Parathyrin (PTH), also known as parathyroid hormone

Transcription

1 Strategies for Laboratory and Patient Management Intraoperative Testing for Parathyroid Hormone A Comprehensive Review of the Use of the Assay and the Relevant Literature Alexis Byrne Carter, MD; Peter J. Howanitz, MD Objective. The rapid intraoperative parathyroid hormone assay is transforming the parathyroidectomy procedure. We present a review of the literature on the use of the assay as an adjunct to surgery. To our knowledge, this is the first review of the literature to encompass and compare all known primary studies of this assay in parathyroidectomy patients. Data Sources. Articles were collected by searching MEDLINE databases using relevant terminology. The references of these articles were reviewed for additional studies. Supplementary articles pertinent to the parathyroidectomy procedure, preoperative parathyroid localization studies, and intraoperative parathyroid hormone assay development also were examined. Study Selection and Data Extraction. One hundred sixty-five references were analyzed and categorized separately into groups. Data Synthesis. The primary studies of intraoperative data on patients undergoing parathyroidectomy were compared when possible. Studies were analyzed by type of assay used, where performed, turnaround time, and efficiency of use. Reviews of the types of parathyroid surgery and preoperative localization were included for educational purposes. Conclusions. The intraoperative parathyroid hormone assay is a useful adjunct to preoperative imaging and parathyroid surgery because of its unique ability to detect an occult residuum of hyperfunctioning parathyroid tissue. Use of this assay will obviate the need for frozen section in most routine cases. The test facilitates minimally invasive parathyroidectomy for single parathyroid adenomas, which, in turn, improves cost-effectiveness and cosmetic outcome. Its use in patients with known preoperative multiglandular disease is promising but requires further study. (Arch Pathol Lab Med. 2003;127: ) Parathyrin (PTH), also known as parathyroid hormone and parathormone, is produced by the parathyroid glands and is critical in maintaining calcium homeostasis. 1 The embryology, location, number, and function of the parathyroid glands and the role of PTH in calcium homeostasis have already been comprehensively reviewed. 2 6 Although most patients have 4 parathyroid glands, some patients only have 2, and others have many more than 4. 7 The superior parathyroid glands are located on the posterior surface of the superior portions of the thyroid lobe close to the entrance of the recurrent laryngeal nerve to the larynx. The inferior parathyroid glands are near the lower pole of the thyroid, are more ventral, and are close to the thyrothymic ligament. 7 Approximately 1% to % of glands have been reported in ectopic sites such as the pharyngeal mucosa and superior mediastinum as well as enclosed within the thyroid parenchyma. 7 When PTH binds to cell-surface receptors on target organs such as the bone or kidney, it triggers the release of calcium into the blood. Accepted for publication May 13, From the Department of Pathology & Laboratory Medicine, Brody School of Medicine, East Carolina University, Greenville, NC (Dr Carter), and the Department of Pathology, State University of New York, Downstate Medical Center, Brooklyn, NY (Dr Howanitz). Dr Carter is now with the Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pa. Reprints: Alexis Byrne Carter, MD, Department of Pathology, University of Pittsburgh Medical Center, 701 Scaife Hall, 30 Terrace St, Pittsburgh, PA 1213 ( carterab@upmc.edu). Conversely, an increase in blood calcium negatively feeds back on the parathyroid tissue to decrease PTH release. In the past, when one or more of these glands began to secrete excessive PTH (hyperparathyroidism), it was believed that this situation forced the remaining normal parathyroid glands to be completely functionally suppressed. However, some evidence indicates that the remaining normal parathyroid glands are suppressed incompletely. 8 Locchi et al 9 confirmed the residual function of normal parathyroid glands by examining the PTH kinetics in patients undergoing parathyroidectomy for hyperparathyroidism. After resection of the hyperfunctioning tissue, the normal parathyroid glands completely recover function in 18 to 40 hours. PTH is first translated in the endoplasmic reticulum of the parathyroid chief cell as a 11-amino acid prepro-pth molecule (see Figure 1). 11 Twenty-five amino acids at the amino-terminal end of the peptide are cleaved as it leaves the endoplasmic reticulum forming pro-pth. The next 6 amino-terminal amino acids are removed as pro-pth leaves the cell cytoplasm. This final product consists of 84 amino acids and is a functionally intact PTH molecule abbreviated as (1 84)PTH. Intact PTH, a term used by many studies in the literature as well as by several manufacturers, recently has been discovered to be somewhat misleading (vide infra). PTH is cleared from the blood biexponentially with an early rapid phase (t ½, usually minutes) and a late slow phase (t ½, several days). 9, The half-life of human PTH 1424 Arch Pathol Lab Med Vol 127, November 2003 Intraoperative Testing for PTH Carter & Howanitz

2 Figure 1. Production and degradation of parathyroid hormone; molecules highlighted in yellow are biologically active. 1 aa indicates amino acids; PTH, parathyroid hormone; npth, amino-terminal fragment of PTH; and cpth, carboxy-terminal fragment of PTH. during the early rapid phase varies depending on the study and ranges from a low of 1.68 minutes 12 to a high of 21. minutes 13 in patients with normal renal function. 1,14 Less than 1% of (1 84)PTH reaches the PTH receptors in target organs after leaving the parathyroid gland. 1 Sixty percent to 70% of (1 84)PTH is cleaved initially between amino acids 33 and 36, and this metabolism is accomplished primarily by the liver. 14 When PTH is metabolized, only the inactive C-terminal portions (cpth) of the molecule, encompassed by amino acids 3 through 84, are re-released into the circulation. cpth fragments also are released directly from the parathyroid gland after intraglandular degradation. 1 This intraglandular degradation probably serves an important regulatory function. 1 The N-terminal products of PTH metabolism (npth), encompassed by the first 34 amino acids, have biologic activity but constitute a much smaller proportion of the circulating PTH population because they are neither re-released into the circulation by the liver 8,1 nor directly secreted by the parathyroid gland. 1 By contrast, cpth is inactive biologically, has a longer half-life, and is cleared primarily by the kidneys. 1,14 Therefore, cpth fragments are elevated in patients with renal dysfunction. This may further confound attempts to measure (1 84)PTH in this setting. 