Evaluation of a specific protein analyser

Clinica Chimica Acta, 46 (1973) 399-405 0 Elsevier Scientific Publishing Company, CCA

Amsterdam

-

Printed in the Nederlands

399

5809

EVALUATION

JOAN MARCROFT

OF A SPECIFIC

AND

IRENE

PROTEIN

ANALYSER

M. NEWLANDS

Department of Clinical Chemistry, and Medical Research Council, Clinical and Population genetics Unit, Western General Hospital, Crewe Road, Edinburgh, LH4 2XU (Scotland) (Received

Cyto-

March 26, 1973)

SUMMARY

A specific protein analyser (Technicon

Autoanalyser

II for Immunoprecipitin

Techniques, Technicon Instruments Corp., Tarrytown, N.Y. 10591, U.S.A.) has been assessed for the estimation of immunoglobulins A and G (IgA and IgG), haptoglobin, and al-antitrypsin ((x,-AT). Values determined by the analyser procedure correlate well with values obtained by the single radial immunodiffusion technique. The effect of haemolysis has been studied and shown to have no effect on the quantitation of IgA, IgG and x,-AT by this method. Reference sera and antisera to IgA and IgG from two sources have been investigated, and good correlations obtained, Normal ranges for IgA and IgG have been determined. The estimation of immunoglobulin M (IgM) h as also been investigated and as no satisfactory results have been obtained studies are continuing.

using the recommended

method,

further

INTRODUCTION

The quantitation

of individual

human serum proteins has been effected by the

use of single diffusion, double diffusion, and radial immunodiffusion techniques1$2. A significant advance has been made with the introduction of automated immunochemical continuous flow techniquess14 employing nephelometric methods to quantitate preprecipitin antigen-antibody complex formation. A commercially available instrument has been developed5, and the present report describes results obtained with this specific protein analyser for the measurement Q-AT.

of IgA, IgG, haptoglobin

and

METHOD AND MATERIALS

The estimation of specific proteins by the analyser method depends upon the formation of stable antigen-antibody complexes, which are formed by reaction of the various proteins with their appropriate monospecific antiserum. The amount of

400

MARCROFT,NEWLANDS

complex so formed is measured using the light scattering effect in a fluoronephelometer. Results are shown as peaks on a strip chart recorder whose heights, when corrected for any significant blank reading, are directly proportional to the concentration of protein in the sample, and may be expressed as mg/roo ml or I.U./ml depending on the standards used. Samples are analysed at a rate of 50 per h, and a sample: wash ratio of I : I is used. Automated Immunoprecipitin (A.I.P.) reference serum, and specific antiserum to IgA, IgG, haptoglobin ments Ltd. (Basingstoke,

and a,-antitrypsin were obtained from Technicon InstruHants.). Human control serum (Batch IIOE), antiserum to

IgA and IgG, and Tri-Partigen immunodiffusion plates for the determination of IgA and IgG were obtained from Behringwerke, Hoechst Pharmaceuticals (Hounslow, Middx.). IgG and IgA Immuno-plate Test Kits were obtained from Hyland, Travenol Laboratories Ltd. (Thetford, Norfolk). Recovery experiments were carried out for each of the four proteins, namely IgA, IgG, a,-AT and haptoglobin. Samples of fresh serum were obtained from six healthy volunteers, and dilutions containing 25, 50 and 75% of Technicon reference standard were prepared from each. These were assayed together with a 100% reference standard and IOO~/'(serum samples using the analyser. Haemolysed samples were prepared as follows. The blood samples were collected into lithium heparin tubes, and from each set of two tubes one was centrifuged, the plasma removed, and stored for use as a control. The other sample was subjected to continuous freezing and thawing to fully haemolyse the blood. The haemolysed sample was centrifuged to remove cell debris and then diluted with the control plasma to give a series of dilutions ranging from I to 10% haemolysis. All samples were centrifuged again and content of the supernatants and the control plasma estimated for IgG, IgA and c(,-AT on the analyser. RESULTS

(I)

