A new microparticle size calibration standard for use in measuring smaller microparticles using a new flow cytometer.

BACKGROUND: Microparticle size measurements are often calibrated on flow cytometers using polystyrene microspheres that forward scatter more light vs. particle diameter than cellular microparticles. METHODS: We compared theoretical with measured forward angle light scattering on the LSRII, FC500 and Apogee A40 using polystyrene and silica microspheres vs. synthetic lipid vesicles and platelets, then compared plasma microparticle counts using different calibration strategies. RESULTS: Polystyrene and silica microspheres with higher refractive indices forward scattered more light with a wavelength of 488 nm for a given size microparticle than did lipid vesicles or platelets. The LSRII and FC500 did not count many, and were unable to separate by size, polystyrene microspheres <0.5 µm in diameter. On the Apogee A40, polystyrene microspheres could be separated by size down to 0.2 µm, and a polystyrene microsphere 0.4 µm in diameter produced the same forward scatter relative intensity as a 1-µm lipid or cellular microparticle. Using the new calibrator, the Apogee A40 found 80 000-4 000 000 µL(-1) total microparticles, 11 000-350 000 µL(-1) annexin V positive microparticles and 6000-350 000 µL(-1) platelet microparticles <1 µm in plasma samples. CONCLUSIONS: The Apogee A40 was able to resolve size differences in polystyrene microspheres down to 0.2 µm and microparticles down to 0.4 µm. On the Apogee A40 we propose using a 0.4-µm polystyrene microsphere as equivalent to a 1-µm cellular microparticle for size calibration. Using this calibrator, the Apogee A40 detected higher numbers of total, platelet and annexin V positive microparticles than were found using a Megamix gate.

Preparation of F-18 labeled annexin V: a potential PET radiopharmaceutical for imaging cell death.

The clinical response to antitumor therapy is measured using imaging, such as CT or MRI, 6-12 weeks following chemotherapy treatment. The images at that time reflect both tumor cell death and new growth. Therefore, the amount of tumor cell death caused by chemotherapy cannot be efficiently quantified with current imaging modalities. A quantitative measurement of tumor cell death immediately following chemotherapy is needed to help validate both new agents and to optimize administration of existing therapies. Annexin V is a 36kD protein that binds to exposed phosphatidylserine (PS) on dying cells. In order to synthesize a probe that can detect cell death in vivo, the positron emitter F-18 was conjugated to annexin V via the compound N- succinimidyl-4-[18F]fluorobenzoate, [18F]SFB. The decay corrected radiochemical yield of F-18 labeled annexin V from 18F fluoride was 17.6 +/- 5.6% (n = 4) in three hours. The stepwise radiochemical yield of the conjugation step with annexin V was as high as 70% when a protein concentration of 5 mg/ml was used. Cancer cells treated with the chemotherapeutic agent, etoposide, showed an 88% increase in the binding of F-18 labeled annexin V compared to untreated cells. We conclude that [18F] labeled annexin V can be readily prepared by the conjugation of annexin V with [18F]SFB and that the positron-emitting compound is biologically active in detecting apoptosis.

Annexin-V imaging for noninvasive detection of cardiac allograft rejection.

Heart transplant rejection is characterized pathologically by myocyte necrosis and apoptosis associated with interstitial mononuclear cell infiltration. Any one of these components can be targeted for noninvasive detection of transplant rejection. During apoptotic cell death, phosphatidylserine, a phospholipid that is normally confined to the inner leaflet of cell membrane bilayer, gets exteriorized. Technetium-99m-labeled annexin-V, an endogenous protein that has high affinity for binding to phosphatidylserine, has been administered intravenously for noninvasive identification of apoptotic cell death. In the present study of 18 cardiac allograft recipients, 13 patients had negative and five had positive myocardial uptake of annexin. These latter five demonstrated at least moderate transplant rejection and caspase-3 staining, suggesting apoptosis in their biopsy specimens. This study reveals the clinical feasibility and safety of annexin-V imaging for noninvasive detection of transplant rejection by targeting cell membrane phospholipid alterations that are commonly associated with the process of apoptosis.

