Abstract
The early years of nuclear medicine included the development and clinical use of several in vitro or nonimaging procedures. The use of radionuclides as replacements for nonradioactive dyes brought improved accuracies and less subjective measurements to indicator dilution studies of body compartments such as the gastrointestinal system, lungs, urinary system, and vascular space. A popular nuclear medicine procedure was the radionuclide dilution method for quantitation of whole-blood volume or red blood cell volume or mass using 51Cr-labeled red blood cells—an important diagnostic element in patients suspected of having polycythemia vera, congestive heart failure, hypertension, shock, syncope, and other abnormal blood volume disorders. The radionuclide dilution method led to improved evaluation of red blood cell survival, which is important for clinical treatment planning in anemia and confirmation of splenic sequestration of damaged red blood cells. Although it was discovered that 51Cr was a chemically stable radiolabel of red blood cells after binding to intracellular hemoglobin, few nuclear medicine departments offered the clinical study for referring physicians because it required laboratory expertise for technologists, patient coordination, and a time-consuming procedure. The introduction of improved methods that are less time-consuming and have clinically acceptable results, along with the discontinuation of the sodium chromate 51Cr injection radiopharmaceutical by manufacturers, has consigned 51Cr red blood cells for red blood cell volume, mass, or survival evaluation to the list of retired nuclear medicine studies.
In vitro medical procedures are those completed outside the natural environment—the body—usually in a controlled setting such as a test tube or petri dish. Nonimaging nuclear medicine studies—in vitro procedures—involved collecting the patient’s blood, urine, or exhaled CO2; analyzing it for certain qualities or inequalities in the laboratory setting; and comparing the results with reference values. Typically, radioactive tracers were not administered to the patient but were added to the samples collected from the patient. The patient never became radioactive in these studies. Examples of early in vitro nuclear medicine studies include radioimmunoassays, thyroid function (triiodothyronine and thyroxin levels) measurements, and confirmation of hepatitis (1).
Modified nuclear medicine in vitro procedures were studies involving laboratory work completed on the patient’s blood, urine, or exhaled CO2 collected after a radioactive tracer (radiopharmaceutical) was administered to the patient and allowed time to be processed by the body. The radiotracer was a radiolabeled chemical that followed or outlined a process in the body. The radiotracer exhibited characteristics of biochemicals in the body system of interest. Nuclear counting of collected biologic samples, including blood, urine, exhaled air, and plasma, allowed evaluation of the body system for disease after comparison with reference values. Nuclear medicine–modified in vitro procedures have included evaluation of vitamin B12 absorption, gastrointestinal urease enzyme function, glomerular filtration rate, plasma volume, and red blood cell volume and survival. These early nuclear medicine–modified in vitro studies included simple procedures that measured dilution of a concentrated radiotracer in a body compartment similar to techniques used in laboratories and industry.
Indicator dilution techniques have been used for over 75 y to measure the unknown volume of a liquid in external containers (e.g., storage drums or underground wells) and in internal body compartments (e.g., cerebral spinal fluid space or blood) (2). The indicator dilution procedure involves the addition of an established volume of a measured concentration of some indicator to an unknown volume of liquid. The concentration of the indicator is measured in a sample taken from the unknown volume after allowing time for dilution or mixing.
Early methods used dyes that allowed measurement of both initial intensity and diluted concentration using colorimetric (visual or instrumental) means. These 2 values, along with the measured volume of the beginning dye solution, were used to calculate the unknown volume. Inaccuracies were associated with technique and problems with measurement of low dye concentrations.
Nuclear medicine procedures involving an indicator (radiotracer) dilution are commonly known as compartment localization or compartmentalization studies, in which the radioactive material is distributed in an enclosed volume or compartment. Examples of body compartments that have been studied using nuclear medicine–modified in vitro and imaging studies include the vasculature, cerebrospinal fluid space, gastrointestinal tract, urinary system, and lung airways. The subject of this review is one of the earliest modified in vitro indicator dilution studies of the blood volume compartment, that is, 51Cr red blood cell volume and survival (3).
