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Two nephelometric methods compared with a radial immunodiffusion method for the measurement of IgG, IgA and IgM
Clin. Biochem. 13 (2) 84-91 (1980)
Two Nephelometric Methods Compared with a Radial Immunodiffusion Method for the Measurement of IgG, IgA and IgM G.C. B L A N C H A R D
AND ROSALIE GARDNER
V e t e r a n s A d m i n i s t r a t i o n M e d i c a l C e n t e r , 150 S o u t h H u n t i n g t o n A v e n u e , B o s t o n , M a s s a c h u s e t t s and the Department of Pathology, Boston University School of Medicine, 75 E a s t N e w t o n S t r e e t , B o s t o n , M a s s a c h u s e t t s 02127 (Accepted
January
1. The IgG, IgA and IgM concentrations of a group of 100 or more human sera found by both the rate nephelometric Beckman Immunochemistry System and the endpoint Behring Laser Nephelometer procedure gave good correlations when compared to results obtained by an endpoint radial immunodiffusion method. The correlation coefficients for the Behring method comparisons were 0.958 for IgG, 0.979 for IgA and 0.966 for IgM when only values under 850 IU of IgM/ml were considered. The Beckman system gave correlation coefficients of 0.984 for IgG, 0.993 for IgA and 0.986 for IgM when IgM concentrations were under 500 I U / m l . Determinations from all three methods agreed well except for very high IgM values which were considerably higher by RID. 2. Both nephelometric methods are fast and precise. The average within-run coefficients of variation were 2.6% for the Behring method, 2.2% for the Beckman system and 2.1% for radial immunodiffusion while average between-run precision values of 6.9% for the Behring method, 3.7% for the Beckman system and 4.9% for radial immunodiffusion were found. The Beckman rate system has the advantage of one point calibration, ease of data processing and elimination of sample blanks. IN RECENT YEARS THERE H A S BEEN CONSIDERABLE ADVANCEMENT in the development of nephelometric methods for the quantitation of specific serum proteins. These methods are based on the measurement of the light scattering produced by antigen-antibody complexes formed in immunoprecipitin reactions. Under antibody excess conditions the concentration of antigen is proportional to both the intensity of the scattered light produced by the reaction and to the rate of change of the scattered light intensity. Procedures using light scattering measurements at equilibrium and rate measurements have been reported. Killingsworth et al.'I-2' used a nephelometric equilibrium approach in a manual and automated continuousflow procedure for the determination of IgG, I g A and IgM. The Centrifugal Fast Analyzer was adapted to light scattering measurements either kinetic or at
equilibrium by T i f f a n y et al. '3~ Buffone et al. '4'51 made use of a laser-modified centrifugal analyzer. Several new instruments have been commercially introduced and evaluated using laser endpoint type nephelometers 's'~' or rate methodology "3,m Correspondence to: Gordon G. Blanchard, Ph.D. Chief of Clinical Immunology Veterans Administration Medical Center 150 South Huntington Avenue Boston, Massachusetts 02130
02130
30, 1980)
We have analyzed 100 or more human serum samples for immunoglobulins G, A and M using two of these new nephelometric techniques, the Behring Laser Nephelometer System which measures light scattering either during the kinetic phase of the reaction or at equilibrium '7'8''') and the Beckman Immunochemistry Analyzer System (ICS) which uses a rate methodology'~3"4'. Results obtained by both nephelometric methods have been correlated by linear regression analysis with results obtained by an endpoint radial immunodiffusion (RID) procedure "5'. MATERIALS AND METHODS
Radial Immunodiffusion W e used Tri-Partigen radial immunodiffusion plates from Calbiochem-Behring Corp. (San Diego, C A 92112) for all the R I D analyses. The standards used were those supplied by Behring for use with the Tri-Partigen plates and had been compared with the World Health Organization ( W H O ) International Reference standard "8~. The IgG standards ranged in concentration from 3.9 to 23.2 IU/ml, the IgA standards from 45-193 IU/ml and the I g M standards from 71-297 IU/ml. To convert the Behring standards from IU/ml to mg/dl, the IU/ml value is multiplied by 100 and divided by the appropriate factor, 11.5 for IgG, 59.5 for IgA and 115 for IgM. The 12 wells per plate were filled with 5 ~I of test samples and standards using a 5 ,~l Micr~dispenser ( D r u m m o n d Scientific Co., Broomall, P A 19008). The sera for IgG analysis were usually diluted 1:10 with isotonic NaCl before use. All patient samples were run in duplicate and the standards were run singly. Determinations were made at endpoint after diffusion times of at least 50 h for IgG and IgA and 80 h for IgM. The diameters of the precipitin rings were measured to 0.1 m m using a Finescale reticle No. 122 magnifying comparator (Laboratory Supplies Co., Hicksville, N Y 11801). Specimens with ring diameters exceeding that of the highest standards were diluted and reassayed. Sample concentrations were found either manually by plotting the squared diameters of the standard precipitin rings against the corresponding concentrations on linear graph paper and reading from the resulting standard curve or by use of the Behring Data Processing Module Model 100 which is programmed for R I D calculations.
Behring Laser Nephelometer For imrnunoglobulin quantitation by the Behring nephelometric system we used the Behring Laser Nephelometer Model 7601 (Calbiochem-Behring Corp., San Diego, C A 92112) and followed the manufacturer's directions with some modification. This instrument uses a helium neon laser, wavelength 632.8 nm, as a light source. The forward
light scattering produced by the antigen-antibody corn-
TWO
NEPHELOMETRIC
METHODS
COMPARED
plexes f o r m e d w h e n a n t i g e n s r e a c t with specific a n t i s e r a is focused onto a photodiode by an optical lens system. The electrical signal g e n e r a t e d by the laser v o l t m e t e r is indicated by a digital display which has a r a n g e of f r o m 0.00 to 19.99. M e a s u r e m e n t s m a y be made at time intervals f r o m 15 to 60 minutes. Precise t i m i n g is necessary for readings taken before endpoint. Six serial dilutions of the B e h r i n g Laser Standard Serum, I g G 109 I U / m l , I g A 115 I U / m l and IgM 99 I U / m l were made using volumetric pipets. The concentrations of the standard dilutions r a n g e d f r o m 0.170 I U / m l to 5.45 I U / m l for IgG, 0.180 I U / m l to 5.75 I U / m l for I g A and 0.155 I U / m l to 4.95 I U / m l for IgM. The same conversion factors as are used for the T r i - P a r t i g e n RID method may be used to change values f r o m I U / m l to m g / d l . The Behring N e p h e l o m e t r i c Grade antisera to human IgG, I g A and IgM were diluted 1"5 and the dilutions were f i l t e r e d t h r o u g h Cathivex disposable filter units, 0.45 ~m (Millipore Corp., Bedford, MA. 01730). P a t i e n t samples were diluted 1:101 using a Gilson P i p e t m a n and a volumetric pipette or the Model 1500 P i p e t t o r - D i l u t o r made by Cavro Scientific Instruments, Los Altos, CA 94022. All dilutions were m a d e using sterile 0.9~ NaC1 i r r i g a t i o n U S P solution obtained f r o m the hospital supply. S e r a which g a v e readings outside of the standard curves were re-diluted and re-assayed. Reactions were carried out in 1 cm × 0.4 cm disposable plastic cuvettes obtained f r o m W a l t e r Sarsted Inc., Princeton, N J 08540 or supplied by Behring. F o r I g G analysis, 10 ~l of standard serum or patient sample dilutions were carefully added to the bottom of a cuvette followed by the addition of 200 ~1 of the appropriate a n t i s e r u m dilution. Mixing was by gentle shaking. F o r the I g A and IgM analyses, 100 ~l of standard or sample dilutions were added to cuvettes again followed by 200 ~1 of the a p p r o p r i a t e antiserum. The B e h r i n g method recommends the use of an e m p t y cuvette r e a d i n g as a blank and the f i l t r a t i o n of any m a r k e d l y turbid serum. We used a sample blank and f i l t e r e d no serum except those used in the f i l t r a t i o n study. The sample blank consisted of the a p p r o p r i a t e volume of sample or standard dilution with 200 ~l of 0.9c/( NaCl i r r i g a t i o n solution added to a cuvette of pre-determined light scatter. R e a d i n g s were taken at 60 minutes and the difference between the two values was used as a sample correction and was applied to the e m p t y cuvette r e a d i n g of the corresponding a n t i g e n - a n t i s e r a mixture. All light s c a t t e r i n g m e a s u r e m e n t s for the correlation data were taken at 60 minutes. The light s c a t t e r i n g in several runs was followed by reading at time intervals f r o m 15 to 240 minutes. We did not encounter any sera in antigen excess as all sera had been previously analyzed by another method and a p p r o p r i a t e dilutions w e r e used. To eliminate the use of two cuvettes for each standard and sample assayed and to obtain a t r u e r r e a g e n t blank, we explored the possibility of t a k i n g an initial light s c a t t e r i n g r e a d i n g on each reaction solution before any increased light s c a t t e r i n g due to antigen-antibody complex f o r m a t i o n was observed. A cuvette containing 10 ,~l of IgG, 5.45 I U / m l and 200 ~l of a 1:5 dilution of IgG antis e r u m was g e n t l y shaken and i m m e d i a t e l y placed in the cell c o m p a r t m e n t of the B e h r i n g L a s e r Nephelometer. R e a d i n g s were t a k e n at 3 second intervals by the Behring Laser D a t a P r o c e s s i n g Module until a sharp and steady increase in light s c a t t e r i n g was obtained. This was repeated with 100 ~l of I g A , 5.75 I U / m l and 200 ~1 1:5 I g A antiserum solution and again with 100 ~1 of IgM 4.95 I U / ml and 200 ~1 of 1:5 I g M a n t i s e r u m solution. The H e w l e t t P a c k a r d Model 9830 Calculator and 9862A Calculator P l o t t e r were used in curve f i t t i n g studies and to obtain the immunaglobulin concentration values. Some c o m p a r a t i v e d a t a was also obtained f r o m the B e h r i n g Data Processing Module Model 100 and f r o m standard curves constructed by m a n u a l l y plotting the standard concentrations vs. l i g h t s c a t t e r i n g r e a d i n g s on linear graph paper.
