Accepted Article
DR. EMRAH YUCESAN (Orcid ID : 0000-0003-4512-8764)
Article type
: Brief Communication
Mail id: eyucesan@bezmialem.edu.tr
Title: Fresh tissue parathyroid allotransplantation with short-term
immunosuppression: one year follow-up
Emrah Yucesan1, Beyza Goncu2, Harun Basoglu3, Nur Ozten Kandas4, Yeliz Emine Ersoy5,
Fahri Akbas6, Erhan Aysan5
1. Bezmialem Vakif University, Institute of Life Sciences and Biotechnology
2. Bezmialem Vakif University, Experimental Research Center
3. Bezmialem Vakif University, Faculty of Medicine, Department of Biophysics
4. Bezmialem Vakif University, Faculty of Pharmacy, Department of Pharmaceutical
Toxicology
5. Bezmialem Vakif University, Faculty of Medicine, Department of General Surgery
6. Bezmialem Vakif University, Faculty of Medicine, Department of Medical Biology
Abstract
Background: Permanent hypoparathyroidism is a serious problem and requires medications
indefinitely. Parathyroid allotransplantation (PA) with short-term immunosuppression is
definitive choice but long-term results are not clear.
Method: We performed PA from two donors to two recipients. Both recipients were 39
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/ctr.13086
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year-old females. Donors were a 32 year-old female and a 36 year-old male, who both have
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chronic kidney disease. Routine tests, viral markers and cross-matches were analyzed
individually. The parathyroid glands were resected from the living donors, fragmented
quickly in the operation room and injected into the left deltoid muscles of the two recipients.
Results: Methylprednisolone was administered on post-PA day one and two. Recipients were
discharged from the hospital without complications. Calcium and PTH levels were observed
throughout one year. We did not observe any complications during the follow-up period.
Medications ceased in posttransplantation week 1 for Case 1 and after one month for Case 2.
Conclusion: Fresh tissue PA with short-term immunosuppression appears to be a promising
technique that is easy to perform, is cost effective, has low risk of side effects and minimial
complications with compatibility for HLA conditions. A longer follow-up period and more
case studies are needed to determine the risks and benefits of this procedure for future cases.
Keywords: parathyroid, allotransplantation, permanent hypoparathyroidism, tissue,
immunosupression
Introduction
Permanent hypoparathyroidism (PH) is a clinical condition accompanied by
hypocalcemia, hyperphospatemia and low parathormone (PTH) levels (1,2). PH is the major
complication post thyroidectomy (1-3). Vitamin D and oral calcium supplementation are the
standard treatment options. Nevertheless some patients due to serious symptoms of tetany
may need parenteral administration of calcium gluconate and/or chloride (4). Additionally,
weakness, muscular dysfunctions, myositis, fasciitis, cataracts, basal ganglia and/or cerebellar
calcifications, teeth malformations and nephropathy are complications of treatment (2,5).
Long term drug therapy for PH also has several side effects; gastritis, urolithiasis,
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nephrocalcinosis, etc., which reduce the quality of life for patients (6). Therefore, treatment
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for PH requires alternative approaches, such as parathyroid allotransplantation (PA).
PA seems an acceptable alternative for patients with PH. Previously several PA
techniques have been described in the literature, such as human leukocyte antigen (HLA)
selection, cellular irradiation, tissue culture manipulation or using fresh tissue from primary
hyperparathyroidic patients to increase the success of transplantation (7-9). Herein, we report
the excision of hyperplastic parathyroid tissue from two living donors and transplantation of
the fragmented fresh tissue onsite to two different PH patients without immunosuppression
via injection with a needle into the deltoid muscle.
Cases
In Turkey, all organ and tissue transplantations are carried out with the permission of
the National Scientific Board for Transplantation, which is part of the Turkish Ministry of
Health. Bezmialem Vakif University is the first and unique center in Turkey that was
officially permitted and approved to conduct PA.
Case 1 was a 39 year old, 0 Rh (+) female. She had undergone total thyroidectomy
two years ago. PH had occured in the postoperative early stage and was treated for two years
with 3000 mg/day oral calcium (Calcimax-D3®, Basel Ilac Co, Turkey), calcitriol 1.5 µg/day
(Rocaltrol®, Deva Ilac Co, Turkey), and levotiroksin sodium 150 µg/day (Levotiron®
100mcg, Abdi Ibrahim Ilac Co, Turkey) to relieve symptoms.
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Case 2 was a 39 year old, 0 Rh (+) female. She had undergone total thyroidectomy
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three years ago. Like Case 1 she was diagnosed with PH in the early postoperative stage and
was also treated for three years with 4000 mg/day oral calcium (Calcimax-D3®, Basel Ilac
Co, Turkey), calcitriol 2 µg/day (Rocaltrol®, Deva Ilac Co, Turkey), and levotiroksin sodium
150 µg/day (Levotiron® 100mcg, Abdi Ibrahim Ilac Co, Turkey) to relieve symptoms.
