aroused by the rather frequent occurrence of certain, well character

13 Pages·2007·1.19 MB·English

CERTAIN CYTOPLASMIC INCLUSIONS OF LIVER CELLS*
ALWIN M. PAPPENHEIMER, M.D., AMS JoHx J. HAwTHoRmE, M.D.
(Fromthe Department ofPathology, College ofPhysicians andSurgeons, Columbia
University, Ne-x IYork City)
On routine examination of liver sections our interest has been
aroused by the rather frequent occurrence of certain, well character-
ized cvtoplasmic inclusions. Although easily seen, even with ordi-
nary stains, they seem to have attracted little attention and to have
escaped the recognition of experienced pathologists. They are not
described in standard textbooks ofpathology, histology or cytology.
Mallory,1 however, in his classical paper on necroses of the liver
(I90I) has accuratelv described and pictured these bodies (Plate
XVI, Fig. i. and Plate XVII, Fig. i), and since we have found but
one other reference to them his description may be cited in full.
"In a majority of the early cases the protoplasm of the liver cells
containsnumeroussmallandlargevacuoles inwhich are single small
hvaline globules; single, sometimes multiple, coarse, and fine
threads; and occasional networks, all of which stain deeply with
eosin and with Weigert's fibrin stain. With other stains thev react
exactly as fibrin does. The hyaline globules are not threads cut
across; whether they really are fibrin is difficult to prove. They
often seem to precede the formation of the threads and networks
of fibrin."
It would seem that MIallory regarded these bodies as an accom-
paniment of early degeneration, ending in necrosis of the liver cells.
Wehave been able tolocate butone other short reference to these
structures. Taniguchi 2 (I93i) describes in liver cells three types
of cytoplasmic indusions as follows:
Type i: Spherical bodies, varying in size from a round mitochon-
drial granule to that of a nucleus, homogeneous and with sharp
contours. They stain deeply with Heidenhain's iron hematoxylin
after fixation in Regaud's fluid and can be distinguished, though
less clearly, in hematoxylin-eosin preparations. They do not stain
with Sudan III.
Type 2: More orless elongated oval bodies which are regarded as
a modification of Type I. They are infrequently found.
* Received forpublication March 25, 1936.
625
626 PAPPENHEIMER AND HAWTHORNE
Type 3: Comparatively narrow, long and short, thread-like,
slightly bent, sometimes spindle shaped bodies. They are derived
from swollen, thread-like mitochondria.
The material for Taniguchi's study was obtained from surgical
biopsies. The bodies described as Type i were found in 27 of 94
cases. The photographic illustrations accompanying the paper
show, though not very clearly, spherules and filaments.
We have been interested in making a somewhat more extended
study of these bodies.
DESCRIPTION OF THE INCLUSIONS
(A) Spherical Bodies: Our observations in the main confirm the
description given by Mallory. The spherules vary in size from that
of a smallmicrococcusup to 4or 5 ,uin diameter, themajority rang-
ingfrom I to 3 ,u. Theylie in a large vacuole which, withthe usual
staining methods, is optically empty or contains only a little shred-
ded material. When the vacuole is contiguous to a nucleus the
nuclearmembranemaybeindentedbyit. Theremaybebutasingle
spherule or the entire cytoplasmmay be riddled with vacuoles, each
containing a single body, so that the cell has a cribriformorporous
structure. Adjacent vacuoles may coalesce into large spaces con-
taining several spherical inclusions. When they are on the surface
they may rupture, discharging the inclusion body into the space
between liver cell and sinus wall.
Staining Reactions
With hematoxylin-eosin staining, the spherical bodies are acido-
philic, but the color is unlike that of the cytoplasm or of the ery-
throcytes, having a brownish yellow cast. They are sharply con-
toured, moderately refractive, and entirely homogeneous without
any suggestion of internal structure.
WiththeGramstainthemethylvioletorgentianvioletisretained,
ifdecolorization isnotpushed toofar. Thedegreeof Grampositive-
ness is about the same as that of the nucleolus.
Mallory's phosphotungstic acid hematoxylin stains the spherules
very selectively a bluish black.
After formalin fixation, staining with phosphotungstic and hema-
toxylin demonstrates that the spherule is surrounded by a homo-
CYTOPLASMIC INCLUSIONS OF LIVER CELLS 627
geneous capsular material which stains a dull brick red and com-
pletely fills the vacuole (Fig. 3).
Heidenhain's iron hematoxylin also brings the bodies out sharply
but they are resistant to decolorization with ferric chloride.
With azocarmine, anilin blue and orange G, the bodies take a dull
orange color similar to that of the erythrocytes.
