Effect of Formalin Fixation on the Immunohistochemical Detection of PRRS Virus Antigen in Experimentally and Naturally Infected Pigs

Abstract

The purpose of this study was to determine the effect of formalin fixation on the immunohistochemical detection of porcine reproductive and respiratory syndrome (PRRS) viral antigen in lungs of experimentally and naturally infected pigs. In separate trials, five 24-day-old pigs and six 10-day-old pigs were housed as separate groups in isolation and inoculated intranasally with 10(5.5) TCID50 of an isolate of PRRS virus (PRRSV; P129). The older and younger pigs were euthanatized at 7 and 10 days post inoculation (dpi), respectively. At necropsy, all pigs had gross and microscopic lung lesions typical of PRRS, and PRRSV was isolated from all pigs. To insure uniform fixation, lungs from each pig were cut into 1-cm-thick slices and immersed into 10% neutral-buffered formalin. After fixation in formalin for 8 hours or 1, 2, 3, 5, 6, 8, 10, and 15 days, 3 lung sections from some or all pigs were processed for histological examination using routine methods. Immunohistochemical staining for PRRSV antigen was positive at the following times (days unless otherwise stated) after fixation (percentage of pigs staining positive for PRRSV in parentheses): 8 hours (100); 1 (100); 2 (100); 3 (80); 5 (33); and 6, 8, 10, and 15 (0-all negative). To further evaluate the effects of formalin fixation on PRRSV immunodetection, 31 field cases of PRRS were selected for immunohistochemistry (IHC). Over a 3-month period, submitted cases were selected from the Purdue University Animal Disease Diagnostic Laboratory, W. Lafayette, Indiana, for IHC if 1) the clinical history included respiratory disease, 2) PRRSV was isolated from lung and/or serum from the submitted pigs or tissues, 3) at least 1 section of lung fixed in 10% neutral-buffered formalin was submitted for IHC, and 4) the duration of fixation could be accurately determined from the case history. Of the 31 PRRSV-infected pig cases meeting the selection criteria, 23 were fixed in formalin for 4 days or less. Twenty-one of these 23 (91%) were positive by IHC. Two of 8 cases fixed for greater than 4 days (25%) were positive by IHC. In practical terms, 1-day shipping of fixed samples to a laboratory followed by routine tissue processing within a laboratory should not adversely affect immunohistochemical detection of PRRS viral antigen. But a delay in shipping or processing of more than 2 days could reduce or prevent the detection of PRRS viral antigen by IHC.

Effects of Prolonged Formalin Fixation on Diagnostic Immunohistochemistry in Domestic Animals

“…These studies demonstrate the importance of optimizing antigen retrieval protocols for each antibody used and evaluating the effects of prolonged fixation in antibody standardization and validation. Van Alstine et al (2002) reported a significantly decreased ability to detect porcine reproductive and respiratory syndrome (PRRS) virus antigen at 5 days of fixation, and were unable to detect any antigen at later time points. Interestingly, using the same antibody, other investigators could detect PRRS virus antigen following 7 days of fixation (Rossow et al 1996).…”

Enhanced detection of Porcine reproductive and respiratory syndrome virus in fixed tissues by in situ hybridization following tyramide signal amplification

“…9,19 In the present study, PRRSV was not detected by IHC in tissue samples fixed for more than 14 days in either 4% PFA or 10% NBF. Prolonged formalin fixation has been shown to lead to masking of epitopes because of cross-linking, which reduces or even abolishes antigen detection.…”

“…[6][7][8]21 Formalin is a commonly used fixative agent that inactivates most infectious agents and inhibits autolysis. 9,19 Formalin fixation results in the cross-linking of tissue protein with DNA or RNA; overfixation can reduce the sensitivity of IHC and ISH. For routine IHC and ISH, solutions of 10% neutral buffered formalin (NBF) or 4% paraformaldehyde (PFA) are commonly used for fixation.…”

Standardization and Validation of an Immunoperoxidase Assay for the Detection of African Horse Sickness Virus in Formalin-Fixed, Paraffin-Embedded Tissues

“…37 The formation of these cross-links is progressive, being dependent on time and temperature, 37,38 and the cross-links may be irreversible, 48 so it is generally accepted that overfixation in formaldehyde can produce false-negative results in IHC. 37 African horse sickness viral antigen could be successfully detected in target tissues after fixation for up to a year in 10% neutral buffered formalin.…”

“…17,39 Routine dewaxing in xylol, followed by rehydration through a graded ethanol and distilled water series, took place inside a fume hood (10 min in xylol and 3 min each in 100%, 96%, and 70% ethanol). 1 Sections were subsequently incubated with 3% hydrogen peroxide in methanol 48,49 for 15 min in a humidified chamber at room temperature (22uC-25uC) to quench endogenous peroxidase activity. Sections were rinsed in distilled water, followed by 0.1 M phosphate buffered saline (PBS; pH 7.6) containing 0.1% bovine serum albumin (BSA) 1,35 for 5 min per rinse.…”