WITH
A RADIAL
IMMUNODIFFUSION
METHOD
85
Beckman Immunochemistry System The Beckman ICS for the d e t e r m i n a t i o n of immunoglobulins by rate n e p h e l o m e t r y uses the I m m u n o c h e m i s t r y A n a l y z e r in conjunction w i t h a F i x e d Ratio Diluter (1:6), a Fixed Volume Dispenser (600 ~1), a 42 ~1 2 position pipet constructed so as to avoid the introduction of air bubbles and a R e a g e n t Test Kit all f r o m Beckman I n s t r u m e n t s , Inc., Fullerton, CA 92634. The i n s t r u m e n t uses a quartziodide lamp and measures the m a x i m u m rate of increase of 70 ° f o r w a r d l i g h t s c a t t e r produced by the f o r m a t i o n of antigen-antibody complexes. The Immunoglobulin R e a g e n t T e s t Kit includes a diluent solution of normal saline, a phosphate b u f f e r e d saline solutio.~ containing a p o l y m e r enhancer, a n t i s e r a to IgG, I g A and IgM and a calibrator serum containing IgG, I g A and I g M concentrations n e a r the midpoints of the measu r i n g r a n g e s of the tests. The concentration values of the standards were established by comparison with a p r i m a r y standard of the W H O . Disposable glass f l a t - b o t t o m e d reaction tubes with stirrers are also provided. The procedure followed was according to the kit directions which were only slightly modified f r o m the detailed description of S t e r n b e r g c14). Successive dilutions 1:6, 1:36 and 1:216 of the calibrator s e r u m and sample sera were made. The a n a l y z e r was calibrated at a single point midw a y in the m e a s u r i n g r a n g e of the immunoglobulin being determined. The m e a s u r i n g r a n g e for IgG is 43.4-446.4 I U / m l , for I g A 42.2-352 I U / m l and for IgM 59-472 I U / m l . Calibration was accomplished by optically r e a d i n g into the i n s t r u m e n t by the insertion of p r o g r a m cards the calibrator value and i n f o r m a t i o n necessary for the analy z e r m i c r o c o m p u t e r to convert peak rate signal to concentration units. This was followed by the introduction of 42 ~l of the a p p r o p r i a t e calibrator dilution and 42 ~l of the specific a n t i - s e r u m into a reaction tube containing 600 ~1 b u f f e r which had been placed in the cell c o m p a r t m e n t of the instrument. The peak r a t e signal produced by the reaction was stored in the computer, compared with a r e p e a t run for s a t i s f a c t o r y a g r e e m e n t and internally calibrated by the a n a l y z e r so t h a t the calibrator serum read at the t a r g e t value. Once calibration was completed, sample dilutions were run in duplicate following a like procedure. Step by step directions were given on the i n s t r u m e n t a l p h a n u m e r i c display. Concentrations units w e r e displayed in m g / d l , g/1 and I U / m l . Conversion of I U / m l to m g / d l m a y be made by m u l t i p l y i n g the I U / m l values by 100 and dividing by the a p p r o p r i a t e factors f o r the Beckman standards, 12.4 for IgG, 70.4 for I g A and 118 for IgM. Out of r a n g e samples were rejected by the i n s t r u m e n t and could be immediately re-analyzed using a n o t h e r dilution. A n t i g e n excess checks were called f o r when determined by the micro-computer. To v e r i f y the linearity of the i n s t r u m e n t , we analyzed solutions of known concentrations which covered the immunoglobulin m e a s u r i n g ranges.
Precision Within-run precision was determined for the nephelometric methods by 20 replicate analyses of each serum. F o r RID within-run precision is the result of 9 replicate analyses on one plate for each serum. The b e t w e e n - r u n precision f o r all three methods was obtained by a n a l y z i n g each serum 20 times in successive runs. Coefficients of v a r i a t i o n were determined for three sera in d i f f e r e n t concentration r a n g e s for each method studied.
Effect of Turbidity Aliquots of 4 m a r k e d l y turbid sera w e r e filtered t h r o u g h Millipore 0.45 ~m f i l t e r s and both the filtered and unfiltered sera were analyzed by the two n e p h e l o m e t r i c methods.
Computations The H e w l e t t - P a c k a r d Model 9830A Calculator and 9862A Calculator P l o t t e r were used for the statistical c o m p u t a tions and f o r d a t a plotting.
86
BLANCHARD AND GARDNER
Fig. 1 - - Linearity of the Beckman Immunochemistry Analyser for the IgM analysis.
I
I
S
~B
I
II
I
I
I
I
I
I ~S
2BB
2~B
3H
3~B
I
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EXPECTED IBM IU/ML
RESULTS Standard Curves The standard curves found with the R I D method at end-point were straight lines w h e n the diameters squared of the precipitin rings were plotted vs the TABLE
c o n c e n t r a t i o n s on l i n e a r g r a p h paper. The p l o t t i n g of s t a n d a r d curves is n o t n e c e s s a r y in the Beckman ICS method. Method l i n e a r i t y was v e r i f i e d when s t r a i g h t lines were o b t a i n e d for IgG, I g A and IgM when k n o w n c o n c e n t r a t i o n s were plotted a g a i n s t c o n c e n t r a t i o n s f o u n d by a n a l y s i s ( F i g . 1). 1
PRECISION OF THE RID, BEHRING LASER NEPHELOMETER AND B E C K M A N ICS M E T H O D S FOR DETERMINATION OF IGG, IGA AND I G M
Within-run Mean, IU/ml RID
Between-run CV,%
Mean, IU/ml
n=9
CV,% n = 10
(4.4)* IgG
208 123 57
2.9 2.1 3.1
200 118 58
7.1 5.2 4.4
IgA
143 92 38
i.I 2.,2 2.3
175 137 88
3.2 3.7 2.3
IgM
289 159 ll0
1.8 1.8 1.2
274 145 104
6.3 6.9 5.0
Behring Laser Nephelometer
n = 20
n = 20 (2.7-4)
IgG
497 152 87
2.7 2.0 2.0
451 177 68
7.3 6.9 6.8
IgA
352 117 27
2.0 1.5 2,7
533 171 62
5.9 5.7 8.9
IgM
418 168 57
1.3 3.1 5.9
458 227 75
7.3 4.7 8.6
Beckman ICS
n
=
20
n
=
20
IgG
369 143 86
2.1 1.9 0.9
(3) (2) (3)
330 138 68
2.9 2.2 (7) 3.4
IgA
278 89 55
2.3 1.6 2.1
(2) (2)
281 162 56
3.2 1.8 (5) 3.9
IgM
336 164 89
2.1 2.6 4.2
(2) (3) (3)
415 154 I01
5.3 4.9 (5) 5.7
* The precision data provided by the manufacturer for approximately the same concentrations are listed in parentheses.
TWO NEPHELOMETRIC METHODS COMPARED WITH A RADIAL IMMUNODIFFUSION METHOD
87
w
I'M
m
v
Fig. 2 - - Behring Laser Nephelometer Standard Curve for IgG. t.~ .J
l
2
3 IGG
4
E
6
IU/ML
::T v
~--
r~
L~ .~
r~
Fig. 3 - - Behring Laser Nephelometer Standard Curve for IgM.