Despite taking optimum daily medications, calcium and PTH levels remained low and
symptoms, such as weakness, transient tetany, and muscle spasms were not relieved for both
patients. After obtaining approval from the local human ethics committee and informed
consents from both patients, we decided to perform PA.
Donor 1 was a 32 year old 0 Rh (+) female. Donor 2 was a 36 year old A Rh (+) male.
Both donors had parathyroid hyperplasia secondary to chronic renal failure. Written informed
consents were obtained from the donors and they were screened for viral markers; anti-HIV,
anti-HCV, anti-HBc, anti-HBs, anti-HBe antibodies, HBV antigen and cytomegalovirus
immunoglobulin
(Ig)
G
and
IgM
antibodies,
Epstein-Barr
virus
IgG,
IgM
and VDRL (venereal disease research laboratory).
We evaluated both Case 1 and Case 2 serum and screened for class I and class II HLA
antibodies with a PRA Kit. Positive samples were further tested for the specificity of
antibodies for HLA-A, -B, -C, -DR, -DP, -DQ using a Single Antigen Kit. Fluorescence
intesity was measured with a Luminex100 flow analyzer and the data were analyzed using
LABScan 100 software. Median fluorescence intensity (MFI) of the PRA bead reactions was
obtained from the output file generated by the flow analyzer and adjusted for the background
signal. Recipients were defined as anti-HLA antibody-positive when they had HLAantibodies with MFI>2000. Results are given in Table 1. In addition we performed Flow
cytometry crossmatch for T and B cells (T-FCXM, B-FCXM respectively) which are
routinely performed in our transplantation unit. For the T-FCXM, 5 x 105 peripheral blood
mononuclear cells were incubated in duplicates with serum of the recipient; negative control
serum and positive control serum (mixture of sera with PRA of > 95%). Then incubated
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groups were labeled with anti-CD3 antibody and measured by flow cytometry. The B-FCXM
groups were labeled with anti-CD20 antibody and measured by flow cytometry. Case 1 and 2
Accepted Article
were both negative for T-FCXM and B-FCXM. The cutoff values were 5% for T cells and
8% for B cells by fluorescence index (FI) which is the percentage shift in the test serum as
compared to the positive control ([test serum FI − negative control FI]/[positive control −
negative control FI] × 100). The microlymphocytotoxicity crossmatch screening for HLA
antibodies was performed using lymphocytes. The recipients serums were treated with
dithiothreitol (DTT- an agent that removes immunoglobulin M) and mixed with donor
lymphocytes. This crossmatch denotes the presence of cytotoxic alloreactive IgG antibodies
against the potential donor HLA antigens. Case 1 and 2 were both negative for this
crossmatch.
Donors and recipients were taken to the operation room at the same time. The
recipients received 250 mg methylprednisolone one hour before the planned transplantation
time.
A standard Kocher incision was made on the anterior neck of the donors. Enlarged
parathyroid glands were found in the regular anatomic locations and standard subtotal
parathyroidectomy operations were performed. Nearly 1cm³ of the parathyroid gland was
separated for transplantation and the other glands were sent to the pathology laboratory in
formaldehyde solution for histopathological evaluation. The separated gland was prepared for
transplantation: the fatty tissues, fibrotic capsule and macroscopic blood vessels were excised
with careful dissections. The pure gland was fragmented into small pieces with a lancet as
much as possible. The fragments were washed with 36 °C saline and mixed with a 2 ml
platelet rich plasma solution obtained from the recipients separately. The fresh parathyroid
gland fragments were injected into the left deltoid muscle of the two recipients using a 14
gauge needle. Wound dressings were applied to the injection sites, and an arm sling was used
to immobilize and rest the deltoid muscles.
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The surgical intervention was successful. Neither the donors nor the recipients showed
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any complications. The recipients were observed in the hospital for the next two days.
Methylprednisolone was administered at a dose of 125 mg on post-PA day one and 60 mg on
post-PA day two to minimize the host’s reaction. Both patients were discharged from the
hospital without any complications. Oral prednisolone of 5 mg per day was prescribed for
one week only. The recipients’ serum calcium and PTH levels were observed throughout the
year. We did not observe any complications during the follow-up period. We ceased
medications (calcium and calcitriol) in post-transplantation week 1 for Case 1 and after one
month for Case 2. No medication was needed after this time and the cases were
asymptomatic. One year follow-up results are presented in Table-2. Histopathological
evaluation of the glands determined paratyhroid hyperplasia for both donors.
Discussion
PH is a severe clinical problem that effects daily life and requires life long treatment.
