固定和脱矿对犬骨髓组织形态学和 DNA 扩增的影响

IF 2.3 2区 农林科学 Q2 PATHOLOGY Veterinary Pathology Pub Date : 2024-11-01 Epub Date: 2024-06-06 DOI:10.1177/03009858241257920
Gabriella M L Diamantino, Janet Beeler-Marfisi, Robert A Foster, William Sears, Alice Defarges, William Vernau, Dorothee Bienzle
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引用次数: 0

摘要

各诊断实验室的骨髓(BM)固定和脱矿方案并不统一。人们对不同方案如何影响组织形态学和 DNA 扩增还不甚了解。本研究对犬骨髓样本进行了 2 种固定剂和 3 种脱矿物质方法的测试。在犬死亡后 24 小时内获得的 20 份重复胸骨样本在醋酸-锌-福尔马林(AZF)或 10%中性缓冲福尔马林(NBF)中固定过夜,并用甲酸脱盐 12 小时。另外 53 份样本在 AZF 中固定,然后用盐酸脱矿 1 小时、甲酸脱矿 12 小时或乙二胺四乙酸(EDTA)脱矿 24 小时。组织切片由 4 位评分员评分,质量分为不足、边缘、良好或优秀。此外,从采用不同固定和脱矿方法处理的切片中提取的 DNA 样本用 3 组引物扩增,引物分别指向 T 细胞受体 gamma 和免疫球蛋白重链基因的保守区。扩增效率根据毛细管电泳图进行分级。用 AZF 或 NBF 固定的切片在组织形态学评分上没有明显差异。不过,与盐酸或甲酸脱矿相比,EDTA脱矿的组织形态学得分更高,而甲酸脱矿的组织形态学得分则高于盐酸脱矿。在 36 个样本中,有 29 个样本(81%)通过 EDTA 除矿获得了 DNA 扩增,而在 72 个样本中,只有 2 个样本(3%)通过两种酸除矿获得了扩增。用 EDTA 进行组织脱矿虽然略微费时费力,但却能获得较好的形态,对于本文所述的 DNA 提取方法进行聚合酶链反应 (PCR) 扩增至关重要。
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Effects of fixation and demineralization on histomorphology and DNA amplification of canine bone marrow.

Fixation and demineralization protocols for bone marrow (BM) across diagnostic laboratories are not standardized. How different protocols affect histomorphology and DNA amplification is incompletely understood. In this study, 2 fixatives and 3 demineralization methods were tested on canine BM samples. Twenty replicate sternal samples obtained within 24 hours of death were fixed overnight in either acetic acid-zinc-formalin (AZF) or 10% neutral-buffered formalin (NBF) and demineralized with formic acid for 12 hours. Another 53 samples were fixed in AZF and demineralized with hydrochloric acid for 1-hour, formic acid for 12 hours, or ethylenediamine tetraacetic acid (EDTA) for 24 hours. Histologic sections were scored by 4 raters as of insufficient, marginal, good, or excellent quality. In addition, DNA samples extracted from sections treated with the different fixation and demineralization methods were amplified with 3 sets of primers to conserved regions of T cell receptor gamma and immunoglobulin heavy chain genes. Amplification efficiency was graded based on review of capillary electrophoretograms. There was no significant difference in the histomorphology scores of sections fixed in AZF or NBF. However, EDTA-based demineralization yielded higher histomorphology scores than demineralization with hydrochloric or formic acid, whereas formic acid resulted in higher scores than hydrochloric acid. Demineralization with EDTA yielded DNA amplification in 29 of 36 (81%) samples, whereas demineralization with either acid yielded amplification in only 2 of 72 (3%) samples. Although slightly more time-consuming and labor-intensive, tissue demineralization with EDTA results in superior morphology and is critical for polymerase chain reaction (PCR) amplification with the DNA extraction method described in this article.

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来源期刊
Veterinary Pathology
Veterinary Pathology 农林科学-病理学
CiteScore
4.70
自引率
8.30%
发文量
99
审稿时长
2 months
期刊介绍: Veterinary Pathology (VET) is the premier international publication of basic and applied research involving domestic, laboratory, wildlife, marine and zoo animals, and poultry. Bridging the divide between natural and experimental diseases, the journal details the diagnostic investigations of diseases of animals; reports experimental studies on mechanisms of specific processes; provides unique insights into animal models of human disease; and presents studies on environmental and pharmaceutical hazards.
期刊最新文献
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