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1 ~E
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~8~
IGM
I
IU/ML
When calculating our data, we found the Behring Laser Nephelometer results processed by the Behring Data Processing Module 100 to be slightly higher especially for IgG and IgA than values obtained from hand plots. We therefore carried out curve fitting studies to consider another method of calculation. Fifteen point standard curves for IgG and 14 point curves for IgA and IgM constructed using data from the Behring Laser Nephelometer showed a third degree polynomial fit for IgG (r = 0.998), for IgA (r----0.997) and for IgM (r---0.996). The IgM standard curve was also described by a quadratic fit (r --- 0.996). The six standard concentrations recommended by the Behring procedure did not clearly define the shape of the cubic curve in the range between the two highest standards, the fifth and sixth standards. A quadratic equation fit the first five points on the IgG and IgA standard curves and followed closely
the third order fit on the 15 and 14 point lines. Sample concentrations for IgG and IgA were calculated from a standard curve fit to a quadratic equation omitting the highest standard value. In the few cases where light scattering readings were above the fifth standard, concentrations were determined by interpolation between standards five and six. IgM values were calculated from 6 point standard curves with quadratic fits. The average measure of fit was 0.998 for the IgG standard curves, 0.996 for the I g A standard curves and 0.997 for the IgM standard curves. Fig. 2 shows an IgG 15 point standard curve fit to a third degree polynomial with a five point standard curve fit to a quadratic equation superimposed. Fig. 3 is a typical standard curve for IgM. Concentrations determined using this method of calculation agreed well with hand plots of the data and were used in all correlation computations. The
B L A N C H A R D AND G A R D N E R
88
+
!. Fig. ~ - - C o m p a r i s o n of the R I D a n d Behring Laser Nephelometer Methods f o r the D e t e r m i n a t i o n of I g M . ( A ) S l o p e : 1.066 f o r values r a n g i n g ]rom 39.1 to 850 I U / m l . ( B ) S l o p e : 0.656 f o r v a l u e s r a n g i n g f r o m 39.1 to 8~67 I U / m l . • V a l u e s 0 to 850 I U / m l q- V a l u e s over 850 I U / m l
,,9
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IBBB
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2[lil~
3~BB
4~EB
5~[[
G~BB
7MB
8BBfl
I
RID 16M IU/ML
plots o b t a i n e d by use of t h e H e w l e t t P a c k a r d p l o t t e r w e r e also h e l p f u l in v i s u a l l y a p p r o v i n g t h e s t a n d a r d curves.
t i o n s a r e g i v e n in T a b l e I. T h e w i t h i n - r u n c o e f f i c i e n t s of v a r i a t i o n (CV) f o r R I D r a n g e d f r o m 1.1 to 3.1% w h i l e t h e R I D b e t w e e n - r u n CV r a n g e d f r o m 2.3 to 7.1%. T h e B e h r i n g L a s e r m e t h o d g a v e w i t h i n run CV of f r o m 1.3 to 5.9% and b e t w e e n - r u n CV o f f r o m 4.7 to 8.9%. T h e CV of t h e B e c k m a n ICS v a r i e d f r o m 0.9-4.2% f o r w i t h i n - r u n and f r o m 1.8 to 5.7% for between-run precision.
Precision
T h e w i t h i n - r u n and b e t w e e n - r u n p r e c i s i o n s f o r t h e RID, B e c k m a n ICS and B e h r i n g L a s e r N e p h e l o m e t e r m e t h o d s f o r IgG, I g A and I g M in t h e s e c o n c e n t r a -
TABLE 2 COMPARISONOF THE RID, BEHRING LASER NEPHELOMETERAND BECKMANICS METHODS FOR DETERMINATIONOF IGG, IGA AND IGM n (no. of data pairs)
Range, IU/ml
r
Slope
y-intercept (IU/ml)
1.137
-5.00
1.111
11.00
0.656
78.5
RID (x)-Behring Laser Nephelometer (y) IgG
109
IgA
102
IgM
108
IgM
103
IgM
100
IgG
112
IgA
108
IgM
110
IgM
108
IgM
101
I&,G
110
IgA
108
IgM
113
IgM
110
IgM
105
x 7.13-554 0.958 y 8.49-613 x 22.6-913 0.979 y 23.7-939 x 39.1-8467 0.996 y 47.2-5471 x 39.1-850 0.966 y 47.2-807 x 39.1-432 0.939 y 47.2-476 RID (x)-Beckman ICS (y) x 7.1-608 0.984 y 9.3-646 x 10.6-913 0.993 y 11.8-1031 x 39.1-8469 0.996 y 59.0-6293 x 39.1-1044 0.992 y 59.0--995 x 39.1-492 0.986 y 59.0-442 Beckman ICS (x)-Behring Laser Nephelometer (y) x 9.3-646 0.950 y 8.5-613 x 5.99-1031 0.980 y 3.11-939 x 30.7-6292 0.995 y 24.1-5472 x 30.7-909 0.977 y 24.2-859 x 30.6-430 0.959 y 24.1-550
1.006
9.38
1.069
- 1.35
1.019
0.179
1.17t
- 1.88
0.768
48.5
0.896
16.32
0.967
3.65
1.050
5.99
0.947
12.5
0.851
39.4
1.033
6.71
1.145
-12.1
TWO NEPHELOMETRIC METHODS COMPARED WITH A RADIAL IMMUNODIFFUSION METHOD
N 3
._i
EB
IBB
IEB
2BB
2EB
TIME (MIN.)
Fig. 5 - - B e h r i ~ g L a s e r N e p h e l o m e t e r I g A S t a n d a r d s - L i g h t scatterb~g f r o m 0-240 min. R e a d i n g s w e r e made at 15, 22, 30, 40, 45, 50, 60, 65,75, 85, 90, 100, 112, 120, 150 and 240 rain.
Correlation Correlation data for the comparison of the RID, Behring Laser Nephelometer and Beckman ICS methods is given in Table 2. Good correlations were found when the Behring Laser method was compared to RID. Correlation coefficients were 0.958 for IgG and 0.979 for IgA with slopes of 1.137 and 1.111 respectively. Slightly higher IgG and IgA values were obtained by the Behring Laser method. For the IgM comparisons there was a marked change in slope from 1.006 to 0.656 when values above 850 I U / m l were included (Fig. 4). The methods did not agree when sera of extremely high IgM concentrations were analyzed, the RID values being considerably higher. For values under 850 IU/ml there was excellent agreement between methods. The correlation coefficients were 0.939, 0.966 and 0.996 reflecting the different ranges of values. The Beckman ICS gave excellent correlation coefficients when compared to RID. The value for IgG was 0.984, 0.993 for IgA and for IgM the correlation coefficient ranged from 0.986 to 0.996 depending on the range of concentrations included. The slope of 1.019 for the IgG comparison indicated a good agreement in results found by both methods. The IgA comparison gave a slope of 1.171. The IgA concentrations determined by the Beckman ICS were slightly higher than those obtained by RID. For the IgM analyses method agreement was good for values under 500 I U / m l (slope 0.967) but as higher values were included, the slope decreased to 0.768. Again, the nephelometric method gave lower results with sera of abnormally high IgM. The Beckman ICS and Behring Laser Nephelometer comparisons showed good correlation and agreement between methods for IgG, IgA and for IgM. The correlation coefficients were 0.950 for IgG, 0.980 for IgA and 0.959 to 0.995 for IgM. The slopes were 1.050 for IgG, 0.947 for IgA and 0.851 - - 1.145 for IgM. The change in the slope when IgM values above 909 I U / m l were included was not as marked as when the nephelometric methods were compared to RID. When the correlation data was expressed in mg/dl
89
r a t h e r than IU/ml, the slopes f o r the RID and Beckman ICS comparisons changed to 0.945 for IgG, 0.990 for IgA and 0.943 for IgM where n=101. For ths Beckman ICS and Behring L a s e r Nephelometer comparisons the slopes became 1.132 for IgG, 1.120 for IgA and 1.060 for IgM, n----ll0. As both Behring methods used the same conversion factors for c h a n g i n g I U / m l to mg/dl, the RID and Behring nephelometric correlation coefficients and slope values remained the same regardless of the change in units. Except for the RID and Beckman IgA comparisons the results from all methods showed better agreement when expressed as IU/ml. The Beckman ICS being a rate method required no sample blank. A correction for significant sample light scatter was necessary in the Behring L a s e r Nephelometer method if the recommended filtration of markedly turbid sera was not followed. Because of reluctance to filter patient serum samples for reasons of safety, economy and possible loss of protein and because we found the Behring Standard L a s e r Serum dilutions to have considerable light scattering not due to the diluent, we decided to include a sample blank in the procedure for all the immunoblobulin analyses by this method. We had also found it difficult to judge by visual inspection which sera should be filtered. Some markedly turbid sera contributed little light scatter to the background while other less turbid sera gave considerable scatter. Filtering did not always clear the serum and some sera were difficult to filter requiring dilution or centrifugation before filtration. Sixteen per cent of the sera used in this study were found to give high background scatter. Two of these were icteric and most of the others were samples which had been received frozen and although not markedly turbid did contain obvious particles. Most fresh sera even though very turbid gave no significant light scatter when diluted for analysis. When filtered and unfiltered samples of f o u r highly turbid and light scattering samples were analyzed by both nephelometric methods, the mean difference between the filtered and unfiltered samples determined by the Beckman ICS was 1.6% for IgG, 1.7% for IgA and 6.6% for IgM. Mean differences in values obtained by the Behring L a s e r Nephelometer were 6.4% for IgG, 3.8% for IgA and 13.8% for IgM. The higher averages for IgM were due to the large difference in one sample possibly due to loss of IgM on filtration of the diluted serum. Laser Nephelometer Method-Reaction Time All the Behring Laser Nephelometer data used in these studies except for the timing experiment were obtained at 60 minutes when the reaction time was at or near equilibrium. When the antigen-antibody complexes became large enough to settle out, there was a rapid decrease in light scattering. We found that sedimentation sometimes occurred in the highest IgA standard before 60 minutes giving a false low light scattering reading to the high (6th) s t a n d a r d (Fig. 5). The f i f t h IgA s t a n d a r d did not decrease in reading until 112 minutes. The IgM reactions gave no such problem but in two experiments in which the light s c a t t e r i n g of the IgG s t a n d a r d s and
BLANCHARD AND GARDNER
90
antisera reactions were followed, the highest standard decreased in light scattering once at 65 minutes and onc~ at ~/5 minutes. Concentrations of sera calculated from 15 minute light scattering readings were in most cases slightly and sometimes markedly lower than values calculated from 60 minute data.
