match is due to
A. Low level class II antibodies due to high concentrations of class II HLA antigens in T Cells
B. Non-HLA antibodies
C. Low level class I antibodies due to high concentration of class II HLA antigens in T Cells
D. Technical errors
E. May need aggressive induction
-T-Cell positive, B -negative FXCM need more work, AS the HLA-class I antigens are expressed on both T and B cells,so I would expect B cell positive as well (T with Class I and B with both class I and II).we need more history of previous positive CDC with previous PRA, Female gender with Pregnancy history and previous bloodtransfusion.
-Technical factors that can influence their clinical interpretation when using the Luminex bead technology, such as variation in antigen density and the presence of denatured antigen on the beads, (A negative B-cell crossmatch in the presence of a positive T-cell crossmatch suggests a technical error).
-Presence of IgG antibodies directed at HLA loci such as Cw, DQA, DPA, and DPB are associated with T-Cell positive, B -negative FXCM. Also, Differential expression of HLA on T cells and B cells and/or differential binding of antibodies to T cells and B cells and Tthe variation in cutoff value used to qualify a FCXM test as positive or negative are potential contributors to T-Cell +ve, B -ve FCXM result.
– Positive T cell FCXM is associated with increased risk of early graft loss due to antibody-mediated rejection and may represent a relative contraindication to transplantation, in one study found that Positive- T-Cell FCXM on background of negative AHG-CDC ,early graft loss in the first two weeks was found in 33% , another 44%,reported SCR in the first month and 11% in 3 months ,acute rejection( ABMR) in 67% and recurrent rejection in another 67%, all underwent a high-risk induction protocol. With OKTA3, patients with previous history of transfusion was found in 89% ,female gender with pregnancyin 89%, cadaveric donor in 89%, previous historical PRA with >10 in 44% .
the answer :
B, D, E
References :
1-T Cell Positive B Cell Negative Flow Cytometry Crossmatch (FCXM):
Frequency, HLA-Locus Specificity, and Mechanisms Among 3073 Clinical FCXM Tests
Prabhakar Putheti,1,2 Vijay K Sharma,1,2 Rex Friedlander,1 Arvind Menon,1 Darshana Dadhania,1,2,3Thangamani Muthukumar,1,2,3 and Manikkam Suthanthiran.1,2,3
2-Flow Cytometric Crossmatching in Primary Renal Transplant Recipients with a Negative Anti-Human Globulin Enhanced Cytotoxicity Crossmatch.
MARTIN KARPINSKI, * DAVID RUSH, * JOHN JEFFERY, * MARKUS EXNER, §HEINZ REGELE, ¶ SILVIA DANCEA, † DENISE POCHINCO, * PATRICIA BIRK, ‡ and PETER NICKERSON*J Am Soc Nephrol 12: 2807–2814, 2001.
the enswer is b and d
a is rejected:
T cell do not express HLA class II on their surface
low levels of class II ab is conflicting with the previous information given as absent DSA
low levels of class II ab will react with B cell surface HLAclass II and produce weak positive B cell XM, and negative T cell XM
c is rejected:
T cell do not express HLA class II on their surface
low levels of class I ab is conflicting with the previous information given as absent DSA
low levels of class I ab will bind HLA class I on T cell and B cell surface will produce weak positive T cell XM as well as weak positive B cell XM
b and d the answer
as both T and B cells express Class I
DSA (anti HLA I will react with both )
DSA (anti HLA II will react with B only)
reaction with T cell in the absence of DSA Nd auto Ab is only explained by non HLA Ab, monoclonal ab, vaccination that causes mutation to the non HLA surface Ag on T cell and binds non specific Ab
or a technical error
e : is rejected
no need for aggressive indiction as there is absent DSA
would you go ahead and transplant her?
yes, one mismatch with No DSA. FCXM can be technical error due to wrong Channel shift setting for positivity for B cell and T cell. I might choose to resend her FCXM or do Solid phase bead assay (Luminex) as it is better method than FXCM .
if you decide to go ahead, what is your immunosupression protocol
i would choose anti-CD 25, basiliximab as induction agent with Mycophenolate acid, tacrolimus and steroid as maintenance therapy
1.Non-HLA antibodies
2.Non-spectic binding of T cells due high level of immunoglobulin in the patient’s sera
3.Not all class I antibody specificities have been identified
Positive T cell crossmatch with negative B cell in the absence of DSA with negative autocross match is due to:
A. Low-level class II antibodies due to high concentrations of class II HLA antigens in T cells
Wrong; because T cell does not express class II HLA
B. Non-HLA antibodies
Yes, non-specific immunoglobulin binding to Ig Fc receptors on lymphocytes, these can be treated by the addition of pronase enzyme which can digest the Fc receptors and other surface proteins (CD20) so, reducing nonspecific binding of patient serum to lymphocytes and reducing the incidence of false-positive reaction in FCXM.
