A short-term assay of myeloid precursor cells in man.

An assessment of the numbers of myeloid precursor cells in human bone marrow, obtainable earlier than with conventional colony assays, would be useful for many reasons. Recently an isotopic assay for murine-colony-stimulating activity has been devised and we have modified this technique for use in man. Bone marrow mononuclear cells are incubated in microtitre plates in the presence of optimal amounts of placental-conditioned medium, pulsed with 3H-galactose for 24 h and the isotope incorporation measured. Isotope uptake by normal bone marrow was found to be proportional to both the number of cells cultured and the amount of conditioned medium added. The cells responsible for isotope incorporation have been characterized partially and found to be nonadherent immature myeloid cells and have a density of less than 1.077. This short-term isotopic assay was also compared to the GM-CFC assay in ten normals and in 24 patients with either neutropenia (of different etiology), myeloid leukemias, or neutrophil leukocytosis. There was good correlation between the two assays in all the patients studied. Thus, our observations suggest that the cell incorporating 3H-galactose in response to conditioned medium has many of the properties of the GM-CFC and its immediate progeny. Although assay specificity has yet to be proven, our early results indicate that it may have use as a rapid, but indirect, assessment of human myeloid precursor cells and thus prove to be a useful adjunct to the standard hematological methods of assessment of certain patients.

A 82Sr-82Rb isotope generator for use in nuclear medicine.

An improved 82Sr-82Rb generator system, based on the complexing ion-exchange resin Chelex-100, has been developed. Columns of this material can be easily and rapidly milked, and the rubidium-strontium separation factor for a fresh generator under the experimental conditions studied was found to be greater than 10-7. Approximately 80% of the 82Rb present can be delivered in a 15-ml volume of aqueous 0.2 M NH4Cl solution. After more than 6 liters of eluant had passed through the generator, the rubidium-strontium separation factor was still observed to greater than 10-5 and no unusual strontium breakthrough behavior was seen in the system over nearly three 82Sr half-lives.

16 alpha-[77Br]bromoestradiol-17 beta: a high specific-activity, gamma-emitting tracer with uptake in rat uterus and uterus and induced mammary tumors.

16 alpha-[77Br]Bromoestradiol-17 beta (Compound 1) has been synthesized by radiobromination of estrone enoldiacetate. Tissue uptake studies performed 1 hr after administration of Compound 1 to immature or mature female rats showed uterus-to-blood ratios of 13, with nontarget issue-to-blood ratios ranging from 0.6 to 2. Co-administration of unlabelled estradiol caused a selective depression in the uterine uptake with no effect on nontarget tissue uptake. In adult animals bearing adenocarcinomas induced by DMBA (7,12-dimethylbenz(a)anthracene), tumor-to-blood ratios of 6.3 were obtained, this uptake also being depressed in animals treated with unlabeled estradiol. The studies demonstrate that Compound 1 has suitable binding properties and sufficiently high specific activity so that its uptake in estrogen target tissues in vivo is mediated primarily by the estrogen receptor. Furthermore, they suggest that this compound may be suitable for imaging human breast tumors that contain estrogen receptors.

Evaluation and application of alumina-based Rb-82 generators charged with high levels of Sr-82/85.

Generator-produced Rb-82, a 75-sec positron emitter with potential for myocardial blood-flow imaging, was studied with various ion-exchange columns to evaluate the characteristics of alumina as an adsorber for the 25-day Sr-82 parent. Test columns of alumina, Bio Rex 70, and Chelex 100 were loaded with multimillicurie amounts of no-carrier-added Sr-82/Sr-85 (Sr-85 is a production contaminant). The breakthrough of Sr-82/Sr-85, and the yield of Rb-82, were determined for long-term elutions from each column with up to 4 liter of 2% NaCl solution at pH 8 to 9. The breakthrough of Sr-82/85 was 10(-6)-10(-5) from aluminal 10(-6)-10(-4) from Chelex 100 and Bio Rex 70. The effects of eluent flow rate and concentration, and of alumina volume, on the breakthrough and yield were also studied. An improved and automated Rb-82 generator was used for myocardial and brain blood-flow studies in experimental animals and in man; it was equipped with solenoid flow-control valves and five in. of lead shielding for the alumina columns, which were charged with 25-50 mCi Sr-82 (100-150 mCi Sr-85). The Rb-82 generator with alumina column provided up to 20-40 mCi of Rb-82 as often as every 5-10 min with less than 10(-5) breakthrough of Sr-82/85 over the 2- to 3-mo, useful life of the generator.