Instrument f5recision The reproducibility of within-batch analysis was tested by assaying a number of sera in duplicate. The coefficients of variation for IgG, IgA, haptoglobin and c+ antitrypsin were found to lie within the range 0.8-3.7~/~ (Table I). Within-batch precision was also determined for IgG and IgA, using immunodiffusion plates produced by Behringwerke and Hyland, and coefficients of variation were found to be 6.7 and 11.0% for IgG, and 4.0 and 6.3% for IgA, respectively. The within-batch precision of the analyser technique is therefore markedly better than that of the manual radial immunodiffusion method. The between-batch precision was measured by assaying a number of sera on successive days. The coefficients of variation were found in the range 1.8~4.8~/~ for the four proteins under investigation (Table II). The above results confirm the manufacturers claim of a reproducibility to 5% for the determination of IgG, IgA, haptoglobin and a,-antitrypsin5.

(2) Recoveries

Results expressed in mg/roo ml were compared with the theoretical based on 100% recovery, and the mean recoveries calculated (Table III).

values

SPECIFIC PROTEIN ANALYSER TABLE

401

I

WITHIN-BATCH

PRECISION

Pvotein

OF THE

Number of Samples (duplicates)

Autoanalyser IgG IgA

30 30 30 30

Haptoglobin

30

a,-Antitrypsin

3o 20

THE

SINGLE

RADIAL

S.D. of diffeerences

Mean value

200

IMMUNODIFFUSION

10.6 11.4 4.2 4.7 4.5 7.6

1010

METHOD

Coefficient of variation (16) 1.1

1230-1750 IOO- 300 300- 500 7op 250 260- 470 125- 240 240- 415

1450 194 381 170 367 201 274

620-1940 60- 630

1228 308

sz.4 12.3

6.7

550-2800 70- 590

1178 297

140.2 16.7

11.9 6.3

Mean value

S.D. of dijferences

Coe&ient variation

Tri-pavtigen plates 40 40

Hyland Immune plates 1gG 39 Igh 34 TABLE

AND

Range mg/roo ml

750-I

3o

Beringwrkr I& IgA

ANALYSER

0.8 2.2 I.2

2.6 2.1 3.7 3.5

7.4 9.6

4.0

II

BETWEEN-BATCH

Protein

PRECISION

ON THE

Number of samples

ANALYSER

Range mgl100

ml

of

(%) I&

800-I

3o 30

IgA

200

1023 1436

27.7 31.1

2.7 2.2

IOO-

300

214

6.1

320~

510

387 181

7.1

2.9 I.8

9.1

1220-1850

3o 3o

Haptoglobin

3o

80-

250

25o125-

470 240

370

a,-Antitrypsin

3o 30

I99

9.6

4.8

20

*4o-

415

276

11.3

4.1

.BLE

3.6 2.j

6.5

III

COVERY

OF STAKDARD

Reference ndard

ON THE

ANALYSER

“/, Recovery: IgG R 106.5

Range 90.4-119.5

IgA f 88.6

x,-A I Range

x

78.1-108.6

Haptoglobwz Range

x

Range

101.0

83.2-111.8

100.8

109.9

104.3-117.9

100.3

91.8-119.7

99.6

87.5-106.8

99.9

94.6-111.4 98.8-100.7

107.6

104.4-114.0

101.3

94.7-113.0

98.9

95.8-102.4

98.4

95.9-101.0

(3) Haemolysis It is inevitable that some blood samples will be haemolysed in varying degrees during collection or separation of the plasma, and the effect of such haemolysis on the IgG, IgA and LX,-AT was investigated (Table IV). It is evident that haemolysis hasno appreciable effect on the estimation of these proteins by this method. (4) Reference sentm and alztiserum A comparison was made between

standard

reference

sera, and also between

YARCROFT,

402 TABLE

NEWLANDS

IV

EFFECT OF HAEMOLYSIS Protein concentration lnzpl100 ml)

9,;

Haemolvsis

o(

:ontroz

~~,”

8

870 925 885 870 875 925 I020

9

880

I22

I42 I25 I25 I32 ‘33 130 130 146

IO

TABLE (A)

5:

COMPARISON

BEHRINGWERKE

ON (y),

THE USING

ANALYSER

(B)

