UNIT OPERATION / PRODUCTION PROCESS


Blood Cell Labeling

Introduction

Radiolabeled red blood cells have played an important role as diagnostic radiopharmaceuticals for many decades. Their current role as the drug of choice for cardiac blood pool imaging has resulted in an evolution in the methods of labeling and a better understanding of labeling mechanisms.

Clinical Indications

The use of radiolabeled red blood cells includes five major areas:

1. Measurement of total red blood cell volume

2. Measurement of red blood cell survival time

3. Identification of sites of red blood cell destruction

4. Blood pool imaging studies including gated cardiac imaging and gastrointestinal bleeding

5. Selective spleen imaging with damaged red blood cells

 

Tc-99m Labeled Red Blood Cells (RBC)

The use of Tc-99m labeled red blood cells as a blood pool imaging agent in nuclear cardiology is well established. Clinical effectiveness of this agent is based on its ability to distribute primarily within the intravascular pool of the body and to leave this compartment at a slow rate. Such behavior allows for the accumulation of high resolution images which can be obtained with the aid of a physiological gating device. Combined with the gamma scintillation camera, this procedure can yield diagnostic information about dynamic processes such as regional myocardial wall motion and left ventricular ejection fraction.

Tc-99m as the pertechnetate ion is not firmly bound to red blood cells and will diffuse into the extravascular fluid compartment, with accumulation in organs such as the stomach, gut and thyroid gland. Such a distribution pattern results in lower blood-to-background activity ratios, poor detection of myocardial borders, interference with GI blood pool imaging and images which are difficult or impossible to interpret. It is, therefore, important that the Tc-99m be firmly and quantitatively bound to the cells and that this labeling persist in vivo during the observation period. In nuclear cardiology this time period may be 1 hour, while in the evaluation of gastrointestinal bleeding, the observation period may be as long as 24 hours.

A labeling method employing stannous chloride as a reducing agent for technetium was introduced with labeling efficiencies of 50 to 60% reported. The method involved the incubation of washed cells with pertechnetate followed by the addition of stannous chloride solution. It was observed that the presence of plasma greatly reduced the labeling efficiency by this method but that the labeled cells exhibited good in vivo and in vitro stability.

General Steps in Labeling RBC with Tc-99m

  1. Treatment of Red Blood Cells with Stannous Ion

Although it is technetium in the +7 (pertechnetate) oxidation state that crosses the intact erythrocyte membrane, only technetium that has been reduced to a lower oxidation state will firmly bind to hemoglobin. Stannous ions are most commonly employed for reduction of technetium and stannous chloride (as a stannous pyrophosphate complex) is preferred. At physiologic pH, stannous ions are subject to hydrolysis and precipitation that causes their rapid clearance from blood by the reticuloendothelial system. When complexed with pyrophosphate (or other soluble chelates), however, stannous ions are sufficiently soluble to be resistant to these effects, yet are not so strongly bound to pyrophosphate as to prevent their dissociation and passage into red blood cells. In the in vivo and modified in vivo methods, treatment with stannous ion is accomplished by the direct intravenous administration of stannous pyrophosphate. Other chelates of stannous ions can also be used (such as pentetate, medronate, etc.) and would yield radiolabeled red blood cells with varying degrees of efficiencies. Pyrophosophate seems nearly ideal, however, because (a) it maintains the solubility of stannous ions in serum until they come into contact with the red blood cells and (b) most kits contain an optimal amount of stannous ion.

For Tc-99m red blood labeling using the in-vivo or the modified in vivo technique, most clinicians utilize 10-20 micrograms Sn+2/kg body weight. Depending upon the commercial formulation chosen, it may be necessary to inject one-third, one-half, or the entire contents of a vial of stannous pyrophosphate to provide required mass of stannous ions. When the in vitro method of radiolabeling is employed, a much smaller number of stannous ions are employed. Currently available in vitro kits contain a stated minimum of approximately 25 micrograms of stannous ion.

  1. Removal of Extracellular Stannous Ions

In either the in vivo or the modified in vivo method, biological clearance of excess stannous pyrophosphate is the method by which the concentration of extracellular stannous ions is reduced. The optimal time between the injection of stannous pyrophosphate and the administration of Tc-99m pertechnetate (in vivo method) or the incubation of the stannous ion pretreated cells with Tc-99m pertechnetate (modified in vivo method) is 20-30 minutes. With the original in vitro labeling method, extracellular stannous ions were removed by centrifugation, a step that physically separates stannous-treated cells from the non-cellular associated stannous ion in serum.

