Misura della Tireoglobulina: specifiche di qualità e utilizzo clinico

Misura della Tireoglobulina: specifiche di qualità e utilizzo clinico Diego Faggian Dipartimento Medicina di Laboratorio Azienda Ospedaliera Universita Padova diego.faggian@sanita.padova.it

AACC-led Collaboration Educates Millions of Patients and Increases Lab Visibility Thyroglobulin The Test 1. How is it used? 2. When is it ordered? 3. What does the test result mean? 4. Is there anything else I should know?

Thyroglobulin: Reference Range Thyroglobulin testing is primarily used as a tumor marker to evaluate the effectiveness of treatment for differentiated thyroid cancer and to monitor for recurrence. The reference range of thyroglobulin in euthyroid persons without thyroglobulin antibody (TgAb) using CRM-457 standards is 3-40 ng/ml in countries with adequate iodide intake. In about 8% of the general population, thyroglobulin values are less than 10 ng/ml. In newborn babies, the thyroglobulin level may be as high as 36-48 ng/ml up to 48 hours after birth.

The Role of Tg in DTC Following thyroidectomy, DTC patients need life-long surveillance to monitor for tumor recurrence. An estimated 10% experience recurrence during the first decade after surgery, and an additional 5% have late recurrences that may develop decades after the initial treatment. Serum Tg measurement and periodic cervical ultrasound are the main tools for long-term surveillance. The reason serum Tg measurement has such value as a tumormarker for DTC is that Tg protein is synthesized uniquely in thyroid follicular cells. Even though the pre-operative Tg concentration is not informative as a biomarker for DTC, preoperative values can provide a gauge of the tumor s efficiency for Tg secretion and validate the utility of postoperative Tg monitoring. Postoperative Tg measurement is likely to be most sensitive when small tumors are associated with a high preoperative Tg concentration, as compared to large tumors being associated with low preoperative serum Tg values. The latter suggests the tumor is not capable of secreting appreciable amounts of Tg.

History of assay (1) A radioimmunoassay (RIA) for measuring serum Tg measurement was first developed in the 1970s. This method was resistant to the presence, in the sample, of Tg autoantibody (TgAb)

IRMA 1 = RADIM, POMEZIA, ITALY IRMA 2 = CISBIO, CEDEX, FRANCE IRMA 3 = BRAHMS AG, HENNIGSDORF, GERMANY

History of assay (2) After 1970s, several different immunometric assay (first-generation IMA and secondgeneration IMA) methods have been introduced and gained in popularity over time. Despite the fact that serum Tg measurement is a wellaccepted test that has been in common use for 4 decades, it still is subject to a variety of analytical challenges, including insensitivity, wide between-method variability, Tg autoantibody (TgAb) interference and HAMA interference

History of assay (3) Clinicians now use serum Tg primarily for DTC monitoring, whereas in the past they also relied on it to investigate non-neoplastic pathologies such as hyperthyroidism, thyroiditis, and goiter. The clinical utility of Tg testing in DTC with the technical limitations related to the test and provide laboratorians with suggested remedies for these analytical challenges.

Ann Clin Biochem. 2002 May;39(Pt 3):196-202. Can we measure serum thyroglobulin? Clark PM, Beckett G. Source Department Of Clinical Biochemistry, University Hospital Birmingham NHS Trust, UK. pmclarkuhb@compuserve.com Abstract Measurement of serum thyroglobulin (Tg) by immunoassay is used to monitor the treatment of differentiated thyroid cancer. An understanding of the disease process and the biochemistry of Tg allows the laboratory to determine the characteristics of suitable Tg assays. There are a number of key analytical issues: standardization, long-term assay stability, minimum detection limit, working range and the effect of endogenous Tg antibodies (TgAb). There are significant limitations to the methods used to determine TgAb interference. Clinicians should be aware of the limitations of immunoassays for this tumor marker.