16,17 Midregion fragments may also be present in the circulation and have characteristics similar to those of cpth. 1,14 Intact PTH assays are 2-site sandwich assays in which the npth and cpth ends of the molecule are bound by separate antibodies. Ideally, measurements of PTH will selectively assay (1 84)PTH. 18 Early in the development of the rapid PTH assay, the antibody for the npth portion was thought to bind the entire first 34 amino acids or (1 34)PTH. Since then, the discovery that the anti-npth antibody is specific for the region encompassed by amino acids 14 through 34, otherwise known as (14 34)PTH, has shown this antibody to be the limiting factor for accuracy in detecting (1 84)PTH. 19,20 Assays that use these antibodies could bind fragments in which a short portion of the amino-terminal end is truncated, a truncation that results in cessation of biologic activity for the molecule. This minimally truncated molecule, first described as the non-(1 84) molecular form of PTH by Brossard et al, 16 has been measured as intact PTH by some assays 16,19,21 and may constitute up to 13% of total intact PTH in patients with renal failure. 16 One of these molecules, the (7 84)PTH fragment, has shown cross-reactivity with the Nichols Allegro Intact PTH assay (Nichols Institute Diagnostics, San Clemente, Calif), the N-Tact Incstar assay (DiaSorin Corporation, Stillwater, Minn), and the Active Intact PTH assay by Diagnostic System Laboratories (Webster, Tex). 19,21 It is currently unknown if (7 84)PTH arises directly from the parathyroid gland, from the metabolism of PTH in the circulation, or both. 20 This cross-reactivity with (7 84)PTH is one possible explanation for the heterogeneity of results from the PTH assay. Therefore, the distinction between intact PTH and (1 84)PTH becomes an important one, since the (1 84)PTH molecule is the best indicator of parathyroid gland activity and is vastly responsible for the signs and symptoms of parathyroid disease. Testing for PTH, therefore, presents several challenges. (1 84)PTH circulates at extremely low levels, approximately to 12 mol/l, in the presence of a much greater number of PTH fragments. 1,22 The ratio of (1 84)PTH to PTH fragments varies widely with the glomerular filtration rate and parathyroid gland activity. 1 Fortunately, the accuracy of immunoassays for (1 84)PTH has markedly improved over time. The specific characteristics of a particular PTH assay must be taken into account when interpreting the results, especially in patients with chronic renal failure. Arch Pathol Lab Med Vol 127, November 2003 Intraoperative Testing for PTH Carter & Howanitz 142

3 STUDY SELECTION AND DATA EXTRACTION Numerous studies regarding the intraoperative PTH assay in patients undergoing parathyroidectomy for hyperparathyroidism are present in the literature. However, to our knowledge, a comprehensive review of these studies has not been performed. Articles were found by searching MEDLINE databases on both the Ovid server and the National Library of Medicine s PubMed server as far back in time as the databases information systems permitted through the end of November The terms rapid, quick, and intraoperative were combined individually with each of the following terms: parathyroid hormone, parathyrin, parathormone, and PTH as search criteria. Parathyroidectomy assay was also used as a search criterion. The resulting articles were collected, and the references of these articles were gathered. Supplementary articles pertinent to the parathyroidectomy procedure, preoperative parathyroid localization, and intraoperative PTH assay development also were assembled and reviewed. One hundred sixty-five references were analyzed and categorized separately into one of the following groups: primary studies of intraoperative PTH assays in patients undergoing parathyroidectomy (n 79), PTH assay development and validation (n 17), information from test manufacturers (n ), studies of alternative uses of rapid PTH assays (n 11), review articles of PTH assays (n 7), review articles and textbook chapters on parathyroid glands (n 6), and references that provided information on parathyroidectomy, preoperative imaging, historic assays used during parathyroidectomy, and other related information (n 3). The primary studies of intraoperative PTH assays were subcategorized into analysis of intraoperative samples by the rapid PTH assay (n 60), retrospective analysis of the intraoperative samples by the standard PTH assay (n 1), and case reports in which intraoperative PTH sampling occurred (n 4). Alternative uses of PTH assays were subcategorized into selective venous sampling (n 7), fine-needle aspiration of parathyroid glands (n 2), and prediction of postoperative hypocalcemia in patients undergoing thyroidectomy (n 2). SYNOPSIS OF PARATHYROIDECTOMY AND PREOPERATIVE IMAGING STUDIES Surgical Techniques The Traditional Bilateral Approach. Primary hyperparathyroidism affects approximately 1 in 700 people. Hyperparathyroidism may be part of several disease processes, but for this review, patients were grouped into those with single gland disease and those with multiglandular disease. Surgery is currently the only curative therapy for parathyroid adenomas and hyperplasia. The first successful parathyroidectomy was performed by Felix Mandl. 23 Since that time, parathyroidectomy traditionally has been accomplished by a bilateral approach (the traditional bilateral approach). After the induction of general anesthesia, the patient s neck is explored bilaterally. The dissection extends to the thyroid cartilage superiorly, to the sternal notch inferiorly, and to the jugular veins laterally. 24 All 4 parathyroid glands are identified in a systematic manner, and any morphologically abnormal parathyroid glands are resected and sent for frozen section confirmation. 7,24,2 A biopsy of an adjacent morphologically normal parathyroid gland is sometimes included. 26 By this approach, a skilled endocrine surgeon may have success in restoring the patient to normocalcemia up to 90% to 98% of the time. 27,28 The most common cause of surgical failure occurs when multiglandular disease (either hyperplasia or multiple adenomas) is missed. 29 The incidence of multiglandular disease varies in the literature from 8% to 3% 30,31 and is more frequent in patients with multiple endocrine neoplasia (MEN) type I, familial hyperparathyroidism, and secondary hyperparathyroidism.,31 Ectopic parathyroid glands such as those located in the thymus, perithymic tissue, and thyroid nodules may complicate surgery in up to 2% of the cases. 2,,18,32,33 Supernumerary glands, surgeon inexperience, and metastatic parathyroid carcinoma have been cited as additional reasons for failed first parathyroid operations. Although frozen section is helpful in the identification of parathyroid tissue, by itself, it cannot indicate the presence or absence of residual disease. In addition, frozen section errors have been identified as a factor contributing to surgical failure in 0.8% of the cases reviewed by Westra et al, 34 in 4.4% of the cases reviewed by Levin and Clark, and in up to 32% of the cases reviewed by Saxe et al. 3 Missed multiglandular disease necessitates reexploration. If the first operation involved a bilateral neck exploration, then the subsequent scar tissue would obscure the delicate and vital structures in this area. Reoperation, therefore, has a substantially increased risk of complications such as permanent hypoparathyroidism (13%), recurrent laryngeal nerve paralysis (4%), and persistent hyperparathyroidism. 