Detection of early atherosclerosis with radiolabeled monocyte chemoattractant protein-1 in prediabeteic Zucker rats.

BACKGROUND: Migration of monocytes into the arterial wall is an early finding of atherosclerosis. Monocytes are attracted to sites of vascular endothelial cell injury, the initiating event in the development of atheromatous disease, by a chemokine known as monocyte chemoattractant protein-1 (MCP-1). Injured vascular endothelial and smooth muscle cells selectively secrete MCP-1. OBJECTIVE: This study was performed to determine if radiolabeled MCP-1 would co-localize at sites of monocyte/macrophage concentration in an experimental model of transplant-induced vasculopathy in diabetic animals. MATERIALS AND METHODS: Hearts from 3-month-old male Zucker rats, heterozygote (Lean) or homozygote (Fat) for the diabetes-associated gene fa, were transplanted into the abdomens of genetically matched recipients. Lean and Fat animals were then fed normal or high-fat diets for 90 days. RESULTS: At 90 days significant increases (P < 0.013) of MCP-1 graft uptake were seen at imaging and confirmed on scintillation gamma well counting studies in Lean (n = 5) and Fat (n = 12) animals, regardless of diet, 400 % and 40 %, above control values, respectively. MCP-1 uptake of native and grafted hearts correlated with increased numbers of perivascular macrophages (P < 0.02), as seen by immunostaining with an antibody specific for macrophages (ED 2). CONCLUSION: Radiolabeled MCP-1 can detect abnormally increased numbers of perivascular mononuclear cells in native and grafted hearts in prediabetic rats. MCP-1 may be useful in the screening of diabetic children for early atherosclerotic disease.

Imaging macrophages and the apoptosis of granulocytes in a rodent model of subacute and chronic abscesses with radiolabeled monocyte chemotactic peptide-1 and annexin V.

Monocytes/macrophages (Mphis), the predominant cell types in subacute and chronic inflammation, are attracted to and activated by monocyte chemotactic peptide-1 (MCP-1). Mphis promote the resolution of inflammation through the induction of apoptosis and phagocytosis of senescent (spent) and bystander (superfluous) granulocytes. We wished to determine whether MCP-1, which selectively binds to Mphis, could be used to image subacute and chronic inflammation. We also sought to image granulocyte apoptosis within these lesions with technetium-99m labeled annexin V, a marker of apoptotic cells. Sterile inflammation was induced in 45 12-week-old male Sprague-Dawley rats by deep intramuscular injection of turpentine into the right thigh. Groups of four to six animals were then imaged 1 h after tail vein injection of 37-148 MBq (1-4 mCi) of 99mTc-labeled MCP-1 or annexin V 1-14 days after turpentine treatment. Image analysis showed significantly greater activity of both MCP-1 and annexin V in inflamed thighs than in control thighs (165%-290% and 188%-313%, respectively; P<0.01) on days 1-5 after turpentine injection. Dual autoradiography in animals co-injected with iodine-125 labeled bovine serum albumin on days 1 and 4 showed specific location of MCP-1 to infiltrating Mphis while annexin V localized to focal zones of apoptosis within granulocytic infiltrates adjacent to abscess cavities. Scintillation well counting on day 5 demonstrated significantly higher (P<0.005) ratios of abscess to control thigh specific activities for MCP-1 (5.83+/-2.17) and annexin V (9.24 +/- 2.8) as compared to 125I-labeled bovine serum albumin (3.11 +/- 0.65). No significant increases in uptake were noted at imaging or ex vivo analyses on days 13 and 14, when lesions were predominately fibrotic. It is concluded that 99mTc-labeled MCP-1 and 99mTc-labeled annexin V both localize in zones of subacute inflammation, reflecting the density of Mphis and the incidence of apoptotic granulocytes, respectively. These agents may be useful in the characterization of subacute inflammation.

Apoptosis and allograft rejection in the absence of CD8+ T cells.

