Improved staining method for determining the extent of thermal damage to cells

Abstract

Background and Objective: Enzyme histochemical stains of frozen sections have been used by investigators to assess thermal damage. The assessment of thermal damage to cells in lipid-rich tissues such as subcutaneous tissue and sebaceous glands can be difficult due to the quality of frozen sections of such tissues. The purpose of this study is to develop an improved method for this type of evaluation. Study Design/Materials and Methods: Thick frozen sections of thermally damaged pig and human skin were stained for lactate dehydrogenase. The sections were fixed in formalin and processed for paraffin-embedded sections. Results: The sections showed well-defined localization of the enzymatic deposits as well as preservation of the tissue architecture. Conclusion: The paraffin-embedded lactate dehydrogenase stained sections provide improved evaluation of thermally damaged tissues, particularly the lipid rich tissues.

Lateral temperature spread of monopolar, bipolar and ultrasonic instruments for robot‐assisted laparoscopic surgery

“…Evaluation of thermal damage at the protein level required use of a modified lactate dehydrogenase (LDH) assay . As a ubiquitous Krebs‐cycle protein, LDH is an established marker for cell damage.…”

“…Compound ® (Sakura, Japan), snap‐frozen, and stored at –80 °C. Cryo‐sected samples of 50 μm were placed on glass slides and incubated at 4 °C in a medium containing 0.1 m Veronal buffer , 1.5 ng β nicotinamide adenine dinucleotide (NADH, Sigma Aldrich, N1511), 15 mg L(+) lactic acid‐sodium salt (Sigma Aldrich, L7022) and 2.5 mg nitroblue tetrazolium chloride (NBTC, Sigma Aldrich, N6876) overnight. The extent of tissue with significant protein damage was assessed by light microscopy (Leica Application Suite, Leica Germany) measuring the distance to the blue‐stained, healthy tissue.…”

Improving the histologic characterization of burn depth

“…A recent study identified the use of LDH in laser‐induced burns as a simplified, reliable, objective method of identifying the depth of thermal injury. This technique works through detection of the LDH enzyme via a colorimetric change of soluble nitroblue tetrazolium to an insoluble blue‐purple formazan salt precipitate, which occurs in cells that are viable at the time of collection . We sought to confirm our clinical suspicion that viable tissue was being removed during tangential excision of deep partial thickness burns using LDH in addition to H&E staining as a first step towards understanding the potential regenerative capacity of deep partial thickness and full thickness burns.…”

“…This technique works through detection of the LDH enzyme via a colorimetric change of soluble nitroblue tetrazolium to an insoluble blue-purple formazan salt precipitate, which occurs in cells that are viable at the time of collection. 12 We sought to confirm our clinical suspicion that viable tissue was being removed during tangential excision of deep partial thickness burns using LDH in addition to H&E staining as a first step towards understanding the potential regenerative capacity of deep partial thickness and full thickness burns. In this study, we hypothesized that for both expert and novice clinicians, H&E staining of formalin-fixed burn tissue as a method for identifying viability of tissue is inconsistent as compared with LDH activity assessment on frozen tissue.…”

Reconsidering the H&E stain as the gold standard in assessing the depth of burn wounds

“…Dr. Gibson discusses in her introduction that there is no agreement on the technique for assessing burn depth and in the literature, there are several approaches to histological assessment that have included observations, such as the demarcation of normal/denatured collagen, inflammation and vascular patency vs vascular congestion with changes in the endothelial cell lining . Unlike the H&E stain, LDH staining highlights viable tissue, as the enzyme is present in all living cells . This assessment of viability is important because viability, in the presence of vascular patency, indicates regenerative potential.…”