1. No category

Primary sample collection systems:
How useful are they in reducing
Preanalytical variability?
Stephen
p
Church
BD Diagnostics - Preanalytical Systems,
Oxford, UK
How good is your sample quality?
• Appropriate Sample Identification?
How good is your sample quality?
• Haemolysis?
How good is your sample quality?
•
Fibrin/Clotting
g Plasma?
How good is your sample quality?
•
Latent Clotting Serum?
How good is your sample quality?
• Incorrect filling/Insufficient Quantity?
1. Petersonn, P & Gottfired, E. The Effect of Inaccurate Blood
Volume Draw on Prothrombin Time (PT) & Activated Partial
Thromoplastin Time, Thrombosis Haemostasis, 47(2)101 103, 1982
How good is your sample quality?
• Poor Barrier Formation?
1
2
1 Spirit
1.
Spiritus,T.
s T Iodinated Contrast Media Interfere with
ith Gel
Barrier Formation in Plasma and Serum Separator Tubes.
Clin. Chem., Jul 2003; 49: 1187 - 1189.
2. van den Ouweland. High Total Protein Impairs Appropriate
Gel Barrier Formation in BD Vacutainer Blood Collection
Tubes Clin. Chem., Feb 2007; 53: 364 - 365.
How good is your sample quality?
• Good or Bad Sample?
Preanalytical error:
• long tourniquet time
• wrong order of draw
• storage at 4°C for hours before centrifugation
• patient came by bicycle and ran up the stairs
because he was late, blood collection was done
immediately
• tube shall be used for monitoring test of tricyclic
antidepressents
• tube has been forgotten over night on the ward
before transport
Consequences:
• elevated proteins
• carry over of additive, e.g. K
• elevated K because RBC Na/K-ATPase was
inhibited
• incorrect lab results because of stress & activity
(Glucose, lactate, creatinin, ...)
• falsely low TDM results
• incorrect
i
t llab
b results,
lt e.g.K
K
Analytical trends
Analyte
Concentration
mmol/L
pmol/L
Does it matter?
1. Foubister, Vida. Cap Today Bench press: The
Technologist/technician shortfall is putting the squeeze on
laboratories nationwide; September 2000
2. Datta, P. Resolving Discordant Samples. Advance for the
Administrators of the Laboratories; July 2005: p.60.
Reducing Variability in the PA Phase
PATIENT
• Patient ID
• In Vivo Hemolysis
due to patient
factors
• Metabolic
Disorders (eg.
Liver disease)
• Chemical Agents
(eg. Medication)
• Physical Agents
(eg. Mechanical
heart valves)
• Infectious Agents
(eg. Bacteria)
PHLEBOTOMY
• Catheter, IV
Collection
• Capillary
Collection
• Needle Gauge
• Position of Arm
• Location of
Venipuncture
• Antiseptic Used for
Phlebotomy
• Tourniquet Time
• Traumatic Draw
• Fist Clenching
• Tube Type
Collected
• Tube Under Filled
• Order of Draw
• Vigorous Mixing
• No Mixing
• Syringe Transfer
SPECIMEN
TRANSPORT
• Origin of Specimen
Maternity,
Emergency &
Intensive Care
• Origin of Specimen
In-patient
• Origin of Specimen
Physician Office
Lab
• Origin of Specimen
Out-patient
• Tubes Transported
Vertical or
Horizontal
• Transport by
Pneumatic Tube
• Courier Transport
• Transport Duration
• Pre-Centrifugation
Pre Centrifugation
and Transport
Temperature
PROCESSING
• Verify Tube with
Request
• Generate
Laboratory
Barcode
• Time between
Collection and
Centrifugation
