American Journal of Transplantation 2010; 10: 2061–2065 C 2010 The Authors Wiley Periodicals Inc. Journal compilation C 2010 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/j.1600-6143.2010.03234.x

Allotransplantation of Cryopreserved Parathyroid Tissue for Severe in a Renal Transplant Recipient

S. M. Flechnera,*,E.Berberb, M. Askarc, Introduction B. Stephanya,A.Agarwala and Mira Milasb Hypercalcemia due to tertiary is fre- aGlickman Urological and Kidney Institute, bEndocrine quently encountered in patients with end stage renal dis- Department, and Metabolism ease (ESRD). It is successfully treated by subtotal parathy- Institute, cAllogen Laboratories, Transplant Center roidectomy, and if needed autotransplant of a small amount Cleveland Clinic Foundation, Cleveland, OH of residual parathyroid tissue. However, in 1–2% of pa- *Corresponding author: Stuart M. Flechner, tients severe hypocalcemia can occur, creating a difficult fl[email protected] clinical problem (1). Herein we report the successful allo- transplantation of cryopreserved parathyroid tissue to re- We report the successful allotransplantation of cryo- verse hypocalcemia in an immunosuppressed kidney trans- preserved parathyroid tissue to reverse hypocalcemia plant recipient. This was done after all standard attempts in a kidney transplant recipient. A 36-year-old male re- to treat symptomatic hypocalcemia failed. ceived a second deceased donor kidney transplant, and 6 weeks later developed severe bilateral leg numb- ness and weakness, inability to walk, acute pain in the left and wrist . His total was Methods 2.6 mg/dL and parathormone level 5 pg/mL (normal 10–60 pg/mL). He underwent allotransplantation of Patient parathyroid tissue cryopreserved for 8 months into A 36-year-old male with ESRD from chronic glomerulonephritis received a his left brachioradialis muscle. in- first renal transplant from his sister in November 2003. The ceased to function after two acute rejection episodes, the second related to fi- cluded (target C0 10–12 ng/mL), mycophe- nolate mofetil and . Within 2 weeks, the left nancial difficulties with the medical regimen, and was removed in March knee pain, leg weakness and numbness resolved, and of 2006. He waited for a deceased donor kidney but had a high PRA of by 1 month he could walk normally. After a peak at 75% to class I HLA. On May 17, 2009 he received a zero HLA mismatched month 2, his (PTH) level fell to deceased donor kidney transplant. Both the lymphocytotoxic and flow cy- <10 pg/mL; therefore at month 3 he received a sec- tometry crossmatches were negative to the kidney donor. Immunosup- ond parathyroid transplant from the same donor. Eight pression consisted of thymoglobulin induction and tacrolimus, mycophe- months later (11 months after initial graft) he has a nolate mofetil and low dose steroids for maintenance. On admission for total calcium of 9.3 mg/dL, PTH level 15 pg/mL and the second kidney transplant his total calcium was 8.5 mg/dL (normal 8.5– is clinically asymptomatic. The amount of parathyroid 10.5 mg/dL). The kidney functioned immediately and he had a nadir crea- tissue needed to render a patient normocalcemic is tinine of 1.6 mg/dL 2 weeks later. Postoperatively, the patient complained not known. In our case, the need for second transplant of progressive left leg weakness and numbness contralateral to the kidney suggests that the amount of tissue transferred for an transplant, which he stated had begun a week prior. He was prescribed allograft may need to be substantially greater than for physical . an autograft. Prior to admission for his second kidney transplant, the patient gave a Key words: Cryopreservation, hypocalcemia, parathy- history of treatment for tertiary hyperparathyroidism (hypercalcemia and roid allotransplantation elevated parathyroid hormone [PTH] levels); for which he underwent subto- tal at another institution. In April 2009, for development of recurrent hyperparathyroidism (PTH level 668 pg/mL, normal range 10– Abbreviations: ABO, blood groups; CMV, cy- 60 pg/mL), he underwent resection of the remaining cervical parathyroid tomegalovirus; EBV, Epstein-Barr virus; ESRD, remnant with immediate into the right deltoid muscle. end-stage renal disease; HIV, human immunode- This operation occurred 1 month prior to his second kidney transplant. ficiency virus; HLA, human leukocyte antigen; PRA, percent reactive antibody; PTH, parathyroid hormone. On June 4, 2009 he was readmitted to the hospital with severe bilateral leg Received 06 May 2010, revised 07 June 2010 and numbness and weakness, inability to walk, acute pain in the left knee and accepted for publication 27 June 2010 clinical tetany of the wrists. His total calcium was 2.6 mg/dL, 1.8 mg/dL, phosphorus 2.5 mg/dL and albumin 4.1 gm/dL. The PTH was 5 pg/mL (normal range 10–60 pg/mL). An EKG demonstrated normal sinus

