Isolation and labelling of human leukocytes with 99mTc

The role of the leukocyte isolation procedure on cell labelling with 99mTc has been evaluated. Separation of leukocytes was performed by two procedures: sedimentation on methyl cellulose, followed by discontinuous gradient centrifugation; methyl cellulose sedimentation and hypotonic haemolysis of residual red blood cells. After washing the cells in saline and incubation with a stannous pyrophosphate agent, the leukocytes were labelled with 5-10 mCi 99mTc. Procedure 1 gave a higher purity but lower recovery of polymorphonuclear leukocytes, and a minor contamination of red blood cells. 99mTc labelling of cells was slightly more efficient with this method, probably due to the presence of red blood cells. Procedure 1 is recommended for in vitro studies on cell kinetics and procedure 2 is recommended for clinical use.     

Evaluation of a new leukocyte labeling procedure with 99mTc-HMPAO

A technique for labeling leukocytes with ^99mTc using hexamethylpropylene-amineoxime (HMPAO) was evaluated in vitro. The labeling procedure resulted in a cell bound fraction of radioactivity of 56% after ^99mTc incubation and 96% after 1 washing of cells. In the final cell suspension 84% of the radioactivity was attached to the polymorphonuclear (PMN) leukocytes constituting 94% of white cells. Only 5% was bound to residual red blood cells. The stability evaluated in autologous plasma showed a decline of cell bound activity from 96% to 84% over 3 h. The chemotactic function of PMN leukocytes was unaffected by the labeling procedure. These findings demonstrate that HMPAO, albeit cell unspecific, is efficient for labeling PMN leukocytes with ^99mTc. The stability of the labeling procedure is high and the technique does not affect cell function. No other current ^99mTc leukocyte labeling technique possesses all these qualities.     

Modifying effect of commercially available stannous pyrophosphate (PYRO-Sn) on the tissue affinity of 99mTc pertechnetate

Investigations were performed to examine the conditions required for in vivo labelling of red blood cells (RBCs) with ^99mTc after injection of stannous pyrophosphate. It was found that good in vivo labelling of RBCs is possible and that the efficacy of this procedure is high. The highest concentration of ^99mTc in the blood occurred in the first 2 h after pertechnetate injection, and the optimum time between stannous pyrophosphate and pertechnetate injection ranged from 10 to 30 min.     

A comparison of radiopharmaceutical labelling efficiency of chromatographic generator vs MEK extraction [99mTc]pertechnetate

Technetium-99m radiopharmaceuticals prepared for routine clinical use, were labelled with [^99mTc]pertechnetate obtained from either a commercial chromatographic generator or from a Winnipeg Health Sciences Centre semi-automated self-shielded methyl ethyl ketone extraction system. The [^99mTc]pertechnetate source for each ^99mTc radiopharmaceutical was selected at random over a 16 month period of time. The routine quality control data (silica-gel thin layer chromatography) was reviewed retrospectively, as an in vitro assessment of the quality of the [^99mTc]radiopharmaceutical prepared from each [^99mTc]pertechnetate source. Bone scans ([^99mTc]pyrophosphate) and wall motion studies ([^99mTc]red blood cells) were evaluated as an in vivo assessment of the [^99mTc]pertechnetate used to label the pyrophosphate or red blood cells. The in vitro studies indicated no difference in the labelling efficiency and radiochemical purity of the ^99mTc radiopharmaceuticals prepared from either source of [^99mTc]pertechnetate and there was also no difference observed in the image quality of either bone scans or wall motion studies obtained with either source of [^99mTc]pertechnetate.     

