Symposium Hematological Cytology
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Symposium Hematological Cytology |
PLENARY LECTURE
1. CURRENT TRENDS IN
LABORATORY DIAGNOSTICS Harald H. Kessler KF-University, Graz,
Austria Molecular assays
basically consist of three main steps: the extraction of DNA or RNA (sample preparation),
nucleic acid amplification, and detection of amplification products. To meet the needs of
routine diagnostic laboratories, simplified automated technologies have recently been
developed. One of the promising future techniques is a real-time PCR. The Light Cycler TM
System, which enables the user to monitor the amplification of the PCR product
simultaneously, in real-time and on-line, has recently been introduced. Because of very
rapid air heating and cooling together with high surface-to-volume ratio of capillaries, a
complete PCR run of 30 to 40 cycles can be performed in 20 to 30 minutes. 2. A
MULTIDISCIPLINARY DIAGNOSTIC APPROACH TO Drago Batinić Laboratory Diagnostics
of Medical Faculty of Zagreb University, Clinical Hospital
Zagreb-Rebro, Zagreb A great contribution to
the development of highly specialized techniques of hematopoietic cell analysis was,
undoubtfully, among the others, the concept of monitoring patients during the various
phases of treatment - determination of minimum of residual disease (MOB).
This means that the development of new technologies for quantification of PCR reaction
products or development of multiparametric flow cytometry is highly
indicative. Apart form this
fundamental task, I believe there are, at least, three more reasons in favour of the need
for extensive researches of hematologic neoplasms: a) broadening of scientific concepts
about biologic features of hematologic neoplasms; b) permanent education of
clinicians/hematologists and experts of particular laboratory professions; and c)
maintaining the interest in laboratory medicine. The mentioned aims were main
promoters for the project of multidisciplinary leukemic cell analysis in our
institution, with special stress on the analysis of cell biologic parametres
of biphenotypic or mixed acute leukemia. Therefore, in the first part of the
exposure, I will present the principles of multidisciplinary approach in the diagnostics
of acute leukemia, while in the second part, I will demonstrate the concept of mixed
acute leukemia analysis within our project. SYMPOSIUM HEMATOLOGICAL CYTOLOGY3. EXPRESSION OF OSTEONECTIN (SPARC) IN HEMATOPOIETIC CELLS Rajko Kušec1,2
, Ika Kardum-Skelin2 , Mara Dominis2 ,
Helen Turley3 1Department
of Molecular Medicine Inst. "Ruđer Bošković" 2Clinical
Hospital "Merkur" Zagreb; Unit of Clinical Cytology and Pathology with Cytology, 3LRF
Immunodiagnostic Unit, University of Oxford SPARC/Osteonectin is a
glicoprotein significant for the bond of a cell and extra-cellular matrix. Its role
in the differentiation, embryogenesis and tissue reparation is well known and its role in
the tumor biology, specially metastasizing is becoming more famous. We have explained the
protein (immunocyto- and histochemical) expression of ON/SPARC in cells of normal and
malignantly changed hematopoiesis. SPARC, tested by antibody SSP2 (author Helene Sage,
Seattle), is present intracytoplasmatically in differentiated cells of myelo- and
lymphopoiesis until it is observed in erythroidic linkeage. Although a few previous
studies described the expression of ON/SPARC in megacaryocytes (western blotom) and
activated thrombocytes, our antibody directed to the domain of calcium bonding in a
