Transcript Funny-coloured serum: what to do? (how to deal with common

Interferences - are some methods
better than others?
Graham Jones
Department of Chemical Pathology
St Vincent’s Hospital, Sydney
Contents
Background
Choosing your instrument
Using your instrument
Introduction
Our aim: to produce timely, accurate results
to allow optimal patient care
Interferences - substances present in a
sample, or events affecting a sample, which
lead to the production of inaccurate results
Accuracy: results which reflect the result
which would have been obtained if the
interference had not been present
Interference Importance
May lead to a clinical error
 Wrong management with bad outcome
Interference-related clinical errors quite rare
 Most clinical errors require several mishaps
concurrently
 Many “near misses”
BUT: can cost time, additional testing, reduced
doctor confidence
Error Importance
Erroneous and Non-believable
 eg potassium of 10.0 due to haemolysis or
EDTA contamination
 Result: ignore or recollect specimen
Erroneous and Believable
 eg potassium of 5.5 due to haemolysis or EDTA
contamination
 result: unnecessarily cease potassium
supplements
Common Interferences
In-vitro haemolysis
Bilirubin
Lipaemia
Drugs
Immunoglobulins
Events (eg delayed separation)
Other (artificial blood)
Common Interferences
In-vitro haemolysis
Bilirubin
The visible
interferences
Lipaemia
Drugs
Immunoglobulins
Events (eg delayed separation)
Other (artificial blood)
Given factors
We wish to have accurate results
We wish to avoid errors due to interferences
We aim to give out results when they are
accurate
We aim to withhold results which are inaccurate
 This implies different cutoff levels for different
analytes
Choosing your instrument
Assesment of Interferents
 Melvin Glick
 Clin Chem (1987) 33: 1453-1458
 Add known amounts of RBC lysate; Intralipid;
bilirubin to normal serum
 Standard procedures
 Plot percent change in result vs interferent
concentration
 “Interferographs”
Final/original result x 100 (%)
Interferographs: Glick
200
110%
100
0
*
90%
* Urea
* Chloride
* Creatinine
0
500
1000
Haemolysate added (as haemoglobin. mg/dL)
Glick
Most work performed in 1980s
Work performed using his own blood (reliable
supply, but limited quantity)
Limited comprehensive third party data available
for current instruments
Data from our own studies
 Haemolysis Interference in Modern Instruments Clin
Biochem Revs 2000;21:124
 Icterus Interference in Modern Instruments Clin Biochem
Revs 2000;21:124
Interferogram
180
160
140
120
100
80
60
40
20
0
500
1000
1500
Na
K
Cl
glu
urea
crea
ca
mg
Phos
bili
alb
prot
alt
ast
ggt
alp
ck
amyl
chol
tg
Roche Modular <P>
Haemolysis
Haemolysate added to
patient samples and
concentrations
measured
Instruments
Instrument
Advia 1650
Aeroset
AU 600
Dimension
Integra
Lx20
Modular
Vitros V250
Manufacturer
Bayer Diagnostics
Abbott
Olympus(1)
Dade-Behring
Roche Diagnostics
Beckman-Coulter
Roche Diagnostics(2)
Ortho Clinical Diagnostics
Table 1. Analysers used in the study.
Except where indicated below manufacturer's reagents were used.
(1) Distributed in Australia by Integrated Sciences. Reagents
from Trace Scientific or Olympus, except for bilirubin from Synermed
(2) Tests performed on D and P modules. The worse performance
of the two was used for the analysis.
