VALIDATION of the COMPLEMENT COMPONENT C1q RID ASSAY
Igor Y. Pavlov, Julio C. Delgado
ARUP Institute for Clinical and Experimental Pathology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah
SUMMARY
RESULTS
Figure 2. Normalized data: ARUP vs National Jewish Health Center
comparison
EP Evaluator
C1q
Research and Development -- ARUP
Alternate (Quantitative) Method Comparison
X Method: ARUP
Y Method: NJH
Scatter Plot
Bias
0.2
Deming Regr
1:1 Line
Percent Bias
15
Mean Bias
10
0.1
1.2
1
Percent Bias
5
Bias (%)
NJH (%)
C1q is a subunit of the C1 complex that activates the classical pathway of the complement system. The Binding Site radial immunodiffusion assay was
validated for the human EDTA plasma samples. It was found that C1q RID assay was accurate and linear in the range between 30 and 230 µg/mL.
Recovery of the spiked samples varied between 99% and 110%. Reproducibility from run to run and within runs was below 10%. Reference interval
was estimated to be from 109 µg/mL to 242 µg/mL. Comparing results of C1q determination by ARUP, Quest and National Jewish Health Center
(NJHC), we found no correlations. Obviously, all 3 kits utilize different proteins as a standard. When results were normalized by the average value of
each laboratory set, normalized data sets correlated to each other. Correlation Coefficient R between ARUP and Quest results was very poor, only 0.4;
NJHC vs ARUP: R=0.87. Bias for ARUP – Quest comparison was twice as high (30% vs 15%) and confidence intervals for slope and intercept were
much wider than for the NJHC – ARUP comparison results. Direct results comparison between different laboratories should not be considered because
of the differences in utilized standards and, accordingly, reference intervals.
0
0
-5
-0.1
-10
0.8
-0.2
0.8
INTRODUCTION
1
ARUP (%)
-15
1.2
0.8
1
ARUP (%)
1.2
0.8
1
ARUP (%)
1.2
Regression Analysis
C1q is a 400kDa hexameric gamma-2 protein that is a subunit of C1 – the first component of complement system. The binding of two or more of C1q’s
six globular domains initiates the classical pathway of complement activation. C1q binds readily to the CH2 domains of aggregated IgG molecules in an
immune complex or the CH3 domain of a single IgM molecule whose conformation has been altered following antigen binding. It can also bind directly
to certain microorganisms and mycoplasmas. The multivalent binding of C1q is believed to lead to a conformational change in C1q complex, activating
C1r, then C1s and thereby initiating the classical complement pathway. The collagen-like tail domain of C1q (which is only exposed once C1 inactivator
dissociates C1r2 and C1s2 from the C1q-activator complex) increases the phagocytosis of particles by monocytes and macrophages. Serum levels of C1q
are reduced in immune complex disease, SLE and meningitis. Hereditary deficiency is also known
Slope:
Intercept:
Std Err Est:
Deming
Regular
1.237 (0.864 to 1.611)
-0.239 (-0.617 to 0.139)
0.100
1.048 (0.692 to 1.403)
-0.049 (-0.409 to 0.311)
0.095
95% Confidence Intervals are shown in parentheses
Supporting Statistics
Corr Coef (R):
Bias:
X Mean ± SD:
Y Mean ± SD:
Std Dev Diffs:
0.8701
-0.001
1.002 ± 0.155
1.001 ± 0.187
0.092
SubRange Bounds:
Points (Plotted/Total):
Outliers:
Scatter Plot Bounds:
CONCLUSIONS
None
15/15
Not Tested
95% CI
Experimentvs
Description
Figure 3. Normalized data: ARUP
QUEST comparison
Expt Date:
Rep SD:
Result Ranges:
Units:
Analyst:
Research and Development -- ARUP
Comment:
EP Evaluator
Figure 1. Accuracy and linearity of the C1q RID assay
Accepted by:
C1q
C1q
1.3
QUEST (%)
Scatter Plot
Percent Recovery
120
1:1 Line
Fitted Overall
110
% Recovery
300
200
Y Method: QUEST
Scatter Plot
Instrument: Plasma*
Accuracy and Linearity
Measured (µg/mL)
08 Mar 2010
1
0.71 to 1.35
%
IP
Bias
Deming Regr
1:1 Line
1.2
0.2
20
1.1
0.1
10
0
-0.1
-10
0.8
-0.2
-20
-0.3
0.7
100
100
0
0.9
0.7
0.8
0.9
1
1.1
ARUP (%)
1.2
1.3
-30
0.7
0.8
0.9
1
1.1
ARUP (%)
1.2
1.3
0.7
0.8
0.9
90
Regression Analysis
0
80
0
100
200
300
400
0
100
Assigned (µg/mL)
200
300
Linearity Summary
N
Slope
Intercept
Error
5
1.026
1.7
4.2%
LINEAR within Allowable Systematic Error of 10.0%
Overall
Deming
Regular
1.058 (0.375 to 1.740)
-0.059 (-0.751 to 0.632)
0.183
0.410 (-0.152 to 0.971)
0.590 (0.021 to 1.159)
0.151
400
Assigned (µg/mL)
Slope:
Intercept:
Std Err Est:
95% Confidence Intervals are shown in parentheses
Supporting Statistics
Statistical Analysis and Experimental Results
Smpl 5
Smpl 4
Smpl 3
Smpl 2
Smpl 1
Assigned Pct
18.7 6.25%
37.4 12.5%
74.8
25%
149.5 50%
299.0 100%
Mean
20.0
41.0
81.0
162.0
296.0
% Rec.
