Acta medica-1-11

MEDICAL UNIVERSITY − SOFIA
This journal is indexed in Global Health Database,
in Bulgarian Medical Literature Database and
in Scopus
ACTA MEDICA
BULGARICA
ambvol. XXXVIII
1/2011
Central Medical Library

Editor in chief
Prof. V. Mitev, MD, Ph. D. DSc
Editorial board
Prof. K. Tsachev, MD, Ph. D., DSc
Prof. M. Marinov, MD, Ph. D., DSc
Prof. D. Ziya, MD, Ph. D., DSc
Prof. N. Lambov, Mag. Ph., Ph. D.
Prof. W. Bossnev, MD, Ph. D., DSc
L. Tacheva, MD

Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 1 3
OSTEOCLASTOMA OF METACARPAL BONES
H. Georgiev, B. Matev, N. Dimitrov and P. Georgiev
University Specialized Hospital for Active Treatment of Orthopaedics “Prof. B. Boychev” –
Sofia
Summary. Osteoclastoma is a locally aggressive tumor, very rarely located in
bones and joints (locomotory system). In 1987, Matev presented his 20-year study
of 502 tumors of the hand, where no osteoclastoma cases were observed. In our
study which tracks a period of 20 years, we found and treated 3 cases of osteo-
clastoma of the metacarpal bone and for the first time in Bulgaria we are presenting
them. We follow consecutively the clinical picture, the applied treatment and the
results. The conclusions show that it is an extremely rare aggressive tumor, com-
plicated to treat, so that early diagnosis and therapy is the key to a comprehensive
treatment, for organ and hand-function preservation.
Key words: osteoclastoma, giant cell tumor, hand
P
ain in the hand and the forearm is a frequent symptom of different dis-
eases: over-tension conditions, sport traumas, neurological diseases
etc. A chronic dull pain in young people localized in the hand area,
although rare, is also the first symptom for primary bone cancer disease. Our study
presents extremely rare cases of osteoclastoma of metacarpal bones, for the first
time described in Bulgaria.
Osteoclastoma is a local aggressive tumor, with strong inclination to recidi-
vate, to give “benign” lung metastases or to transform into a real malignant blasto-
ma. This is the reason for this tumor to be a specific clinical-X ray and pathological
anatomy unit, filed in a separate group of the bone cancer classification [12, 13].
This giant cell tumor received this name and was described in 1818 by Sir Astley
Cooper. In 1922, Steward introduced the British term osteoclastoma. Jaffe defined this
primary bone tumor as quasi-malignant, because approximately 20% of the lesions are
malignant, while 80% are benign. In 1940, the same author divided osteoclastoma in
three stages depending on the histological picture [14]. It is assumed that the single-
nucleus cells, the basic ones in the tumor, originate from the undifferentiated mesen-
chymal cells or the supporting connective tissue of the marrow [7, 11, 12, 13, 16, 18].

Osteoclastoma of metacarpal bones4
CLINICAL MATERIAL
Three patients with osteoclastoma of the metacarpal bones have been diag-
nosed and treated at “Prof. B. Boychev” University Ortopaedics Hospital – Sofia,
for over a twenty-year period. The patients were 34 years and 6 months old at the
average, women prevailing by 2:1.
The clinical picture of the three cases was identical: complaints of a dull and
constant pain localized in the arm. Presence of a swelling with hard consistence,
painful at palpation. In some of the cases, the complaints were accompanied by a
minimal reduction of the movements in the adjacent joint.
X-ray picture: Roentgenography of two surfaces showed metaepiphyseal os-
teolytic focus without periostal reaction. In one of the cases, the lesion was con-
fined to the bone, and the corticalis was strongly thinned and inflated. In the other
two cases, due to its aggressive character, the tumor had destroyed the cortex
and spread over the adjacent soft tissues and joint. In one of the cases, the tumor
had a characteristic X-ray cell-like look – “soap bubbles”. The lytic character was
expressed in the other two cases.
Pathological anatomy picture: The intraoperative finding was a tissue of dark-
red to brownish color with zones of yellow-grayish color resulting from occurred
necroses. The tumor had soft consistency, was fragile and friable. The histologi-
cal pictures showed presence of single-nucleus cells of round, oval and spindle
shapes, as well as giant multi-nucleus cells situated in loose intercellular stroma.
The treatment strategy in every case was surgically individualized. In one of
the cases, recidivating occurred.
Patient: K.T.K. – male, aged 41, with osteoclastoma of the fourth metacarpal
bone on the right, and affected proximal 2/3 of the bone. Histology – Ist degree after
Jaffe: Abundant giant multi-nucleus cells of osteoclastic type and sinlge-nucleus
cells – oval and spindle-shaped, with nidus accumulation of xanthoma cells. Sev-
eral consecutive stages of surgical treatment were applied: block resection of IV
th metacarpal bone with auto-osteoplastics and carpo-metacarpal arthrodesis. The
next stage included falangeal endoprosthesis with Matis bipolar joint. Fig 1.
A B C
A 3D – CT scan – primary diagnostic view; B Radiography after autoosteoplastics from fibula;
C Finally radiography – endoprosthesis of carpometacarpal joint
Fig. 1. Patient: K.T.K. – male, 41 age

Patient: D.O.S. – female, aged 25 with giant cellular tumor in the Vth meta-
carpal bone. After biopsy, the histological diagnosis was osteoclastoma, locally
aggressive of II-III degree: Soft tissues – skin with focused inflammatory changes
in the papillary layer of the derma and a blastoma – well restricted by the hypo-
derma, and hypercellular due to the prevailing single-nucleus cells with big round
vesicular nuclei and distinct small nuclei in part of them and presence of mitoses.
Giant multi-nucleus cells of osteoclastic type. Focal bone formation and infiltra-
tion in the skeleton muscles as well as in the adjacent spongioid bone. Due to the
aggressive character of the tumor, disarticulation of the whole fifth ray of the right
hand was performed. Two years later – no data for recidivating and “benign” lung
metastases. Fig 2.
A B C
A Diagnostic primary radiography; B One year later radiography;
C Postoperative radiography – Disarticulation of the V th ray
Fig. 2. Patient: D.O.S. – female, 25 age
Patient: Sht.A.D. – female, aged 38 with metacarpal osteoclastoma – Ist and
IInd degree after block resection of the bone. Reoperated after six months fol-
lowing recidivation. A larger block resection of the bone with aloosteoplastics was
performed. Two years later – no data for recidivation.
DISCUSSION
The giant cell tumor is a primary neoplasma representing 5-8% of all primary
malignant bone tumors and 15% of all primary benign bone tumors [19]. According
to Andreev, the osteoclastoma rates at 6, 6% of all primary new formations and
tumor-like diseases of the skeleton [1]. The most frequent localizations of affection
are bones around the knee joint, the distal end of the radius, the proximal humerus,
the proximal part of the femoral bone and so on in this order, with reducing fre-
quency. Localization of the tumor in hand bones is quite rare, about 2-3% [1, 8, 16,

20]. Still rarer is osteoclastoma isolated only in metacarpal bones, less than half
of all hand osteoclastomas [8]. In an extensive study by Matev and co., treating
502 patients with tumors of the hand, there is no case of osteoclastoma [3]. As a
consequence, we treat our three cases not as exotic casuistic, but as an important
differential diagnosis direction.
The age diapason of osteoclastoma is 20-40 years – the most active age.
Frequency in males and females is almost identical, benign osteoclastomas prevail
in females corresponding to 3:2, while malignant prevail in males at 3:1 [19]. All our
three cases are typical of the known statistics.
The clinical expression of the tumor localized in metacarpal bones, especially
at the beginning, is not typical – complaints of a dull, constant pain in the area
of the hand. Complaints are similar as in light chronic hand trauma, pressure of
n.medianus or n.ulnaris syndrome or mm. lumbricales et interosei insertionites.
Only later swelling of hard consistence, painful at palpation and restriction of move-
ments in the adjoining joint appear. Sometimes the tumor is discovered only after a
pathological fracture occurs.
X-ray is basic for the diagnosis. The tumor is visualized as an eccentrically
situated metaepiphyseal osteolytic focus. Periostal reaction is lacking. The lesion
can be reduced to the bone, the corticalis could be strongly thinned and inflated or
due to its aggressive character, the tumor can destroy the cortex and spread over
the adjoining soft tissues and joint [4, 21]. In about 60% of the cases, the lesions
have a purely lytic picture, while in 40% the picture is “soap bubbles” type as an
expression of the reactive bone trabeculi formed in the tumor [6, 9]. On this basis
the M. Campanacci et al. classification is built, which divides osteoclastomas into
latent, active and aggressive forms, as well as the adopted in Bulgaria Andreev
classification, describing the tumors as quite, cell-like and osteolytic types [4, 5].
The histological diagnosis confirms the X-ray and the intraoperative findings
and is basic for the adoption of the therapeutic plan.
Prognosis of the benign lesions is good, but only 10% of patients with the
malignant form of osteoclastoma are still alive after the 5th year. That is why timely
and adequate surgical treatment is of vital importance. Several methods for osteo-
clastoma treatment are proposed: curetage, curetage and plombage with cortical
bone or biomaterial, resection, resection with bone graft, amputation and disarticu-
lation [2, 8].
The curetage is easily performable and organ-preserving intervention, but
carries a big risk for recidivation – in some studies from 47 to 80% recidivated [9,
15]. Curetage as an independent procedure is recommended only for cases with
very small lesions and unaffected cortex of the respective bone. We have not ap-
plied this technique on our patients.
En block resection of the affected part of bone is the safest way to avoid
recidives, leading to cure. M. Campanacci et al. report only 13% recidivated after
resection [5, 6]. The necessity to further surgery interventions is taken into con-

sideration here, for replacing one of the joint poles which is also a joint surface,
with an autotransplant and performing arthrodesis or replacing the defection with
endoprosthesis. In two of our cases, we chose the surgical method of resection of
affected bone, the first – with autograft and arthrodesis to the wrist joint, followed by
metacarpal-falangeal endoprosthesis and the second recidivated, imposing a wider
resection and allotransplant.
Amputation or desarticulation of the entire ray of the hand is applied when
the malignant form of osteoclastoma tumor has been proven and has affected a
big part of the bone and the adjacent soft tissues [10]. Desarticulation of the entire
Vth ray was performed in one of the cases of our clinical material with histological
results for local aggressive osteoclastoma of II-III degree.
CONCLUSION
Osteoclastoma is an extremely rare tumor, which nevertheless provokes quite
difficult issues to solve. For the first time in Bulgaria we present three cases of
osteoclastoma of the metacarpal bone. Prognosis, treatment and its results are
directly dependent on early diagnosis and adequate therapy. We treat the pre-
sented cases of osteoclastoma with metacarpal localization as a contribution to the
differential diagnosis field of hand diseases. Our cases demonstrate the necessity
for early diagnosis thus giving the chance for organ-preserving surgery to keep the
functions of the hand.
REFERENCES
A n d r e e v, Iv. et R. Raychev. [Tumors of the bones.]. Sofia, Med. i fizk., 1993, 97-113. (in Bulgarian)1.
A n d r e e v, Iv. et R. Raychev. [Operative treatment of the giant cell tumor of the bones.] – Ortop.2.
Traum., 10, 1973, № 3-4, 208. (in Bulgarian)
M a t e v, Iv. [Tumors of the hand. – In: Diseases of the hand. Bossnev, V., Matev, I. (eds.)] Sofia,3.
Med. i fizk., 1989, 131-137. (in Bulgarian)
H a d j i d e k o v, G., Iv. Andreel et L. Velikov. [Rentgenological particularities of osteolytic variant of4.
osteoclastoma of the long tubular bones.]. – Onkologiya (Sofia), 7, 1970, № 3, 117. (in Bulgarian)
C a m p a n a c c i , M., A. Guini еt R. Olmi. Giant cell tumors of bone. A Study of 209 cases with5.
long follow-up in 1130. – Ital. J. Orthop. Traumatol., 1, 1975, 249.
C a m p a n a c c i , M. et al. Giant cell tumor of bone. – J. Bone Joint Surg.,6. 59A, 1987, 106-114.
F u j i w a r a , Y. Tissue culture study of the giant-cell tumor of bone. – Arch. Jap. Chir.,7. 36, 1967, 803.
G l i c e n s t e i n , J., J. Ohana et C. Leclercq. Tumors of the Hand. Berlin, Heidelberg, Springer-8.
Verlag, 1988, 189-191.
G o l d e n b e r g e r, R. R., C. J. Campbell et M. Bonfiglio. Giant cell tumor of bone. An analysis of9.
218 Cases. – J. Bone Joint Surg., 52A, 1970, 619-663.
G u p t a , S., A. Kumar et I. Gupta. Giant cell tumor of the first metacarpal bone. – Hand,10. 12, 1980,
288-292.
H u t t e r, R. V. P. et al. Benign and malignant giant cell tumors of bone. A Clinical-pathological11.
analysis of the natural history of the disease. – Cancer, 15, 1962, 65.