  1. Addition of Tc-99m Pertechnetate

Actual red blood cell labeling with Tc-99m occurs whenever Tc-99m pertechnetate is brought into contact with RBCs that have been previously treated with stannous ions. This can be accomplished by either the in vivo or in vitro addition of Tc-99m pertechnetate to RBCs that have been pretreated with stannous ions.

 

CURRENT Tc-99m RBC LABELING METHODS

  1. In Vitro Kits

Although the stannous chloride method of labeling autologous red blood cells resulted in a clinically useful radiopharmaceutical, the procedure was long and required multiple washing steps as well as the extemporaneous compounding of a stannous chloride solution suitable for intravenous injection. These disadvantages were partially eliminated with the introduction of simple kits for the preparation of Tc-99m red blood cells using stannous citrate and stannous glucoheptonate (gluceptate). The introduction of these kits, although not widely available, greatly simplified the labeling procedure. One major advantage was that reagents could be prepared in advance and stored while quality control testing was undertaken.

  1. In Vivo Method

In this method, labeling is accomplished with two consecutive intravenous injections. The first injection of non-radioactive stannous pyrophosphate is followed in 20-30 minutes by a second injection containing Tc-99m pertechnetate. Reported results for average labeling efficiency using the in vivo method vary widely from 71-96%. The interval between pyrophosphate and pertechnetate injection also affects the composition of the plasma Tc-99m activity. With a short interval, the plasma activity is primarily Tc-99m pyrophosphate while as the interval increases to 30 minutes the technetium is equally divided between pertechnetate and pyrophosphate.

  1. Modified In-Vivo Methods

This method evolved from observations that the rate of incorporation of Tc-99m pertechnetate into human red blood cells in vivo proceeds at a measurable rate. During the time interval between i.v. injection of Tc-99m pertechnetate and firm binding to red blood cells, the Tc-99m is free to distribute to extracellular compartments and localize in organs such as thyroid and stomach. A standard in vivo technique was, therefore, modified so as to isolate pretinned red blood cells and Tc-99m pertechnetate from other body compartments during labeling. If sufficient time is allowed for the reaction to proceed to completion, approximately 90% of the total Tc- 99m present will be firmly bound to the red blood cells at the time of injection. This results in increased intravascular retention and improved image quality.

PHARMACOKINETICS OF Tc-99m RBCs

The pharmacokinetics of technetium-99m red blood cells has been studied in patients and normal volunteers. After intravenous injection of pertechnetate during in vivo labeling, maximum whole blood activity was not reached until at least 30 minutes after injection. This suggests that pertechnetate freely diffuses into the extracellular fluid space, then re-enters the intravascular pool as blood levels of pertechnetate fall.

Drug interference with Tc-99m red blood cells for equilibrium blood pool imaging can be classified into two general categories: (1) agents that alter, by a direct pharmacological effect, cardiac function and have the potential to interfere with the interpretation of equilibrium blood pool images, or (2) agents that inhibit or diminish the radiolabeling or red blood cells by Tc-99m. Agents that induce an alteration in cardiac function include (a) the beta adrenergic blockers, such as propranolol (b) calcium channel blockers, including verapamil and (c) the nitrates, notably, nitroglycerin. Studies performed in patients receiving these pharmaceuticals may not detect the presence of coronary artery disease or accurately reflect its severity. It has been proposed that these interfering drugs be withdrawn from patients prior to exercise ventriculography. For beta blocking medications a 48-hour interval between withdrawal of the drug and the nuclear medicine study has been suggested, while for the calcium channel blockers the proposed interval is 48-72 hours, and 12 hours has been suggested for the nitrates

Drugs Suspected of Interfering with Labeling of

Red Blood Cells with Tc-99m

Drug/Device

Possible Mechanism

Heparin

Formation of Tc-99m labeled Heparin

Methyldopa, Hydralazine

Oxidation of Sn 2+

Chemotherapeutic Agents

Unknown

Whole Blood Transfusions

Formation of free Tc-99m hemoglobin; presence of anti-RBC antibodies

Intravenous Catheters

Binding of stannous ions to tubing

Digoxin, prazocin, propranolol

Unknown

Iodinated Contrast media

Competition between iodide and pertechnetate for transport by the band-3 anion transport system

 

MECHANISMS OF LABELING

It has been shown that in red blood cells labeled with Tc-99m, the majority of radioactivity is associated with hemoglobin. Further investigation has shown that 87% of the activity is associated with the globin portion of the molecule and 10% with the heme. It was therefore concluded that Tc- 99m in the lower valence state (probably technetium +4) binds irreversibly with globin, with the highest specific activity found in the beta-chain, most probably by coordinate covalent bond formation.