Comparing Interference Performance:
Amylase and Haemolysis
80
60
160
40
Aeroset
Advia
Mod-P
20
Integra
0
Lx20
0
-20
-40
500
1000
Haemoglobin (mg/dL)
1500
V250
AR
990
-60
Using RCPA-AACB Allowable Limits of Performance
Advia
Aeroset AU600 Dim AR Integra
ALB
ALP
ALT
AMYL
AST
BILI
CA
CHOL
CK
CL
CREA
GGT
GLU
K
MG
NA
PHOS
PROT
TG
UA
UREA
609
244
1300
544
144
58
804
841
1300
1300
650
51
60
787
923
410
233
780
650
1300
1300
573
650
377
135
309
600
1207
350
1300
1300
858
1300
63
95
873
384
158
927
96
1300
Average:
649
674
650
734
650
Lx20
Modular
V250
641
44
257
463
280
1300
988
1300
400
1226
70
1300
1300
674
189
1300
1300
1300
422
66
182
875
40
736
1089
1300
1300
759
588
1300
1300
113
88
1300
1000
128
1300
969
1300
1300
58
1300
790
395
677
1300
504
1300
1300
501
1300
160
132
76
969
1300
258
1300
1300
590
1300
64
594
820
650
650
337
649
1300
1300
937
662
977
105
1300
1300
1257
157
1300
650
947
1300
66
1300
596
330
799
581
1300
1300
257
90
248
377
91
56
400
1215
282
1300
1300
620
1300
70
1300
1168
276
190
1300
1300
1300
710
827
846
740
879
688
60
1298
1300
1169
1300
1300
275
1000
64
Instrument Comparison
Some Instruments are better than others
but
All are affected by interferences
Data is NOT transferable between instruments
There is room for improvement by
manufacturers
Effect of Haemolysis - methods
2
Aeroset
100
Advia
50
Mod-D
Integra
0
-50
0
500
1000
1500
Lx20
V250
AR
-100
AU600
Phosphate (mmol/L)
Bilirubin (umol/L)
150
1.5
1
0.5
0
-150
Haemoglobin (mg/dL)
0
500
1000
1500
Haemoglobin (mg/dL)
Examples of tests where different instruments show wide
variations in response to haemolysis (Data from 2000).
Method Comparison
Some methods better than others
Suggest choosing methods which are less prone
to interference
May require third party supplier
Using your instrument
Using Your Instrument
Once the instrument is chosen the fun begins
A protocol must be set which allows appropriate
response to samples with interferences
requires detailed knowledge of your method /
instrument
Sources:
 Manufacturer
 Literature
 Own studies
TEST: Cholesterol
9
Raw Data
8
7
6
l
5
m
4
n
g
3
i
2
1
200
400
600
800
1000
1200
1400
Exp change*
0.93
Exp change*
0.58
1600
Absolute Change
-0.66 to 2.52
-0.41 to 1.58
* if linear
Dec.Pl.
2
Data Set
l
m
n
g
i
0
0
Cuttoffs
Current
800
Proposed
500
CV(wi)
5.3%
CV(a)
2.5%
Instrument
AU2700
AU2700
AU2700
Modular
Modular
Date
1-Apr-05
1-Apr-05
1-Apr-05
1-Jan-04
1-Jan-04
Dashed
horizontal
lines:
ALP
1.4
1.2
1
0.8
0.6
0.4
0.2
0
-0.2 0
200
400
600
800
1000
1200
1400
1600
-0.4
Vertical
lines:
Green current
Black proposed
-0.6
Change per 1000H
Average
STDEV
#N/A
#N/A
#N/A
#N/A
Slope*1000
Correl
A
1.257
0.99
B
0.911
0.97
C
1.165
1.00
Data set
D
E
Relative Change
1.3
Dashed
horizontal
lines:
ALP
1.2
1.1
Yellow
+/- CVwi
1
0.9
0.8
0.7
0
200
400
600
800
1000
1200
1400
1600
Vertical
lines:
Green current
Black proposed
Available Data Sets:
A
AU2700
B
AU2700
C
AU2700
D
AU2700
E
AU2700
F
AU2700
G
Modular
H
Modular
I
Modular
J
H917
K
H917
l
AU2700
m
AU2700
Cholesterol
2-May-04
2-May-04
2-May-04
5-May-04
5-May-04
1-Apr-02
1-Jan-04
1-Jan-04
1-Jan-04
1-Jan-00
1-Jan-00
1-Apr-05
1-Apr-05
Olympus Results
Absolute Change
1.4
h
1.2
1
0.8
0.6
V
0.4
Modular Results
0.2
0
-0.2 0
200
400
600
800
1000
1200
1400
1600
-0.4
-0.6
Olympus Cholesterol Reagent and Modular Cholesterol Reagent
G
c
B
pr
-0.2 0
200
400
600
-0.4
800
1000
1200
1400
1600
Olympus Results: 10% at 500
2.5, 4.1 and 6.0 mmol/L
-0.6
current
Black proposed
Relative Change
1.3
Dashed
horizonta
lines:
ALP
1.2
1.1
Yellow
+/- CVwi
Modular Results
10% at 700
Vertical
3.5 mmol/L
lines:
1
0.9
0.8
0.7
0
200
400
600
800
1000
1200
1400
1600
Response best expressed as absolute (not not percentage)
Green current
Black propose
Data Sources
Best data is from your own instrument
 No factors
 Full data set
 Perform experiment as needed.