The Importance of Direct TgAb Measurement Guidelines recommend measuring TgAb in all specimens prior to Tg testing because the qualitative TgAb status (positive or negative) determines the risk for interference with Tg measurement. The immune system is sensitive to circulating Tg antigen, so for the approximately 20% of DTC patients with detectable TgAb. Specifically, when the antigenic stimulus is removed by thyroidectomy, TgAb concentrations typically decline by approximately 50% within the first year and eventually disappear within a median of 3 years when patients are rendered disease-free. Conversely, TgAb concentrations rise in response to increased antigen concentrations following second surgeries, fine needle biopsy, or radioiodine therapy as well as with recurrence. Serial monitoring of TgAb concentrations can overcome the problem of unreliable Tg IMA measurements.

Endocrine. 2008 Feb;33(1):40-4. doi: 10.1007/s12020-008-9053-2. Epub 2008 Mar 29. In vitro assays to test the interference of anti-thyroglobulin antibodies on thyroglobulin measurement. Madureira D, Prazeres S, Pedro MS, Pereira T, Font AP, Bugalho MJ. Laboratório de Endocrinologia, Instituto Português de Oncologia de Lisboa, Francisco Gentil E.P.E., Rua Professor Lima Basto, 1099-023, Lisbon, Portugal. dmadureira@ipolisboa.min-saude.pt Abstract OBJECTIVE: To assess the interference of anti-thyroglobulin antibodies (TgAb) on serum thyroglobulin (Tg) measurement by in vitro experiments. Design Re-evaluation of Tg concentration after dilution with different TgAb-positive sera. On a first step, dilutions of the same Tg with different TgAb sera were performed and on a second step, different Tgs were diluted with the same TgAb serum. METHODS: Tg measurements were performed using an immunometric (IMA) chemiluminescence assay. TgAb measurements were performed using two methods: immunoflurimetric assay (UNICAP 100) and IMA chemiluminescent assay (IMMULITE 2000). RESULTS: Dilution of a known concentration of Tg with different TgAb-positive sera resulted in a variation of the final concentration of Tg ranging from -24 to -79%. A weak correlation was observed between the TgAb concentration and the percentage of the Tg deviation. Dilution of different Tgs with the same TgAb-positive serum illustrated how the same TgAb positive serum may determine a high interference or a neutral effect. CONCLUSIONS: Present results suggest that the interference on Tg measurement observed in the presence of TgAb may result not only from the anti-thyroglobulin antibodies, but also from the thyroglobulin itself

Countering Interferences HAMA in the specimen can interact with one of the monoclonal antibody reagents to create a false signal that simulates the presence of a high antigen (Tg) concentration. HAMA interference usually results in a falsely high serum Tg that may prompt unnecessary imaging or radioiodine treatment for presumed disease. In contrast, TgAb interference causes falsely low or undetectable serum Tg that can have more serious consequences because it can mask the presence of disease. HAMA and TgAb interference can be investigated by re-testing using RIA methodology or LC-MS/MS that are not influenced by HAMA and TgAb.

Determinazione della Tireoglobulina in Spettrometria di Massa La misura di proteine particolarmente grandi in LC-MS/MS, prevede uno step addizionale, cioè la digestione proteolitica della proteina in peptidi che siano abbastanza piccoli per essere testati dallo spettrometro di massa: 1) Digestione di tutta la proteina in peptidi 2) Separazione dei peptidi in cromatografia liquida 3) Determinazione delle molecole intatte e dei frammenti in MS 4) Scelta del/dei frammenti proteina specifici (nel caso della TG esiste solo 1 frammento: VIFDANAPVAVR) Metodo che non risente della presenza di AbTg e HAMA - Perdita di sensibilità (elevato n di peptidi, variabilità della digestione proteica) - Mutazioni genetiche e post-traslazionali - TAT elevato - Metodo indaginoso

