Version May 2024
INTRODUCTION — During its approximately four-month lifespan, the human red blood cell (RBC) travels approximately 300 miles, making approximately 170,000 circuits through the heart, enduring cycles of osmotic swelling and shrinkage while traveling through the kidneys and lungs, and an equal number of deformations while passing through capillary beds [1,2]. It has been speculated that accumulated damage to the RBC, especially to its membrane, renders the aging RBC unfit to circulate, leading to its destruction, via mechanisms that are poorly understood.
The normal time of RBC senescent (age-related) death in adults is approximately 110 to 120 days. Hemolysis can therefore be arbitrarily defined as a shortening in the survival of circulating RBCs to a value of less than 100 days.
This topic will review the mechanisms of normal RBC destruction and the methods used to measure RBC survival, which may be used in the evaluation of patients with suspected hemolysis [3]. Approaches to the patient with hemolytic anemia and with anemia in general are presented separately. (See "Diagnosis of hemolytic anemia in adults" and "Diagnostic approach to anemia in adults".)
MECHANISMS OF RBC DESTRUCTION — RBCs are normally destroyed by two different mechanisms, one related to increasing RBC age (senescence) and the other to a random process that destroys intact RBCs, or portions of an intact RBC (eg, vesicles) independent of their age (random hemolysis). These two processes may not be fully independent of one another [4].
Senescence — Virtually all RBCs die of processes associated with "wear and tear" associated with prolonged circulation within the intravascular space, unless another cause of RBC destruction intervenes first. These processes include, but are not limited to [5]:
●Shearing forces across high pressure areas in the circulation, such as the aortic valve.
●Multiple deformations subsequent to passage through capillaries and splenic cords.
●Multiple cycles of osmotic swelling and shrinkage when passing through the lung and kidneys [6].
●Oxidative challenges to the RBC from transported oxygen molecules and circulating oxidant molecules and immune-mediated mechanisms. As an example, age- and in vitro storage-dependent oxidation of band 3, the major membrane transmembrane protein, increases the affinity of anti-band 3 antibodies [7,8]. Such antibodies, when membrane bound, lead to partial complement activation on the red cell surface, which may be associated with decreased red cell deformability [9]. These complement components in turn are recognized by complement receptors on macrophages and removed from the circulation [10].
●A slow decline in RBC enzyme activity with advancing age, since the initial endowment of a RBC cannot be replaced during its lifespan.
●Decreased RBC deformability and increased sphericity due to progressive loss of surface area (membrane) with time.
Senescent death takes place mainly in the spleen, within the cords of Billroth. These unique vascular channels end blindly, unlike others in the body. The only way for an RBC, which has a diameter of 7 to 8 microns, to escape from these cords and return to the general circulation is to deform sufficiently to pass through 2 to 4 micron slits in the walls of the cords (picture 1). Cells that negotiate this process survive; those that cannot find themselves in a hostile environment, surrounded by cells of the monocyte-macrophage system. It is at this stage that senescent signals on the RBC membrane are detected by phagocytic cells [11]. Aging cells that fail this quality control step lose part or all of their membrane, with approximately two to three million RBCs being destroyed per second through these processes.
Senescent death or survival of RBCs can be approximated mathematically by a Gaussian distribution (the "bell shaped" curve), with a mean time to senescent death (mean potential RBC lifespan) of approximately 110 to 120 days in adults [5,12-14]. Senescence signals most likely arise from alterations in membrane proteins on the surface of aging RBC [15]. Candidate processes include exposure of cryptic alpha-galactosyl residues or phosphatidylserine on the RBC surface, alterations in the anion transporter (band 3) protein, altered expression or recognition of CD45 or CD47 on the red cell surface, increased membrane immunoglobulin, and accumulated membrane damage and stiffness from the effects of reactive oxygen species [16-24].
Random RBC death — The rate of random or age-independent RBC destruction is normally quite low, in the range of less than 0.05 to 0.5 percent per day; this value can be appreciably increased in hemolytic states, especially when destruction in an enlarged spleen is part of the hemolytic process [12]. It has been estimated that, in healthy individuals, up to 20 percent of the RBC's content of hemoglobin is lost during its lifespan secondary to loss of membrane-bound hemoglobin by the process of vesiculation [4,25].
ESTIMATION OF RED CELL SURVIVAL FROM THE RETICULOCYTE COUNT — A less accurate but clinically useful estimate of RBC survival can be obtained from easily available information such as the reticulocyte percentage (or preferably, the absolute reticulocyte count) and hemoglobin or hematocrit. This is based upon the fact that, to maintain a constant RBC mass, the bone marrow must produce as many new RBCs (reticulocytes) per day as are being destroyed. (See "Overview of hemolytic anemias in children", section on 'Reticulocyte count' and "Diagnosis of hemolytic anemia in adults", section on 'High reticulocyte count'.)
