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Abbreviations: ATG: Anti-Thymocyte Globulin; CDC: Complement Dependant Cytotoxicity;
DBD: Donation After Brain Stem Death; DCD: Donation After Circulatory Death;
DSA: Donor Specific Antibody; FCXM: Flow Cytometry Cross-match; GIFT:
Granulocyte Immunofluorescence Test; HLA-Ab: Human Leucocyte Antigen specific
Antibodies; HLA-DSA: Human Leucocyte Antigen Donor Specific Antibodies; HNA:
Human Neutrophil Antigens; LIFT: Lymphocyte Immunofluorescence Test; LKD:
Living Kidney Donor; NAIN: Neonatal Alloimmune Neutropenia; MICA: Major
Histocompatibility Complex Class I Polypeptide-Related Sequence A; MICB: Major
Histocompatibility Complex Class I Polypeptide-Related Sequence B; RMF:
Relative Mean Fluorescence; TRALI: Transfusion Associated Acute Lung Injury
INTRODUCTION
Antibodies directed against endothelial and non-Human Leucocyte Antigen
(HLA) targets are known to account for positive cross-matches and cause graft
dysfunction in recipients of kidney transplants both in the absence of HLA
specific antibodies (HLA-Ab) and where no donor HLA specific antibodies
(HLA-DSA) can be detected [1].
Non-HLA antigens to which antibodies have been detected include the
Major Histocompatibility Complex class I polypeptide-related sequence A and B
(MICA and MICB) [2], vimentin [3], angiotensin II type I receptor antibody [4]
and glutathione-S-transferase [5]. These antigens are both polymorphic (MICA
and MICB) and non-polymorphic (vimentin). Antibodies directed against these
antigens have been reported to be associated with antibody mediated rejection
in solid organ transplantation.
The Human Neutrophil Antigen (HNA) system currently consists of five
identified groups (HNA 1-5), with limited polymorphism, present on diverse
extracellular structures including Fc-γ receptor IIIb (HNA-1) and choline
transporter-like protein 2 (HNA-3) [6]. HNA-3 is expressed on neutrophils,
lymphocytes, platelets, endothelial cells, kidney and spleen. HNA-3 is a
bi-allelic system comprising HNA-3a (SLC44A2*01) and 3b (SLC44A2*02)
Alloimmunisation to HNA occurs mainly in pregnancy where the mother produces
neutrophil specific antibodies to mismatched paternal antigens present in the
foetus. Around 5% of the Caucasian population are homozygous for HNA-3b and
thus at risk of allosensitisation to HNA-3a. HNA specific antibodies are known
to be clinically relevant and are a cause of transfusion associated lung injury
and neonatal alloimmune neutropenia [7,8]. The expression of HNA on lymphocytes
has the potential to result in a positive
MATERIALS AND METHODS
Seven kidney transplant recipients with identified HNA-3a antibodies and
transplanted between 2006 and 2018 were analysed as a series of case studies.
All recipients were Caucasoid females, with a sensitisation history of
pregnancy, pregnancy and blood transfusion or in one case a previous kidney
transplant.
All HNA testing was performed retrospective to the transplant. All recipients
and donors were HNA genotyped by sequence-based typing methods. Recipients were
tested for HNA-Ab by indirect granulocyte immunofluorescence test (GIFT),
lymphocyte immunofluorescence test (LIFT) utilising recombinant HNA-3a positive
cell lines and Luminex®. Recipient HLA specific antibodies were
detected and characterised using a combination of Luminex® based
assays (One Lambda Inc LABScreen® single antigen, screen and I.D,
analysed using a LABScan™ 100 flow analyser). Pre-transplant crossmatch was by
3 colour flow cytometry (FCXM) and in addition in case 2 complement dependant
cytotoxicity (CDC). Positivity in the FCXM was determined by centre specific
criteria and reported as relative mean fluorescence (RMF). Histological
analysis was performed using the current Banff classification of renal
allograft pathology in use at the time of the kidney transplant biopsy.
Maintenance immunosuppression consisted of standard triple therapy using
tacrolimus, mycophenolate mofetil and prednisolone. Induction therapy was with
either anti-thymocyte globulin (ATG) or Basiliximab.
RESULTS AND CASE
REPORTS
The kidney transplants were performed at five different UK centres (2 of
the centres each transplanted 2 recipients). Four of the seven recipients had
at least one previous unexplained positive FCXM with other potential donors
which had resulted in non-proceeding transplants (range 1-6). The donors
included both living donors (LKD) and deceased donors (DCD and DBD). All
recipients except patient 7 received induction therapy at the time of
transplant due to the positive pre-transplant donor cross-match. Patient 7 was
found to have a positive cross-match on retrospective testing following a
virtual cross-match (Table 1).
DISCUSSION
A range of non-HLA proteins have been documented to be
potential targets for antibodies in the setting of solid organ transplantation.
Not all are expressed on the surface of leucocytes and hence may remain
undetected in the context of the usual cross-match assays for transplantation.
They can be identified in some cases using endothelial cell cross-match
techniques and solid phase assays. Some are however expressed on leucocytes and
therefore, may give rise to an unexplained positive cross-match in patients
where there is no HLA specific antibody present or no HLA DSA. Where auto
reactive antibodies are not present the laboratory should seek to identify the
cause of the positive cross-match.