Method and Biological Variability Although a Tg reference preparation (CRM-457) has been available for at least 15 years, method-related variabilities in Tg measurement persist. However, even in the absence of interfering TgAb, the betweenmethod coefficient of variation is about 30%, more than twice the within-person biologic variability. Complicating matters, serum Tg obtained from DTC patients is very heterogeneous, and different from the glandular Tg preparations used as standards. Abnormalities in the post-translational maturation of Tg protein involving glycosylation, phosphorylation, sulfation, and iodination cause this heterogeneity. Indeed, some tumors secrete immature, poorly iodinated, and/or conformationally abnormal Tg molecules that are detected with different specificities by different assays depending on the monoclonal antibody reagents they use

SENSIBILITA ANALITICA valore minimo quantificabile con un determinato metodo, cioè primo valore sicuramente diverso da 0 20 determinazioni del calibratore a concentrazione 0 o di un campione che non contenga l analita in esame nella stessa serie analitica: Media + 2 DS nel referto < limite di sensibilità SENSIBILITA FUNZIONALE concentrazione alla quale il CV delle 20 misure ripetute è uguale al 20% (al diminuire delle concentrazioni di analita, i CV aumentano e quindi, i metodi sono più imprecisi)

The Benefits of Functional Sensitivity (1) A realistic determination of Tg assay sensitivity is critical for the effective management of DTC patients following thyroidectomy, when very little Tg-producing tissue is left. FS is a clinically relevant parameter based on low-end, between-run precision. The guidelines define it as the Tg concentration that can be measured with 20% coefficient of variation determined from multiple measurements of a human serum pool containing a low Tg level made across a clinically-relevant time-span (6 12 months) and employing at least two calibrator lots and two reagent lots.

Functional Sensitivity: Impatto sul FOLLOW-UP L'obiettivo più importante del follow-up è identificare, il più precocemente possibile, la presenza di malattia (più frequente per High Risk), distinguendo contemporaneamente i pazienti liberi da malattia (più frequente per Low Risk). Per ottenere questi obiettivi, le più recenti strategie di follow-up si basano, non solo sul rischio iniziale, ma anche sulla ridefinizione del rischio

La tireoglobulina dopo stimolo con rh-tsh L importanza del dosaggio della tireoglobulina dopo stimolo con rh-tsh, raccomandato in tutte le linee guida, è principalmente legata all elevato valore predittivo negativo del test, che se negativo (Tg dopo rh-tsh <0.5) assicura l eradicazione del tumore in quasi il 100% dei pz (rischio di recidiva è inferiore allo 0.5%.) all elevato valore predittivo positivo del test, che se positivo (Tg dopo rh-tsh >2) prevede il riscontro di recidiva/persistenza di malattia nell 80% dei pz a 5 anni

dosaggio della Tg basale e dopo rh-tsh EUROPA

dosaggio della Tg basale e dopo rh-tsh USA

The Benefits of Functional Sensitivity (2) As stressed in current guidelines it is critical that assay sensitivity be assessed in terms of functional sensitivity and not analytical sensitivity The guidelines committee developed this definition to realistically represent the sensitivity of the test used in clinical practice, and to replace descriptive terms like ultrasensitive and supersensitive that manufacturers favor for marketing.

First-generation FS (Functional Sensitivity) - RIA methodology is only capable of first-generation FS ranging from 0.5 1.0 µg/l - First-generation assays are too insensitive clinically to use for basal Tg monitoring without TSH stimulation. -By consensus, a 72-hour rhtsh-stimulated Tg above 2.0 µg/l is considered a risk factor for disease. (In reality, this fixed rhtsh-tg cutoff depend on the method used. A Tg value of 2.0 µg/l could be reported as being anywhere between 1.5 and 3.2 µg/l using different methods ). Second-generation FS (Functional Sensitivity) - More recently, second-generation IMAs have become available with FS ranging from 0.05 0.1 µg/l. (Fs 10-fold higher than the first-generation assays) - Second generation IMAs also facilitate the monitoring of basal Tg trends that improve positive and negative predictive values for assessing risk for disease, as compared with the rhtsh-tg cut-off value of 2.0 µg/l. Second-generation IMA may eliminate the need for expensive and inconvenient recombinant human TSH (rhtsh) stimulation.