Non-HLA and auto-antibodies can explain
C. Low level class I antibodies due to high concentration of class II HLA antigens in T cells.
T cell does not express HLA class II. Class I has different concentrations between T and B cells, HLA-C is expressed more on T cells and this can explain weak positive T-cell FCXM.
D. Technical errors
Yes, can be
E. May need aggressive induction
In absence of DSA there is no need for aggressive Induction, but we need to correlate with patient history of sensitization
-Cross matching 100 mismatch means that HLA A is incompatible which has less impact on graft survival while HLA B ,HLA DR are compatible
No DSA and positive T cell and negative B cell cytometery indicates multiple possibiliteies as
technical error (possibly related to B-cell viability) and the test should be repeated
Another explanation is that there would be a higher false positive rate in non-sensitized patients than in sensitized patients also another factor determining the significance of the result is the cut-off values used to determine a positive test
-In unsensitized recipients, an isolated positive flow cytometry crossmatch in the presence of negative autocrossmatch and no DSA is considered clinically insignificant and transplant can proceed
Induction agent IL2R antagonist as she could be standard risk patient
Maintenance with triple therapy Tacrolimus ,MMF and steroids
Positive T cell crossmatch with negative B cell in the absence of DSA with negative autocross match is due to
answer
B and D
positive T-cell flow crossmatch suggests that there is a DSA against HLA class I antigen ,
the cell surface expression of HLA-class I antigens is higher on B than T cells Thus, a T+B+ FCXM, rather than a T+B-, is the expected result in the presence of HLA-class I antibodies.
Justification
Could be that IgG antibodies directed at HLA- A, B and/or C locus determined antigens are associated with a T+B- result. Differential expression of HLA on T cells and B cells and/or differential binding of antibodies to T cells and B cells and Control Serum used to qualify a FCXM test as positive or negative are potential contributors to the surprising T+B- FCXM result.(1)
or non-complement fixing antibody,
or a non-HLA antibody include MICA, angiotensin type I receptor antibody, anti-vimentin and anti-collagen antibody which can be identified by Luminex solid phase assays
or a low-level antibody.
Or technical error and variable cut off values used
1- Putheti P et al T Cell Positive B Cell Negative Flow Cytometry Crossmatch (FCXM): Frequency, HLA-Locus Specificity, and Mechanisms Among 3073 Clinical FCXM Tests. medRxiv preprint doi: https://doi.org/10.1101/2021.05.20.21257541; this version posted May 24, 2021
Sera with anti-HLA-Cw IgG had also been shown by others to cause T+B FCXM .
Possible confounding due to the presence of non-HLA antibodies.
A positive CDC-XM result can be due to autoantibodies, and these are usually IgM.
Autoantibodies are found in autoimmune diseases.
An auto-crossmatch (recipient serum tested against recipient lymphocytes) can be used
to exclude the presence of autoantibodies.
IgM autoantibodies are normally ignored in kidney transplantation
The influence of IgM antibodies on the crossmatch result can be eliminated by the
following maneuvers:
i. Adding dithiothreitol (DTT)
ii. Heating the recipient’s serum to 55°C.
iii. Washing (Amos-modified CDC-XM)
iv. Autocross match.
v. Prolonged incubation period.
Answer is D and B.
Reference:
Martin Chari, Mohsen El Kos,Jon Kim Jim,Ajay Sharma, Ahmed Halawa.
in Renal Transplantation by Non-Immunologists for Non-Immunologists.
Urology & Nephrology Open Access Journal, ISSN: 2378-3176
as we all know a positive T cell crossmatch with CX as done with Flowcytometry which is sensitive to low titers of antibodies reflects anti HLA due to class I antigens. but because B FCX is negative this is not accepted and we need further workup. It could be due to technical error but speaking in general, it may be true due to autoantibodies (so we need the history of autoimmune disease). The patient may be treated before with monoclonal antibodies lite rituximab etc. History of vaccination. as in history, it is reflux nephropathy and nothing evident or no clue for autoimmune disease etc. recent vaccination could be a reason.
this is an excellent crossmatch with just one mismatch so it is low risk. many centers may prefer 1 low dose rATG because the history of 16 years carries many possibilities of being transfused sensitized etc. we may need auto flow to crossmatch ( by mixing recipients lymphocyte and serum to clarify this phenomenon). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4961613/
,,, I will prefer 1 dose of rTG may be 1.5 mg/kg as this is a low risk transplant for ABMR
Basically, the main cause of a positive XM result is the presence of preformed antibodies that recognize and react specifically to donor HLA molecules.
So, a Positive T cells FCXM with negative B cells FCXM and no DSA and no history of autoimmune diseases
may be due to:
The right answers are:
B: Prescence of non HLA antibodies (Anti-angiotensin II type 1 receptor
IgG,MICA,MICB) non-HLA antibody-mediated rejections have been increasingly
reported so they have to be tested. Also, presence of incompatible ABO can cause
positive FCXM through anti blood type Ig G antibodies detected as anti-donor Ig
G antibodies.