Scintigraphic demonstration of subcutaneous abscesses with 99mTc-labeled leukocytes

Our initial experience with the clinical application of autologous leukocytes labeled in vitro with technetium 99m is reported. An injection of 3.6×10⁸ leukocytes (83% of which were polymorphonuclear) labeled with 6.4 mCi ^99mTc was administered to a patient with subcutaneous abscesses in order to evaluate the in vivo stability of the labeling and cellular function. Scintigraphic examinations demonstrated marked accumulations of radioactivity at the sites of inflammation, suggesting that ^99mTc was efficiently bound to the cells and that the migration of polymorphonuclear leukocytes was not disturbed by the labeling procedure.     

Animal and human studies of a new 99mTc labelled phosphine-isocyanide complex with possible applications to radionuclide ventriculography

A new ^99mTc-phosphine-isocyanide complex with the general structure [^99mTc (DEPE)₂ (CNR)₂]⁺ has been synthesised and tested in animals and one human. In three animal species (rat, rabbit, dog), the complex is an efficient myocardial imaging agent, while in humans it remains in the blood pool. The complex is 100% protein bound in animals and humans, but whereas in humans it is attached to a 51.5 kdalton protein (probably prealbumin), in rabbits it appears to be bound to a larger macromolecule (M.W.> 100 kdalton). The efficiency of the complex for blood pool labelling was tested in a human volunteer and compared with the standard in vivo red cell labelling technique with stannous pyrophosphate. A satisfactory radionuclide angiogram could be performed with less than 370 MBq of the complex. The count rate for the complex (cps/MBq) was 15% higher than that obtained with the labelled red cells and the absence of splenic activity was notable. In humans this complex appears to be an efficient blood pool labelling agent which might be useful for radionuclide ventriculography.     

The behavior of99mTc-hexamethylpropyleneamineoxime (99mTc-HMPAO) in blood and brain

^99mTc-hexamethylpropyleneamineoxime (^99mTc-HMPAO) is a reagent for scanning cerebral blood flow. We investigated how^99mTc-HMPAO changed in the blood and brain. The^99mTc-HMPAO, which was prepared by adding of^99mTcO _- ⁴ to HMPAO and Sn(II), consisted of primary and secondary complexes, reduced hydrolyzed^99mTc, and^99mTc0pertechnetate. The percentage of the primary complex in^99mTc-HMPAO decreased with time after preparation. The primary complex converted to the secondary one very rapidly in the presence of plasma. When^99mTc-HMPAO was injected into patients,^99mTc activity was immediately partitioned in the plasma fraction, with approximately 60% in whole blood. In plasma,^99mTc was found to be associated with proteins such as albumin and globulin.^99mTc trapped in red cells was not washed out with either plasma or saline. Biodistribution studies showed that the less lipophilic compounds of^99mTc-HMPAO could not pass through the blood brain barrier (BBB), and therefore did not accumulate in the brain. The results of gel chromatography and equilibrium dialysis indicated that no specific^99mTc binding protein was present in the brain. Considering the instability of^99mTc-HMPAO in vivo, we proposed that the speed at which the primary complex converted to the less lipophilic compounds was important in allowing^99mTc-HMPAO to pass through the BBB and to be fixed in the brain.     

Technetium-99m mercaptoalbumin as a potential substitute for technetium-99m labelled red blood cells