NEI PAZIENTI CON us-tg <0.05 IL TEST DI STIMOLAZIONE CON rhtsh SEMBREREBBE NON NECESSARIO

NEI PAZIENTI CON ustg > 0.1 e 1.0 ng/ml IL TEST DI STIMOLO CON rhtsh è IN GRADO DI DISCRIMINARE PAZIENTI LIBERI DA MALATTIA da PAZIENTI CON PERSISTENZA/RECIDIVA DI MALATTIA

Labs on the Front Lines Given the importance of life-long postoperative monitoring of DTC patients, labs have a vital responsibility to ensure that Tg measurements are as accurate as possible, and that they keep abreast of and address the analytical limitations of their Tg assay methods. The technical pitfalls of Tg measurement include between-method variability, inappropriate reference ranges, suboptimal functional sensitivity (FS), hook effects, and human anti-mouse antibody (HAMA), as well as TgAb interferences. Ongoing dialogue with endocrinologists and oncologists likewise is essential, so these clinicians can be well-informed of any method changes and confer readily with laboratorians about any discrepant results, and decide toghether the optimal FS to use.

Sistema Immunoenzimatico : AIA-2000 TOSOH

Sistema Immunoenzimatico : AIA-2000 TOSOH Sistema immunoenzimatico. La tg presente nel campione si lega con l anticorpo monoclonale immobilizzato sulle particelle paramagnetiche (anticorpo monoclonale anti-tg mouse) e un anticorpo monoclonale mouse Ab-tg coniugato all alcalino-fosfato bovina con sodio azide come conservante presente nella cuvetta di reazione. Substrato fluorogenico 4-metillumbelliferil fosfato. Calibratore riferito allo standard internazionale CRM457

CONFRONTO TRA METODI UNICEL DXI BECKMANN VS TOSOH AIA MEDIA DEI VALORI DELLA TOSOH = 9.713 ng/ml; MEDIA DEI VALORI DEL UNICEL DXI = 5.97 ng/ml. ANALISI DI BLAND ALTMAN : EVIDENZIA UN BIAS POSITIVO MA NON SIGNIFICATIVO CLINICAMENTE: [ + 3.739 ( - 1.135 to 8.612)]. 100 Difference Plot Identity 250 Scatter Plot with Passing & Bablok Fit Difference (TIREO AIA ng/ml - TIREO DXI ug/l 80 60 40 20 0-20 Bias (3.7) 95% Limits of agreement (-25.3 to 32.8) TIREO AIA ng/ml 200 150 100 50 Identity Passing & Bablok (I) fit (-0.06 + 1.71x) -40 0 50 100 150 200 250 0 0 50 100 150 200 250 Mean of All TIREO DXI ug/l

RIPETIBILITA USANDO 5 POOL DI SIERO A DIVERSA CONCENTRAZIONE - POOL I POOL II POOL III POOL IV POOL V MEDIA = 0.26 ng/ml CV%=10.2 MEDIA = 0.79 ng/ml CV%= 4.7 MEDIA = 1.42 ng/ml CV%= 4.3 MEDIA = 26.36 ng/ml CV%= 2.8 MEDIA = 67.92 ng/ml CV%= 4.2

COEFFICIENTI DI VARIAZIONE USANDO COME MATRICE I CONTROLLI ESTERNI DELLA DITTA BIORAD ( LOTTO 54550) INTRA-ASSAY 20 REPLICATI BIORAD 54551 BIORAD 54552 BIORAD 54553 MEDIA = 13.70 ng/ml CV% = 2.5% MEDIA = 83.80 ng/ml CV%= 3.0 MEDIA = 189.98 CV=3.2%