• Type of Centrifuge
• Centrifuge
Calibrated
• Centrifuge
Temperature
Extremes
• Speed of
Centrifuge
• Duration of
Centrifugation
• Poor Separator
Barrier Integrity
• Cells on Stopper
• Automated
Decapping
• Specimen ReCentrifugation
• Aliquot
Ali
t Labeling
L b li
• Specimen
Aliquoted
ANALYSIS
• Long Time after
Centrifugation
• Serum vs. Plasma
vs. Whole Blood
• Tube mixed prior
to analysis
• Re-run Specimen
(Same Day)
• Verify Instrument
Cal & Controls
• Identify Instrument
Used for Testing
• Identity Tech
Performing Testing
• Verify Report Value
SPECIMEN
STORAGE
• Re-Centrifugation
Add-On
• Post-Analysis
Storage
Temperature
• Duration of
Storage
The Sample Collection System
Vascular Access Device
+ Sample Container
Cannula material
L bi i
Lubrication
Cannula gauge
Fluid Path
Closure
Cl
Rubber Stopper
Tube Material
Additives
Label
Evacuation
Sterilization
Reducing PAV: Sample ID
CAP Q Probes
CAP-Q
P b Study
St d outcomes
t
*
# of labs included in the study
120
Total # of errors recorded in the survey period
6.705
Total # of errors for which it has been possible to identify a precise cause
4.852 (72%)
Cause of the error
#
Incidence
Wrong identification (labeling) of primary samples
2.691
55,5 %
Wrong Lab check-in (prescription)
1.088
22,4 %
Data transcription
604
12,4 %
Wrong identification (labeling) of secondary tubes
184
3,8 %
80
1,7 %
205
4,2 %
Test results in
in-put
put
Others (miscellaneous)
1. CAP-Q Probes Study (Valenstein PN, et al. Identification
Errors Involving Clinical Laboratories Arch Pathol Lab Med
2006;130:1106-1113)
Reducing PAV: Sample Collection
•
•
Ensuring the correct sample ID:
Minimum requirements for patient identification are defined:
– Patient full name, address, Patient ID, Patient DOB1
– Solutions:
•
Accurate label placement is key for barcodes
– Solutions:
1. Clinical and Laboratory Standards Institute, Procedures for
the Collection of Diagnostic Blood Specimens by Venipuncture;
Approved Standard. 6th ed. H3-A6, Wayne, PA: CLSI; 2007
Reducing PAV: Sample Collection
Other CLSI/ISO1,2
•
•
Choosing
g the
correct sample type
Solution: Use of
colour codes
Serum non g
gel
Serum Separator*
Heparin
Heparin Separator*
EDTA
Citrate
Glucose
ESR
* Consult Manufacturer
1. Clinical and Laboratory Standards Institute, Tubes and
Additives for Venous Blood Specimen Collection; Approved
Standard. 5th ed. H1-A5, Wayne, PA: CLSI; 2003
2. ISO 6710, Single use containers for venous blood specimen
collection
Reducing PAV: Sample Collection
•
•
•
Additive Carry
y
Over
Carry-over is
extremely
y unlikely
y
with vacuum blood
collection systems,
but cannot be
excluded
completely 1,2
Solution: Order of
draw definition
Blood Cultures
Sodium Citrate
Draw tubes
in
descending
order
ESR
Serum
Lithium Heparin
EDTA
Glucose
Trace element
1. Sharratt CL, Gilbert CJ, Cornes MC, Ford C, Gama R. EDTA
sample contamination is common and often undetected, putting
patients at unnecessary risk of harm. Int J Clin Pract 2009; Vol.
63: 1259-62.
2. Davidson DF. Effects of contamination of blood specimens with
liquid potassium EDTA anticoagulant. Ann Clin Biochem. 2002,
Vol. 39, 273-280.