36,37 Any one of these complications can be devastating for a patient; hence, techniques that have equivalent or better rates of cure have been sought to prevent the necessity of bilateral neck exploration. The Minimally Invasive Parathyroidectomy. In an effort to reduce the extent of neck exploration in patients with single gland disease, a minimally invasive, or concise, parathyroidectomy procedure (minimally invasive parathyroidectomy) was developed and is the procedure of choice in many hospitals for patients who meet certain criteria These criteria include a clear diagnosis of primary hyperparathyroidism, no history of familial parathyroid disease, no coexisting thyroid disease, a skilled endocrine surgeon with experience in the minimally invasive parathyroidectomy and the traditional bilateral approach, the availability of a high-quality preoperative imaging scan that clearly demonstrates a single enlarged gland, and laboratory capability to perform the rapid intraoperative PTH test. 4,47 49 The minimally invasive parathyroidectomy encompasses the minimally invasive open procedure, 4 the video-assisted gasless parathyroidectomy, 46,0 3 the pure closed endoscopic approach from the anterior neck, and the endoscopic approach from the axilla ,3 The open procedure seems to be the most common and either uses a standard collar incision with unilateral dissection only or uses a minimal incision just over the location of the abnormal parathyroid gland. In general, the minimally invasive parathyroidectomy has a shorter operating time, may be performed on an outpatient basis, and has reduced total costs associated with each of these elements. 27,43,4, In contrast to the traditional bilateral approach, this procedure can be performed with cervical block anesthesia ipsilateral to the abnormal gland. 6 These procedures are beneficial for elderly or debilitated patients 1426 Arch Pathol Lab Med Vol 127, November 2003 Intraoperative Testing for PTH Carter & Howanitz

4 who otherwise may not tolerate general anesthesia. 43,7 Local anesthesia also allows the surgeon to hear the patient s voice during the procedure and monitor for potential damage to the recurrent laryngeal nerve. 46 Patients also have a quicker recovery and a more cosmetically pleasing scar. 44,8,9 The current drive toward outpatient surgery and cost containment is another factor. 9 Prager et al 60 demonstrated that the minimally invasive parathyroidectomy by open dissection was successful in patients with thyroid disease and who had undergone previous neck operations. Video-assisted parathyroidectomy, however, cannot be used with local anesthesia or in patients with thyroid disease. 49,0,61,62 Moore et al 8 pointed out that the minimally invasive parathyroidectomy is not without limitations. Imperfect preoperative localization, the intrinsic rate of bilateral multiglandular disease, and operative complications have been implicated as causes of a failed minimally invasive parathyroidectomy resulting in either conversion to an open bilateral procedure or a repeat operation. 8 Specifically, the authors cited an increased potential for recurrent laryngeal nerve injury because of the limited exposure involved in a minimally invasive parathyroidectomy. 8 The cost of adding the preoperative adjunctive studies may also be a limiting factor in some settings. The costs are further discussed later in this article. Because the surgeon cannot visually examine all of the parathyroid glands during the operation, adjunctive studies are necessary to ensure that all abnormal parathyroid tissue is removed. These will ideally include a means to localize the abnormal glands preoperatively as well as to determine if the resection is complete prior to closure of the surgical incision. To this end, several techniques have been developed that assist the procedure in different ways. Preoperative Imaging Studies The ideal preoperative imaging study is inexpensive, safe, and readily available; it also gives 3-dimensional information and images the neck as well as ectopic sites. 46 Ultrasound, Technetium-99 (Tc-99m) sestamibi, and magnetic resonance imaging with short tau inversion recovery sequencing have been shown to be beneficial in localizing enlarged parathyroid glands, with sensitivities ranging from 78% to 0%. 39,40,63 Ultrasound is highly operator-dependent and cannot detect mediastinal adenomas. 40 However, it does provide information regarding the relationship of the gland to important anatomic structures and may be the best modality for imaging intrathyroidal parathyroid adenomas. 46 Sestamibi scintigraphy is the preferred method of preoperative imaging. 46,64,6 Sestamibi is a monovalent lipophilic cation that easily crosses the lipid bilayer and accumulates almost exclusively in mitochondria. 6 Tc-99m sestamibi is injected into the bloodstream and absorbed by many tissues, including highly active parathyroid glands. Tissues with increased numbers of mitochondria, such as heart and parathyroid glands, retain the sestamibi for a longer period than other tissues. 2 Abnormal parathyroid glands have a relative lack of p- glycoprotein, a transmembrane transporter included in the multidrug resistance gene complex. In theory, since the abnormal parathyroid chief cells cannot pump out the Tc- 99m sestamibi adequately, it is retained within these cells for a longer time. 66 After injection of the Tc-99m sestamibi, a radiosensitive camera is placed over the patient s neck, and the first image produced shows uptake in the thyroid and other tissues as well as the parathyroid glands. After a delay of 2 to hours, a second image is obtained. The area of uptake that remains indicates an abnormal gland. 6 This is dualphase scintigraphy. For regions in which the incidence of nodular goiter is high, scintigraphy with a second thyrotropic radioactive tracer is recommended in order to delineate the parathyroid glands by a subtraction technique. 64,6 The sestamibi scan can be performed with or without single proton emission computerized tomography. During single proton emission computerized tomography, multiple images are obtained from different angles around the patient s head and neck. These can be reconstructed by a computer into a 3-dimensional picture. The advantage of single proton emission computerized tomography is increased detection of abnormal parathyroid glands that may be masked by adjacent superimposed structures in the anterior planar view. 6,6 Therefore, single proton emission computerized tomography is most useful in defining ectopic and deep neck adenomas. 64 Although sestamibi scanning is more sensitive for mediastinal and other ectopic adenomas than other imaging methods, this technique may miss small ( 00 mg) adenomas. 40,67,68 The most frequently encountered problem with the sestamibi scan is undetected multiglandular disease. Several studies reported that sestamibi scintigraphy had a single region of uptake, suggesting the presence of a single adenoma, when, in fact, the patient had multiglandular disease. 