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Serum or plasma sample –
which one is better?
Presented by:
Jeffrey Chance, PhD
04/2011
Presentation Overview
•
•
•
•
•
•
•
•
Introduction
Turnaround Time
Sample Quality
H dli & T
Handling
Temperature Eff
Effects
Test Results / Analyte Stability
Advantages & Disadvantages
Which Sample is Better ?
Summary
2
What Type of Plasma ?
• Lithium heparin is the preferred anticoagulant in
clinical
c
ca cchemistry.
e st y
• EDTA, citrate and oxalate cannot be used for routine
chemistry testing since they bind calcium and have
commonly measured counterions (K, Na).
• The lithium salt is more commonly used than the
sodium salt to avoid interference with sodium
determinations.
determinations
3
Which Sample is Better ?
1 Serum
1.
2. Heparin
p
Plasma
3. It Depends / Equal
4
Turnaround Time
• Recommended clotting times for serum blood
collection
co
ect o tubes ge
generally
e a y range
a ge from
o 30
30-60
60 minutes.
utes
• Use of plasma allows laboratories to process and test
specimens upon receipt
receipt, while avoiding latent fibrin
formation due to incomplete clotting.
• Plasma advantage where specimen must be
centrifuged without delay, eg. ER/STAT, end of day
courier pickup
pickup.
5
Time from
phlebotomy
t
to
centrifugation
> 30 min
Yes
Serum or
Plasma
No
Are serum
specimens
held to ensure
minimum clot
time?
Yes
Use of
plasma can
improve TAT
No
Use of
plasma can
reduce fibrin
6
Sample Quality
• Serum specimens are subject to latent fibrin
formation
o at o when
e cclotting
ott g is
s inadequate.
adequate
- insufficient clotting time
- patients receiving anticoagulant
or thrombolytic therapy
• Fibrin can range from thin strands
to large cloud-like masses.
7
Issues Due To Fibrin
• Physical obstruction of sampling probe
• Insufficient sampled volume
• Gradual deposition of fibrin in reaction chambers or
pathways
• Interference with measurement systems or reagents
• Potential consequences: instrument downtime,
failure to provide test results, or erroneous test
results.
8
Addressing Fibrin Issues
• Approaches to minimize the impact of fibrin in serum
specimens
spec
e so
often
te require
equ e use
user intervention,
te e t o , increase
c ease
TAT, and may not be recommended.
• To help reduce these issues
issues, some laboratories have
switched to plasma.
• However,
However plasma specimens also have unique
characteristics concerning specimen quality and
integrity.
integrity
9
Sample Quality
• Serum almost “cell free”.
• Separation of anticoagulated blood based on density
gradient: platelets (least dense) > white blood cells >
red blood cells (most dense)
• Platelets most abundant in plasma followed by WBC.
10
Sample Quality
• "Clean" plasma (low cell/platelet counts) can be
obtained
obta
ed with
t p
proper
ope ce
centrifugation.
t ugat o
• Non-gel tubes – cells/platelets near bulk cell
interface Plasma often aliquoted
interface.
aliquoted.
• Gel tubes – rapid gel movement traps cells/platelets
before sedimentation is complete
complete. Some
cells/platelets above gel surface. Plasma not
aliquoted.
aliquoted
11
serum
plasma
12
Gel Movement
• The presence of a solid clot in serum gel tubes leads
to a d
difference
e e ce in tthe
e movement
o e e to
of ge
gel du
during
g
centrifugation.
• Serum: Gel must move up and around the clot
clot,
against the tube wall.
• Plasma: Gel moves up in pieces similar to a ‘lava
lava
lamp’.
13
Gel Movement (simulated)
serum tube
plasma tube
14
Test Results
• In general, most assays in clinical chemistry are
compatible
co
pat b e with
t bot
both serum
se u a
and
d heparin
epa p
plasma,
as a, a
and
d
test results are sufficiently equivalent that the same
reference ranges
g can be used.
• However, for certain assays or test methods, plasma
specimens may be unacceptable
unacceptable, or differences in
results may be sufficient to warrant a change in
reference range
range.
15
Potassium
• Potassium and phosphorus increased in serum due
to release
e ease from
o ce
cells/platelets
s/p ate ets du
during
g cclotting.
ott g
• A linear correlation has been shown between platelet
count and the increase in serum potassium
potassium.
World Health Organization. Use of anticoagulants in diagnostic laboratory investigations. WHO/DIL/LAB/99.1 Rev.2, 2002.
