The Analysis of Mascara from Eyelashes
Written by Kristi Mayo   

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WHEN AN EYELASH dropped onto her microscope’s stage, Kristen Wiley did what any microscopist would do: she gave it a thorough examination.

Wiley is a senior research microscopist at McCrone Associates (the analytical division of the McCrone Group) in Westmont, Illinois, and an instructor in sample preparation, isolation, and manipulation at the Hooke College of Applied Sciences (the educational division of the McCrone Group).

“As I was looking at it, I realized there was quite the abundance of mascara on my eyelash,” said Wiley. “With that, I started picking at the mascara and realized that I could come up with different techniques to remove the mascara from the eyelash.”

She began to play with a relatively simple concept: Trace-evidence examiners could pair the availability of trace eyelashes at a scene with a tendency toward brand loyalty among mascara users in order to link a victim or suspect to a crime.

Deciding to move forward with this concept, Wiley then asked two of her lab colleagues to donate their own mascara-coated eyelashes. She removed the mascara from all three eyelashes and mounted each sample for examination under a polarized light microscope (PLM); each sample was also prepared for analysis with Fourier transform infrared spectroscopy (FTIR).

The FTIR spectra revealed detectible differences between the three samples, so Wiley took the next step and prepared samples from each of the eyelash donors’ personal tubes of mascara. “At that time, I gave the samples to the IR operator blindly and asked him if he could match the samples from the tubes with the three that I originally gave him,” Wiley said, “and he had no problem with matching them up.”

Eyelashes: Voted least likely to succeed?

Among the various kinds of hair evidence, head hair and pubic hair hold the most potential to yield enough nuclear DNA or mtDNA for analysis. Because of its diminutive size, the eyelash is likely to be ignored by trace-evidence examiners and DNA analysts.

“I am certain that if one looks closely, they would find things like eyelashes on pillows and bed clothing and people’s clothing, particularly in assault situations,” said Richard Bisbing, executive vice president and director of services at McCrone Associates, and an instructor at the Hooke College of Applied Sciences. “But the hair examiners normally would ignore them, because they are not useful for comparison—and most of the time they are not sufficient for doing DNA analysis. They are just very small.”

As Wiley’s initial work with eyelashes and mascara has shown, however, these little bits of hair could provide an investigation with good comparative evidence.

“Rather than summarily ignoring the little hairs, I would suggest that investigators and forensic scientists keep in mind the potential here,” said Bisbing. “This is something that the microscopist in the crime laboratory can learn to do. It is not rocket science. It only requires knowing how to do it, practice, and the infrared spectroscopy that most laboratories can do.”

Developing techniques for analyzing and removing mascara

The first step in analyzing the mascara on an eyelash begins under the microscope, where the mascara is carefully scraped off the hair. To remove the mascara, Wiley utilizes fine, one-time-use tungsten needles that are made in-house at McCrone Associates. The needles are made exothermically with sodium nitrate or electrolytically with KOH solution. “Both procedures are controlled, so depending on the application in which the needle is going to be used, you can alter the tip size as you see fit,” said Wiley. For this procedure, Wiley uses a tungsten needle with a 5- to 10-micron tip.

Using a stereo microscope to visualize the procedure, the tungsten needle is held at a low angle and dragged across the length of the eyelash. “You are not using the tip of the needle,” Wiley clarified, “you are using the side of the needle.”

If there is enough mascara on the eyelash, it will transfer to the needle and the sample can then be mounted on a different substrate for analysis, such as a glass slide or a KBr crystal.

Sometimes, only very small amounts of mascara may be present on the eyelash, and it may not be possible to remove very much with the tungsten needle. In this case—or, in cases where mascara is smeared on a pillowcase, for example—Wiley has found that a water-soluble adhesive can be very useful in recovering enough material for analysis.

“The water-soluble quality of the adhesive is important because, if you want to do any DNA work on the sample, the water-soluble adhesive does not interfere with the PCR profile or the buffer solutions for the PCR,” explained Wiley.

She said McCrone Associates tested literally hundreds of adhesives that could be used for this purpose, and finally arrived at a product manufactured by 3M called Water Soluble Wave Solder Tape.

The use of the adhesive would be particularly applicable in a situation where, under closer examination, the analyst discovered blood on the hair. “All you really need to do is touch that area with the water-soluble adhesive and then put that into the PCR tube,” said Wiley. “The DNA laboratory that works with us has been successful in getting a profile on that.”

Understanding the nature of mascara

In 2005, Michigan State University graduate student JoAnn I. Bilek worked with advisor Dr. Jay Siegel on the forensic analysis of black mascara. In the study, Bilek analyzed 24 black mascara samples utilizing four analysis techniques: traditional light microscopy; Fourier transform infrared spectroscopy (FTIR); scanning electron microscopy-energy disper-sive spectroscopy (SEM-EDS); and visible microspectrophotometry (MSP). The objective was to see if mascara formulations differed enough to be of forensic value. Bilek’s study did not investigate the collection of mascara from eyelashes, fabrics, or other materials.

