Journal of Veterinary Parasitology
Year : 2005, Volume : 19, Issue : 1
First page : ( 9) Last page : ( 14)
Print ISSN : 0971-6157.

Evaluation of chick red blood cells stabilized with various aldehydes in the indirect haemagglutination (IHA) test for diagnosis of neurocysticercosis

Parija Subhash Chandra, Sahu Priyadarshi Soumyaranjan

Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry-605 006, India

Abstract

The present study was carried out to evaluate the use of chick cells in the indirect haemagglutination (IHA) test for the diagnosis of neurocysticercosis. Patients attending epilepsy clinic, Jawaharlal Institute of Postgraduate Medical Education and Research, (JIPMER) Hospital, Pondicherry were included in the present study. The study included 15 cases of neurocysticercosis with single small cystic lesion, 15 cases of neurocysticercosis with multiple cystic lesions, 15 cases of neurological disorders other than cysticercosis (meningitis, etc.) and 30 normal healthy people as controls. Results of the study showed that agglutinated chick red blood corpuscles (RBCs) settled after 30 to 45 min of incubation at room temperature and demonstrated a definite pattern of haemagglutination with the cysticercus positive sera. In case of most of the sera from patients with neurocysticercosis, the IHA using double aldehyde stabilized (DAS) chick cells gave 2–4 times higher titer than the chick cells stabilized by single aldehydes such as formaldehyde, glutaraldehyde or pyruvic aldehyde. The IHA employing DAS chick cells was found to be equally sensitive with that of the IHA using DAS human RBCs. A total of 27 out of 30 sera from the cases of neurocysticercosis were positive for cysticercus antibodies by the IHA employing the chick DAS as well as human DAS cells sensitized with cysticercus antigen. In conclusion, results of the present study show that the IHA using DAS chick RBCs instead of human ‘O’ RBCs will be a useful procedure with equal sensitivity for the diagnosis of neuerocysticercosis.

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Keywords

Indirect haemagglutination, Chick RBCs, Double aldehyde stabilized cells, Haemagglutination, dehydrogenase, Neurocysticercosis.

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Introduction

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Materials and Methods

Patients

Patients attending epilepsy clinic, JIPMER Hospital, Pondicherry were included in the present study. The following groups of the patients and controls were included:

Group I

It included 15 cases of neurocysticercosis with single cystic lesion in the brain on CT scan; Group II: it included 15 cases of neurocysticercosis with multiple small cystic lesions in brain on CT scan; Group III: included 15 cases of neurological disorders (meningitis, etc.) other than cysticercosis; and Group IV: included 30 normal healthy people as controls.

Blood samples were collected from these cases and controls, sera were separated and stored at −20°C till use.

Cysticercus antigen

Cysticercus cellulosae cysts, from infected pig muscles were collected for antigen. Preparation of complete homogenate. Cysticercus cellulosae antigen was prepared as per the procedure given by Sreenivasamurthy et al. (1999). Briefly, cysts were homogenized in a glass tissue homogenizer with phosphate buffered saline (PBS, pH 7.2) containing phenyl-methyl sulphonyl fluoride (PMSF) (0.1 mM), the suspension was then sonicated 8 times (each time for 1 min duration) at 12 kH with 30 sec cooling interval; then centrifuged at 4°C for 30 min at 14,000 rpm. The supernatant was collected as the complete homogenate antigen and stored at −20 °C.

Red blood cells (RBCs)(RBCs)(RBCs)

Chick RBCs

Blood was collected from chicks into Alsever’s solution. The cells were washed thrice in PBS, pH 7.2, (0.15 M) and treated with the stabilizing agent within 48 h.

Human RBCs

Blood was collected from human group ‘O’ into Alsever’s solution. The cells were washed thrice in PBS, pH 7.2, (0.15 M) and treated with the stabilizing agent within 48 h.

Stabilization of chick RBCs

Stabilization of the chick RBCs with different aldehydes was carried out as per the procedures described below:

Stabilization of chick RBCs with formaldehyde

Chick RBCs were stabilized by the method of Fulthrope et al. (1961) as cited by Herbert (1978). To a suspension of chick RBCs (10 ml of packed cells in 500 ml of PBS, pH 7.2). 50 ml of formalin (aqueous formaldehyde, 40% w/v) was added slowly from a burette, over a period of 30 min. The mixture was left overnight at 4 °C and further 50 ml of formalin was then added in one lot. Twenty-four hours later the preserved cells were washed thrice in PBS, pH 7.2 and stored at 4 °C as a 50% v/v suspension in a 1 in 200 dilution of formalin in PBS, pH 7.2.