BACKGROUND: The requirement for cytotoxic T lymphocytes during allograft rejection is controversial. We previously demonstrated that CD8+ T cells are not necessary for allograft rejection or for the induction of apoptosis in rat small intestinal transplantation. In this study, we examined the mechanisms of apoptosis and rejection after liver transplantation in the absence of CD8+ T cells. METHODS: Either Lewis or dark agouti rat liver grafts were transplanted into Lewis recipients to create syngeneic and allogeneic combinations. CD8+ T cells were depleted in an additional allogeneic group by treatment with OX-8 mAb on day -1 and day 1 after liver transplant. RESULTS: Apoptosis and rejection were observed in both the CD8+ T cell-depleted allogeneic and allogeneic grafts by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and radiolabeled-annexin V in vivo imaging. Granzyme B and FasL were expressed in all allogeneic transplants, including those depleted of CD8+ T cells, indicating that a mononuclear cell other than a CD8+ T cell can be the source of these molecules during allograft rejection. Activation of the caspase cascade was detected in all rejecting allografts. Caspases 3, 8, and 9 were activated at similar significantly elevated levels in both allogeneic and CD8+ T cell-depleted liver grafts. CONCLUSION: These data indicate that in the absence of CD8+ T cells an alternative pathway, associated with granzyme B and FasL expression and activation of the caspase cascade, can mediate apoptosis and graft rejection.

Imaging cyclophosphamide-induced intramedullary apoptosis in rats using 99mTc-radiolabeled annexin V.

UNLABELLED: Intramedullary apoptosis of hematopoietic tissue is believed to play a major role in the pathophysiology of myelodysplastic syndrome. Annexin V, a specific marker of the early to intermediate phases of apoptosis, has been applied to the in vitro study of bone marrow aspirates. A noninvasive measure of intramedullary apoptosis in vivo that could serially monitor the clinical progression of myelodysplastic syndrome may be helpful. METHODS: We used 99mTc-radiolabeled annexin V and radionuclide gamma camera imaging to serially study the sites, extent, and severity of intramedullary apoptosis induced by cyclophosphamide treatment. RESULTS: Intravenously administered radiolabeled annexin V localized preferentially in the femur, pelvis, vertebrae, and spleen; increased uptake in these organs was easily visualized as early as 8 h after injection of 100 mg/kg cyclophosphamide in 8- to 10-wk-old animals. Higher doses of cyclophosphamide (150 mg/kg) in animals of the same age increased annexin V uptake in the bone marrow and splenic tissue and delayed recovery of these organs as seen histologically compared with lower doses. Older animals, 5-6 mo old, showed a slower response to cyclophosphamide treatment and delayed recovery of bone marrow and splenic tissues. CONCLUSION: Radiolabeled annexin V can be used to detect and directly quantify the degree of intramedullary and splenic apoptosis in a noninvasive fashion using current clinical radionuclide imaging equipment. Annexin V imaging may be useful clinically in the diagnosis and management of myelodysplastic syndrome.

In vivo imaging of acute cardiac rejection in human patients using (99m)technetium labeled annexin V.

Annexin V binds phosphatidylserine moieties on apoptotic cells. This study reports the initial experience at Stanford University Medical Center with 99mTc-labeled annexin V imaging as a noninvasive measure of apoptosis in acute cardiac rejection. Ten cardiac transplant patients had 99mTc Annexin V imaging and endomyocardial biopsy (EMB) performed within 24 h. No complications related to 99mTc annexin V administration occurred. Eight patients had ISHLT grade of acute rejection of 1A or less. Five patients had two or more areas of uptake noted in the right ventricle on imaging studies. Two of these patients had positive biopsies: one patient had grade 2 rejection with two focal uptake areas and another had grade 3A rejection with three foci. An additional five patients had either one or zero hot spot areas and corresponding negative EMBs. 99mTc-annexin V appears to be well tolerated and may identify patients with acute cardiac rejection.

99mTc annexin V imaging of neonatal hypoxic brain injury.