molecule, has not confirmed that finding. The tumor blasts of acute myeloic leukemia did
not show reactivity. However, in the acute promyelocytic leukemia a part of the population
of immature cells was positive. Normal and plasma cells of multiple myeloma exprimated
ON/SPARC a lot. For that reason, we initiated a research of possible application of this
protein as a marker for the assessment of multiple myeloma tumor mass size, i.e. its
value in monitoring the minimum residual disease. SYMPOSIUM
HEMATOLOGICAL CYTOLOGY 4. LACTOFERIN IN
PATIENTS WITH MYELOPROLIFERATIVE DISEASES AND
MYELODYSPLASIA Sučić M1 ,
Boban D1 , Marković-Glamočak M1 ,
Batinić D1 , Užarević B1,
Zadro R1 , Mrsić S1 ,
Ries S1 , Gjadrov-Kuvedžić K1 , Labar B2,
Nemet D2 , Mrsić M2 , Stavljenić-Rukavina A1 Clinical Department of
Laboratory Diagnostics1 and Department of
Hematology2 Clinical Hospital
Zagreb Lactoferin, a protein
found in the secondary corpuscles of more mature granulocytopoiesis cells, has a
bacteriostatic activity. Recent reports have shown that the role of lactoferin is still
not completely known. In patients with acute
myeloic leukemia (AML) or myelodysplasia, besides immature malignant cells, very often,
at least a part of other mature granulocytopoiesis cells shows certain deviations
(cytomorphological, biochemical, immunophenotypic, cytogenetic and molecular
changes), and therefore, less lactoferin. The aim of the work is
to compare the percentage of lactoferin positive mature granulocytopoiesis cells in the
bone-marrow in patients with chronic myeloproliferative disease, in patients with MDS, and
in patients with AML during the remission of disease with the same values of
the control group. In the patients´ group
there were 11 patients with AML, 9 patients with chronic myeloproliferative disease and 6
patients with MDS. The control panel, consisted of 18 non-hematologic patients. The
disease diagnosis was established after the cytomorphological and cytochemical analyses of
bone-marrow and peripheral blood according to the FAB and WHO classification.
Lactoferin is determined in granulocytopoiesis cells by the APAAP immunocytochemical
method. The results of the comparison have shown that all the patients´ groups have
statistically significantly smaller percentage of lactoferin positive mature
granulocytopoiesis cells in relation to the control group (p<0.001). In the majority of
patients of all the three groups, certain characteristic cytogenetic and molecular changes
were determined. The working results suggest that mature granulocytopoiesis cells, in
patients with MDS and in patients with chronic myeloproliferative disease, reveal
some deviations from the mature granulocytopoiesis cells of normal hematopoiesis,
and that the granulocytopoiesis cell differentiation in patients with AML is changed
even during disease remission. SYMPOSIUM
HEMATOLOGICAL CYTOLOGY 5. MORPHOLOGICAL
CHARACTERISTICS OF ACUTE LEUKEMIA WITH CHROMOSOMAL
ABNORMALITIES Šušterčić D1
, Kardum-Skelin I1 , Borovečki A1 , Anić V1
, Minigo H1 , Vrhovac R1 ,
Hitrec V2 , Lasan R2, Kušec R3
, Jakšić B1 Clinic of Internal
Diseases KB "Merkur"1, Department of Cytogenetics of
Clinic for Childhood Diseases KBC Rebro2 , Institute "Ruđer
Bošković"3 Zagreb Certain specific
chromosomal abnormalities in acute leukemia are joined with characteristic morphology and
have different clinical picture (WHO 2000). Specific categories are defined as: AML
t(8;21)(q22;q22), 2. acute promyelocytic leukemia with t(15;17)(q22;q11-12) and variant,