Technetium-99m labelled red blood cells (^99mTc-RBCs) are far superior to ^99mTc-labelled human serum albumin (^99mTc-HSA) for radionuclide ventriculography, but their labelling is more complex, time consuming and risk bearing (in vitro labelling) or suffers from interference by some medications (in vivo labelling). We have now modified HSA by the introduction of mercapto groups with the purpose of preparing stable and practical ^99mTc-mercaptoalbumin with long retention in the vascular system, that could replace ^99mTc-RBCs. HSA was incubated with N-succinimidyl S-acetylthioacetate (SATA) or N-succinimidyl 2,3-di(S-acetylthio) propionate (SATP) to introduce a chain containing one or two protected sulfhydryl groups on some of the lysine amino groups. After purification by size-exclusion chromatography (SEC), the mercapto groups were deprotected by incubation at alkaline pH or by treatment with hydroxylamine. The reaction products were used with or without SEC purification for direct or exchange labelling experiments with ^99mTc at neutral pH. SEC-HPLC was used to determine labelling yields and to isolate pure ^99mTc-mercaptoalbumin. Stable ^99mTc-mercaptoalbumin complexes could be formed in 90%–95% yield after coupling albumin with SATA or SATP in all molar ratios used followed by deacetylation in one of the mentioned conditions. The most favourable results were obtained after reaction of SATA or SATP with HSA in a 25: 1 ratio and deprotection with NH₂OH. The stability of the resulting ^99mTc-mercaptoacetyl-albumin (^99mTc-MAHSA) and ^99mTc-dimercaptopropionyl-albumin (^99mTcDMP-HSA) and their retention in vivo in plasma of mice and rabbits are clearly higher than that of conventional ^99mTc-HSA preparations. ^99mTc-DMP-HSA approaches the behaviour of ¹²⁵I-HSA quite well in both animal species. A preliminary study with ^99mTc-DMP-HSA in a volunteer showed a retention in the vascular compartment almost identical to that of ^99mTc-RBCs and clearly higher than that of a common ^99mTc-HSA preparation. The results indicate that these ^99mTc-mercaptoalbumins and especially ^99mTc-DMP-HSA are very promising as a practical alternative to ^99mTc-RBCs.K.A. Verbeke is a Research Assistant for the Belgian National Fund for Scientific Research     

Leukocyte labeling with technetium-99m

Erythrocyte labeling with ⁵¹Cr or ^99mTc is a well-established procedure in nuclear medicine. The labeled cells are useful in the measurement of red cell mass, in erythrocyte survival studies, and in the localization of erythrocyte pools. Attempts to label the leukocyte with the same radionuclides have not been as successful.This study reports on various laboratory parameters of labeling leukocytes with ^99mTc. Microgram quantities of stannous chloride are used in the labeling process to reduce ^99mTc-pertechnetate to a lower valence state. Other reducing agents such as ferrous ion and penicillamine are not effective. Pertechnetate itself is only loosely bound to the leukocyte. An electrophoretic technique is used to determine labeling yield. Differences in labeling efficiency between the cells from normal donors and the cells from chronic myelocytic leukemia (CML) donors have been detected. Leukocytes from normal donors generally label with greater efficiency than leukocytes from CML donors, but subject age is probably a contributing factor, with cells from young normal donors labeling more like cells from CML donors. The leukocyte and erythrocyte can be labeled under identical conditions in about the same yield. Labeled leukocytes retain good viability and phagocytic capability. Although autoradiographic studies are not completely conclusive because of the poor radiation characteristics of ^99mTc, it appears possible that the -Sn-^99mTc label is on the surface of the cell and is not intracellular. The labeling process is being adapted to an aseptic process for future experimentation in animals and in human subjects.     

In vitro labeling of human polymorphonuclear leukocytes with 99mTc

Preliminary experience with ^99mTc labeling of isolated polymorphonuclear leukocytes is reported. Leukocytes efficiently purified from 100 ml peripheral blood were labeled in vitro with ^99mTc after pretreatment with small amounts of a stannous agent to reduce pertechnetate. A labeling yield of 1.3 mCi equivalent to 12.5% of the added dose of ^99mTc and a cell binding efficiency of more than 90% was achieved under sterile conditions. Viability assays showed no influence of the labeling procedure on cell function. The technique may prove a valuable alternative for assessment of local leukocyte accumulation by scintigraphy.     

