Effects of Histological Staining on the Analysis of Human DNA from Archived Slides*

Abstract

Archived slides of cell smears treated with histological stains for sperm detection are often the only source of DNA available when cold cases are reopened. There have been conflicting reports as to the negative effects of particular histological stains on DNA recovery and quality from human cells, making stain selection an important consideration for forensic laboratories. This study investigates the effect of several staining systems on DNA recovery from histological slide samples stored from 0 to 10 weeks. DNA profiles obtained after analysis of these samples with AmpFlSTR(®) Identifiler™ and increased cycle AmpFlSTR(®) SGM Plus™ short tandem repeat (STR) profiling systems and the effects that these stains have on DNA quantity and quality over time are described. Results indicate that Christmas Tree and Hematoxylin and Eosin stains do not have significantly different effects on DNA quality after 10-week storage of slides. This research will assist scientists to select staining systems that have minimal deleterious effects on the DNA recovered.

Cold Case Homicides: DNA Testing of Retained Autopsy Sexual Assault Smears

“…There have been conflicting results as to whether particular histological stains are better or worse for DNA testing thus making stain selection an important consideration for forensic laboratories (17,18). One study found that Christmas Tree and Hematoxylin and Eosin stains do not have different effects on DNA quality after 10-week storage of slides (17). Sanders et al used a modified H&E staining method with reduced exposure times, in conjunction with laser microdissection and STR analysis and demonstrated adequate sperm identification and improved PCR product acquisition compared to other evaluated staining protocols (19).…”

An easy, reproducible and cost-effective method for andrologists to improve the laboratory diagnosis of non-obstructive azoospermia: a novel microcentrifugation technique

“…The NF-PICS or Christmas Tree stain is one of the most widely used for histological tests for the identification of sperm in sexual assault cases ( 10 ). With this stain it is possible to color both the post-acrosomal region of the sperm head, that stains pink, and the acrosome itself, that stains light pink, while cell debris are colored green, allowing easy differentiation of spermatozoa from cellular debris and thus facilitating the reading of the slide.…”

“…This method of diluting the specimen is preferable to using a premixed dilution and minimizes cell-sampling errors, and should be used on specimens in which “no spermatozoa” were detected in a wet preparation. After centrifugation the cells are deposited in a uniform monolayer in a compact area (32mm 2 ) and even with the use of stains such as nuclear fast red and picroindigocarmine (described as NF-PICS or Christmas Tree stain; Sigma-Aldrich, St. Louis MO, USA) ( 9 , 10 ), is not always easy to identify isolated sperm heads owing to the large amount of cellular debris. Therefore, our group has developed a simple and cost-effective technique as an alternative to the Cytospin method: the microcentrifugation technique to confirm azoospermia (Labnet, Woodbridge NJ, USA).…”

Simple Protocol for DNA Extraction from Archival Stained FNA Smears, Cytospins, and Thin Preparations

“…The DNA damaging effect of hematoxylin has been described by some authors [3,4,5]. However, more recent studies describe no significant differences between the quality of DNA obtained from slides stained with hematoxylin versus unstained material [6,7], but these latter studies do not specifically look at samples that have been archived for long periods of time. Although additional studies are warranted to investigate the variation among tissue stains in the preservation of DNA in archived material, we have observed that, despite lower quality, testing can be successfully performed in most cases, regardless of the stain used.…”