HISTORICAL DEVELOPMENT
Understanding fluid balance in the blood was an early medical interest for improved diagnosis and treatment of disease. Early 20th-century attempts at blood volume measurement included the use of plasma diluent dyes such as Congo red and Evans blue and eyeball colorimetric detection. A measured volume of the dye was injected into one arm vein. After a few minutes had been allowed for appropriate mixing, blood was withdrawn from the opposite arm. Laboratory methods for isolation of the plasma from whole blood allowed a comparison of the quantity of dye from blood to a known volume standard. This allowed the calculation of plasma volume using a dilution formula and blood volume with a plasmacrit correction (4):
Further studies identified several errors using dye dilution in plasma as a measurement of blood volume. Problems associated with dye leakage from the vasculature, absorption, and diffusion, as well as inaccuracies of the plasmacrit correction, led to attempts to improve blood volume measurements. While using 32P in a study of phosphates in blood, Hevesy et al. discovered that red blood cells could be labeled with the radionuclide in vitro and reinjected and that isotope dilution could be calculated, providing a blood volume estimate (5).
Although small compared with the leakage of nonradioactive dyes from plasma, loss of 32P from radiolabeled red blood cells during circulation provided an improved but flawed method for blood volume measurement. Further development of the availability of radionuclides in medicine allowed the evolution of several nuclear medicine procedures. The discovery of the labeling of red blood cells with 51Cr remarkably changed blood volume estimation (6). The technique used by Gray and Sterling was simple. A few milliliters of a patient’s whole blood were incubated at room temperature with 51Cr sodium chromate. After a few minutes to allow for equilibrium of the intracellular and extracellular 51Cr, ascorbic acid was added to end the transfer of 51Cr across the cell membrane. A final washing of the cell suspension with saline to remove plasma and extracellular 51Cr was completed before reinjection of a measured quantity of 51Cr-radiolabeled red blood cells. After a few minutes of circulation to allow for mixing, a sample was removed for counting and used in the formula developed by the group for determination of the red blood cell volume (7):
Radiolabeled 51Cr red blood cells provided a uniform dispersion within the blood pool allowing for determination of red blood cell volume or red cell mass. Recommended by an international panel for standardization of studies involving measurement of red blood cell volume, the 51Cr red blood cell procedure provided reliability, reproducibility, and ease of adoption for routine use (8).
CHROMIUM AND ITS RADIONUCLIDES
Chromium is a naturally occurring mineral discovered in the mid-18th century and used early as a pigment in paints and tanning salts in the production of leather goods (9,10). Chromium has been used for electroplating car parts, plumbing, and furniture parts since the early 1920s (11).
Chromium is a member of group 6 of the periodic table, along with molybdenum, tungsten, and seaborgium. It has the atomic number 24 and occurs in nature as a hard, steely-gray, brittle transition metal. Although naturally occurring chromium is composed of 4 stable isotopes, 22 radionuclides of chromium have been created and characterized in the laboratory setting. Most radionuclides of chromium have half-lives of less than 1 min, with 51Cr remaining the only radionuclide of chromium with nuclear decay properties that allow industrial and medical use (12).