Rapid measurement of blood leakage during regional chemotherapy

In order to avoid complications after regional chemotherapy (isolated hyperthermic perfusion) of the extremities, rapid measurement of blood leakage from the extracorporeal to the systemic circulation is important. A method using technetium-99m in vivo red blood cell (RBC) labelling is reported that provides results within 3 min. Blood samples drawn from the systemic and the extracorporeal circulation were measured for ^99mTc activity using a mobile well counter, and the leakage values calculated. The mean result was 7.6%±6.5%/15 min (n=209). The corresponding flow rate was 100.2±85.7 ml/15 min (mean ± SD). The values for isolation perfusion of the upper and the lower extremities are compared. The leakage results using ^99mTc RBC labelling were correlated with other blood pool markers. Iodine-125 human serum albumin and indium-113 m transferrin were administered in subgroups of 4 and 19 patients simultaneously. Using linear regression, the coefficient of correlation was 0.72 for ^99mTc/^113mIn and 0.58 for ^99mTc/¹²⁵I. Comparison with the alternatives suggests that the rapid method of leakage measurement after ^99mTc RBC labelling can be considered one of the most practicable and reliable methods available.This paper is dedicated to Prof. E. Oberhausen, Homburg/Saar, on the occasion of his 65th birthday Correspondence to: C. Alexander     

The use of technetium-99m labelled heat-damaged red cells for the quantitative measurement of splenic function

A comparison of the rates of clearance of ⁵¹Cr labelled and ^99mTc labelled heat-damaged red cells in 25 patients and 4 control subjects is reported. Very little correlation was found between the clearance half-times of the two types of labelled cells when the cells were labelled with ^99mTc prior to heat damaging. The correlation was improved when the labelling step occurred after the cells had been damaged. Urinary excretion measurements revealed that the rate of excretion of ^99mTc could be as much as nine times that of the ⁵¹Cr label. ^99mTc labelled heat-damaged red cells were found not to be sufficiently stable a preparation for use in quantitative clearance studies.     

Failure in labelling of red blood cells with99mTc: Interaction between intravenous cannulae and stannous pyrophosphate

The effect of different methods of stannous pyrophosphate (SnPYP) administration on a modified in vivo technique for labelling red blood cells (RBCs) with^99mTc has been studied. Retention of^99mTc in the bloodstream was measured and parallel in vitro RBC labelling experiments were performed. After administration of SnPYP or a mixture of SnPYP and heparin via the metal needles of intravenous cannulae, the mean 30-min retentions of^99mTc were 96.8% (SD 8.6) and 95.3% (SD 12.4) respectively and the mean in vitro labelling efficiencies were 86.9% (SD 5.0) and 81.9% (SD 18.2) respectively. When the SnPYP was administered via teflon cannulae, the mean 30-min retention of^99mTc was 40.0% (SD 16.7) and the in vitro labelling efficiency was 18.7% (SD 27.0). Reduced RBC labelling and rapid clearance of^99mTc from the bloodstream were not explained by adsorption of stannous ion or pyrophosphate onto the teflon cannulae or retention of SnPYP in the cannulae. When labelling RBCs with^99mTc it is important that SnPYP is not injected via a teflon cannula.     

Tc Nanocoll: A radiopharmaceutical for sentinel node localisation in breast cancer—In vitro and in vivo results

This study evaluated labelling efficiency and radiochemical purity of ^99mTc colloid albumin to identify an optimal labelling protocol for sentinel node detection. Results indicate that a 72 h eluate is not recommended for high specific labelling of ^99mTc colloid albumin. Ex vivo, significantly higher count rates were reached using a 2 h eluate in vacuum or nitrogen. Labelling 26 MBq/μg ^99mTc colloid albumin with a 2 h eluate under nitrogen is recommended because of the ease of labelling.     

Can technetium 99m bisdiethylphosphinoethanebis-t butylisocyanide (99mTc-DEPIC) be used for routine radionuclide ventriculography?