The process of pertechnetate binding to the red blood cell essentially involves passive diffusion of pertechnetate into the cell. More recently it has been shown that the pertechnetate ion is transported across the red blood cell membrane by the band-3 anion transport system. This system is responsible for maintaining the transmembrane concentrations of chloride and bicarbonate. Since there is no mechanism inside the cell to reduce pertechnetate in the absence of a reducing agent, pertechnetate is readily transported out of the cell by this system when the red blood cells are suspended in a vehicle containing chloride or bicarbonate as exchangers. The role of intracellular reduction of pertechnetate, which results in binding of the Tc-99m to hemoglobin, has been well documented.

 

FACTORS AFFECTING LABELING EFFICIENCY

Using a method to stop the labeling reaction between Tc-99m and red blood cells at the time of sampling, it has been shown that Tc-99m is incorporated into red blood cells in vivo at a measurable rate, reaching a value of 91.4% at 10 minutes following injection. This suggests that significant amounts of non-red blood cell-bound Tc-99m, probably as pertechnetate, is available for distribution to the extravascular compartments.

The incorporation of Tc-99m pertechnetate into pretinned red blood cells in a system isolated from other body compartments was shown to be affected significantly by the temperature and hematocrit of the reaction mixture, the dose of stannous ion administered and the presence of plasma. The volume of whole blood, activity of Tc-99m, and patient population have no significant effect on the rate or extent of Tc-99m labeling.

  1. Temperature

There is a direct relationship between the temperature of the reaction mixture and rate and extent of labeling. The temperature data suggest that increased labeling and shorter incubation times could be obtained if, in the modified in vivo method, the syringes were maintained at 37ºC rather than allowed to slowly cool during the incubation period. Elevation of the syringe temperature to 49-50ºC for 35 minutes has been shown to sufficiently damage the red blood cells so as to be able to do selective spleen imaging.

  1. Hematocrit

Whole blood hematocrit has a major effect on the rate and extent of red blood cell labeling. Normal values for the hematocrit vary with an individual’s age and sex. The normal hematocrit value for adults is 36 to 46% for women and 42 to 52% for men. There is a slight decrease in the hematocrit level after 50 years of age. However, in patients with anemia and in patients with significant blood loss, hematocrit values as low as 12 to 15% can be seen. Thus, these individuals would be expected to show decreased labeling efficiency with resultant increase in extravascular concentration of Tc-99m activity. This effect may be partially overcome by increasing incubation time when patients with known low hematocrit values are studied with the modified in vivo method.

  1. Volume of Whole Blood

Increasing the volume of whole blood from 1.5 to 4.5 ml in the modified in vivo method did not significantly alter the labeling parameters. The normal range of red blood cell count in men is 4.5- 6.5E6/uL and in women it is 3.9-5.6E6/uL.

It has been shown that the presence of plasma exerts a competing effect on labeling. The effect of diluting red blood cells with saline has less of an effect on relative labeling than when dilutions are done with plasma. This suggests that the effects of hematocrit on labeling efficiency is due partially to the concentration of red blood cells and partially to the presence of plasma.

  1. Stannous Ion Dose

Changes in blood disappearance of Tc-99m pertechnetate at stannous ion doses as low as 1 μG/kG have been reported. A plateau of labeling efficiency at 10 μG/kG has been reported in several studies, which have also shown this to be the minimum dosage of stannous ion that resulted in satisfactory red blood cell labeling. Decreases in labeling efficiency have been reported at doses in the 35-40 μG/kG range.

Optimal amounts of stannous ion may be decreased in several situations to levels that result in suboptimal radiolabeling of red blood cells. These include oxidation of stannous ion in PYP vials reconstituted with normal saline and not used immediately for in vivo labeling; infiltration of the stannous pyrophosphate injection for in vivo or modified in vivo methods, resulting in inadequate Sn+2 delivered into the blood; injection of stannous solution through an IV catheter or tubing for in vivo or modified in vivo methods, resulting in binding of Sn+2 to the device; and premature addition of sodium hypochlorite during the in vitro method, thereby oxidizing Sn+2 before it enters red blood cells.

 

References:

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