3. AML with abnormal eosinophiles in bone-marrow inv (16)(p13:q22) or t(16;16)(p13;q2), 4.
AML with 11q23 (MLL) abnormalities, 4. Burkitt cell leukemia and translocation 8; 14 etc. In Clinical
Hospital "Merkur", 317 acute leukemia cytogenically typified have been treated
in the Department for Cytogenetics of the Clinic for Childhood Diseases of KBC
"Rebro". In only 10 cases (3.1%) the analysis was not possible or it was
insufficient. 47% of acute leukemia cases were without chromosomal abnormalities. In 53%
of all leukemia cases (similar rate for AML and ALL), cytogenetic abnormalities were
revealed. Good correlation
between morphological picture and pertaining abnormality has been confirmed in acute
promyelocytic leukemia and translocation 15;17, as well as in Burkitt cell leukemia and
translocation 8;14 and in acute myeloic leukemia with translocation 8;21, a little smaller
in AML with abnormal eosinophiles in bone-marrow and inversion 16, while the acute
leukemia with chromosomal change 11q23 is impossible to be morphologically identified. The morphology and
immunophenotyping themselves are not sufficient for the classification of acute leukemia,
and cytogenetic analysis is obligatory in the subtyping of specific
categories. SYMPOSIUM HEMATOLOGICAL CYTOLOGY 6. PCR IN SITU IN DIAGNOSTICS OF ACUTE MYELOID LEUKEMIA WITH MATURATION Gjadrov-Kuvedžić K,
Sučić M, Boban D, Marković-Glamočak M,
Ries S, Zadro R, Batinić
D, Mrsić S,
Stavljenić-Rukavina A, Labar B Clinical Department of Laboratory Diagnostics, Department of Cytology, KBC Zagreb The classification of the World Health Organization of
myeloid and lymphoid neoplasms includes acute myeloid leukemias bearing t(8;21), t(15;17),
inv (16) and reorganizations of MLL
gene in chromosome 11 into separate subgroups. It is clinically extremely important to identify patients,
satisfying the criterion to be classified into
these subgroups of acute myeloid leukemias, because, as a rule, they demonstrate better reaction to the therapy, faster entrance
into the disease remission which means longer life. The cytomorphological and cytochemical
results are the basis for diagnosis of acute
leukemia, but, by PCR in situ it is possible
to diagnose even leukemias that, apart from the recognizible morphological phenotype, also
contain genome changes. In our research, we
have analysed 21 patients with cytomorphologically and
cytochemically diagnosed acute myeloid leukemia with marks of maturation (M2 according to
FAB), and through the application of PCR in situ we
have diagnosed 5 leukemias positive on
t(8;21). Translocation causes the formation of fussional gene AML1/ETO. We have also
monitored the patients positive on AML1/ETO by PCR in situ during the remission of disease, which enabled us to differ malignant
immature cells from the immature cells of
normal hematopoiesis, created during the bone-marrow recovery. The application of PCR in
situ, as a morphological molecular method,
rises the sensibility of cytomorphological analysis. SYMPOSIUM HEMATOLOGICAL
CYTOLOGY 7. ACUTE LEUKEMIA WITH CORPUSCLES - CASE PRESENTATION Ries S, Boban
D, Marković-Glamočak M, Sučić M, Gjadrov-Kuvedžić K, Batinić D, Mrsić S, Zadro R, Labar
B Department of Clinico-Laboratory Diagnostics, Department of Hematology, KBC Zagreb The patient is directed to the Department of Hematology
with the diagnosis of acute myeloid leukemia. Upon the reception, the bone marrow is aspirated and the specimens for
cytomorphology, immunotyping, cytogenetics, molecular analysis and ultrastructural
analysis are collected. The blasts are MPO, sudan and ANAE negative, and PAS positive. The
metachromic staining by toluidine is shown non-specific. Immunocytochemical lymphatic
markers are positive in majority of blasts. The immunotyping finding reveals neoplasm of B-cells
precursor of "common-ALL"
immunophenotype with coexpression of myeloid markers. The cytogenetic finding excludes (15;17). SYMPOSIUM HEMATOLOGICAL CYTOLOGY 8. TRANSITION OF SYSTEMATIC MASTOCYTOSIS INTO MASTOCYTIC LEUKEMIA - CASE PRESENTATIONBoljkovac S1 , Kardum
Skelin I2 , Šušterčić D2 , Borovečki