51Cr DECAY
51Cr is produced in a nuclear reactor by the neutron bombardment of enriched 50Cr. After capture of the neutron, the compound nucleus reacts by emitting a γ-photon via the nuclear reaction 50Cr (n,γ) 51Cr. Synthesis of radiochemical forms of 51Cr must accommodate the low specific activity of the material since chemical separation of the 51Cr radionuclide from the nonradioactive 50Cr is not possible. 51Cr decays by electron capture with a physical half-life of 27.7 d when a K-shell electron is brought into the nucleus, where it combines with a proton. This begins the path toward nuclear stability by reducing the positive charge of the nucleus by creation of a neutron and neutrino. Figure 1 illustrates the decay of 51Cr, in which 90% of the atoms decay directly to the ground state (electron capture 2) of stable 51V through the emission of a neutrino from the nucleus with an energy of 753 keV. The remaining 10% of 51Cr atoms emit a neutrino of only 433 keV (electron capture 1), with the remaining 320 keV of energy emitted as a γ-photon (γ1) (13). The emission of the 320-keV photon allows for the detection of 51Cr using classic radiation detection instrumentation found in nuclear medicine. Similar to the energy of the primary photon emission of 131I, the 320-keV γ-photon of 51Cr easily penetrates tissue, allowing external detection. However, the low 10% abundance of the 320-keV photon would require megabecquerel (millicurie) quantities of the radionuclide to provide reasonable count densities and imaging time for the creation of diagnostic-quality images. The absorbed radiation doses to the patient after the administration of the required radioactivity doses of 51Cr for imaging prohibit the use of the radionuclide for imaging.
Decay scheme for 51Cr. EC = electron capture.
51Cr RED BLOOD CELL LABELING
To facilitate the modified in vitro study of the clinical behavior of cells, tagging of specific cells using tracer quantities of a radionuclide has enabled nuclear medicine studies involving radionuclide-labeled blood cells of all types. Over the years, radiolabeled erythrocytes, leukocytes, and platelets have served an important role in defining pathophysiologic factors of various diseases and disorders. An early nuclear medicine procedure involving 51Cr-labeled red blood cells proved helpful in defining factors in hematologic diseases. Introduced in the early 1950s, random labeling of the circulating red blood cell population with 51Cr met requirements for in vivo use (Table 1) (14). 51Cr quickly became the most common radionuclide label for red blood cells after publication of its use for determination of red blood cell volume in 1950 (7). Nuclear medicine–modified in vitro studies continued to use 51Cr for erythrocyte labeling into the 1980s. Ultimately, the introduction of mechanized cell-sorting instruments provided more accurate cell counts without lengthy study requirements involving radioactive materials and handling of blood components; these advances led to a significant reduction in nuclear medicine 51Cr red blood cell studies.
Requirements of Radiolabeled Red Blood Cells for In Vivo Studies
Radiolabeling with 51Cr sodium chromate met the criteria of nontoxicity to erythrocytes, adequate specificity for targeting hemoglobin-rich erythrocytes, radionuclide properties that accommodate clinical studies, and acceptable elution of intracellular 51Cr properties. Although early studies demonstrated an initial rapid loss of 5% of the 51Cr radiolabel from red blood cells followed by a slower elution of 1% per day, improved radiolabeling procedures using washing of suspended cells after labeling and before patient infusion led to more accurate red blood cell survival studies (15).
Several modifications to 51Cr sodium chromate erythrocyte radiolabeling procedures added to the accuracy and reliability of nuclear medicine procedures to determine red blood cell survival and volume (Tables 2–4). Revisions were incorporated into the 1980 recommended techniques by the International Committee for Standardization in Haematology Expert Panel on Diagnostic Applications of Radio-Isotopes in Haematology (8). Several changes to 51Cr sodium chromate erythrocyte radiolabeling procedures included attention to whole-blood handling techniques to improve cell viability and final washing techniques to remove unbound 51Cr.
