Apoptotic abscess imaging with 99mTc-HYNIC-annexin-V

Objectives Infection upregulates neutrophils (PMN) in circulation and at the site driven by many inflammatory mediators. At the infection site, following bacterial ingestion, PMN apoptosis is elevated. Annexin-V (AnxV) is commonly used to identify apoptotic cells through high affinity binding to externalized phosphatidylserine. When modified with hydrazinonicotinamide (HYNIC), AnxV can be chelated with ^99mTc and used to image apoptosis. We hypothesized that ^99mTc-HYNIC-AnxV will target high concentrations of apoptotic PMNs and image infection.

Methods Apoptosis was induced in isolated human PMNs by incubation for 17 hrs and 22 hrs with 2% saline [1]. HYNIC-AnxV (40 mg) was labeled with ~4.00 mCi of ^99mTcO₄^- following reduction with 20 mg of SnCl₂^.2H₂O. Labeling efficiency was determined by ITLC. Apoptotic and normal PMNs were incubated with ^99mTc-HYNIC-AnxV at 37^oC, 5% CO₂ and association with ^99mTc was determined. Infection was induced in mice by i.m. injection of bacteria. Following i.v. administration of ^99mTc-HYNIC-AnxV, mice were imaged and tissue distribution was performed at 4 hrs and 24 hrs.

Results ^99mTc-HYNIC-AnxV labeling efficiency was 86.0 + 3%. ^99mTc-HYNIC-AnxV bound to apoptotic PMNs was 71.6 + 0.01% and 74.9 + 0.02% at 17 hrs and 24 hrs, respectively. The ^99mTc-HYNIC-AnxV retained by normal controls was 48.6 + 0.01% and 47.2 + 0.02%, respectively. ^99mTc-HYNIC-AnxV in abscesses was 1.3% ID/g and 1.1 % ID/g as compared to controls which was 0.3% ID/g and 0.1% ID/g at 4 hrs and 24 hrs. Abscess to muscle ratios measured 5.3 and 12.9 at 4 hrs and 24 hrs.

Conclusions ^99mTc-HYNIC-AnxV represents a minimally invasive and promising agent to image sites of infection.

Research Support We thank Dr. Francis Blackenberg for his gift of HYNIC-AnxV. 1. Fanning, N.F., et al., Iodinated contrast media induce neutrophil apoptosis through a mitochondrial and caspase mediated pathway. British Journal of Radiology, 2002. 75(899): p. 861-73.