A2, Dominis M3 , Minigo H2 , Vrhovac R2 , Jakšić B2 Private Cytological Clinic Karlovac1 , Clinic of Internal Diseases2 and Clinical Pathology and Cytology KB "Merkur"3 Zagreb A patient, 39- year old was admitted to hospital MC
Karlovac and the Clinic of Internal Diseases of KB "Merkur" for several times
because of justified suspicion on mastocytosis. Clinically, the patient suffered attacks repeated for
several times during the week, starting with the sense of suffocation,
continued by heart beating, face redness accompained by fainting. After the attack, usual
appearances were temperature, gynecological bleeding and uncontrolled bowels. The patient
lost 10 kg in 6 months with the skin itchness, specially palms and soles. The status is
regular. Among the results, there are accelerated sedimentation, leukocytosis (21×10
9/L), anemia. In the leukogram, 14% eosiphilic granulocytes were found, and by the
bone-marrow FNA 36% eosinophilic granulocytes and 24% toluidine positive cells -
mastocytes. Together with the ordinary therapy with corticosteroids, antihistamines, after
8 months the disease worsened with the everyday attacks and leukocytosis (64 - 107 × 10 9/L). The worsened condition was verified in the
aspirated fragments and bioptic bone samples - the rise in
number of atypic mastocytes for 44%, accompained by mastocytes in peripheral
blood. The karyogram of bone-marrow cells did not reveal clonic abnormalities. Cytostatics
were introduced into the therapy as well, and the patient died after 3 months under the
picture of hemorrhagic diathesis and sepsis. The systematic mastocytosis is very often combined with
eosinophilia, whilst the occurrance of lymphadenopathy, absence of skin infiltration and
combination with myeloproliferative and myelodysplastic diseases, indicates a bad
prognosis. The systematic mastocytosis can turn to acute
leukemia, usually into M1, M2 or M4, more
rarely into M6 and M7 and the most seldom into mastocytic leukemia. Mastocytic leukemia, most frequently, develops de novo, and more rarely from pigmentosis
urticaria, i.e. systematic mastocytosis. SYMPOSIUM HEMATOLOGICAL CYTOLOGY 9. AIDS-RELATED BODY-CAVITY-BASED LYMPHOMA: CASE PRESENTATIONA. Vince1 , J.
Begovac1 , H.
Kessler2 ,
Z.Šiftar1 , S.Židovec1 , M.Poljak3 , T.Jeren1 University Hospital for Infectious Diseases, Zagreb1 , Institute of Hygiene, Karl-Franzens University, Graz2 , Microbiological Institute, Medical Faculty, Ljubljana3 Background:Body-cavity-based lymphomas are rare
malignancies in HIV infected patients but because of their unusual clinical, morphological
and immunophenotypic features, they are recognized as a distinct subgroup of lymphomas
connected to human herpesvirus 8 (HHV-8) infection. Case presentation:A 39-years old HIV-positive
homosexual man was admitted to hospital
because of left sided pleural effusion which contained malignant lymphoid cells. He
responded partially to a low dose CHOP regimen and died 5 months after the diagnosis of
lymphoma. Cytology: The sediments of pleural effusion
contained exclusively large round neoplastic lymphoid cells with abundant basophilic
cytoplasm, large round nuclei with prominent nucleoli. Many cells had immunoblastic
features and some plasmocytoid differentiation, mitotic figures were numerous. Flow-cytometry:The homogenous population of large
cells expressed CD45, CD38, HLA-DR and unexpectedly CD7 positivity. Other specific T,
B, NK cells markers tested negative. PCR proved EBV and HHV-8 presence in malignant
effusion. Conclusion: Primary effusion lymphoma with
molecular evidence of HHV-8 and EBV coinfection represents a distinct clinical and
morphological entity in AIDS patients. However, immunophenotypic markers of malignant
clone can be diverse in different cases. SYMPOSIUM HEMATOLOGICAL CYTOLOGY 10. CYTODIAGNOSTICS OF NECK LYMPHADENOPATHY Branka Lončar, Blaženka
Staklenac, Biljana Pauzar, Irena Hihlik-Babić, Marija Pajtler Clinical Hospital Osijek The findings of aspirational cytodiagnostics of neck
lymphadenopathy in 271 patients have been displayed. In 188 aspirated fragments, the
benign cytological diagnosis was rendered,
and in 53 aspirated fragments malignant,
while 30 aspirated fragments were unsatisfactory for giving cytological opinions.