2061 Flechner et al. rhythm with a prolonged Q-T interval. He was given intravenous and oral 12 ng/mL the mycophenolate mofetil was increased to 1 gm b.i.d and the calcium for several weeks. prednisone kept at 10 mg daily for 3 months.

On June 27, 2009 the patient was receiving daily 7 gm of oral calcium × 5, Monitoring of parathyroid cell allograft 3 gm of intravenous calcium and oral up to 2 mg. The symptoms Levels of circulating PTH (pg/mL) were measured weekly in both the left of tetany and lower extremity numbness were much improved, but he still and right arm. Both total calcium (mg/dL) and ionized calcium (mmole/L) could not walk without a walker. The total calcium was 5.6 mg/dL; ionized were measured twice weekly. calcium 0.82 mmole/L (normal range 1.08–1.30) with a pH of 7.36 and the PTH was 6 pg/mL. Development of de novo posttransplant donor specific antibodies to the parathyroid donor class I and class II HLA mismatched antigens was moni- The previous week a request was made to the Cleveland Clinic Innovative tored using the Luminex bead assay (One Lambda, Canoga Park, CA, USA). Practice Committee, a subcommittee of the Cleveland Clinic Institutional Tests were run at each follow-up visit (Table 2). Review Board (irb.ccf.org), for permission to perform a third-party parathy- roid allotransplant. Permission was granted since this patient was a failure of maximal medical therapy for hypocalcemia using oral and i.v. calcium Results and calcitriol. A repository of stored cryopreserved parathyroid tissue at our center was screened for a suitable donor. There were several constraints Written informed consent was obtained from both the re- on the donor source of stored parathyroid tissue, which included frozen cipient and the parathyroid tissue donor. The donor se- storage for less than a year, the absence of exposure to Human Immun- lected was a 40-year-old male on hemodialysis with ESRD odeficiency Virus (HIV), Hepatitis B and C, as well as HLA phenotypes, from glomerulonephritis who was waiting for a deceased to which the recipient was not already sensitized, and ABO compatibility. 1 donor kidney. He had undergone 3 /2 gland parathyroidec- Since such antibody screening and tissue typing were not done routinely on tomy for tertiary hyperparathyroidism with cryopreserva- parathyroidectomy patients, the donor pool was limited to four individuals who were either on the kidney transplant wait list or had already received tion of the excised parathyroid tissue on November 8, a kidney transplant at our center. 2008. He had negative serology for the HIV, Hepatitis B and C and Cytomegalovirus (CMV), but was Epstein Barr The first parathyroid allotransplant was performed on July 1, 2009. The Virus (EBV) positive. The recipient was both CMV and EBV thawed parathyroid tissue was placed into the brachioradialis muscle in seropositive. the left forearm under local anesthesia and sedation. About 20 single 1- to 3-mm fragments of parathyroid tissue were placed into individual muscle Within 2 weeks of the first parathyroid cell allograft the pockets. The implanted parathyroid tissue was confirmed to be both benign symptoms of left knee pain, and leg weakness and numb- and hypercellular during pathological evaluation when it was recovered prior ness resolved, and the patient began walking with a cane to cryopreservation. for support. By 1 month postparathyroid transplant, the Immunosuppression patient could walk normally, absent any support. After the kidney transplant in May 2009 the recipient was maintained on tacrolimus 3 mg b.i.d (target C0 8–10 ng/mL); mycophenolate mofetil Total and ionized calcium levels remained in the 5–8 mg/dL