With methyl green-pyronin, both spherules and rods take only a
faint yellowish stain. With mucicarmine the globules do not take
the mucin stain.
Masson's trichrome stain shows the spherules to be red, in con-
trast to the dark violet gray of the cytoplasm. The nucleoli are
bluish black. Contents of the vesicle are pale blue. There is no un-
stained vacuole. The bluish material is sometimes homogeneous,
sometimes finely granular (Fig. 4). The rods stain orange-red and
lie in a dear vacuole without the bluish staining matrix.
Mallory's anilin blue collagen stain shows about thesamepicture
as that of Masson's, except that the spherules are orange-red and
the bluish matrix is less sharply brought out.
With Mann's stain the spherules are dull red to bluish purple,
depending on the degree of differentiation. Filaments stain about
the same. Contents of vacuoles remain unstained (Fig. i).
With Giemsa, they are stained a rose pink.
Themost selective stain that we have found is Laidla-uw's method
for demonstrating virus inclusions.* By careful differentiation with
orange Galcohol the cytoplasm of theliver cellsmay bedecolorized
to a grayish blue: the spherical inclusions retain the fuchsin and
stain an intense crimson. The red blood cells are a tawny orange-
red (Fig. 2). NVhile Laidlaw's method calls for fixation in acetic
* LAIDLAW'S METHODFOR ST.AIING INCLUsION BODIES
irF.ixation in gslaatcuarlataecdetaiqcueacoiuds c.5orrcocs.ivesublimate ............... IOOcc.
2. Embedinparaffinandcutat 3 .
3. Deparaffinize in xylol, absolute and 9 7% alcohol andrinse inwater.
4. Stainin Weigert's ironhematoxylin (2%) 5 minutes.
5. Differentiate ino.,7c acidalcohol.
6. Rinsein tapwaterfollowedbydistilled.
7. Stain in I%c aqueous acidfuchsin 5-I5 minutes.
8. Rinseindistilledwater.
9. Mordant in i7c phosphomolybdic acdfor30 seconds.
io. Rinsein distilled water.
ii. Differentiatein0.25%c ornge Gin 70% alcohol.
12. Dehydrate, clearand mountin balsam.
628 PAPPENHEIMER AND HAWTHORNE
acid bichloride, Zenker's fixation without acetic acid gives equally
brilliant pictures.
The bodies are not stained by scharlach R.
(B) Rod Shaped and Filamentous Structures: These occur almost
always in association with the spherical bodies, but far less fre-
quently (only in about one-fifthof the cases). They also lie in clear
vacuoles and assume a variety of forms-simple bacillus-like rods,
beaded or fuzzy straight rods, or long curving fibrils, sometimes
looped upon themselves. Small crossing or interlacing filaments
may be seen within the same vacuole.
Not infrequently thefibrils seem to be formed directly on orfrom
the spherical body; the spherules may lie in the middle or at the
end of the rod. But in many of the vacuoles containing rods or
filaments, no spherical body can be discerned.
The staining reaction of these filaments and rods, as Mallory
pointed out, is the same as that of fibrin. They retain the gentian
violet in the Gram-Weigert stain, and stain bluish black with phos-
photungstic acid hematoxylin. They are also brought out by the
Heidenhain iron hematoxylin method and by the Laidlaw stain.
General Incidence
In all, 562 sections of liver from human cases have been studied.
The spherical inclusions were found in 175 cases, or 31.I per cent.
Many blocks were recut and stained with phosphotungstic acid
hematoxylin, and in these preparations the bodies are more easily
recognized. Their incidence in the hematoxylin-eosin sections was
therefore somewhat lower (23.7 per cent) than in the I24 sections
stained with phosphotungstic acid hematoxylin, which gave 43.6
per cent of positives.
Includedinthe 562 casesare39I adults, I24children, and47 new-
born infants and stillbirths. The percentage incidence in the differ-
ent age groups is shown in Table I.
TABLEI
wks. 2wks.- I-5 6-io II-20 21-30 31-40 41-50 5I-60 61-70 7I-80 80+
Age 2WS. Iyr. yrs. yrs. yrs. yrs. yrs. yrs. yrs. yrs. yrs. yrs.
No. 47 46 30 i8 30 42 66 79 104 65 32 3
No.+ 14 6 7 6 9 14 27 23 40 17 IO 2
%+ 29.7 I3.0 23.3 33-3 30.0 33.3 4I.0 29.1I 38.4 26.I 31.2 66.6
CYTOPLASMIC INCLUSIONS OF LIVER CELLS 629
There is, as the analysis shows, no significant difference in the
various age groups. The spherules were found once in a stillbom
full'term infant, and in several prematures.