Value of standard Value of standard COMPARISON

REFERENCE

OF

BETWEEN

TECHNICON

Pvdein

I‘+ IgG

246 248 243 243 242 243 234 239 243 241 239

150 II.5

ANTISERUM

REFERENCE

TECHNICON

(X)

Reference sevum

Correlation between vefwence seva

Trchnicon

y = ax+b

Behringwerke

580 2300 FROM

270 1300

TECHNICON

(X)

AND

Antiserum Technicon

IgG

FROM

AND

a

b

Corvelatzon coejicient = v

0.99 I.03

- 5.43 - 40.64

0.99 0.99

BEHRINGWERKE

(y),

USING

TECHNICON

SERUM

Protei?z

IgA

SERUM

ANTISERUM

Dilution* Sample line size Dilution* Sample line size

* Antiserum

I :40 0.015” I :40 0,007y

dilution in 0.994 NaCl containing

Cowelation between antiserum y = ax+b a

b

Correlation coejicient = v

I : 25

0.89

31.15

0.97

1:20

I.04

- 29.82

0.98

Behringwrke 0.01” 0.0075” Tween-ao.

antisera to IgG and IgA, obtained from Technicon Instruments Ltd. and Behringwerke using the analyser technique. The results are shown in Table V and it is obvious that good correlations were obtained. Antiserum from Behringwerke is of lower titre than that produced by Technicon, and antiserum dilution and sample line size have to be reduced accordingly. This results in slightly increased costs per test (Table VIII). Behringwerke reference serum (Batch IIOE) has an IgA value of only 270 mg/roo ml, which falls in the middle of the normal range and as a result sera having an IgA value in the upper normal range must be repeated on dilution, and this results in increased time and cost per assay. It is essential that a control serum, intended for use as a primary standard on the analyser, has a concentration greater than that of the highest normal concentration encountered. A very high degree of reproducibility is required of both reference standard and

SPECIFIC PROTEIN ANALYSER

antiserum,

403

as well as reasonable

but by no means

and identifiable

shelf lives and expiry dates. Certain

all firms meet these requirements

and it is hoped that the others

will soon raise their standards. (5) Comparison of methods IgA and IgG values of sera were determined in duplicate on the analyser, Behringwerke Tri-Partigen Immunodiffusion plates, and Hyland Immuno-plates. Twenty fresh sera were investigated, and a further batch of twenty were stored at -20°, thawed once and quantitated. Means of the IgA and IgG values were compared and correlations determined (Table VI). Immunoglobulin concentrations in Technicon, Behringwerke and Hyland reference sera are quoted in both mg/roo ml and I.U./ml, the latter based on the WHO international reference preparations (67186, 67195 or 67197). The correlations in Table VI calculated in I.U./ml are thus related to a common standard, which is imperative if comparisons are to be made between different methods. Correlation coefficients for IgA and IgG estimation on fresh serum between the analyser and Behringwerke Tri-Partigen immunodiffusion methods are good. Poorer correlation is observed between the analyser and Hyland Immuno-plates. Comparisons obtained between results using fresh sera are significantly better than those calculated for frozen sera. It is therefore recommended that sera be stored at 4”, and the immunoglobulins quantitated as early as is possible.

(6) Normal range IgA and IgG values for a group of 80 male and 60 female adults were estimated

TABLE

VI

COMPARISONBETWEEN TKE ANALYSER AND RADIAL IMMUNODIFFUSION Protein

Manual method

IgA

Behringwerke

plates

Correlation between results with Analyser (x) and Manual method (y) y = ax+ b: correlation coejicient (r) (i)

fresh serum

a

b

0.81

16.09

o.6g*

Hyland plates

I@

Behringwerke

Hyland plates

plates

(ii) frozen serum

0.81

(i) fresh serum

o.6g* I.19

v 0.98

o.g8*

9.85*

0.80 0.80*

55.97 33.21*

0.91 *

0.97*

::::i

(ii) frozen serum

0.84 o.6g*

79.49 45.87*

(i)

I.11

fresh serum

0.91:

0.80 0.80*

1.02*

112.4 12.57

0.97 0.97*

(ii) frozen serum

0.58 0.54*

339.1 3*.3*

0.88 o.go*

(i)

1.04 1.04*

- 88.6 - 10.87*

0.85 0.85*

1.06

-68.4 7.83*

0.74 0.73*

fresh serum

(ii) frozen serum

1.06~ * Calculated

METHODS

using results expressed in I.U./ ml. All other results in mg/Ioo

ml.

