The Use of Cryopreserved Human Thyroid Tissue for the In Vitro Assay of Thyroid Stimulators1 ANDREW
J. S. KEOX,
CHRISTIAN
VOS
WESTARP,
VAS 1’. ROW,
ASI) ROBERT
\‘OLl%
The Endocrine Rcseurcll L&oratory, The Wcllcsler~ Hospital, and the Dcpmtment Medicine, Udcelaity of Toronto, Toronto, Of~tario, Can&
The long-acting thyroid stimulator (LATS) was first identified in the sera of patients with Graves’ disease by Adams and Purves in 1956 (1). This immunoglobulin ( IgG ) is routinely assayed by the McKenzie mouse bioassay (7). Recently, a number of other immunoglobulins have been discovered in the sera of patients with Graves’ disease which specifically stimulate the human thyroid but are undetectable in animal bioassay systems. These have been variously immunoglobulins termed LATS protector (LATSP) by Adams and Kennedy (2), human thyroid stimulator (HTS) by Onaya et al. (a), and thyroid stimulating immunoglobulin (TSI ) by Smith and Hall (12). The assays described for these immunoglobulins are dependent on fresh human thyroid tissue obtained at operation or within a few hours of death. Kendall-Taylor (4) has shown that tissue obtained routinely at autopsy is unresponsive to stimulation. The use of fresh thyroid tissue, although feasible for rcsearch purposes, is precluded in routine assays because of Iimited availability. The measurement of these human thyroid stim---_Rcccived May 29, 1976. 1 This work forms part of a thesis submitted to the University of London by A. J. S. Knox, in partial fulfillment of the degree of MS. 2 Reprint requests to: Dr. Robert Volp15, Endocrine Research Laboratory, The Wellesley Hospital, 160 WelIcsIey Street East, Toronto, Ontario, Cnnadn, M4Y lJ3.
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
2
of
ulating immunoglobulins (HTSI) has become of increasing importance in the understanding, diagnosis, and management of Graves’ disease as demonstrated by Hall
et al. (3). It was the purpose of this study to determine whether or not human thyroid tissue stored for weeks at low temperatures (-196’C) could be substituted for fresh tissue in these assays, since even fresh tissue left in the refrigerator at 4-7°C for longer than 3 hr after surgical removal was unresponsive to thyroid stimulators. The assay system chosen was that involving stimulation of the adenylate-cyclasecyclic-AMP system in human thyroid slices. The assay was calibrated using bovine thyroid stimulating hormone (TSH). However, thyroid stimulating immunoglobulin preparations were also tested in the assay system together with control immunoglobulin preparations. hlATERL4LS
AND
METHODS
(a) Storage and Revival of Human Thyroid Tisszce Fresh human thyroid tissue at surgery was collected in a sterile beaker surrounded with ice and taken to the laboratory. The specimen was cleaned of all adipose and cartilaginous tissue on ice. The tissue was immediately cut into approximately 0.5mm slices with a Stadie-Riggs microtome, and the slices were floated on cold sterile saline
ISSN 0011 -2240
:44
KSOS 1
J. S. KEOX,
CHRISTIAN
VOS
WESTARP,
VAS 1’. ROW,
ASI) ROBERT
\‘OLl%
The Endocrine Rcseurcll L&oratory, The Wcllcsler~ Hospital, and the Dcpmtment Medicine, Udcelaity of Toronto, Toronto, Of~tario, Can&
The long-acting thyroid stimulator (LATS) was first identified in the sera of patients with Graves’ disease by Adams and Purves in 1956 (1). This immunoglobulin ( IgG ) is routinely assayed by the McKenzie mouse bioassay (7). Recently, a number of other immunoglobulins have been discovered in the sera of patients with Graves’ disease which specifically stimulate the human thyroid but are undetectable in animal bioassay systems. These have been variously immunoglobulins termed LATS protector (LATSP) by Adams and Kennedy (2), human thyroid stimulator (HTS) by Onaya et al. (a), and thyroid stimulating immunoglobulin (TSI ) by Smith and Hall (12). The assays described for these immunoglobulins are dependent on fresh human thyroid tissue obtained at operation or within a few hours of death. Kendall-Taylor (4) has shown that tissue obtained routinely at autopsy is unresponsive to stimulation. The use of fresh thyroid tissue, although feasible for rcsearch purposes, is precluded in routine assays because of Iimited availability. The measurement of these human thyroid stim---_Rcccived May 29, 1976. 1 This work forms part of a thesis submitted to the University of London by A. J. S. Knox, in partial fulfillment of the degree of MS. 2 Reprint requests to: Dr. Robert Volp15, Endocrine Research Laboratory, The Wellesley Hospital, 160 WelIcsIey Street East, Toronto, Ontario, Cnnadn, M4Y lJ3.
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
2
of
ulating immunoglobulins (HTSI) has become of increasing importance in the understanding, diagnosis, and management of Graves’ disease as demonstrated by Hall
et al. (3). It was the purpose of this study to determine whether or not human thyroid tissue stored for weeks at low temperatures (-196’C) could be substituted for fresh tissue in these assays, since even fresh tissue left in the refrigerator at 4-7°C for longer than 3 hr after surgical removal was unresponsive to thyroid stimulators. The assay system chosen was that involving stimulation of the adenylate-cyclasecyclic-AMP system in human thyroid slices. The assay was calibrated using bovine thyroid stimulating hormone (TSH). However, thyroid stimulating immunoglobulin preparations were also tested in the assay system together with control immunoglobulin preparations. hlATERL4LS
AND
METHODS
(a) Storage and Revival of Human Thyroid Tisszce Fresh human thyroid tissue at surgery was collected in a sterile beaker surrounded with ice and taken to the laboratory. The specimen was cleaned of all adipose and cartilaginous tissue on ice. The tissue was immediately cut into approximately 0.5mm slices with a Stadie-Riggs microtome, and the slices were floated on cold sterile saline
ISSN 0011 -2240
:44
KSOS 1
