Original article

Blood clearance of HowellJolly bodies in an experimental

autogenic splenic implant model
R. G. Marques1 , S. B. S. G. Lucena2 , C. E. R. Caetano3 , V. Oliveira de Sousa3 , M. C. Portela4
and A. Petroianu5
Departments of 1 General Surgery and 2 Internal Medicine, and 3 Laboratory of Experimental Surgery, Rio de Janeiro State University, Rio de Janeiro,
4
National School of Public Health, Oswaldo Cruz Foundation, and 5 Minas Gerais Federal University, Belo Horizonte, Brazil
Correspondence to: Dr R. G. Marques, Department of General Surgery, Rio de Janeiro State University, Boulevard 28 de Setembro 77, Vila Isabel,
20.551-030, Rio de Janeiro RJ, Brazil (e-mail: rmarques@uerj.br)

Background: Autogenic splenic implant (ASI) is one of the few alternatives for preservation of splenic

tissue when total splenectomy is inevitable. The aim of this study was to determine the morphological
and functional regeneration of ASIs, as indicated by the clearance of HowellJolly (HJ) bodies, in an
experimental model.
Methods: Ninety-nine male Wistar rats were divided into three groups: sham-operated (group 1), total
splenectomy alone (group 2), and total splenectomy combined with ASI (group 3). Animals in group 3
were further allocated to nine subgroups of nine rats each, and analysed at different time points (1, 4, 8,
12, 16, 20, 24, 28 and 32 weeks after surgery). Blood smears were prepared at predetermined times for
detection of HJ bodies. Morphological regeneration of tissue in the ASI was analysed by histology.
Results: At 1 week, the regenerated mass corresponded to about 7 per cent of the tissue implanted,
reaching approximately 54 per cent at 24 weeks. The HJ body levels were increased in groups 2 and
3 until 8 weeks after surgery, following which levels in the ASI group became similar to those in the
sham-operated group. HJ bodies were difficult to detect when a level of 225 per cent of regenerated
ASI mass was reached.
Conclusion: Functional regeneration of ASIs occurred from 8 weeks after surgery. When 225 per
cent of regenerated ASI mass was reached almost no HJ bodies could be observed in the bloodstream,
resembling a spleen in situ.

Surgical relevance
Splenectomy has been practised routinely, both in the emergency setting and as a therapeutic elective procedure. There is
a correlation between asplenia/hyposplenia and the occurrence
of fulminant sepsis, underlining the importance of developing
surgical methods for preserving splenic function.
Both clinical and experimental studies have shown at least
partial morphological and functional regeneration of autogenic
splenic implants (ASIs). Experimental studies investigating the

immunoprotective effect of ASIs, based mostly on exposure of

animals to various bacteria, have demonstrated that ASIs can
increase the rate of bacterial clearance and decrease mortality
from sepsis. Clinical studies have shown their ability to remove
colloidal substances and altered erythrocyte corpuscular inclusions, such as HowellJolly, Heinz and Pappenheimer bodies,
from the bloodstream. In this experimental study the functional
and morphological regeneration of ASIs was studied over time
in rats.

Paper accepted 10 February 2014

Published online in Wiley Online Library (www.bjs.co.uk). DOI: 10.1002/bjs.9496

Introduction

Total splenectomy has been practised routinely, both in

the emergency setting, such as abdominal trauma, as well as
in therapeutic or diagnostic elective procedures for splenic
disorders caused by haematological, oncological, metabolic
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and immune disorders, and in portal hypertension1,2 . For

a long time it was believed that removal of the spleen did
not have any harmful consequences for the patient. It was
only from the mid-20th century, with the recognition of
overwhelming postsplenectomy infection, that the spleen
was recognized to have multiple functions, notably those
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R. G. Marques, S. B. S. G. Lucena, C. E. R. Caetano, V. Oliveira de Sousa, M. C. Portela and A. Petroianu

