6/29/2016
Angelique Jermac, CG(ASCP)CM
Lane Wilcox, CG(ASCP)
Daniel L. Van Dyke, PhD
Mayo Clinic
AGT 41st Annual Meeting
June 25, 2016
INTRODUCTION
• Acute Lymphocytic
Leukemia (ALL)
• 80% childhood
leukemias
• Genetic
aberration
• Proliferation &
lack of
differentiation
INTRODUCTION
• Cytogenetic
testing
• Challenging
• Well spread,
high quality
metaphase
chromosomes
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INTRODUCTION
• Improve quality
• COG Conference
• South Australia
Cancer Cytogenetics
Unit (SACC)
• Validation
INTRODUCTION
Comparison of clinical harvest
method with SACC “validation”
method
Clinical
Validation
MATERIALS & METHODS
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MATERIALS & METHODS
Mayo
SACC
Clinical Method
Validation Method
Part I
Part I
Chang BMC
HTB-9
Conditioned
Media
Media
MATERIALS & METHODS
• Supplemented Media
•
•
•
•
•
•
•
Cell line 5637-ATCC HTB-9 bladder carcinoma
T-25 flask- complete media
Split - 8 T-150 flasks
Add media
Ultracentrifuge
Sterilize & freeze at -80C
Supplement
MATERIALS & METHODS
CHANG BMC
•
•
•
•
•
•
•
G-CSF
GM-CSF
M-CSF
IL-1
IL-6
TPA
human erythroid
enhancing activity
• prostaglandin E
HTB-9 SUPPLEMENTED
MEDIA
• G-CSF
• GM-CSF
• M-CSF
• IL-1
• IL-6
• IL-7
• IL-8
• TNF-alpha & beta
• SCF
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MATERIALS & METHODS
Mayo Clinical
Method
SACC Validation
Method
PART I
PART I
Chang BMC
HTB-9 media
24 hr culture
FudR + U
BrDU
EtBr
Colcemid
KCl hypotonic
Cold 3:1 fixative
24 & 48 hr cultures
Colcemid
KCl Hypotonic
Room temp 2:1
fixative
MATERIALS & METHODS
PART I
•Cultured &
Clinical & harvested
Validation •Slide prep
Samples •Baked &
stained
MATERIALS & METHODS
PART I
• PRE-ASSESSMENT
• Blinded Analysis
• Five cases
• Mitotic index
• Bands on ten
• Metaphase quality
(rating scale 1-5)
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MATERIALS & METHODS
Metaphase Quality
Rating Scale
Example: Rating of 1 (poor)
Example: Rating of 5
(excellent)
MATERIALS & METHODS
Part I
• Blinded analysis
–20 cases
–Completely analyzed
• Comparison
–Abnormal cells
MATERIALS & METHODS
Mayo Clinical
SACC Validation
Method
Method
PART II
PART II
CHANG BMC
CHANG BMC
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MATERIALS & METHODS
Mayo Clinical
Method
SACC Validation
Method
PART II
PART II
Chang BMC
24 & 48 hr cultures
Colcemid
KCl Hypotonic
Room temp 2:1
fixative
Chang BMC
24 hr culture
FudR + U
BrDU
EtBr
Colcemid
KCl hypotonic
Cold 3:1 fixative
MATERIALS & METHODS
PART II
•Cultured &
Clinical & harvested
Validation •Slide prep
Samples •Baked &
stained
MATERIALS & METHODS
Part II
• Blinded analysis
–18 cases
–Completely analyzed
• Comparison
–Abnormal cells
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RESULTS
PART 1- PRE-ASSESSMENT
Table 1. Bands on Ten Averages
• Bands on ten
• Validation
method
RESULTS
PART 1 - PRE-ASSESSMENT
Table 2 Mitotic Index
• Mitotic index