BACKGROUND AND PURPOSE: Delayed cell loss in neonates after cerebral hypoxic-ischemic injury (HII) is believed to be a major cause of cerebral palsy. In this study, we used radiolabeled annexin V, a marker of delayed cell loss (apoptosis), to image neonatal rabbits suffering from HII. METHODS: Twenty-two neonatal New Zealand White rabbits had ligation of the right common carotid artery with reduction of inspired oxygen concentration to induce HII. Experimental animals (n=17) were exposed to hypoxia until an ipsilateral hemispheric decrease in the average diffusion coefficient occurred. After reversal of hypoxia and normalization of average diffusion coefficient values, experimental animals were injected with (99m)Tc annexin V. Radionuclide images were recorded 2 hours later. RESULTS: Experimental animals showed no MR evidence of blood-brain barrier breakdown or perfusion abnormalities after hypoxia. Annexin images demonstrated multifocal brain uptake in both hemispheres of experimental but not control animals. Histology of the brains from experimental animals demonstrated scattered pyknotic cortical and hippocampal neurons with cytoplasmic vacuolization of glial cells without evidence of apoptotic nuclei by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. Double staining with markers of cell type and exogenous annexin V revealed that annexin V was localized in the cytoplasm of scattered neurons and astrocytes in experimental and, less commonly, control brains in the presence of an intact blood-brain barrier. CONCLUSIONS: Apoptosis may develop after HII even in brains that appear normal on diffusion-weighted and perfusion MR. These data suggest a role of radiolabeled annexin V screening of neonates at risk for the development of cerebral palsy.

Zinc-mediated reduction of apoptosis in cardiac allografts.

BACKGROUND: Apoptosis is thought to occur during immune-mediated acute rejection of cardiac allografts. In vitro studies have shown that zinc inhibits the activity of the proapoptotic enzyme caspase-3. We hypothesized that ZnCl(2) would reduce acute cardiac rejection in vivo via the blockade of caspase-3-dependent apoptosis. (99m)Tc-labeled annexin V was used to measure apoptosis in cardiac allografts through nuclear imaging. Annexin V binds to phosphatidylserines, which are externalized to the outer membrane of apoptotic cells. METHODS AND RESULTS: Twenty-seven PVG rat hearts were transplanted heterotopically into the abdomen of untreated ACI rats as controls (group 1). Fifteen were scanned and euthanized on postoperative day 4, and 12 were assessed for graft survival. Group 2 and 3 rats (n=15 each) received 1 and 5 mg/kg ZnCl(2) BID IP, respectively. Nine of each of these groups were scanned and euthanized on postoperative day 4, and 6 were studied for allograft survival. Group 4 rats (n=3) received isografts. Region-of-interest analysis demonstrated a dose-dependent reduction in (99m)Tc annexin uptake in ZnCl(2)-treated allografts: 2.43+/-0.37% for group 1, 1. 97+/-0.41% for group 2, 1.21+/-0.47% for group 3, and 0.55+/-0.19% for group 4 (ANOVA, P:=0.001). Graft survival times of 6.4+/-1.7, 9. 3+/-3.0, and 11.5+/-3.4 days for groups 1, 2, and 3, respectively, were also observed (ANOVA, P:=0.001). Caspase-3 activity in the allografts showed a 3.7-fold reduction in group 3 animals compared with group 1 animals (P:=0.004). CONCLUSIONS: Apoptosis that occurs in acute cardiac allograft rejection is reduced with ZnCl(2) in a dose-dependent manner via caspase-3 inhibition.

Development and characterization of annexin V mutants with endogenous chelation sites for (99m)Tc.

[(99m)Tc]Annexin V can be used to image organs undergoing cell death during cancer chemotherapy and organ transplant rejection. To simplify the preparation and labeling of annexin V for nuclear-medicine studies, we have investigated the addition of peptide sequences that will directly form endogenous chelation sites for (99m)Tc. Three mutant molecules of annexin V, called annexin V-116, -117, and -118, were constructed with N-terminal extensions of seven amino acids containing either one or two cysteine residues. These molecules were expressed cytoplasmically in Escherichia coli and purified to homogeneity with a final yield of 10 mg of protein/L of culture. Analysis in a competitive binding assay showed that all three proteins retained full binding affinity for erythrocyte membranes with exposed phosphatidylserine. Using SnCl(2) as reducing agent and glucoheptonate as exchange agent, all three proteins could be labeled with (99m)Tc to specific activities of at least 50-100 microCi/microg. The proteins retained membrane binding activity after the radiolabeling procedure, and quantitative analysis indicated a dissociation constant (K(d)) of 7 nmol/L for the annexin V-117 mutant. The labeling reaction was rapid, reaching a maximum after 40 min at room temperature. The radiolabeled proteins were stable when incubated with phosphate-buffered saline or serum in vitro. Proteins labeled to a specific activity of 25-100 microCi/microg were injected intravenously in mice at a dose of 100 microg/kg, and biodistribution of radioactivity was determined at 60 min after injection. Uptake of radioactivity was highest in kidney and liver, consistent with previous results obtained with wild-type annexin V. Cyclophosphamide-induced apoptosis in vivo could be imaged with [(99m)Tc]annexin V-117. In conclusion, annexin V can be modified near its N-terminus to incorporate sequences that form specific chelation sites for (99m)Tc without altering its high affinity for cell membranes. These annexin V derivatives may be useful for in vivo imaging of cell death.