Radionuclide ventriculography is a useful investigation in the evaluation of cardiac function. Generally, in vivo technetium 99m-labelled red blood cells (RBC) yield good quality images in ventriculography. However, it is widely believed that some drugs have an adverse effect on RBC labelling. Zanelli et al. (1987) developed a radiopharmaceutical (technetium 99m bisdiethylphosphinoethanebis-t-butylisocyanide,^99mTc-DEPIC) to obtain better results in patients using such drugs. We untertook a prospective study of 6 patients with cardiovascular and/or pulmonary disease using several kinds of drugs to evaluate imaging of the cardiac blood pool with^99mTc-DEPIC and in vivo labelled^99mTc-RBC. After injection, blood samples were taken, and gated equilibrium blood pool studies were performed. The radiochemical purity of the injected^99mTc-DEPIC varied from 76.4 to 93.6% (mean 86.4%, SD 5.7%). The protein (pre-albumin) binding was 100%. Biological half-life in blood varied from 3.3 to 4.7 h (mean 4.1 h, SD 0.5 h). For^99mTc-RBC no significant blood disappearance was seen for 8 h. The percentage of RBC-bound^99mTc varied from 96.9% to 98.3% (mean 97.0%, SD 0.5%) and was stable for at least 8 h. The heart-to-lung, heart-to-spleen, and heart-to-liver ratios were higher for^99mTc-RBC than for^99mTc-DEPIC. Furthermore,^99mTc-DEPIC showed a significant decline of the ejection fraction with time. Visually, the images with^99mTc-RBC were superior to those with^99mTc-DEPIC, especially a few hours after injection. According to our findings, in vivo labelling of^99mTc-RBC is still the method of choice for routine radionuclide ventriculography. The decline of the ejection fraction, the short blood half-life, and the intense liver uptake make^99mTc-DEPIC less suitable for this purpose.     

First evaluation of technetium-99m dimercaptopropionyl albumin as a possible tracer agent for ventriculography in a volunteer

Technetium-99m labelled red blood cells (^99mTc-RBCs) are the standard radiopharmaceutical for radionuclide ventriculography but suffer from some practical disadvantages such as risk of viral contamination and lengthy preparation (in vitro labelling) or poor labelling efficiency due to patient medication interactions (in vivo labelling). ^99mTc-labelled human serum albumin (HSA) is not really a valuable alternative as the activity diffuses too rapidly out of the vascular space due to the weak binding of the radionuclide. We have modified HSA by reaction with N-hydroxysuccinimidyl 2,3di(S-acetylmercapto)propionate (SAMP) to introduce a varying number of 2,3-dimercaptopropionyl (DMP) side chains. The resulting DMP-HSA can be rapidly labelled with ^99mTc at room temperature by simple addition of stannous ions and eluate of a ^99mTc generator. After evaluation in mice and rabbits, two different ^99mTc-DMP-HSA preparations — obtained after reaction of SAMP with albumin in a molar ratio of, respectively, 8:1 and 16:1 — were tested in a volunteer and compared to ^99mTcRBCs. The blood time-activity curves of the three preparations were quite similar but both ^99mTc-DMP-HSA preparations were excreted much less into the urine than ^99mTc-RBCs. Ventriculography was performed with the three tracer agents, each time with a 1-week interval. In the three studies, the heart was clearly visualized and the left and right ventricle could be easily delineated. The ejection fractions obtained after administration of the three preparations were similar. With both ^99mTc-DMP-HSA preparations the low activity in the spleen was a distinct advantage and facilitated delineation of the left ventricle. However, a slightly higher liver uptake was seen with ^99mTc-DMP-HSA 16:1, whereas the liver uptake of ^99mTc-DMP-HSA 8:1 and ^99mTc-RBCs was the same. These first results suggest that ^99mTc-DMP-HSA, and in particular ^99mTc-DMP-HSA 8:1, could be used for ventriculography as a practical and reliable alternative to ^99mTc-RBCs.     