related to the immune system and blood ltering. With

increasing knowledge of these important splenic functions,
alternative treatments were developed aimed at preservation of the spleen after trauma and elective procedures.
The active role of the spleen in functions related to
immunity and phagocytosis is highlighted by the different
types of cell found temporarily or permanently in the
organ, by its anatomical and histological organization,
which favours opsonization, phagocytosis and destruction
of foreign antigens, among others, and by the abundance
of its blood supply3 6 .
Blood ltration is one of the main functions of the
spleen. The organ acts as a voluminous lter, through
which up to 6 per cent of the total blood volume passes
per minute (about 300 ml/min), removing senescent and
altered erythrocytes, and particles such as HowellJolly
(HJ), Heinz and Pappenheimer bodies, a procedure of
great importance for maintenance of the morphology and
function of red blood cells6 8 .
HJ bodies are basophilic DNA remnants of the
erythrocyte precursor nucleus. Typically, erythrocyte
precursors expel their nucleus, but some retain a small
portion of DNA, usually observed as small cytoplasmic
corpuscles (approximately 1 m in size). The ltering
function of normal spleen promotes the removal of cells
containing these corpuscles from the bloodstream, but
when the spleen is absent or is functioning inadequately
they remain in the circulation9 . The presence of HJ
bodies in peripheral blood smears, although not pathognomonic, is considered suggestive of splenic dysfunction
or aspleny10 12 . Although HJ bodies may not be present
in blood in patients with slight hypospleny, when they are
present they denote a degree of hypospleny representing
risk of onset of fulminant infection3,13 .
When a total splenectomy cannot be avoided, an
autogenic splenic implant (ASI) is among the only
alternatives for preservation of splenic tissue and function.
This procedure is based on clinicopathological ndings
observed in spontaneous splenic implants or splenosis,
after accidental or surgical severe splenic trauma2,5,13 15 ,
and has been further studied in an experimental setting.
Complete regeneration of autologous spleen slices
implanted in the greater omentum of 6-week-old rats
has been reported16 . After 16 weeks, the implanted splenic
tissue appears to be morphologically indistinguishable from
normal spleen16 18 . However, the functional regeneration
and blood ltering function can be partial or may not
occur, or take place over a different time interval. In
experimental studies it has been shown that HJ bodies
remain indenitely in the blood of animals submitted to
total splenectomy alone, but they disappear completely

over the course of weeks in animals with an ASI16 . Similar

ndings were described in an experimental rabbit model
after 12 weeks19 . At the same time, immunoglobulin M
concentrations increased to normal levels, suggestive of
functional regeneration of the implants19 .
Morphological and functional regeneration of ASIs,
assessed by the presence of the bacterial phagocytic function of macrophages in young and adult rats of both sexes,
was demonstrated by 16 weeks after implantation17,18 . In
addition, the critical mass of ASI needed for the recovery
of phagocytic activity in young adult rats was studied. If
26100 per cent of the total splenic mass was regenerated,
there was no difference in the bacterial index in the blood
of rats undergoing total splenectomy combined with ASI
compared with that in sham-operated animals20 .
The evidence that ASIs are effective for blood clearance
of HJ bodies is of major importance, as it provides an
indication of functional regeneration. The objective of
the present study was to determine the evolution of
morphological and functional regeneration of ASIs in
young rats over time, especially regarding blood clearance
of HJ bodies.

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Methods

The study was approved by the Ethics Committee

on Animal Research of the Biology Institute Roberto
Alcantara Gomes, Rio de Janeiro State University, Brazil
(protocol number 027/2008). All procedures rigorously
followed Animal Research: Reporting In Vivo Experiments
(ARRIVE) guidelines for animal experimentation21,22 .
Ninety-nine 21-day-old male Wistar rats weighing
4548 g were allocated to three groups: sham-operated
(group 1), total splenectomy alone (group 2), and total
splenectomy combined with ASI (group 3). Animals in
group 3 were subsequently allocated to nine subgroups of
nine rats each, corresponding to the time after surgery
at which they were analysed: 1, 4, 8, 12, 16, 20, 24, 28
and 32 weeks. The animals were housed in appropriate
cages, ve to a cage at most, under conditions of controlled
temperature and humidity, on a 12-h light/12-h dark cycle.
Free access to water and standard laboratory chow was
allowed.

Surgical procedures
After fasting for 12 h, the animals were anaesthetized
intramuscularly with 80 mg/kg ketamine and 12 mg/kg
xylazine. The entire procedure was performed under
aseptic conditions. In all animals, the procedure was started
with a supraumbilical midline incision, approximately 3 cm
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Blood clearance of Howell-Jolly bodies with splenic implant

Illustration of procedure

Autogenic spleen implant

Autogenic splenic implant. a Implantation of three segments of spleen into the greater omentum of a 21-day-old rat. Stitches
were introduced alternately into the omentum and the slices to permit interposition of omental tissue between the splenic slices.
b Illustration of the surgical procedure

Fig. 1

in length. In group 1 animals (sham-operated), the spleen

was just mobilized to the surgical eld, and subsequently
returned to its usual place. In groups 2 and 3, the splenic
vessels were ligated and the spleen was removed.
In group 3, after total splenectomy the spleen was
weighed and cut transversely into three segments, each
about 5 mm thick. The splenic slices were implanted into
the greater omentum using continuous 4/0 polyglycolic
acid sutures. Stitches were introduced alternately into the
omentum and the splenic slices to permit interposition of
omental tissue between the slices of spleen (Fig. 1). Finally,
the abdomen was closed with continuous polyglycolic acid
3/0 sutures in all animals.