69,70 Hyperplastic parathyroid glands have been reported to have inconsistent uptake of the sestamibi marker. 39,69 The scan is further complicated if the patient has undergone a previous neck operation, because the subsequent scar formation from the previous surgical procedure can distort the anatomy and hide enlarged parathyroid glands in unusual locations. 71 In addition, false-positive scans may occur when the patient has thyroid nodules 37,63,72 or when the tracer localizes in lymph nodes. 69 Similar problems occur with magnetic resonance images. 63 USE OF RAPID PTH ASSAYS Use of the Rapid PTH Test During Parathyroidectomy Rapid intraoperative PTH assays are primarily used to determine whether all hyperfunctioning parathyroid tissue has been removed from the patient. Preoperative localization of hyperfunctioning parathyroid tissue is crucial to the success of limited exploration. 4,73 If preoperative imaging shows an area of increased uptake suggestive of an abnormal parathyroid gland and no contraindications are identified, then the patient is eligible for a minimally invasive parathyroidectomy On the day of operation and preferably just prior to incision, a baseline rapid PTH level is obtained. The abnormal gland is located and removed. Time elapses to allow the circulating PTH to degrade, and a second rapid PTH level is collected and sent for rapid analysis. When serum levels of PTH fail to decline promptly after removal of an abnormal parathyroid gland, this indicates either (1) that the presence of residual hyperfunctioning parathyroid tissue was missed by preoperative localizing studies, or (2) that the tissue removed was not the abnormally functioning parathyroid gland. Frozen section is used only if there is uncertainty about the identity of the resected tissue. A significant drop in the postresection PTH levels can give the surgeon confi- Arch Pathol Lab Med Vol 127, November 2003 Intraoperative Testing for PTH Carter & Howanitz 1427

5 Figure 2. Algorithm of minimally invasive parathyroidectomy using rapid intraoperative parathyroid hormone (PTH) in conjunction with preoperative imaging and frozen section. 26 dence that further exploration is unnecessary. 4,74 Ageneral outline of the basic method for using the rapid PTH assay during the minimally invasive parathyroidectomy is described in Figure 2. Communication between the laboratory and the surgeon is essential to the proper planning and execution of intraoperative PTH tests. The laboratory should be informed on a daily basis of any changes made to the operating room schedule regarding these procedures. 2 The rapid PTH assay has been used in a variety of minimally invasive parathyroidectomy procedures. Criteria to Predict a Surgical Cure Ideally, the cutoff to indicate a surgical cure depends on the half-life of the molecule and the time at which the postresection specimen is collected. The criteria used to indicate a surgical cure have varied considerably among the studies we examined. Fifty-eight of 79 primary studies strictly defined criteria by which the PTH test predicted a cure,* and 12 of these toggled 2 sets of criteria to determine which was more indicative of a cure. The percent decline in PTH used as a cutoff varied from 0% to 8%, and some criteria required that the PTH level return to normal. The time after resection of the gland at which the percent decline should meet or exceed the defined cutoff included, 8,, 1, 20, 30, and 90 minutes as well as any time after resection. The 4 sets of criteria (percent decline at minutes postresection) that were studied the most were 0% at minutes (n 19), 0% at anytime after resection (n ), 0% at or minutes (n 8), and 0% at minutes (n 8). Kinetic analysis of PTH degradation was also examined as a possible method of determining a cure. 77,84,92 The times at which the baseline PTH samples were drawn included the day before operation, the day of operation but before induction of anesthesia, induction of anesthesia, after induction but prior to neck incision, at incision, after mobilization of the thyroid gland but before identification of the abnormal gland, after isolation of the abnormal gland but before excision, and at the moment of excision. Thirty studies collected more than one baseline sample on each patient, and others did not specify exactly when the baseline PTH sample was drawn. Eight studies used the higher of 2 separate baseline values for the calculation of percent decline. This is of concern because, as will be described in detail later in this article, the percent decline in PTH may change depending on when the baseline sample is drawn. The number of samples obtained after parathyroid resection also ranged widely among studies. The number of postresection samples drawn was 1 (n 20), 2 (n 20), 3 (n 14), 4 (n 4), and or greater (n 6) in studies in which this number was static. In other studies, more samples were obtained when the PTH level did not decline past the cutoff. The minimum number of samples drawn in these studies was 1 (n ),2(n 6), and 3 (n 2). The lack of standardization regarding criteria and sample times has been discussed previously. 9,83,92 Yang et al 83 stressed the importance of standardizing the timing of baseline PTH samples. Confusing Terminology The application of the rapid PTH assay for clinical decision making in the studies we examined generated some confusion regarding the use of terms such as true positive, true negative, false positive, and false negative. Traditionally, a true-positive result is defined as a positive test in the presence of disease, and a true-negative result is defined as a negative result in the absence of disease. 1 Clinical sensitivity is the proportion of diseased subjects with test results higher than a particular cutoff value, and clinical specificity is the proportion of disease-free subjects with results lower than a particular cutoff value. 111 In the case of intraoperative PTH testing, we may define a positive test as a decline in PTH greater than the cutoff value and a negative test as a decline less than the cutoff value. * References 12, 26 28, 30 32, 36 44, 48 1,, 6, 8 62, 69 71, References 28, 32, 39, 40, 42, 9, 70, 77, 82, 92, 93, 0. References 9,, 26, 27, 30, 31, 49, 1,, 9, 62, 70, 71, 74, 80 83, 89, 92, 93, 1 9. References 12, 26, 44, 49, 1, 9, 62, Arch Pathol Lab Med Vol 127, November 2003 Intraoperative Testing for PTH Carter & Howanitz