Medication is the first approach for these patients, however it is only palliative and long-term
medication use has several side effects (2). PA may represent a definitive treatment option for
PH (1). PA may be divided into two classes: cell type PA and tissue type PA. There are many
methods for cell type PA e.g. direct transplantation, cryopreservation and cultivation, and
microencapsulation (1,7,10). Each method has positive and negative outcomes. Direct tissue
transplantation requires less equipment, and is easy to perform, but it requires long term
immunosupressant adminstration. In our previous reports, including animal and human
models, cryopreservation and cultivation seem a better approach rather than medication
(12,13). Cryopreservation and cultivation is a useful method to avoid immune response
throughout cultivation; however, because of the freeze thaw process, sustaining cell viability
is difficult, and also cultivation causes cell death in viable parathyroid tissue (10-13). Success
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rates are approximately 17-83% and depend on viability and functional capacity of the cells
Accepted Article
(16). Microencapsulation technique is another suitable method for avoiding an immune
response because of capsule formation, but costs are relatively high. We have evaluated all of
this information not only from the literature, but also from our experience from previous
studies (12-15).
Fresh tissue transplantation is a promising tissue type for the PA technique which was
described almost three decades ago by Zeng et al. (21). However it has not been widely
implemented. In 1991 Kunori et al. used the same method for treatment for an 18-year-old
male who had severe PH due to elective thyroid surgery. In the same study, parathyroid tissue
was obtained from two unrelated living donors (22). In 2005 Torregrosa et al. used the same
method in a kidney transplant patient with severe hypocalcemia. Tissues were recieved from
a patient with secondary hyperparathyroidism (8). In the last decade PA studies for both
living and cadaver donors have accelareted but long-term follow up has not occured. To our
knowledge only two studies have been published that include long term follow-up (1,23).
Hermosillo et al. performed PA for five cases. One case showed no increase for PTH and also
an imaging study did not show functionality and the remaining four cases were followed-up
for two years with an immunosuppressive regimen (23). Agha et al. performed PA for a 32
year-old female who had intractable PH after thyroid surgery for papillary cancer. They used
two healthy parathyroid glands from living donors and transplanted them to the recipient’s
forearm. The case was followed-up for three-years with immunsuppressive drugs. Adverse
effects of immunosuppressive drug adminstration for long-term is known, but these two
studies did not state any.
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In our study we used immunsuppressants initially (medications ceased in post-
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transplantation week 1 for Case 1 and after one month for Case 2) but they were not required
permanently. Before transplantation we evaluated donors and recipients for immunological
compliance with complement-dependent lymphocytotoxicity (CDC-XM) and flowcytometric (FCXM) cross-match tests. During one year of follow-up no side effects or
complications were detected in the recipients. Hypocalcemic symptoms gradually diminished
and oral supplementation of calcium and calcitriol ceased in post-transplantation week 1 for
Case 1 and after one month for Case 2. Both cases were asymptomatic and neither case
required medications. We assume that our long term success without immunosuppression
may depend on compatibility for HLA between donors and recipients.
In conclusion, fresh tissue PA with short-term immunosuppression appears to be a
promising technique that is easy to perform, is cost effective, has low risk of side effects and
minimial complications with compatibility for HLA conditions. A longer follow-up period
and more case studies are needed to determine the risks and benefits of this procedure for
future cases.
Conflict of Interest
The authors declare no conflict of interests.
Acknowledgement
We highly appreciate the efforts of Monica Ann Malt, MSN, RN, and CPAN (Bezmialem
Vakif University, Turkey) in language editing of this paper.
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Accepted Article
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Accepted Article
Class I
Class II
Case 1
A26 (MFI:1810), A36 (MFI:1301), B39
(MFI:1192), B48 (MFI:1382), B58
(MFI:1295), B64 (MFI:1174), B78
(MFI:1065).
DR1 (MFI:1164), DR10 (MFI:1192),
DR15 (MFI:1439), DR51 (MFI:1439),
DR103 (MFI:1046), DQ2 (MFI:1577).
Case 2
A36 (MFI:343), B52 (MFI:341), B60
(MFI:208), B7 (MFI:446), B77
(MFI:179).
Negative
Case
1
Case
2
Before
Day 1
PA
After
Week 1
Month 1
Month 3
Month 6
Month 12
After
After
After
After
After
PA
PA
PA
PA
PA
PA
PTH
2.5
6.9
10.9
22.5
5.3
3.9
6.6
(pg/ml)
(+)
(+)
(-)
(-)
(-)
(-)
(-)
+2
8.7
8.4
8.8
8.7
7.9
8.4
9.8
(mg/dl)
(+)
(+)
(-)
(-)
(-)
(-)
(-)
PTH
6.0
7.2
10.6
27.2
31.2
11.9
10.2
(pg/ml)
(+)
(+)
(+)
(-)
(-)
(-)
(-)
+2
8.5
9.1
7.6
8.5
7.2
7.4
7.4
(mg/dl)
(+)
(+)
(+)
(-)
(-)
(-)
(-)
Ca
Ca
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