Postoperative monitoring
Rats in groups 1 and 2 were followed daily for 32 weeks.
For those in group 3, the duration of monitoring depended
on the subgroup to which the animal was allocated. At
1, 4, 8, 12, 16, 20, 24, 28 or 32 weeks after surgery,
smears were prepared from peripheral blood obtained by
tail vein puncture under anaesthesia. After collection of
blood, the splenic implants were removed through a median
laparotomy for macroscopic and microscopic studies, and
the rats were then killed by overdose with ketamine and
xylazine.
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Staining of blood smears and counting of

HowellJolly bodies

Blood smears were stained by the MayGrunwaldGiemsa

method and examined under light microscopy. In each
smear, ten microscopic elds were used for counting
HJ bodies, with analysis of 100 erythrocytes in each,
giving a total of 1000 red blood cells per animal. Results
are reported as the percentage of erythrocytes with HJ
bodies.

Histology
Splenic implants were removed and placed in a solution
containing buffered 10 per cent formalin. The tissue was
processed with increasing concentrations of alcohol and
xylene, embedded in parafn, and cut into 4-m sections
on to slides. The slides were stained with haematoxylin and
eosin, and assessed by light microscopy.

Statistical analysis
The non-parametric KruskalWallis test was used to
compare weights of animals weekly during the experiment.
Means(s.d.) and median values were calculated for
implanted and regenerated splenic mass, as well as the
percentage of regenerated mass relative to implanted
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R. G. Marques, S. B. S. G. Lucena, C. E. R. Caetano, V. Oliveira de Sousa, M. C. Portela and A. Petroianu

Table 1 Analysis of implanted splenic mass, regenerated mass and percentage of regenerated mass in rats that received autogenic splenic
implants
Implanted mass (g)

Regenerated mass (g)

Regenerated mass/implanted mass (%)

Time after
implantation (weeks)

Mean(s.d.)

Median

Mean(s.d.)

Median

Mean(s.d.)

Median

1
4
8
12
16
20
24
28
32

9
9
9
9
9
9
9
9
9

021(002)
021(002)
021(002)
022(002)
021(002)
021(002)
021(002)
021(002)
021(002)

021
021
021
022
020
021
021
021
022

002(000)
003(000)*
005(000)*
007(001)*
009(001)*
010(000)*
011(002)*
012(002)
012(002)

002
003
005
007
009
010
012
011
012

709(095)
1199(123)*
2248(147)*
3397(210)*
4047(286)*
4505(300)*
5404(585)*
5412(602)
5428(700)

699
1207
2237
3388
4134
4500
5528
5356
5448

1000

< 0001

< 0001

*P < 0050 versus previous measurement (Wilcoxon test); KruskalWallis test.

mass, for the different ASI subgroups of group 3.

The KruskalWallis test was used to compare changes
throughout the course of the experiment. The Wilcoxon
test was used to determine whether the mean regenerated
splenic mass (absolute and relative) at each measurement
from week 4 onwards was signicantly higher than that of
the previous measurement.
Mean percentages of erythrocytes with HJ bodies in the
peripheral blood were compared at each time point during
the experiment using Students t test. The sham-operated
group was compared with the total splenectomy group, and
each of these groups was compared with the ASI subgroups.
The level of signicance was set at = 005. Analyses
were performed using SAS version 9.2 (SAS Institute,
Cary, North Carolina, USA).

Results

Regeneration of the ASI was observed in all animals in

group 3. At 8, 16 and 24 weeks after surgery, only two
splenic slices could be recovered from two animals in each
subgroup. These slices lacked interposed omental tissue,
leading to the impression that fusion had occurred between
two of the three slices originally implanted. No such fusion
was observed at other time points, and three splenic slices
were recovered from all remaining animals.
The masses of the ASI were similar at surgery. A significant growth of the ASI, expressed in both absolute terms
and relative to the implanted mass, was observed until the
24th week after surgery (Table 1). One week after implantation, the mass of regenerated splenic tissue corresponded to
about 7 per cent of the splenic tissue originally implanted,
reaching 54 per cent at 24 weeks. From week 24 onwards,
there was no further signicant increase in regenerated
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splenic mass (regenerated mass, P = 1000; regenerated

mass as a percentage of implanted mass, P = 0954).