Comparing cytological and pathohistologic results (41 patients), the correspondence was
found in 90,2% cases, in 2,4% cases, the
results were false positive, and in 4,9% the results were false negative. The sensitivity
of method is 93,3%, and specific quality 90,9%. SYMPOSIUM HEMATOLOGICAL CYTOLOGY 11. IMMUNOCYTOCHEMISTRY IN DIAGNOSTICS AND SUBTYPING OF LYMPH NODE DISEASESKardum-Skelin I1 , Šušterčić
D1 , Borovečki
A1 , Križaj
B1 , Kardum MM2 , Šiftar Z2 ,
Planinc-Peraica A1 , Minigo H1 , Ostojić A1, Vrhovac R1 , Radić-Krišto
D1 ,
Marić-Bešić K1 , Džebro S3 , Jakšić
B1 Clinic of Internal Diseases1, Department of Clinical Chemistry2 and Clinical Pathology and Cytology KB "Merkur"3 , Zagreb Immunophenotyping in diagnostics of lymph node diseases
holds a significant position. From one point of view, it is a great help in
differentiation of reactive hyperplasia from
B NHL, and on the other hand in lymphoma subtyping which is based on the determination of
lymphatic cell differentiation markers. The aim of the work has been: 1. to analyse the adequacy
of aspirated lymph node fragment specimens on flow cytometry; 2. to analyse the presence
of clonality in order to separate the reactive from the B clonal disturbances; 3. to
analyse the correspondence of cytological and histological subtyping with immunophenotype
performed on flow cytometry; 4. to analyse the possibility of subtyping of ALCL from HB,
and largecellular lymphomas from weakly differentiated tumors of different cellular
origins through immunocytochemical analysis on smears using the combination of cellular
markers. In KB
"Merkur", during a 3-year period, 201 analyses of aspirated lymph node fragments
on flow cytometry have been performed. As a priority, the clonality was determined, and in
the cases of monoclonal neoplasms, additionally subtyping. The specimen adequacy for
analysis was 95,1%. The polyclonality in cytologically diagnosed reactive hyperplasias was
98,9% (91/93) which is a great security in the separation of malignant lymphoma of B
phenotype from reactive hyperplasias. The correspondence of immunophenotype (separation of
T and B neoplasms) in relation to a cytological diagnosis was 97,1% (102/105), and 94.2
(65/69) to a pathohistological diagnosis. The compatibility of lymphoma subtyping
immunologically, cytologically and pathohistologically has shown significantly lower
reproductibility than only 50,7% (35/69). Immunophenotyping through flow cytometry did not help us in the diagnostics
of peripheral T lymphomas and Hodgkin´s disease, In the cases when the lymph node is unaccesable to excision and pathohistological analysis in the infiltration of body liquids with lymphatic
cells, during following-up the disease, the immunocytochemical analysis on cytological
smears, with the combination of cellular markers used, helps us in making
distinction between ALCL and HB as well as largecellular
lymphomas and poorly differentiated tumors of another
biological origin. SYMPOSIUM HEMATOLOGIC CYTOLOGY 12. COMPARISON OF RESULTS OF CYTOMORPHOLOGICAL AND IMMUNOPHENOTYPING CELL ANALYSIS IN BONE-MARROW ASPIRATED FRAGMENTS IN B-NON HODGKIN LYMPHOMA (B-NHL) Šiftar Z1 , Kardum MM1 , Nazor A1 , Kardum-Skelin
I2 , Šušterčić
D2, Department of Clinical Chemistry1, Zagreb and Clinic of Internal Diseases2 KB"Merkur", Zagreb B-Non Hodgkin Lymphoma belongs to heterogenic group of immunologic system diseases which are
morphologically and clinically presented in a different way. The most frequently affected
localizations are lymph nodes, spleen, bone-marrow, but also, due to the lack of
immunological reaction, other organs can be
affected. The utility of cytomorphological analysis and
immunophenotyping by the flow cytometry method on bone-marrow
aspirated fragments has been examined in the cases of justified suspicion on bone-marrow infiltration with malignant cells. In
the period from July, 1977 to the end of
February, 2000 on 41 bone-marrow aspirated fragments, both analyses were performed at the
same time in order to detect bone-marrow
infiltration with B-NHL cells which was diagnosed simultaneously or immediately before in
the cytological aspirated lymph node. Out of the total number of aspirated bone-marrow fragments, in 35 (88%) of them bone-marrow affection with malignant clone was