• Validation
method
RESULTS
PART 1 - PRE-ASSESSMENT
Table 3. Metaphase Quality
• Metaphase quality
• Validation method
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RESULTS
PART I
• 20 cases
–13 Normal
–Clinical: 7 abnormal
–Validation: 5 abnormal
• Missed 2 abnormals
RESULTS
PART 1
Clinical Results
1
2
3
4
5
6
7
Validation Results
46,XY,add(3)(q12)[7]
/46,XY[13]
46,XY[14] 61,X,+X,+4,+5,+6,+8,
+der(10)t(1;10)(q21;q24),+11,+14,+17,
+18,+21, +21,+22[cp20]
46,XX,t(9;22;11)(q34;q11.2;q13)[2]
/46,XX[18]
46,XY,t(1;19)(q23;p13.3)[15]
/46,XY[5]
46,XY,del(9)(p13),t(11;14)(p11.2;q11.2)[cp20]
45,XY,der(7;17)(q10;q10),+8
dic(9;12)(p11;p12),[cp18]
/46,XY[2]
46,XX,t(8;11)(q24.1;p13)[19]
46,XY[10]
46,XY[7]
46,XX,t(9;22;11)(q34;q11.2;q13)[1]
/46,XX[13]
46,XY,t(1;19)(q23;p13.3)[15]
/46,XY[2]
46,XY,del(9)(p13),t(11;14)(p11.2;q11.2)[19]
/46,XY[1]
45,XY,der(7;17)(q10;q10),+8,
dic(9;12)(p11;p11.2)[20]
46,XX,t(8;11)(q24.1;p13),del(16)(q22)[cp15]
/46,XX[5]
RESULTS - PART I
Patient #1
Abnormal
Chang BMC
Clinical Harvest
Normal
HTB-9 Media
vs Validation Harvest
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RESULTS - PART I
Patient #2
Abnormal
Chang BMC
Clinical Harvest
Normal
HTB-9 Media
vs Validation Harvest
RESULTS
Part II
• 18 cases
–12 Normal
–Clinical: 5 abnormal
–Validation: 5 abnormal
• Each method missed one
abnormal case
RESULTS
PART II
Clinical Results
2
46,XY,t(2;8)(p21;q24.1),add(5)(q13),
del(7)(p11.2),der(7;11)(q10;q10),
add(10)(p11.2),add(12)(p11.2),
-17,add(18)(q21),[cp13]/
/46,XY[7]
47,XY,+10[2]
/46,XY[18]
3
46,XX[20]
4
46,XX,t(9;22)(q34;q11.2)[18]
/46,XX[2]
47,XY,+add(1)(p13),del(11)(q13q23)[16]
/46,XY[4]
1
5
6
56,XY,+X,dup(1)(q25q32),+4,+6,+10,
+add(14)(q32),+add(17)(p11.2),+18,+21,+21,
+mar[16]
/46,XY[4]
Validation Results
45-46,XY,-6,del(7)(q22),add(17)(p11.2),
der(19)t(6;19)(p21.3;p13.1),[cp3]
/45-46,XY,add(17)(p11.2)[cp9]
/46,XY[8]
46,XY[12]
46,XX,inv(5)(p13q11.1)[2]
/46,XX[18]
46,XX,t(9;22)(q34;q11.2)[20]
47,XY,-11,+1-3mar[cp3]
/46,XY[17]
56,XY,+X,dup(1)(q23q32),+4,+6,+10,
+add(14)(q32),+del(17)(p11.2),+18,+21,+21,
+mar[18]
/46,XY[2]
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RESULTS –PART II
Patient #3
Abnormal
Chang BMC
Clinical Harvest
Normal
Chang BMC
vs Validation Harvest
RESULTS –PART II
PATIENT #4
Normal
Abnormal
Chang BMC
Chang BMC
Clinical Harvest
vs Validation Harvest
CONCLUSION
• Chang BMC appears to stimulate abnormal
cells somewhat better than HTB-9 media.
• Validation method
– Bands on ten
– Mitotic index
– Metaphase quality
• Unsure about using HTB-9 conditioned media/
validation method as a 3rd culture –ALL
• Continue to experiment and collaborate with
SACC to improve the quality of our ALL
samples.
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Acknowledgements
• Dr. Van Dyke
• Lane Wilcox
• Mayo Clinic Cytogenetics Laboratory
ANY QUESTIONS?
ANY QUESTIONS?
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