6 Table 1. New, Unambiguous Terminology Regarding Intraoperative Parathyroid Hormone (PTH) Results True decline Diagnostic PTH decline* in a patient free of hyperfunctioning parathyroid tissue False decline Diagnostic PTH decline* in a patient with residual hyperfunctioning parathyroid tissue True failure to decline Failure of the PTH to decline past the cutoff in a patient with residual hyperfunctioning parathyroid tissue False failure to decline Failure of the PTH to decline past the cutoff in a patient free of hyperfunctioning parathyroid tissue * Diagnostic PTH decline: a decline in the PTH level from the baseline that meets or exceeds the cutoff, indicating surgical cure. Using all of these definitions, a positive test in a diseased patient is a true positive, and a negative test in a diseasefree patient is a true negative. Twelve of 79 primary studies reported the results of intraoperative PTH testing as true or false positives and false negatives. Eight of these clearly defined how they used these terms in the text, and 3 indicated the definition of the terms by their use in the text. 26,9,71 All except for one 7 of these studies defined the terms as follows: true positive positive test in a cured (disease free) patient, true negative negative test in an uncured patient (diseased), false positive positive test in an uncured patient, and false negative negative test in a cured patient. Hence, these terms have meanings completely opposite to the traditional terms previously described. The remaining study defined false positives as those patients who had delayed declines in PTH that subsequently returned to normal without further intervention. 7 Two articles reported sensitivity and specificity but did not categorize their results as true or false positives and negatives. 30,31 For readers unfamiliar with how these terms have been used for the rapid intraoperative PTH assay, incorrect conclusions about the test could be drawn. For instance, an exchange of the negative and positive results in this manner results in a complete reversal of the numbers associated with clinical sensitivity and specificity as reported. That only 12 of 79 primary studies reported results using these terms may indicate that the confusion is not just limited to the authors. The intraoperative PTH test is unique because the test is based on a change between values instead of one simple value. Therefore, in Table 1, we propose new, unambiguous terminology that clearly reflects the results of the PTH test. Using this terminology, a true decline is a true negative by traditional definitions but is a true positive according to most authors in the previously described literature. Similarly, a true failure to decline is a true positive by traditional definitions but is a true negative according to the authors already cited. The use of our terminology is intended to avoid confusion in the remainder of this review and may be useful to future authors comparing results across different studies. Advantages of the Intraoperative PTH Assay The rapid PTH assay appears to be quite accurate in detecting residual parathyroid disease, and most authors favorably regarded the use of the assay when they studied References 12, 26, 27, 9, 71, 7, 76, 83, 87, 88, 0, 2. References 12, 27, 7, 76, 83, 87, 88, 0. the results from multiple patients.# Use of the rapid PTH assay changed the course of patients operations in many of the cases reported in the literature.** Continuation of surgical exploration was mandated when the assay suggested the presence of residual hyperfunctioning parathyroid tissue. In most cases, the surgeon found one or more abnormal parathyroid glands that were missed by preoperative imaging. Most of these patients were cured because the surgeon took action based on the test results. When the preoperative image shows a second small area of uptake contralateral to the enlarged gland, the PTH assay may help the surgeon determine whether the second small area is an artifact. 121 The addition of rapid PTH analysis increased the accuracy of various preoperative localization studies by 9% to 1%. 73 The assay may be more beneficial in patients undergoing reoperation, since scar tissue can limit the sensitivity of preoperative imaging. 113 In review, the intraoperative PTH assay has prevented early termination of surgery as well as necessity for reoperation. 37,38,80 Boggs et al 120 demonstrated that their overall surgical failure rate decreased by 7% (from 6% to 1.%) after they introduced adjunctive modalities such as preoperative sestamibi scintigraphy and intraoperative PTH testing. We cannot rule out the possibility of bias in these publications. Factors to Consider When Interpreting the Results of the Rapid PTH Assay Certain difficulties exist in the interpretation of the rapid PTH assay results when determining whether a complete resection has taken place. As with any laboratory test, erroneous results have been reported. Thompson and colleagues 37 reported that a patient s PTH decreased 0% to 60% at 20 minutes after the resection of the gland, and this resulted in termination of the procedure. The patient was discovered soon afterward to have symptomatic multiglandular disease. This result would be classified as a false decline using our terminology. Therefore, some hypersecretory tissue may be missed if a 0% PTH decline is used as a cutoff. 27 Dackiw et al 74 have suggested that fewer false declines occur when a 70% cutoff is used. Thirty-five of the studies that we examined either reported that a mean decrease in PTH level occurred at specific times postresection or that a mean decrease could be calculated by the authors on the basis of the data available in the # References 8, 12, 13, 17, 18, 26 28, 30 32, 36 40, 42, 43, 48, 49, 1, 4,, 9, 60, 62, 69 71, 73 77, 79 86, 89 91, 93 9, 98, 99, 2 7, 9, ** References 27, 28, 37 39, 42, 48 1, 4, 6, 69 71, 74, 7, 79, 80, 82, 94, 2, 7, 11, 119, 120. Arch Pathol Lab Med Vol 127, November 2003 Intraoperative Testing for PTH Carter & Howanitz 1429

7 Table 2. Reference No. Average Percent Decline in Parathyroid Hormone (PTH); Data Collected From 3 Intraoperative PTH Studies (See Also Figure 3) Type of Disease* No. of Patients Postexcision, min Average Decline, % Standard Deviation (Range), % (2 93) (82 9) (6 89.8) ( ) 1.49 ( ) ( ) Arch Pathol Lab Med Vol 127, November 2003 Intraoperative Testing for PTH Carter & Howanitz (2 91) ( 99) (70 0) (37 90) ( 92) ( ) ( ).66 ( ) 2.2 ( ) ( ) (47.7 0)

8 Reference No. Type of Disease* No. of Patients Table 2. Postexcision, min Continued Average Decline, % * indicates single gland disease;, multigland disease; and, results of combined and patients. Averages that were calculated from the data in the article. Article has results from multiple PTH assays. Two separate groups of patients with were studied. Standard Deviation (Range), % (1 98) (2 98) paper (see Figure 3 and Table 2). Of these, 31 studies reported specimens at minutes or greater postresection, and in all of the studies except for 4, the means of the PTH declines were greater than 70% after resection of the last abnormal gland. Three of the exceptions were in groups of patients with multiglandular disease. 18,88,9 This variability in clearance of the hormone from the serum represents another potential source of error. The metabolism and clearance of PTH vary greatly between patients and are influenced by the rate of the production of PTH as well as by its metabolism, the patient s renal function, the type of parathyroid disease that is present, and the concentration of PTH in the patient s serum at baseline prior to resection. 