COEFFICIENTI DI VARIAZIONE USANDO COME MATRICE I CONTROLLI ESTERNI DELLA DITTA BIORAD ( LOTTO 54550) INTRASSAY 20 REPLICATI [TG] 15 14 13 CONTROLLO BIORAD 54551 12 LOTTO 0.3 VS LOTTO 0.4 BIORAD 54551 BIORAD 54552 BIORAD 54553 LOTTO TG 03 LOTTO TG 04 MEDIA = 13.70 ng/ml MEDIA = 13.458 ng/ml CV% = 2.5 CV% = 4.3 MEDIA = 83.80 ng/ml CV%= 3.0 CV%= 2.9 MEDIA = 84.96 ng/ml MEDIA = 189.98 MEDIA = 199.91 CV%=3.2 CV%=2.8 [TG] 92.5 90.0 87.5 85.0 82.5 80.0 77.5 CONTROLLO BIORAD 54552 LOTTO 0.3 VS LOTTO 0.4 CONTROLLO BIORAD 54553 210 200 [TG] 190 180 170 LOTTO 0.3 VS LOTTO 0.4

TEST DI LINEARITA DILUIZIONI ESEGUITE : 1; 1/2; 1/4 ; 1/8 ; 1/16; 1/32; 1/64; 1/128; 1/256 n 9 R 2 1,00 35 30 Linearity Plot Adjusted R 2 1,00 SE 0,23417 n 9 Concentrations 9 Media TG 25 20 15 10 5 Linear fit (0.1619 +1.004x) Polynomial fit (0.01169 +1.073x -0.002256x²) Range 0.1271 to 32.55900 0 0 5 10 15 20 25 30 35 Valore Atteso Valore Atteso Replicates Mean % Scatter Plot with Fit 32.559 2 32.559 0.00 35 Linear fit (0.1619 +1.004x) 16.2795 2 16.966 4.2 30 95% CI 8.1397 2 8.488 4.3 4.0698 2 4.3305 6.4 2.0349 2 2.2305 9.6 Media TG 25 20 15 10 95% Prediction interval 1.0174 2 1.124 10.5 5 0.5087 2 0.5485 7.8 0 0.2543 2 0.299 17.6-5 0 5 10 15 20 25 30 35 0.1271 2 0.136 7. Valore Atteso

TEST DI LINEARITA DILUIZIONI ESEGUITE : 1; 1/2; 1/4 ; 1/8 ; 1/16; 1/32; 1/64; 1/128; 1/256 Scatter Plot with Fit n 9 14 12 10 Linear fit (0.04484 +1.001x) 95% CI 95% Prediction interval R 2 1,00 Adjusted R 2 1,00 SE 0,00.6393 Media TG 8 6 4 2 0-2 0 5 10 n 9 Valore Atteso Concentrations 9 Linearity Plot Range 0,04980 to 12,77000 Valore Atteso Replicates Mean % discrepanza 14 12 12.77 2 12.77 0 6.385 2 6.524 2.2 3.1925 2 3.233 1.3 1.59625 2 1.756 10 Media TG 10 8 6 Linear fit (0.04484 +1.001x) Polynomial fit (0.01417 +1.037x -0.002994x²) 0.7981 2 0.82 2.7 0.399 2 0.435 9 4 0.1995 2 0.2155 8 2 0.0997 2 0.1055 5.3 0.0498 2 0.0692 8.8 0-0,9502 4,0498 9,0498 Coded Concentration

TEST DI RECUPERO n 22 Range 0,3390 to 199,5715 Valore atteso Replicates Mean % Recovered 0.339 2 0,3620 106,8% è stato eseguito miscelando due volumi uguali di sieri a concentrazione diversa di Tg 0.8315 2 0,8220 98,9% 2.4815 2 2,6230 105,7% 3.112 2 3,1620 101,6% 10.2165 2 10,7660 105,4% 10.847 2 10,5010 96,8% Scatter Plot with Fit 12.497 2 12,6020 100,8% 13.8675 2 14,1690 102,2% 14.498 2 14,9110 102,8% 16.148 2 17,1160 106,0% 23.883 2 24,3070 101,8% 200 150 Linear fit (1.097 +0.9333x) 95% CI 95% Prediction interval 81.7485 2 74,9690 91,7% 82.3655 2 74,6940 90,7% 84.0155 2 78,2000 93,1% 91.7505 2 97,4680 106,2% 94.4015 2 92,7000 98,2% 118.037 2 107,9110 91,4% 118.668 2 107,9290 91,0% 120.318 2 107,1640 89,1% 128.053 2 130,1930 101,7% 131.704 2 126,4650 96,0% 199.571 2 183,8520 92,1% Valore ottenuto 100 50 0-50 0 50 100 150 200 Valore atteso