proved by flow cytometry method, and at the
same time, by a cytomorphological
examination, the same thing was proved in 30 cases (73%). The correspondence of opinions
in both methods has been achieved in 35 cases (85) among which 30 samples were declared
positive and 5 negative. Disagreement existed in 6 cases; 5 were revealed positive through
the flow citometry method, with negative
cytomorphological results, while one was cytomorphologically positive, and negative in
immunophenotyping. According to the REAL lymphoma classification, out of 30 bone-marrow
fragments with the proved B-NHL cells by both methods, 19 (63%) were identically characterized, while in 11 cases
there was a certain disagreement in opinions, which indicated the possibility of
insufficient usage of the REAL classification, i.e. limitation of the applied methods. The basic application of both methods in detection of bone-marrow infiltration with malignat B-NHL cells
has been proved as a very successful one,
with high correspondence in results, and with methods mutually compensating. SYMPOSIUM HEMATOLOGICAL CYTOLOGY13. CHROMOSOMAL ABNORMALITIES IN LYMPH NODES FROM CYTOLOGICal ASPIRATED FRAGMENTS Šušterčić D1 , Kardum-Skelin
I1 , Borovečki
A1 , Hitrec
V2,
Lasan R2, Kušec R3 , Planinc-Peraica
A1 , Ostojić
S1 ,
Radić-Krišto D1 , Jakšić
B1 Clinic of Internal Diseases KB"Merkur"1, Department of Cytogenetics of Clinic for Childhood Diseases KBC "Rebro"2, Institute "Ruđer Bošković"3, Zagreb The division of lymphoid neoplasms
according to classification of the World Health Organization (WHO) is based on
morphological, immunophenotypic, genetic and clinical indicators. The aim of the work was to 1. analyse the
adequacy of lymphatic cell cultures obtained by cytological lymph node FNA; 2. compare
chromosomal aberations with cytological diagnosis of neoplastic disturbances in a lymph
node. The results have shown that in 22/25 specimens (88%) there
was enough material for a chromosomal analysis. The valid karyotype was found in 24% of
specimens; in reactive hyperplasia (3/3); Non Hodgkin lymphoma T peripheral type (2/2) and
lymphoblastic NHL (1/4). Chromosomal abnormalities were found in 64% of the examined lymph
nodes: hyperdysploidias in lymphoblastic lymphoma and immunocytoma. The specific
chromosomal translocations corresponded the cytological diagnosis in 82% of the cases. At
the same time, complicated abnormalities, characteristic for various NHL subtypes, were
found in the same specimen. The cell culture from a lymph node aspirated fragment is
an adequate method for chromosomal analysis which provides additional information in a
more precise diagnosis of lymph node disturbance, specially in particular NHL subtypes, as
well as in the separation of complicated abnormalities as bad prognostic subgroups. SYMPOSIUM HEMATOLOGICAL CYTOLOGY 14. T-gama - LYMPHOPROLIFERATIVE DISEASE (T-gama LPB)- CASE PRESENTATION Kardum MM1 , Šiftar Z1 ,
Planinc-Peraica A2 , Kardum-Skelin
I2 , Šušterčić D2 , Borovečki
A2 , Nazor A1 , Jakšić
B2 Department of Clinical Chemistry1 and Clinic of Internal Diseases2 KB "Merkur", Zagreb A patient, 31- year old applies in the
surgery of the Clinic of Internal Diseases KB "Merkur" in January, 1998 because
of higher temperatures, frequent infections and sore throat in the last year (in 1986
recovered from toxoplasmosis). The laboratory findings demonstrated normal number of
leukocytes (L=6.9; Hb 147; Tr 203), but DKS indicates absolute lymphocytosis and
neutropenia (Ly=75%, Seg=9%). Suspected of lymphoproliferative disease, the patient is
directed to the Infectious Clinic because of
infectous examination (documentation uncompleted). In June, the same year, he is
ambulatory treated in the Hematological Surgery in the Clinic of Internal Diseases, where,
cytologically processed, infiltration
of peripheral blood (Ly=76%) and bone-marrow (Ly=29%)
with multiplied lymphocytes
with azurophilic corpuscles in cytoplasma (T-gama-LPS) is detected. By cellular immunophenotyping of peripheral blood and bone-marrow, the
T-lymphocytes of phenotype: CD2+CD3+CD7+CD8+CD4-CD57+CD38+CD56- wtih the aberrancy of CD5
marker (positive on approx. 65% mature T-lymphocytes) and with the domination of cytotoxic-surpressing T-lymphocytes
(CD3+CD57+) with Cd3+CD8+>>CD3+CD4+) (phenotype T-gama lymphocytes) are revealed.