Morphological analysis of regenerated splenic

implants
Macroscopically, the regenerated ASIs were smaller than
the original implants, and the splenic tissue was restricted
to the periphery at 1 week after surgery. The centre of
the implants became a large necrotic mass. From 4 weeks
onwards, larger masses of regenerated splenic tissue were
recovered, without any remaining signs of central necrotic
tissue (Fig. 2). From 12 weeks onwards, the recovered ASI
showed an anatomical conformation resembling that of the
original slices implanted and resembled a portion of normal
spleen in situ, especially when they were fused together.
On palpation, their consistency was elastic and similar to
that of normal splenic tissue.
Microscopically, at 1 week after implantation, lymphoid
aggregates with a follicular aspect were observed, with
a large amount of coagulation necrosis in the central
part of the implanted fragments. There was structural
disturbance, without clear demarcation of white and
red pulp. Blood vessels with numerous red blood cells
and other cell types dispersed in the interstitial spaces
were already evident in the periphery of the implant. At
4 weeks, the focal concentrations of lymphocytes around
blood vessels increased, with some already initiating the
formation of follicles without central arterioles, and with
sinusoids lled with red cells between them, resembling
the outline of regenerating red pulp. At 8 weeks, these
follicles were further developed, featuring regenerated
white pulp, with central arterioles and a red pulp of better
structure.
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Blood clearance of Howell-Jolly bodies with splenic implant

1 week

4 weeks

8 weeks

12 weeks

Fig. 2 Histological sections of autogenic splenic implants, showing a lymphocytic aggregate of follicular aspect (arrow) at 1 week;
b lymphocytic inltrate (arrow) at 4 weeks; c lymphoid follicle with central arteriole (arrow), and the outline of red and white pulp at
8 weeks; and d well dened red and white pulp (arrow and asterisk respectively) and a marginal zone (MZ) at 12 weeks (haematoxylin
and eosin stain, scale bar 50 m)

At 12 weeks, the focal concentrations of lymphocytes

around blood vessels increased, although they were slightly
smaller than those observed at 8 weeks. The red and white
pulp, as well as the marginal zones, were now more clearly
dened. Numerous macrophages containing cytoplasmic
haemosiderin pigments were observed in the splenic
parenchyma. Blood vessel walls were preserved without
signs of vasculitis or thrombosis. From 16 weeks onwards,
developed lymphoid follicles with precise delimitation
of the white and red pulp, and the marginal zone
were observed, with the implant resembling a neospleen
(Fig. 2).
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Blood clearance of HowellJolly bodies

The percentage of erythrocytes containing HJ bodies was
similar in the total splenectomy (group 2) and ASI (group
3) groups at weeks 1 and 4, with a high frequency of
HJ bodies compared with sham-operated animals. From
week 8 onwards, however, these two groups began to differ
signicantly, with the ASI group beginning to resemble
the sham-operated group (Fig. 3).
In the ASI group, analysis of the percentage of
erythrocytes with HJ bodies in relation to the percentage
of regenerated splenic mass showed that the HJ body level
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% of erythrocytes with HJ bodies

R. G. Marques, S. B. S. G. Lucena, C. E. R. Caetano, V. Oliveira de Sousa, M. C. Portela and A. Petroianu

040

035
030
025

Total splenectomy alone

020

Autogenic splenic implant

015

Sham-operated

010
005
0

12

16

20

24

28

32

Percentage of erythrocytes with HowellJolly (HJ) bodies in peripheral blood over time in the three experimental groups.
*P < 0010 versus sham-operated, P < 0010 versus autogenic splenic implant (Students t test)

