Necropsy and Tissue Collection Services

Necropsy is the point at which an in vivo study becomes a set of samples, and almost every downstream endpoint depends on decisions made during that hour. Tissue fixed for histology cannot subsequently be used for protein work. Tissue frozen for molecular analysis cannot be used for flow cytometry. An organ collected late in a long necropsy has been at ambient temperature longer than one collected first, and for labile analytes that difference is measurable.

These are not recoverable errors. A study that generated excellent animal data and collected its tissue poorly has lost information that no subsequent analysis restores, and the loss is usually discovered weeks later when the assay fails.

Altogen Labs conducts necropsy and tissue collection to a protocol-defined plan in which every tissue has a designated preservation method matched to the endpoint it will support. To discuss a collection plan, request a quote.

Planning the collection

The collection plan is built backward from the endpoint list. Each analysis is identified first, then the tissue it requires, then the preservation method and the quantity needed, and only then the sequence in which tissues are taken. Organs containing labile analytes or destined for the most sensitive assays are collected earliest.

Where one tissue must support several endpoints, the division is specified in advance: which portion is fixed, which is flash frozen, which is placed into stabilization reagent, and which is processed fresh for flow cytometry. Deciding this at the bench with a tumor in hand produces inconsistent divisions between animals, and inconsistent division is itself a source of variance in the results.

Preservation methods

Formalin fixation for histopathology and immunohistochemistry requires controlled fixation time, since both over-fixation and under-fixation alter epitope accessibility and a staining difference between groups can originate in handling rather than treatment.

Flash freezing preserves tissue for nucleic acid and protein isolation, with the interval between excision and freezing minimized. Tissue destined for phosphorylation analysis is handled to preserve modification state, which requires collection directly into appropriate buffer within a defined interval. RNA stabilization reagent is used where immediate freezing is impractical. Fresh processing is required for most flow cytometry panels, which is why the flow aliquot is taken first when a tumor must support both.

Gross observations

Necropsy also generates data in its own right. Gross observations, organ weights, macroscopic lesion description, and metastatic burden assessment are recorded systematically rather than noted informally. Organ weights relative to body weight identify changes that absolute weights conceal, and macroscopic metastatic counts in metastasis studies are frequently a primary endpoint rather than an observation.

Applied examples

Full endpoint panel from one tumor. A single tumor is divided at necropsy into a fresh aliquot for flow cytometry, a fixed portion for histology and immunohistochemistry, and a flash frozen portion for transcript and protein analysis, supporting four endpoint classes from one animal rather than four cohorts.

Labile analyte preservation. Tumors destined for phosphoprotein analysis are collected first in the necropsy sequence and placed directly into inhibitor-containing buffer, preserving the modification state that a standard collection order would have partially lost.

Metastatic burden. Lungs are collected, inflated, fixed, and step sectioned to a defined protocol, with surface nodule counts recorded before fixation, providing both macroscopic and microscopic burden measures from the same tissue.

Archival material

Tissue beyond the immediate endpoint list is archived under defined conditions and retained for a specified period. This is inexpensive at the time and frequently decisive later, since a question arising after a study closes can often be answered from archived tissue rather than by repeating the animal work. What is archived, how, and for how long is agreed before the study opens.

Conduct, documentation, and welfare

Necropsy is performed to a written plan specifying tissue list, collection sequence, preservation method per tissue, aliquot sizes, and labeling convention, with deviations recorded. Sample identity is tracked from collection through analysis. All procedures are conducted under active IACUC protocols, including method of euthanasia appropriate to the species and to the downstream endpoints, since some methods affect tissue analytes and the choice is made with that in mind. GLP standards are applied where a study is formally designated as GLP.

Send the endpoint list and tissue requirements for a planned study, or request a quote.