Behring Laser Nephelometer Method Initial Blank Reading The longer reaction time in the Behring Laser Nephelometer method due to the absence of polyethylene glycol or other reaction enhancer made possible an initial reading for background light scattering. The IgG 5.45 IU/ml and IgG anti-serum reaction solution gave reasonably steady light scattering for 30 seconds before there was a sharp increase in readings. Initial blank readings could be made during the first 183 seconds for the IgA 5.75 IU/ml anti-IgA reaction solution and for 144 seconds for the IgM 4.95 I U / m l anti-IgM solution.
Analysis Time The analysis of a batch of 18 samples for IgG, IgA and IgM including antigen excess checks was easily accomplished in 3 hours by both the Beckman ICS and the Behring Laser Nephelometer method. Two hours of technician time and a period of 2 to 4 days was necessary for determinations to be made on the same number of samples by RID. Using the Beckman ICS, immunoglobulin concentrations may be determined on a single sample in the m a t t e r of a few minutes. I f an incubation time of 15 minutes is used in the Behring Laser method, samples may be processed relatively rapidly especially if related to a previously run standard curve. We found, however, that light scattering in the lowest standard of IgM was not evident at 15 minutes making a longer incubation time necessary for IgM determinations in this range. DISCUSSION Both nephelometric methods gave good precision generally in the same ranges as we found for RID. The Behring Laser Nephelometer method within-run precisions were similar to those stated by the supplier and to the 3.2-5.4% CV found by Conrad et al.'" and the 3.8% CV of W~.lsh et al. '~''. The between-run precisions agreed well with the 4.4-8.3% CV of Conrad et al. '7', the 6.8-8.5% CV reported for IgG by Walsh et al. '11~ and the 5.6 and 1.8% CV for IgG and IgA respectively found by Schliep et al. '1~. The coefficients of variation for the Beckman ICS agreed favorably with the precisions claimed by the m a n u f a c t u r e r and with the 2-3% CV for the withinrun found by Sternberg 'm. The precisions of the Beckman system were slightly better than those of the Behring procedure and than the CV values reported from evaluations of a nephelometric method using another commercial instrument, the Hyland laser nephelometer ~1°-~. When the Behring L a s e r Nephelometer method was compared to RID, we found correlation coefficients which showed good agreement with the 0.967 to 0.981 values of Conrad et al. '7~, with the 0.996 to
0.998 coefficients reported by Daigneault and Lemieux 'a~ and the 0.97 value for IgG of Walsh et al. 'm We found the method agreement to be not quite as good as indicated by the slopes of 0.98 to 1.05 found by Daigneault and Lemieux '8', the slopes of 0.840 - 0.986 (x, Behring L a s e r ; y, RID) found by Conrad et al. '7', and the 0.96 slope for IgG determined by Walsh. et al. 'H', when results from both methods were compared by linear regression analysis. The correlations obtained when results from the Beckman ICS were compared to results found by RID were similar to the 0.939 to 0.952 values of Sternberg ''4~. When our data was expressed in mg/dl, the slopes of the regression lines from comparison studies showed better method agreement for IgA and similar method agreement for IgG and IgM when compared to the slopes of 1.316 for IgA, 0.952 for IgM and 1.035 for IgG reported by Sternberg ''4~. The correlation coefficients and method agreement with RID which we found in this study with the Behring and Beckman systems were close to ''°''~' and somewhat better than 's'''' values reported from similar studies using the Hyland laser nephelometer. The considerably higher IgM concentration values obtained by RID for some sera may be due to the presence in the sera of rapidly diffusing low molecular weight IgM giving a false result ~'8~. Virella and F u n d e n b e r g ''s' found a nephelometric method (Hyland) to give substantially lower results than RID in two cases of patients with macroglobulinemia and Deaton et al. '~°~ found slope improvement when abnormal I g A and IgM sera values were excluded from a comparison of data obtained by a nephelometric method and RID. In the course of this work we encountered few grossly turbid sera. We found that the sample blank correction made no difference in the IgG analyses using the Behring Laser Nephelometer method due to the instrument sensitivity and the dilution used. In most cases a sample blank serum correction for IgA and IgM analyses by this method also made little difference. However, background light scattering corrections for some sera and often for the standards was necessary. We suggest the initial blank reading as a possible method modification. The addition of at least one more point in the high range of the standard curve to better define a cubic fit line and the reading of the IgG and IgA analyses at time intervals less than 60 minutes before any antigen antibody complex sedimentations occur are also suggested improvements to the Behring L a s e r Nephelometry method. The two nephelometric procedures studied both o f f e r good alternative methods to RID for immunoglobulin determinations. The rate method has the advantage of single point calibration, automatic antigen excess check and no necessity for sample blank. ACKNOWLEDGEMENTS We thank Calbiochem-Behring Corp. and Beckman Instruments, Inc. for supplying reagent kits and for the use of their instruments. We are also most grateful to Dr. Eldon A. Boling for his assistance in the data
TWO
NEPHELOMETRIC
METHODS
COMPARED
c o m p u t a t i o n s and to Dr. Beni H o r v a t h for s u g g e s t i o n s to the m a n u s c r i p t .
WITH
A RADIAL
10.
REFERENCES
1. Killingsworth, L.M., and Savory, J. Manual nephelometric methods for immunochemical determinations of immunoglobulins IgG, IgA and I~M in human serum. Clin. Chem. 18, 335-339 (1972). 2. Killingsworth, L.M., and Savory, J. Automated immunochemical procedures for measurement of immunoglobulins IgG, IgA and IgM in human serum. Clin. Chem. 17, 936-940 (1971). 3. Tiffany, T.O., Parella, J.M., Johnson, W.F., and Burtis, C.A. Specific protein analysis by light scatter measurement with a miniature centrifugal fast analyzer. Clin. Chem. 20, 1{)55-1060 (1974). 4. Buffone, G.J., Savory, J., Cross, R.E., and Hammond, J.E. Evaluation of kinetic light scattering as an approach to the measurement of specific proteins with the centrifugal analyzer. I. Methodology. Clin. Chem. 21, 1731-1734 {1975). 5. Buffone, G.J., Savory, J., and Hermans, J. Evaluation of kinetic light scattering as an approach to the measurement of specific proteins with the centrifugal analyzer. II. Theoretical considerations. Clin. Chem. 21, 1735-1746 (1975). 6. Bruver, R.M., and Salkie, 'M:L. A comparative study of three approaches to the routine quantitation of human serum proteins. Clin. Biochem. 1l, 112-116 (1978). 7. Conrad, A., Sehurmann, J., and Kreutz, F.H. Elaboration of a method for the quantitative determination of proteins by laser nephelometry in the clinical routine laboratory. J. Clin. Chem. Clin. Biochem. 16, 299305 (1978). 8. Daigneault, R. and Lemieux, D. Evaluation of a Behring Laser-Nephelometer prototype in the measurement of IgG, IgA and IgM. Clin. Bioehem. 11, 2831 (1978). 9. Deaton, C.D., Maxwell, K.W., and Smith, R.S. In Automated lmmunoanalysis ( P a r t 2), R.F. Ritchie,
11.
12.
13. 14. 15.
16.
17.
18. 19.
I M M U N O D I F F U S I O N METHOD
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Ed., Marcel Dekker, New York, N.Y., 1978, pp. 375408. Deaton, C.D., Maxwell, ICW., Smith, R.S., and Creveling, R.L. Use of laser nephelometry in the measurement of serum proteins. Clin. Chem. 22, 14651471 (1976). Walsh, R.L., Coles, ,M.E., and Geary, T.D. Evaluation of two commercially available Laser Nepbelometers and their recommended methods for measurement of serum immunoglobulin G. Clin. Chem. 25, 151-156 (1979). Whieher, J.T., Perry, D.E., and Hobbs, J.R. An evaluation of the Hyland Laser nephelometer PDQ system for the measurement of immunoglobulins. Ann. Clin. Biochem. 15, 77-85 (1978). Anderson, R.J., and Sternberg, J.C., In Automated Immunoanalysis ( P a r t 2), R.F. Ritchie, Ed., Marcel Dekker, New York, N.Y., 1978, pp. 409-470. Sternberg, J.C. A rate nephelometer for measuring specific proteins by immunoprecipitin reactions. Clin. Chem. 23, 1456-1464 (1977). Mancini, G., Carbonara, A.O., and Heremans, J.F. Immunochemical quantitation of antigens by single radiai immunodiffusion. Immunochemistry 2, 235-254 (1965). Rowe, D.S., Grab, B., and Anderson, S.G. An international reference preparation for human serum immunoglobulins G, A, and M; content of immunoglobulins by weight. Bull. W.H.O. 46, 67-79 (1972). Sehliep, G., and Felgenhauer, K. Rapid determination of proteins in serum and eerebrospinal fluid by lasernephelometry. J. Clin. Chem. Clin. Bioehem. 16, 631635 (1978). Jones, D. Low molecular weight IgM as a pitfall in immunoglobulin quantitation by radial diffusion. Clin. Chim. Aeta 29, 551-556 (1970). Virella, G., and Fudenberg, H.H. Comparison of immunoglobulin determinations in pathological sera by radial immunodiffusion and laser nephelometry. Clin. Chem. 23, 1925-1928 (1977).
