Molecular Haematology for Haematologists
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Transcript Molecular Haematology for Haematologists
Molecular Haematology
Alberto Catalano
email: [email protected]
10 Mar 2011
Outline of topics
Molecular biology refresher
Molecular biology in haematology
Quality control issues
Laboratory layout and equipment
Case studies
Our wish list
Molecular biology
DNA or RNA: when and why
Genes and gene structure
Methodology
Nucleic acid extraction
DNA electrophoresis
Polymerase chain reaction (PCR)
Quantitative PCR
High resolution melting (HRM)
DNA sequencing
Chromosomes are DNA
Sugar + Phosphate + Base
Sugar + Phosphate
form the backbone
DNA: R = H
RNA: R = OH
Base-pairing
Purines
Pyrimidines
DNA or RNA: when and why
One chromosome = 1 dsDNA molecule
Autosome pair = 2 dsDNA molecules
Mitochondrial DNA (many copies/cell)
DNA is more stable for analysis
DNases are easily heat denatured
DNA autolysis is minimal under normal pH & temp
RNA is less stable
RNases are ubiquitous & difficult to remove
RNA autolysis in mildly acidic pH
The three bones from Vindija from which Neandertal DNA was sequenced.
38,310yo
44,450yo
DNA or RNA: when and why
Gene level:
DNA there whether expressed or not
RNA copies depend on level of expression in cell
Different cell types: Different expression levels
Messenger RNA is an “edited” version of DNA
Shorter
Without introns
… because of large introns and variable
genomic breakpoints
mRNA lacks the introns present in gDNA
Fusion genes are more easily amplified from a
shorter sequence
Breakpoint clustering and multiple breakpoint
clusters
Sizes of PCR products indicate the types of
breakpoints present
However, RNA is less stable than DNA
Gene structure
Promoter
Transcription start site
Exons
Poly adenylation site
Open reading frame
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
7-Methylguanosine cap
Poly A tail
ORF
Alternative splicing
One gene : many mRNA transcripts: many protein isoforms
DNA
Primary RNA transcript
Skipped exon
Skipped exon
RNA splicing
Altered reading frame
Genomic Breakpoints in BCR-ABL
Sample preparation
Purity of sample
Hypotonic lysis of blood or bone marrow
Ficoll density fractionation
Speed & sample turnover
RNA stabilization
DNA extraction
Quick for robust assays (e.g. blood boiling)
High purity DNA for troublesome assays
RNA Stabilization
• Guanidine isothiocyanate
• Trizol reagent
– mono-phasic solution of phenol and guanidine
isothiocyanate
• RNA-Later
– Ammonium sulphate protein precipitation
– Stabilises RNA at ambient temperature
– Suitable for transport with minimal packaging (post)
RNA-based assays
RNA purification from Trizol lysed cells
Synthesis of copy DNA (cDNA) from RNA
Gene-specific PCR of:
Target gene (leukaemia specific fusion gene)
Control gene (“housekeeping” gene) to assess the
quality of the RNA
For quantitative assays:
Result = fusion gene copies / control gene copies
RNA preparation from cells using
Trizol® reagent
Inherited disorders
Thalassaemias
Hereditary Haemochromatosis
Factor V Leiden, MTHFR (Ala677Val),
Prothrombin gene 20210 mutation
Clonal disease
Clonal markers
X chromosome inactivation (HUMARA assay)
Gene rearrangements
Antigen receptor genes
T-cell receptor
Immunoglobulin
Abnormal fusion genes
Mutations
Loss of heterozygosity & uniparental disomy
(acquired)
Qualitative nested PCR
FIP1L1-PDGFRA for CEL
a
b
Patient samples
10-3 10-4
Patient samples
10-3 10-4
M - N 1 2 3 4 5 6 + + W M - N 1 2 3 4 5 6 + + W
Assays run in duplicate
Slight differences due to stochastic factors e.g. Pt # 4
Controls to give an estimate of assay sensitivity
Transferrin receptor TFRC
RNA quality control
Patient samples
M
-
N
1
2
3
4
5
6
Positive TFRC indicates RNA quality is acceptable
ABL qPCR
RNA quality control
110
0.60
100
0.55
90
0.50
80
0.45
Fluorescence
Norm. Fluoro.