Radiolabeling of Red Blood Cells with 51Cr Using Citrate Washing Procedure
International Committee for Standardization in Haematology 51Cr Radiolabeling of Red Blood Cells
Sodium Chromate 51Cr Injection USP Prescribing Information Radiolabeling Procedure
The prescribing information available from the 2 manufacturers, until the radiopharmaceutical was discontinued, provided red blood cell labeling procedures very similar in content. To meet U.S. Pharmacopoeia (USP) compendial requirements and avoid toxic effects on the red blood cells, the initial specific activity was required to be greater than 370 MBq (10 mCi) per milligram of sodium chromate. 51Cr in the chemical form of sodium chromate (Na2CrO4) exists in the +6 valence state similar to 99mTc in the pertechnetate (NaTcO4) form. The hexavalent 51Cr chromate ions readily cross the red blood cell membrane, where they undergo reduction to the +3 valence state and irreversibly bind to intracellular hemoglobin. This radiolabeling procedure has shown greater than 90% binding of 51Cr to hemoglobin within 10 min (15). Studies have shown minimal loss (<1% per day) of the 51Cr from the radiolabeled red blood cells. 51Cr that leaves the red blood cells remains in the +3 valence state, is unable to repenetrate the red blood cell membrane, and is quickly removed by the kidneys (16). Table 4 lists the basic steps involved in the radiolabeling of red blood cells using sodium chromate 51Cr injection USP. Some instructions for radiolabeling using 51Cr sodium chromate do not include the addition of ascorbic acid to reduce the +6 valence state 51Cr ions to the +3 trivalent state, which does not penetrate the red blood cell membrane (Tables 2 and 3). The centrifugation step to wash the red blood cells functions to remove any unbound 51Cr, therefore making the addition of ascorbic acid an optional step. When used as a radiolabeled cell-washing procedure, centrifugation and 51Cr red blood cell resuspension should be done carefully so as not to damage the cells before patient infusion. Collection of the whole blood from the patient and handling of the blood during the radiolabeling procedure should be done with caution to ensure the red blood cells are not damaged. Damaged 51Cr red blood cells are quickly sequestered by the spleen and liver, providing errors associated with red blood cell volume and survival determinations. Over the years, variations in the procedure for the 51Cr labeling of red blood cells appeared as improvements were introduced in the radiopharmaceuticals, additives, techniques, and methods (8,17–19).
RADIOPHARMACEUTICALS
The prescribing information or package insert for sodium chromate 51Cr injection indicated it was a radiopharmaceutical diagnostic agent used in the determination of red blood cell volume or mass, the study of red blood cell survival time, and evaluation of blood loss. The radiopharmaceutical was available from 2 manufacturers.
Chromitope sodium (sodium chromate 51Cr injection USP; Bracco, formerly Squibb Diagnostics) was available in 7.4 MBq/mL (200 μCi/mL) and 74 MBq/mL (2 mCi/mL) radioactive concentrations. This Food and Drug Administration–approved radiopharmaceutical has been discontinued and is no longer available (20).
Sodium chromate 51Cr (sodium chromate 51Cr injection USP; Curium, formerly Mallinckrodt) was available in a 3.7 MBq/mL (100 μCi/mL) radioactive concentration. This Food and Drug Administration–approved radiopharmaceutical has been discontinued and is no longer available (21).
Sodium chromate 51Cr injection USP was made available as a sterile solution of 51Cr in the form of sodium chromate in water for injection. Sodium chromate 51Cr injection USP contained not less than 90.0% and not more than 110.0% of the labeled amount of 51Cr as sodium chromate. Other chemical forms of 51Cr must not exceed 10% of the total radioactivity. The specific activity of sodium chromate 51Cr injection USP was not less than 370 MBq (10 mCi) per milligram at the end of expiry (22).