Rapid radioiodination of rose bengal at room temperature.

Published methods for radioiodination of rose bengal require reaction times of 1 hr or more at temperature from 50 to 120 degrees C. Through the use of an acidified ethanol solvent and potassium iodate oxidant, purified rose bengal is radioiodinated at room temperature within 15 min with chemical yields ranging between 93 and 97%. Radiochemical impurities are sufficiently minimized to permit preparation in a single 10-ml serum vial, requiring no additional purification steps. The method reported here is readily adaptable to cold-kit preparation.

Rubidium-82 generators for imaging studies.

Strontium-82, produced by spallation reaction with medium-energy proton beams, was used to evaluate Bio-Rex 70 and Chelex-100 ion-exchange resins for use in a compact Rb-82 generator. Adsorption of Sr-82 to the resin column, Rb-82 elution yields, Sr breakthrough, and 82Rb-Sr separation factors were determined for newly prepared columns and for longterm elution conditions. Separation factors of 10(7) to 10(8) were obtained with 2% NaCl elutions from Bio-Rex 70 resin columns while the separation factors was about 5 X 10(4) with the Chelex-100 resin column.

Brain tumor evaluation using Rb-82 and positron emission tomography.

Positron emission tomography of the brain with 75-sec rubidium-82 obtained from a portable generator (25-day Sr-82 leads to Rb-82) was used to evaluate the integrity of the blood-brain barrier (BBB) in patients with brain tumors. Rubidium is normally excluded from the central nervous system by the intact BBB, but when the BBB is disrupted by a tumor. Rb enters and pools in the extravascular spaces of the central nervous system. Since Rb is also rapidly cleared from the blood, a high tissue-to-blood ratio of the Rb-82 tracer is achieved in regions of BBB disruption after intravenous injection. With dynamic positron emission tomographic imaging, the extravasation of the Rb tracer can be evaluated independent of the intravascular Rb concentration, and very small changes in the BBB permeability can be detected. The results of our studies in eight patients show that this technique is a promising method for evaluation of the BBB integrity in brain-tumor patients.U

Myocardial perfusion with rubidium-82. I. Measurement of extraction fraction and flow with external detectors.

Accurate measurement of the regional extraction of a diffusible radiopharmaceutical is essential for the quantifying of regional blood flow, and may also provide an important physiologic or diagnostic indicator of the cellular viability of an organ in man through external detection by positron emission tomography. However, extraction fraction of a diffusible tracer usually decreases as flow increases, and thus noninvasive methods for measuring flow are nonlinear unless the extraction fraction can be measured independently. This report describes the theoretical basis and documents the applicability of this theory for determining, with external detectors, the first-pass regional extraction fraction of rubidium-82 by the heart, following a single intravenous bolus injection of the tracer. Measurement of extraction fraction was found to be independent of flow, thereby making it possible to determine accurately with a single intravenous bolus injection of rubidium-82, the regional blood flow in the myocardium at up to five times normal resting flow.

Myocardial perfusion with rubidium-82. II. Effects of metabolic and pharmacologic interventions.

In order to validate a new method for quantifying coronary blood flow, we injected intravenously a bolus of rubidium-82 (Rb-82) into 28 open-chested dogs under a wide range of flow and physiologic conditions, using beta probes to monitor myocardial radioactivity. Extraction fraction and perfusion were measured using a functional model that separates the data into the free and trapped myocardial rubidium. Extraction and uptake of rubidium were lower during acidosis than during alkalosis and were unchanged by glucose-insulin, digoxin, or propranolol. Myocardial flow, as indicated by rubidium, correlated linearly with simultaneous measurements of flow by microspheres in the same sample volume over a wide range of flow (r = 0.97, n = 106, range 0.02-7.76 ml/min/g). Regional myocardial blood flow can be accurately determined using generator-produced Rb-82. Studies using current state-of-the-art, fast positron-emission tomographic cameras are required to determine the utility of this approach in man.