A micro-autoradiographical study of the localization of 99mTc(Sn)-MDP and 99mTc-MDP in undecalcified bone sections

The localization of ^99mTc(Sn)-MDP in bone tissue was compared with ^99mTc-MDP by means of microautoradiography of undecalcified bone sections. Sections of good histological quality were obtained by a rapid embedding method in methylmethacrylate. No differences were found in the localization of these radiopharmaceuticals in fetal rat calvariae after incubation in vitro or in rat femora after administration in vivo. In the incubation experiment, hydrolyzed ^99mTc was formed. The uptake was high in areas of new bone formation. No uptake was seen in cells or in resorbing areas. In compact bone ^99mTc(Sn)-MDP was predominantly taken up in the vicinity of blood vessels.     

Anti-MUC1 aptamers: radiolabelling with Tc and biodistribution in MCF-7 tumour-bearing mice

IntroductionAptamers previously selected against the protein core (AptA) or the tumour glycosylated (AptB) MUC1 glycoprotein have been conjugated to MAG2 and labelled with ^99mTc, for the potential use as radiopharmaceuticals for diagnostic imaging of breast cancer.MethodsThe conjugation was achieved in high yield using standard peptide coupling reactions between an amino modification on the aptamer and the activated carboxylic group on the ligands. The retention of the affinity of the MAG2 modified AptA for the MUC1 protein core was confirmed using a fluorescent intercalator displacement binding assay. The labelled aptamers were separated from free ^99mTc using ultrafiltration and monitored by high-performance liquid chromatography at all stages, to ensure that only radiolabelled aptamers were produced. The biodistribution properties of the two aptamer-radionuclide conjugates were analysed in MCF-7 tumour bearing mice and compared.ResultsEfficient and convenient labelling of the two aptamers with ^99mTc was achieved as the last step of the synthesis (post-conjugation labelling). Both the aptamer-chelator conjugates had strong ^99mTc binding properties and the resulting complexes were stable in vivo, both in terms of nuclease degradation and leaking of the metal. The radiolabelled aptamers showed a high renal clearance and a high uptake in the intestine.ConclusionsAptA and AptB have been successfully conjugated in high yield to the ligand MAG2 and labelled with ^99mTc. The radiolabelled aptamers showed different tumour uptake and clearance, but will require further development prior to diagnostic use.     

99mTc-pertechnetate in the detection of thyroid carcinoma in a ten year period

During a 10-year period, 63 patients with thyroid malignancies were imaged with ^99mTc-pertechnetate (^99mTc) and 9 of them also underwent imaging with ¹³¹I. To evaluate ^99mTc in the detection of thyroid carcinoma, the scans were blindly analyzed and compared with the reports of surgeons and pathologists. The carcinomas were located in hypoactive nodules in 60 cases, there were cold nodules in the three remaining thyroids, but accurate localization of the carcinomas was not possible, however, it seemed that only one of these could have been situated either in a hot or a cold nodule. The ^99mTc and ¹³¹I images were almost the same. The most common carcinomas were papillary (46%) and follicular (38%) forms. More than one hypoactive nodule was detected in 48% of patients, two or more carcinoma nodules were noted in 17%, and multinodular goitre in 29% of patients. Our study confirms the usefulness of ^99mTc in carcinoma detection, we suggest that reimaging of all the functioning nodules on the ^99mTc scan with radioiodine, as recommended by many authors, is neither necessary nor justifiable.     

Oxygen bubbling can improve the labelling of pentavalent technetium-99m dimercaptosuccinic acid

It has previously been reported that almost all of the trivalent technetium-99m dimercaptosuccinic acid (^99mTc (III) DMSA) present in the labelling product of pentavalent technetium-99m DMSA (^99mTc (V) DMSA) can be changed into^99mTc (V) DMSA by bubbling with pure oxygen. We therefore performed studies in animals (mice) and humans to investigate the effect of such oxygen bubbling on the labelling efficiency of and on the renal uptake of^99mTc. The method of labelling of^99mTc (V) DMSA was that of Hirano. It was found that oxygen bubbling oxidized the contaminated^99mTc (III) DMSA into^99mTc (V) DMSA in vitro and decreased the uptake of radioactivity in the kidney in both animals and humans.