Reducing PAV: Sample Collection
•
Collection induced haemolysis
•
Solution range of partial draw tubes
1. Sixsmith DM, Weinbaum F, Ann Chan SY, Nussabaum M &
Magdich K. Reduction of Hemolysis of blood specimens
drawn from ED patients for routine chemistry tests by use of
low vacuum collection tubes. Academic Emergency Medicine
2000; 7(5): 524
Reducing PAV: Sample Collection
Örebro Hospital Emergency Department
Falun Hospital Emergency Department
18%
16%
14%
12%
10%
8%
6%
4%
2%
0%
New blood collection system introduced
3
25
2.5
percent
pe
ercent
New blood collection system introduced
2
1.5
1
3
5
7
9
11
13
15
17
19
21
23
25
27
29
31
33
35
1
0.5
w eek
0
Percentage of Potassium Results not reported because
of haemolysis (n=approx 400 samples/week)
Ortho Diagnostics Vitros 5.1 Biochemistry
Mean 4% before conversion 10% after
conversion
The author hypothesizes that increase in
haemolysis may be attributed to increased
speed
d off the
th blood
bl d flow
fl
into
i t the
th tube
t b (Tube
(T b
Fill Rates) noted (0.8mL/sec) 4ml versus
(0.6mL/sec) 3.5ml
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43
week
Percentage of Potassium Results Not Reported because
Of Haemolysis (n=approx 300 samples/week)
Abbott ci8200
Mean 0.3%% before conversion 1.5% after
conversion
1. Swedish Society for Clinical Chemistry’s Members
Magazine, Klinisk Kemi, No 2, 2009
Reducing PAV: Sample Collection
• Collection induced haemolysis
• Solution: Selection of tube type
yp & training
g
1. A Monitoring Process To Assess The Impact Of Preanalytical
Variables On Sample Quality Following A Change In Blood
Collection System, Muller C et al, Ahead of Publication
Euromedlab 2011
Reducing PAV: Transportation & Stability
• Sample degradation
during transport
• Glucose,, K,, LDH < 2
hours contact whole
blood 1
• Separation of
supernatant from
cellular
ll l mass
• Solution: gel based
separator tubes
1. Nadja N. Rehak and Betty T. Chlang. Storage of Whole Blood: Effectof
Temperature on the Measured Concentrationof Analytes inSerum Clin
Chem 1985;31:2005-6.
2. BD White Paper: Comparison of BD Vacutainer™ SST™ Plus Tubes
with SST™ II Plus Tubes Common Analytes on the Toshiba/Abbott
Aeroset, VS5780, 2001
3. BD White Paper: Gel Barrier Stability Comparison of BD Vacutainer™
SST™ II PLUS,SST™ PLUS and SST™ PLUS Transport Tubes in
Post Centrifugation Transport, VS5824, 2001
Reducing PAV: Transportation & Stability
• Sample degradation during transport
• Solutions: Control & monitoring of time and temperature
Reducing PAV: Transportation & Stability
• Sample degradation during transport
• Stability APTT citrate <1 hour 1
• Solutions: CTAD provides stability for up 4 hours
at RT 2,3
1. Collection, Transport and Processing of Blood Specimens for
Testing Plasma-Based Coagulation Assays and Molecular
Hemostasis Assays; Approved Guideline - Fifth Edition. CLSI
document H21-A5. 2008
2. G. Contant: Use of CTAD versus sodium citrate in different
haemostasis assays. Serbio, March 1995.
3. van den Besselaar: Photochemical decomposition of
dipyridamole in aqueous solution and the utilisation of CTAD for
monitoring heparin, Int. Jnl. Lab. Hem. 2010(32):265-267
Reducing PAV: Transportation & Stability
• Sample degradation during transport
• Stability of Glucose
• Solutions: Glycolytic inhibitors
1. Guder WG, Narayanan S, Wisser H, Zawta B. Samples: From
the Patient to the Laboratory. Wiley-VCH 2003.
2. BD White Paper: A Comparative Evaluation of BD Vacutainer®
Plus Fluoride/EDTA Tubes for Glucose & HbA1c Preservation
After 24 Hours Storage at Room Temperature, VS7594, 2007
3. Gambino, R. Acidification of Blood Is Superior to Sodium
Fluoride Alone as an Inhibitor of Glycolysis, Clinical Chemistry
55(5)1019–1021 (2009)
Reducing PAV: Sample Processing
•
•
•
Incorrect centrifugation results in poor separation & sample
instability
S l ti
Solutions:
G
Gell ttubes
b with
ith a b
broad
d range off centrifugation
t if
ti conditions
diti
Solutions: Coagulation plasma centrifuged at a speed and time to
consistently product platelet-poor
platelet poor plasma (platelet count <
10,000/µL)
1. BD White Paper: A Comparative Evaluation of BD Vacutainer®
Plus Fluoride/EDTA Tubes for Glucose & HbA1c Preservation
After 24 Hours Storage at Room Temperature, VS7594, 2007
2. Collection, Transport and Processing of Blood Specimens for
Testing Plasma-Based Coagulation Assays and Molecular
Hemostasis Assays; Approved Guideline - Fifth Edition. CLSI
document H21-A5. 2008
Reducing PAV: Sample Processing
•
•
•
•
Latent Fibrin/Fibrin Mass Formation:
Solutions: To clot or not to clot?