Comparative serum stability of radiochelates for antibody radiopharmaceuticals.

Serum incubation of monoclonal antibodies chelate labeled by DTPA, benzyl-EDTA and benzyl-TETA with 111In, 57Co, and 67Cu demonstrated marked differences in their stability. In serum, 111In-benzyl-EDTA-antibody was more stable than 111In-DTPA-antibody. Cobalt-57 or 67Cu chelated antibody were less stable than either 111In chelated antibody; 67Cu was only firmly attached to the antibody as 67Cu-benzyl-TETA-antibody. The relative stability of the radiometal chelated antibodies was paralleled by the relative stability in serum of the radiometal chelates themselves. These in vitro studies suggest that in vivo behavior of metal chelates exposed to a complex protein environment cannot be predicted by classical equilibrium constants.

Character of transient ischemia in angina pectoris.

There is growing interest in the possible therapeutic and prognostic significance of silent myocardial ischemia in coronary artery disease (CAD) and its detection by ambulatory electrocardiographic (ECG) monitoring. In 100 apparently healthy normal subjects (20 with angiographically normal coronary arteries), Holter monitoring revealed significant ST-segment depression in only 2 (both over 40 years, one with positive treadmill test, the other with risk factor for CAD). No significant ECG changes were found in those with normal coronary vessels. In 30 patients with documented CAD, significant ST-segment depression during 1,934 episodes over 446 days of monitoring over 18 months was found. Only 24% of the episodes were associated with angina. Asymptomatic and symptomatic episodes were associated with comparable changes in perfusion detected by positron emission tomography. Heart rate increases greater than 10 beats/min preceding the onset of the ST-segment changes occurred in only 23% of the episodes. There was considerable variability in the ST-segment changes in the same patient monitored serially over long periods of time. The data indicate that it is extremely uncommon for patients without CAD to exhibit silent myocardial ischemia, whereas patients with stable angina exhibit frequent, variable and often asymptomatic ECG evidence of myocardial ischemia rarely triggered by increases in heart rate. These findings are likely to be of therapeutic and prognostic significance.

Relation between regional myocardial uptake of rubidium-82 and perfusion: absolute reduction of cation uptake in ischemia.

Experiments were undertaken using rubidium-82 and position tomography to examine the relation between myocardial perfusion and cation uptake during acute ischemia. Rubidium-82 was repeatedly eluted from a strontium-82-rubidium-82 generator. In six dogs emission tomograms were used to measure the delivered arterial and myocardial concentrations at rest and after coronary stenosis, stress and ischemia. There was a poor overall relation between regional myocardial uptake and flow measured by microspheres and a large individual variability. Extraction of rubidium-82 was inversely related to flow. Significant regional reduction of cation uptake was detected in the tomograms when regional flow decreased by more than 35 percent. This reduction was significantly greater when ischemia was present. A small but significantly greater when ischemia was present. A small but significant decrease (33.0 +/- 9.1 percent, mean +/- standard deviation) in the myocardial uptake of rubidium-82 was detected only when flow was increased by more than 120 percent in relation to a control area after administration of dypiridamole. The technique using rubidum-82 and tomography was applied in five volunteers and five patients with angina pectoris and coronary artery disease. Myocardial tomograms recorded at rest and after exercise in the volunteers showed homogeneous uptake of cation in reproducible and repeatable scans. In contrast, the patients with coronary artery disease showed an absolute mean decrease of 36 +/- 14 percent in regional myocardial uptake of rubidium-82 after exercise. These abnormalities persisted in serial tomograms for more than 20 minutes after the symptoms and electrocardiographic signs of ischemia.

A simple assay of intrinsic factor-vitamin B12 complex employing the binding intrinsic factor antibody.