J a c o b s , Th. et al. Giant cell tumor in Paget’s disease of bone. – Cancer,12. 20, 1979, 742.
J a f f e , H. L. Tumors and tumorous conditions of bones and joints. Philadelphia, Lea & Febiger,13.
1958.
J a f f e , H. L., L. Lichtenstein et B. Portis. Giant cell tumor of bone. Its pathologic appearance,14.
grading, supposed variants and treatment. – Arch. Pathol., 30, 1940, 993.
R o c k , M.G. et al. Secondary malignant giant cell tumor of bone: Clinical pathological assess-15.
ment of 19 patients. – J. Bone Joint Surg., 68A, 1986 1073-1079.
S a i k i a , K. C. et al. A. Rare site giant cell tumors: report of two cases on phalanges of the finger16.
and review of literature. – J. Orthop. Traumatol., 10, 2009, № 4, 193-197.
S c h a i o w i c z , F. Giant cell tumors of bone (Osteoclastoma). A pathological and histochemical17.
study. – J. Bone Joint Surg., 43-A, 1961, 1.
S p j u t , H. J. et al. Tumors of Bone and Cartilage.18. – In: Tumor Pathology. Washington, D. C.,
Armed Forces Inst. Pathology, 1971.
Ya s u d a , T. et al. Multicentric diffuse-type giant cell tumor of the hand. – Mod. Rheumatol.,19. 18,
2008, № 1, 67-71.
Y i p , K. M., P. C. Leung et S. M. Kumta. Giant cell tumor of bone. – Clin. Orthop. Relat. Res.,20.
323, 1996, 60-64.
Yo c h u m , T. R. et L. J. Rowe. Essentials of skeletal radiology. Ed.2. Vol. 2. Baltimore, Williams21.
& Wilkins, 1996, 1054-1059.
Addresses for correspondence:
Assoc.prof. Dr. Hristo Georgiev, PhD
Clinic of Children’s Orthopaedics
USHATO “Prof. B. Boychev” – Sofia
Sofia, 56 N. Petkov Blvd.
00359887621513
е-mail: titigeorgiev@yahoo.com

HEMOSTATIC MONITORING OF THE PERIOPERATIVE
FIBRINOLYTIC ACTIVITY DURING LIVER
TRANSPLANTATION
A. Stancheva1
, L. Spassov1
and K. Tzatchev2
1
University Hospital “Lozenets”, Sofia, Bulgaria
2
Medical University, Sofia, Bulgaria
Summary. Rotation thrombelasthometric analysis (ROTEM®
) is a global he-
mostatic method, initiating the coagulation process both by contact surface activa-
tion and by additional activating reagents. The graphical and numerical ROTEM®
results reflect the whole clot formation process and the next-coming fibrinolysis, as
well as a great number of hemostatic and therapeutic factors, changing the kinetics
of the hemostatic process.The performance of an extended perioperative fibrin-
olytic and therapeutic control during liver transplantation becomes possible with
the development of the rotation thrombelastometry method ROTEM®
. Perioperative
hemostatic monitoring was performed to 30 patients undergoing orthotopic liver
transplantation (13 male – 42% and 17 female – 58%), age (mean ± SD): 21±17
years. A STA Compact instrument (Diagnostica Stago-Roche, France, chronometric
method) was used to determine prothrombin time (PT/INR), activated partial throm-
boplastin time (APTT) and fibrinogen (FIB). Rotation Thrombelastometry analyses
were performed on ROTEM®
analyzer (Pentapharm GmbH, Munich, Germany) in
citrated blood, based on reagent activation of the hemostatic processes with throm-
boplastin tissue factor (TF) for EXTEM test and aprotinin for APTEM test. The cor-
relation between MCF and A15 parameters was assessed to explore the possibility
for application of A15 during the monitoring. The correlation between MCF and A15
parameters was excellent for EXTEM, INTEM and FIBTEM tests (r=0,98 p<0,001).
The highest percentages of patients from the general group with increased fibrin-
olytic activity (33,33%) and with hyperfibrinolytic activity (8,7%) were determined
during the anhepatic period R4. 16 % from the group with heavy coagulopathy
displayed hyperfibrinolytic activity in the anhepatic period R4, while no patient from
the moderate coagulopathy group developed hyperfibrinolysis during all periop-
erative periods R1-R6. During the anhepatic period (R4), the percentage of adults
with increased fibrinolytic activity was significantly higher than the percentage of
children (adults 41,66% and children 25,00%). Conclusions are that EXTEM and

Hemostatic monitoring of the perioperative...10
APTEM parameters could be used for determing the increased fibrinolytic and hy-
perfibrinolytic activity during the preanhepatic, anhepatic and reperfusion periods
(R3, R4, R5) of liver transplantation. Patients with considered preoperative heavy
coagulopathy are more risky to develop hyperfibrinolysis during the preanhepatic
and anhepatic periods ( R3-R4). The established excellent correlations between
MCF and A15 parameters suggest the application of A15 as satisfactory for the
monitoring of fibrinolytic activity.
Key words: rotation thrombelastometry ROTEM®, fibrinolysis, liver transplantation
INTRODUCTION
T
he perioperative hemostatic monitoring during liver transplantation in-
cludes performing clinical-laboratory diagnostics of the arising coagul-
opathies [4, 3], suggesting adequate medicamentous and substitutive
therapy [6] and following up the effect of the applied therapy [14].
The possibilities of standard coagulation and fibrinolytic parameters for diag-
nostics of the perioperative states of hyperfibrinolysis or increased coagulability,
are estimated as not sufficiently quick or informative, with no prognostic value for
hemostatic bleeding and necessity for hemotransfusion, when concerning the dy-
namic coagulation changes during liver transplantation.
The performance of an extended perioperative fibrinolytic and therapeutic con-
trol during liver transplantation becomes possible with the development of the ro-
tation thrombelastometry method ROTEM®
(3). The ROTEM®
EXTEG and APTEM
activated tests are used for the diagnostics of fibrinolitytic activity. Increased values
of CT and CFT parameters of EXTEM , with normal values of APTEM indicate in-
creased or hyperfibrinolytic activity and are treated with antifibrinolytics [1, 8, 9, 17].
The aim of the study was to assess the fibrinolytic and hyperfibrinolytic activity
during the perioperative periods (R1-R6) of orthotopic liver transplantation by rota-
tion thrombelastometric method ROTEM®
.
MATERIALS AND METHODS
Materials
Perioperative hemostatic monitoring was performed to 30 patients undergoing
orthotopic liver transplantation (13 male – 42% and 17 female – 58%), age (mean
± SD): 21±17 years.
The patients were classified in the following groups:
General group (n=30)1.
According to age – up to 10 years (n=15); up to 48 years (n=15)2.
According to the type of the preoperative coagulopathy – heavy coagul-3.
opathy (n=16); moderate coagulopathy (n=14)

(With heavy coagulopathy were considered patients with PT(INR) > 1,6; APTT
> 1,5 ratio, PLT< 100 х 109/L, FIB < 1,0 g/l), one hour preoperatively [18].
Blood samples were collected according to a study design protocol:
One hour preoperatively :•
the perioperative period (R1)
Four times intraoperatively:•
10 min. after the intervention – the dissection period (R2)
10 min. before clamping – the preanhepatic period (R3)
10 min. before declamping – the anhepatic period (R4)
30 min. after grafts’ reperfusion – the reperfusion period (R5)
Two hours postoperatively:•
the postoperative period (R6)
The correlation between MCF and A15 parameters was explored for EXTEM,
INTEM and FIBTEM tests in six groups: a reference group (n=20), the general
group for liver transplantation (n=30), two groups according to age (n=15) and two
groups – with heavy or moderate preoperative coagulopathy (n=15).
A STA Compact instrument (Diagnostica Stago-Roche, France, chronometric
method) was used to determine prothrombin time (PT/INR) (STA Neoplastin®
plus, Di-
agnostica Stago, France) and activated partial thromboplastine time (APTT) (STAaPTT
Diagnostica Stago, France, chronometric method). Levels of fibrinogen (FIB) were as-
sayed according to Clauss technique (STA Fibrinogen, Diagnostica Stago, France).
Platelets (PLT) and red blood cells (RBC) were determined in EDTA plasma
on Cell Dyn 3700 (Abbott Diagnostics, Germany, MAPSS technology).
Rotation Thrombelastometry analyses were performed on ROTEM®
analyzer
(Pentapharm GmbH, Munich, Germany) in citrated blood.
The fibrinolytic activity was assessed by ROTEM®
method – a global hemo-
static method for fast diagnostics of hemostatic disorders with acute bleeding,
leading to hypercoagulation, hypocoagulation or hyperfibrinolysis [3]. Like classi-
cal thrombelastography, ROTEM®
analysis is expressed graphically (by a reaction
curve – TEM-ograme) and numerically (by thrombelstometric parameters).
The new ROTEM®
methodology is based on reagent activation of the global
haemostatilc processes in a whole blood assay, using supplements like thromboplas-
tin tissue factor (TF) for test EXTEM, contact activator (ellagic acid) for test INTEM,
anti-platelet reagents (anti IIb/IIIa, cytochalasin D) for test FIBTEM, aprotinin for test
APTEM, heparinase for test HEPTEM and ecarine activator for test ECATEM.
STATISTICAL ANALYSIS
Statistical analysis was performed using SPSS 17.0.1. software. To determine
the perioperative fibrinolytic activity, the International Guidelines of the Expert Group
on Rotation Thrombelastometry 2005-2009, Munich, Germany were followed.

Thrombelastometric indications for increased ﬁbrinolytic activity were deter-
mined in cases of:
APTEM_CT<EXTEM_CT ≥ ∆ 10%,
APTEM_MCF >EXTEM_MCF ≥ ∆ 10% ≥ ∆ 10%
(p<0,05)
Thrombelastometric indications for hyperﬁbrinolytic activity were determined
in cases of:
АРТЕМ_СТ / ЕХТЕМ_CT < 0.75
АРТЕМ_MCF/ ЕХТЕМ_MCF > 1,25
or
АРТЕМ_CT < ЕХТЕМ_CT ≥ Δ 25%
АРТЕМ_MCF > ЕХТЕМ_MCF ≥ Δ 20%
>20%
( MCF_ EXTEM < 35 mm; p < 0,05)
RESULTS
The correlations established between MCF and A15 parameters were excel-
lent and very good for EXTEM, INTEM and FIBTEM tests in the reference group
and in all groups of liver transplanted patients. (Tables 1, 2, 3).