500 mg b.i.d (target C0 2–4 mg/L) and prednisone 5 mg daily. After the and 0.7 to 1.0 mmole/L range, respectively, for sev- parathyroid allotransplant the tacrolimus target was increased to C0 10– eral months (Figure 1). During this time the patient was

Figure 1: Parathormone blood levels (pg/mL) during the first year. Measure- ments were taken above the graft in the left arm and in the right arm.

2062 American Journal of Transplantation 2010; 10: 2061–2065 Allotransplantation of Cryopreserved Parathyroid Tissue

Figure 2: Blood calcium levels dur- ing the first year. Measurements were made both as total calcium (mg/dL) and as ionized calcium (mmole/L). receiving calcium carbonate 500 mg hourly while awake. 11-month follow-up the patient did not experience any re- Serum PTH levels quickly increased from baseline, but jection episodes of the renal allograft, and has maintained stayed in the 10–15 pg/mL range for several months a creatinine of 1.6–1.8 mg/dL. (Figure 2). The patient was given daily oral calcitriol 0.5–2 mg since his kidney transplant, and had hydroxy- Throughout the postoperative period the recipient was levels of 35–80 ng/mL throughout the follow-up monitored for any de novo donor specific HLA antibodies. period. The parathyroid donor was HLA mismatched for HLA-A30, −B58, Cw3, Cw7, DR4 and DQ8 (Table 1). There was no The patient had magnesium measured on alternate antibody detected to any of these mismatched antigens at months during follow-up. He received oral magnesium any time postparathyroid transplant. DQ8 antibodies were oxide 800 mg b.i.d since the first parathyroid transplant, positive presecond kidney transplant, but became nega- and had levels maintained between 1.6–2.4 mg/dL. Serum tive postkidney transplant and remained negative on sub- phosphorus was measured monthly during the 10-month sequent posttransplant monitoring (up to day 329), Table 2. follow-up period and varied between 3.7–6.6 mg/dL. All The second kidney donor was a zero HLA-A, −Band−DR but two determinations were in the normal range (2.5– mismatch deceased donor. 4.5 mg/dL). Discussion Although he felt well, the patient could not be weaned off oral calcium, and by 3 months the PTH had fallen While severe hypocalcemia after parathyroidectomy for hy- <10 pg/mL. Therefore, on October 7, 2009 (day 97) a re- perparathyroidism is uncommon, it has been observed in peat parathyroid allograft was placed just below the first about 1–2% of patients (1). Those at risk are usually pa- in the left brachioradialis muscle under local anesthesia tients who have had total or more exten- and sedation. The parathyroid cells came from a larger sive resection of parathyroid glands, whether for primary frozen aliquot from the same donor as the first graft. Im- hyperparathyroidism or for typically as- munosuppression remained the same except for a bolus sociated with renal disorders (secondary or tertiary hyper- of 500 mg i.v. methylprednisolone. In a pattern similar to parathyroidism). As early as 1976, it was recognized that the first parathyroid allograft, PTH levels in the ipsilateral parathyroid autotransplantation is a successful interven- arm rose to the 10–15 pg/mL range for 2 months, but tion to prevent permanent hypoparathyroidism (2). How- then increased further to 25 pg/mL during the third month ever, this unintended complication can nevertheless occur, (Figure 2). This was accompanied by a rise in total cal- causing dependence on high-dose calcium and vitamin D cium over 8 mg/dL and ionized fraction over 1 mmole/L supplementation with long-term risks of , mul- (Figure 1). After 26 weeks the patient was weaned to tiorgan calcinosis and renal failure. one TUMS (regular strength, calcium carbonate 500 mg) p.o. t.i.d, and has remained on this dose due to his os- Parathyroid cell allotransplantation has been described spo- teoporosis. At 44 weeks he maintains a total calcium of radically over the last 30 years as a potential therapy 9.3 mg/dL and has a PTH of 15 pg/mL. During the entire for permanent hypoparathyroidism (3–10). A number of

American Journal of Transplantation 2010; 10: 2061–2065 2063 Flechner et al.

