Evaluation of Passive Hemagglutination Techniques in Detection of Australia Antigen (HBsAg) G . SCALISE, M.

s. MURA,AND s. DELIA

From the Infectious Diseases Department. University ofSassari, and the Infectious and Tropical Diseases Department. University of Rome, Italy

Some techniques for HBsAg detection in the serum were evaluated. The direct hemagglutination test with turkey erythrocytes appears to be a suitable test for speed, simplicity, and cost.

TECHNIQUES for determination of Hepatitis B surface antigen (HBsAg) have been improved in recent years becoming more and more sensitive. l 2 However certain problems still remain to be solved. The most sensitive tests are not entirely satisfactory owing to the s1ownes.s of procedure, high costs and lack of ~pecificity.~.” Other tests lack sufficient sensitivity and often reproductiveness. lmmunodiffusion (ID) belongs to the “first generation.” Even though lacking in sensitivity, it has the advantage of specificity. The “second generation” includes immunoelectrophoresis (CEP)‘ which is still employed in certain transfusion centers, Latex (LA)’ which has been cited for its aspecificity, and complement fixation (CF)x which is quite sensitive but takes too much time and lacks reproductiveness. The “third generation” includes the inverse hernagglutination techniques (IH) using the red blood cells of sheep (IHS) or turkeys (IHT),5 and radioimmunoassay (R1A).I2 T h e IH with recently added modifications (turkey’s red blood cells) is proving to be the quickest, simplest, least expensive and only little less sensitive te~t.’.‘.~Therefore, we shall compare I H with all techniques more commonly used in H BsAg studies.

Materials and Methods We examined 229 cases of acute hepatitis, 490 patients with other diseases, 51 HBsAg positive healthy carriers and a group of 88 negative controls. We carried out ID and C E P using Prince techniques7 and C F as described by Shulman.” The IHS,’ IHT’ and RIA12 techniques were checked by neutralization or by control agglutination. Four times concentration of HBsAg negative sera was done using Minicon S filters. The sera were heated at 70 C for 10 minutes or treated with 2,mercaptoethanol (0.1 M) for 24 hours and dialyzed against 2,iodoacetamide (0.02 M).

Received for publication May 12, 1976; accepted August 16, 1976. This work was supported by a grant from C N R Rome No 75.581.04

Results As can be seen in Table 1 the sensitivity of techniques used in HBsAg research progressively increases according to the techniques used. In the group with a c u t e hepatitis we go from a percentage of positivity of 15.6 with ID to 38.8 per cent with RIA. In the group hospitalized for nonhepatic diseases we see an increase from 3.6 per cent with ID to 6.8 per cent with IHT or RIA. The sensitivity of tests was checked in different ways as shown in Table 2. I f C F positive sera were subdivided according to the titer the most sensitive techniques, capable of always detecting positivity of the serum, are RIA and IHT. Of course this grade of sensibility was seen only in sera with a titer of 1:4(CF), since at I:512(CF) all four techniques used showed equal sensitivity. Special “negative” sera were concentrated four times by ultrafiltration with Minicon S filters. The specificity of CEP,IHS,IHT was related with antigammaglobulinemic factors (Ra test). The results are shown in Table 3. Fourteen sera, positive for both HBsAg and the Ra test, were heated to 70 C for 10 minutes and submitted to the action of 2,mercaptoethanol. These two procedures are capable of inactivating IgM to

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626

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SCALISE ET A L .

which category most “rheumatoid factors” belong. In spite of neutralization o f the Ra test in this way, all the sera maintained their HBsAg reactivity with CEP or IHS. The specificity of positive reactions obtained with IHS and IHT are shown in Table 4.The sera positive by IHS were retested after thorough absorption of serum with sheep erythrocytes. The sera positive by IHT were retested after adsorption with turkey red b!ood cells sensitized with immunoglobulins not containing anti-HBs. The results show that false positivity lay between 3.4 per cent for I HS and I .6 per cent for I HT.

Table 2.

Ncr\..-I>c.c. 1017

Table 1.

