Either inadequate or excessive apoptosis (programmed cell death) is associated 
with many diseases. A method to image apoptosis in vivo, rather than requiring 
histologic evaluation of tissue, could assist with therapeutic decision making 
in these disorders. Programmed cell death is associated with a 
well-choreographed series of events resulting in the cessation of normal cell 
function, and the ultimate disappearance of the cell. One component of apoptosis 
is signaling adjacent cells that this cell is committing suicide by 
externalizing phosphatidylserine to the outer leaflet of the cell membrane. 
Annexin V, a 32-kDa endogenous human protein, has a high affinity for 
membrane-bound phosphatidylserine. We have coupled annexin V with the 
bifunctional hydrazinonicotinamide reagent (HYNIC) to prepare technetium-99m 
HYNIC-annexin V and demonstrated localization of radioactivity in tissues 
undergoing apoptosis in vivo. In this report we describe the results of a series 
of experiments in mice and rats to characterize the biologic behavior of 
(99m)Tc-HYNIC- annexin V. Biodistribution studies were performed in groups of 
rats at 10-180 min after intravenous injection of (99m)Tc-HYNIC-annexin V. In 
order to estimate the degree of apoptosis required for localization of 
(99m)Tc-annexin V in vivo, mice were treated with dexamethasone at doses ranging 
from 1 to 20 mg/kg, 5 h prior to (99m)Tc-HYNIC-annexin V administration, to 
induce thymic apoptosis. Thymus was excised 1 h after radiolabeled HYNIC-annexin 
V injection; thymocytes were isolated, incubated with Hoechst 33342 followed by 
propidium iodide, and analyzed on a fluorescence-activated cell sorter. Each 
sorted cell population was counted in a scintillation counter. To test 
(99m)Tc-HYNIC-annexin V as a tracer for external radionuclide imaging of 
apoptotic cell death, radionuclide imaging of Fas-defective mice (lpr/lpr mice) 
and wild-type mice treated with the antibody to Fas (anti-Fas) was carried out 1 
h post injection. Rat biodistribution studies demonstrated a blood clearance 
half-time of less than 10 min for (99m)Tc-HYNIC-annexin V. The kidneys had the 
highest concentration of radioactivity at all time points. Studies in the mouse 
thymus demonstrated a 40-fold increase in (99m)Tc-HYNIC-annexin V concentration 
in apoptotic thymocytes compared with the viable cell population. A correlation 
of r=0.78 was found between radioactivity and flow cytometric and histologic 
evidence of apoptosis. Imaging studies in the lpr/lpr and wild-type mice showed 
a substantial increase of activity in the liver of wild-type mice treated with 
anti-Fas, while there was no significant change, irrespective of anti-Fas 
administration, in lpr/lpr mice. Excellent images of hepatic apoptosis were 
obtained in wild-type mice 30 min after injection of (99m)Tc-HYNIC-annexin V. 
The imaging results were consistent with histologic analysis in these animals. 
In conlusion, these studies confirm the value of (99m)Tc-HYNIC-annexin V uptake 
as a marker for the detection and quantification of apoptotic cells in vivo.