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CHAPTER 33 in cell surface-bound immunoglobulins and complement components
All of these changes have been investigated as signals for
(Chap. 54).
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ERYTHROCYTE TURNOVER recognition by the macrophages.
MEASUREMENT OF RED CELL DESTRUCTION

Perumal Thiagarajan and Josef Prchal The original method for the measurement of the red cell life span con-
sisted in the transfusion of cells that were compatible but identifiable
immunologically—the Ashby technique; type O red cells were infused
into individuals with type A or B cells. The differential agglutination
SUMMARY technique used anti-A or anti-B antiserum to measure the life span of
type O red cells that were transfused to type A or type B recipients and
The survival of red cells in the circulation can be measured in a variety of ways: the recipients’ own cells were removed using anti-A or anti-B serum.
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(1) by labeling with radioactive isotopes, particularly chromium-51 ( Cr), During World War II and shortly after, this method was used extensively,
and assessing the disappearance of the radioactive tag from the circulation but in recent years, because of the hazards associated with the admin-
over time; (2) by labeling the erythrocytes with biotin or a fluorescent dye istration of allogeneic erythrocytes, it has been completely replaced by
and measuring this marker over time; (3) by determining the disappearance techniques based on labeling of autologous blood. Furthermore, this
of transfused antigen-matched allogeneic erythrocytes using immunologic method could not be applied to autologous red cells.
In 1946, Shemin and Rittenberg demonstrated that the incorpora-
markers; and (4) by measuring the excretion of carbon monoxide, a product tion of nitrogen ( N)-labeled glycine into heme could be used to mea-
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of heme catabolism. sure the life span of the red cells. Since then a number of other isotopic
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Such studies show that normal human red cells have a finite life span aver- methods have been developed. These can be divided into three groups:
aging 120 days, with very little random destruction. The mitochondrial and (1) those that label a cohort of cells, (2) those that label cells randomly,
ribosomal removal highlighting maturation of the reticulocyte is accompanied and (3) those that use indirect measurements such as the rate of produc-
by increasing cell density, but after a few days of intravascular life span there tion of red cells or the rate of heme breakdown. The first two method-
is little further increase in density or other changes in the physical property ologic approaches yield information about the nature of the shortening
of the red cells. Thus, cell density is not a good marker for aged red cells. This of the red cell life span, age-dependent or random. The methodology
has made the senescent changes in the red cell that mark it for destruction yields only mean life span.
difficult to study. Candidates for such changes include changes in membrane
band 3 and exposure of phosphatidylserine on the membrane, which may be COHORT METHODS
of major importance. Cohort methods depend on the biosynthetic incorporation of the
label into the developing red cells. In these methods, a group of cells
of approximately the same age is labeled. The labels used are glycine-
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containing labeled N, radioactive carbon ( C), or radioactive iron
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RED CELL LIFE SPAN (either Fe or Fe). 16–18 The main disadvantage of cohort labeling is the
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need for prolonged periods of sampling, especially if the life span is only
Normal human red blood cells have a life span of approximately 120 days, moderately reduced (Fig. 33–1). In addition, radioiron from destroyed
after which they are engulfed by macrophages. This is an extremely red cells may be reused, making it difficult to interpret results. Further-
efficient process as macrophages phagocytose approximately 5 million more, the increasing restrictions on use of radiochemicals has drasti-
erythrocytes every second without a significant release of hemoglobin cally decreased availability of these two previously widely used nuclear
into the circulation. The precise molecular mechanism by which mac- medicine tests.
rophages recognize senescent red blood cells for phagocytosis remains A simple double-labeling technique that allows nonradioactive
largely unknown. As red blood cells age, several physiologic changes cohort labeling was described using two distinct labeling steps separated
occur that may serve as signals for recognition by macrophages. These by a defined time interval. Cells are subsequently evaluated by the rela-
1,2
include a decrease in the activity of enzymes, a progressive decrease tive proportions of these labels. The initial labeling step uses biotin that
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of ATP content, a loss of lipid asymmetry with exposure of phosphat- binds to all circulating cells (the red blood cells accounting for most of
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idylserine, an accumulation of lipid peroxidation products, a desialy- the label) the second administered labeling substance at later time digox-
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lation of membrane glycoprotein, an exposure of cryptic senescent igenin then distinguishes erythrocyte subpopulation of known age. 19
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antigens, aggregation of band 3 protein (Chap. 46), a decrease in
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deformability as the result of increased oxidative stress, and an increase
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RANDOM-LABEL METHODS
The random-label methods are the Ashby differential agglutination
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Acronyms and Abbreviations: ADP, adenosine diphosphate; AMP, adenosine technique, which uses an immunologic marker, and or the use of var-
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monophosphate; BNIP3L, an hypoxic regulated gene that facilitates mito- ious red cell labels such as chromium-50 ( Cr), chromium-51 ( Cr),
diisopropylfluorophosphate (DFP) labeled
or chromium-53 ( Cr),
20–22
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chondrial autophagy; C , third component of complement; C, radioac- with phosphorus-32 ( P), C, or C cyanate, a lipophilic dye, 26,27
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23 14
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tive carbon; CD44, cell differentiation antigen; CO, carbon monoxide; Cr, or biotin. 28,29
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chromium-50; Cr, chromium-51; DFP, diisopropylfluorophosphate; Fe or
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59 Fe, radioactive iron; G6PD, glucose-6-phosphate dehydrogenase; HO, heme Chromium-51 Method
oxygenase; Ig, immunoglobulin; In, indium-111; N, nitrogen; PK, pyru- By far the most commonly used radioactive isotope for the measurement
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99m
vate kinase; Tc, technetium-99m. of the red cell life span is Cr. As the chromate ion penetrates the red cell
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membrane it binds to the β and γ chains of globin. Unfortunately, these
Kaushansky_chapter 33_p0495-0502.indd 495 9/17/15 6:10 PM