Fig. 3

was close to zero when 225 per cent of the regenerated

mass (measured relative to the implanted mass) was
achieved.
Discussion

Besides its immune function, the predominant functional

activity of the spleen is blood ltration, removing senescent
and abnormal erythrocytes, as well as foreign particles.
Thus, it is of importance to demonstrate that an ASI is
effective in blood clearance of HJ bodies, as this is an
indication of the functional regeneration of the spleen.
Studies in humans with ASIs have shown restoration of
some splenic functions. These include the ability to remove
colloidal substances and altered erythrocyte corpuscular
inclusions, such as HJ, Heinz and Pappenheimer bodies,
from the bloodstream12,23,24 . ASIs also result in normalization of the production of antibodies against pneumococcal
polysaccharides, along with levels of immunoglobulins,
complement, platelets and lymphocytes14,23 25 . How this
splenic regeneration evolves over time is not well known.
Experimental studies16 18,20,26 30 investigating the
immunoprotective effect of ASIs have been based mostly
on exposing animals to various bacterial species, then
assessing the level of clearance of these microorganisms
from the bloodstream, and death from bacterial sepsis.
Blood ltering16 and the potential benets of immunization combined with ASI have also been investigated,
especially after exposure to pneumococci; this combination
increased the rate of bacterial clearance and decreased
mortality from sepsis14,28 30 . In general, the regenerative
capacity of an ASI varies according to animal species and
age16,31 35 . In young animals, ASIs seem to regenerate better, with well developed splenic compartments resembling
the structure of a normal spleen, and showing better
recovery of functional activity than in adults16,31,35 40 .
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Although analysing blood clearance of HJ bodies in rats

seems to be a suitable model for monitoring the functional
regeneration of an ASI, there is some controversy about
the reliability of its use as an indicator of splenic function,
especially in cases of low-grade hyposplenism, because the
number of cells with HJ bodies is very low (0111 per
cent of erythrocytes in splenectomized patients). However,
others9,10,31,32 have reported that blood clearance of HJ
bodies is a good marker of functional splenic regeneration.
A small percentage of erythrocytes with HJ bodies has
been found in the peripheral blood of healthy young rats
(21 days after weaning)41 . This is in line with ndings of
HJ bodies in neonates, especially in premature infants9,41 ,
suggestive of splenic dysfunction during the rst weeks
of life.
Previous experimental studies17,18 demonstrated morphological regeneration and phagocytic activity of
macrophages in ASIs implanted on the greater omentum. It was concluded that ASIs provide protection against
bacteraemia. Subsequently, a similar experimental model,
with thin splenic slices sutured to the greater omentum
and intercalated omental tissue, was used to allow better
vascularization. In this model the critical mass needed to
achieve efcacious phagocytic activity in macrophages in
adult rats occurred when 260 per cent of the total splenic
mass had regenerated20 .
As ASI regeneration starts in a centripetal way from
the periphery to the centre of the graft16,33,39 41 , the
present experimental model also had three thin splenic
slices sutured to the greater omentum with omental
tissue intercalated between them. Omental implants led to
more complete morphological and functional regeneration
than implants at other sites, showing the relevance
of maintaining venous drainage into the portal vein,
resembling the spleen in situ16,33,34 .
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Blood clearance of Howell-Jolly bodies with splenic implant

All implants showed morphological regeneration with

the presence of red and white pulp, lymphoid follicles and
a marginal zone from 12 weeks after surgery onwards. The
mass of implanted spleen was the same in each subgroup,
but the regenerated mass as a percentage of the implanted
mass increased signicantly with each experimental week.
Regeneration occurred in a centripetal direction and
gradually replaced the central necrotic zone, in agreement
with previous ndings33,41 . One week after completion
of the ASI, the regenerated mass was only 7 per cent
of the implanted mass, but signicant linear growth was
subsequently observed, reaching 40 per cent at 16 weeks.
By week 24, 54 per cent regeneration was attained, after
which no further signicant regeneration was found.
From week 8 of the experiment, HJ bodies in the
peripheral blood of animals with an ASI decreased
signicantly, to reach levels comparable to those in shamoperated animals; when 225 per cent of regenerated mass
was achieved, the proportion of erythrocytes with HJ
bodies tended to approach zero. At this time point, a more
structured morphological regeneration was also seen. This
indicates that, besides a requirement for a critical minimal
mass of splenic tissue to be implanted20 , a critical level of
regenerated splenic tissue may be needed for restoration of
splenic function.
Acknowledgements

The authors thank A. Viana de Oliveira for her support in

reviewing and analysing morphological data on regenerated splenic implants. This work was supported partially
by a grant from Fundaca o Carlos Chagas Filho de Amparo
a` Pesquisa do Estado do Rio de Janeiro (FAPERJ), Brazil.
Disclosure: The authors declare no conict of interest.
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R. G. Marques, S. B. S. G. Lucena, C. E. R. Caetano, V. Oliveira de Sousa, M. C. Portela and A. Petroianu

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