*Another explanation: As HLA-Cw antigen expression was found to be higher on T cells than B cells, antibodies directed at HLA-Cw antigens may contribute to a T+B- FCXM
D: Technical errors can be the cause of positive cross match in absence of DSAs.
E: May need aggressive induction: according to repeat FCXM and presence of absence of other causes that can cause acute graft rejection.
*Needs to do Solid phase assay (Luminex) to rule out the presence of very low level DSAs.
A & C wrong because T cell expressed only class I HLA
Referene:
Yu S, Huh HJ, Lee KW, Park JB, Kim S-J, Huh W, et al. Pre-transplant angiotensin II type 1 receptor antibodies and anti-endothelial cell antibodies predict graft function and allograft rejection in a low-risk kidney transplantation setting. Ann Lab Med. 2020;40:398–408. doi: 10.3343/alm.2020.40.5.398
Factors affecting positivity of B and T cross match :
I- HLA antigen expression
T cell express only HLA class I while B cell (as it act as APC) express HLA class I and II, So ideally…
II- Differential expression
III- Differential binding of antibodies to T cells and B cells
IV- Channel shift
V- Non HLA abs
VI- Autoantibodies
VII- Vaccination
VIII- Rituximab
IX- Technical error
X- Inter-center, inter-laboratory variability in performing FCM
So…
T cell + B cell + cross match indicates
1- DSA directed to class I, or both class I, II
2- Non HLA AB directed to B and T cells
T cell – B cell + cross match indicates one of the following
1- True DSA directed to only class II
2- DSA directed to both class I and II and T cell cross match is negative due to
3- Rituximab intake which is monoclonal AB directed B cells only
4- Auto AB directed against B cell
5- Non HLA AB directed to B cells
6- Vaccination altering the surface of B cell
T cell + B cell – cross match may indicate one of the following:
1- True DSA against HLA class I and B cell cross match is negative due to :
2- Technical error
3- Non HLA AB directed to B cells
Auto AB directed against T cell and vaccination altering the surface of T cell are extremely unlikely causes of T cell + B cell – cross match
Positive T cell cross match has the worst prognosis and donor should be excluded If proved to be due to DSA directed to HLA class I, It was found that DSA directed against HLA-I antigens are associated with high risk of hyper acute and acute rejection.
Positive T cell crossmatch with negative B cell in the absence of DSA with negative autocross match is due to
1- Low level class II antibodies due to high concentrations of class II HLA antigens in T Cells
Wrong, as T cells has no class II antigens
2- Non-HLA antibodies
Correct if directed to T cells only
3- Low level class I antibodies due to high concentration of class II HLA antigens in T Cells
Wrong, as T cells has no class II antigens,
4- Technical error
Correct
5- May need aggressive induction
Wrong, as there is no DSA, but if DSA present desensitization and aggressive induction are required
REFERANCES
1- Pellegrino MA, Belvedere M, Pellegrino AG, Ferrone S. B peripheral lymphocytes express more HLA antigens than T peripheral lymphocytes. Transplantation. 1978;25(2): 93-95.
2- Honger G, Krahenbuhl N, Dimeloe S, Stern M, Schaub S, Hess C. Inter-individual differences in HLA expression can impact the CDC crossmatch. Tissue Antigens. 2015;85(4): 260-266.
3- Badders JL, Jones JA, Jeresano ME, Schillinger KP, Jackson AM. Variable HLA expression on deceased donor lymphocytes: Not all crossmatches are created equal. Hum Immunol. 2015;76(11): 795-800
Yes , transplantation is better than leaving her more on hemodialysis. .mismatch at A locus has no significant effect on graft survival1.However, if the graft fails, her odds of rejection of the second transplant will be high 2
Induction : basiliximab because the most likely possibility is the presence of non-HLA antibodies. These might be non pathogenic . The risk is still there so we need to monitor their level and consider protocol biopsy to avoid subclinical AMR 3
Maintenance : tacrolimus, mycophenolate mofetil and prednisone
1.Shi, X., Lv, J., Han, W. et al. What is the impact of human leukocyte antigen mismatching on graft survival and mortality in renal transplantation? A meta-analysis of 23 cohort studies involving 486,608 recipients. BMC Nephrol 19, 116 (2018). https://doi.org/10.1186/s12882-018-0908-3
2. Vasilios Kosmoliaptsis,Olivera Gjorgjimajkoska,Linda D. Sharples,Afzal N. Chaudhry,Nikolaos Chatzizacharias,Sarah Peacock,Nicholas Torpey,Eleanor M. Bolton,Craig J. Taylor,J. Andrew Bradley, Impact of donor mismatches at individual HLA-A, -B, -C, -DR, and -DQ loci on the development of HLA-specific antibodies in patients listed for repeat renal transplantation, CLINICAL INVESTIGATION, VOLUME 86, ISSUE 5, P1039-1048, NOVEMBER 01, 2014
A& C : wrong ,class II are present only in B cells.