Table 1. Correlation (r) between INTEM_MCF and INTEM_ A15 in different groups
Parameter Group
INTEM_ A15
Reference
group
General
patients’
group
Moderate
coagulopathy
group
Heavy
coagulopathy
group
Children Adults
INTEM_MCF
Reference
group
0,937***
General
patients’ group
0,980***
Moderate
coagulopathy
group
0,913***
Heavy
coagulopathy
group
0,986***
Children 0,947***
Adults 0,991***
*** – p < 0.001
Table 2. Correlation (r) between EXTEM_MCF and EXTEM_A15 in different groups
Parameter Group
EXTEM_ A15
Reference
group
General
patients’
group
Moderate
coagulopathy
group
Heavy
coagulopathy
group
Children Adults
ЕХTEM_MCF
Reference
group
0,878***
General
patients’ group
0,981***
Moderate
coagulopathy
group
0,955***
Heavy
coagulopathy
group
0,993***
Children 0,985***
Adults 0,988***
***– p < 0.001

Table 3. Correlation (r) between FIBTEM_MCF and FIBTEM_A15 in different groups
Parameter Group
FIBTEM_ A15
Reference
group
General
patients’
group
Moderate
coagulopathy
group
Heavy
coagulopathy
group
Children Adults
FIBTEM_MCF
Reference
group
0,830***
General
patients’ group
0,892***
Moderate
coagulopathy
group
0,819***
Heavy
coagulopathy
group
0,985***
Children 0,844***
Adults 0,901***
***– p < 0.001
The highest percentage of patients with increased fibrinolytic activity from the
general group (33,33%) was determined in the anhepatic period (R4), with calcu-
lated ROTEM parameters APTEM_CT < EXTEM_CT > Δ 10% and APTEM_A10
>EXTEM_A10 > Δ 10%.
During the anhepatic period (R4), in the group with heavy coagulopathy a sig-
nificantly higher percentage of patients with increased fibrinolytic activity (46,15%)
was determined, than in the group with moderate coagulopathy (18,8%). This per-
centage decreased in the course of the operation for both groups – (30,7% and
28% (R5); 15,38% and 11,8% (R6) - and the difference was insignificant (fig. 1).
Fig. 1. Patients from the corresponding groups with activated fibrinolysis during the perioperative peri-
ods R1-R6 (APTEM_CT < EXTEM_CT > Δ 10% and APTEM_A15 >EXTEM_A15 > Δ 10%)

The childrens’ group didn’t present increased fibrinolytic activity in the first two
periods (R1, R2). The percentage of the adults with increased fibrinolytic activity
was significantly higher than the percentage of the children in the preanhepatic
(R3) and anhepatic period (R4). (adults 41,66% and children 25,00%). The dif-
ference between the two groups was insignificant during next periods R5 (adults
33,33% and children 25,00%) and R6 (fig. 2).
Fig. 2. Patients from the corresponding groups with activated fibrinolysis during the perioperative peri-
ods R1-R6 (APTEM_CT < EXTEM_CT > Δ 10% and APTEM_A15 >EXTEM_A15 > Δ 10%)
The highest percentage of patients with established hyperfibrinolytic activ-
ity from the general group (8,7%) was also determined in the anhepatic period
(R4), and decreased to 4,35% during the next R5, R6 periods, with caculated СТ_
АРТЕМ < СТ_ЕХТЕМ > Δ 25% and АРТЕМ_СТ/_ЕХТЕМ CT < 0,75; АРТЕМ_
MCF/ЕХТЕМ_MCF > 1,25. 16 % of the patients from the group with heavy co-
agulopathy displayed hyperfibrinolytic activity in the anhepatic period R4, while no
patient from the moderate coagulopathy group developed hyperfibrinolysis during
the perioperative periods (fig. 3).
Fig. 3. Patients from the corresponding groups with activated hyperfibrinolysis during the perioperative
periods R1-R6 ( СТ_ АРТЕМ < СТ_ЕХТЕМ >Δ 25%) and АРТЕМ_ СТ/_ЕХТЕМ CT<0,75),
(АРТЕМ_ A15/ЕХТЕМ_ A15 > 1,25)

In the childrens’ group, the percentage of patients with hyperfibrinolytic activity
(12%) was less than the percentage in the adults’ group (13,8%) (fig. 4).
Fig. 4. Patients from the corresponding groups with activated hyperfibrinolysis during the perioperative
periods R1-R6 ( СТ_ АРТЕМ < СТ_ЕХТЕМ > Δ 25%) and АРТЕМ_ СТ/_ЕХТЕМ CT<0,75),
(АРТЕМ_ A15/ЕХТЕМ_ A15 > 1,25)
DISCUSSION
The assessment of the ROTEM®
amplitude on the 15th min. (INTEM_A15,
ЕXTEM_A15 or FIBTEM_А15) is performed in a fixed and shorter interval when
compared to MCF. The established excellent correlations between MCF and A15
parameters in our study suggest the application ofA15 as satisfactory for monitoring
the fibrinolitical activity. This is extremely important in cases of urgency with acute
bleeding, when the diagnosis is established on the 15th min. before the themogram
has reached the maximum clot firmness (MCF). Rugeri et al have determined
similar correlations in patients with severe trauma [18].
In our study, the fibrinolytic activity under the urgency conditions of liver
transplantation was determined through calculating rotation thrombelastometric
parameters of APTEM and EXTEM tests, which was in concordance with the
investigations of Goerlinger et al. and the Guidelines of the Expert Group [7].
The highest percentage of patients with increased fibrinolytic activity in all
groups was detected during the anhepatic period (R4), perhaps due to the reduced
biosynthesis of fibrinolytic inhibitors and the absence of the purge out function of
the reticuloendotelium system of the liver in this period. The determined in our study
most frequent manifestation of activated fibrinolysis during the anhepatic period
confirms the observations of several contemporary authors [2, 5, 12, 15, 16].
The displayed significant difference between the groups with heavy and
with moderate coagulopathy in developing hyperfibrinolysis, could be possibly

due to the differences in the hemostatic preoperative status. The heavy
preoperative coagulopathy could be regarded as a possible reason for a next-
coming manifestation of intraoperative hyperfibrinolysis. The established by us
paraoperatively activated hyperfibrinolysis, expressed mostly during the anhepatic
period, confirms the investigations of J. H. Levis [11], Y. G. Kang [10], D. J.
Loskutoff [13] who explained the established hyperfibrinolysis with the increased
production of tPA from the endothelium cells and lack of clearance in the same
period (R4). Less children developed increased fibrinolytic activity in the early
R1-R3 transplantation periods, when compared to the adults’ group. A probable
reason for this could be the better preoperative general hemostatic status in this
group. In childrens’ group, hyperfibrinolysis was unexpectedly established only
during the preanhepatic period (R3).
CONCLUSSION
According to the ROTEM®
guided hemostatic monitoring, patients with
preoperative heavy coagulopathy are more risky to develop hyperfibrinolysis
during the preanhepatic and anhepatic periods (R3-R4). This could determine the
application of antifibrinolytic premedication as prophylactics for patients with heavy
preoperative coagulopathies.
The established excellent correlation between MCF and A15 parameters
in our study suggests the application of A15 as satisfactory for the monitoring of
fibrinolytical activity.
For determing the increased fibrinolytic activity the following relations between
EXTEM and APTEM parameters could be used: APTEM_CT < EXTEM_CT > Δ 10%
andAPTEM_A15 > EXTEM_A15 >Δ10%. Hyperfibrinolysis could be defined through
the relations: СТ_АРТЕМ < СТ_ЕХТЕМ > Δ 25%; АРТЕМ_A15 > ЕХТЕМ_A15 ≥ Δ
20% and АРТЕМ_СТ/_ЕХТЕМ CT < 0,75; АРТЕМ_A15/ЕХТЕМ_A15 > 1,25.
REFERENCES
Av i d i a n , M. et al. The effect of aprotinin on thrombelastography with three different activators. –1 .
Anesthesiology, 95, 2001, 1169-1174.
B e c h s t e i n , W. O. et P. Neuhaus. Blutungsproblematik in der Leberchirurgie und Lebertrans-2 .
plantation. – Chirurg, 71, 2000, 363-368.
C a l a t z i s , A. N. et al. Fast and specific coagulation monitoring through modified thrombelastog-3 .
raphy. – Ann. Hematol., 1996, Suppl 1., P 92.
C a l a t z i s , A. N. et al. Thrombelastographic coagulation monitoring during cardiovascular sur-4 .
gery with the ROTEG coagulation analyzer. – Cardiovasc. Surg., 119, 2000, 215-226.
F i n d l y, J. Y. et S. Retteke. Aprotinin reduces blood cell transfusion in orthotopic liver transplanta-5 .
tions. – Liver Transpl., 7, 2001, 808-810.
G o e r l i n g e r, K. et al. ROTEM-based management for diagnosis and treatment of acute haemor-6 .
rhage during liver transplantation. – Eur. J. Anaesth., 23, 2006, 85.
G o e r l i n g e r, K. ROTEM- Erweitertes perioperatives Gerinnungsmanagement. – JAI,7 . 12, 2005,
53-58.

G r o s s e , H. et L. van Obbereh. Platelet aggregates in small blood vessels and death during liver8 .
transplantation. – Lancet, 338, 1991, 532-534.
H i m m e r l e i c h , G. et al. Different approtinin application influencing hemostatic changes in9 .
orthotopic liver transplantation. – Transplantation, 53, 1992, 132-136.
K a n g , Y. G. et al. Intraoperative changes in blood coagulation and thrombelastographic monitoring1 0 .
in liver transplantation. – Anesth. Analg., 64, 1985, 888-896.
L e w i s , J. H. et al. Liver transplantation: intraoperative changes in coagulation factors in 100 first11 .
transplants. – Hepatology, 9, 1989, 710-714.
L l a m a s , P. et R. Cabrera. Haemostasis and blood requirements in orthotopic liver transplantation.1 2 .
– Haemostageologica, 83, 1998, 38-46.
L o s k u t o ff , D. J. et T. E. Edgington. Synthesis of a fibrinolytic activator and inhibitor by endothelial1 3 .
cells. – Proc. Natl. Acad. Sci. USA, 74, 1977, 3903-3907.
M a h l a , E. et al. Thrombelastography for monitoring prolonged hypercoagulability after major1 4 .
abdominal surgery. – Anesth. Analg., 92, 2001, 563-564.
P i e t s c h , U. C. et L. Schaffrainietz. Anaesthesiologisches Vorgehen bei OTX. – Ergebnisse einer1 5 .
Umfrage. – AINS, 41, 2006, 21-26.
P o r t e , R. J. et I. Q. Molenaar. Aprotinin and transfusion requirements in OLT. EMSALT Study1 6 .
Group. Lancet, 355, 2000, 1289-90.
R a m s a y, M., H. Randall et E. Burton. Intravascular thrombosis and thromboembolism during1 7 .
liver transplantation: Antifibrinolitic therapy indicated. – Liver Transplant., 10, 2004, 310-314.
R u g e r y, L. et al. Diagnosis of early coagulation abnormalities in trauma patients by rotation1 8 .
thrombelastography. – J. Thromb. Haemost., 5, 2007, 289-295.
Address for correspondence:
Assia Stancheva, MD
Universtity Hospital "Lozenets"
1 Koziak str.
1407 Sofia
e-mail: assiastancheva@yahoo.com

UNCOMMON CLINICAL PRESENTATION
OF FIBROSARCOMA OF THE THYROID GLAND
IN A PATIENT WITH FATAL OUTCOME: THE ROLE
OF IMMUNOHISTOCHEMISTRY FOR CONFIRMATION
OF THE DIAGNOSIS
J. Ananiev1
, M. Gulubova2
, I. Manolova3
, G. Tchernev4
, V. Ramdan5
, V. Velev6
and J. Gerenova7
1,6
Department of General and Clinical Pathology, Medical Faculty – Trakia University,
Stara Zagora
3
Laboratory of Clinical Immunology, University Hospital, Stara Zagora
4
Department of Dermatology and Venereology, Medical Faculty, Trakia University,
Stara Zagora
5
Department of Anesthesiology and Intensive Care Medicine, University Hospital,
Stara Zagora
7
Department of Internal Medicine, Clinic of Endocrinology, University Hospital, Stara Zagora
Summary. We report a case of a rare thyroid gland tumor – fibrosarcoma,
and autopsy findings. A 79-year-old woman presented with a twenty-year history
of a thyroid gland enlargement. Since twenty days she had respiratory failure and
dull pain in the anterior neck region. Preliminary cytological diagnosis confirmed
undifferentiated thyroid gland tumor. The patient died four days after hospitalization
with symptoms of complete respiratory failure, hypoxemia and suspected inflam-
mation of the lungs. Autopsy revealed primary neoplasm оf the thyroid gland area,
infiltrated trachea and adjacent tissues; acute tracheitis and lobular pneumonia.
Histopathologically, the primary tumor showed proliferation of spindle-shaped tu-
mor cells and was ultimately confirmed by immunohistochemistry as a primary fib-
rosarcoma of the thyroid gland.
Key words: fibrosarcoma, thyroid gland, immunohistochemistry, vimentin
INTRODUCTION
F
ibrosarcomas are relatively uncommon tumors and account for 12-19%
of soft tissue sarcomas. More than half of all tumors arise in the lower
extremities; approximately 10% occur in the head and neck, most com-