18,28,77,4 Individual parathyroid glands probably do not secrete the same amount of PTH or secrete PTH at the same rate. 18 Ryan et al 9 and Kobayashi et al 116 found good correlation between weight and secretion of PTH. However, when Mózes et al 122 retrospectively compared the PTH level to the weight of the excised parathyroid gland as well as to the type of disease in the patient (single gland disease or multigland disease), they found that the PTH level had no correlation with single gland disease or multiglandular disease and that only extremes of PTH concentration correlated with the size of the gland in a direct manner. Moderate ranges of PTH had no correlation with gland size. Hormone clearance may be especially sluggish in cases of multigland hyperplasia and in patients with MEN. 18,77,78 Tonelli et al 18 noted that patients with a single adenoma are more likely to have a rapid and marked drop in PTH level than are patients with multiglandular disease. Except for the PTH level obtained just after resection of the first parathyroid gland, a stepwise decrease in PTH levels was recognized after removal of each subsequent parathyroid gland in patients with parathyroid hyperplasia. 18 Degradation of PTH may be slowed in patients with tertiary hyperparathyroidism, and this must be taken into account when determining whether these patients are cured after resection. 8 For this reason, we classify a delayed drop in PTH as a false failure to decline. Miura et al 73 had significant difficulty with these false failures to decline, as have other authors. Less commonly, false declines may occur. One report 96 described a resection of a parathyroid adenoma with a subsequent decrease in the PTH to normal. After 1 month, the patient s PTH level increased again, and a second adenoma was found at reoperation. Speculation about whether the first adenoma was dominant and suppressed the secretion of the second was considered. These factors complicate the use of strict percent cutoff criteria for indicating whether a complete resection has taken place in an individual patient. Libutti et al 77 studied the kinetics of PTH by rapid assay in patients undergoing parathyroidectomy for hyperparathyroidism and found that patients with a high baseline PTH had a more rapid decay curve than patients with a low baseline PTH. The percentage of PTH remaining at minutes postresection was determined to be approximately the inverse log of the baseline PTH. 77 Another study had similar findings. 4 Bieglmayer et al 92 and Libutti et al 77 tested this hypothesis retrospectively to investigate this method of determining a cure in patients with minimally elevated levels of PTH at baseline. Bieglmayer et al 92 tested kinetic analysis by 2 mathematic methods. Although the data obtained by one method seemed superior to a strict percent cutoff, a single baseline level was insufficient for kinetic calculations. 92 In addition, at least 4 separate PTH measurements were needed for the most accurate calculations. These requirements diminish cost-effectiveness. However, Libutti et al 77 found that 2 of their patients, who would have been incorrectly treated using the 0% cutoff criterion, were successfully treated by kinetic analysis. Interpretation of PTH values based on kinetic analysis also permits more freedom regarding the times at which samples are drawn. An interesting controversy was raised by several authors when they discovered that some patients with multiple morphologically abnormal parathyroid glands had a diagnostic drop in PTH just after removal of the first abnormal gland. 30,88,90,123 Several postulated that some patients could have morphologically abnormal but not hyperfunctioning parathyroid glands. 30,88 Recognition of a PTH level within the reference range might prevent unnecessary resection of an enlarged but not hyperfunctioning gland. 27 The incidence of multiglandular disease was used by Carneiro and Irvin 90 to compare the surgeon s visual estimation of glandular size with the results of the intraoperative PTH assay. After removal of the first abnormal gland, the remaining glands were visualized, and the first postresection PTH test was performed at the prescribed time. If any additional glands were enlarged by visual inspection, then the surgeon determined that the patient had multiglandular disease and removed the enlarged gland. The patient s first postresection PTH value, if it did not meet cutoff criteria, was used to determine that the patient had functional multiglandular disease. When the surgeon s visual estimation was used, 9% of patients had multiglandular disease. However, when the intraoperative PTH assay was used, only 3% of patients had more than one hyperfunctioning gland. 90 Unfortunately, since all morphologically abnormal glands were removed, the authors could not determine whether the declines in PTH were true or false. The controversy over what truly constitutes a hyperfunctioning gland, on the basis of characteristics such as size and PTH secretion, also was ad- Arch Pathol Lab Med Vol 127, November 2003 Intraoperative Testing for PTH Carter & Howanitz 1431

9 Figure 3. Average decrease in parathyroid hormone (PTH) by time elapsed since resection of abnormal gland in 3 different reports (data listed in Table 2). A, Patients with single gland disease (). B, Patients with multigland disease (). C, All patients with parathyroid disease (). dressed by Wilkinson et al 124 ; however, they, too, were unable to reach a satisfactory conclusion. Most studies have validated the use of this assay in patients with single gland disease. The results have been less clear in patients with multiglandular disease. 48 Reasons for this include the increased incidence of renal failure in these patients as well as uncertainty about the kinetics of PTH production between multiple hyperfunctioning glands. Chou et al 93 studied the assay in 24 patients with multiglandular disease and found that PTH elimination 1432 Arch Pathol Lab Med Vol 127, November 2003 Intraoperative Testing for PTH Carter & Howanitz

10 Table 3. Reported Reasons for Erroneous Results in Rapid Intraoperative Parathyroid Hormone Testing* False Decline False Failure to Decline Other Errors Additional morphologically enlarged glands remained despite decrease in PTH beyond cutoff 27,32,62,76,83,88 Occult was present that would have been detected if more stringent criteria for the cutoff had been employed 37 Perioperative temporary decrease in PTH of unknown etiology 89 Initial decline that never reaches the normal range in MEN type I patients 6 Rupture of a parathyroid cyst early in the procedure, which caused falsely increased baseline PTH 120 Unknown 96 Tumor manipulation during surgery 18,26,,7,83,6,123,147 Normal or only mildly elevated baseline PTH; not enough room for a significant decline 32,7,78 Patients closed despite high postresection PTH, but PTH declined after surgery 32,7,78 Slow rate of PTH decline of unknown cause 27,41,6,70,7,80,2 Slow rate of decline secondary to renal failure when using a PTH assay with a low specificity for (1 84)PTH 16,19,21,9 Slow rate of decline in patient with clear cell ( wasserhelle ) adenoma 80 Slow rate of decline due to rupture of a parathyroid cyst after the baseline PTH is drawn 30,70 