B :right ,FCXM can detect all types of antibodies including non HLA 3
D : right :
Other causes Aberrant expression of class I on B cells
autoantibodies against T cells1
antibodies against A, B or Cw loci 2
E : wrong , could be non pathogenic antibodies
ref:
1.Prabhakar Putheti, VijayK Sharma, Rex Friedlander, Arvind Menon, Darshana Dadhania, Thangamani Muthukumar, Manikkam Suthanthiran, T Cell Positive B Cell Negative Flow Cytometry Crossmatch (FCXM): Frequency, HLA-Locus Specificity, and Mechanisms Among 3073 Clinical FCXM Tests ,medRxiv 2021.05.20.21257541; doi: https://doi.org/10.1101/2021.05.20.21257541
2.Putheti, Prabhakar & Sharma, Vijay & Friedlander, Rex & Menon, Arvind & Dadhania, Darshana & Muthukumar, Thangamani & Suthanthiran, Manikkam. (2021). T Cell Positive B Cell Negative Flow Cytometry Crossmatch (FCXM): Frequency, HLA-Locus Specificity, and Mechanisms Among 3073 Clinical FCXM Tests. 10.1101/2021.05.20.21257541.
3. Zachary A ,Leffell M, transplant immunology , human press,2ond edition .287
Positive T cell crossmatch with negative B cell may occur in :
Laboratory error and should be repeated
History of autoimmune disease, with autoantibodies directed against T but not B cells which is not in our case
Received monoclonal antibodies that specifically bind to T cells
Antibodies directed against HLA-Cw antibodies were found in almost all patients with T+ B- FCXM in one study.
Or non-HLA ab
Judith Desoutter et al. reported donor and recipient investigations that revealed unexpected positive B-cells crossmatch, probably due to donor cells, as the donor had received rituximab therapy shortly before organ harvesting 1.
It might be also H-Y minor histocompatibility
Study on the effect of minor H-Y HLA showed transplantation of male donor kidneys into female recipients was associated with an increased risk of graft failure during the first year 3.
A positive T-lymphocyte CDC-XM in the absence of IgM autoantibodies is currently an absolute contraindication to kidney transplantation. One the other hand, the importance of a positive B-lymphocyte crossmatch should be interpreted together with the Luminex test. This is because as much as 50% of B-cell CDC-XM results can be false positive. Thus, a positive B-cell result is considered as a relative contraindication to transplantation 2.
For this case i would go for induction with ATG and triple IS.
1Judith Desoutter et al. Case Rep Transplant. 2016.False Positive B-Cells Crossmatch after Prior Rituximab Exposure of the Kidney Donor.
2 Eng HS, Bennett G, Tsiopelas E, et al. Anti–HLA donor–specific antibodies detected in positive B–cell crossmatches by Luminex predict late graft loss. Am J Transplant. 2008;8(11):2335–2342.
3Alois Gratwohl et al. Lancet. 2008.H-Y as a minor histocompatibility antigen in kidney transplantation: a retrospective cohort study
B and D
Dear All, please respond to the question, select the best possible answer, and importantly Justify your decision.
1- Non HLA Ab against T cell only.
2- Autoantibodies ( not expected in this case as no history of autoimmune disease or sensitization history)
3- Anti HLA antibodies against HLA-A, B or Cw locus are associated with a T+B- result (1)
4- Lab error.
To confirm the result
1- Do Luminex SAP to exclude HLA class A, B , Cw .
2- Auto crossmatch to exclude autoantibodies.
Ref
1-Putheti P, Sharma VK, Friedlander R, Menon A, Dadhania D, Muthukumar T, Suthanthiran M. T Cell Positive B Cell Negative Flow Cytometry Crossmatch (FCXM): Frequency, HLA-Locus Specificity, and Mechanisms Among 3073 Clinical FCXM Tests. medRxiv. 2021 Jan 1.
B- this patient is a good candidate for kidney transplantation as
1- No history of sensitization
2- No DSA
3- Living related donor with good HLA matching 100
Immunesuppression protocol
As she is standard immunological risk
Induction with basiliximap and maintenance triple IS ( steroids, CNI, MMF).
Well done
Case interpretation and management
This patient is a good kidney transplant candidate because: (a) she has no history of sensitization, (b) there is only one out of six HLA mismatch, (c) negative DSA. However, she has a weakly positive T cell on FCXM with a negative B cell cross match. A T cell positive FCXM where there is no antibody binding with B cells suggests that the antibody may not be HLA-specific.