Ta b l e 1 : HLA antibody status of both donors and the recipient Patient ABO HLA A HLA B HLA Bw HLA Cw Dr Dq Parathyroid/kidney recipient HLA O 2, 29 44, 62 2, 16 7, 11 2, 7 phenotypes 52, 53 Parathyroid/kidney recipient anti-HLA O 3, 34 7, 27, 42, 61, 1, 9, 51,103 4, 5, 6, 8, 9 antibodies (pretransplant) 73, 81, 8201 Kidney donor HLA phenotypes O 2, 29 44, 62 3, 16 7,11 52, 53 7, 8 Parathyroid donor HLA phenotypes O 2, 30 58, 62 4, 6 3, 7 4, 11 52, 53 2, 7 methodologies have been tried, including microencapsu- abnormal parathyroid cells were capable of further up- lation of parathyroid cells and allotransplantation without regulated expression of HLA antigens when cocultured immunosuppression. The results have not been durable, with interferon-c (14). These findings suggest that donor as the cell implants are subsequently rejected through parathyroid tissue recovered from abnormal glands may the usual mechanisms of the host alloimmune response. be more immunogenic. If severe hypocalcemia due to de- In vitro techniques to diminish the immunogenicity of ficient circulating PTH is known in advance of kidney trans- parathyroid tissue have been attempted by digestion with plantation, recovery from the same de- collagenase and filtering the tissue though a nylon mesh. ceased donor may be possible. However, this ap- Such dispersal can reduce the numbers of accompany- proach is unlikely or not possible in cases of live donor ing macrophages and dendritic cells that are rich in sur- kidney transplant or delayed onset of severe hypocalcemia face HLA class I and class II antigens (11). Additional after a prior renal allograft, as in our case. novel approaches have included dispersal of cells in cul- ture with HLA antibody coated microspheres to remove The use of tissue banking to cryopreserve parathyroid tis- HLA-rich stromal cells (5); a temporary transfer of irradi- sue after parathyroidectomy for future autografting is not ated human parathyroid cells to a murine ‘interim host,’ universally available. The cryopreservation technique has by placing them under the mouse kidney capsule for sev- been reported by several investigators, and the method eral weeks prior to clinical transfer (4); and encapsulating utilizes standard processes of tissue handling and step- parathyroid cells in engineered microcapsules of degassed wise preparation for freezing and storage in liquid nitro- 2% sodium-alginate (8). These techniques may prolong sur- gen that have not significantly changed with time (15–17). vivals compared to unmodified tissue. Nevertheless, a recent publication reported that this re- source is available to fewer than 30% of surgeons who Simultaneous kidney and parathyroid transplantation from perform parathyroid surgery (18). Where available, these the same deceased donor has been reported with the re- tissue banks provide a source of parathyroid tissue for covery of circulating PTH (10,12). If available, this can be a those who have insufficient parathyroid hormone produc- viable approach to the problem of both renal and parathy- tion and are traditionally reserved for potential autotrans- roid organ failure, and presents no additional histocom- plantation (17,19). For example, patients with secondary patibility challenge to the recipient. In such cases, healthy hyperparathyroidism related to kidney failure and long-term and fresh donor parathyroid tissue may be advantageous dialysis inevitably develop secondary hyperparathyroidism for allograft implantation, since the expression of class I with multigland parathyroid hyperplasia. At time of parathy- and II HLA phenotypes on normal parathyroid tissue ap- roid surgery, the subtotal or near-total parathyroidectomy pears to be downregulated (13). Whereas parathyroid tis- excises parathyroid tissue in quantities that are many times sue from patients with either hyperplastic or adenomatous larger than any quantity that might be needed for possible glands demonstrated increased expression of class I and autotransplantation (20). Cryopreserved tissue can theo- class II (DP, DQ, DR) antigens in culture. In addition, retically be used for allotransplantation but since the stored tissue is intended for use in the same patient, screening Ta b l e 2 : Posttransplant screening for donor-specific antibody for transmissible viral pathogens and tissue typing is rarely Time after DQ8 bead DQ8 bead DQ8 bead performed. This would require specific permission and de- parathyroid Tx 1 MFI 2 MFI 3 MFI ferral of any additional expense from the parathyroid donor. Some of these issues could be obviated if the parathyroid Week 1 1090 839 342 Week 2 965 747 270 donor was a transplant wait list candidate that had previ- Month 1 624 415 135 ously undergone such screening, as in our case. Month 2 1060 864 338 Month 3 902 715 294 An additional important consideration for allotransplanta- Month 6 721 495 156 tion using stored cryopreserved parathyroid tissue is the Month 9 1152 871 383 quantity of tissue necessary for successful reversal of Month 11 1169 863 292 hypoparathyroidism. While generous amounts of excess Luminex HLA detection assay: controls <500 MFI (mean fluores- parathyroid tissue are usually frozen for future autograft- cent intensity). ing, sufficient sample size would need to be available for