Anticoagulant Lithium Heparin
Rapid collecting additive such as thrombin or heparin neutralisers
such
h as protamine
t i
Visible clotting was achieved
rapidly in RST specimens
within 5 min where participants
had received a total of 0
to 5000 units of heparin) but
not from participants who
received a total of 7000 units
of heparin
p
((APTT >150 s).
) 2
1. BD internal data to be published
2. Dimeski et al. Evaluation of the Becton-Dickinson rapid
serum tube: does it provide a suitable alternative to
lithium heparin plasma tubes? Clin Chem Lab Med
2010;48(5)
Reducing PAV: Analysis
Reducing PAV: Analysis
•
Assay & sample compatability
•
TDM & Immunoassays with gel separators
Factors Influencing Analyte Stability – TDM & Special Chemistry
Manufacturer Control
• Chemical nature of
resin
• Surface area of the gel
prior
i to
t centrifugation
t if
ti
User Control
No Control
• Duration of gel:sample contact both
before and after centrifugation
• Storage temperature
• Actual volume of sample on the gel
• Tube orientation before centrifugation
• Surface area of gel after centrifugation
• Chemical
nature of the
analyte or
drug
1. Extracted from Quality of Diagnostic Samples:
Recommendations of the Working Group on Preanalytical
Quality of the German Society for Clinical Chemistry and
Laboratory Medicine, 2010
Reducing PAV: Analysis
•
•
•
•
Solutions:
Monitoring of quality critical parameters during the manufacturing
processes
Cooperation with IC during assay development
Demonstrate analyte compatibility across a broad spectrum of sample
types, pathologies & instrumentation platforms:
1. BD White Paper: Therapeutic Drug Compatibility in BD Vacutainer®
SST™ II Plus Tubes, VS7050, 2004
2. BD White Paper: Performance of BD Vacutainer® SST™ II Plus
Tubes for Special Chemistry Testing, VS7051, 2004
Reducing PAV: Analysis
Reducing PAV: Post Analysis
•
•
Stability - Freezing primary sample
Often used for blood banking and sometimes for ‘add-on’ tests
–
–
–
–
•
No sample aliquoting
Reduced identification errors
Reduce biohazard risk
R d
Reduced
d costt
Solutions:
– Airflow around the tube, ensure that the samples are in wire racks that
allow airflow rather than EPS/styrofoam trays.1
– Gradual freezing of the tubes, refrigerated 4-8C for 4 hours, then -20C
for 24 hours then final temperature. 1
– Most analytes in aliquoted serum are stable when frozen (≤ −20°C).2
– Even repeat freeze/thaw cycles at -20°C or -70°C seem to have little
effect on many analytes. 3
1
– Majority
M j i off routine
i chemistries
h i i are stable
bl iin serum separator tubes.
b
1. A Jaap Bakker et al, An Evaluation Of The Integrity of BD
Vacutainer® SST™ II and Analyte Stability when Subject to
g at -20°C.,EUREGIO,
,
, 2003.
Freezing
2. Heins Met al. Storage of serum or whole blood samples?
Effects of time and temperature on 22 serum analytes. Eur J
Clin Chem Biochem. 1995; 33:231-38.
3. DiMagno EP et al, Effect of long-term freezer storage, thawing
and refreezing on selected constituents in serum. Mayo Clinic
Proc 1989; 64:1226-34.