0.40
70
0.35
60
0.30
50
0.25
40
0.20
30
0.15
20
0.10
Threshold
10
0.05
0.00
0
55
10
10
15
15
20
20
25
25
Cycle
Cycle
30
30
35
35
40
40
45
50
50
ABL qPCR
RNA quality control
Cycling
CyclingA.Green
A.Green(ABL):
(ABL):
R=0.99993
R=0.99993
R^2=0.99986
R^2=0.99986
M=-3.529
M=-3.529
B=40.693
B=40.693
Efficiency=0.92
Efficiency=0.92
30
30
29.5
29.5
29
29
28.5
28.5
28
28
CT
CT
27.5
27.5
27
27
26.5
26.5
26
26
25.5
25.5
25
25
24.5
24.5
24
24
23.5
23.5
23
23
3
3
10 10
3.5 3.5
10 10
4 4
1010
Concentration
Concentration
4.5
4.5
10
10
5
10
10
Reduction of T315I mutation
16/2/2011
~100% T315I
25/1/2011
10%-30% T315I
Residual disease monitoring
Detection of fusion gene or clonal TCR or IgH
Highly sensitive
Very early detection of molecular relapse
Quantitation of fusion gene
Allows monitoring of treatment effect
Detection of relapse
Chimerism in post-transplant patients
Sensitivity of TCRg PCR analysis
Norm. Fluoro.
BCR-ABL qPCR
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
Threshold
0.00
5
10
15
20
Cycle
25
30
35
40
BCR-ABL IS calculation
BCR-ABL copies (average of 2 cDNA)
IS=
BCR copies (average of 2 cDNA)
X laboratory correction factor
Correction factor checked annually against reference laboratory
(IMVS Royal Adelaide Hospital)
Laboratory layout and
equipment
Measures to ensure quality
Laboratory Automation
Reducing manual handling reduces
chances of sample mix-up
Promega Maxwell 16
Automated DNA extraction system
Uses magnetic bead cartridges containing lysis and wash reagents
CAS-1200 Robot
Real time PCR instrumentation
Rotor Gene™ 6000 (Corbett Research)
36 / 72/ 100 well rotor format
Thermal uniformity ±0.01°C, Resolution ±0.02°C,
HRM data acquisition (read) rate: 20 reads for each 0.02°C
increment
5-20 µl Capacity
15 minutes per run (after amplification)
HRM, real time PCR and allelic discrimination (5 colours)
Rotor Gene™ 3000 (Corbett Research)
36 / 72 well rotor format
5-20 µl Capacity
Real time PCR and allelic discrimination (4 colours)
LABORATORY FACILITIES
• Laboratories configured to minimise the risk of
contamination of samples and reagents by
amplified material or other samples in the
laboratory
• Minimum Standards
• Additional Standards for Nested PCR
Minimum Standards
• area for the extraction of nucleic acids from
samples and for the addition of sample DNA
• dedicated clean area for the preparation of
reagents (including dispensing of the master
mix)
• a dedicated, contained area for amplification and
product detection
Workflow
Sample
preparation
Reagent
preparation
Template
addition
Detection
Amplification
Laboratory Layout
Sample preparation
Reagent preparation
Template addition
Amplification
Detection
1
2
3
Laboratory Layout
Sample preparation
Reagent preparation
Template addition
Amplification
1
2
Detection
Quality assurance
Undertaking a volume of testing that is
sufficient to maintain the knowledge, experience
and expertise of staff
Benefits of centralisation versus those of
developing local expertise and autonomy
Associations or collaborations between
diagnostic laboratories and research laboratories
are encouraged for small volume testing
RCPA QAP programme
JAK2 V617F
BCR-ABL
PML-RARA
DNA Chimerism
Factor V Leiden, Prothrombin 20210,
MTHFR (A677V)
BCL1, BCL2, TCR, IGH
Thal a, Thal b
Haemochromatosis Cys282Tyr, His63Asp
Inter-laboratory sample
exchanges
Informal regular exchanges of samples with
other laboratories
Blinded
Comparison of sensitivities
Not generally surveyed by RCPA QAP
For establishment of new methods
Contamination
specimen collection or transport
handling or testing in the testing or referring
laboratory before nucleic acid detection
during:
extraction of nucleic acids from the sample
amplification
product detection
by contamination from the reagents used for the
test
Contamination Sources
positive samples (cross contamination);
amplified nucleic acid (e.g. contamination of
stock reagents or equipment, or in aerosol
droplets);
Measures to Control Contamination
• the competency of staff at performing
laboratory tasks