HISTORICAL METHODS FOR DETERMINING RBC LIFESPAN — Three general methods are available for accurate determination of the RBC lifespan:
●Random labeling of RBCs
●Cohort labeling of RBCs
●Kinetic studies of iron, bilirubin (and other bile pigments), or carbon monoxide metabolism
An historical review of RBC survival methodologies is available [3], beginning with the Ashby differential RBC agglutination studies initially published in 1919 [26].
Utility of RBC survival studies — With few exceptions, all of the above methods for determination of RBC survival entailed the use of radioactive or nonradioactive isotopes, were time-consuming, expensive, and required the patient to be in a steady state throughout the study. However, such studies were instrumental in our gaining an accurate understanding of RBC production and physiologic destruction, as well as mechanisms of increased turnover in various disorders of hematopoiesis such as aplastic anemia, pernicious anemia, thalassemia, and the various hemolytic anemias.
RBC survival studies are rarely employed in the 21st century for studies in individual patients, as routine laboratory studies for detecting intravascular and extravascular hemolysis are routinely available (eg, LDH, indirect bilirubin, haptoglobin, urine for cell-free hemoglobin and hemosiderin). A candidate for such studies might be the rare patient with anemia in whom the underlying mechanism is unclear. (See "Diagnosis of hemolytic anemia in adults".)
However, research applications of RBC survival methods have produced important evidence for the evaluation of RBC storage media, the effects of agents employed to improve RBC survival in the hemolytic anemias, and understanding the relationship between RBC survival and hemoglobin A1c levels in people with diabetes [3,27-30]. As an example, a study that measured the relative survival of RBCs stored at refrigerator temperature for different durations (one to five weeks versus six weeks) by transfusing six volunteers with 51Cr labeled red cells and determining the recovery of bilirubin and non-transferrin-bound iron markers found that RBC survival decreased and hemolysis increased progressively with longer storage times [31]. (See "Structure and function of normal hemoglobins", section on 'Glycated hemoglobins (hemoglobin A1C)'.)
Random label RBC survival methods
●Biotin/avidin method – This method has been used in clinical research studies when it becomes important to determine the lifespan of the entire population (or selected subpopulations) of RBCs. It is not used clinically. An aliquot of RBCs was labeled in vitro with biotin, washed to remove unreacted reagent, reinfused into the subject, and activity periodically monitored by reacting a sample of blood with fluorescein-labeled avidin or streptavidin, followed by flow cytometric quantitation of fluorescein-labeled RBC as a percentage of the total [3,32-36]. This method had the added advantage that serial studies could be performed in the same subject by utilizing differing concentrations of biotin.
●Radioactive labeling – In this method, which is of historic interest, a sample of circulating RBC, containing cells of all ages, was labeled in vitro with a radioactive isotope (such as 51Cr or 32P-labeled di-isopropylfluorophosphate) and reinfused into the patient. Blood samples were removed periodically, and survival was measured from the resulting plot of RBC radioactivity versus time after injection. While these techniques could also be used to determine blood volume, two major parameters of RBC survival could be obtained: the RBC half-time and the time of RBC senescence.
●Differential agglutination – The Ashby differential agglutination method, which is also mainly of historic interest, was based upon administration of type O blood into a subject with type A or B blood and serially measuring the number of non-agglutinated RBC as a function of time following injection [26]. This method cannot be used to determine the survival of autologous RBC.
RBC half-time — The RBC half-time is the time at which activity in the patient's circulating RBCs falls to one-half of the initial (or 24-hour) value. For a "perfect" label, such as 32P-labeled di-isopropylfluorophosphate (DFP) or biotin, this occurs at 55 to 60 days (ie, the normal RBC lifespan divided by two). However, the most commonly used radioactive isotope in the past for this purpose (51Cr) was not a perfect label, and eluted from the RBC at a rate of approximately 1 percent per day [37]. Thus, when employing 51Cr, the observed half-time was shorter than the theoretical value, in the range of 28 to 37 days for healthy adults [32,37], with values as low as 5 to 10 days in individuals with sickle cell anemia [38,39].
While corrections can be made to adjust for label elution, the 51Cr half-time was used directly as a semiquantitative measure of RBC lifespan. Its advantage over the cohort method described below (see 'Cohort labeling technique' below) was that an estimate of RBC survival could be obtained in less than two weeks.