107.0
109.7
108.4
108.4
99.0
Est
20.8
40.0
78.4
155.1
308.6
Resid
-0.8
1.0
2.6
6.9
-12.6
Linear?
Pass
Pass
Pass
Pass
Pass
Measured Concentrations
18
22
43
39
81
81
162
162
296
296
See User's Specifications for Pass/Fail criteria
0.4007
-0.001
1.002 ± 0.155
1.001 ± 0.158
0.172
20.0%
50%
10.0%
SubRange Bounds:
Points (Plotted/Total):
Outliers:
Scatter Plot Bounds:
X: Excluded from calculations
Experiment Description
Supporting Data
User's Specifications
Allowable Total Error:
Systematic Error Budget:
Allowable Systematic Error:
Corr Coef (R):
Bias:
X Mean ± SD:
Y Mean ± SD:
Std Dev Diffs:
Analyst:
Date:
Value Mode:
Units:
Lot Number:
IP
02 Apr 2010
Pct-Assigned
µg/mL
Expt Date:
Rep SD:
Result Ranges:
Units:
REFERENCES
Percent Bias
Mean Bias
1
Complement component C1q radial immunodiffusion assay was validated and ARUP reference
interval defined. Direct comparison to other laboratories results should not be considered because of
differences in utilized standards and, accordingly, reference intervals.
30
Percent Bias
Research and Development -- ARUP
400
08 Mar 2010
1
0.73 to 1.30
%
IP
X Method: ARUP
EP Evaluator
Radial immunodiffusion assay from (The Binding Site Limited, Birmingham, U.K) was
applied to 81 healthy donor plasmas for reference interval determination, 29
serum/plasma pairs of the healthy donor specimens, and to samples with low, medium and
high C1q concentrations to access accuracy , linearity and reproducibility of the assay. EP
Evaluator software was applied to the result interpretation. Eighteen EDTA plasma
samples were sent to the Quest Diagnostics, National Jewish Health Center (NJHC), and
run at ARUP for C1q assays. Both Quest and NJHC are utilizing home-brewed radial
immunodiffusion kits.
Y Method
Alternate
(Quantitative) Method Comparison
Signature
Date
Bias (%)
MATERIALS and METHODS
X Method
During the validation study we found that C1q RID assay was accurate and linear in the range between
30 and 230 µg/mL with allowable error of 20% with 50% systematic error. Recovery of the spiked
samples varied between 99% and 110%. Reproducibility from run to run and within runs was within
10%. By the nature of radial immunodiffusion assay, detection of C1q concentration below 50µg/mL
(corresponding to the ring diameter below 5.0 mm) is not reliable: potential reading error of 0.1 – 0.2
mm generates 15% – 30% error in C1q concentration. Reference interval calculated with transformed
parametric was estimated to be from 109 µg/mL (90% confidence limits from104 to117 µg/mL) to 242
µg/mL (90% confidence limits from 227 to 258 µg/mL). Comparing results of C1q determination by
ARUP, Quest and NJHC, we found no correlations. Obviously, all 3 kits utilize different proteins as a
standard. When results were normalized by the average value of each laboratory set, normalized data
sets correlated to each other: confidence intervals for slopes included 1, and confidence intervals for
the intercept included 0. Correlation Coefficient R between ARUP and Quest results was very poor,
only 0.4; NJHC vs ARUP: R=0.87. Bias for ARUP – Quest comparison was twice as high (30% vs
15%) and confidence intervals for slope and intercept were much wider than for the NJHC – ARUP
comparison results.
X Method
Y Method
08 Mar 2010
1
0.73 to 1.30
%
08 Mar 2010
1
0.76 to 1.25
%
None
15/15
Not Tested
95% CI
1
1.1
ARUP (%)
1.2
1.3
1. Cooper NR (1985). The classical complement pathway: Activation and regulation of the first
complement component. Adv. Immunol. 37, 151-216
2. Arlaud, GJ et al (1987). A functional model of the human C1 complex. Immunol. Today, 8,
106-111.
3. Ross, SC & Densen, P. (1984). Complement deficiency states and infection: Epidemiology,
pathogenesis and consequences of neisserial and other infections in an immune deficiency.
Medicine, 63, 243-273
4. Schifferli, GA et al (1986). The role of complement and its receptor in the elimination of
immune complexes. New Eng. J. 315, 488-495