AMeX(acetone, methyl benzoate and xylene)방법을 이용한 피부 기저막대 항원의 면역 조직 화학적 염색

Abstract

[한글]

면역조직 화학적 연구를 위해서는 생검조직의 처리 과정중 항원성과 항원이 존재하는 조직의 형태가 양호하게 보존되어야 한다. 흔히 사용되는 포르말린 고정과 파라핀포매 과정을 거친 조직 표본은 조직의 형태를 비교적 양호한 상태로 보존시킬 수 있는 장점은 있

지만 조직내의 많은 단백질 항원을 파괴시키므로 항원성을 약화시키는 반면, 냉동 조직은 항원성의 보존은 양호하지만 냉동 과정 중 결빙에 의한 조직손상으로 인해 염색 조직의 형태가 불량하여 항원과 조직과의 관계를 명확하게 파악할수 없는 단점이 있다.

본 연구에서는 AMeX (acetone, methylbenzoate and xylene)방법, 즉 조직을 저온 아세톤에 고정시킨 후 methyl benzoate와 xylene 세척 과정을 거쳐 파라핀에 포매시키는 방법을 적응하여 피부 기저막대에 존재하는 Ⅳ형 교원질 항원, Ⅶ형 교원질 항원, 수포성 유천포창 항원, 후천성 표피 박리증 항원의 항원성 보존 정도를 면역 조직 화학적 염색 방법을 통해 냉동 조직과 포르말린 고정 파라핀 포매 조직의 결과와 비교하고, 형태학적 보존 정도를 hematoxylin-eosin염색으로 비교하여 다음과 같은 결과를 얻었다.

1. AMeX방법으로 처리한 조직은 포르말린 고정 조직에 비해 일부 각질 형성세포의 핵이 위축되고 진피 교원질의 균일화가 관찰되었지만 전체적인 형태는 양호하였으며, 냉동 조직은 현저한 각질 형성 세포의 공포화와 변성 ,기저 세포의 액화 변성, 진피 교원질의 분열이 관찰되었으며 부분적인 기저막대의 분열이 관찰되어 불량한 결과를 나타내었다

2. Ⅵ형 교원질과 Ⅶ형 교원질에 대한 특이성이 높은 단크론 항체를 일차 항체로 사용한 경우 AMeX 처리 조직은 냉동조직과 같거나 더 강하게 염색됨을 관찰할 수 있었고 유천 포황과 후천성 표피 박리증 환자의 혈청 항체를 사용한 경우에는 냉동조직보다 다소 약하게 염색되었다. 포르말린 고정 조직은 Ⅳ형 교원질에 대한 단크론 항체에만 반응하였으며 AMeX처리 조직이나 냉동조직에 비해 약하게 염색되었다.

이상의 결과 AMeX 방법으로 처리한 조직은 포르말린 고정 조직이나 냉동 조직에 비해 피부 기저막대 항원의 항원성 보존과 형태학적 보존 정도가 모두 우수하였다. 또한 본 방법은 조직을 파라핀에 포매시켜 보관하므로 조직의 장기간 보관이 용이하여 향후 보관된 조직을 이용한 후향적 연구영역에서 다양하게 응용될 수 있을 것으로 사료된다.

Immunohistochemical demonstration of the skin basement membrane antigens by the

AMeX (acetone, methylbenzoate and xylene) method

Won Hur

Department of Medical Science The Graduate School, Yonsei University

(Directed by Assistant Professor Soo-Chan Kim)

Immunohistochemical labelling of molecules with antibodies is essential to the

study of protein antigens, some of which play important roles in the elucidation of

the pathogenesis of immunologic disorders. However, some antigens lose their

immunoreactivity during the routine process of pathology fixation and paraffin

embedding.

Conventional formalin fixation and paraffin embedding procedures are useful in

preserving tissue architecture and cytologic detail, however, they destroy the

antigenicity of many proteins in the tissue sample. On the other hand, fresh frozen

section preserve the antigenicity of most proteins, but yield poor morphological

preservation. To circumvent the problems that are associated with routine tissue

fixation and cryofixation, AMeX method(cold acetone fixation with subsequent

methylbenzoate and xylene treatment and routine paraffin embedding) was developed

for better preservation of antigenicity as well as morphologic details.