Manufacturer information best when all results
available
 Beware of “No Interference” limits (eg 10%)
 Format of limits may not be useful
How accurate do we need to be?
RCPA-AACB Quality Assurance limits
Change greater than 2 SD of analytical
precision
Change related to biological variation
10%
Other fixed percentage or absolute values
A difference that may lead to a change in
clinical management - subjective*
Error Budget
Int. error
Other errors
Total error
The Accuracy - Utility Balance
More accuracy
More rejections
More recollections
More delays
Unhappier ptns
and Drs
Fewer clinical
errors
Less accuracy
Fewer rejections
Fewer recollections
Shorter TAT
More clinical errors
Interference Limits
No easy solution
Take all factors into account
Likely clinical effects is the main parameter
 (personal opinion)
Include pathologist / clinician in decision
making
Hitachi 917/747 Haemolysis Index
The haemolysis index (H) should be measured on all samples analysed on the Hitachi 747 or 917
In response to the H index, results should be handled as indicated by the table. When the H index is greater
than the value indicated for that analyte, the results for that analyte should not be released:
Haem
(mg/dL)
30
40
100
130
160
350
500
600
700
800
1000
1200
1500
2000
Analyte
LDH
LD1
Potassium
AST, CK
Bilirubin, c.Bili
Phosphate
Fructosamine, Iron
Protein
Triglycerides
Albumin, ALT, GGT,
Cholesterol
Amylase, ALP, Bicarbonate,
Calcium, CK-MB, Lipase,
HDL, Magnesium
Sodium
Uric Acid
Chloride, Creatinine, Glucose,
Transferrin, Urea
Trace Haemolysis (H1)
30-90
Mild haemolysis (H2)
90-170
Moderate Haemolysis (H3)
170-850
Gross Haemolysis (H4)
>850
- Clinicom will hold samples with an H index >100 as well as all requests for LD and LD1. Thus all
samples containing LD or LD1 must have the H index reviewed.
-In place of the result, “HAEM” should be placed in Clinicom which will generate the comment “H’lysed”
in place of the result in Pathnet. A footnote should be inserted as follows:
H1 (No result due
interference from trace haemolysis) if H index between 30 and 90; H2 (…mild haemolysis) if H index
between 90 and 170, H3 (…moderate haemolysis) if H index between 170 and 850, and H4 (…gross
haemolysis) if H index greater than 850.
- A footnote is only used if a result is withheld due to haemolysis.
SydPath
Haemolysis
Protocol
Other quality factors
Sample type:
 Serum, heparin plasma, EDTA plasma, fluoride
oxalate, Citrate, gel separators.
Sample stability
 As whole blood, as serum/plasma
 At RT, 4 degrees, -20 degrees
What can I do
that will make a
difference to
your business?
A supplier’s question…
Suppliers…..
Quality data on interferences, sample types and
analyte stability can:
 Reduce recollections
 Reduce unnecessary recollections
 Reduce repetition in multiple laboratories
Head office, literature watch, local data
Conclusions
Interferences and our response to them are part
of providing a quality laboratory service
 Choose methods and instruments with low
interference
Choose methods where data is available about
interferences or generate local data
Implement a policy for responding to
interferences