Time of RBC senescence — When a random label technique was employed, the time of RBC senescence could be measured by sampling the patient's blood until activity was no longer detected on circulating RBCs. The time when the last labeled RBC has left the circulation was called the "extinction time"; this represents the time of death, by senescence, of labeled reticulocytes present in the original sample. This value is equal to the mean potential lifespan of the person's RBCs, averaging 120 days (range: 100 to 132 days) in adults without hemolytic disorders [32,35,37].
Similar reasoning applied to the determination of the lifespan of fetal red cells transfused into the maternal circulation via an unplanned, large volume fetomaternal hemorrhage [40]. In one report, fetal red cells, as measured by blood type-based flow cytometry, remained within the maternal circulation for 15 to 17 weeks (105 to 119 days) following delivery, indicating a lifespan similar to that of adult red cells [41].
Sites of RBC destruction — Chromium-51 studies were also used to detect the sites of RBC destruction, by placing radiation-sensitive probes over the liver and spleen. This technique was used to document the presence of dominant splenic (rather than hepatic) sequestration in patients with hemolytic anemia [42,43].
Cohort labeling technique — The cohort labeling technique, also of historical interest, involved the administration of a radioactive precursor of hemoglobin, such as 14C-labeled glycine or a non-radioactive precursor such as 15N-labled glycine. These metabolic precursors were rapidly incorporated into hemoglobin heme, producing a group (or cohort) of labeled RBC of defined age [13,14]. Their survival could be determined by repeated sampling of blood for labeled heme or stool for labeled bile pigments (eg, stercobilin). The rate of random hemolysis and the mean potential RBC lifespan could be obtained via analysis of the resulting curves of activity versus time after isotope injection [14]. Such studies are no longer available.
Quantitative erythrokinetic studies — Erythropoiesis could be quantitated following the intravenous injection of labeled transferrin-bound iron (59Fe or 55Fe) [44]. Using appropriate kinetic models, rates of erythropoiesis and estimates of RBC survival could be obtained [44-46]. These studies could provide information concerning the presence or absence of ineffective erythropoiesis, hepatic and splenic erythropoiesis, and splenic sequestration [46].
SUMMARY
●Modes of RBC destruction – Red blood cells (RBCs) are destroyed by two different mechanisms. These two processes may not be fully independent of one another. (See 'Mechanisms of RBC destruction' above.)
•Senescence – Destruction related to age-related alterations in the RBC.
-The average time for senescence death of the RBC (also called the mean potential lifespan) in adults is 110 to 120 days. (See 'Senescence' above and 'Time of RBC senescence' above.)
-When random RBC labeling techniques are used, the RBC half-time (when half of the labeled cells have left the circulation) is 55 to 60 days for a "perfect" (non-eluting) label such as biotin and 28 to 37 days for an eluting label such as 51Cr. (See 'RBC half-time' above.)
•Random hemolysis – Random hemolysis is due to processes that destroy intact RBCs or portions of an intact RBC independent of cell age. Most clinical conditions associated with shortened RBC lifespan are of this type, including autoimmune hemolytic anemia and RBC destruction in sickle cell disease. (See 'Random RBC death' above and "Diagnosis of hemolytic anemia in adults", section on 'Conceptual framework'.)
The rate of random hemolysis has been estimated to be <0.5 percent per day in adults. (See 'Random RBC death' above.)
●Methods for estimating RBC survival – RBC survival studies were frequently used during the second half of the 20th century and were instrumental in fostering understanding of RBC production and destruction in health and disease. They have limited clinical utility in the modern era and are mainly of historical interest. (See 'Utility of RBC survival studies' above.)
Examples include the following:
•Random or cohort RBC labeling techniques. (See 'Random label RBC survival methods' above and 'Cohort labeling technique' above.)
•Determination of iron or bilirubin turnover. (See 'Quantitative erythrokinetic studies' above.)
●Reticulocyte counts – A less accurate but clinically useful estimate of RBC survival can be obtained from the patient's reticulocyte percentage (or preferably, absolute reticulocyte count) and hemoglobin or hematocrit, along with an estimate of the reticulocyte lifespan within the circulation. (See 'Estimation of red cell survival from the reticulocyte count' above.)
ACKNOWLEDGMENTS
UpToDate gratefully acknowledges Stanley L Schrier, MD (deceased), who contributed as Section Editor on earlier versions of this topic and was a founding Editor-in-Chief for UpToDate in Hematology.
The UpToDate editorial staff also acknowledges the extensive contributions of William C Mentzer, MD, to earlier versions of this and many other topic reviews.
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