Two Nephelometric Methods Compared with a Radial Immunodiffusion Method for the Measurement of IgG, IgA and IgM G.C. B L A N C H A R D
AND ROSALIE GARDNER
V e t e r a n s A d m i n i s t r a t i o n M e d i c a l C e n t e r , 150 S o u t h H u n t i n g t o n A v e n u e , B o s t o n , M a s s a c h u s e t t s and the Department of Pathology, Boston University School of Medicine, 75 E a s t N e w t o n S t r e e t , B o s t o n , M a s s a c h u s e t t s 02127 (Accepted
January
1. The IgG, IgA and IgM concentrations of a group of 100 or more human sera found by both the rate nephelometric Beckman Immunochemistry System and the endpoint Behring Laser Nephelometer procedure gave good correlations when compared to results obtained by an endpoint radial immunodiffusion method. The correlation coefficients for the Behring method comparisons were 0.958 for IgG, 0.979 for IgA and 0.966 for IgM when only values under 850 IU of IgM/ml were considered. The Beckman system gave correlation coefficients of 0.984 for IgG, 0.993 for IgA and 0.986 for IgM when IgM concentrations were under 500 I U / m l . Determinations from all three methods agreed well except for very high IgM values which were considerably higher by RID. 2. Both nephelometric methods are fast and precise. The average within-run coefficients of variation were 2.6% for the Behring method, 2.2% for the Beckman system and 2.1% for radial immunodiffusion while average between-run precision values of 6.9% for the Behring method, 3.7% for the Beckman system and 4.9% for radial immunodiffusion were found. The Beckman rate system has the advantage of one point calibration, ease of data processing and elimination of sample blanks. IN RECENT YEARS THERE H A S BEEN CONSIDERABLE ADVANCEMENT in the development of nephelometric methods for the quantitation of specific serum proteins. These methods are based on the measurement of the light scattering produced by antigen-antibody complexes formed in immunoprecipitin reactions. Under antibody excess conditions the concentration of antigen is proportional to both the intensity of the scattered light produced by the reaction and to the rate of change of the scattered light intensity. Procedures using light scattering measurements at equilibrium and rate measurements have been reported. Killingsworth et al.'I-2' used a nephelometric equilibrium approach in a manual and automated continuousflow procedure for the determination of IgG, I g A and IgM. The Centrifugal Fast Analyzer was adapted to light scattering measurements either kinetic or at
equilibrium by T i f f a n y et al. '3~ Buffone et al. '4'51 made use of a laser-modified centrifugal analyzer. Several new instruments have been commercially introduced and evaluated using laser endpoint type nephelometers 's'~' or rate methodology "3,m Correspondence to: Gordon G. Blanchard, Ph.D. Chief of Clinical Immunology Veterans Administration Medical Center 150 South Huntington Avenue Boston, Massachusetts 02130
02130
30, 1980)
We have analyzed 100 or more human serum samples for immunoglobulins G, A and M using two of these new nephelometric techniques, the Behring Laser Nephelometer System which measures light scattering either during the kinetic phase of the reaction or at equilibrium '7'8''') and the Beckman Immunochemistry Analyzer System (ICS) which uses a rate methodology'~3"4'. Results obtained by both nephelometric methods have been correlated by linear regression analysis with results obtained by an endpoint radial immunodiffusion (RID) procedure "5'. MATERIALS AND METHODS
Radial Immunodiffusion W e used Tri-Partigen radial immunodiffusion plates from Calbiochem-Behring Corp. (San Diego, C A 92112) for all the R I D analyses. The standards used were those supplied by Behring for use with the Tri-Partigen plates and had been compared with the World Health Organization ( W H O ) International Reference standard "8~. The IgG standards ranged in concentration from 3.9 to 23.2 IU/ml, the IgA standards from 45-193 IU/ml and the I g M standards from 71-297 IU/ml. To convert the Behring standards from IU/ml to mg/dl, the IU/ml value is multiplied by 100 and divided by the appropriate factor, 11.5 for IgG, 59.5 for IgA and 115 for IgM. The 12 wells per plate were filled with 5 ~I of test samples and standards using a 5 ,~l Micr~dispenser ( D r u m m o n d Scientific Co., Broomall, P A 19008). The sera for IgG analysis were usually diluted 1:10 with isotonic NaCl before use. All patient samples were run in duplicate and the standards were run singly. Determinations were made at endpoint after diffusion times of at least 50 h for IgG and IgA and 80 h for IgM. The diameters of the precipitin rings were measured to 0.1 m m using a Finescale reticle No. 122 magnifying comparator (Laboratory Supplies Co., Hicksville, N Y 11801). Specimens with ring diameters exceeding that of the highest standards were diluted and reassayed. Sample concentrations were found either manually by plotting the squared diameters of the standard precipitin rings against the corresponding concentrations on linear graph paper and reading from the resulting standard curve or by use of the Behring Data Processing Module Model 100 which is programmed for R I D calculations.
Behring Laser Nephelometer For imrnunoglobulin quantitation by the Behring nephelometric system we used the Behring Laser Nephelometer Model 7601 (Calbiochem-Behring Corp., San Diego, C A 92112) and followed the manufacturer's directions with some modification. This instrument uses a helium neon laser, wavelength 632.8 nm, as a light source. The forward
light scattering produced by the antigen-antibody corn-
TWO
NEPHELOMETRIC
METHODS
COMPARED
plexes f o r m e d w h e n a n t i g e n s r e a c t with specific a n t i s e r a is focused onto a photodiode by an optical lens system. The electrical signal g e n e r a t e d by the laser v o l t m e t e r is indicated by a digital display which has a r a n g e of f r o m 0.00 to 19.99. M e a s u r e m e n t s m a y be made at time intervals f r o m 15 to 60 minutes. Precise t i m i n g is necessary for readings taken before endpoint. Six serial dilutions of the B e h r i n g Laser Standard Serum, I g G 109 I U / m l , I g A 115 I U / m l and IgM 99 I U / m l were made using volumetric pipets. The concentrations of the standard dilutions r a n g e d f r o m 0.170 I U / m l to 5.45 I U / m l for IgG, 0.180 I U / m l to 5.75 I U / m l for I g A and 0.155 I U / m l to 4.95 I U / m l for IgM. The same conversion factors as are used for the T r i - P a r t i g e n RID method may be used to change values f r o m I U / m l to m g / d l . The Behring N e p h e l o m e t r i c Grade antisera to human IgG, I g A and IgM were diluted 1"5 and the dilutions were f i l t e r e d t h r o u g h Cathivex disposable filter units, 0.45 ~m (Millipore Corp., Bedford, MA. 01730). P a t i e n t samples were diluted 1:101 using a Gilson P i p e t m a n and a volumetric pipette or the Model 1500 P i p e t t o r - D i l u t o r made by Cavro Scientific Instruments, Los Altos, CA 94022. All dilutions were m a d e using sterile 0.9~ NaC1 i r r i g a t i o n U S P solution obtained f r o m the hospital supply. S e r a which g a v e readings outside of the standard curves were re-diluted and re-assayed. Reactions were carried out in 1 cm × 0.4 cm disposable plastic cuvettes obtained f r o m W a l t e r Sarsted Inc., Princeton, N J 08540 or supplied by Behring. F o r I g G analysis, 10 ~l of standard serum or patient sample dilutions were carefully added to the bottom of a cuvette followed by the addition of 200 ~1 of the appropriate a n t i s e r u m dilution. Mixing was by gentle shaking. F o r the I g A and IgM analyses, 100 ~l of standard or sample dilutions were added to cuvettes again followed by 200 ~1 of the a p p r o p r i a t e antiserum. The B e h r i n g method recommends the use of an e m p t y cuvette r e a d i n g as a blank and the f i l t r a t i o n of any m a r k e d l y turbid serum. We used a sample blank and f i l t e r e d no serum except those used in the f i l t r a t i o n study. The sample blank consisted of the a p p r o p r i a t e volume of sample or standard dilution with 200 ~l of 0.9c/( NaCl i r r i g a t i o n solution added to a cuvette of pre-determined light scatter. R e a d i n g s were taken at 60 minutes and the difference between the two values was used as a sample correction and was applied to the e m p t y cuvette r e a d i n g of the corresponding a n t i g e n - a n t i s e r a mixture. All light s c a t t e r i n g m e a s u r e m e n t s for the correlation data were taken at 60 minutes. The light s c a t t e r i n g in several runs was followed by reading at time intervals f r o m 15 to 240 minutes. We did not encounter any sera in antigen excess as all sera had been previously analyzed by another method and a p p r o p r i a t e dilutions w e r e used. To eliminate the use of two cuvettes for each standard and sample assayed and to obtain a t r u e r r e a g e n t blank, we explored the possibility of t a k i n g an initial light s c a t t e r i n g r e a d i n g on each reaction solution before any increased light s c a t t e r i n g due to antigen-antibody complex f o r m a t i o n was observed. A cuvette containing 10 ,~l of IgG, 5.45 I U / m l and 200 ~l of a 1:5 dilution of IgG antis e r u m was g e n t l y shaken and i m m e d i a t e l y placed in the cell c o m p a r t m e n t of the B e h r i n g L a s e r Nephelometer. R e a d i n g s were t a k e n at 3 second intervals by the Behring Laser D a t a P r o c e s s i n g Module until a sharp and steady increase in light s c a t t e r i n g was obtained. This was repeated with 100 ~l of I g A , 5.75 I U / m l and 200 ~1 1:5 I g A antiserum solution and again with 100 ~1 of IgM 4.95 I U / ml and 200 ~1 of 1:5 I g M a n t i s e r u m solution. The H e w l e t t P a c k a r d Model 9830 Calculator and 9862A Calculator P l o t t e r were used in curve f i t t i n g studies and to obtain the immunaglobulin concentration values. Some c o m p a r a t i v e d a t a was also obtained f r o m the B e h r i n g Data Processing Module Model 100 and f r o m standard curves constructed by m a n u a l l y plotting the standard concentrations vs. l i g h t s c a t t e r i n g r e a d i n g s on linear graph paper.