2064 American Journal of Transplantation 2010; 10: 2061–2065 Allotransplantation of Cryopreserved Parathyroid Tissue both the intended recipient and for a potential allograft 3. Feind CR, Weber CJ, Derenoncourt F, Williams G, Hardy MA, recipient. There is an expected loss of viable parathyroid Reemtsma K. Survival and allotransplantation of cultured human cells in the freeze-thaw process. It has been reported that parathyroids. Transplant Proc 1979; 11: 1011–1016. about 71% of cryopreserved parathyroid tissue will be vi- 4. Sollinger HW, Mack E, Cook K, Belzer FO. Allotransplantation of able for the first 24 months of frozen storage, with viability human parathyroid tissue without immunosuppression. Transplan- tation 1983; 36: 599–602. inversely proportional to the length of time stored (19). The 5. Anton G, Decker G, Stark JH, Botha JR, Margolius L. Allotrans- negative effect of cryopreservation no doubt contributes to plantation of parathyroid cells. Lancet 1995; 345: 124. the relatively modest success rates reported with delayed 6. Wozniewicz B, Migaj M, Giera B et al. Cell culture preparation of parathyroid autotransplantation of 40–60% (15,17,21). Al- human parathyroid cells for allotransplantation without immuno- ternatively, the freezing of parathyroid tissue may con- supression. Transplant Proc 1996; 28: 3542–3544. tribute to a loss of surface HLA expression and contribute 7. Tolloczko T, Wozniewicz B, Sawicki A, Gorski A. Allotransplantation to reduced immunogenicity. The precise amount of cryo- of cultured human parathyroid cells: Present status and perspec- preserved parathyroid tissue needed to render a patient tives. Transplant Proc 1997; 29: 998–1000. normocalcemic is not known. In our case, the need for 8. Hasse C, Klock G, Schlosser A, Zimmermann U, Rothmund second transplant points out that the amount of tissue M. Parathyroid allotransplantation without immunosuppression. Lancet 1997; 350: 1296–1297. transferred for an allograft may need to be substantially 9. Nawrot I, Wozniewicz B, Tołłoczko T et al. Allotransplantation of greater than for an autograft. cultured parathyroid progenitor cells without immunosuppression: Clinical results. Transplantation 2007; 83: 734–740. At the present time, durable results have only been re- 10. Chapelle T, Meuris K, Roeyen G et al. Simultaneous kidney- ported in parathyroid allotransplantation when immuno- parathyroid allotransplantation from a single donor after 20 suppression to prevent rejection is administered. A years of tetany: A case report. Transplant Proc 2009; 41: 599– commonly used regimen in of 600. tacrolimus, mycophenolate mofetil and steroids appears to 11. Rudberg C, Grimelius L, Johansson H et al. Alteration in den- permit the recovery of PTH secretion. The optimal agents sity, morphology and parathyroid hormone release of dispersed needed for parathyroid allotransplantation are not known. parathyroid cells from patients with hyperparathyroidism. Acta Pathol Microbiol Immunol Scand 1986; 94: 253–261. The