CLINICAL USE
Whole blood is composed of an extracellular fluid or liquid phase (plasma) and a solid (cellular) component. The cellular component includes red blood cells, white blood cells, and platelets, with the red blood cells comprising most of the cellular mass (Fig. 2) (23). Normal blood volume is dependent on the height, weight, and sex of the individual, with approximate values available in published nomograms (24). The average adult whole blood volume is 5 L, consisting mostly of plasma (55%), with red blood cells composing most of the remaining (45%) volume (25). Under normal clinical conditions, blood volume is maintained with a combination of blood pressure influence, cardiac output, and kidney function. Blood loss leads to a decrease in cardiac output and blood pressure. The kidneys respond by retaining fluid, slowly restoring a normal blood volume. An increase in blood volume causes cardiac output and blood pressure to increase, leading to increased kidney output and restoration of normal blood volume. Several drug therapies and diseases can greatly influence these normal control mechanisms. An accurate measurement of red blood cell volume and survival is often the foundation of an accurate diagnosis and positive clinical outcome. Clinical indications for red blood cell volume determination include evaluation of hemolytic anemia, common in renal failure; evaluation of extensive trauma, burns, shock, and hypertensive volume overload; evaluation of possible polycythemia, an underlying cause of congestive heart failure; evaluation of blood loss and blood replacement therapy in transfusion medicine; and preoperative evaluation of elderly or compromised patients.
Composition of blood and components of blood volume. Hematocrit (arrow) is fractional relationship between plasma volume and red cell volume. (Reprinted from (23).)
Red Blood Cell Volume or Mass
Blood volume analysis continues to hold an important application in the management of various conditions in hematology, cardiology, pre- and postoperative surgical cases, and critical care medicine (26). Although newer methods to determine blood volume have improved accuracy, reliability, and study completion times, the clinical importance of blood volume analysis in cardiac and renal failure, hypertension, shock, screening for occult anemia, and evaluation of therapies in burn patients remains relevant (27).
Measurement of blood volume in nuclear medicine uses the indicator dilution method, in which the indicator is the patient’s red blood cells labeled with 51Cr. After 51Cr labeling of the red blood cells, a known volume (5–10 mL) containing a measured amount of the patient’s 51Cr red blood cells (0.37–1.11 MBq) is given as an intravenous infusion. After allotting 10–20 min for the 51Cr red blood cells to distribute uniformly throughout the vascular space, blood sampling can begin. According to the indicator dilution technique, the known volume of 51Cr red blood cells (V1), known radioactivity concentration of 51Cr red blood cells (C1), and measured radioactivity concentration of the blood sample (C2) are used to calculate the patient’s blood volume using the equation C1V1 = C2V2 (23,28). Below is a simplification of the calculation and a patient case example after the administration of 1 mL of 51Cr red blood cells radiolabeled using 5.55 MBq (150 μCi):
Red Blood Cell Survival or Blood Loss
After its introduction in the 1950s, there was widespread use of 51Cr red blood cells for the study of erythrocyte survival. Further study demonstrated loss of the 51Cr label by circulating red blood cells due primarily to loss of nonviable cells from the circulation produced during early radiolabeling procedures. This finding led to the study of splenic sequestration using methods to damage 51Cr red blood cells. Improved 51Cr radiolabeling of red blood cells that included washing the erythrocytes with normal saline solution or citrate led to improved 51Cr red blood cell lifespans.
A case study describing 51Cr red blood cell survival and splenic sequestration in a patient with hemolytic anemia and hypersplenism aided in establishing a diagnosis in a patient with an unanticipated clinical history on laboratory tests (26).
The red blood cell survival study evaluates the lifespan of the cell in patients with suspected hemolytic anemia and splenic sequestration, providing information on the rate and mechanism of hemolysis. The 51Cr red blood cell survival study has been used to confirm the diagnosis and evaluate the effectiveness of therapy.
The 51Cr red blood cell survival study was used to indicate the lifespan of the erythrocyte. Although the primary clinical use involved patients with suspected hemolytic anemia, the study provided a measure of red blood cell destruction expressed in other causes of red blood cell defects. The method involved administering 51Cr red blood cells, collecting blood samples at various times, and counting and plotting the counts over time, which allowed construction of a red blood cell survival time. Although the true mean half-life of the normal red blood cell is 50–60 d, the 51Cr red blood cell normal mean half-life is reduced to 25–30 d because of the 1% loss of the 51Cr radiolabel from the red blood cell into the vascular space, where it is cleared by the kidneys. A measured half-life of less than 20 d would indicate a faster blood loss or red blood cell destruction (27).