Uncommon clinical presentation...20
monly in the sinonasal tract and neck. Moreover fibrosarcoma is exceptionally rare
neoplasm of thyroid gland [1]. Depending on differentiation, tumor cells may re-
semble mature spindle-shaped fibroblasts, secreting collagen, with rare mitoses.
The prognosis as fibrosarcoma greatly depends on the extent of the disease, the
size and location of the tumor, presence or absence of metastases and the tumor’s
response to therapy.
The data of this type of tumor in literature are scanty and we did not find any
thorough publications about thyroid fibrosarcoma in our country.
In this article, we consider the existence of true fibrosarcoma originating in the
thyroid gland and confirm that fibrosarcomas can arise de novo in the thyroid gland.
CASE REPORT
A 79-year-old woman presented with a swelling in her anterior neck. Her med-
ical history was unremarkable. On physical examination, a non well-circumscribed,
elastic firm soft tissue mass was palpated. The mass was attached to the deep tis-
sues and was not mobile. Its maximum width was about 66 mm. The skin over the
swelling was slightly reddish and showed no signs of either ulceration or necrosis.
Laboratory tests revealed no abnormalities.
From fine needle aspiration cytology (before exitus letalis) we suspected cells
like of an anaplastic – undifferentiated carcinoma or fibrosarcoma of the thyroid
(Fig. 1). The neoplastic cells were randomly distributed and dyshesive. Occasional
small aggregates occured. The majority of cells in this low-grade tumor are spindle-
shaped, with occasional tadpole, stellate, and polygonal forms. Nuclear pleomor-
phism and atypia is obviously.
Fig. 1. Fine needle aspiration cytology – the majority of cells are spindle-shaped, with occasional
tadpole, stellate, and polygonal forms. Nuclear pleomorphism and atypia are obviously
(Magnification x 400)

Against the background of this neoplasm process, the patient developed hy-
postatic pneumonia with fever and enhancement of WBC which increased to 12.6 x
103
ml. The clinicians were unable to make a definite diagnosis of the type of tumor,
and the patient died four days after admission due to respiratory failure.
Autopsy was performed and after fixation of the necropsy specimens in 10%
formaldehyde solution, tissue samples were taken from different areas and em-
bedded in paraffin. Multiple 3-μm sections were obtained by means of a standard
microtome and then stained with haematoxylin and eosin, and by Von Gieson’s
mixture.
Immunohistochemistry was performed on de-parafinized by polimer-peroxi-
dase method using commercially available monoclonal antibodies against Cytok-
eratin (IR053, DAKO, ready-to-use), desmin (IR606, DAKO, ready-to-use) and Vi-
mentin (IR630, DAKO, ready-to-use). Paraffin sections 5 μm thick were dewaxed
in two xyllenes at 56° C for 1 h, and were rehydrated in ethanol. Later, they were
washed in 0.1 M phosphate buffered saline (PBS), pH 7.4, incubated in 1.2% hy-
drogen peroxide in methanol for 30 min, and rinsed in 0.1 M PBS, pH 7.4, for 15
min. The detection system immunostaining kit used was DAKO LSAB®
2 System,
HRP (K0675, DAKO) and DAKO®
DAB Chromogen tablets (S3000, DAKO).
Autopsy, histology and immunohistochemistry findings
The tumor was whitish and with a size of – 58×36×29 mm. The adjacent struc-
tures – trachea, soft tissues, and the skin, were involved (Fig. 2). The tumor cut
surface had a firm, solid, grey-white to yellow-brown appearance and checkered with
areas of necrosis and little or occult hemorrhagies. We didn’t found evident capsule.
Fig. 2. Solitary fibrous tumor of the thyroid gland with infiltrative growth pattern

The other significant finding was in the lungs. Both lungs were large, thick
with weighty tumescence. The cut surface demonstrated the typical appearance
of a bronchopneumonia with areas of tan-yellow consolidation. These areas con-
fluented in the left basal lobe and on the middle and basal right lobe. The areas of
consolidation were firmer than the surrounding lung.
Histological examination of the tumor revealed a malignant mesenchymal tu-
mor, and primary fibrosarcoma of the thyroid gland was ultimately confirmed. We
found that the tumor was composed of malignant spindle cells and rare pleomorphic
cells that vary markedly in size and shape (Fig. 3). It was difficult to determine them
because of their tendency to be poorly differentiated or anaplastic. Mitotic figures and
foci of necrosis were observed. The peripheral thyroid parenchyma showed atrophic
changes due to the tumor pressure and invasion. The background of fibrosarcoma
with abundant collagen fibers was stained in red by Van Gieson stain (Fig. 4).
Fig. 3. Fibrosarcoma of thyroid gland – atrophic changes due to the pressure of fibrosarcoma.
HE stain (Magnification x 200)
Fig. 4. The background of fibrosarcoma with abundant collagen fibers stain red. Van Gieson stain
(Magnification x 100)

Immunohistochemical analysis showed the tumor cells to be strong posi-
tive for vimentin, but negative for other markers – desmin and cytokeratin (Fig.
5a-c).
From histology of lung specimens we confirmed bronchopneumonia. The al-
veoli were filled with a neutrophilic exudate that corresponded to the areas of con-
solidation seen grossly with the bronchopneumonia. We found also diffuse inflam-
matory process in trachea responding on acute tracheitis.
A B
C
Fig. 5. Fibrosarcoma. Spindle cells exhibit: a) positive reaction for vimentin; b) negative reaction for
cytokeratin; c) negative reaction for desmin. (Magnification x 200)
DISCUSSION
Fibrosarcoma of thyroid gland is uncommon neoplasm observed in middle
aged adults, with a slight predominance of females [2, 3]. Solitary fibrous tu-
mors of this organ are characteristically immunoreactive for vimentin and some
other markers. However, negative reactions were observed for cytokeratin and
desmin [4]. It is important that clinical symptoms and radiological signs are not
characteristic.

The histological examination of the tumor of our patient revealed medium cel-
lular density with a slight polymorphism, abundant intervening collagen fibers, 4 mi-
toses per 10 high power fields and foci of necrosis. These features allowed to clas-
sify this tumor as moderate differentiated fibrosarcoma. In differential diagnostic
plan we discussed some other lesions such as fibromatosis, malignant spindle cell
carcinoma, malignant fibrous histiocytoma and anaplastic carcinoma. Fibromatosis
has more collagen fibers, is less cellular, with less mitoses and shows no necrosis
or nuclear overlap or hyperchromasia. Negative immunohistochemical reaction for
cytokertain and positive for vimentin of neoplastic cells allowed to rule out spindle
cell carcinoma, malignant fibrous histiocytoma, but not convincingly – anaplastic
carcinoma.
The clinical data about thyroid fibrosarcoma reported in literature are scarce.
Unusual locations make the diagnosis of fibrosarcoma difficult, especially when it
occurs in the thyroid. Most reports in the literature have demonstrated anaplastic
thyroid carcinomas by use of immunohistochemical studies and electron micros-
copy, stating that the sarcoma-like tumors of the thyroid gland are in fact of epi-
thelial histogenesis, i.e., anaplastic carcinomas [5, 6, 7]. Differential diagnosis with
anaplastic carcinomas is very difficult due to their similar clinical, histological and
immunohistochemical features.
Again, the World Health Organization classification of thyroid tumors indicates
that it is very difficult or impossible to distinguish some thyroid sarcomas from un-
differentiated carcinomas. The recommendation is that primary thyroid sarcoma
should only be diagnosed when there is a complete lack of all epithelial differentia-
tion and there is definite evidence of specific fibrosarcomatous differentiation [8],
as was the case of our patient.
In conclusion, the case reported here shows a fibrosarcoma of thyroid gland
confirmed by imunohistochemistry, rarely previously reported in the literature.
These findings may contribute to broadening of the spectrum of differentiation of
this unusual neoplasm.
REFERENCES
F i s h e r, C., E. Van den Berg et W. M. Molenaar. Adult fibrosarcoma. – In: Tumors of Soft Tissue1 .
and Bone. Ed. Fletcher, D. M., Unni, K. K., Mertens, F. Lyon, IARC Press, 2002, 100-101.
C a m e s e l l e -Te i j e i r o , J. et al. Solitary fibrous tumor of the thyroid. – Am. J. Clin. Pathol., 1994,2 .
№ 4, 535-538.
R o d r i g u e z , I. et al. Solitary fibrous tumor of the thyroid gland: report of seven cases. – Am. J.3 .
Surg. Pathol., 25, 2001, № 11, 1424-1428.
Ti t i , S., K. Sycz et M. Umiński. Primary fibrosarcoma of the thyroid gland-a case report. – Pol. J.4 .
Pathol., 58, 2007, № 1, 59-62.

C a r c a n g i u , M. L. et al. Anaplastic thyroid carcinoma:a study of 70 cases. – Am. J. Clin. Pathol.,5 .
83, 1985, 135-158.
T h o m p s o n , L. D. et al.. Primary smooth muscle tumors of the thyroid gland. – Cancer,6 . 79, 1997,
№ 3, 579-587.
N i s h i y a m a , R. H., E. L. Dunn et N. W. Thompson. Anaplastic spindle-cell and giant-cell tumors7 .
of the thyroid gland. – Cancer, 30, 1972, 113-127.
H e d i n g e r, C., E. D. Williams et L. H. Sobin. Histologic Typing of Thyroid Tumors. – In:World8 .
Health Organization International Histologic Classiﬁcation of Tumors. 2nd ed. Berlin, Springer,
1988, 13-15.
Georgi Tchernev, Associated Professor
Dermatology and Venerology
Department of Dermatology and Venereology
Trakian University
Medical Faculty
Armeiska 11,
6000 Stara Zagora
Bulgaria
00359 885 588 424
e-mail: georgi_tchernev@yahoo.de