WITH
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IMMUNODIFFUSION
METHOD
85
Beckman Immunochemistry System The Beckman ICS for the d e t e r m i n a t i o n of immunoglobulins by rate n e p h e l o m e t r y uses the I m m u n o c h e m i s t r y A n a l y z e r in conjunction w i t h a F i x e d Ratio Diluter (1:6), a Fixed Volume Dispenser (600 ~1), a 42 ~1 2 position pipet constructed so as to avoid the introduction of air bubbles and a R e a g e n t Test Kit all f r o m Beckman I n s t r u m e n t s , Inc., Fullerton, CA 92634. The i n s t r u m e n t uses a quartziodide lamp and measures the m a x i m u m rate of increase of 70 ° f o r w a r d l i g h t s c a t t e r produced by the f o r m a t i o n of antigen-antibody complexes. The Immunoglobulin R e a g e n t T e s t Kit includes a diluent solution of normal saline, a phosphate b u f f e r e d saline solutio.~ containing a p o l y m e r enhancer, a n t i s e r a to IgG, I g A and IgM and a calibrator serum containing IgG, I g A and I g M concentrations n e a r the midpoints of the measu r i n g r a n g e s of the tests. The concentration values of the standards were established by comparison with a p r i m a r y standard of the W H O . Disposable glass f l a t - b o t t o m e d reaction tubes with stirrers are also provided. The procedure followed was according to the kit directions which were only slightly modified f r o m the detailed description of S t e r n b e r g c14). Successive dilutions 1:6, 1:36 and 1:216 of the calibrator s e r u m and sample sera were made. The a n a l y z e r was calibrated at a single point midw a y in the m e a s u r i n g r a n g e of the immunoglobulin being determined. The m e a s u r i n g r a n g e for IgG is 43.4-446.4 I U / m l , for I g A 42.2-352 I U / m l and for IgM 59-472 I U / m l . Calibration was accomplished by optically r e a d i n g into the i n s t r u m e n t by the insertion of p r o g r a m cards the calibrator value and i n f o r m a t i o n necessary for the analy z e r m i c r o c o m p u t e r to convert peak rate signal to concentration units. This was followed by the introduction of 42 ~l of the a p p r o p r i a t e calibrator dilution and 42 ~l of the specific a n t i - s e r u m into a reaction tube containing 600 ~1 b u f f e r which had been placed in the cell c o m p a r t m e n t of the instrument. The peak r a t e signal produced by the reaction was stored in the computer, compared with a r e p e a t run for s a t i s f a c t o r y a g r e e m e n t and internally calibrated by the a n a l y z e r so t h a t the calibrator serum read at the t a r g e t value. Once calibration was completed, sample dilutions were run in duplicate following a like procedure. Step by step directions were given on the i n s t r u m e n t a l p h a n u m e r i c display. Concentrations units w e r e displayed in m g / d l , g/1 and I U / m l . Conversion of I U / m l to m g / d l m a y be made by m u l t i p l y i n g the I U / m l values by 100 and dividing by the a p p r o p r i a t e factors f o r the Beckman standards, 12.4 for IgG, 70.4 for I g A and 118 for IgM. Out of r a n g e samples were rejected by the i n s t r u m e n t and could be immediately re-analyzed using a n o t h e r dilution. A n t i g e n excess checks were called f o r when determined by the micro-computer. To v e r i f y the linearity of the i n s t r u m e n t , we analyzed solutions of known concentrations which covered the immunoglobulin m e a s u r i n g ranges.
Precision Within-run precision was determined for the nephelometric methods by 20 replicate analyses of each serum. F o r RID within-run precision is the result of 9 replicate analyses on one plate for each serum. The b e t w e e n - r u n precision f o r all three methods was obtained by a n a l y z i n g each serum 20 times in successive runs. Coefficients of v a r i a t i o n were determined for three sera in d i f f e r e n t concentration r a n g e s for each method studied.
Effect of Turbidity Aliquots of 4 m a r k e d l y turbid sera w e r e filtered t h r o u g h Millipore 0.45 ~m f i l t e r s and both the filtered and unfiltered sera were analyzed by the two n e p h e l o m e t r i c methods.
Computations The H e w l e t t - P a c k a r d Model 9830A Calculator and 9862A Calculator P l o t t e r were used for the statistical c o m p u t a tions and f o r d a t a plotting.
86
BLANCHARD AND GARDNER
Fig. 1 - - Linearity of the Beckman Immunochemistry Analyser for the IgM analysis.
I
I
S
~B
I
II
I
I
I
I
I
I ~S
2BB
2~B
3H
3~B
I
I
H!
H~B
EXPECTED IBM IU/ML
RESULTS Standard Curves The standard curves found with the R I D method at end-point were straight lines w h e n the diameters squared of the precipitin rings were plotted vs the TABLE
c o n c e n t r a t i o n s on l i n e a r g r a p h paper. The p l o t t i n g of s t a n d a r d curves is n o t n e c e s s a r y in the Beckman ICS method. Method l i n e a r i t y was v e r i f i e d when s t r a i g h t lines were o b t a i n e d for IgG, I g A and IgM when k n o w n c o n c e n t r a t i o n s were plotted a g a i n s t c o n c e n t r a t i o n s f o u n d by a n a l y s i s ( F i g . 1). 1
PRECISION OF THE RID, BEHRING LASER NEPHELOMETER AND B E C K M A N ICS M E T H O D S FOR DETERMINATION OF IGG, IGA AND I G M
Within-run Mean, IU/ml RID
Between-run CV,%
Mean, IU/ml
n=9
CV,% n = 10
(4.4)* IgG
208 123 57
2.9 2.1 3.1
200 118 58
7.1 5.2 4.4
IgA
143 92 38
i.I 2.,2 2.3
175 137 88
3.2 3.7 2.3
IgM
289 159 ll0
1.8 1.8 1.2
274 145 104
6.3 6.9 5.0
Behring Laser Nephelometer
n = 20
n = 20 (2.7-4)
IgG
497 152 87
2.7 2.0 2.0
451 177 68
7.3 6.9 6.8
IgA
352 117 27
2.0 1.5 2,7
533 171 62
5.9 5.7 8.9
IgM
418 168 57
1.3 3.1 5.9
458 227 75
7.3 4.7 8.6
Beckman ICS
n
=
20
n
=
20
IgG
369 143 86
2.1 1.9 0.9
(3) (2) (3)
330 138 68
2.9 2.2 (7) 3.4
IgA
278 89 55
2.3 1.6 2.1
(2) (2)
281 162 56
3.2 1.8 (5) 3.9
IgM
336 164 89
2.1 2.6 4.2
(2) (3) (3)
415 154 I01
5.3 4.9 (5) 5.7
* The precision data provided by the manufacturer for approximately the same concentrations are listed in parentheses.
TWO NEPHELOMETRIC METHODS COMPARED WITH A RADIAL IMMUNODIFFUSION METHOD
87
w
I'M
m
v
Fig. 2 - - Behring Laser Nephelometer Standard Curve for IgG. t.~ .J
l
2
3 IGG
4
E
6
IU/ML
::T v
~--
r~
L~ .~
r~
Fig. 3 - - Behring Laser Nephelometer Standard Curve for IgM.