Assay less sensitive at lower concentrations 32 Unknown 73 Different rates of PTH decline in parathyroid adenoma versus hyperplasia 18,28,77,4 Premature devascularization of the tumor during surgery, either before or after the baseline value is drawn 3 Technical error 71 * PTH indicates parathyroid hormone;, multigland disease; and MEN, multiple endocrine neoplasia. Surgeon misinterpretation of the test results 60 Specimen integrity problems in the presence of hemolysis 7 Preanalytic errors: incorrect determination of time of gland removal, postresection sampling, and specimen collection 28 Sampling from neck veins near tumor 94 kinetics were similar to those seen in single gland disease if postresection PTH levels were drawn after resection of the last abnormal gland. Kivlen et al 98 observed similar results in patients undergoing reoperation for MEN type I. Physical manipulation of the parathyroid glands during surgery is impossible to avoid and may result in false increases in serum PTH levels. If the baseline PTH is obtained after incision, then this falsely increased baseline PTH, when compared to the PTH level drawn at the prescribed time after resection, may appear to meet cutoff criteria. However, the second PTH level may actually represent the patient s true baseline PTH. 8,83 This may give the surgeon false confidence that all the abnormal parathyroid tissue has been removed. Samples obtained at incision result in PTH values that are an average of 61% higher than those obtained just after induction of anesthesia. 26 Libutti and colleagues 77 caution against drawing the sample minutes or less from the time of resection because of the variability in half-life of PTH among individuals. Falsely decreased baseline PTH levels may be obtained after incision because of occult devascularization. 18,83,3 These factors stress the importance of drawing a baseline PTH at the induction of anesthesia. Sokoll et al 7 determined that propofol, a short-acting sedative-hypnotic agent used during anesthesia, negatively interferes with the PTH assay; thus, they now recommend discontinuing the drug infusion approximately minutes prior to drawing the first baseline PTH specimen. Sampling PTH in central neck veins may have falsely increased levels because of the proximity of these veins to the abnormal glands. 94 Parathyroid cyst fluid contains high quantities of PTH. If a parathyroid cyst is ruptured during surgery, then this may result in falsely elevated PTH levels. 12 Postulated reasons for erroneous PTH results are summarized in Table 3. Some of these problems have led several authors to conclude that the intraoperative PTH assay does not significantly improve the overall success rate of the operation. One of these studies stated that intraoperative PTH testing did not improve the success rate of bilateral neck exploration. 8 All patients in this study underwent the traditional bilateral approach but were split into 2 groups, one with and one without intraoperative PTH sampling. The authors stated that the use of the test did not improve the outcomes of surgery, since 3 operative failures, defined as patients with postoperative hypercalcemia, were reported in each group. However, a careful examination of the article revealed that all 3 patients in the group with PTH testing had hypercalcemia due to factors other than parathyroid disease. Conversely, one of the patients in the group without PTH testing was a true surgical failure. Sprouse et al 126 found that most minimally invasive parathyroidectomies could be conducted successfully with preoperative localization alone, but they reported a failure rate of 6.2%. All of the failed procedures were due to occult multiglandular disease. If they had used the rapid PTH assay results for intraoperative decisions, then an additional 13 patients would have been converted unnecessarily to a traditional bilateral approach. However, the criteria for making this determination were not described. Agarwal et al 0 also do not support the view that the rapid PTH test is a necessary part of minimally invasive parathyroidectomies because of the higher rate of unnecessary conversions to a bilateral neck procedure on the basis of a false failure to decline in PTH. The Effect of the Rapid PTH Assay on Intraoperative Frozen Sections The traditional bilateral approach parathyroidectomy includes the use of frozen section as the only adjunctive References 8, 78, 87, 88, 0, 8, 123, 126. Arch Pathol Lab Med Vol 127, November 2003 Intraoperative Testing for PTH Carter & Howanitz 1433

11 modality to the operation. The authors of a large retrospective study of patients undergoing parathyroidectomy determined that, when used alone as an adjunct to surgery, frozen section can be a significant factor leading to surgical failure in up to 0.8% of cases. 34 Frozen section artifact, follicular parathyroid growth patterns, cellular thyroid nodules, oncocyte-rich nodules, and brown fat have all contributed to diagnostic problems. 2,34 Distinguishing these lesions from parathyroid tissue may even be difficult on examination of the permanent microscopic sections. 34 With the development of the minimally invasive parathyroidectomy and its new adjunctive studies, some anatomic pathologists may be concerned that the rapid PTH assay will completely eradicate the use of frozen sections during parathyroidectomy. Only a few of the published reports compared the use of frozen sections with the rapid PTH assay. 27,34,36 Johnson et al 41 compared a study group of patients who underwent minimally invasive parathyroidectomies along with preoperative imaging and rapid PTH assays to a control group of patients that were treated with minimally invasive parathyroidectomies alone before the advent of these adjunctive techniques. Their use of frozen sections in the study group decreased significantly from 2. to 1.4 per patient, and of the patients in the study group had no frozen sections at all. Other authors preferred to use frozen section because the turnaround time for frozen section in their study was less than that for the rapid PTH assay. 123 Although the number of frozen sections may decrease because of the use of the rapid PTH assay, this assay and frozen section are not truly comparable because the information that the 2 modalities generate is very different. With the advent of intraoperative PTH testing, frozen sections of parathyroid glands are being used in a complementary manner. For example, a surgeon may send resected tissue for frozen section after a patient s PTH concentration does not decline as expected in order to confirm that the specimen was actually parathyroid tissue. Frozen sections may also be obtained when the surgeon is truly uncertain about the nature of the tissue before the results of the postresection PTH level have been returned. These uses are outlined in Figure 2. Effects on the Cost of Care Several authors have compared the costs of the traditional bilateral approach to the minimally invasive parathyroidectomy with its adjunctive studies. Patients undergoing minimally invasive parathyroidectomies with preoperative imaging and intraoperative rapid PTH testing were described as having shorter operating times, fewer complications, the option of local anesthesia, and the possibility of undergoing surgery as outpatients. When used, these factors significantly reduced the overall costs of the procedure. According to a computer-generated model devised by Fahy et al, 127 the use of rapid PTH confirmation decreases costs by $ (US Dispensary [USD]) per patient, and the use of preoperative localization decreases costs by $ (USD) per patient. The cost of the rapid PTH assay depended on the exact test used, and these tests will be described in detail later in this article s section on Test Methodology. In general, References 28, 38, 41, 6, 62, 7, 99. the portable test kits are more expensive than tests performed on automated analyzers in the laboratory. The difference in cost is made wider with the recognition that the instrumentation used to perform the rapid assays in the laboratory is often multifunctional, capable of performing assays on different analytes. 