Tireoglobulina SENSIBILITA FUNZIONALE DICHIARATA = 0.09 ng/ml SENSIBILITA FUNZIONALE OTTENUTA = 0.08 ng/ml

INTERFERENZA CON GLI ANTICORPI Ab-Tg ID Valore Atteso Valore Ottenuto % discrepanza 1 0,50025 0,469-6,2 2 0,50425 0,4965-1,53 3 0,447 0,4435-0,78 4 0,476 0,525 10,29 5 18,80725 13,734-26,97 6 71,20325 57,49-19,25 ID Valore Atteso Valore Ottenuto % discrepanza 7 0,122 0,114-6,55 8 0,197 0,2 1,52 9 15,79 9,921-37,16 Campione aggiunto con titolo anticorpale di 2868 KU/L e [Tg]= 0,804 Campione aggiunto con titolo anticorpale di 1388 KU/L e [Tg]= 0,000

Casistica Dell U.O.C. di Medicina di Laboratorio, Az. Ospedaliera di Padova Anno 2012 - Test di Tireoglobulina effettuati Test di stimolo rhtsh Totali 5054 Totali 248 (744) Test con concentrazione di Tg < 0,1 ng/ml 2152 Posiviti Concentrazione di Tg > 2 ng/ml 17 Test con concentrazione di Tg >0,1 <2 ng/ml 1225 Negativi Concentrazione di Tg < 2 ng/ml 231 Test con concentrazione di TG > 2 ng/ml 1677 131 con tutti e tre i prelievi con concentrazione di Tg < 0,1

Casistica Dell U.O.C. di Medicina di Laboratorio, Az. Ospedaliera di Padova Da Gennaio a Ottobre -Anno 2013- Test di Tireoglobulina effettuati Test di stimolo rhtsh Totali 5441 Totali 452 (1356) Test con concentrazione di Tg < 0,1 ng/ml 2423 Posiviti Concentrazione di Tg > 2 ng/ml 36 Test con concentrazione di Tg >0,1 <2 ng/ml 1358 Negativi Concentrazione di Tg < 2 ng/ml 416 Test con concentrazione di Tg > 2 ng/ml 1660 273 con tutti e tre i prelievi con concentrazione di Tg < 0,1

ACCESS DXI 800 BECKMAN COULTER Dosaggio immunoenzimatico in una unica fase La miscela biotinilata anticorpale è formata da 4 anticorpi monoclonali anti-tg Standardizzato contro lo standard Internazionale di riferimento CRM 457 Sensibilità funzionale dichiarata: 0.1 ng/ml Sensibilità funzionale ottenuta: 0.05 ng/ml

ACCESS DXI 800 AIA-2000 LA

COEFFICIENTI DI VARIAZIONE USANDO COME MATRICE I CONTROLLI ESTERNI DELLA DITTA BIORAD ( LOTTO 54550) INTRA-ASSAY 20 REPLICATI BIORAD 54551 BIORAD 54552 BIORAD 54553 MEDIA = 13.70 ng/ml CV% = 2.5% MEDIA = 83.80 ng/ml CV%= 3.0 MEDIA = 189.98 ng/ml CV=3.2%

HIGHLIGHTS 1) Armonizzazione dei risultati su standard internazionale CRM-457 2) Richiedere al mercato materiali di controllo a concentrazione adeguata 3) Referto con riportato il valore di sensibilità funzionale 4) Privilegiare per il follow-up metodiche con minima/assente interferenza da AbTG

Grazie Per l attenzione

UN RINGRAZIAMENTO GRANDE LO DEVO ALLA MIA COLLABORATRICE CHIARA COSMA PER LA SUA PAZIENZA.