Pre‑analytical variables introduced during tissue collection and fixation are a major source of variability in histology and immunohistochemistry (IHC). Careful control of these early steps is essential to preserve tissue morphology, maintain antigen integrity, and ensure reliable and reproducible results.

Below we outline best‑practice considerations for tissue handling prior to fixation or freezing, as well as during fixation, to support high‑quality IHC and related downstream applications.

Tissue collection handling

Avoid mechanical trauma

Tissues should be excised gently to minimize crushing, stretching, or tearing. Mechanical trauma can disrupt cellular architecture and accelerate enzymatic degradation, leading to artefacts ^[1]and loss of antigenicity that negatively affect staining quality and interpretation.

Best practice:

• Use sharp instruments
• Minimize handling
• Avoid compression or force during removal

Prevent specimen drying

Once removed, tissues should never be allowed to dry out. Even short periods of desiccation can result in cellular shrinkage, morphological distortion, and reduced antigen preservation.

Best practice:

• Transfer tissue immediately into fixative whenever possible
• If fixation must be delayed, cover the specimen with saline‑moistened gauze to maintain hydration until fixation^[1,2]

Minimize ischemic time and delay to fixation

Ischemic time, the interval between loss of blood supply and initiation of fixation, is a critical pre‑analytical variable. Prolonged or inconsistent ischemic times have been shown to alter IHC detection of clinically relevant biomarkers, including estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki‑67^[3,4].

Delayed fixation allows continued metabolic activity and enzymatic degradation, which can reduce antigen detectability and contribute to inter‑sample variability.

Best practice:

• Standardize time to fixation wherever possible
• Initiate fixation as rapidly as possible to preserve protein and enzyme integrity

Optimize fixation conditions

Fixation is one of the most significant contributors to variability in IHC reproducibility. Over‑fixation can cause excessive protein cross‑linking, leading to irreversible epitope masking and reduced antibody binding. Under‑fixation, on the other hand, may result in poor tissue preservation and uneven staining.

Best practice:

• Use validated fixation protocols
• Control fixation time according to tissue size and type
• Avoid prolonged fixation beyond recommended limits

Use sufficient fixative volume and appropriate containers

Adequate fixative volume is essential for uniform fixation. A tissue‑to‑fixative ratio of at least 1:10 (ideally up to 1:20) should be used to ensure complete penetration and consistent preservation.

Best practice:

• Select containers large enough to prevent tissue distortion
• Ensure specimens are fully submerged and not folded or compressed

Check fixative quality and pH

Fixative quality directly influences histological outcomes. Formalin should be fresh, properly stored, and buffered to near‑neutral pH. Acidic formalin reacts rapidly with hemoglobin to form formalin pigment^[5], which can obscure tissue detail and interfere with staining.

Best practice:

• Use high‑quality, buffered formalin
• Monitor fixative pH
• Remove formalin pigment prior to staining when present

Handling larger or bloody specimens

Expedite processing of large specimens

Fixative penetration is limited in thick tissue samples. To avoid uneven fixation and central autolysis, large specimens should be promptly sectioned.

Best practice:

• Slice unfixed tissue to a thickness of no more than ~0.5 cm^[6] before fixation
• Fix sections immediately after grossing

Wash bloody samples before fixation

Blood proteins can inactivate fixatives, reducing fixation efficiency and contributing to artefacts. Heavily blood‑contaminated specimens are particularly susceptible to poor preservation.

Best practice:

• Gently rinse bloody specimens with saline ^[6]prior to fixation
• Avoid prolonged washing that may damage tissue integrity

References

1. William E. Grizzle, Cecil R. Stockard, Paul E. Billings. the effects of tissue processing variables other than fixation on histochemical. staining and immunohistochemical detection of antigens. The Journal of Histotechnology. 2001, 24:213-219.
2. Amanda F Marsch, Jonathan N Truong, Melissa M McPherson et al.  A dermatopathologist's guide to troubleshooting immunohistochemistry--part 2: troubleshooting immunohistochemical tests in the laboratory. American Journal of Dermatopathology. 2015,37(9):665-676.
3. Isil Z Yildiz-Aktas, David J Dabbs, Rohit Bhargava. The effect of cold ischemic time on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast carcinoma. Modern Pathology. 2012, 25(8):1098-1105.
4. Thaer Khoury. Delay to formalin fixation alters morphology and immunohistochemistry for breast carcinoma. Appl Immunohistochem Mol Morphol. 2012, 20(6): 531-542.
5. P Pizzolato. Formalin pigment (acid hematin) and related pigments. American Journal of Medical Technology. 1976 , 42(11):436-440.
6. John D. Bancroft, Marilyn Gamble et al. Theory and practice of histological techniques (sixth edition), Churchill Livingstone, 2008.