-I-
I
~
I
I
l
i
I
I
1
J
I
I ME
1 ~E
28D
2~E}
3B~
3~8
4El~
H~B
~8~
IGM
I
IU/ML
When calculating our data, we found the Behring Laser Nephelometer results processed by the Behring Data Processing Module 100 to be slightly higher especially for IgG and IgA than values obtained from hand plots. We therefore carried out curve fitting studies to consider another method of calculation. Fifteen point standard curves for IgG and 14 point curves for IgA and IgM constructed using data from the Behring Laser Nephelometer showed a third degree polynomial fit for IgG (r = 0.998), for IgA (r----0.997) and for IgM (r---0.996). The IgM standard curve was also described by a quadratic fit (r --- 0.996). The six standard concentrations recommended by the Behring procedure did not clearly define the shape of the cubic curve in the range between the two highest standards, the fifth and sixth standards. A quadratic equation fit the first five points on the IgG and IgA standard curves and followed closely
the third order fit on the 15 and 14 point lines. Sample concentrations for IgG and IgA were calculated from a standard curve fit to a quadratic equation omitting the highest standard value. In the few cases where light scattering readings were above the fifth standard, concentrations were determined by interpolation between standards five and six. IgM values were calculated from 6 point standard curves with quadratic fits. The average measure of fit was 0.998 for the IgG standard curves, 0.996 for the I g A standard curves and 0.997 for the IgM standard curves. Fig. 2 shows an IgG 15 point standard curve fit to a third degree polynomial with a five point standard curve fit to a quadratic equation superimposed. Fig. 3 is a typical standard curve for IgM. Concentrations determined using this method of calculation agreed well with hand plots of the data and were used in all correlation computations. The
B L A N C H A R D AND G A R D N E R
88
+
!. Fig. ~ - - C o m p a r i s o n of the R I D a n d Behring Laser Nephelometer Methods f o r the D e t e r m i n a t i o n of I g M . ( A ) S l o p e : 1.066 f o r values r a n g i n g ]rom 39.1 to 850 I U / m l . ( B ) S l o p e : 0.656 f o r v a l u e s r a n g i n g f r o m 39.1 to 8~67 I U / m l . • V a l u e s 0 to 850 I U / m l q- V a l u e s over 850 I U / m l
,,9
| I
IBBB
I
I
I
I
I
I
I
2[lil~
3~BB
4~EB
5~[[
G~BB
7MB
8BBfl
I
RID 16M IU/ML
plots o b t a i n e d by use of t h e H e w l e t t P a c k a r d p l o t t e r w e r e also h e l p f u l in v i s u a l l y a p p r o v i n g t h e s t a n d a r d curves.
t i o n s a r e g i v e n in T a b l e I. T h e w i t h i n - r u n c o e f f i c i e n t s of v a r i a t i o n (CV) f o r R I D r a n g e d f r o m 1.1 to 3.1% w h i l e t h e R I D b e t w e e n - r u n CV r a n g e d f r o m 2.3 to 7.1%. T h e B e h r i n g L a s e r m e t h o d g a v e w i t h i n run CV of f r o m 1.3 to 5.9% and b e t w e e n - r u n CV o f f r o m 4.7 to 8.9%. T h e CV of t h e B e c k m a n ICS v a r i e d f r o m 0.9-4.2% f o r w i t h i n - r u n and f r o m 1.8 to 5.7% for between-run precision.
Precision
T h e w i t h i n - r u n and b e t w e e n - r u n p r e c i s i o n s f o r t h e RID, B e c k m a n ICS and B e h r i n g L a s e r N e p h e l o m e t e r m e t h o d s f o r IgG, I g A and I g M in t h e s e c o n c e n t r a -
TABLE 2 COMPARISONOF THE RID, BEHRING LASER NEPHELOMETERAND BECKMANICS METHODS FOR DETERMINATIONOF IGG, IGA AND IGM n (no. of data pairs)
Range, IU/ml
r
Slope
y-intercept (IU/ml)
1.137
-5.00
1.111
11.00
0.656
78.5
RID (x)-Behring Laser Nephelometer (y) IgG
109
IgA
102
IgM
108
IgM
103
IgM
100
IgG
112
IgA
108
IgM
110
IgM
108
IgM
101
I&,G
110
IgA
108
IgM
113
IgM
110
IgM
105
x 7.13-554 0.958 y 8.49-613 x 22.6-913 0.979 y 23.7-939 x 39.1-8467 0.996 y 47.2-5471 x 39.1-850 0.966 y 47.2-807 x 39.1-432 0.939 y 47.2-476 RID (x)-Beckman ICS (y) x 7.1-608 0.984 y 9.3-646 x 10.6-913 0.993 y 11.8-1031 x 39.1-8469 0.996 y 59.0-6293 x 39.1-1044 0.992 y 59.0--995 x 39.1-492 0.986 y 59.0-442 Beckman ICS (x)-Behring Laser Nephelometer (y) x 9.3-646 0.950 y 8.5-613 x 5.99-1031 0.980 y 3.11-939 x 30.7-6292 0.995 y 24.1-5472 x 30.7-909 0.977 y 24.2-859 x 30.6-430 0.959 y 24.1-550
1.006
9.38
1.069
- 1.35
1.019
0.179
1.17t
- 1.88
0.768
48.5
0.896
16.32
0.967
3.65
1.050
5.99
0.947
12.5
0.851
39.4
1.033
6.71
1.145
-12.1
TWO NEPHELOMETRIC METHODS COMPARED WITH A RADIAL IMMUNODIFFUSION METHOD
N 3
._i
EB
IBB
IEB
2BB
2EB
TIME (MIN.)
Fig. 5 - - B e h r i ~ g L a s e r N e p h e l o m e t e r I g A S t a n d a r d s - L i g h t scatterb~g f r o m 0-240 min. R e a d i n g s w e r e made at 15, 22, 30, 40, 45, 50, 60, 65,75, 85, 90, 100, 112, 120, 150 and 240 rain.
Correlation Correlation data for the comparison of the RID, Behring Laser Nephelometer and Beckman ICS methods is given in Table 2. Good correlations were found when the Behring Laser method was compared to RID. Correlation coefficients were 0.958 for IgG and 0.979 for IgA with slopes of 1.137 and 1.111 respectively. Slightly higher IgG and IgA values were obtained by the Behring Laser method. For the IgM comparisons there was a marked change in slope from 1.006 to 0.656 when values above 850 I U / m l were included (Fig. 4). The methods did not agree when sera of extremely high IgM concentrations were analyzed, the RID values being considerably higher. For values under 850 IU/ml there was excellent agreement between methods. The correlation coefficients were 0.939, 0.966 and 0.996 reflecting the different ranges of values. The Beckman ICS gave excellent correlation coefficients when compared to RID. The value for IgG was 0.984, 0.993 for IgA and for IgM the correlation coefficient ranged from 0.986 to 0.996 depending on the range of concentrations included. The slope of 1.019 for the IgG comparison indicated a good agreement in results found by both methods. The IgA comparison gave a slope of 1.171. The IgA concentrations determined by the Beckman ICS were slightly higher than those obtained by RID. For the IgM analyses method agreement was good for values under 500 I U / m l (slope 0.967) but as higher values were included, the slope decreased to 0.768. Again, the nephelometric method gave lower results with sera of abnormally high IgM. The Beckman ICS and Behring Laser Nephelometer comparisons showed good correlation and agreement between methods for IgG, IgA and for IgM. The correlation coefficients were 0.950 for IgG, 0.980 for IgA and 0.959 to 0.995 for IgM. The slopes were 1.050 for IgG, 0.947 for IgA and 0.851 - - 1.145 for IgM. The change in the slope when IgM values above 909 I U / m l were included was not as marked as when the nephelometric methods were compared to RID. When the correlation data was expressed in mg/dl
89
r a t h e r than IU/ml, the slopes f o r the RID and Beckman ICS comparisons changed to 0.945 for IgG, 0.990 for IgA and 0.943 for IgM where n=101. For ths Beckman ICS and Behring L a s e r Nephelometer comparisons the slopes became 1.132 for IgG, 1.120 for IgA and 1.060 for IgM, n----ll0. As both Behring methods used the same conversion factors for c h a n g i n g I U / m l to mg/dl, the RID and Behring nephelometric correlation coefficients and slope values remained the same regardless of the change in units. Except for the RID and Beckman IgA comparisons the results from all methods showed better agreement when expressed as IU/ml. The Beckman ICS being a rate method required no sample blank. A correction for significant sample light scatter was necessary in the Behring L a s e r Nephelometer method if the recommended filtration of markedly turbid sera was not followed. Because of reluctance to filter patient serum samples for reasons of safety, economy and possible loss of protein and because we found the Behring Standard L a s e r Serum dilutions to have considerable light scattering not due to the diluent, we decided to include a sample blank in the procedure for all the immunoblobulin analyses by this method. We had also found it difficult to judge by visual inspection which sera should be filtered. Some markedly turbid sera contributed little light scatter to the background while other less turbid sera gave considerable scatter. Filtering did not always clear the serum and some sera were difficult to filter requiring dilution or centrifugation before filtration. Sixteen per cent of the sera used in this study were found to give high background scatter. Two of these were icteric and most of the others were samples which had been received frozen and although not markedly turbid did contain obvious particles. Most fresh sera even though very turbid gave no significant light scatter when diluted for analysis. When filtered and unfiltered samples of f o u r highly turbid and light scattering samples were analyzed by both nephelometric methods, the mean difference between the filtered and unfiltered samples determined by the Beckman ICS was 1.6% for IgG, 1.7% for IgA and 6.6% for IgM. Mean differences in values obtained by the Behring L a s e r Nephelometer were 6.4% for IgG, 3.8% for IgA and 13.8% for IgM. The higher averages for IgM were due to the large difference in one sample possibly due to loss of IgM on filtration of the diluted serum. Laser Nephelometer Method-Reaction Time All the Behring Laser Nephelometer data used in these studies except for the timing experiment were obtained at 60 minutes when the reaction time was at or near equilibrium. When the antigen-antibody complexes became large enough to settle out, there was a rapid decrease in light scattering. We found that sedimentation sometimes occurred in the highest IgA standard before 60 minutes giving a false low light scattering reading to the high (6th) s t a n d a r d (Fig. 5). The f i f t h IgA s t a n d a r d did not decrease in reading until 112 minutes. The IgM reactions gave no such problem but in two experiments in which the light s c a t t e r i n g of the IgG s t a n d a r d s and
BLANCHARD AND GARDNER
90
antisera reactions were followed, the highest standard decreased in light scattering once at 65 minutes and onc~ at ~/5 minutes. Concentrations of sera calculated from 15 minute light scattering readings were in most cases slightly and sometimes markedly lower than values calculated from 60 minute data.