Reducing PAV: The Future
•
•
Reducing sample collection volumes - Why
Advances in instrument technology should be reflected in the
collection requirements
•
Blood collection 45 times greater than the volume required (Range
2 102 times)
2-102
ti
)1
Patients in Intensive Care Units (ICU) are phlebotomised three
times as often as patients on the wards. 2
5% of the patients undergoing intensive care, the volume of blood
collected for laboratory testing was >200 mL and for 0.7% was >600
mL during their hospital stay.3
Complications: Investigational anaemia, neonatal anaemia
•
•
•
•
Solution instrumentation compatible low draw blood collection
tubes 0.5 – 2 mL
1 Dale JC
1.
JC, Pruett SK.
SK Phlebotomy - a minimalist approach
approach. MayoClin
Proc. 1993; 68: 249-255.
2. Smoller BR, Kruskall MS. Phlebotomy for diagnostic tests in adults:
pattern of use and effect on transfusion requirements. N Engl J Med.
1986; 314: 1233-1235
3. Wisser D, Van Ackern K, Knoll E, et al. Blood loss from laboratory
tests. Clin. Chem. 2003;49:1651-1655
Reducing PAV: The Future
• Alternatives to gel separators
• Provide improved sample purity
purity, analyte stability
stability,
eliminate analyte adsorption
1.
BD Data on file
Reducing PAV: The Future
Time “0”
EDTA-0\0_K1\1\1SRef
EDTA
4
3
2
1537.001
1
P100-0\0_K4\1\1SRef
P100
4
1211.958
882.674
2
1896.312
863.709
1536.964
1465.965
1865.227
1
1348.998
1606.140
Intens. [a.u.]
x104
3
1060.802
2021.388
1778.279
0
x104
1051.964 1166.010
EDTA-15m\0_K2\1\1SRef
1537.067
4
1465.941
0
x104
P100-15m\0_K5\1\1SRef
4
1060.921
1536.936
1896.428
2
2
2021.534
1166.174
1349.065
0
x104
1617.213
863.689
1
1060.803
1466.285
EDTA-2h\0_L1\1\1SRef
1896.644
4
1865193
1865.193
1465.891
1778.377
Intens. [a.u.]
882.944
1
Intens. [a.u.]
2021.312
1778.219
3
3
15 min
Intens. [a
a.u.]
x104
Intens. [a.u.]
Intens. [a
a.u.]
• Proteomic: Protein stabilisation
• Solution: P100
P100, Anticoagulant: K2EDTA,
EDTA Optimized
protease inhibitor mix & Mechanical plasma separator
1350.893
202129
2021.295
1778.162
0
x104
4
P100-2h\0_L4\1\1SRef
3
3
2 hr
1537.076
1537.347
1061.104
2
1
1207.183
882.994
1466.022
2
1896.470
1
1351.262
1762.781
863.790 948.964
2021.756
0
1206.896
1350.950
2021.568
0
800
1000
1200
1400
1600
1800
2000
2200
800
1000
1200
1400
m/z
1. Jizu Yi,* Changki Kim, and Craig A. Gelfand
Inhibition of Intrinsic Proteolytic Activities Moderates
Preanalytical Variability and Instability of Human
PlasmaJournal of Proteome Research 2007, 6, 17681781
1600
1800
2000
2200
m/z
Reducing PAV: The Future
•
•
Molecullar: Stabilisation against gene induction and degradation
TM for
S l ti
Solution:
PAX
PAXgene
f isolation
i l ti off cellular
ll l RNA ffrom whole
h l bl
blood
dRNA is stabilized immediately at blood collection and remains stable
for days at room temperature
Gene Induction
PAXgeneTM
EDTA
1. Lynne Rainen,et al Stabilization of mRNA Expression in
Whole Blood Samples Clin. Chem., Nov 2002; 48: 1883
- 1890.
Reducing PAV: Conclusions
•
•
•
•
•
•
•
•
Selection of the appropriate sample collection system
Together with correct handling and procedures
Minimises Preanalytical variability
Optimum sample quality
Enable the generation of the true in-vivo result
Maximise laboratory efficiency
Reduce costs
Assure patient treatment and care