What is Toluidine Blue (TB) Dye?
TB dye is a basic thiazine metachromatic dye, which has a high affinity for acidic tissue components, thereby staining tissues rich in DNA and RNA. The epithelium of the external genitalia does not have nucleated cells and prevents contact of stain with nuclei. Where the epithelium is damaged and the underlying nucleated cells are exposed, the nuclei stain blue.
Does TB Dye interfere with evidence collection?
Toluidine Blue stain is easy and safe to use, available, inexpensive and does not interfere with medico-legal evidence. There are no adverse effects on DNA detection and identification with the use of toluidine blue.
Where do most injuries occur?
The most likely structures to be injured during genital penetration are the posterior fourchette, the fossa navicularis, the hymen and the labia minora. The genital and anal injuries generally found during the clinical exam are subtle, shallow and do not typically show blood on the surface. Peri-anal injuries are generally in the form of multiple radial tears of the perianal area inside and outside the midline and extending into the anus. The color of the normal tissue in these structures are typically similar to that of injured tissue. Injuries from genital penetration show a distinctive distribution. Tears appeared most often on the posterior fourchette and fossa; abrasions appear on the labia minora.
Is TB Dye safe?
It is safe for use on humans. Although a slight stinging sensation may occur, it does not have any long-term adverse effects and it does not interfere with biological evidence.
Can you have injuries with consensual intercourse?
According to a study by Slaughter, it found that injuries due to cooperative, consensual genital penetration are generally limited to only one small injury to one genital anatomical site. If more than one genital anatomical site is injured, the likelihood that the penetration took place with consent and cooperation decreases.
Increased detection of injury with TB dye
There is very little research regarding the use of TB dye in forensics to detect injury, but one study by McCauley found that the detection of vaginal lacerations increased from 1 in 24 to 14 in 24 in reported adult sexual assaultl cases when TB dye was used. In another study by Lauber and Souma, injuries were detected in 1 out of 22 patients after consensual intercourse, but after examined by TB dye within 48 hours after reported rape, 70% of nulliparous females and 40% of the total number of patients had positive TB dye uptake. In Slaughter and her co-workers study, genital injuries were found in 11% of females after consensual intercourse and 89% after sexual assault.
False-Positives
TB dye stains all exposed nucleated cells and yields positive results in vulvitis, herpes and other ulceration, self-inflicted injury (like scratching), dermatological conditions and more. In cases where the cause of the disruption is not clear, the history should be used to suggest a differential diagnosis, which should be evaluated as indicated. This is also why we do the speculum exam after applying TB dye, as the speculum can cause small injuries to the tissue from insertion.
False-Negatives
Injuries may be missed if there is a barrier between the tissue and the stain like lubricating gel, or oozing. Healing can also interfere with the staining of injuries. Experience shows that injuries may still be highlighted more than 48 hours after injury occurred despite 48 hours being the cut-off in previous studies.
Pointers to distinguish between positive findings due to penetration vs. other causes
The distribution and nature of the damaged area differs between the different causes of epithelial damage.
In herpetic and other ulcerative conditions, the stain will show up in the typical minor breaks in the muco-cutaneous epithelium.
Vulvitis presents as a patchy uptake of dye and is unlikely to be symmetrical.
Self-inflicted injury due to scratching may be present in the presence of a history of itching. It will unlikely present as single visible abrasions and will have an irregular appearance. Dermatological conditions or infection causing itching should be excluded.
The distribution of tears is linear and generally occurs at the fossa navicularis and the posterior fourchette in the area behind the 3-9 o'clock location of the genitalia.
Abrasion due to genital penetration appears as a diffuse blue area in a symmetrical distribution.
Generally, genital injuries due to genital penetration of the female are located between 5 o'clock and 7 o'clock on the fossa navicularis and the posterior fourchette.
Problems with TB Dye
When applied poorly, it is a concern as injury may be overcalled because all of the pooling of dye is not recognized as such. TB dye should be used like a highlighter, not a searchlight.
