Origin and Composition of Cabot Rings
in Pernicious Anemia
LAWRENCE KASS,
M.D.
Department of Internal Medicine (Simpson Memorial Institute), The University of Michigan,
Ann Arbor, Michigan 48104
ABSTRACT
Kass, Lawrence: Origin and composition of Cabot rings in pernicious
anemia. Am J Clin Pathol 64: 5 3 - 5 7 , 1975. Cytochemical studies were
performed on erythrocytes containing Cabot rings from the peripheral
blood of two patients with severe untreated pernicious anemia. These
studies demonstrated that the Cabot ring contained arginine-rich histone
and non-hemoglobin iron. Structures that may represent precursors of
Cabot rings were found in stippled late intermediate marrow megaloblasts.
T h e Cabot ring may result in part from abnormalities in metabolism of both
iron and arginine-rich histone that are known to occur in pernicious anemia.
(Key words: Cabot rings; Pernicious anemia; Erythrocytes.)
50 patients with untreated pernicious
anemia were carefully reviewed. In only
three instances, patients with erythrocytes
containing Cabot rings were detected.
From two of these patients, sufficient
material was obtained prior to treatment
to permit a variety of cytochemical
studies. These studies were performed in
an attempt to ascertain the composition of
the Cabot ring.
Peripheral blood films from these patients were stained for "reticulum" using
brilliant cresyl blue as a supravital stain,
DNA using the Feulgen reagent, 21 RNA
and DNA using methyl-green pyronin, 21
glycogen using the PAS (periodic acidSchiff) reagent, 8 mitotic spindle fibers
using Heidenhain's iron hematoxylin
stain, 16 metachromatic substances using
toluidine blue, 21 iron using the Prussian
blue reagent 6 and 1% aqueous neutral red
counterstain, histone using the alkaline
fast green stain, 1 and arginine-rich and
lysine-rich histone using the ammoniacal
silver stain. 3 With this stain, arginine-rich
THIS REPORT describes cytochemical
properties of Cabot rings in erythrocytes
obtained from peripheral blood of two
patients with classic untreated pernicious
anemia. In the bone marrows of these two
patients and in the peripheral blood of
another patient who also had Cabot rings
in erythrocytes of peripheral blood, apparent precursors of Cabot rings and actual
Cabot rings were found in heavily stippled late intermediate megaloblasts. T h e
results of these studies may afford insight
into a possible pathogenetic basis for the
formation of these unusual structures.
Materials and Methods
Wright-stained films of capillary peripheral blood and bone marrow from
Received October 15, 1974; received revised manuscript December 3, 1974; accepted for publication
December 3, 1974.
Supported by the Elizabeth Roodvoets Memorial
Grant for Cancer Research of the American Cancer
Society (#CI-79) and by the National Cancer Institute Grant USPHS CA 14428-02.
Address reprint requests to Dr. Kass.
53
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KASS
A.J. C.P. — Vol.64
FIG. 1. T o p row, two erythrocytes containing prominent Cabot rings. These rings appear as a "necklace" of
stippled granules joined by a red-staining strand on Wright-stained film. Bottom row, what may be
precursors of these stippled "necklaces" in peripheral blood erythrocytes within the cytoplasm of heavily
stippled late megaloblasts in the bone marrow. Arrows point to loop of ring-shaped structures. In the
megaloblast at bottom left, an actual ring form may be seen within the cytoplasms, x 1,800.
histone appears brown or black, and
lysine-rich histone appears yellow.
Bone marrows from these patients were
carefully studied in an attempt to identify
possible precursors of Cabot rings in the
megaloblastic erythroid precursors. In the
instance where material was available,
marrow films were also stained with the
Prussian blue reagent and counterstained
with 1% neutral red.
in the patients' peripheral bloods. These
rings were composed of either basophilic
or acidophilic stippled granules connected
to one another by a red, strand-like
material, giving the ring the appearance
of a stippled "necklace." At times, "figure
8" configurations of the ring were observed. In most instances, the ring was
associated with heavy basophilic and
acidophilic stippling of the erythrocyte
cytoplasm. Even when no gross stippling
Results
was seen, at least several coarse granules
Cabot rings (Fig. 1, top row) were found could be observed adherent to the ring.
in approximately 1 per 500 erythrocytes Cabot rings were not observed in cells that
July 1975
55
CABOT RINGS IN PERNICIOUS ANEMIA
FIG. 2. Left, enlargement of the ring
form in the cytoplasm of the cell illustrated in Fig. 1, bottom left, showing
coarse granules connected by strandlike material. X 1,800. Right, late
megaloblast stained with Prussian blue
and counterstained with neutral red. A
red-stained loop is seen in the cytoplasm, and siderotic granules adhere to
the loop. This loop may be the precursor of or an actual Cabot ring, and
suggests that it may be related to
abnormalities in iron metabolism,
x 1,500.
A**
contained reticulum as visualized by supravital staining with brilliant cresyl blue.