MARCROFT, NEWLANDS

404

on the analyser (Table VII). The group of males were taken from a general practice by means of a random number system. The female group, however, although taken from a general practice, was not randomly chosen and had attended the practice for a variety of reasons. Average values for IgG of 1065 and 1012 mg/roo ml were found for the female and male groups respectively, with an overall mean of 1034 mg/Ioo ml (128 I.U./ml). This results in a normal range of 710-1360 mg/roo ml (88-170 I.U./ml)

IgA

Male Female Total

80 60 140

hZale Female

80 60

W

Total

TABLE

140

247

329 276

75- 4’7

‘74

463 120- 430 I95-

232

194

1012

I25

106.5 IO34

I32 128

690-1333 73x-1390 710-1360 .~.

53-294

137~326 83-300 8j-16j 92-172

88-170

\‘I11

Reagent

Reagent

Technicon reference serum and antiserum Technicon reference serum and Behringwerke antiserum Behringwerkc Tri-partigcn plates Hyland Immune-plate Test Kit

cost pev assay

(pence)

Ig ‘4

IgG

Hapto&bin

cqantitrypsin

15.6

15.6

15.6

19.4

18.4

17.5

2X.0 23.2

26.0 23.2

50.0

jO.0

which is markedly lower and narrower than Technicon’s suggested normal range of 800-1800 mg/roo ml. blean values of IgA for males and females were 279 and 329 mg/roo ml, respectively. The reason for such a large difference in the means is not clear, and while it was thought that the differences may be due to the fact that the two groups were selected in different ways, this does not appear to be the case. There is no apparent difference between the malt and female IgG group means, although this may be due to the higher levels and broader range of IgG values. (7) Reagent cost The cost of reagents per test serum has been evaluated for the analyser, based on runs of 40 sera. Cost of the immunodiffusion method is also given for comparison. CONCLUSIONS

The x,-AT and number of the initial

analyser has a great deal to commend it for the determination of IgG, IgA, haptoglobin, the main advantages over the manual methods being the samples that can be analysed per day, the small overall reagent cost after outlay of purchasing the instrument and the small volume of samples re-

SPECIFIC PROTEIN ANALYSER

quired. The most important

405 advantage

of all is the precision

with which results can

be obtained (i 5 TO). The main disadvantage of the system is the fact that small bore pump tubes are used, and these are readily liable to block. The samples should be completely free from particulate matter and must be centrifuged before use. The effect of haemolysis has been studied and it has been shown that even with a degree of haemolysis as great as IO%, there is no apparent effect upon the results obtained. Similarly, it has been shown that the quantitation of immunoglobulins A and G, by the analyser technique and by the radial immunodiffusion methods, correlate significantly better for fresh sera than for sera that have been stored in the deep freeze at -20’. ACKNOWLEDGEMENTS

We wish to thank Dr. W. H. Price and Dr. D. B. Horn for their help and advice, and we are grateful

to Miss W. D. Langmuir

for technical

assistance.

REFERENCES G. MANCINI, A. D. CARBONARA AND J. F. HEREMANS, Immunochemistry, 2 (1965) 235. K. Lou AND E. SHANBRON, J. Am. Med. Ass., ax (1967) 323. I. ECKMAN, J, B. ROBBINS, C. J, A. VAN DER HAMER, J. LENTZ AND I. H. SCHEINBERG, C&Z. Chem., 16 (1970) 55X. L. hf. KILLINGSWORTH AND J. SAVORY, Clin. Chem.. I7 (1971) 936. C. LARSON, P. ORENSTEIN AND R. F. RITCHIE, An automated method for quantitation of proteins in body fluids. Advances in Automated Analysis. Technicon International Congress, 1970, I, Clinical Analysis, (1971) 19I.