Occurrence in the Human Fetus
Through the courtesy of Dr. Coler of the Sloane Hospital, we
havebeen abletoobtainmaterial fromhumanfetusesfrom5months
to term. The sections stained withphosphotungstic and hematoxy-
lin showed an astonishingly high incidence of inclusions.
AgeofFens No.Ezaimied Positive Negative or Dou.5tfil
5mos. 7 7
6mos. 7 4 3
7mos. 7 6 I
8mos. 6 , I
Ful term 7 4 3
34 26 8
The failure to identify the bodies in a few of the cases may have
been due to poor preservation of the tissue. In 8 of the 34 cases,
that is in about the same proportion as in the postnatal livers, the
spherules were associated with rods and filaments.
Relation to Possible Postmortem Changes
The bodies seem to be relatively little affected by autolytic
changes. and they do not becomemore numerousafter death. They
have been found as early as 45 minutes portmortem and as late as
53 hours. The incidence of positive findings at various periods after
death is shown in Table II.
TABLE II
Hourspostmortem 2 .2-6 6-12 12-24 Morethan 24
Totalautopsies .............. 26 1i65 1I30 I46 43
No. showingglobules ......... 6 4 39 i6
No. showingrods ............ I 12 I3 4 9
%C globules + ............... 23.I 30.3 34.6 26.7 37-2
%rods+ ................... 3.8 7.2 I0 6.2 9.3
Although there are slight variations in the different groups, there
is no trend toward adefinite increase ordecrease in the incidence, as
the period elapsing after death becomes greater.
630 PAPPENHEIMER AND HAWTH0ORNE
Relation to Other Diseases
We have attempted to correlate the occurrence of the spherules
with various general diseases such as arteriosclerosis, nephritis, and
acute and chronic infections. As might have been anticipated from
theratheruniform age distribution andthepresenceof thebodiesin
the fetal liver, no such correlation seems to exist. The bodies have
been present in all sorts of pathological conditions, and a detailed
analysisofourmaterialfromthispointofviewwouldbefutile. The
only reservation to the above statement is in the case of malignant
disease. Amongthe 562 casesexamined, iii hadmalignant tumors,
and of these 24 showed liver inclusions- an incidence of 21.6 per
cent as against an incidence of 33 per cent in the 45I non-tumor
controls. Thesignificanceofthisloweredoccurrenceisnotapparent,
butthedifferencewouldappeartobetoo greatto ascribe to chance.
Not only is there no clear-cut correlation with general diseases,
but we have been able to find no constant association with any
particular type of liver lesion. The inclusions are found frequently
in otherwise normal appearing cells. When they are very numerous
thecytoplasmofthecellscontainingthemhasaspongyorcribriform
appearance. They are alwaysmore abundant in the central portion
of the lobule and often seem to be especially numerous when the
liver cellsare atrophic, or inthepresenceof central congestion. But
they are quite definitely not associated with necrosis of the cells
and we cannot interpret their presence as an indication of early
degeneration or cell death. On the other hand, they may often be
recognized in cells that have recently undergone necrotic changes.
We have notfound them in the neoplastic cellsof primary liver cell
carcinoma.
Occurrence in Laboratory Animals
In monkeys the spherical inclusions have been found in I of 5
livers examined. In guinea pigs they have been seen in 8 out of
44livers- slightlylessfrequently than in human livers. In 7 fer-
ret livers they were present in every instance. Rods and fila-
ments, however, have not been observed.
On the other hand, we have searched in vain for these bodies in
the liversof rabbits, dogs, cats, pigs, rats, mice, chickens or ducks.
CYTOPLASMIC INCLUSIONS OF LINER CELLS 63I
DIscussIoN
It has not been possible to arrive at any final conclusion as to
the nature of the spherical inclusions. Several possible interpreta-
tions come to mind and may be discussed briefly:
i. They are some unknown structural component of the cell, like
the Golgi net, centrosome, or mitochondria.
2. They are secretory products.
3. They are degenerative products, akin to the colloid droplets in
degenerative renal epithelial cells.
4. They are cytoplasmic "virus inclusions."
It is dlifficult to believe that these structures represent some un-
known organs of the cell. Were such the case, one would expect to
find them in every cell, certainlv in everv liver and in every species.
Nor is there any reason to believe them to be derived from known
cv-toplasmic constituents. They stain quite differently from mito-
chondria, persist long after the mitochondria have been destroyed
by autolvsis, and are not affected by acid. There is nothing to sug-
gest that they are related to the Golgi net or centrosome, andthey
are most certainly not due to extrusion of nuclear chromatin.