Putheti P et al, suggests antibodies directed at HLA-Cw antigens may contribute to a T+B- FCXM. Additionally, antibodies directed at HLA-A and antibodies directed at HLA-B are also associated with a T+B- FCXM. A repeat crossmatch should be considered. She is considered a standard risk for transplant.
I will consider induction with either ATG or basiliximab. Immunosuppressive medications are triple; Tacrolimus, MMF, and steroid.
Reference:
1. Putheti P, Sharma VK, Friedlander R, Menon A, Dadhania D, Muthukumar T, Suthanthiran M. T Cell Positive B Cell Negative Flow Cytometry Crossmatch (FCXM): Frequency, HLA-Locus Specificity, and Mechanisms Among 3073 Clinical FCXM Tests. medRxiv. 2021 Jan 1.
2. Chari M, Kosi ME, Jim JK, Sharma A, Halawa A (2017) Crossmatching in Renal Transplantation by Non-Immunologists for Non-Immunologists. Urol Nephrol Open Access J 5(2): 00166.
A 41-year-old CKD 5 lady secondary to reflux nephropathy received a kidney offer from her cousin with 100 mismatch. FCXM showed negative B cell cross match, T cell was weakly positive, no DSA. She is on the waiting list for 16 years.
What is the explanation of this crossmatch?
Would you go ahead and transplant her?
FCXM assay:
This test performed by adding the recpient serum to the donor lymphocytes (T or B) in the presence of anti-IgG fluorescein-labelled antibodies. when donor specific antibodies are absent no binding will occur
While when donor-specific antibodies bind to lymphocytes that are detected by flow cytometry once the anti-IgG fluorescein labelled antibodies tag the lymphocytes. and are quantified by detectors in the impedance flow cytometer.
Interpretation of weak Positive T CELL-FCXM, negative B cell FCXM, No DSA:
1-non-complement fixing antibodies
2-non-HLA antibodies
3-Low-level antibodies
4- the cut-of values are not uniform cross laboratories. Low cut offs would increase
sensitivity while affecting specificity (3).
Report from large retrospective study showed that CDC crossmatch negative patients who had positive T-cell flow cytometry results had significantly poorer
absolute 5-year graft survival rates compared to those who were both CDC and
flow cytometry crossmatch negative [4)
this patient was on long waiting list(16 years) and if she is on hemodialysis which is likely yes the HD dialyzer membrane could be immunogenic and lead to an increased risk of graft loss,In one report they found that despite using the biocompatible membranes, patient survival on pre-transplant PD was still superior to the HD counterparts [2]
If you decided to go ahead, what is your immunosuppression protocol?
so in her case despite she is one mismatch from LD with negative DSA which may be due to low titer or non HLA antigens , not detected by FXM Preferred to do luminex single bead antigen (SAB) which is more sensitive if negative by SAB will go ahead with transplantation with basiliximab induction followed by triple IS with tacrolimus based, MMF, steroid and DSA monitoring post transplantation .
References:
1- An Update on Crossmatch Techniques in Transplantation
Kumar A1,2, Mohiuddin A2,3, Sharma A2,3, El Kosi M2,4 and Halawa A2,5*
2–Van Biesen W, Veys N, Vanholder R, Lameire N. The impact of the pre-transplant renal replacement modality on outcome after cadaveric kidney transplantation: the ghent experience.Contrib Nephrol 2006; 150: 254-258.
3-Tait BD, Hudson F, Cantwell L, Brewin G, Holdsworth R, et al. (2009)
Review article: Luminex technology for HLA antibody detection in organ
transplantation. Nephrology (Carlton) 14: 247-254.
4- Graf RJ, Buchanan PM, Dzebisashvili N, Schnitzler MA, Tuttle-Newhall
J, et al. (2010) Te clinical importance of fow cytometry crossmatch in
the context of CDC crossmatch results. Transplant Proc 42: 3471-3474.
B cell negative and t cell positiv crosshatch indicate technical error possibly related to become cell viability
Test should be repeated
What are the other possibilities?
Presence of Non-HLA-specific antibodies. Antibodies directed against HLA-Cw antigens, HLA-A or HLA-B are also associated with a T+B- FCXM.
positive T Cell crossmatch with negative B cell explained by autoantibodies, non HLA antibodies or recent vaccination, In this case ,weekly positive T cell crossmatch & negative B cell(B cell has class 1 & 2 while T cell has class 1 ), we can repeat crossmatch & auto crossmatch if negative we can proceed for transplant.
the transplant is considered low risk as 100 mismatch , negative B cell crossmatch & no DSA , the immunosuppressive medication : induction basiliximab & triple Tacrolimus ,MMF& steroid.
negative B-cell with positive T-cell cross matches seen if there is a technical error(as B cell has more concentrated class 1 than T cell) , presence of autoantibodies, non-HLA antibody or recent vaccinations.
2- I will repeat the cross match again to confirm the technique is done appropriately.
