Assessment of monoclonality in large B cell non-Hodgkins lymphoma Lotfi N. M.Sc Rastin M, Ph.D Memar...
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Transcript of Assessment of monoclonality in large B cell non-Hodgkins lymphoma Lotfi N. M.Sc Rastin M, Ph.D Memar...
Assessment of monoclonality in large B cell non-Hodgkins
lymphoma
Lotfi N. M.ScRastin M, Ph.DMemar B, MD
Mahmoudi M. MD, Ph.D
Immunology Research Center, BuAli Research Institute, Mashhad University of
Medical Sciences
Centroblastic lymphoma
Immunoblastic lymphomas
Subdivision of large B-cell lymphomas by morphology
Composed of large cells
Resemble centroblasts or immunoblasts
Often with a mixture of the two
Clinical significance is yet debated
Centroblastic type (80% of the cases) is composed of :
- cells resembling germinal center centroblasts
- with one to three peripheral nucleoli
- a narrow rim of basophilic cytoplasm
-mixture of both immunoblasts and centroblasts
-In crucial cases:
-Morphological criteria do not help distinguish
-Diagnosis of NHL still represents a challenge
-In reactive and malignant lymphoproliferations
- Molecular Diagnostic methods are useful tools
-Clonality assays are helpful
- Demonstration of monoclonality in gene level
- In B cell lymphomas is of great importance
-Distinguish lymphomas from reactive lymphoid lesions
-When morphological and immunophenotypic features are difficult to interpret
Southern blot analysis of Ig genes:
Is the established method for monoclonality demonstration
However it is:
Slow
Complex
Requires fresh samples
Expensive
Polymerase chain reaction overcome limitations
- Offers :
Sensitive
Rapid
Simple
Flexible
Detection of B cell monoclonoclonality
PCR
VH-N-DH-N-JH
FR1 CDR1 FR2 CDR
2 FR3 N D N FR4
CDR3
3´5´
5´ 3´
CHJHDHVH
DH-N-JH
100-150 bp
Methods & Materials
DNA ExtractionPCR with Specific primers
Electrophoresis and staining
Method
Receiving sample blocks from Pathology
archive
Reading OD and PCR with beta actin
primer
Samples obtained from:
-archives of : Imam RezaGhaem Omid Hospitals private laboratories in Mashhad
Samples include 44 DLBCL specimens
20 samples from tonsil and appendix as controls
These cases were reviewed by a pathologist for confirmation.
5 µm sections were cut from paraffin blocks
Samples were deparraffinized
DNA extracted using proteinase K digestion method
ß -Actin gene amplified To confirm extracted DNA quality
PCR reaction
To analyze the PCR products :
15µl of each PCR product was electrophoresed on
8% PAGE
Stained with AgNo3
50 bp
FR3 N D N JH
FR3 N D N JH
FR3 N D N JH
MW 1 2 3 4 5 6
FR3 N
FR3 N
FR3 N
D N JH
D N JH
D JHN
50 bp
1 2 3 4 MW
100 bp
1 2 (c) 3 4 5 6 MW
125bp
Samples
Number
Positive Results
Monoclonality
DLBCL
4433%75
Control
200%0
Results:- all samples had adequate DNA quality
- using primers amplifyng FRIII-JH region
- clonal IgH gene rearrangements
- demonstrated in 75%(33/44) of the cases.
Disccusion and conclusion
Previous studies YearResults
Fc- Linng et al199356%
Yon- Chin- Tai et al
200254.3%
Adan-Begg et al200247%
Timeap. Gurbitty et al
200355%
Thanks for Your Attention
Thanks for Your Attention
100 bp
1 2 3 4 M 5 6 7 8 9 10 1 2 3 4 M 5 6