We can find no particular evidence for or against the possibility
that they represent a secretory product of the liver cells. The fact
that with Gram and phosphotungstic acid hematoxylin the staining
is like that of fibrin. brings up the thought that they may be asso-
ciated with the production of fibrinogen. But the stainingforfibrin
is far from being specific and it would require much more evidence
to justify such an assumption.
Against the secretory nature of the product is the observation
that only a single body may be present in the cell. We can recall
no instance inwhich secretory granules were notmultiple. Perhaps
it may be taken asa point also against the secretory nature of these
bodies that they are not found in all human livers, as one might
expect if they represent a normal activity of the liver cells, and that
they are not demonstrable in the livers of dogs, rats, rabbits, cats,
chickens or ducks.
Against the viewthatthespherules representadegenerative alter-
ation in the cvtoplasm is the fact that they are often found in cells
that shownootherevidenceofinjury. It is true that they aremost
numerous in the central portions of the liver lobules, where atrophy
632 PAPPENHEIMER AND HAWTHORNE
or necrosis accompanying central congestion is so frequently seen.
Doubtless also, their presence in great number, with the resulting
spongy rarefaction of the cytoplasm, may lead to the distintegra-
tion of some of the cells. On the other hand, the bodies may be
foundin cellsadjacent totheportal spaces, and in thissituation the
liver cells often show no evidence whatever of damage.
The last possibility which comes to mind is that the bodies are
of the nature of virus inclusions. This assumption would lead of
necessity to the view that alargeproportion of human livers harbor
a non-pathogenic virus or viruses, and that similar viruses occur in
the livers of monkeys, guinea pigs and ferrets. The fact that we
have found the inclusions in the liver cells of the fetus would also
logically involve the assumption that the hypothetical virus is
transmitted to the embryo.
The only evidence in favor of these bodies being virus inclusions
is (i) their morphology, which is not unlike that of certain cyto-
plasmic indusions, such as the Guarnieri bodies (cf. Cowdry, E. V.,
J. Exper. Med., 1922, 36, 666, Fig. 33); and (2) the staining reac-
tions. We have hadopportunity to compare theliver cell inclusions
with cytoplasmic virus indusions in the various tissues of ferrets
experimentally infected with a virus now being studied by Dochez,
Slanetz and Smetana.* The staining of the liver cell inclusions is
practically identical with that of the virus inclusions, but the latter
are not surrounded by a stainable halo.
It would be of interest to obtain experimental support for the
virus theory, as the demonstration of such a frequently occurring,
latent, non-pathogenic virus would be of great theoretical impor-
tance. Unfortunately wehave thoughtof noobviouswayofattack-
ing the problem experimentally at the present time.
As regards the rod-like and filamentous inclusions, we agree with
Mallory that they are probably fibrinous in nature. They occur in
about one-fifth of the livers containing the spherical inclusions, but
only in association with them. They may occupy the same vacuole
as the spherical bodies, and indeed one frequently gets the impres-
sionthatfilamentshaveformedonthespherical bodiesasanucleus.
But often the rods and filaments occur in vacuoles that contain
no spherical body. The only plausible explanation for their occur-
rence is that coagulable fluid from the plasma has seeped into the
* Weareindebted toDr. Smetanaforplacingthisferretmaterialatourdisposal.
CYTOPLASMIC INCLUSIONS OF LIVER CELLS 633
vacuoles from the adjoining sinusoids and has crystallized out
within the vacuoles. Why this does not occur in every case is not
easy to understand.
SUIBARY AN-D CONCLUSIONS
A study has beenmade of certain spherical, rod-likeand filament-
ous cytoplasmic inclusions that occur frequently in the liver cells
of man, monkeys, ferrets and guineapigs. Theyhave notbeen seen
intheliversofotherlaboratory animals. Whilenodecisionhasbeen
reached as to their nature and significance, various possible inter-
pretations are discussed.
REFERENCES
i. Mallory, F. B. Necroses of theliver. J. M. Research, igoi, 6, 264-279.
2. Taniguchi, A. Cytological studies of the liver cells. Tr. Jap. Palh. Soc.,
I93I, 21, 260.
DESCRIPTION OF PLATES
PLATE II7
FIG. i. Livercellscontainingsphericalandfilamentouscytoplasmicinclusions.
Mann's stain.
FIG. 2. Spherical inclusions. Laidlaw's stain.
FIG. 3. Spherical indusions surrounded by a homogeneous, slightly reddish
substance. Formalin fixation, phosphotungstic acid hematoxylin stain.
FIG. 4. Spherical inclusions surrounded bybluish staining material. Masson's
trichrome stain.