Effects of fixation and demineralization on histomorphology and DNA amplification of canine bone marrow

Abstract

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.

Keywords: EDTA; PARR; bone; clonality; decalcification; dog; formalin; formic acid; hematopoietic tissue; hydrochloric acid; lymphocyte antigen receptor genes.

Figure 1.

The mean ± standard deviation of histomorphology scores for 20 bone marrow samples fixed with acid-zinc-formalin (AZF) and neutral-buffered formalin (NBF) were (a) not significantly different (P = .566) from each other and (b) significantly correlated with each other (P < .001, r = 0.730).

Figure 2.

Histomorphology of sternal bone marrow sections from a dog. Histomorphology scores did not differ significantly between samples fixed with the different fixatives. All samples were decalcified with formic acid and fixed with (a, c, e) acid-zinc-formalin or (b, d, f) neutral-buffered formalin. Hematoxylin and eosin.

Figure 3.

The mean histomorphology scores of 20 bone marrow samples prepared with 2 different fixatives were not significantly correlated with the time-after-death. AZF, acid-zinc-formalin; NBF, neutral-buffered formalin.

Figure 4.

The mean ± standard deviation histomorphology scores for 53 bone marrow samples decalcified with hydrochloric acid (HCl), formic acid (FA), or EDTA. HCl vs FA, P = .001; HCl vs EDTA P < .001, FA vs EDTA P < .001.

Figure 5.

Histomorphology of sternal bone marrow sections from a dog. (a, b) Samples decalcified with hydrochloric acid (HCl) showed trabecular bone fragmentation, while (e, f) samples decalcified with EDTA had better bone and hematopoietic tissue preservation. All samples were fixed with acid-zinc-formalin and decalcified with (a, b) HCl, (c, d) formic acid, or (e, f) EDTA. Hematoxylin and eosin.

Figure 6.

Amplification scores for bone marrow samples fixed with acid-zinc-formalin (AZF) or neutral-buffered formalin (NBF), decalcified with hydrochloric acid (HCl), formic acid (FA), or EDTA, and amplified with primers targeting (a) conserved regions of the T cell receptor gamma (TRG) and (b, c) the immunoglobulin heavy chain (IgH) genes. (b) IgH framework 2, FR2. (c) IgH framework 3, FR3. Amplification scores of samples decalcified with EDTA were significantly higher than those of samples decalcified with HCl or FA (P < .001).