• the routine use of controls to detect
contamination
• Splitting samples
• Uracil-N-glycosylase (UNG)
• the design of the laboratory
Uracil-N-glycosylase
• If dUTP is used instead of TTP in PCR
• Uracil-N-glycosylase (UNG) cleaves
contaminating PCR products prior to PCR
• Real template lacks dU and therefore is not
degraded
• Prevents amplification of minor amounts of
contaminating DNA
• Cannot prevent gross contamination
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UNG
UNG treatment
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dUTP incorporation
Nested PCR
• Products from the 1st round of PCR are used as
templates for 2nd round of PCR
• Requires 4th isolated area
– Laboratory
– Class 2 biosafety cabinet within area 2
• Uracil-N-glycosylase (UNG) :
– 1st round: + UNG , - dUTP
– 2nd round: - UNG , + dUTP
Sample processing
Hypotonic lysis:
Ficoll purification of mononuclear cells:
For low level JAK2 V617F
Granulocyte and T-cell isolation:
PML-RARA
Ficoll purification of granulocytes:
BCR-ABL, AML-ETO, CBFB-MYH11,
FIP1L1-PDGFRA, JAK2
DNA chimerism
DNA-based assays: JAK2, chimerism
RNA-based assays: all others
DNA Sequencing
Four different fluorophores incorporated
into primer or dideoxy-NTP
A dideoxy-NTP terminates strand
extension
Cycle sequencing with thermostable DNA
polymerase
Four bases electrophoresed in same gel
capillary
Multiple capillaries
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52
Molecular Haematology
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•Random incorporation of ddNTP
•Fragments separated by electrophoresis
•Fluorescent signals one base apart
•Colour indicates sequence of
complementary strand
ATTAGCGCACGCGATATTCCGGGACAT
ATG
ATGTCCCGGAATATCGCGT
ATGTCCCGGAATATCGCG
ATGTCCCGGAATA
ATGTCCCGGAATATCG
ATGTCCCGGAA
ATGTCCCGG
ATGTCCCGGAATATCGCGTGCG
ATGTCCCGGA
ATGTCCC
ATGTCCCGGAATATCGCGTGCGCTA
ATGTCCCGGAATATCGC
ATGTCCCGGAATATCGCGTGCGCTAAT
ATGTCCCGGAATAT
ATGT
ATGTCCCGGAATATC
ATGTCCCG
ATGTCCCGGAATATCGCGTGC
ATGTCCCGGAATATCGCGTGCGCTAA
ATGTCCCGGAAT
ATGTCCCGGAATATCGCGTGCGCT
ATGTC
ATGTCCCGGAATATCGCGTGCGC
ATGTCC
ATGTCCCGGAATATCGCGTG
DNA template
DNA Sequencing
Sequencing results
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Somatic cell genetics is complicated by the background of normal cells
and clonal evolution
Polymerase chain
reaction
(PCR)
Steps of PCR
DENATURATION
PRIMER ANNEALING
PRIMER EXTENSION BY POLYMERASE
20 to 21 (i.e. 1 copy to 2 copies)
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21 to 22 (i.e. 2 copies to 4 copies)
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22 to 23
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(i.e. 4 copies to 8 copies)
…and so on,
the number of copies doubling with each cycle
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PCR amplifies a gene of interest
Each cycle of PCR doubles the number of
copies of the gene
10 cycles… approx 1000 fold (210)
20 cycles… approx 1,000,000 fold (220)
30 cycles… approx 1,000,000,000 fold (230)
“Invisible” amounts of DNA become “visible”
Post-PCR analysis
Electrophoresis
DNA
Sequencing
Hybridization
Restriction digestion
Denaturing HPLC
High resolution melting
Loading the samples in the wells
of an agarose gel
Apply voltage to electrophoresis
apparatus
Ethidium bromide stained DNA
Ethidium bromide stained DNA
under ultraviolet light
Real-time and quantitative PCR
Detection of labelled PCR products while
cycling
By using internal standard (gene dilutions)
can be used for quantitation
Large variety of detection technologies
and instrument platforms
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Common rtPCR
Chemistries
SYBR Green I
TaqMan (5’-nuclease) probes
dsDNA
+ Dye
+ light
= Fluorescence
more DNA
more incorporated dye
more fluorescence
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Taqman probe example
TET fluorescence quenched by BHQ-1
BHQ-1
TET
AAGACCCGAC C A A G C A C T A G T C C A T C T
Probe is complementary to PDGFRA exon 12/13 junction
exon 13
GTCGGGTCTTGGGGTCTGGAGCGTTTGGGA...