Apoptotic cell death: its implications for imaging in the next millennium.

Apoptosis, also known as programmed cell death, is an indispensable component of normal human growth and development, immunoregulation and homeostasis. Apoptosis is nature's primary opponent of cell proliferation and growth. Strict coordination of these two phenomena is essential not only in normal physiology and regulation but in the prevention of disease. Programmed cell death causes susceptible cells to undergo a series of stereotypical enzymatic and morphologic changes governed by ubiquitous endogenous biologic machinery encoded by the human genome. Many of these changes can be readily exploited to create macroscopic images using existing technologies such as lipid proton magnetic resonance (MR) spectroscopy, diffusion-weighted MR imaging and radionuclide receptor imaging with radiolabeled annexin V. In this review the cellular phenomenon of apoptotic cell death and the imaging methods which can detect the process in vitro and in vivo are first discussed. Thereafter an outline is provided of the role of apoptosis in the pathophysiology of clinical disorders including stroke, neurodegenerative diseases, pulmonary inflammatory diseases, myocardial ischemia and inflammation, myelodysplastic disorders, organ transplantation, and oncology, in which imaging may play a critical role in diagnosis and patient management. Objective imaging markers of apoptosis may soon become measures of therapeutic success or failure in both current and future treatment paradigms. Since apoptosis is a major factor in many diseases, quantification and monitoring the process could become important in clinical decision making.

Erythroid phosphatidyl serine exposure is not predictive of thrombotic risk in mice with hemolytic anemia.

Thrombosis is a major complication of human hemolytic anemias such as sickle cell disease, thalassemia, and severe hereditary spherocytosis (HS). Mice with severe HS and severe hereditary elliptocytosis (HE) also suffer from thrombosis, with incidences ranging from 15 and 22% in beta-spectrin- and ankyrin-deficient mice, respectively, to 85 to 100% in alpha-spectrin-deficient and band 3 knockout mice. A contributing factor to thrombosis could be loss of phospholipid asymmetry of the mutant red blood cells (RBCs), with concomitant exposure of the aminophospholipid phosphatidylserine (PS). Increased PS exposure occurs in RBCs from sickle cell and thalassemia patients and in RBCs from band 3-deficient mice. To determine if increased PS exposure correlates with thrombotic risk in HS and HE mice with ankyrin, beta-spectrin, and alpha-spectrin deficiencies, measurements of FITC-labeled annexin V binding to externalized PS on RBCs were performed. PS exposure is elevated in all mice with HS and HE, but the percentage of RBCs with exposed PS does not correlate with thrombotic risk in these mice.

Radionuclide imaging of acute lung transplant rejection with annexin V.