As a very general summary, the study found:

  • Traditional light microscopy allowed the 24 mascara samples to be separated into four groups.
  • FTIR separated the samples into 22 groups.
  • SEM-EDS “excluded and distinguished between 99.67% of the 600 comparisons based on calculated weight percentage ratios.”
  • And MSP “did not yield any discriminatory data for the mascara samples in the study.”

Siegel, who is now director of the Forensic & Investigative Science Program at Indiana University-Purdue University Indianapolis, said that while he has not done any additional work on mascaras since advising Bilek’s study, the concept does deserve the attention of crime-scene investigators and trace-evidence examiners.

“Suppose this was a real case where an eyelash with mascara was found at a sexual-assault scene, and you wanted to find out if the source could have been this particular bottle of mascara,” said Siegel. “So, you do FTIR—or perhaps Raman these days would be better—and you get what we will call a match for now: two spectra that seem to have all of the same major peaks and no unexplainable differences. Then you could say, Well, this bottle could have been the source of that mascara.

“I think that could be done, and I think that’s very valuable… as long as you keep in mind that you will never be able to drill down any further and say that it definitely came from that particular bottle.”

Siegel warned that there are a number of factors that need to be taken into consideration when working with not just mascara, but also any cosmetic. In particular, one must understand the nature of the cosmetics industry.

“I did a lot of work in lipsticks back when I was at Michigan State University, and also some work with face powders,” said Siegel. “We found—especially with lipsticks—that the manufacturers change these darn things almost every day. It’s really hard to keep up with lipstick technology. So, you build what you think is a nice database of different kinds of lipsticks, and then you go to a drugstore three months later and the whole thing is out the window because a lot have been discontinued and new ones have come out.”

Also, the formula of mascaras may differ significantly—even within bottles bearing the same label. And mascaras with different labels may have very similar formulas.

“In some cases, the companies treat this stuff like a commodity,” said Siegel. “They go out on the market and buy gallons of mascara and then they put it in their mascara containers and sell them. You see this a lot in inks, too: What’s in a Bic pen might not be Bic ink at all. Someone just went out on the marketplace and bought 50,000 gallons of some blue ink and they stuff it in their Bic pens; then next week they will get 50,000 gallons of ink from somebody else and stuff it in the same kind of pen.

“So, whenever you have a commodity, this always causes these issues of continuity from one container to another, even with the same brand and material.”

Consideration should also be given to the chemistry of mascara. In her thesis, Bilek found that mascaras can be separated into three groups: water-based, solvent-based, and water/solvent hybrid-based. These bases keep the mascara in semi-liquid form so that it can easily be coated onto the eyelashes. Once applied, the water or solvent evaporates—and as a result, noted Siegel, the mascara’s chemical characteristics change.

“The known sample needs to duplicate the unknown as much as possible,” said Siegel. “This doesn’t eliminate, but it does minimize, differences that may occur with evaporation or with reactions to whatever the matrix is.”

Bilek’s work with mascara included a drying study. As a result of that study, the thesis recommended a two-day drying period for samples taken from a known source.

One final issue to consider, said Siegel, is the presence of other materials on the eyelash. “If there is some other material on the hair and you scrape that off, too, you can get a false negative,” said Siegel. “So that would be one other concern.”

Learning to look beyond DNA

While the comparison of mascaras may not provide the specific “match” that one would hope to get with DNA analysis, it does provide a potential piece to a puzzle when DNA cannot help the investigation.

“I appreciate the value of DNA typing. It has revolutionized forensic science,” said Siegel. “But it is not applicable in all cases. So, where you have just the trace evidence, then you need to go back and start focusing on the chemistry again.”

Wiley emphasized the importance of trace-evidence examiners working closely with forensic scientists in the DNA laboratory to be certain that important bits of evidence—such as mascara, blood, or other materials that could be found on the hair itself—are not ignored.

“I think that collaboration between the trace groups in the forensic lab and the DNA people needs to be open-ended,” said Wiley. “When teaching, I have heard from quite a few students—who are working in labs as the initial trace-evidence examiners—that they are simply tasked with, Is it a hair or it not a hair? Is there a root or is there not a root? And that’s it.

“But if they were to actually look closer at the hair and check to see if they are potentially missing any of this ‘micro-trace’ evidence that can be on the hair itself—then communicating that to the DNA people could be very beneficial.”

Siegel agreed. “The tendency is to ignore the trace evidence and concentrate on the DNA,” he said. “Crime-scene investigators are doing this more and more: they are looking for evidence with DNA on it, and they ignore the rest.

“But scraping mascara off an eyelash and comparing it with mascara from a bottle that belongs to a victim or suspect: that’s a good test. It adds information. And a good comparison between the two says, ‘This could be the source of that mascara. It might not be, but it certainly could be, and therefore it is probative evidence.’ It is good evidence.

“There is no such thing as too much evidence,” Siegel concluded, “as long as it is interpreted properly.”

Additional Reading

Bilek, JoAnn I. (2005). Forensic analysis of black mascara via microscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS), and visible microspectrophotometry (MSP) (Master’s thesis). Available online from ProQuest Dissertations & Theses database.

About the Author

Kristi Mayo is the editor of Evidence Technology Magazine.

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