Fatty acid composition of fish...26
FATTY ACID COMPOSITION OF FISH SPECIES FROM
THE BULGARIAN BLACK SEA
M. Stancheva, A. Merdzhanova and L.Makedonski
Department of Chemistry, Medical University of Varna
Summary. The total lipids and fatty acid profile in the edible tissue of two
traditionally consumed fish species from Bulgarian Black Sea coast – shad and
red mullet in two seasons are determined. The fatty acid composition was analy-
sed by GC/MS. The total content of omega-3 fatty acids was significantly higher
than the total content of omega-6 fatty acids in shad whereas red mullet showed
opposite trend. The omega-3/omega-6 FA ratio, an useful indicator for evalua-
tion the relative nutritional value of a given fish, was within the recommended
range for the studied Black Sea fish species. Obtained results for FA composi-
tion, omega-3/omega-6 and polyunsaturated /saturated fatty acids ratios indicate
that these Black Sea fish species in both seasons – spring and autumn are good
sources of essential fatty acids.
Key words: omega-3, omega-6, fatty acids, GC/MS, fish species
INTRODUCTION
N
owadays marine food and especially marine fish are an important part
of healthy diets. This is because marine fish provides us with a large
number of nutrients especially omega-3 (ω 3) fatty acids such as ei-
cosapentaenoic (EPA, C 20:5) and docosahexaenoic (DHA, C 22:6) fatty acids. A
large body of scientific paper shows that fish consumption has beneficial effects
on coronary heart disease, growth and development, blood lipids and other health
issue [4, 12].
Fish consumption in Bulgaria is lower than recommended by World Health
Organization (WHO) and the Food and Agricultural Organization (FAO) (at least
15-20 kg fish per capita) [5]. Their is a lack of information on the fatty acid (FA)
composition of local fish species consumed in Bulgaria. The shad (Alosa pontica)
and red mullet (Mullus barbatus ponticus) are traditionally consumed fish species

in our country. However, the fat content and FA composition of fish lipids are not
constant. They vary according to fish and its characteristic such as catching place,
in response to their habitats as water temperature change, salinity and dietary lip-
ids [1, 6, 7, 9, 13]. Some Turkish articles presented information for a proximate
composition of the Black Sea species living in South Eastern part of Black Sea but
they do not show similar results [6, 9, 13]. Significant differences were also reported
in the fat content and FA composition of several marine fishes during the seasons
[15]. The aim of the present study is to determine the fatty acid composition in the
edible parts of shad and red mullet. The influence of seasonality on total lipids and
FA profile are followed in order to find in which season these species are the best
source of essential FA.
Sampling
Shad was caught from region of Kavarna, the North-Eastern Black Sea,
whereas red mullet was caught from region of Varna (Tracata). They were pur-
chased from Varna local fish markets during two commercial catching seasons
– spring (March-April) and autumn (Octomber-November) of 2009. The biometric
and biologic characteristics of analysed Black Sea fish species are presented in
Table 1.
Table 1. Biometric and biologic characteristics
Fish species
Mean total
weight
(g) ± SD
Spring
Mean total
weight
(g) ± SD
Autumn
Mean stand.
length
(cm) ± SD
Spring
Mean stand.
length
(cm) ± SD
Autumn
Habitat Food habits
Pelagic Demersal Carnivorous
Shad (n=4) 325.00±5.00 315.00±5.20 25.50±2.10 28.50±3.50 + – +
Red mullet
(n=12)
35.00±3.00 47.50±4.00 14.50±1.50 16.00±2.00 – + +
n – number of specimens
The edible fish tissue was filleted with the skin left on and homogenized. The
freshly prepared homogenates (5,00 g) were extracted by the method of Blight and
Dyer [10] using chloroform/methanol/water in a ratio 2:2:1. After phase separation,
the chloroform extracts were evaporated to dry residue and were quantified by
weight. Three replicate total lipid analyses were performed gravimetrically and the
results were present as g.100g-1 raw tissue.
The dry residue of the chloroform fraction was methylated by base-catalyzed
transmethylation using 2M methanolic potassium hydroxide and n-hexane [11]. Af-
ter centrifugation, the hexane layer was separated and analysed by GC-MS.

GS-MS condition
Gas chromatography analyses were performed by Gas Chromatograph
with autosampler, equipped with Polaris Q MS detector (Thermo Scientific,
USA). The capillary column used was a TR-5 MS (Thermo Scientific, USA)
universal column 30 m length and 0.25 mm i.d. Helium was used as a carrier
gas at flow rate 1 ml/min. Peaks were identified according to retention time
based on available fatty acid methyl esters mix standard (37 FAME mix C4-
C24, SUPELCO) and mass spectra (ratio m/z). Three replicate GC analyses
were performed and the results were expressed in GC area % as mean values
± standard deviation (SD).
Statistical analysis
Statistical analysis was performed using Graph Pad Prism 5 program. The
descriptions of the data are given as mean ± Standard Deviation (SD) in Table 2.
To assess the statistically significant levels (P >0.05), a one way ANOVA (nonpara-
metric test) was employed.
RESULTS AND DISCUSSION
The total lipid (TL) content and FA composition of analysed fish species are
presented in Table 2. As it can be seen from the above table, red mullet can be
classified as moderately oily fish in spring season until red mullet (autumn) and
shad (over two seasons) are typical fatty species. Our results are similar to other
relevant publications describing seasonal changes in TL of pelagic species and in
Mediterranean red mullet [10, 15].
A lipid analysis enabled the classification and quantitative determination
of FA as well as the sum of saturated fatty acids (SFA), monounsaturated fatty
acids (MUFA), polyunsaturated fatty acids (PUFA). FA contents in red mullet fol-
lowed a relative pattern with SFA > MUFA > PUFA in both seasons. In contrast
to the red mullet, shad (autumn) contained a higher amount of SFA than that of
MUFA (e.g. SFA > PUFA> MUFA) whereas the shad (spring) contained a dif-
ferent pattern of FA composition as evidenced by the relatively greater MUFA
compared to PUFA (e.g. MUFA > SFA > PUFA). Figure 1 presented significant
differences obtained when comparing the FA groups in each analysed fish spe-
cies. These FA groups presented significant differences in their values during
both seasons. Those visible FA variations observed in the red mullet in both
seasons are in accordance with the data presented by Polat et al. [10] for the
Mediterranean red mullet. Fig. 1 shows that the amount of FA groups varies
widely among the species and seasons.

Table 2. Total lipids and fatty acid composition in edible ﬁsh tissue (mean ± SD)
Fatty Acid
% of total FA
Red mullet
Spring
Red mullet
Autumn
Shad
Spring
Shad
Autumn
TL (g.100-1
g r.w) 5.38±0.65 14.69±0.85 13.15±0.70 12.70±0.80
C 12:0 0.53±0.02 1.10±0.05 0.05±0.01 0.84±0.02
C 14:0 4.27b
±0.70 5.85±0.75 2.68b
±0.22 3.46±0.65
C 15:0 0.02±0.01 0.01±0.01 0.00 1,19±0.25
C 16:0 21.43a,b
±1.15 30.05b
±1.75 26.02a
±1.40 27.74a±2.01
C 17:0 0.63±0.02 0.31±0.01 0.24±0.01 1,43±0.30
C 18:0 6.30±0.050 6.23±0.045 1.54b
±0.20 3.02b
±0.60
C 20:0 0.65±0.02 0.28±0.01 0.10b
±0.01 2.47b
±0.80
C 21:0 0.39±0.01 0.15±0.01 0.02±0.01 0,00
C 22:0 0.69±0.02 0.24±0.01 0.06b±0.01 2.52b
±0.20
C 23:0 0.37±0.01 0.13±0.01 0.02±0.01 0,00
C 24:0 0.65±0.02 0.22±0.01 0.05b
±0.01 2.87b
±0.50
Σ SFA 35.44 43.90 30.81 46.03
C 14:1 2.06±0.50 1.15±0.40 0.03±0.01 1.32±0.10
C 16:1 9.36b
±1.02 12.32±1.10 16.82b
±0.65 7.72b
±1.01
C 17:1 0.68±0.05 0.24±0.01 0.29±0.02 1,40±0.30
C 18:1 n9 c 20.9b
1±1.50 16.75±1.20 9.35b
±0.50 6,76±0.85
C 18:1 n9 tr 0.17±0.01 0.06±0.01 1.03a
±0.02 1,17a
±0.20
C 20:1 2.06±0.40 1.73±0.30 2.45a
±0.30 1,89a
±0.20
C 22:1 n9 1.85b
±0.40 0.90±0.05 3.10b
±0.30 1,68b
±0.15
C 24:1 0.50±0.02 0.22±0.01 1.49±0.02 1,44±0.20
Σ MUFA 37.56 39.45 34.56 25.39
C 18:3 ω 6 1.57±0.10 1.01±0.40 0.04±0.01 1,32±0.20
C 18:2 ω 6 c 3.07b
±0.15 2.14±0.20 5.21b
±0.25 4,43±0.25
C 18:2 ω 6 t 0.02±0.01 0.01±0.01 0.22±0.01 1,98±0.50
C 18:3 ω 3 1.20±0.80 0.63b
±0.05 0.41b
±0.02 2,11b
±0.20
C 20:5 ω 3 1.13±0.50 0.68±0.02 0.13b
±0.01 1,33b
±0.40
C 20:4 ω 6 5.11b
±0.85 4.41±0.90 0.15b
±0.01 2,50±0.20
C 20:2 0.96±0.05 0.34±0.02 0.15±0.01 1,42±0.50
C 20:3 ω 3 0.90±0.05 0.45b
±0.04 0.07±0.02 1,86b
±0.80
C 20:3 ω 6 0.96±0.03 0.34±0.01 0.00 1.42±0.20
C 22:6 ω 6 7.01b
±0.97 4.53±0.65 21.04b
±0.30 9,30b
±1.05
C 22:2 0.83±0.02 0.43b
±0.01 0.06±0.01 1,73b
±0.80
Σ PUFA 21.40 15.65 28.25 29.60
SFA – saturated FA; MUFA – monounsaturated FA; PUFA – polyunsaturated FA.
value without superscript – P=0.01; for value with superscript- a
– P=0.001; b
– P= 0.0001

0
20
40
60
SFA
MUFA
PUFA
red mullet
spring
red mullet
autumn
shad
spring
shad
autumn
%oftotalFA
Fig. 1. Seasonal changes in fatty acid groups in red mullet and shad
In the SFA group, C 16:0 (predominated in all groups), C 18:0 and C 14:0
were found in large quantities. Availability of high levels of this fatty acid supports
the results published in many similar studies conducted on seawater fish [7, 14].
We found that the increases in total SFA in the autumn in two species were directly
related to growth quantities of C 16:0 and C 14:0.
The amounts of unsaturated FA as MUFA vary especially in wild fish [7, 15].
The percentage of this FA in the observed Bulgarian Black Sea fish during two sea-
sons is not similar. The highest total MUFAs value was determined for red mullet
(autumn), while the lowest one was for shad (autumn). That was due to the high
concentration of C 16:1,C 18:1 ω 9 and C-20:1 ω 9. C16:1 was the main MUFA
in the sad, especially in spring season which is in accordance with FA profile pre-
sented for some Mediterranean and Aegean fish species as mullet (Mugul cepha-
lus) and sardine (Sardinella aurita) [9]. Many studies [1, 6, 7, 13] report that C 18:1
is the main MUFA in seawater fish species. Our results for red mullet confirmed
these informations. This fatty acid has exogenous origin and usually reflects the
type of diet of the fish. Some Turkish papers report that the highest level of C18:1
(up to 13.00%) is measured in the Black Sea turbot [1, 6, 7, 9, 13]. Some investiga-
tions supposed that because of the decline in populations of zooplankton and the
increase of phytoplankton mass, as a response to the eutrophication in the Black
Sea, the fish in that region have low levels of MUFA as C 20:1 and high levels of ω
3 PUFA [14]. Contrary results were obtained in our study where C 20:1 levels were
high (for red mullet in both seasons) and low for C 22:1 ω 9 especially in red mullet
(autumn). The shad presented high level of C 22:1 ω 9 (spring samples).
The major FA identified as PUFA is DHA. Other important long-chain fatty
acids such as linoleic acid (C 18:2 ω6), EPA and arahidonic acid (C 20:4 ω6) were
found in significant amounts too. The maximum value of DHA was defined in shad
(74.50% of total PUFA) and the minimum – in red mullet (48.80 % of total PUFA).