This report on seven individuals includes the first reported
case (patient 2) of HNA-3a specific antibodies retrospectively detected in an
individual with delayed graft function, vascular and cellular rejection at 5
days and persistent vascular rejection and positive C4d staining at 13 days.
Following treatment, the patient did become dialysis independent but ultimately
lost their graft after 1 year [9]. In the cases presented here all patients had
a positive FCXM on the day of transplant with their respective kidney donors.
We retrospectively investigated possible causes of this and found all the
patients had circulating antibodies to HNA-3a. Antibodies to HNA-3a are the
likely cause of the positive FCXM in these allosensitized individuals
who are
homozygous for HNA-3b. Such patients potentially face
long waiting times for transplantation because around 95% of donors will
express HNA-3a and these recipients are therefore likely to have multiple
positive FCXMs which can lead to a decision to not proceed with the transplant.
The high frequency of HNA-3a expressing individuals also accounts for the
observed phenomena of previous positive cross-matches often with multiple
potential apparently HLA compatible donors.
In this series of cases we report a range of outcomes although
in only one case (patient 6) did the post-transplant course appears to be
incident free. In the cases of patient 2 and 5 both grafts were lost within a
year of transplant following deterioration in renal function. Patients 1, 3 and
4 and 7 had evidence of graft dysfunction including episodes of ABMR, although
in these cases the kidneys are still functioning at 11 years, 3 years and 15
months and one-month post-transplant. It should be noted despite induction
therapy four patients had severe early ABMR and one has chronic transplant
glomerulopathy.
We estimate that the frequency of patients with HNA antibodies
on a transplant waiting list would be less than 1%. The incidence of HNA-3a
specific antibodies in a female donor population has been reported to be 0.26%;
this was defined whilst screening apheresis donors as part of the TRALI
reduction programme in the UK [10].
The role for HNA-3a antibody testing is initially likely to be
of most value in cases of non-HLA/unexplained positive FCXM. In the LKD setting
there is clearly an appropriate time frame to investigate, whereas in the
deceased donor setting this would not be currently possible. The frequency of
HNA incompatible donors in these scenarios means a patient sensitised to HNA-3a
is very unlikely to be offered a HNA compatible deceased donor kidney. One
option maybe to investigate family members particularly siblings for a LKD with
HNA compatibility. Where no realistic choice of a compatible HNA donor exists
then pre-transplant antibody removal and augmented immunosuppression should be
carefully considered. Further investigation is required to assess the
pathological significance and prevalence of HNA antibodies in kidney
transplantation. However, of note two patients with HNA-3a specific antibodies
lost their transplanted kidney within a year.
1.
Alvarez-Márquez A, Aguilera I, Gentil MA, Caro JL, Bernal
G, et al. (2005) Donor-specific antibodies against HLA, MICA and
GSTT1 in patients with allograft rejection and C4d deposition in renal
biopsies. N Engl J Med 352:
558-569.
2.
Carter V, Shenton BK, Jaques B, Turner D, Talbot D, et al. (2005)
Vimentin
antibodies: A non-HLA antibody as a potential risk factor in renal
transplantation. Transplant Proc 37: 654-657.
3.
Curtis BR, Cox NJ, Sullivan MJ, Konkashbaev A, Bowens K, et al.
(2010) The neutrophil alloantigen HNA-3a (5b) is located on choline
transporter-like protein 2 and appears to be encoded by an R>Q154 amino acid
substitution. Blood 115: 2073-2076.
4.
Davoren A, Curtis BR, Shulman IA, Mohrbacher AF, Bux J, et al. (2003)
TRALI
due to granulocyte-agglutinating human neutrophil antigen-3a (5b)
alloantibodies in donor plasma: A report of 2 fatalities. Transfusion 43: 641-645.
5.
Davoren A, Saving K, McFarland JG, Aster RH, Curtis BR (2004) Neonatal neutropenia and bacterial sepsis
associated with placental transfer of maternal neutrophil-specific
autoantibodies. Transfusion
44: 1041-1046.
6.
Day S, Lucas G (2014) Possible role of HNA-3a antibodies in graft rejection
following kidney transplantation. IJI 41: 413-436.
7.
Dragun D, Müller DN, Bräsen JH, Fritsche L,
Nieminen-Kelhä M, et al. (2009) Angiotensin II type 1-receptor activating
antibodies in renal-allograft rejection. Transplantation 87: 94-99.
8.
Lucas G, Win N, Calvert A, Green A, Griffin E, et al.
(2012) Reducing the incidence of TRALI in the UK: The results of screening for
donor leucocyte antibodies and development of national guidelines. Vox Sanguin
103: 10-17.
9.
Sumitran-Karuppan S, Tyden G, Reinholt F, Berg U, Moller
E (1997) Hyperacute rejections
of two consecutive renal allografts and early loss of the third transplant
caused by non-HLA antibodies specific for endothelial cells. Transpl Immunol 5: 321-327.
10.
Zwirner NW, Marcos CY, Mirbaha F, Zou Y, Stastny P (2000) Identification of MICA
as a new polymorphic alloantigen recognized by antibodies in sera of organ
transplant recipients. Hum Immunol 61: 917-924.
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