3- immunosuppressive(Basiliximab and Tacrolimus, MMF and prednisolone. some centres if the recipient has 0 mismatches at DR , avoid the anti-proliferative.
I would not consider this patient a low immunological risk. This kidney failed after 4 years. Do you know why?
I guess he has non HLA antibody.
What is the explanation of this crossmatch?
1. Presence of auto antibodies that bind T cell only
2. Technical error
3. Monoclonal AB binding T cell
4. Vaccination altering surface ag on the surface of T cells
Would you go ahead and transplant her?
Yes as all these causes does not involve HLA antigens
If you decided to go ahead, what is your immunosuppression protocol?
Yes if ABO compatible, this patient is considered low risk patient so the recommended protocol will be induction with basiliximab, maintenance triple therapy and fu
Thanks Dr Sherif
Unlikely due to auto-antibodies, What else it could be?
You did answer similar question before.
The HLA matching is 100 mismatch that mean there’s one mismatch in A loci and it may be minor antibodies for epitope in A loci this make this weak positive cross match. So I wouldn’t go ahead for transplant until did desensitization and get a negative cross match result .
Immunosuppressive protocol will be induction with ATG and maintenance by CNI,MMF and predinsilone
What are you going to desensitise Dr Nazik?
T cell cross match was positive so the patient may had non DSA antibodies that why am thinking about desensitization
It is difficult to desensitise as we do not know the HLA antibodies causing positive crossmatch. We desensitise what we know and have a level.
A crossmatch with negative B cell crossmatch and a weakly positive T cell signifies a technical error (might be related to non-viable B cells) and should be repeated ideally.
Putheti et al showed that HLA antibodies directed at HLA-A, B or Cw locus are associated with a T cell positive and B cell negative crossmatch result. They also postulated mechanisms for such results.
There is no DSA. 100 mismatch and negative B cell FCXM with weak positive T cell FCXM.
Yes. I would go ahead with the transplant.
If no history of sensitization, It is standard risk transplant. Induction with Basiliximab and maintenance with triple drug immunosuppression (Tacrolimus, MMF and steroids)
Reference:
1) Altaf MM, Kossi ME, Jin JK, et al. Human leukocyte antigen typing and crossmatch: a comprehensive review. World J Transplant 2017;7:339-348.
2) Putheti P, Sharma VK, Friedlander R, et al. T cell positive B cell negative flow cytometry crossmatch (FCXM): frequency, HLA locus specificity, and mechanisms among 3073 clinical FCXM tests. medRxiv 2021.05.20.21257541
What else it could be?
You did answer similar question before
Non-HLA antibodies
What is the explanation of this crossmatch?
Would you go ahead and transplant her?
If you decided to go ahead, what is your immunosuppression protocol?
Correction Ibrahim T cells carry ONLY class I HLA.
sorry, it was by mistake.
thank you
What else it could be?
What is the explanation of this crossmatch?
As we already learned that the presence of HLA-class I antibodies result in a T+B+ FCXM, rather than a T-B+, knowing that HLA class I is more expressed on the surface of B cells comparing to the T cells. A T-B+ FCXM is expected in the presence of HLA-class II antibodies because HLA-class II antigens are expressed on B cells but not on T cells.
The result of T+B- crossmatch is extremely rare, unexpected and uncommon and needs further testing. The introduction of SAB assay in the detection of preformed anti-human leukocyte antigen (HLA) antibodies has improved transplantation success and helped in identification of new antibodies against previously unidentified antigens.
The result of T+B- crossmatch could be explained by following factors:
-Lab error and should be repeated for confirmation
– History of autoimmune disease, with autoantibodies directed against T but not B cells
– History of monoclonal antibodies that specifically bind to T cells
– History of vaccination, for example COVID-19 vaccine was shown to alter the surface expression of some antigens and make the antibodies bind nonspecifically.
– Antibodies directed against HLA-Cw (antibodies directed at HLA-Cw were found in almost all patients with T+ B- FCXM in one study recently published)
– Could be partially caused by CREG2 reactivity in addition to non-HLA interference
Would you go ahead and transplant her?
In this patient with history of reflux nephropathy, surgical anatomical correction at UVJ should be performed initially. From immunological point of view, as the flow cytometry is only weakly positive for T cells, negative DSA and living related kidney donor of 100 mismatch, the risk of rejection is not high.
I would recommended induction therapy with Anti IL-2 and pulse steroids followed by maintenance therapy CNI based+ MMF with or without steroids
REFERENCES
Lecture by ASNRT on crossmatching
doi.org/10.1101/2021.05.20.21257541
doi.org/10.1016/j.humimm.2017.06.295
doi.org/10.1016/j.humimm.2012.07.305
What else it could be?