Saglik et al. [11] have analysed ω 3 FA in some Turkish seawater fish and reported
that DHA occurred in higher amounts. In comparison with our results, significant dif-
ferences for EPA and DHA between Black Sea fishes and presented Mediterranean
fishes were found. In our investigation for all species, EPA levels were lower than
those of C 20:4 ω 6 in both seasons.
The obtained results (Table 3) for omega-3/omega-6 ratios for both species
varied according to seasons.
Table 3. Total sum of omega-3 and omega-6 content (% of total FA)
Fatty Acids Red mullet
Spring
Red mullet
Autumn
Shad
Spring
Shad
Autumn
ω 3 9.34±1.20 5.78±0.90 21.66±1.65 14.60±1.35
ω 6 10.73±1.10 7.64±1.52 5.60±0.94 10.35±1.05
ω 3/ ω 6 0.87 0.75 3.87 1.41
The data indicate that shad was characterized by high level of omega-3 FA
series and low levels of omega-6 series while red millet presented reverse version.
Nevertheless, we can conclude that these fish species are a valuable source of
essential fatty acids, especially DHA. The ω 3 / ω 6 ratio has been suggested to be
a useful indicator for comparing the relative nutritional value of fish. The seasonal
changes observed in ω 3 and ω 6 PUFA for both species are presented in Figure
2. The ratio 0.20-3.80 recommended by UK Department of Health [3, 12] would
constitute a healthy human diet and our study proved that this ratio for all Black
Sea fish species is within the recommended level. Due to its diet prior to breeding
season shad showed unbalanced omega-3/omega-6 ratio.
r m S r m A s S s A
0
5
10
15
20
25
omega 3
omega 6
red mullet
spring
red mullet
autumn
shad
spring
shad
autumn
%oftotalFA
Fig. 2. Content of omega-3 and omega-6 fatty acids

In Bulgaria, people have a relatively low intake of omega-3 FA compared with
omega-6 FA. A moderate to low consumption of fish and other seafood (especially
fatty fish), combined with limited conversion of ALA to EPA and DHA in particular,
contributes to a relatively high level of omega-6 fatty acids in the body compared
with the marine omega-3 fatty acids. By combining a lower intake of omega-6 fatty
acids with a higher intake of omega-3 fatty acids, e.g. through the consumption of
seawater fish, a relatively significant effect will be achieved [12]. Values for PUFA/
SFA ratio greater than 0.45 are recommended [5 ,12]. Our results are in agreement
with this requirement showing higher PUFA/SFA ratios for three studied fish species
(Table 3). The highest PUFA/SFA ratio was observed in the shad (spring), whereas
the lowest value was found for red mullet (autumn). The most balanced PUFA/SFA
ratio was obtained for the shad.
CONCLUSION
The present study provides useful information about the seasonal variation of
total lipid content and fatty acid composition of two Black Sea fish species – shad
and red mullet:
The total lipids in shad had similar values in both seasons while in red mullet•
a threefold increase in quantity in the autumn period was observed.
Seasonal variations were observed in all FA groups as the most significant•
were in SFA and PUFA – increasing amounts of SFA at the expense of reduction of
PUFAs in the autumn in both species.
The most important omega-3 fatty acids in PUFAs group are DHA and they•
were presented with the highest concentration in all species and in all observed
seasons. The ω 3/ ω 6 ratio varies in the range of 0.75 up to 3.87. The most bal-
anced PUFA/SFA ratio was obtained for the shad.
In conclusion, regarding the lipid contents, the omega-3/omega-6 and PUFA/
SFA ratios, we may assume that these Black Sea fish species were found to be a
valuable source of the essential PUFA for human diet in both seasons.
Acknowledgments
The authors would like to thank the National Science Fund, Ministry of
Education and Science of Bulgaria for their financial support (Project DVU 440 /
2008).
REFERENCES
B a y i r, A., H. I. Haliloğlu. et N. Sirkecioğlu. Fatty acid composition in some selected marine fish1 .
species living in Turkish waters. – J.. Sci. Food Agric., 86, 2006, 163-168.
BDS EN ISO 5509. Animal and vegetable fats and oils-preparation of methyl esters of fatty acids.2.
2000.
B l i g h E. et W. J. Dyer. A rapid method of total lipid extraction and purification. – Can. J. Biochem.3 .
Physiol., 37, 1959, 913–917.

C u l y e r, A. Supporting research and development in the NHS: a report to the Minister of Health.4 .
London, HMSO, 1994.
FAO/WHO: Scientific and ethical challenges in agriculture to meet human needs. http://www.fao.5.
org/docrep/003/X8576M/x8576m05.htm
G u n e r, S. et al. Proximate composition and selected mineral content of commercially important6 .
fish species from the Black Sea. – J. Sci. Food Agric., 78, 1998, 337.
I m r e , S. et S. Saglik. Fatty acid composition and cholesterol content of some Turkish fish species.7 .
– Turk. J. Chem., 22, 1998, 321-324.
M u r r a y, J. et J. R. Burt. The Composition of Fish. Ministry of Technology, Torry Research Station,8 .
2001, Torry Advisory Note No. 38. http://www.fao.org/wairdocs/tan
Ö z o g u l , Y. et F. Özogul. Fatty acid profile of commercially important fish species from the9 .
Mediterranean, Aegean and Black Seas. – Food Chem., 100, 2007, 1634-1638.
P o l a t , A., S. Kuzu et G. Özyurt. Fatty acid composition of red mullet: A seasonal differentiation.1 0 .
– J. Muscle Foods, 20, 2009, 70-78.
S a g l i k , S. et S. Imre. ω3 fatty acids in some fish species from Turkey. – J. Food Sci.,11 . 66, 2001,
210-212.
S i m o p o u l o s , A. et L. Cleland (eds). Importance of the Ratio of Omega-6/Omega-3 Essential1 2 .
Fatty Acids: Evolutionary Aspects. Omega-6/Omega-3 Essential Fatty Acid Ratio: The Scientific
Evidence. – World Rev. Nutr. Diet., 92, 2003, 1-22.
Ta n a k o l , R. et al. Fatty acid composition of 19 Species of fish from the Black Sea and the1 3 .
Marmara Sea. – Lipids, 34, 1999, 291-294.
Z a i t s e v, Y. Recent Change in the Trophic Structure of the Black Sea. – Fish. Ocean.,1 4 . 2, 1992,
180-189.
Z l a t a n o s , S. et K. Laskaridis Seasonal variation in the fatty acid composition of three Mediterra-1 5 .
nean fish – sardine (Sardina pilchardus), anchovy (Engraulis enchrasiholus) and picarel (Spicara
smaris). – Food Chem., 103, 2007, 725-728.
A. Merdzhanova
Department of Chemistry
Medical University of Varna
55 Marin Drinov Str.
9002 Varna
Bulgaria
035952 650019
e-mail: albenamerdjanova@mu-varna.bg

Rheumatoid arthritis in the general...34
RHEUMATOID ARTHRITIS IN THE GENERAL
PRACTITIONER'S RACTICE
V. Paskaleva-Peycheva1
, M. Panchovska-Mocheva2
and E. Kavrakov3
1
UMBAL “Sveti Ivan Rilski” – Sofia, Rheumatology Clinic, Medical University – Sofia
2
Military Medical Academy – Plovdiv, Clinic of Internal Diseases, Medical University – Plovdiv
3
Medical student, MU – Plovdiv
Summary. Rheumatoid arthritis (RA) is a inflammatory joint disease with a
variety of manifestations, decursus and treatment approaches. It is essential to
stress the importance of the early proven diagnosis and early treatment which pro-
tect from serious complications in the musculoskeletal system and internal organs.
This requires some highly specialized help from a rheumatologist. There has to be
a good dialogue between general practitioner and rheumatologist who must work
together. Such an approach would lead to quick and efficient treatment of patients
with RA.
Key words: rheumatoid arthritis, patients, general practitioner
R
heumatoid arthritis is an autoimmune rheumatic disease with unknown
etiology and a complex pathogenesis. It is characterized by the gradual
development of chronic erosive arthritis (synoviitis) and appearance of
systemic manifestations – lesions of different internal organs.
The frequency of RA is 0,8-1,0%, with male to female ratio of 2-3 : 1. The
disease onset is earlier in women (it starts at the age of 30-50) while in men the
onset is later but course is more severe.
This disease is associated with some antigens: HLA-DR3, DR4, DRW52
General practitioners (GPs) and RA
GPs are the first who get to know about a patient’s problem. Having in mind
that RA happens to be a disease following the patient all his life with its chronic
course, the interaction of the physician and the patient is very important.
The best way to manage the disease is to diagnose and treat it early enough.
The early referral of patients to specialists in rheumatology may prevent disability
and provide a higher quality of life [1].

The American College of Rheumatology developed the following criteria (in
1987) for diagnosing RA:
1) Morning stiffness lasting at least 1 hour
2) Arthritis of 3 or more joint areas
3) Arthritis of hand joints
4) Symmetric arthritis
5) Rheumatoid nodules
6) Serum RF
7) Radiographic changes typical of RA
*The diagnosis is accepted if at least 4 out of 7 criteria are present.
**Criteria 1) – 4) have to last at least 6 weeks and to be observed by a physician [2].
Differential Diagnosis:
1. Osteoarthritis: “mechanical” nature of pain, morning stiffness is continuing
less than 30 min., etc.
2. Systemic Lupus Erythematosus: characteristic ﬁndings in immunological
tests, lack of erosive radiographic changes in impaired joints.
3. Gout: a chronic disease more distributed among females; increased uric
acid level, cystose radiographic changes
4. Psoriatic arthritis: visible psoriatic lesions, genetic predisposition
5. Bechterew’s Disease (Ankylosing spondylitis): HLA – B27, other cases in
the family, sacroileitis, enthesopathia.
6. Reactive arthritis: after a short-lasting acute urogenital ot intestinal infection
7. Other types of arthritis
Which are the indications for a hospitalization of patients with RA?
1. Diagnosis establishment and prognostic evaluation of the disease
2. Selection of a disease-modifying antirheumatic drug (DMARD) for treat-
ment
3. Disease activity evaluation
4. If infections are developed – for example pneumonia, cholecystitis, septic
arthritis, also in case of complications of RA like amyloidosis or complications of the
selected treatment (drug ulcers, toxic drug hepatitis, interstitial nephritis, etc)
5. Surgical treatment (carpal tunnel syndrome, endoprosthesis)
6. Visceral manifestations such as “rheumatoid” lungs, glomerulonephritis, ac-
companying vasculitis
The treatment’s purpose is to:
1. Decrease the activity of the disease
2. Prevent destruction of joints and deformity, as much as functional impair-
ment
3. Reach a remission state
4. Improve the overall quality of life
5. Increase life expectancy (the mean life expectancy of the population has to
be reached) [3, 4]

Rheumatoid arthritis in the general...36
The behavior of GPs when working with patients with RA has to unify the fol-
lowing options:
1. Interdisciplinary treatment – participation of rheumatologist, neurologist,
physiotherapist and orthopedist in the treatment plan
2. Avoidance of those factors that can lead to exacerbations – social and living
conditions, professional harmful factors
3. Smoking and alcohol intake have to be given up and, in addition, a BMI <
25 has to be achieved and supported
4. A suitable balanced diet rich in Ca, proteins and omega-3-fatty acids must
be recommended.
5. Education of the patient; active kinesitherapy
6. Physiotherapy in case activity is low or RA is in a stage of remission
7. Orthopedic tools – corsettes, walking sticks and other gait facilitating tools
8. Sanatorium treatment during periods of remission
9. Treatment of accompanying diseases: focal infections, gastroduodenal
problems, thyroid dysfunctions
BIOLOGIC AGENTS are used in the treatment schemes of RA in the last
years [5].
Biologic agents are protein molecules directed against other molecules, par-
ticipating in the inflammatory process (i.e.the cytokines).
Following groups of biologic agents are directed against the respective target
molecules:
1) Remicade, Enbrel, Humira – vs. TNF-α
2) Anakinra – vs. IL-1
3) Tocillizumab – vs. IL-6
4) Rituximab (Mabthera) – vs. B cells
Biologic agents are indicated in the cases stated below:
1. In cases of insufficient effect from nonbiologic treatment: methotrexate, hy-
droxychloroquine, leflunomide, sulfasalazine
2. TNF-α inhibitors may be prescribed in the beginning of treatment of newly
diagnosed patients before methotrexate
3. Biologic agents combined with methotrexate are prescribed if RA activity is
very high
4. Combinations of more than one biologic agent are not recommended
5. Treatment contraindications to biologic agents include: bacterial infections,
H.zoster, active and latent TBC, acute or chronic hepatitis B and C
6. High-risk patients should be advised to undergo vaccinations against influ-
enza, pneumococcus infection and HBV
GPS AND CONTROL OF PATIENTS WITH RA
GPs have the following specific tasks concerning patients with RA:
1. Arterial blood pressure monitoring due to a tendency to increased values as
a side effect of NSAIDs and corticosteroids