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5’ nuclease assay
exon 13
......
GTCGGGTCTTGGGGTCTGGAGCGTTTGGGA...
TET
BHQ-1
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3’-primer
69
Quantitation
by PCR
Determining when
to determine how much
Analysing quantitative data
Absolute Quantitation
Unknown samples are compared to a
standard curve
Standard is a known DNA sample whose
absolute concentration is known
Relative Quantitation
Two or more genes are compared to each
other; result is a ratio
Endogenous control or a housekeeping gene
is compared to a gene of interest
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the
CT
CT= threshold cycle:
the calculated fractional cycle
number at which the PCR product
crosses a threshold of detection
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Real time quantitative PCR
qPCR standard curve
...
.
High Resolution Melt Analysis
Use Saturating dye in PCR
Analysis of fluorescence as amplicon is melted
Heteroduplexes and homoduplexes will melt with different profiles
Molecular diagnostics in the era of
targeted therapies
Diagnosis and suitability of targeted therapy
Monitoring of MRD and detection of early relapse
CML, Ph+ ALL, CEL and tyrosine kinase inhibitors:
imatinib, dasatinib, nilotinib, ponatinib
ATRA, arsenic and APL
Detection of resistance by mutation screening (e.g.
BCR-ABL T315I, KIT D816V)
Prognostic indicators (e.g. FLT3 ITD & NPM1 mutation)
Case studies
Molecular monitoring in
APL
a case study
Molecular monitoring in APL
a case study
PETHEMA
induction
0.03%
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Molecular monitoring in APL
a case study
PETHEMA
consolidation
PETHEMA
induction
started
maintenance
0.03%
19/3/2009
returned to
Australia
neg
Molecular Haematology
80
Molecular monitoring in APL
a case study
PETHEMA
consolidation
PETHEMA
induction
started
maintenance
0.03%
19/3/2009
returned to
Australia
neg
0.04%
0.02%
Molecular Haematology
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Molecular monitoring in APL
a case study
PETHEMA
consolidation
PETHEMA
induction
started
maintenance
0.03%
19/3/2009
returned to
Australia
neg
CNS
relapse
0.04%
1.45%
0.02%
Molecular Haematology
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Molecular monitoring in APL
a case study
PETHEMA
consolidation
PETHEMA
induction
started
maintenance
0.03%
19/3/2009
returned to
Australia
neg
modified CARE
+ PBSC harvest
CNS
relapse
0.04%
IT chemo
+ ATRA
+ As2O3
cyclo-TBI
autograft
1.45%
neg
0.02%
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Molecular monitoring in
CML
2 case studies
Molecular monitoring in CML
a case study: 1
BCR-ABL Quantitative PCR
IM 400mg/d
100
BCR-ABL/BCR (%)
log
reduction
from std
baseline
IM 800mg/d
10
1
2-log
0.1
3-log
IM resistance
4-log
0.01
0.001
06
1/
00
/2
5
/1
0
3
0
20
/
1
5
/0
1
3
0
20
/
5
6
/1
9
2
0
20
/
1
6
/0
0
3
0
20
/
5
7
/1
8
2
IS ratio %
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Molecular Haematology
0
20
/
1
7
/0
8
2
0
20
/
5
8
/1
6
2
0
20
/
1
8
raw ratio %
85
Mechanisms of Resistance to
Imatinib
BCR-ABL dependent
BCR-ABL independent
• gene amplification
• drug sequestration by
1-acid glycoprotein
• kinase domain
point mutations
• OCT-1 mediated drug
uptake
• MDR1-mediated drug
efflux
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Suboptimal response to maximal dose imatinib
BCR-ABL : BCR > 0.1 %
700
740
710
750
720
760
base 760: T
codon 253: TAC
19/3/2009
CAC
770
780
C
Tyrosine
Molecular Haematology
730
Histidine ( Y253H )
87
Hughes et al. , 2006
Blood 108:28-37.