STUDY OBJECTIVES: Early detection and treatment of lung transplant rejection is critical for preservation of pulmonary graft function. Damage to pulmonary allografts is mediated by apoptotic cell death induced by the alloreactive T lymphocytes that infiltrate lung grafts. Previous studies demonstrate that acute cardiac allograft rejection can be visualized using radiolabeled annexin V. This study was done to determine whether this technique could visualize acute rejection in a rodent model of unilateral orthotopic lung transplantation. DESIGN: Eighteen Sprague-Dawley ACI rats underwent removal of their left lung followed by orthotopic transplant of either an allogeneic (PVG, immunologically mismatched; N = 10) or a syngeneic (ACI, immunologically matched) pulmonary graft (N = 8). Animals were imaged 1 h after IV injection of 1 mCi (37.0 MBq) of (99m)Tc-annexin V 1 to 7 days after transplantation. RESULTS: Lungs receiving the allograft demonstrated moderate to marked mononuclear infiltration of the perivascular, interstitial, and peribronchial tissues. No mononuclear infiltrates were noted in the native right lungs nor in the syngeneic transplants. Region of interest image analysis revealed significant (p < 0.0005) increases of transplant to normal lung activity ratios 3 to 7 days after allograft surgery. The increased annexin V uptake in these lungs was confirmed at biodistribution assay (allograft 151% greater than isograft activity, p < 0.005). CONCLUSIONS: Acute experimental lung transplant rejection can be noninvasively identified using (99m)Tc-annexin V. Radiolabeled annexin V may be a clinically useful noninvasive screening tool for acute rejection.

Radiolabeled annexin V imaging: diagnosis of allograft rejection in an experimental rodent model of liver transplantation.

PURPOSE: To assess the value of imaging rejection-induced apoptosis with technetium 99m and annexin V, a human protein-based radiopharmaceutical used in the diagnosis of acute rejection of a liver transplant, in a well-characterized rodent model of orthotopic liver transplantation. MATERIALS AND METHODS: 99mTc-radiolabeled annexin V was intravenously administered to six allografted (immunologically mismatched) and five isografted (immunologically matched) recipient rats on days 2, 4, and 7 after orthotopic liver transplantation. Animals were imaged 1 hour after injection of 0.2-2.0 mCi (8.0-74.0 MBq) of radiolabeled annexin V by use of clinical nuclear scintigraphic equipment. RESULTS: All animals in the allografted group demonstrated marked increases of 55% and 97% above the activity in the isografted group in hepatic uptake of annexin V on days 4 and 7, respectively. Severe acute rejection was histologically detected in all allografted livers on day 7. There was no histologic evidence of acute rejection in isografted animals. Dynamic hepatobiliary imaging with 99mTc and mebrofenin, an iminodiacetic acid derivative, demonstrated no correlation with the presence or absence of acute rejection or with annexin V uptake. CONCLUSION: Noninvasive imaging with radiolabeled annexin V is more sensitive and specific than imaging with 99mTc-mebrofenin in the diagnosis of acute rejection of a liver transplant.

Technetium-99m HYNIC-annexin V: a potential radiopharmaceutical for the in-vivo detection of apoptosis.

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.

Frequency of problems during clinical molecular-genetic testing.

Concerns have been raised about the quality of DNA-based genetic testing, but few data are available on the problems that occur during clinical genetic testing. We sought to determine the frequency and severity of such problems in US laboratories. Problems were defined as events that could or did impair patient care significantly. Data on the frequency and severity of adverse events during genetic testing were collected from laboratories by anonymous mail questionnaire and detailed on-site inspection. The surveyed laboratories (n = 42) reported significant problems in 0.33% of tests performed; the corresponding value in the inspected laboratories (n = 2) was 0.38%. Sixty percent of problems occurred in the pretest phase, 32% in the laboratory phase, and 8% in the posttest phase or multiple phases. The average level of actual harm resulting from these problems was low. Moderate or high levels of harm occurred in only 0.008% of total cases. No lawsuits, judgments, or disciplinary actions were taken against the laboratories in 277,000 tests performed. The overall frequency of problems in a given laboratory did not correlate with laboratory age, test volume, accreditation status, proficiency testing performance, or institution type (academic, private nonprofit, private for profit). In conclusion, significant problems during genetic testing occur infrequently (< 0.5% in most laboratories), and problems resulting in moderate or high levels of harm to patients are rare (0.008%).

Imaging of apoptosis (programmed cell death) with 99mTc annexin V.