Harr R, Bond L, Trumbull D. A comparison of results for serum versus heparinized plasma for 30 common analytes. Lab Med.
1987;18:449-55.
Ciuti R, Rinaldi G. Serum and plasma compared for use in 19 common chemical tests performed in the Hitachi 737 analyzer. Clin Chem.
1989;35:1562-3.
Guder WG, Narayanan S, Wisser H, Zawta B. Samples: from the patient to the laboratory. 3rd ed. Darmstadt, Germany: Wiley-VCH;
2003:32-3.
Miles RR, Roberts RF, Putnam AR, Roberts WL. Comparison of serum and heparinized plasma samples for measurement of chemistry
analytes. Clin Chem. 2004;50:1704-5.
B ti CA,
Burtis
CA A
Ashwood
h
d ER
ER, eds.
d Tietz
Ti t fundamentals
f d
t l off clinical
li i l chemistry.
h i t 4th ed.
d Phil
Philadelphia,
d l hi PA
PA: W
W.B.
B S
Saunders
d
C
Company; 1996
1996:499.
499
Hartland AJ, Neary RH. Serum potassium is unreliable as an estimate of in vivo plasma potassium. Clin Chem. 1999;45:1091-2.
16
Potassium
• Patients with essential thrombocythaemia and
significant
s
g ca t tthrombocytosis
o bocytos s were
e e found
ou d to have
a e se
serum
u
pseudohyperkalemia (> 5.5 mmol/L) that corrected
when measured on plasma.
p
• It has been suggested that when there is no obvious
explanation for an elevated serum potassium
potassium, the
plasma potassium level should be measured,
particularly in patients with elevated platelet counts
counts.
Howard MR, Ashwell S, Bond LR, Holbrook I. Artefactual serum hyperkalaemia and hypercalcaemia in essential thrombocythaemia.
J Clin Pathol. 2000;53:105-9.
Fort JA. Thrombocytosis and hyperkalemia revisited. Am J Pediatr Hematol Oncol. 1989;11:334-6.
17
Total Protein
• Slightly increased in plasma due to presence of
fibrinogen.
b oge
World Health Organization. Use of anticoagulants in diagnostic laboratory investigations. WHO/DIL/LAB/99.1 Rev.2, 2002.
Harr R, Bond L, Trumbull D. A comparison of results for serum versus heparinized plasma for 30 common analytes. Lab Med.
1987;18:449-55.
Ciuti R, Rinaldi G. Serum and plasma compared for use in 19 common chemical tests performed in the Hitachi 737 analyzer. Clin Chem.
1989 35 1562 3
1989;35:1562-3.
Guder WG, Narayanan S, Wisser H, Zawta B. Samples: from the patient to the laboratory. 3rd ed. Darmstadt, Germany: Wiley-VCH;
2003:32-3.
18
Other Tests
• Differences in certain enzymes (e.g., LD, ALKP, AST)
may
ay be see
seen.
• Lithium/sodium increased with use of lithium or
sodium heparin
heparin.
• Interference from fibrinogen may also make plasma
an unsuitable specimen for certain protein analysis
methods (e.g., protein electrophoresis).
• Heparin
H
i may iinterfere
t f
with
ith certain
t i iimmunoassays.
Lee DC
L
DC, Kl
Klachko
hk MN
MN. F
Falsely
l l elevated
l
t d lithi
lithium llevels
l iin plasma
l
samples
l obtained
bt i d iin lithi
lithium containing
t i i ttubes.
b
JT
Toxicol
i l Cli
Clin T
Toxicol.
i l
1996;34:467-9.
19
Handling Effects
• Mixing/agitating plasma gel tubes after centrifugation
will re-suspend
e suspe d ce
cells
sa
and
dp
platelets
ate ets
• Specimen may appear slightly darker or cloudy
20
unmixed
mixed
21
Temperature Effects
• Temperature can also influence plasma sample
qua ty
quality.
• Cold activation of clotting factors
• Refrigeration may drive reaction towards clotting -->
>
increase in fibrin and cell aggregate formation
• Re-centrifugation
R
t if
ti off stored
t d plasma
l
aliquots
li
t can h
help
l
clean up sample prior to testing.
22
Analyte Stability
• Heparin plasma samples with increased cell/platelet
concentrations
co
ce t at o s e
exhibit
b t reduced
educed stab
stability
ty o
of ce
certain
ta
common analytes.
• Analytes affected are involved in cell/plateletmediated metabolic processes and/or are present in
higher concentration in cells or platelets
platelets.