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ABL kinase domain
Clinical Hematology (eds: Young, Gerson & High; pub: Elsevier). From Tauchi & Ohyashiki: Leuk Res 28[Suppl 1]:S39–S45, 2004, with
permission; based on data compiled from Shah, Nicoll, Nagar et al: Cancer Cell 2:117, 2002; and Druker: Semin Hematol 40:50, 2003.
In vitro activity of nilotinib and dasatinib against imatinib-resistant mutations
O’Hare et al, Cancer Research 65:4500, 2005
ABL WT
280
1
15
1
0.6
1
300
1
Y253H
>5,000
>18
400
27
1.8
3
>5,000
>17
T315I
>5,000
>18
>16,667
>5,000
>17
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>5,000
>333
>10,000
Molecular Haematology
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1
0.8
1
190
27
1.4
2
>5,000
>714
>1,000
>1,250
90
Molecular monitoring in CML
a case study: 1
BCR-ABL Quantitative PCR
IM 400mg/d
100
BCR-ABL/BCR (%)
log
reduction
from std
baseline
IM 800mg/d
Das 70mg bd
10
lymphadenopathy
1
2-log
0.1
3-log
0.01
4-log
0.001
06
1/
00
/2
5
/1
0
3
0
20
/
1
5
/0
1
3
0
20
/
5
6
/1
9
2
0
20
/
1
6
/0
0
3
0
20
/
5
7
/1
8
2
IS ratio %
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Molecular Haematology
0
20
/
1
7
/0
8
2
0
20
/
5
8
/1
6
2
0
20
/
1
8
raw ratio %
91
An answer…
Reactive lymphoid hyperplasia with giant
follicles associated with post-therapeutic state
of hematological malignancies:
A report of six cases
Masaru Kojima et al. (2006)
Leukemia and Lymphoma 47(7):1404 -1406
Molecular monitoring in CML
a case study: 1
BCR-ABL Quantitative PCR
IM 400mg/d
100
BCR-ABL/BCR (%)
log
reduction
from std
baseline
IM 800mg/d
Das 70mg bd
10
lymphadenopathy
1
2-log
0.1
3-log
IM resistance
4-log
0.01
0.001
06
1/
00
/2
5
/1
0
3
0
20
/
1
5
/0
1
3
0
20
/
5
6
/1
9
2
0
20
/
1
6
/0
0
3
0
20
/
5
7
/1
8
2
IS ratio %
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Molecular Haematology
0
20
/
1
7
/0
8
2
0
20
/
5
8
/1
6
2
0
20
/
1
8
raw ratio %
93
Molecular monitoring in CML
a case study 2
BCR-ABL/BCR (%)
100
IM serum trough levels
10
mutation?
No
1
0.1
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Molecular Haematology
-0
8
28
-D
ec
8
29
-J
un
-0
-0
7
30
-D
ec
ul
-0
7
1J
-0
6
31
-D
ec
ul
-0
6
2J
1Ja
n06
0.01
94
Imatinib plasma concentration
(ng/mL)
4000
3000
2000
1000
500
270
0
Median
value
300
400
600
n=7
n=57
n=21
813 ng
1135 ng
1709 ng
Daily dose
(mg)
Figure courtesy of Dr Francois Xavier Mahon (ref. Picard, S., et al., Blood, 2007. 109(8): p. 3496-9).
Demonstrating the achievable plasma trough level for imatinib at 3 doses, 300, 400 and 600 mg per
day in 75 patients.
19/3/2009
Molecular Haematology
95
Molecular monitoring in CML
a case study 2
100
BCR-ABL/BCR (%)
10
A case of non-compliance
1
0.1
0.01
nd
19/3/2009
Molecular Haematology
-0
8
28
-D
ec
8
29
-J
un
-0
-0
7
30
-D
ec
ul
-0
7
1J
-0
6
31
-D
ec
ul
-0
6
2J
1Ja
n06
0.001
96
The molecular lab’s wish list
Blood or bone marrow samples in EDTA
Plenty of material (1mL BMA; 9mL PB)
Pre-treatment specimens
Consider WCC and send more PB if low WCC
Unclotted! Mix well and immediately
Cytogenetics at diagnosis doesn’t tell us molecular
breakpoint of gene rearrangement
Assists with future MRD
Tubes labelled with specimen type, collection times,
dates, collector, etc.
The End