39 If the PTH analysis is performed within the operating room, the cost of not having the medical technologist contributing to the workflow in the central laboratory must be considered. 2 This can be an important issue in laboratories where staffing shortages are already a problem. 7 Another concern is that instruments taken to the point of care are not available for general use in the laboratory during that period. On the other hand, analytic turnaround times are much shorter in studies that have performed the test in the operating room (see Figure 4). Shorter analytic turnaround times contribute to shorter operative times with an associated cost savings. In addition to its influence on cost, the turnaround time of the test is critical to its usefulness in the operating room. However, a specific comparison of the overall cost savings between the shorter operative time for an assay performed at the point of care and the decreased test cost associated with the central laboratory assay has yet to be performed. Of course, cost savings is a moot issue if the operation is not successful. 2 Other potential advantages and disadvantages of the point-of-care assay versus the central laboratory rapid PTH assay are listed in Table 4. To determine how the current trend toward cost containment affected the location of rapid PTH testing in hospitals, a questionnaire about participating laboratories use of the rapid PTH assay was attached to the College of American Pathologists Surveys Program 2001 Y-A Special Ligand Survey. 1 The results of this survey have been reported in detail. 128 Eighty percent of the respondents perform less than 6 rapid PTH assays per month, with most hospitals performing only one per month. Thirty-nine percent of laboratories reported that rapid PTH testing was requested in all parathyroid surgeries performed. In 31% of hospitals, the test was used in greater than 0% of parathyroid surgeries. Thirty percent of reporting hospitals were using the rapid assay only in selected surgical cases ( 0%). Seventy-one percent of respondents performed the test within the laboratory, 6% within a satellite special purpose laboratory, and 23% in the operating room. The data reinforce that a current focus is on controlling costs in the laboratory but also suggest less understanding of the true value of the test and its application on the part of most surgeons performing parathyroid operations. Indeed, the most current edition of a standard general surgery text only briefly mentions intraoperative PTH testing and minimally invasive parathyroidectomies. 7 The ability of the rapid PTH assay to reduce overall patient costs lies in its facilitation of outpatient parathyroidectomy by minimally invasive parathyroidectomy. One study found no significant cost savings when the minimally invasive parathyroidectomy was used, but only 36% of the patients of this study were discharged home on the day of surgery. 99 Conversely, studies with higher discharge rates had much more dramatic cost savings. Dr Udelsman 119 recently reported on his experience with 66 consecutive explorations for primary hyperparathyroidism. This included 401 patients who underwent the traditional bilateral approach and 2 patients who underwent the minimally invasive parathyroidectomy. Patients who underwent minimally invasive parathyroidectomies 1434 Arch Pathol Lab Med Vol 127, November 2003 Intraoperative Testing for PTH Carter & Howanitz

12 Figure 4. Turnaround times in minutes from the collection of the sample to the reporting of the result in published studies. * References in which only the upper limits of turnaround times were given. References in which multiple assays were used in a single study. Gland aspirate analysis (no serum separation step required). PTH indicates parathyroid hormone. Table 4. Potential Advantages and Disadvantages of Performing Rapid Parathyroid Hormone Testing in the Central Laboratory Versus at the Point of Care Point-of-Care Testing Shorter turnaround time ( min*), thereby reducing the duration of the operation and anesthesia Surgeons do not have to wait as long for results Medical technologist directly participates in patient care Sample is directly processed (less chance of unintended delay in transport and processing) Reduced risk of preanalytic and postanalytic errors Medical technologist has to leave laboratory to perform test Equipment usually cannot be used for other types of testing Individual test kits are currently expensive Central Laboratory Testing Advantages More cost-efficient use of medical technologists More cost-efficient use of laboratory equipment Can use the instrument for more than one type of test Disadvantages Longer turnaround time ( min*) with longer duration of the operation Transporting and acquiring results in higher probability of unintended delay in processing High risk of preanalytic and postanalytic errors * Method-independent average time 1 standard deviation calculated from individual turnaround times by report. See references 12,18,2,26,28 30,32,36,38 40,42,43,49,4 6,8,69 71,74,7,77,79 82,84,8,92,93,99,2,3,6,8,9,112,11,130,147,11,12,18. See graph in Figure 4. had equivalent complication rates but had significantly less time in the operating room, under anesthesia, and in the hospital. Patients who underwent successful minimally invasive parathyroidectomies had an average cost savings of $ (USD) compared to patients who had the traditional bilateral approach. A different approach to the cost-effectiveness of the assay was undertaken by Agarwal et al. 87 They looked at the cost of measuring a sample by a dedicated Immulite analyzer and technologist versus measuring PTH samples later that same day after the operation was completed. If the rapid PTH assay was used during every parathyroidectomy procedure, the cost to prevent a single failed surgical operation for primary hyperparathyroidism was $ (USD). Analyzing the specimen with other routine specimens on the same day decreased that cost to $ (USD). This study did not Arch Pathol Lab Med Vol 127, November 2003 Intraoperative Testing for PTH Carter & Howanitz 143