The sum of mature T-lymphocytes subpopulations ( approx. 77%) is far smaller than the
demonstrated marker positivity of these cells (CD3=90.4%). In the pateint´s plasma,
neither antigranulocyte nor leukoaglutinating antibodies are confirmed, and the abdomenal
ultrasound describes liver and spleen valid
structure. The lymphadenopathia is not detected. The bone-marrow karyogram is valid (46xy)
and without clonal disturbances. The histological results, with the description of
normocellular bone-marrow with interstitially slightly increased mature lymphatic cells,
do not confirm positive cytological and immunophenotyping findings of T-gama-LPS. Although
the majority of T-gama-LPB is cytomorphologically homogeneous,
and the demonstrated immunophenotype of T-lymphocytes suggests the phenotype
T-gama-lymphocyte, a decreased sum of subpopulations of mature T-lymphocytes, in relation
to their total number, could suggest the presence of "atypic" T-gama-lymphocytes
within T-gama-LPB. POSTER SECTION 15. LYMPHOCYTIC PROLIFERATION ASSAY BY FLOW CYTOMETRY M.Rudolf, B.Užarević,
D.Batinić Department of Immunology, Clinical Department of Laboratory Diagnostics, KBC Zagreb The ability of lymphocytic proliferation under the
influence of non-specific mitogenes or antigenes in cultures in vitro was determined by
various methods. One of the methods, being lately used,
is the analysis of cellular cycle by propidium iodide on flow cytometry.
Propidium iodide is a stain which is stechiometrically
linked to DNK double helix which makes it
suitable for monitoring the DNK contents in a cellular cycle. Through a DNK
analysis on flow cytometry, it is possible to
determine different phases of cellular cycle such as G0/G1, S, and G2/M. Monitoring a DNK
synthesis (S-phase), we determine cellular proliferation, i.e. the quantity of de novo
formed DNK, after the stimulation of lymphocytes by mitogenes or antigenes. The
lymphocytic proliferation assay is based on the Hartzman and alt. method. Cells are
isolated from peripheral blood on the ficoll-paque gradient. After the lavation in media
199, lymphocytes are adjusted to the
concetration of 6-8 × 10 6/mL. Inactivated AB serum, purified lymphocytes, media 199, and optimally diluted mitogenes or antigenes are added
into sterile plates for microculture. Three specimens with each mitogen or antigen plus
three conrols without mitogen or antigen are processed at the time. The cultures are incubated 64-72 hours at 37
degrees centigrades with constant conditions of Co2, air and humidity flow. After incubation, the
cells are collected, and stained with Vindel´s colour that contains
propidium iodide, and in that way they are prepared for flow cytometer measuring in the
Cellquest programme. The results are analysed
in the ModFit programme and expressed in relative percentages. The lymphocytic proliferation assay has a broad
application in basic and clinical researches. It is used in the research of congenital
immunological defects, in monitoring the immunosurpressive
therapy, for determination of different cytokines, detection of serum factors which can
prevent or initiate immunological reaction, for monitoring the patient's immunological status in different
clinical states such as surgery operation, anestesia, transplatation, malignant diseases
etc. POSTER SECTION 16. BONE TISSUE CHARACTERISTICS IN MYELODYSPLASTIC SYNDROME Vesna Kušec, Mara Dominis, Rajko Kušec, Mladen
Petrovečki Clinical Department of Laboratory Diagnostics KBC Zagreb, Department of Pathology KB Merkur, Institute Ruđer Bošković, Department of Biochemistry KB Dubrava, Zagreb The changes in bone-marrow
substance in myelodysplastic syndrome (MDS) influence
activities in bone tissue. The
aim of this work is to evaluate the bone
tissue characteristics and their connection to survival. The examination comprised 51
patient with MDS (33 M, 18 F, aged 20-83, monitored 1-55 months) in which the evaluation
of bone-marrow and histomorphometrical analysis of bone biopsy were performed. The bone
volume, surface with osteoid and trabecular obesity were averagely decreased, and the