2. Control of drug doses because there is a risk of toxicity and complications
3. Due to risk of NSAIDs-induced gastroduodenopathia selective COX-2 in-
hibitors are prescribed (Celebrex, Arcoxia)
4. Monitoring of peripheral blood and biochemistry tests – creatinin, amin-
otransferases; endoscopy of the gastric and duodenal mucosa.
5. Risk factors in treatment of patients with RA include: age > 65; cardiovas-
cular diseases; anticoagulant intake; H. pylori infection.
6. In cases of allergy towards sulphonamidic drugs, Sulfasalazin and Cele-
coxib should not be used.
CONCLUSION
RA is an inflammatory joint disease which is characterized by a large variety
of manifestations, course and therapeutic approaches. Early diagnosis and early
adequate treatment are essential as they prevent disability in these patients. The
necessity of providing highly specialized medical help from a rheumatologist de-
termines the importance of the collaboration between specialists and GPs. Such
an approach would ensure right professional behavior towards patients with RA,
higher quality of life and preserved life expectancy.
REFERENCES
Va n d e r L i n d e n , M. P. et al. Long-term impact of delay in assessment of patients with early1 .
arthritis. – Arthritis Rheum., 62, 2010, № 12, 3537-3546.
S o k o l o v e , J. et V. Strand. Rheumatoid arthritis classification criteria – it’s finally time to move2 .
on. – Bull. N. Y. Univ. Hosp. H. Dis., 68, 2010, № 3, 232-238.
R o b i n s o n , P. C. et M. J. Taylor. Time to treatment in rheumatoid arthritis: factors associated3 .
with time to treatment initiated and urgent triage assessment of general practitioner. – J. Clin.
Rheumatol., 16, 2010, № 6, 267-273.
N a r a n j o , A. et al. Cardiovascular disease in patients with rheumatoid arthritis: results from the4 .
QUEST-RA study. – Arthritis Res. Ther., 10, 2008, 1186.
A m e r i c a n College of Rheumatology. Recommendations for use of nonbiological and biological5 .
disease – modifying antirheumatic drugs in Rheumatoid arthritis. – Arthritis Rheum., 59, 2008,
№ 6, 762-784.
Assoc. Prof. Veneta Paskaleva-Peycheva, MD
Rhematology Clinic
UMBAL "Sveti Ivan Rilski"
13 Urvich str.
1612 Sofia
958-25-53

Immunobiology of endometriosis38
IMMUNOBIOLOGY OF ENDOMETRIOSIS
N. Manolova, D. Zasheva and M. Stamenova
Department of Immunochemistry, Institute of Biology and Immunology of Reproduction,
Bulgarian Academy of Sciences
Summary. Endometriosis is a condition in which tissue similar to the lining of
the uterus (endometrial cells and glands, which should appear only in the uterus)
is found in other parts of the body. This tissue can be implanted itself and grow
throughout the abdominal cavity. As a disease, it is a unique clinical and scientific
challenge. This disease called “enigma wrapped in mystery”, is still etiologic and
pathogenetic phenomenon. According to some authors in women with infertility, the
rate of genital endometriosis is estimated at 20-55% and 30 to 50% of women with
endometriosis are infertile. The disease leads to poor success rate in patients who
undergo assisted reproductive technologies. Endometriosis is a benign, chronic,
estrogen – dependent gynecological disease accompanied by pelvic pain. Because
of the clinical, psychological and social significance of the problem, endometriosis
is a widely studied disease. It affects the quality of life of women in their most active
age and has serious economic consequences. For accurate diagnosis and proper
treatment of endometriosis and prevention it is necessary to perfect knowledge of
the mechanisms of emergence and development of pathological process. Basic
environment for emergence and development of endometriosis is peritoneal fluid.
Middle of the peritoneal fluid surrounding the endometrial implants is biochemically
dynamic and it becomes a meeting of reproductive and immune systems.
Key words: endometriosis, implantation, infertility, peritoneal fluid, serum, reproduc-
tive system, immune cells, humoral immunity
IMMUNE CELLS AND THEIR FUNCTION IN ENDOMETRIOSIS
I. Cell immunity
Monocytes/macrophages1. – peripheral blood monocytes (PBMs) and
macrophages in peritoneal fluid are major cellular components of the immune sys-
tem. Women with endometriosis have an increased circulating monocyte activa-

tion status, which has been found by chemiluminescence [3]. Under disincentives
(basal) and stimulated conditions in women with endometriosis, PBMs produced
high levels of tumor necrosis factor (TNF-α), interleukin (IL)-6 and IL-8, but not
IL-10 compared with monocytes of healthy controls [4]. Macrophages in the perito-
neal cavity remove red blood cells, damaged tissue fragments, apoptotic cells and
endometrial cells most probably penetrating into the peritoneal cavity through the
fallopian tubes. In endometrial peritoneal fluid, the concentration and the number of
macrophages are significantly increased [5]. They are large activated macrophag-
es that produce high levels of TNF-α, IL-6 and IL-8 and IL-10 in comparison with
macrophages in healthy women [6]. They also produce high levels of smooth-mus-
cle-contracting prostaglandins (PGs), such as PGE2 and PGF2α [7]. Endometrial
macrophages are also a source of other cytokines, growth factors, adhesion mol-
ecules, complement components, hydrolytic enzymes, reaction O2
products stimu-
lating eutopic and ectopic endometriosis cell proliferation in vitro and decrease
endometriotic-cell apoptose [8]. It is assumed that in healthy women, phagocytic
activity of peritoneal macrophages is crucial to perform the elimination of menstrual
detritus. This activity is mediated via surface scavenger (cleaning) receptors, which
are regulated by different cytokines and growth factors. It has been shown that
these scavenger-receptors play a role in cell adhesion and that non-adherentive
macrophages do not express type-A scavenger-receptor. Thus the increase in non-
adherentive macrophages without scavenger-receptor may contribute to the patho-
genesis of endometriosis [9].
NK-cells:2. NK-cells are large granular lymphocytes that participate in the
destruction of abnormal cells derived from viral infections, malignant changes or
aging. One of the mechanisms by which NK-cells kill target cells is through an-
tibody-dependent cellular cytotoxicity. To this end, NK-cells have receptors that
bind immunoglobulin-G (Ig G) and kill then covered with IgG target cells. Another
mechanism involving recognition of target cells is through the so-called KAR (killer
activating receptors) and KIR (killer inhibitory receptor). If KAR are employed, i.e.
are activated, NK-cells have cytotoxic activity, when KIR are vacant, i.e. are inacti-
vated, cytotoxic activity is inhibited. Cytotoxic activity of NK-cells can be enhanced
by lymphokines such as IL-2, a function known as LAK-activity (lymphokine-acti-
vated killer) [5].
Several studies have demonstrated lower NK-cell cytotoxicity against autolo-
gous and heterologous endometrial cells in women with endometriosis [10]. This
decrease in NK-cell cytotoxicity against autologous endometriotic cells may also
reflected increased resistance of endometrial cells to NK-mediated cytolysis, which
was first presented by Oosterlynck et al. [11].
If the NK-cell cytotoxicity is one component of the immune “disposal” system
of menstrual detritus, NK-cell deficiency may facilitate the development of disease.
Changes in NK-cell cytotoxicity in endometriosis appear more functional than quan-
titative. The percentage of peripheral NK-cells do not change [5].

Several studies demonstrate increased expression of KIRs family of NK-cells in
women with endometriosis. These molecules interact with the main complex for tis-
sue compatibility class-I (MHC-I) of the potential target cells to block the “killing” abil-
ity of NK-cells. Cells with no expression of MHC-I treat waste program activated NK-
cell [12, 13]. Studies of Maeda et al. [14] showed that endometriosis has increased
the proportion of NK-cells that express KIR2DLI phenotype compared with normal
controls. This was demonstrated both in circulating and peritoneal NK-cells.
T-lymphocytes:3. T-lymphocytes derived from pluripotent stem cells in fetal
liver and bone marrow. From these places they travel to the thymus, where complete
their development in the two main subpopulations characterized by expression of
the glycoprotein CD4 and CD8, functioning as co-receptors for MHC-II class and
MHC-I class molecules. CD4 T-lymphocytes can later be turned into subclasses
CD4 Th1 and Th2 CD4 cells. Th1 cells enhance differentiation of CD8 cells to killer-
cells and activated monocytes/macrophages system to facilitate cell-mediated im-
munity. Th2 cells amplify B-cell differentiation to antibody-secreting cells. Th1 and
Th2 cells can be distinguished by their cytokine characteristics. Fully activated CD8
T- lymphocytes can eliminate intracellular pathogens by destroying virus-infected
cells simultaneously with the activation of monocytes-macrophages system [5].
Quantitative studies provide T-lymphocytes and their subclasses in peripheral
blood and peritoneal fluid in women with endometriosis. Changes were not detect-
ed in the total number of T-lymphocytes, and CD4/CD8 ratio in peripheral blood, but
peritoneal fluid observed elevated absolute number of CD4 and CD8 subclasses,
and their ratio. In eutopic endometrium, total lymphocytes and CD4/CD8 ratio were
similar both in endometriosis and in healthy control patients. In ectopic endometri-
um, although the number of T-lymphocytes was increased compared with the pro-
liferative and secretory eutopic endometrium, CD4/CD8 ratio remained unchanged
[5]. With regard to functional changes in peripheral lymphocytes in women with
endometriosis and adenomyosis, there were submitted data in 1980 (Startseva
[15]). A little later, using peripheral blood lymphocytes and autologous-Cr-labeled
endometriotic target cells in women with endometriosis was found decreased cell
lysis to target cells compared with controls and patients with moderate and severe
endometriosis [16].
II. Humoral immunity
B-lymphocytes:1. B-cells are precursors of plasma cells which are antibody-
producing cells of the immune system. It is suggested that CD5+ B-cells, which are
presented in 10-20% of B-cell population, are responsible for the production of au-
toantibodies. The number of CD5+ B-cells is increased in patients with autoimmune
diseases such as systemic lupus erythematosus, rheumatoid arthritis and others.
Autoantibodies and location of endometriosis as an autoimmune disease:−
The idea for the autoimmune nature of endometriosis was first presented by
Gleicher et al. [17], demonstrating that endometriosis is responsible for most of the
criteria to be classified as autoimmune diseases, as presented in Table 1.