Behring Laser Nephelometer Method Initial Blank Reading The longer reaction time in the Behring Laser Nephelometer method due to the absence of polyethylene glycol or other reaction enhancer made possible an initial reading for background light scattering. The IgG 5.45 IU/ml and IgG anti-serum reaction solution gave reasonably steady light scattering for 30 seconds before there was a sharp increase in readings. Initial blank readings could be made during the first 183 seconds for the IgA 5.75 IU/ml anti-IgA reaction solution and for 144 seconds for the IgM 4.95 I U / m l anti-IgM solution.
Analysis Time The analysis of a batch of 18 samples for IgG, IgA and IgM including antigen excess checks was easily accomplished in 3 hours by both the Beckman ICS and the Behring Laser Nephelometer method. Two hours of technician time and a period of 2 to 4 days was necessary for determinations to be made on the same number of samples by RID. Using the Beckman ICS, immunoglobulin concentrations may be determined on a single sample in the m a t t e r of a few minutes. I f an incubation time of 15 minutes is used in the Behring Laser method, samples may be processed relatively rapidly especially if related to a previously run standard curve. We found, however, that light scattering in the lowest standard of IgM was not evident at 15 minutes making a longer incubation time necessary for IgM determinations in this range. DISCUSSION Both nephelometric methods gave good precision generally in the same ranges as we found for RID. The Behring Laser Nephelometer method within-run precisions were similar to those stated by the supplier and to the 3.2-5.4% CV found by Conrad et al.'" and the 3.8% CV of W~.lsh et al. '~''. The between-run precisions agreed well with the 4.4-8.3% CV of Conrad et al. '7', the 6.8-8.5% CV reported for IgG by Walsh et al. '11~ and the 5.6 and 1.8% CV for IgG and IgA respectively found by Schliep et al. '1~. The coefficients of variation for the Beckman ICS agreed favorably with the precisions claimed by the m a n u f a c t u r e r and with the 2-3% CV for the withinrun found by Sternberg 'm. The precisions of the Beckman system were slightly better than those of the Behring procedure and than the CV values reported from evaluations of a nephelometric method using another commercial instrument, the Hyland laser nephelometer ~1°-~. When the Behring L a s e r Nephelometer method was compared to RID, we found correlation coefficients which showed good agreement with the 0.967 to 0.981 values of Conrad et al. '7~, with the 0.996 to
0.998 coefficients reported by Daigneault and Lemieux 'a~ and the 0.97 value for IgG of Walsh et al. 'm We found the method agreement to be not quite as good as indicated by the slopes of 0.98 to 1.05 found by Daigneault and Lemieux '8', the slopes of 0.840 - 0.986 (x, Behring L a s e r ; y, RID) found by Conrad et al. '7', and the 0.96 slope for IgG determined by Walsh. et al. 'H', when results from both methods were compared by linear regression analysis. The correlations obtained when results from the Beckman ICS were compared to results found by RID were similar to the 0.939 to 0.952 values of Sternberg ''4~. When our data was expressed in mg/dl, the slopes of the regression lines from comparison studies showed better method agreement for IgA and similar method agreement for IgG and IgM when compared to the slopes of 1.316 for IgA, 0.952 for IgM and 1.035 for IgG reported by Sternberg ''4~. The correlation coefficients and method agreement with RID which we found in this study with the Behring and Beckman systems were close to ''°''~' and somewhat better than 's'''' values reported from similar studies using the Hyland laser nephelometer. The considerably higher IgM concentration values obtained by RID for some sera may be due to the presence in the sera of rapidly diffusing low molecular weight IgM giving a false result ~'8~. Virella and F u n d e n b e r g ''s' found a nephelometric method (Hyland) to give substantially lower results than RID in two cases of patients with macroglobulinemia and Deaton et al. '~°~ found slope improvement when abnormal I g A and IgM sera values were excluded from a comparison of data obtained by a nephelometric method and RID. In the course of this work we encountered few grossly turbid sera. We found that the sample blank correction made no difference in the IgG analyses using the Behring Laser Nephelometer method due to the instrument sensitivity and the dilution used. In most cases a sample blank serum correction for IgA and IgM analyses by this method also made little difference. However, background light scattering corrections for some sera and often for the standards was necessary. We suggest the initial blank reading as a possible method modification. The addition of at least one more point in the high range of the standard curve to better define a cubic fit line and the reading of the IgG and IgA analyses at time intervals less than 60 minutes before any antigen antibody complex sedimentations occur are also suggested improvements to the Behring L a s e r Nephelometry method. The two nephelometric procedures studied both o f f e r good alternative methods to RID for immunoglobulin determinations. The rate method has the advantage of single point calibration, automatic antigen excess check and no necessity for sample blank. ACKNOWLEDGEMENTS We thank Calbiochem-Behring Corp. and Beckman Instruments, Inc. for supplying reagent kits and for the use of their instruments. We are also most grateful to Dr. Eldon A. Boling for his assistance in the data
TWO
NEPHELOMETRIC
METHODS
COMPARED
c o m p u t a t i o n s and to Dr. Beni H o r v a t h for s u g g e s t i o n s to the m a n u s c r i p t .
WITH
A RADIAL
10.
REFERENCES
1. Killingsworth, L.M., and Savory, J. Manual nephelometric methods for immunochemical determinations of immunoglobulins IgG, IgA and I~M in human serum. Clin. Chem. 18, 335-339 (1972). 2. Killingsworth, L.M., and Savory, J. Automated immunochemical procedures for measurement of immunoglobulins IgG, IgA and IgM in human serum. Clin. Chem. 17, 936-940 (1971). 3. Tiffany, T.O., Parella, J.M., Johnson, W.F., and Burtis, C.A. Specific protein analysis by light scatter measurement with a miniature centrifugal fast analyzer. Clin. Chem. 20, 1{)55-1060 (1974). 4. Buffone, G.J., Savory, J., Cross, R.E., and Hammond, J.E. Evaluation of kinetic light scattering as an approach to the measurement of specific proteins with the centrifugal analyzer. I. Methodology. Clin. Chem. 21, 1731-1734 {1975). 5. Buffone, G.J., Savory, J., and Hermans, J. Evaluation of kinetic light scattering as an approach to the measurement of specific proteins with the centrifugal analyzer. II. Theoretical considerations. Clin. Chem. 21, 1735-1746 (1975). 6. Bruver, R.M., and Salkie, 'M:L. A comparative study of three approaches to the routine quantitation of human serum proteins. Clin. Biochem. 1l, 112-116 (1978). 7. Conrad, A., Sehurmann, J., and Kreutz, F.H. Elaboration of a method for the quantitative determination of proteins by laser nephelometry in the clinical routine laboratory. J. Clin. Chem. Clin. Biochem. 16, 299305 (1978). 8. Daigneault, R. and Lemieux, D. Evaluation of a Behring Laser-Nephelometer prototype in the measurement of IgG, IgA and IgM. Clin. Bioehem. 11, 2831 (1978). 9. Deaton, C.D., Maxwell, K.W., and Smith, R.S. In Automated lmmunoanalysis ( P a r t 2), R.F. Ritchie,
11.
12.
13. 14. 15.
16.
17.
18. 19.
I M M U N O D I F F U S I O N METHOD
91
Ed., Marcel Dekker, New York, N.Y., 1978, pp. 375408. Deaton, C.D., Maxwell, ICW., Smith, R.S., and Creveling, R.L. Use of laser nephelometry in the measurement of serum proteins. Clin. Chem. 22, 14651471 (1976). Walsh, R.L., Coles, ,M.E., and Geary, T.D. Evaluation of two commercially available Laser Nepbelometers and their recommended methods for measurement of serum immunoglobulin G. Clin. Chem. 25, 151-156 (1979). Whieher, J.T., Perry, D.E., and Hobbs, J.R. An evaluation of the Hyland Laser nephelometer PDQ system for the measurement of immunoglobulins. Ann. Clin. Biochem. 15, 77-85 (1978). Anderson, R.J., and Sternberg, J.C., In Automated Immunoanalysis ( P a r t 2), R.F. Ritchie, Ed., Marcel Dekker, New York, N.Y., 1978, pp. 409-470. Sternberg, J.C. A rate nephelometer for measuring specific proteins by immunoprecipitin reactions. Clin. Chem. 23, 1456-1464 (1977). Mancini, G., Carbonara, A.O., and Heremans, J.F. Immunochemical quantitation of antigens by single radiai immunodiffusion. Immunochemistry 2, 235-254 (1965). Rowe, D.S., Grab, B., and Anderson, S.G. An international reference preparation for human serum immunoglobulins G, A, and M; content of immunoglobulins by weight. Bull. W.H.O. 46, 67-79 (1972). Sehliep, G., and Felgenhauer, K. Rapid determination of proteins in serum and eerebrospinal fluid by lasernephelometry. J. Clin. Chem. Clin. Bioehem. 16, 631635 (1978). Jones, D. Low molecular weight IgM as a pitfall in immunoglobulin quantitation by radial diffusion. Clin. Chim. Aeta 29, 551-556 (1970). Virella, G., and Fudenberg, H.H. Comparison of immunoglobulin determinations in pathological sera by radial immunodiffusion and laser nephelometry. Clin. Chem. 23, 1925-1928 (1977).