The purpose of this study is to evaluate the AMeX method as to ability to

preserve both antigenicity and morphologic details of the skin basement membrane

zone so that precise localization of antigens can be attained in

immunohistochemistry.

Tissues were fixed in acetone at -20℃ over night, then cleared in methyl

benzoate and xylene, consecutively, and embedded in ordinary paraffin at 58-60℃.

Sections made from this paraffin-embedded tissue were stained with hematoxylin and

eosin for a morphologic study and immunolabelled with antibodies against type Ⅳ

collagen, type Ⅶ collagen, bullous pemphigoid antigen, and epidermolysis bullosa

acquisita antigen which are major basemert membrane antigens, to evaluate

antigenic preservation. The staining intensity and preservation of the morphology

by the AMeX method were compared with conventional formalin processed tissues and

frozen tissues. The results are summarized as follows:

1. Morphological preservation of the AMeX method-processed sections was good

throughout the epidermis, basement membrane, and dermis, and as good as that of

routinely formalin-fixed paraffin-embedded sections. Frozen sections usually

revealed various degrees of damage by ice crystal formation throughout the

epidermis to the dermis.

2. The AMeX method-processed sections showed better or same antigenic

preservation comparing the frozen sections when the sections were immunolabelled

with specific monoclonal antibodies but when the sections were immunolabelled with

patient's sera, the AMeX method showed less antigenic preservation than the frozed

sections. The anti-type Ⅳ collagen monoclonal antibody exbibited immunoreactivity

only in conventional formalin- fixed paraffin-embedded skin sections, but the

intensity of the staining was weaker than the AMeX processed sections and the

frozen sections.

Our results suggest the AMeX method can be utilized for the demonstration of skin

basement membrane antigens and is superior to the fresh-frozen method in that the

histologic figures are more distinct and antigencity can be preserved for a long

time.

[영문]

Immunohistochemical labelling of molecules with antibodies is essential to the study of protein antigens, some of which play important roles in the elucidation of the pathogenesis of immunologic disorders. However, some antigens lose their

immunoreactivity during the routine process of pathology fixation and paraffin embedding.

Conventional formalin fixation and paraffin embedding procedures are useful in preserving tissue architecture and cytologic detail, however, they destroy the antigenicity of many proteins in the tissue sample. On the other hand, fresh frozen

section preserve the antigenicity of most proteins, but yield poor morphological preservation. To circumvent the problems that are associated with routine tissue fixation and cryofixation, AMeX method(cold acetone fixation with subsequent methylbenzoate and xylene treatment and routine paraffin embedding) was developed for better preservation of antigenicity as well as morphologic details.

The purpose of this study is to evaluate the AMeX method as to ability to preserve both antigenicity and morphologic details of the skin basement membrane zone so that precise localization of antigens can be attained in immunohistochemistry.

Tissues were fixed in acetone at -20℃ over night, then cleared in methyl benzoate and xylene, consecutively, and embedded in ordinary paraffin at 58-60℃. Sections made from this paraffin-embedded tissue were stained with hematoxylin and eosin for a morphologic study and immunolabelled with antibodies against type Ⅳ collagen, type Ⅶ collagen, bullous pemphigoid antigen, and epidermolysis bullosa acquisita antigen which are major basemert membrane antigens, to evaluate antigenic preservation. The staining intensity and preservation of the morphology by the AMeX method were compared with conventional formalin processed tissues and frozen tissues. The results are summarized as follows:

1. Morphological preservation of the AMeX method-processed sections was good throughout the epidermis, basement membrane, and dermis, and as good as that of routinely formalin-fixed paraffin-embedded sections. Frozen sections usually revealed various degrees of damage by ice crystal formation throughout the

epidermis to the dermis.

2. The AMeX method-processed sections showed better or same antigenic preservation comparing the frozen sections when the sections were immunolabelled with specific monoclonal antibodies but when the sections were immunolabelled with patient's sera, the AMeX method showed less antigenic preservation than the frozed sections. The anti-type Ⅳ collagen monoclonal antibody exbibited immunoreactivity only in conventional formalin- fixed paraffin-embedded skin sections, but the intensity of the staining was weaker than the AMeX processed sections and the

frozen sections.

Our results suggest the AMeX method can be utilized for the demonstration of skin basement membrane antigens and is superior to the fresh-frozen method in that the histologic figures are more distinct and antigencity can be preserved for a long time.