23
Analyte Stability
• Heparin plasma stability data for routine chemistry
analytes,
a
a ytes, 24 hours
ou s at room
oo te
temperature:
pe atu e
- Aliquot from non-gel tube: STABLE
- Plasma stored in gel tube: decreases in glucose
and CO2; increases in phosphorus, K, LD
• Plasma
Pl
stored
t d in
i contact
t t with
ith cells
ll – significant
i ifi
t
changes in glucose and other analytes over time†
BD White Paper VS7172. Comparison of BD Vacutainer® PST™ II Plus Tubes With BD Vacutainer® Lithium Heparin Glass Tubes for
Routine Chemistry Analytes. 2004.
BD White Paper VS7174. Comparison of BD Vacutainer® Lithium Heparin Plus Tubes With BD Vacutainer® Lithium Heparin Glass
Tubes for Routine Chemistry Analytes. 2004.
†B
Boyanton
t BL JJr, Bli
Blick
k KE
KE. Stability
St bilit studies
t di off ttwenty-four
t f
analytes
l t in
i h
human plasma
l
and
d serum. Cli
Clin Ch
Chem. 2002
2002;48:2242-7.
48 2242 7
24
Analyte Stability
• Some mechanisms are temperature dependant –
e.g.,
e
g , low
o te
temperature
pe atu e may
ay cause higher
g e increase
c ease in
K due to inhibition of Na+,K+-ATPase activity.
• Laboratories should evaluate the need for delayed or
add-on testing when considering the use of heparin
plasma samples
samples.
Guder WG, Narayanan S, Wisser H, Zawta B. Samples: from the patient to the laboratory. 3rd ed. Darmstadt, Germany: Wiley-VCH;
2003:57.
Heins M, Heil W, Withold, W. Storage of serum or whole blood samples? Effects of time and temperature on 22 serum analytes. Eur J
Clin Chem Clin Biochem. 1995;33:231-8.
25
Analyte Stability
• Remember that courier pick-up?
(plasma
(p
as a ad
advantage
a tage for
o immediate
ed ate ce
centrifugation)
t ugat o )
• Was the plasma aliquoted or in a gel tube?
• What tests were ordered?
• What is the transit time/delay until testing?
26
Ad
Advantages
t
Serum
– nearly cell-free
– good storage stability for most analytes
– wide range of assays available
Pl
Plasma
– shorter
h t TAT:
TAT can be
b centrifuged
t if
d immediately
i
di t l
– faster gel movement in gel tubes/ more reproducible gel
barrier formation
– more representative of in vivo state
– available plasma yield slightly higher than serum
27
Di d
Disadvantages
t
Serum
– may cause pseudohyperkalemia
– longer TAT
– instrument or test interference from fibrin, esp. with
anticoagulation therapy
Pl
Plasma
– higher
hi h cell/platelet
ll/ l t l t counts;
t potential
t ti l ffor instrument
i t
t or test
t t
interference
– reduced storage stability for certain analytes; fibrin
f
formation
i during
d i storage
– interference from anticoagulant
– interference from fibrinogen
g
– some tests may not be supported
28
Serum vs. Plasma
• Serum usage generally still predominant but varies
by region.
eg o
• Many laboratories try to standardize to one sample
type and will rely heavily on that one type
type.
• A main reason for not using plasma samples in
laboratories currently is because laboratory
processes are standardized for serum samples.
• The
Th main
i driver
di
ffor using
i plasma
l
samples
l iis th
the
shortened TAT.
29
Serum vs. Plasma
• Top Serum Advantages / Plasma Disadvantages:
Assay
ssay co
compatibility
pat b ty
• Top Plasma Advantages / Serum Disadvantages:
Turnaround time, fibrin, (hemolysis)
30
Which Sample is Better ?
• How much weight does each factor carry in your
institution
st tut o or
o sett
setting?
g
31
Which Sample is Better ?
Fibrin
TAT
Stability
Assay
Compatibility
32
Which Sample is Better ?
Fibrin
TAT
Stability
Assay
Compatibility
33
Which Sample is Better ?
Fibrin
TAT
Stability
y
Assay
Compatibility
34
Summary
• Serum and heparin plasma both have benefits and
limitations
tat o s in clinical
c ca chemistry.
c e st y
• The selection of serum vs. heparin plasma may be
dependent on the specific setting/population.
setting/population
• Standardizing on one sample type may be desirable
but not always practical
practical.
35
Thank You
36