Of the various cytochemical tests, only
three demonstrated structures consistent
with Cabot rings. Peripheral films stained
with the Prussian blue reagent and counterstained with neutral red showed a thin
red ring to which blue-stained particles
adhered. Films stained with the alkaline
fast green stain for histone showed a
green ring surrounded by green-stained
particles, some of which adhered to the
ring. In peripheral blood films stained
with the ammoniacal silver stain to identify lysine-rich and arginine-rich histones,
a black ring or "figure-8" ring form was
seen. Orange, brown, and black particles
were seen attached to the ring and isolated within the cytoplasm of the erythrocyte. Notably, structures resembling Cabot
rings were not observed in erythrocytes
stained with reagents specific for DNA
(Feulgen stain and methyl green stain).
T h e second row of Figure 1 illustrates
bone marrow erythroid precursors obtained from the patients. As delineated by
the arrows, ring and loop structures could
be observed within the cytoplasm of certain late intermediate megaloblasts. T h e
megaloblasts that showed these ring structures most frequently were those that
showed unusually abundant and coarse
cytoplasmic basophilic and acidophilic
stippling. As in the Cabot rings in the
peripheral blood, the rings and loops in
I
/
late stippled megaloblasts in the bone
marrow also appeared as a "necklace" of
coarse granules connected to each other
by a red-staining strand. Partial loops of
this appearance were seen most frequently, but on several occasions, as seen
in Figure 1, bottom row, left, and in
Figure 2 (enlargement of the area in
Figure 1), an actual ring or "figure-8"
stippled "necklace" could be observed in
the cytoplasm. In most instances, the
"necklace" did not seem to show continuity with the nuclear chromatin, nor
did it have the tinctorial properties of
nuclear chromatin as visualized with ordinary Wright's stain.
In one instance, a megaloblast stained
with Prussian blue to demonstrate iron
and counterstained with neutral red demonstrated a ring structure in the cytoplasm (Fig. 2). T h e ring appeared to consist
of a red-staining thread to which bright
blue siderotic granules adhered.
Discussion
Since Cabot's original report of ring
bodies in pernicious anemia in 1903, 5 the
nature of these structures has been a
subject of lively controversy. In his original report, Cabot 3 illustrated stippled
"necklace"-shaped and "figure-8" ring
bodies similar to those found in the
present study, and he believed but could
not prove that these rings were nuclear
remnants. Bostrom 4 postulated that two
56
KASS
erythroid precursors in pernicious anemia
were joined by a red-staining fiber of
nuclear origin that became enclosed in a
pseudopod of the erythroid precursor.
This pseudopod then appeared as an
erythrocyte containing a ring or "figures'' structure.
Discombe 7 and Picard 18 were unable to
demonstrate structures consistent with
Cabot rings using stains for DNA. Picard 18
and van Oye 22 believed that Cabot rings
were remnants of the mitotic spindle
apparatus. In the present report, structures analogous to Cabot rings could not
be demonstrated using Heidenhain's iron
hematoxylin, a reagent commonly used
to demonstrate the mitotic spindle apparatus. 16 In contrast to the postulates
of the authors mentioned, other investigators 2,15,20 have concluded that Cabot
rings are laboratory artifacts.
A.J.C.P.—
Vol.64
arginine-rich histone and nonhemoglobin iron. Although histone and iron were
found to be the only substances demonstrable in Cabot rings in the present
report, it is possible that there may be
additional substances within the ring
that might be demonstrable by other
cytochemical reagents not utilized in these
studies.
The basis for the presence of argininerich histone and non-hemoglobin iron in
the Cabot ring and the pathogenetic implications of these cytochemical findings
can only be speculated upon at present. It
is known that arginine-rich histones are
synthesized within the cytoplasm of cells
on polysomes 19 and that histone biosynthesis is abnormal in pernicious anemia. 12
In pernicious anemia there is also a defect
in the metabolism and mobilization of
iron, and this disorder has been considered a type of secondary acquired
sideroblastic anemia.9"13,14 In the present
study, Cabot ring precursors were found
in late intermediate megaloblasts that
showed unusually coarse cytoplasmic
basophilic stippling. This type of coarse
stippling is believed to be composed
largely of non-hemoglobin iron. 17
In the present study, Cabot rings and
their presumed precursors were found in
late intermediate marrow megaloblasts.
Other authors have also found these
structures in megaloblasts in pernicious
anemia and in erythroid precursors from
patients with other types of anemias. In
1938, Isaacs 10 described and illustrated a
late intermediate megaloblast from a paOn the basis of the available informatient with pernicious anemia that con- tion, it is possible that the spatial proximtained a large Cabot ring "entirely sepa- ity of iron and arginine-rich histone in the
rate from the nucleus." Isamboulas and cytoplasm of the late i n t e r m e d i a t e
Malikoosis,11 in 1939, also illustrated a megaloblast, combined with the known
Cabot ring in an erythroid precursor abnormalities of both iron and histone in
from a child who had typhus. Apparent pernicious anemia,9>12,13,17 facilitates an inprecursors of or actual Cabot rings have teraction between these two substances.
also been observed in erythroid precur- Why this interaction should result in a
sors by Picard 18 and by van Oye. 22
structure having the shape of a loop or a
T h e results of cytochemical tests for "figure-8" is unknown.
DNA in this study agree with the findings
of others, 7,8 namely that the Cabot ring
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CABOT RINGS IN PERNICIOUS ANEMIA
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