You did mention this before
If we are talking about the presence of non-IgG antibodies, for example IgM antibodies against non-HLA components of T lymphocytes, but this is not CDC cross match. Here the crossmatch is FCXM which only detects IgG. Recipients that have only anti-HLA IgM antibody have a negative flow cytometric crossmatch since the fluorochrome-labeled detection antibody is selective only for IgG, so this option is not applicable here.
Another option, the presence of low titer of non-HLA antibodies to T lymphocytes which is not detected as significant DSA because of low MFI.
The CDC-XM may be applied to both T cells and B cells. The former reflects the presence of HLA class I antibodies, while the latter reflects both HLA class I and II antibodies. Since B cells express higher amounts of class I antigens, a positive B cell CDC-XM associated with a negative T cell CDC-XM may indicate low levels of class I antibodies.
HLA-class I antigens are expressed on T cells and B cells, and some but not all studies suggest that the cell surface expression of HLA-class I antigens is higher on B than T cells{Pellegrino MA, Belvedere M, Pellegrino AG, Ferrone S. B peripheral lymphocytes express more HLA antigens than T peripheral lymphocytes. Transplantation. 1978;25(2): 93-95.} {Honger G, Krahenbuhl N, Dimeloe S, Stern M, Schaub S, Hess C. Inter-individual differences in HLA expression can impact the CDC crossmatch. Tissue Antigens. 2015;85(4): 260-266.}. Thus, a T+B+ FCXM, rather than a T+B-, is the expected result in the presence of HLA-class I antibodies. A T-B+ FCXM is the anticipated result in the presence of HLA-class II antibodies only since HLA-class II antigens are expressed on B cells but not on resting T cells {Daar AS, Fuggle SV, Fabre JW, Ting A, Morris PJ. The detailed distribution of MHC Class II antigens in normal human organs. Transplantation. 1984;38(3): 293-298.}
A mechanism for the T+B- FCXM test result is the differential expression of HLA-class I antigens on T cells and B cells. HLA-Cw antigen expression, as detected using mAb DT9, was found to be higher on T cells than B cells, and in this study, seven of 10 FCXM T+B- FCXM were observed with sera containing HLA-Cw DSA. Sera with anti-HLA-Cw IgG had also been shown by others to cause T+BFCXM. Our findings are consistent with the earlier findings that antibodies directed at HLA-Cw antigens may contribute to a T+B- FCXM. Importantly, we report that antibodies directed at HLA-A and antibodies directed at HLA-B are also associated with a T+B- FCXM.{T Cell Positive B Cell Negative Flow Cytometry Crossmatch (FCXM): Frequency, HLA-Locus Specificity, and Mechanisms Among 3073 Clinical FCXM Tests Prabhakar Putheti et al,: https://doi.org/10.1101/2021.05.20.21257541; }
A mechanism for the T+B- FCXM:
Frequency of T+ B- FCXM ( a look at study):
Result A total of 3609 clinical FCXM tests were performed in our laboratory from February 2014 to April 2018 and data from 3073 FCXM tests were included in downstream data analysis .
({T Cell Positive B Cell Negative Flow Cytometry Crossmatch (FCXM): Frequency, HLA-Locus Specificity, and Mechanisms Among 3073 Clinical FCXM Tests Prabhakar Putheti et al,: https://doi.org/10.1101/2021.05.20.21257541; })
Would you go ahead and transplant her?
Exellent explanation of the impact of T+ B- FCXM
But your +\- anticipation of DSA ? is not needed.
thanks for motivation
+/- based on DSA detection
Auto-antibodies against T cells & HLA-Cw Ab ( HLA-Cw Ag expression is higher in T cells than B cells) can give T+B- FCXM result.
This patient can precede to transplantation with T cell depleting agent as induction therapy, & triple immunosuppression (CNI+MMF+Steroids) as maintenance.
T cells express only class I antigens while B cells express both. Hence, a positive T cell and B cells cross match would indicate presence of antibodies to HLA type I and II antigens while a positive B cell cross match could indicate either (i) DSAs to type II antigens alone or (ii) Low levels of DSAs to type 1 antigens. A positive T cell crossmatch alone is usually due to technical error.
IF CDC IS NEGATIVE, A POSITIVE FLOW CYTOMETRY COULD BE INTEEPRETED AS FOLLOWING
1-NON COMPLAEMENT FIXING ANTIBODIES
2-NON HLA ANTIBODIES
3-LOW LEVEL ANTIBODIES
YES I WILL GO AHEAD FOR TRANSPLANTION .
I WILL GO FOR BASILIXMAB AS INDUCTION IMMUNSUPPRESSION , LOW IMMUNOLOGICAL RISK PT .
MAINTINACE AS RECOMMENED BY ALL CENTRES (CNS , ANTIMETABOLITE, PRED)
REFFERNCE
Kumar A, Mohiuddin A, Sharma A, El Kosi M, Halawa A (2017) An Update on Crossmatch Techniques in Transplantation. J Kidney 3: 160. doi:10.4172/2472-1220.1000160
What is the explanation of this crossmatch?