use of these agents, which are associated with signif- 12. Alfrey EJ, Perloff LJ, Asplund MW et al. Normocalcemia thirteen icant morbidity and toxicities, would therefore be limited years after successful parathyroid allografting in a recipient of a to those patients with hypoparathyroidism accompanied renal transplant. Surgery 1992; 111: 234–236. by an additional need for transplantation and immunosup- 13. Bjerneroth G, Juhlin C, Klareskog L, Rastad J, Akerstrom G, pression. The primary transplant organ would dictate the Klareskog L. Major histocompatibility complex class II expression regimen employed. Finally, if a local cryobank is not avail- and parathyroid autoantibodies in primary hyperparathyroidism. able, frozen parathyroid tissue could be shipped to alter- Surgery 1998; 124: 503–509. nate sites for allotransplant with appropriate arrangements 14. Bjerneroth G, Juhlin C, Rastad J, Akerstrom G, Klareskog L. MHC and screening. class I and II antigen expression on parathyroid cells and prospects for their allogenic transplantation. Transplantation 1993; 56: 717– 721. Acknowledgments 15. Cohen MS, Dilley WG, Wells S, Jr. et al. Long-term functional- ity of cryopreserved parathyroid autografts: A 13-year prospective The authors appreciate the technical support of John Fung, MD, PhD, and analysis. Surgery 2005; 138: 1033–1040. Lina Lu, PhD, to the parathyroid tissue bank, and assistance of Barbara 16. Wagner PK, Seesko HG, Rothmund M. Replantation of cryopre- Mastroianni, RN, and Kathy Savas, RN, as kidney transplant coordinators. served human parathyroid tissue. World J Surg 1991; 15: 751– 755. Funding: No source 17. Caccitolo JA, Farley DR, van Heerden J Grant C, Thompson GB, Sterioff S. The current role of parathyroid cryopreservation and Disclosure autotransplantation in parathyroid surgery: an institutional experi- ence. Surgery 1997; 122: 1062–1067. The authors of this manuscript have no conflicts of inter- 18. Greene AB, Butler R, McIntyre S et al. National trends in parathy- est to disclose as described by the American Journal of roid surgery from 1998 to 2008: a decade of change. J Am Coll Transplantation. Surg 2009; 209: 332–343. 19. Guerrero MA, Evans D, Lee JE et al. Viability of cryopreserved parathyroid tissue: when is continued storage versus disposal in- References dicated? World J Surg 2008; 32: 836–839. 20. Milas M and Weber CJ. Near-total parathyroidectomy is benefi- 1. Udelsman R. Six hundred fifty-six consecutive explorations for pri- cial for patients with secondary and tertiary hyperparathyroidism. mary hyperparathyroidism. Ann Surg 2002; 235: 665–672. Surgery 2004; 136: 1252–1260. 2. Wells SA, Ellis GJ, Gunnells JC, Schneider A, Sherwood LM. 21. Feldman AL, Sharaf RN, Skarulis MC et al. Results of heterotopic Parathyroid autotransplantation in primary parathyroid hyperplasia. parathyroid autotransplantation: A 13-year experience. Surgery N Engl JMed 1976; 295: 57–62. 1999; 126: 1042–1048.

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