Stabilization with glutaraldehyde

The RBCs were stabilized with glutaraldehyde as per the method described by Avermeas et al. (1969) as cited by Parija et al. (1986). Briefly, 10 ml of 5 % v/v aqueous glutaraldehyde was added to 1 ml of 10% suspension of chick RBCs in PBS, and the mixture was stirred by a magnetic stirrer for 4 h as a 30% v/v suspension in PBS.

Stabilization with pyruvic aldehyde

The RBCs were stabilized by pyruvic aldehyde as per the method described by Parija et al. (1986). A reaction mixture consisting of the following substances: 1.7% NaCl, 12 ml; 40% v/v aqueous pyruvic aldehyde, 4 ml; 1% Na₂Co₃ 35 ml; Soresen's phosphate buffer (pH 7.2) 7 ml added in the order given. The reaction mixture thus prepared was kept in the cold and to this 10 ml of a 50% suspension of chick RBCs was added. The mixture was stirred for 30 min and stored for 24 h at 4°C, the cells were washed thrice in PBS and stored in this as a 2.5% v/v suspension at 4°C.

Double aldehyde stabilization (DAS) with pyruvic aldehyde and glutaraldehyde

Double aldehyde stabilization (DAS) of chick RBCs was carried out, as per the method described by Parija and Ananthakrishnan (1985). The cells were first stabilized with pyruvic aldehyde (as above). To a 2.5 % suspension of these an equal volume of cold tannic acid (1 in 25,000 in PBS, pH 7.2) was added and the mixture kept at 4°C for 30 min. The cells were then washed thrice in PBS and made upto a 4% suspension in the same diluent. To this was added an equal volume of 2% v/v aqueous glutaraldehyde in PBS, pH 7.2 over a magnetic stirrer which was run for 2 h. The cells were then washed thrice with PBS, pH 7.2 and stored as a 50% v/v suspension in this diluent.

Sensitization of double aldehyde stabilized chick cells with parasite antigen

The DAS chick cells were sensitized with the Cysticercus antigen, as described by Farshy and Kagan (1972). Briefly, one volume of packed DAS cells was suspended in 10 volumes of an optimum sensitizing dose (OSD) of the antigen prepared in PBS, pH 6.4. The OSD determined for the particular batch of antigen was a dilution 1:80 in PBS, pH 6.4. The cell plus antigen mixture was incubated at 50 ° C in water batch of antigen for 5 min. The sensitized cells were washed thrice with PBS, pH 7.2 and stored in small volumes as a 10% suspension in PBS, pH 7.2 at 4°C until used.

Sensitization of single aldehyde stabilized chick cells with parasite antigen

Single aldehyde stabilized chick cells were first tanned, as described above (in case of DAS) just before to Sensitization. Then rest of the procedure for Sensitization was same as per the DAS cells.

Stabilization and Sensitization of human ‘O’ RBCs

The human ‘O’ RBCs were stabilized by double aldehyde stabilization methods in the same way as described for chick RBCs. The stabilized human ‘O’ RBCs were sensitized with the OSD of cysticercus antigen in the same ways as described for the stabilized chick RBCs. The sensitized cells were stored in small volumes as a 10% suspension in PBS, pH 7.2 at 4°C until used.

Indirect haemagglutination test

Stored sensitized chick RBCs were used to prepare a 1 % suspension in PBS, pH 7.2 bovine serum albumin (BSA) (0.1%). This suspension was used in the IHA test on the same day and after storage at 4°C for 7, 14, 31 and 45 days. The test was performed as described by Parija and Ananthakrishnan (1985). The results were read by observing the settling patterns of the cells in the plate after incubation for 15, 30, 45, 60 and 120 min at room temperature and after holding the plates overnight at 4°C.

The IHA employing the stored sensitized DAS human RBCs were carried out on the same batch of sera for demonstration of serum antibodies.

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Results and Discussion

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Tables