surface with osteo-resorption and number of
trabeculae increased in relation to the control panel. The age, sex or diagnostic subgroup
did not seem to impact the results of histiomorphometrical analysis or other indicators in
this research. Patients, with no atypic blast
rise for diagnostic MDS subgroup observed,
lived longer. In these patients, longer survival was also connected to increased bone
resorption, i.e. resorption higher than 8% was a precondition for survival during the
monitored period. These findings suggest the existance of bone-marrow changes in MDS
patients during establishing the diagnosis. Patients without atypically high rate of
blasts in the marrow and with increased bone
resorption lived longer, which indicates the
existance of favourable conditions in the bone-marrow for differentiation and acitivity of
osteoclasts, cells originating from the moncyte-macrophagal lineage. POSTER SECTION 17. PRESENTATION OF A PATIENT WITH ACUTE LEUKEMIA AND MALIGNANT SCHWANNOMA Ries S, Hutinec
Z, Boban D,
Marković-Glamočak M, Sučić M, Gjadrov-Kuvedžić
K, Podolski P,
Kovačević-Metelko J Department of Clinico-Laboratory Diagnostics, Department of Hematology, KBC Rebro A patient applies, in 1995, because of lymphadenopathy and
hypersplenism. The lymph node FNA is performed and the elements of granulomatous
inflammation are revealed The bone-marrow FNA demonstrates scanty hematopoietic tissue,
morphologically within the normal limits. Pathohistologically: tuberculous lymphadenitis Treated by antitubercolotics. The spleen still enlarged. At the beginning of 1998, pancytopenia occurs. In the
bone-marrow there are 14% of blasts and in the peripheral blood smear 10% of blasts. Due
to MDS suspected, a bone biopsy has been
performed. Considerable hematoma occurrs and it is surgically processed with the sample
collected for PHV. The pathohistological findings correspond
to malignant Schwannoma which infiltrates
ischium and spreads into bone-marrow. The patient is undergoing an appropriate chemo-therapy. During the control bone-marrow
FNA, AML (70% blasts) was diagnosed entering remission
after the therapy. POSTER SECTION 18. NON-HODGKIN LYMPHOMA OF ADRENAL GLANDS WITH CEREBRAL INFILTRATION Borovečki A1 , Kardum-Skelin
I1 , Šušterčić
D1 , Sabljar-Matovinović M1 , Prkačin I1 , Ostojić S1 , Planinc-Peraica A1, Drinković I2 , Škegro
D1 ,
Gašparov S3 , Žarković
K4 Clinic of Internal Diseases1, Department of Radiology2 and Clinical Pathology and Cytology KB "Merkur"3, Department of Neuropathology KBC Rebro4, Zagreb A patient, 67- year old was admitted to the Clinic of
Internal Diseases KB "Merkur" in October, 1999. Three months earlier he had
started to complain about weakness and fatigue accompained by loss of 15 kg, and the
feeling of instability in walk and speech. By ultrasound
and CT examination, a both-sided magnification of adrenal glands (on the right 48×60 mm,
and on the left 36×36 mm) was identified. A cerebral
CT suggested atrophic changes
frontally. The laboratory results did not suggest a primary process of adrenal glands, by
pulmonological processing a lung primary process was excluded as well, and gastroscopic
results were valid. Upon the admittance to our Clinic, the patient was difficult to
contact, afebrile. It was
heteroanamnestically learned he intensively perspired with
coreotic movements observed. The diagnosis of B largecellular Non-Hodgkin
lymphoma - DLBCL (cells are positive on CD 45 and CD 20, and negative on CD 30 and Cd 3)
was rendered by cytological FNA under the
ultrasound control based on cellular
morphology and cellular markers on cytological slides. The patient´s condition was
temporarly improved after an ordinary therapy, but still
extremely difficult. Two months after the admittance, the patient died. The
autopsy findings confirmed infiltration of
both suprarenal glands and clinical suspicion of cerebral
infiltration with largecellular B lymphoma
cells. The
localization of NHL in adrenal glands and brain is extremely rare. The cytological
analysis, completed by cellular markers, can independently isolate that type of tumor from
other adrenal gland tumors. |
