Reference tissue in receptor studies

Reference tissue is a region where

Reference tissue can be used as a substitute of arterial plasma curve as input function in quantitative analysis of PET studies, applying compartmental models or multiple-time graphical analyses, or in semi-quantitative analysis applying tissue-to-reference tissue ratio. Ex vivo studies are needed to validate that reference tissue does not express the target. Ex vivo and in vivo blocking or displacement studies can verify that the region is suitable as reference region for a specific radioligand. In addition, the in vivo human studies should be able to demonstrate that the distribution volume (VT) of the radioligand is low and similar in the reference region in patient and healthy volunteer groups.

Estimation of the VT requires a metabolite-corrected plasma input function, and application of compartmental model or Logan plot. In optimal situation the ratio of reference tissue and plasma input curves reaches a steady level, and may even be used to estimate the fractions of parent radiopharmaceutical and labelled metabolites in the plasma (Wong et al., 1986). In some cases, low and unchanged SUV in the proposed reference region could be used for verification (de Vries et al., 2021).

Reference tissue vs arterial plasma as input function

Reference tissue Arterial plasma
  • noninvasive method
  • can provide binding potential in receptor studies
  • optimal reference tissue is available only for a few brain receptor radioligands
  • radioactivity spillover from adjacent tissues or due to scatter
  • specific analysis models required
  • represents the true input function (gold standard)
  • can be corrected for labelled metabolites
  • invasive
  • not accessible from all subjects or small animals
  • labour-intensive and error-prone metabolite analysis

If reference tissue has specific binding

If the reference region has specific binding, the binding potential will be underestimated (Gunn et al. 1997):

Specific binding in reference region will also lead to bias in Scatchard analysis (Litton et al. 1994). However, correction methods have been developed (Turkheimer et al., 2012).

Specific binding in reference tissue may lead to severe underestimation of receptor occupancy.

If reference tissue contains labelled metabolite

If label-carrying metabolite passed the blood-brain barrier, then reference tissue input model may lead to severe bias in results, and plasma input models may be preferable (Zoghbi et al., 2006). Specific model needs to be developed, where the concentrations of the metabolite in plasma is used as second model input (Matsubara et al., 2014).

For certain radiopharmaceuticals it may be possible that the concentration of labelled metabolite at late time points is minimal and can be ignored (Kim et al., 1999).

Extracerebral reference region

When no suitable cerebral reference region is available, it may be possible to quantify brain receptors using muscle as reference region (Le Foll et al. 2007). However, there are several caveats in using this approach:

  1. Radioactive label carrying metabolites that can not penetrate the blood brain barrier may well enter extracellular tissues and prevent their use as reference region
  2. Blood flow is much lower in muscle than in the brain, therefore becoming the limiting factor for K1 in the reference tissue
  3. Furthermore, blood flow in muscle is highly variable in awake state
  4. Non-displaceable distribution volume is probably different in the brain and in extracerebral reference region; therefore it has to be measured, and only if proven to be constant the binding potentials can be corrected for it (Le Foll et al. 2007)
  5. Additional requirements may be set by the model, for example, SRTM requires that reference region kinetics can be reasonably well described by 1-tissue compartment model
Tumour In oncology, the concentration in tumour is frequently divided by concentration in normal tissue, which often is muscle (T/N or T/M). This mimics visual analysis, but gives a numerical value to the target-to-background difference.
Problem is that radioligand uptake in muscle may multiply if patient is nervous or position is hard to keep.

Tissue-to-plasma ratio may be a better alternative in these cases, if at least one or two (arterialized) venous blood samples can be taken during the late-scan.

"Positive" reference tissue

If radioligand uptake is very high in a certain brain region, it may take up all ligand that is available in the plasma. In that case the tissue curve is proportional to the AUC (integral) of arterial plasma curve. "Positive" reference tissue can be found and used with few radioligands, including certain AChE radiopharmaceuticals.