Would you go ahead and transplant her?
As long as there is no DSA , I will transplant her
If you decided to go ahead, what is your immunosuppression protocol?
Induction with ATG and maintenance therapy with Tac, MMF and tapered doses of steroid.
References:
explanation:
1- lab error, to be repeated , if still positive do:
2- detect autoantibodies which are IgM, then you have to repeat XM after neutralization of IgM
yes , i will go for transplantation safely because no DSA.
immunosuppressive protocol induction with ATG, maintenance on CNI, MMF, steroid with slow tapering with close follow up.
Nalaka Gunawansa, Roshni Rathore, Ajay Sharma and Ahmed Halawa. Crossmatch Strategies in Renal Transplantation: A Practical Guide for the Practicing Clinician. Journal of Transplant Surgery. REVIEW ARTICLE. Oct 2017 | VOLUME 1 | ISSUE 1.
presence of non-HLA antibodies also can cause positive XM
T cells have only HLA class I surface antigen, while B cells have both HLA class I and II. Therefore, a Negative B cell crossmatch with weak positive of T cell crossmatch with No DSA, as in this case scenario, may have several possible causes other than a genuine immunological incompatibility. Possible causes include:
– Technical errors (in This case, repeating crossmatch may solve the problem).
– Autoimmune disease (Auto crossmatch for the recipient will also be positive due to autoantibodies).
– History of monoclonal antibodies against T cell surface antigens (History of monoclonal antibody therapy will explain the situation).
– Occasionally post-vaccination (some cases were noted to have positive crossmatch post-COVID-19 vaccination, and again a careful history can explain this uncommon crossmatch result).
The transplantation workup for this patient should cover immunological and non-immunological risk factors.
The original kidney disease in this case scenario is reflux nephropathy. Therefore, we should have a detailed history of any symptoms or previous lab results documenting recurrent UTI. In addition, a voiding cystourethrogram may be required to evaluate the presence and severity of reflux (1).
Native kidney nephrectomy is not routinely required in such cases. However, it may be a valuable option if the patient has recurrent infection secondary to reflux. Considering the potent immune suppression during the induction and the need for livelong maintenance immune suppression mandates eliminating any confirmed infection source pre-transplantation (1).
I will consider this case is at low immunological risk. On the other hand, the patient is 41 years old lady who was on dialysis for 16 years, which most probably exposed her to long term complications of dialysis (e.g. osteoporosis).
I suggest potent induction using ATG to allow early steroid withdrawal in order to avoid the additive side effects of long-term steroids on the patient health. The maintenance immune suppression can be with dual agents (Tacrolimus and MMF)
References:
1) Ana P Rossi and Christina L Klein. If you decided to go ahead, what is your immunosuppression protocol? © 2021 UpToDate. (Accessed on 4 December 2021)
What else it could be?
What else it could be?
Dear Dr Ahmed,
Do you ask about non-HLA antigens? (as it will be associated with positive CDC and flow cytometry crossmatch. Meanwhile, No detected DSA by Solid-phase assay, which is only specific for HLA antigens)
Other possibilities as summarized in the attached table with a red arrow marking the situation with negative antibody detection by solid-phase assay while there is positive flow cytometry crossmatch (1). causes include:
– Non-HLA antibodies.
– Antibodies against specific loci which were not listed by the DSA assay kits.
– Newly formed DSA which were absent in the last serum examined for DSA (a new event that led to sensitization).
References:
1) Melissa Y Yeung. Kidney transplantation in adults: Overview of HLA sensitization and crossmatch testing. © 2021 UpToDate. (Accessed on 7 December 2021)
Low level class II antibodies due to high concentrations of class II HLA antigens in T Cells
Wrong as T cells have only class I antigens.
Non-HLA antibodies
Right.
Low level class I antibodies due to high concentration of class II HLA antigens in T Cells
Wrong as T cell have only class I antigens
Technical error
Right. As T cells have class I antigens and B cells have both class I and II antigens ,positive T cell and negative B cell FCXM can be a technical error
May need aggressive induction
Since DSA is negative ,Induction with ATG and maintenance with Tac, MMF and Prednisolone.
Other causes of a positive T cell and negative B cell crossmatch
1)differential expression of HLA class I antigens like Cw, A, B on T and B cells .
2)aberrant expression of HLA class I on B cells
3)impaired binding of anti HLA antibodies.
4) Higher CS used to classify a B cell FCXM.
5)Pronase treatment used for FCXM may unmask cryptic epitopes on T cells leading to false positive T cell FCXM.
6)autoantibody against T cell( ruled out by negative auto cross match)
7)source of cells( HLA class I expression is lower on B cells isolated from deceased donor blood compared to B cells isolated from spleen, lymph node or B cells isolated from living donor blood.