The reaction between binding intrinsic factor antibody and intrinsic factor-vitamin B(12) complex has been studied. Initially in the zone of antibody excess, the relationship between the amount of antigen present and the amount of antigen-antibody complex adsorbed onto zirconium phosphate gel was linear. With increasing amounts of antigen, the curve departed from linearity and reached a plateau. The linear portion of the reaction forms the basis of a simple and reproducible assay for quantitating intrinsic factor to which vitamin B(12) has already been bound. The assay provides a method for studying the fate of intrinsic factor-vitamin B(12) complex during digestion and absorption. In two normal subjects given radioactive vitamin B(12) orally, aspiration of ileal contents showed that only 50 to 70% of the radioactivity was bound to intrinsic factor at that level.

High colony stimulating activity in a patient with disseminated carcinoma and neutrophil leucocytosis.

The case of a man with widespread bronchogenic carcinoma associated with pronounced neutrophil leucocytosis is presented. There was no evidence of infection or metastatic bone marrow infiltration. Increased levels of colony stimulating activity were shown in the patient's serum using three methods. Findings in this patient suggest that the leukaemoid blood picture was related to inappropriate tumour associated production of colony stimulating factors.

Transfer of copper from metallothionein to nonmetallothionein proteins in cultured cells.

Copper uptake and distribution with time among cytoplasmic proteins were followed in cultured cells under several conditions: (1) CHO cells, which cannot synthesize metallothioneins, were labeled with(67)Cu in the presence of 100 µM ZnCl2; (2) Cd(r)30F9 cells, which contain some constitutive metallothionein (MT), were labeled in the absence of additional ZnCl2 and; (3) Cd(r)30F9 cells were labeled in the presence of ZnCl2, under which conditions they synthesized additional metallothioneins. The exogenous(67)Cu and ZnCl2, where present, were then removed, and the distributions of(67)Cu among size fractions of the cellular proteins were observed at intervals for 16 h. In addition, a culture identical to condition (3) above was also treated with 100 µM ZnCl2 during the redistribution period. The(67)Cu was initially resolved into three peaks by Sephadex G-75 chromatography: high molecular weight, intermediate molecular weight, and MT. The(67)Cu in the MT fraction decreased with at 1/2 of 10-12 h. In contrast to this, generally, in cells with a higher initial(67)Cu bound to metallothionein, there was a progressive increase in the amount of(67)Cu eluting with the high- and intermediate-molecular-weight fractions. Since no other source of(67)Cu was available, these experiments suggest that copper stored in MT can be transferred to other proteins in these cells.

Sweet's syndrome associated with trans-retinoic acid treatment in acute promyelocytic leukaemia.

A case of acute promyelocytic leukaemia is reported in which Sweet's syndrome developed at the time of an improvement in white blood cell count due to transretinoic acid. Acute febrile neutrophilic dermatosis (Sweet's syndrome) is a disorder characterized by acute onset of inflammatory skin nodules associated with systemic features which include malaise, fever and neutrophilia. Many triggers and associated disorders have been identified since the syndrome was described, the most important being the association with haematological diseases including leukaemias and other myeloproliferative disorders. We describe a case apparently provoked by drug therapy.

Granulopoiesis in the neutropenia of negroes.

A number of reports have shown that the peripheral blood neutrophil count in many negroes is considerably lower than in white populations. This neutropenia has been ascribed previously to both genetic and nutritional causes. We have studied bone marrow reserve function and myelopoiesis in 3 negroes with neutropenia, and found these to be normal.

A radioisotope dilution assay for unlabelled vitamin B12-intrinsic factor complex employing the binding intrinsic factor antibody: probable evidence for two types of binding antibody.

A new radioisotope dilution assay for vitamin B12-intrinsic factor complex is described. The method is based on the use of the binding type intrinsic factor antibody (the binding reagent), which when combined with the intrinsic factor-vitamin B12 complex (labelled ligand), is quantitatively adsorbed onto zirconium phosphate gel at pH 6.25. The new assay has been shown to provide a measure of intrinsic factor comparable with other intrinsic factor assays, but it has the important advantage of being able to measure the unlabelled vitamin B12-intrinsic factor complex (unlabelled ligand), and will, therefore, be valuable in the study of physiological events in the gastrointestinal tract. During the study, it was found that there is some evidence for a least two types of binding intrinsic factor antibody: One which combines preferentially with the intrinsic factor-vitamin B12 complex and one which combines equally well with this complex or with free intrinsic factor.