See also:


Cunningham VJ, Hume SP, Price GR Ahier RG, Cremer JE, Jones AKP. Compartmental analysis of diprenorphine binding to opiate receptors in the rat in vivo and its comparison with equilibrium data in vitro. J Cereb Blood Flow Metab. 1991; 11: 1-9. doi: 10.1038/jcbfm.1991.1.

Gunn RN, Lammertsma AA, Hume SP, Cunningham VJ. Parametric imaging of ligand-receptor binding in PET using a simplified reference region model. Neuroimage 1997; 6:279-287. doi: 10.1006/nimg.1997.0303.

Kim SE, Szabo Z, Seki C, Ravert HT, Scheffel U, Dannals RF, Wagner HN Jr. Effect of tracer metabolism on PET measurement of [11C]pyrilamine binding to histamine H1 receptors. Ann Nucl Med. 1999; 13(2): 101-107. doi: 10.1007/BF03164885.

Lammertsma AA, Hume SP. Simplified reference tissue model for PET receptor studies. Neuroimage 1996; 4:153-158. doi: 10.1006/nimg.1996.0066.

Le Foll B, Chefer SI, Kimes AS, Shumway D, Goldberg SR, Stein EA, Mukhin AG. Validation of an extracerebral reference region approach for the quantification of brain nicotinic acetylcholine receptors in squirrel monkeys with PET and 2-18F-fluoro-A-85380. J Nucl Med. 2007; 48(9): 1492-1500. doi: 10.2967/jnumed.107.039776.

Litton J-E, Hall H, Pauli S. Saturation analysis in PET - Analysis of errors due to imperfect reference regions. J Cereb Blood Flow Metab. 1994; 14: 358-361. doi: 10.1038/jcbfm.1994.45.

Matsubara K, Ikoma Y, Okada M, Ibaraki M, Suhara T, Kinoshita T, Ito H. Influence of O-methylated metabolite penetrating the blood-brain barrier to estimation of dopamine synthesis capacity in human L-[β-11C]DOPA PET. J Cereb Blood Flow Metab. 2014; 34: 268-274. doi: 10.1038/jcbfm.2013.187.

Mille E, Cumming P, Rominger A, La Fougère C, Tatsch K, Wängler B, Bartenstein P, Böning G. Compensation for cranial spill-in into the cerebellum improves quantitation of striatal dopamine D2/3 receptors in rats with prolonged [18F]-DMFP infusions. Synapse 2012; 66:705-713. doi: 10.1002/syn.21558.

Slifstein M, Laruelle M. Models and methods for derivation of in vivo neuroreceptor parameters with PET and SPECT reversible radiotracers. Nucl Med Biol. 2001; 28: 595-608. doi: 10.1016/S0969-8051(01)00214-1.

Sossi V, Holden JE, Chan G, Krzywinski M, Stoessl AJ, Ruth TJ. Analysis of four dopaminergic tracers kinetics using two different tissue input function methods. J Cereb Blood Flow Metab. 2000; 20: 653-660. doi: 10.1097/00004647-200004000-00002.

Turkheimer FE, Selvaraj S, Hinz R, Murthy V, Bhagwagar Z, Grasby P, Howes O, Rosso L, Bose SK. Quantification of ligand PET studies using a reference region with a displaceable fraction: application to occupancy studies with [11C]-DASB as an example. J Cereb Blood Flow Metab. 2012;32:70-80. doi: 10.1038/jcbfm.2011.108.

Wong DF, Gjedde A, Wagner HN Jr. Quantification of neuroreceptors in the living human brain. I. irreversible binding of ligands. J Cereb Blood Flow Metab. 1986; 6: 137-146. doi: 10.1038/jcbfm.1986.27.

Zoghbi SS, Shetty HU, Ichise M, Fujita M, Imaizumi M, Liow J-S, Shah J, Musachio JL, Pike VW, Innis RB. PET imaging of the dopamine transporter with 18F-FECNT: a polar radiometabolite confounds brain radioligand measurements. J Nucl Med. 2006; 47(3): 520-527. PMID: 16513622.

Tags: , , , ,

Updated at: 2021-11-30
Written by: Vesa Oikonen