Sadhana S Kode1, Shailesh D Pawar2, Deeksha S Tare1, Jayati Mullick1. 1. ICMR-National Institute of Virology, Microbial Containment Complex, 130/1, Sus Road, Pashan, Pune, 411021, India. 2. ICMR-National Institute of Virology, Microbial Containment Complex, 130/1, Sus Road, Pashan, Pune, 411021, India. Electronic address: shaileshpawarniv@gmail.com.
Abstract
INTRODUCTION: Hemagglutination (HA) and hemagglutination inhibition (HI) assays are conventionally used for the detection and identification of influenza viruses, using red blood cells (RBCs) from mammalian and avian sources. However, there could be limitations for availability of fresh RBCs due to situations such as pandemics, public health emergencies, outbreaks in avian species, lack of animal facilities, animal ethics concerns; or resource-constrained laboratories, and laboratories which do not carry out HA and HI assays routinely. Turkey RBCs (tRBCs) are widely used for HA and HI assays of influenza viruses. The present study explored the possibility of the use of glutaraldehyde-fixed tRBCs, which could be stored at -80 ºC and readily used for HA and HI assays. MATERIALS AND METHODS: A total of nine subtypes of human and avian influenza viruses, A H1N1, H3N2, H4N6, H5N1, H6N1, H7N9, H9N2, H11N1 and type B, were used in the study. Turkey RBCs were fixed with glutaraldehyde. The HA and HI assays were performed three times by two different operators using fresh and glutaraldehyde fixed tRBCs. The significance of difference in HA and HI titers between fixed and fresh RBCs was compared using 't-test'. The performance of fixed RBCs was evaluated before and after storing at -80 ºC for three weeks. RESULTS: There was no significant difference (p > 0.05) between mean HA and HI titers using fresh and glutaraldehyde-fixed turkey RBCs. In addition, the HA and HI titers using fixed tRBCs before and after storing at -80 ºC were equivalent, indicating suitability of the fixed and stored RBCs. CONCLUSIONS: This is the first report of the use of fixed and stored tRBCs for HA and HI assays of influenza viruses, highlighting their applicability as a ready-to-use reagent for laboratory diagnosis of influenza.
INTRODUCTION: Hemagglutination (HA) and hemagglutination inhibition (HI) assays are conventionally used for the detection and identification of influenza viruses, using red blood cells (RBCs) from mammalian and avian sources. However, there could be limitations for availability of fresh RBCs due to situations such as pandemics, public health emergencies, outbreaks in avian species, lack of animal facilities, animal ethics concerns; or resource-constrained laboratories, and laboratories which do not carry out HA and HI assays routinely. Turkey RBCs (tRBCs) are widely used for HA and HI assays of influenza viruses. The present study explored the possibility of the use of glutaraldehyde-fixed tRBCs, which could be stored at -80 ºC and readily used for HA and HI assays. MATERIALS AND METHODS: A total of nine subtypes of human and avian influenza viruses, A H1N1, H3N2, H4N6, H5N1, H6N1, H7N9, H9N2, H11N1 and type B, were used in the study. Turkey RBCs were fixed with glutaraldehyde. The HA and HI assays were performed three times by two different operators using fresh and glutaraldehyde fixed tRBCs. The significance of difference in HA and HI titers between fixed and fresh RBCs was compared using 't-test'. The performance of fixed RBCs was evaluated before and after storing at -80 ºC for three weeks. RESULTS: There was no significant difference (p > 0.05) between mean HA and HI titers using fresh and glutaraldehyde-fixed turkey RBCs. In addition, the HA and HI titers using fixed tRBCs before and after storing at -80 ºC were equivalent, indicating suitability of the fixed and stored RBCs. CONCLUSIONS: This is the first report of the use of fixed and stored tRBCs for HA and HI assays of influenza viruses, highlighting their applicability as a ready-to-use reagent for laboratory diagnosis of influenza.