UNLABELLED: Apoptosis (programmed cell death) is a critical element in normal physiology and in many disease processes. Phosphatidylserine (PS), one component of cell membrane phospholipids, is normally confined to the inner leaflet of the plasma membrane. Early in the course of apoptosis, this phospholipid is rapidly exposed on the cell's outer surface. Annexin V, an endogenous human protein, has a high affinity for membrane-bound PS. This protein has been labeled with fluorescein and has been used to detect apoptosis in vitro. We describe the use of radiolabeled annexin V to detect apoptosis in vivo. The results are compared to histologic and flow cytometric methods to identify cells and tissues undergoing apoptosis. METHODS: Annexin V was coupled to hydrazinonicotinamide (HYNIC) and radiolabeled with 99mTc. Bioreactivity of 99mTc-HYNIC annexin V was compared with fluorescein isothiocyanate (FITC)-labeled annexin V in cultures of Jurkat T-cell lymphoblasts and in ex vivo thymic cell suspensions undergoing apoptosis in response to different stimuli. In addition, the uptake of FITC annexin V and 99mTc-HYNIC annexin V was studied in heat-treated necrotic Jurkat T-cell cultures. In vivo localization of annexin V was studied in Balb/c mice injected with 99mTc-HYNIC annexin V before and after induction of Fas-mediated hepatocyte apoptosis with intravenously administered antiFas antibody. RESULTS: Membrane-bound radiolabeled annexin V activity linearly correlated to total fluorescence as observed by FITC annexin V flow cytometry in Jurkat T-cell cultures induced to undergo apoptosis in response to growth factor deprivation (N = 10, r2 = 0.987), antiFas antibody (N = 8, r2 = 0.836) and doxorubicin (N = 10, r2 = 0.804); and in ex vivo experiments on thymic cell suspensions with dexamethasone-induced apoptosis from Balb/c mice (N = 6, r2 = 0.989). Necrotic Jurkat T-cell cultures also demonstrated marked increases in radiopharmaceutical (4000-5000-fold) above control values. AntiFas antibody-treated Balb/c mice (N = 6) demonstrated a three-fold rise in hepatic uptake of annexin V (P < 0.0005) above control (N = 10), identified both by imaging and scintillation well counting. The increase in hepatic uptake in antiFas antibody-treated mice correlated to histologic evidence of fulminant hepatic apoptosis. CONCLUSION: These data suggest that 99mTc-HYNIC annexin V can be used to image apoptotic and necrotic cell death in vivo.

Phosphatidylserine receptors: role of CD36 in binding of anionic phospholipid vesicles to monocytic cells.

Exposure of phosphatidylserine (PtdSer) has been implicated in the recognition and phagocytosis of senescent and apoptotic cells, and CD36 has been proposed as one receptor protein that recognizes PtdSer and other anionic phospholipids. We investigated the binding of phospholipid vesicles to the monocytic leukemia cell lines THP-1 and J774A.1 with a flow cytometric assay; vesicles contained 50 mol% PtdSer, phosphatidylinositol (PtdIns), or phosphatidylglycerol (PtdGro), with the balance being phosphatidylcholine. Specific, high affinity binding was observed for vesicles containing PtdSer, PtdIns, or PtdGro. Specificity of the assay was confirmed by control experiments with erythrocytes, which showed minimal vesicle binding, and with annexin V, which blocked the binding of PtdSer, PtdGro, and PtdIns vesicles to the THP-1 cells. However, O-phospho-L-serine (to 1 mM) had no effect on the binding of PtdSer vesicles, indicating that high affinity binding requires a surface containing multiple phosphoserine groups rather than a single molecule. A monoclonal antibody to CD36 blocked up to 60% of the specific binding of PtdSer vesicles but had minimal to no effect on the binding of PtdGro or PtdIns vesicles. This antibody also selectively inhibited the phagocytosis of PtdSer-containing vesicles as measured by fluorescence microscopy, indicating that CD36 is functionally significant for phagocytosis of this vesicle type. In addition, collagen and thrombospondin, two other putative ligands of CD36, were unable to inhibit the binding of PtdSer vesicles. We conclude that CD36 is the primary protein responsible for the high affinity binding of PtdSer vesicles to these monocyte-like cells. In addition, CD36 appears to be specific for PtdSer among anionic phospholipids, and non-phospholipid ligands of CD36 do not share binding sites with PtdSer on CD36.