Table 1. Common characteristics between autoimmune diseases and endometriosis
(Nothnick. Endometriosis and autoimmunity. Fertil Steril 2001)
1. Tissue damage 7. Multiorgan complications
2. Polyclonal B-cell activation 8. Family history
3. T-lymphocyte immunological disorder 9. Potential effects of environmental factors
4. B-lymphocyte immunological disorder 10. Possible genetic basis
5. Association with autoimmune diseases 11. Altered apoptosis
6. Prevalent in women
Like the classic autoimmune disease endometriosis was associated with poly-
clonal B-cell activation, first described in 1980 [15]. The disease is associated with
frequent abortions and infertility [18] which can be explained by the presence of au-
toimmune abnormalities. Further evidence for the role of autoimmunity or autoan-
tibodies in infertility, concomitant endometriosis, comes from studies showing that
medical treatment with Danazol [19] or gonadotropin-releasing hormone (GnRH)-
analogues [20] suppresses levels of autoantibodies associated with endometriosis.
To be a disease characterized, however, really as autoimmune in nature, it should
be exercised in healthy animals after the implementation of transfer of immuno-
globulins from serum or from affected tissues of sick. Until now such studies were
not conducted. However, there is uncertainty whether endometriosis is an autoim-
mune disease. It has a similar pathophysiology with other autoimmune diseases
(rheumatoid arthritis, Crohn’s disease, psoriasis) by: increasing inflammation, in-
creased levels of components of tissue remodeling, altered apoptosis, increased
local and/or systemic levels of cytokines [21].
In 1980, a group of Weed and Arguembourg reported for C3 and immunoglob-
ulin G (IgG) accumulation in the uterine endometrium in women with endometriosis
and lowering total serum complement manifested through antigen-antibody reac-
tions in the endometrium [22].
Mathur et al. first described in patients with endometriosis autoantibodies that
recognize endometrial antigens [4] (ranging between 34-140 KDa), eventually au-
toantigens the immune response [23]. IgG and IgA autoantibodies described by
these authors in serum and cervical and vaginal secretions of women are with
specificity against endometrial and ovarian tissues. There are other data from the
same research group, indicating that in normal fertile controls and in women with
endometriosis, there were observed decreased levels in the circulation and perito-
neal fluid of antibodies against endometrial antigens with different molecular weight.
It is believed that this may be a mechanism for clearing the reproductive tract of
menstrual detritus. Only against endometrial antigens with molecular weights of
26 KDa and 34 KDa auto-antibodies in endometriosis were identified. There were
also antibodies to endometrial transferrin, α2 Heremans-Schmidt glycoprotein (α2-
HSG) and carbonic anhydrase in women with endometriois [24]. Antibody response

against carbohydrate epitop (Thomsen-Friedenreich antigen) suggests that the au-
toimmune response may have direct contact with the disease process or reflect an
abnormality in glycosylation in endometriosis [25].
During the early stages of endometriosis, the disease is associated with el-
evated serum and peritoneal liquid levels of anti-phospholipid antibodies against
inositol, kardiolipin, ethanolamine and β2-glycoprotein I, while in patients with stage
I-II disease more autoantibodies compared with III-IV stage were observed [26].
Other researchers presented circulating autoantibodies against subcell items (in-
cluding antinuclear antibody) or against chemical substances, inseparable from
the cellular structures (including anti-DNA or anti-phospholipid antibodies). The
high frequency of these autoantibodies was also reported in women with autoim-
mune diseases and various forms of reproductive failure, and unexplained infertil-
ity and recurrent miscarriage [5]. Gleicher et al. reported that among 31 women
with endometriosis, 65% had IgG and 45% – IgM autoantibodies against at least
one of the sixteen tested antigens [27]. These findings are most often associated
with autoantibodies to phospholipid (especially phosphatidylserine), histones and
nucleotides. In support of the theory to unlock the humoral autoimmune response
in endometriosis are the data for the high prevalence of thyroid autoimmunity in
women with endometriosis as a result of significantly higher serum levels of thy-
roid peroxidase-antibody [28, 29] . In infertile patients with the disease, IgG anti-
laminin-I antibodies have been found [30]. There were proven also auto-antibodies
against oxidative stress-induced antigens, such as malone dialdehyde-modified
low density lipoproteins and oxidized LDL (low density lipoprotein) are elevated in
the serum of patients with endometriosis [31].
In support of the theory of autoimmune nature of endometriosis, data about
apoptotic deregulation process in the disease may be adapted. Breach in apoptosis
may trigger an autoimmune process through persistent self-reactive lymphocytes
and/or the inclusion of so-called death receptors and/or their ligands in tissue de-
struction [32]. Inability of cells to transmit “signal death” or the ability of cells to
avoid cell death is associated with other autoimmune diseases. There are studies
demonstrating that the endometrial cells in ectopic and eutopic endometrium in
women with endometriosis have altered apoptotic mechanisms [33, 34, 35]. Fur-
thermore, it is assumed that the change in apoptosis allows retrograde endometrial
tissue to “escape” from cell death. The precise mechanisms responsible for these
changes of apoptosis in infected women are unknown. It has been proven that this
changing the pattern of cell death can be “reversed” by GnRH-analog therapy, sug-
gesting that this peptide could play an important role in the reorganization of normal
endometrial cells during the menstrual cycle [33].
Growth factors:2. It is known that during the inflammatory response, mac-
rophages release a variety of inflammatory mediators. Some may be associated
with ectopic implantation of endometrial cells, for example: fibroblast-like growth
factor (FGF), epidermal growth factor (EGF), transforming growth factor-α (TGF-α),

transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α) [30].
It has been proven that these growth factors stimulate proliferation of endome-
trial stromal cells in vitro. This implies that they can improve the implantation of
endometrial cells [36]. Several studies have reported increasing activity of perito-
neal macrophages in endometriosis, which is associated with production of vari-
ous growth factors such as thrombocytic growth factor-β (TGF-β) and epidermal
growth factor (EGF) [37, 38]. It is also found that levels in serum and peritoneal
fluid of vascular endothelial growth factor (VEGF) are increased in women with
endometriosis compared with healthy controls [39]. But other authors argue that
there is no correlation between serum levels of VEGF and endometriosis [40]. El-
evated levels of the receptor of epidermal growth factor (EGF-R), which is involved
in angiogenesis imply an active role of the EGF in the development of disease [41].
Serum insulin-like growth factor-I (IGF-I) in patients with early stage endometriosis
and in healthy controls were significantly reduced compared with levels in patients
with severe stage of endometriosis, suggesting that IGF-I is an important mediator
in the development and/or maintenance of endometriosis or progression to late
stage of the disease [42].
Cytokines:3. Cytokines are proteins or glycoproteins released mainly in the
intercellular environment from leukocytes or other cells, exert their effects on cells
which have secreted (autocrine action) or on nearby cells (paracrine action). Some
of these proteins may circulate or pass through the cavities of the body, thus exert
its endocrine action. In some cases, cytokines are found in cell membrane-associ-
ated forms, where they act on adjacent cells [43]. Cytokines are key mediators of
intercellular communication in the immune system. They act pleyotropic on a vari-
ety of target cells, exerting proliferative, cytostatic, or differentiating chemoattrac-
tant effect. Cytokines have biological activity at high concentration range and are
associated with intracellular signaling and function of secondary mediators through
specific, high affinity receptors on target cell membranes [43].
RANTES (Regulated on Activation, Normal T-cell Expressed and Secret-
ed): RANTES is a cytokine of β or “C-C” chemokine family. RANTES is hemoat-
tractant for monocytes and memory cells. Besides being identified by the secre-
tion of chematopoietic cells, this cytokine is also secreted by some epithelial and
mesenchymal cells. RANTES may be an important mediator of acute and chronic
inflammation. Numerous potential binding sites for transcription factors regulate the
expression of the gene for RANTES. In normal endometrium, RANTES-protein is
presented primarily in stromal compartment. In vitro stromal cell cultures synthesize
RANTES-mRNA and RANTES-protein, respectively, when they impact with TNF-α
and INF-γ, while epithelial cells synthesize neither transcripts nor protein [44]. But
there is a significant difference between normal endometrial cell cultures and those
derived from endometriotic lesions. Placed under similar conditions, endometrial
stromal cell cultures secrete significantly greater amounts of RANTES-protein [45].
It is believed that secretion of RANTES by ectopic implants provides a mechanism

for recovery of peritoneal leukocytes. Expression of RANTES-gene has a high level
of regulation in endometrial stromal cells in response to IL-1β (released by mac-
rophages) acting pleyotropic. This in turn triggers inflammation, “loop” in which IL-
1β, by activated macrophages, may lead to RANTES production and subsequent
monocytic chemotaxis [43]. Figure №1 shows that macrophages occupy a central
role in immunobiology of endometriosis.
Fig. 1. Central roll of macrophages in immunobiology of endometriosis. Interactions between endo-
metriotic cells and macrophages are mediated by growth factors, cytokines and chemokines leading to
survival of implants instead of their death
(Lebovic. Immunobiology of endometriosis. Fertil Steril 2001)
IL-1: This is a cytokine that plays a central role in the regulation of inflammation
and immune responses. Originally recognized as a product of activated monocytes
and macrophages, IL-1 is now regarded as an activator of T-lymphocytes and dif-
ferentiation of B-lymphocytes. Two receptor agonists, IL-1α and IL-1β, are encoded
by different genes, and although both proteins have only 18- 26% amino acid-sim-
ilarity, they bind to the same receptors and have similar biological activities. IL-1ra,
receptor antagonist of IL-1, is an endogenous inhibitor that blocks binding of IL-1α
and IL-1β to IL-I receptor type. IL-1 was isolated from the peritoneal fluid of patients
with endometriosis and its concentration was significantly increased compared to
healthy controls [46, 47, 48]. Mori et al. observed increased levels of IL-1β in perito-
neal macrophages mRNA in women with “mild” endometriosis, but increased levels
of IL-1ra-mRNA in moderate and severe endometriosis. Coordinating activities of
these proteins are not sufficiently studied [49]. The role of IL-1β in strengthening
the process of angiogenesis in endometrial lesions is represented by induction of
angiogenic factors VEGF and IL-6, which was observed in endometriotic stromal
cells but not in normal endometrial stromal-cells [50]. Vigano et al. found that IL-1β
increased secretory form of ICAM-1 (sICAM-1) separated from endometrial cells,

but it can intercept, allowing refluxing endometriotic tissue to avoid destruction in
the peritoneal antrum [51].
IL-6: The pleiotropic removable cytokine IL-6 is an important regulator of in-
flammation and immunity, which serves as a physiological link between endocrine
and immune systems. IL-6 also modulates the secretion of other cytokines, in-
creasing T-cell activation and B-cell differentiation, inhibits growth of various hu-
man cell lines. It is 23-26 KDa, phosphoglycoprotein that exists in multiple iso-
forms and is produced by many cell types, including monocytes, macrophages,
fibroblasts, endothelial cells, smooth-muscle cells and endometrial epithelial and
stromal cells [52, 53]. IL-6 is also produced by several endocrine glands including
the pituitary gland and pancreas. Endometrial stromal and epithelial cells produce
IL-6 in response to hormones and other immune activators. Endometrial stromal
cells release IL-6 protein under the action of IL-1α or IL-1β, TNF-α, TGF and IFN-γ
[50, 53]. Since, usually estrogen increases proliferation of endometrial epithelium
suggests that estrogen causes proliferation by limiting the synthesis of epithelial
cell inhibitors, such as IL-6. Parmakoupis et al. found that IL-6 inhibits proliferation
of human endometrial stromal cells, this inhibition depends on the density of cells,
suggesting that IL-6 may play a role in epithelial-stromal interactions governing the
regulation of normal uterine function [54]. Tabibzadeh et al. suggest that fluctua-
tions of IL-6 during the menstrual cycle show feedback to estrogen action; estrogen
concentrations are elevated during the proliferative phase, while the levels of IL-6
are decreased. Serum levels and levels of IL-6 in peritoneal fluid were significantly
elevated in women with endometriosis compared with controls, higher levels of IL-6
were found in women with “chocolate” cysts [55, 56]. But other results reported
back that the levels of IL-6 in peritoneal fluid in patients with endometriosis are
decreased [57, 58].
TNF: This are pleyotropic acting cytokines with a range of beneficial and
harmful effects, depending on the quantity produced, their tissue localization, lo-
cal activity of TNF-binding proteins and their hormonal and cytokine environment.
TNF-α is produced by neutrophils, activated lymphocytes, macrophages, NK-cells,
several nonhomeopathic cellular branches, while TNF-β is produced by lympho-
cytes. Although these TNF were initially identified by their ability to kill certain cell
lines, their main function is in conjunction with IL-1 to initiate a cascade of cytok-
ines and other factors associated with inflammatory responses. TNF have similar
biological activities, but regulation of expression and processing is quite different.
There is speculation that TNF-α facilitates endometrial cell adhesion to peritoneal
mesothelium which provides a potential mechanism for initiation of endometriosis
[59]. Other studies found that levels of TNF-α in the serum and peritoneal fluid were
significantly elevated in the early stages of the disease and decreased in severe
stages [60], i.e. concentration of TNF-α correlates with the stage of disease [61].
IL-8 and MCP-I: Interleukin-8 is a chemoattractant for neutrophils and a potent
angiogenic factor. It is produced by number of cells types, including monocytes, en-

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