Results of HBsAg Determinations with Different Techniques

~~~~~

~

% Positive for HBsAg

Total Examined Acute Hepatitis

Technique

Other Diseases

_________~

~~

134 229 134 229 119 134

ID CEP FC

IHS IHT RIA

Acute Hepatitis

490 490 490 490 88 90

Other Diseases

~

156 28 3 34 3

3 4 5 6 6 6

27 9 35 2 38 8

6 8 1 1 8 8

Cornparison of the Sensitivity of the Techniques ~~

~~~

Dilution of the sera positive in CF: 1 : 4 (at CF) 1:512(atCF)

CEP 4 13

IHS 7 14

I HT 9 14

RIA 10 15

Concentration 4 times of ”negative” sera:

CEP

IHS

IHT

RIA

+4.1% .f20.8%

+lo% +40.2%

+12.5% +60.9%

+14.1%

Acute hepatitis HBsAg negative Sera positive only in RIA

Comparison of the Specificity for CEP. IHS. IHT Related to Ra Test

Table 3.

Acute HBsAg+ hepatitis HBsAg carriers Other diseases with HBsAg

-

Cases positive for Ra test and CEP. IHS. IHT

CEP

IHS

IHT

Ra test

CEP

IHS

IHT

Ratest

5 4

5 4

5 4

4 4

0 1

5 4

5 4

5 4

0 1

5

5

5

4

1

5

5

5

1

Table 4.

After Treatment at 7 0 C

After Treatment with 2,mercaptoethanol

Comparison of the Specificity between IHS and IHT

HBsAg Carriers

Acute Hepatitis

Positive at the beginning Positive after control’ Difference

IHS

IHT

IHS

IHT

39.2% 35.8% 3.4%

36.8% 35.2% 1.6%

100% 100% 0

100% 97.0% 3.0%

‘After adsorption with sheep red blood cells (1%) globulin without HBsAb (IHT).

and after absorption with turkey red blood cells sensitized with immuno-

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PASSIVE H E M A G G L U T I N A T I O N

Discussion

Results obtained clearly show that, a t present, inverse hemagglutination and radioimmunoassay for HBsAg a r e the most sensitive techniques to be used. Inverse hemagglutination is easily and rapidly carried out with a limited expense while RIA requires more time and more expensive equipment. RIA is more sensitive than is IHS, but t h e a d v a n t a g e is b a l a n c e d o u t by t h e presence of certain number of false positives. The IHT, which possess a more rapid sedimentation rate, reduces reading times to 30 to 60 minutes. Also results show that the presence of rheumatoid factors does not int e r f e r in any way with hemagglutination reaction. We believe that the I H T technique using the turkey’s red blood cells will find a useful place in blood transfusion centers. A further possible perfection of I H T t e c h n i q u e , consisting of the introduction of a neutralization test with specific antibodies capable of confirming positive reactions, would greatly increase its significance. References 1.

Cayzer, I., D. S. Dane, C. H. Cameron, and J. V. Denning: A rapid haemagglutination test for hepatitis-B antigen. Lancet 1:947, 1974.

2.

3.

4.

5. 6.

7.

8.

9.

10.

I I.

12.

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Chrystie, I. L., M. N. Islam, J. E. Banatvala, and 1. Cayzer: Clinical evaluation of the turkeyerythrocyte passive-haemagglutination test for hepatitis-B surface antigen. Lancet 2:l 193, 1974. Cossart, Y. E., E. Boxall, and T. H. Flewett: False negative radioimmunoassay tests for Australia antigen. Lancet 2:1333, 1973. -, A. M. Field, S. P. March, and A. A. Porter: Latex test for Au antigen. Lancet 2:379, 1972 Hadziyannis, S. J.: Detection of HBsAg by passive haemagglutination. Lancet 2:344, 1974. Klossner, M. L., and K. Willman: Latex test, complement fixation a n d i m m u n o e l e c t r o o s m o phoresis in detection of Australia antigen. Lancet 1:322, 1973. Prince, A. M.: A n antigen detected in the blood during the incubation period o f serum hepatitis. Proc. Nat. Acad. Sci. U.S.A.60:814, 1968. Purcell, R. P., P. Holland, D. Walsh, D. Wong, A. Morrow, and R . Chaneck: A complement-fixation test for measuring Australia antigen and antibody. J. Infect. Dis. 120:383, 1969. Reesink, H. W., W. J. Duimel, and H. C. J. Brumelhuis: Evaluation of a new haemagglutination technique for the demonstration of hepatitis-B antigen. Lancet 2:1351, 1973. Shulman, N.. and L. Barker: Virus-like antigen, antibody and antigen-antibody complexes in hepatitis measured by complement fixation. Science 165:304, 1969. Vyas, G. N., H. A. Perkins, and R. Schmid: Hepatitis and Blood Transfusion. New York, Grune and Stratton, 1972. Walsh, J. M., R. Yalow, and S. Berson: Detection of Australia antigen and antibody using radioimmunoassay techniques. J. Infect. Dis. 12550, 1970.