Splenic sequestration of 51Cr red blood cells was a diagnostic procedure that used external probe counts over the heart and spleen. A heart-to-spleen count ratio of 1:1 indicated normal localization parameters. A spleen-to-heart ratio of 2:1 or greater indicated hemolytic anemia leading to destruction of red blood cells (26).
Confirmation of suspected gastrointestinal bleeding using 51Cr red blood cells was reported using counts from collected stools and blood over time. Blood loss in stools was detected and quantified, allowing a diagnosis of anemia (28,29).
Although rarely included in the current list of diagnostic procedures performed by nuclear medicine professionals, 51Cr red blood cell studies can play a vital role in a definitive diagnosis when clinical history and other laboratory tests fail (30). Along with a definitive test for hemolytic anemia, 51Cr red blood cell volume measurement is considered an important clinical test for evaluation of blood loss, anemia, polycythemia vera, syncope, and congestive heart failure. Referred to as the gold standard technique for measurement of blood volume, the 51Cr red blood cell volume method performed well when compared with other laboratory studies of blood volume (31).
In search of improved accuracies, blood volume measurement involving simultaneous use of 51Cr red blood cells and 125I human serum albumin was instituted by some nuclear medicine departments. Red cell volume measurement with 51Cr red blood cells and plasma volume assessment using 125I human serum albumin and differential counting of the 2 distinctive radionuclides in collected blood samples provided extremely precise results (32). Because of the time required (4–6 h) and the complexity of the radiolabeling, blood collection, processing, counting, and generation of results (presenting prospects for errors), few nuclear medicine sections offered the study for referring practitioners. In late 1998, an automated system for blood volume analysis was approved by the Food and Drug Administration. The BVA-100 blood volume analyzer (Daxor Corp.) uses radiolabeled 131I human serum albumin as the injectate in an indicator dilution technique to measure plasma volume. The measured hematocrit of the patient is used to calculate the red cell volume and total blood volume (23,30).
CONCLUSION
Red blood cells radiolabeled with 51Cr have been used for clinical studies in nuclear medicine since the early 1950s (7). However, 51Cr red blood cell studies of blood volume and red cell destruction and survival were labor-intensive; were inconvenient to schedule in a busy nuclear medicine department; and required a cooperative patient, an experienced technologist with focused laboratory techniques, and a clinician familiar with blood volume parameters with comparison to reference values. These factors, along with low volume, lack of interest by referring physicians, and improved methods of diagnosing diseases of the blood, have relegated this once-popular nuclear medicine procedure to one rarely performed (33). A recent nuclear medicine technology job task analysis reported that almost 40% of respondents excluded total blood volume, plasma volume, and red cell mass from their “Knowledge and Performance of Nuclear Medicine Procedures” classification (34).
The introduction of improved methods that are less time-consuming and have clinically acceptable results, along with the discontinuation of the sodium chromate 51Cr injection radiopharmaceutical, has moved studies of 51Cr red blood cell volume and survival to the list of retired nuclear medicine procedures.
DISCLOSURE
No potential conflict of interest relevant to this article was reported.
Footnotes
CE credit: For CE credit, you can access the test for this article, as well as additional JNMT CE tests, online at https://www.snmmilearningcenter.org. Complete the test online no later than September 2027. Your online test will be scored immediately. You may make 3 attempts to pass the test and must answer 80% of the questions correctly to receive 1.0 CEH (Continuing Education Hour) credit. SNMMI members will have their CEH credit added to their VOICE transcript automatically; nonmembers will be able to print out a CE certificate upon successfully completing the test. The online test is free to SNMMI members; nonmembers must pay $15.00 by credit card when logging onto the website to take the test.
Published online Aug. 13, 2024.
REFERENCES
- Received for publication February 29, 2024.
- Accepted for publication June 19, 2024.
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