Court Considerations
The presence of anogenital injuries in sexual assault cases is of major importance in court hearings. The general perception is that rape should result in injuries. Since prosecutors only prosecute rape cases when they expect a conviction, they may be reluctant to prosecute cases where there are no injuries. With the use of TB dye, injuries are demonstrated more often in victims. The defense is generally that the sexual act was consensual and the regular question is, "Can you rule out consensual intercourse on grounds of the medical examination?" You have to concede that the scenario is possible regardless of the clinical picture. What the prosecutor needs in order to reason with authority is the additional remark "yes, it is possible, but in this case it is unlikely since I found injuries of more anatomical sites than I would expect from consensual intercourse", for example.
Rambow and colleagues reported on 182 sexual assault cases, 53 of which had the potential for successful prosecution with a victim willing to testify and an identified assailant. They found that 34% of the cases resulted in a successful prosecution, and evidence of genital or non-genital injury were associated with a successful legal outcome.
Process for applying TB dye
Collect all of the external genital specimens as indicated by examination before application of the dye.
Before speculum examination, apply 1% toluidine blue to the vulva. The anus may also be stained. Do not use the dye in the vaginal vault or mucous membranes (avoid the hymen). Toluidine blue dye may be used on labia majora, labia minora, posterior fourchette, fossa navicularis, perineal body, and perianal area. (Focus on the areas between 3 o'clock and 9 o'clock as this is where most injuries occur if patient was in supine position during assault).
Allow to dry for a 30-60 seconds.
Remove excess dye with a water-soluble lubricant jelly by gently blotting the area until the excess dye is removed. This may take several gauze pads and lubricant.
DO NOT RUB the area.
Photograph the area if indicated (and consent obtained).
The dye will fade in 1 to 2 days.
TB dye History and other uses
Toluidine blue has been known for various medical applications since its discovery by William Henry Perkin in 1856, after which it was primarily used by the dye industry. There are two techniques of vital staining, namely, intravital staining in the living body (in vivo) and supravital staining outside the body usually applied to slide preparation of detached cell. TB was first applied for in vivo staining by Reichart in 1963 for uterine cervical carcinoma in situ6. During 1960s suggestion was made that TB may stain malignant epithelia of the mucous membrane in vivo, whereas normal tissue failed to retain the dye.
Vital staining of the oral epithelium has been suggested as a means of surveillance in patients who are at a risk of developing oral cancer and for those who had confirmed neoplasms of other parts of aerodigestive tract. TB has been used as a vital stain to highlight potentially malignant oral lesions and may identify early lesions, which could be missed out on clinical examination. It is useful in obtaining the marginal control of carcinoma and in selecting the biopsy sample site in premalignant lesions.
The dye is sometimes used by surgeons to help highlight areas of mucosal dysplasia (which preferentially take up the dye compared to normal tissue) in premalignant lesions (e.g. leukoplakia). This can be used to choose the best site of the lesion to biopsy, or during surgery to remove the lesion to decide whether to remove more tissue from the margins of the excision defect or leave it behind. It is also commonly used to identify mast calls.
Resources
Kotze JM, Brits H (2018). Do we miss half of the injuries sustained during rape because we cannot see them? An overview of the use of toluidine blue tissue stain in the medical assessment of rape cases, South African Family Practice, doi:10.1080/20786190.2017.1386868
Slaughter L, Brown CR, Crowley S, et al. Patterns of genital injury in female sexual assault victims. Am J Obstet Gynecol. 1997; doi: 10.1016/S0002-9378(97)7-556-8.
Jones, et al. (2004). Significance of toluidine blue positive findings after speculum exam for sexual assault. Retrieved at www.ncbi.nlm.nih.gov/pubmed/15138957
Sommers, M. (2007). Defining Patterns of Genital Injury From Sexual Assault. Retrieved from www.ncbi.nlm.nih.gov/pmc/articles/PMC3142744/...
Sachs, C., Wheeler, M. (2015). Examination of the Sexual Assault Victim, Emergency Medicine. Retrieved from clinicalgate.com/
Sridharan, G., & Shankar, A. A. (2012). Toluidine blue: A review of its chemistry and clinical utility. Journal of oral and maxillofacial pathology : JOMFP, 16(2), 251–255. doi:10.4103/0973-029X.99081
Great information Christine. One thing to add is that it can be beneficial to take a photo of where the dye was applied prior to removal so that during peer review, the reviewer has a better idea of where to assess for uptake. I appreciate you posting this.