2_analysis in body fluids.pdf

2. Composition of body uids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Forensic Science International 188 (2009) 117

Article history:

Received 16 December 2008

Received in revised form 2 February 2009

Body uid traces recovered at crime scenes are among the most important types of evidence to forensic

investigators. They contain valuable DNA evidence which can identify a suspect or victim as well as

exonerate an innocent individual. The rst step of identifying a particular body uid is highly important

Contents lists available at ScienceDirect

Forensic Science International3. Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3.1. Current techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3.1.1. Presumptive tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3.1.2. Conrmatory tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

3.2. Emerging techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

4. Semen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7


4.1.1. Presumptive tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

4.1.2. Conrmatory tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

4.2. Emerging techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

5. Saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9


5.2. Emerging techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

* Corresponding author. Tel.: +1 518 591 8863.

E-mail address: [email protected] (I.K. Lednev).

0379-0738/$ see front matter 2009 Elsevier Ireland Ltd. All rights reserved.Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Accepted 8 February 2009

Available online 27 March 2009

Keywords:

Body uid identication

Forensic biology

Non-destructive analysis

Raman spectroscopy

Biospectroscopy

Portable spectrometer

since the nature of the uid is itself very informative to the investigation, and the destructive nature of a

screening test must be considered when only a small amount of material is available. The ability to

characterize an unknown stain at the scene of the crime without having to wait for results from a

laboratory is another very critical step in the development of forensic body uid analysis. Driven by the

importance for forensic applications, body uid identicationmethods have been extensively developed

in recent years. The systematic analysis of these new developments is vital for forensic investigators to

be continuously educated on possible superior techniques. Signicant advances in laser technology and

the development of novel light detectors have dramatically improved spectroscopic methods for

molecular characterization over the last decade. The application of this novel biospectroscopy for

forensic purposes opens new and exciting opportunities for the development of on-eld, non-destructive,

conrmatory methods for body uid identication at a crime scene. In addition, the biospectroscopy

methods are universally applicable to all body uids unlike themajority of current techniques which are

valid for individual uids only. This article analyzes the current methods being used to identify body

uid stains including blood, semen, saliva, vaginal uid, urine, and sweat, and also focuses on new

techniques that have been developed in the last 56 years. In addition, the potential of new

biospectroscopic techniques based on Raman and uorescence spectroscopy is evaluated for rapid,

conrmatory, non-destructive identication of a body uid at a crime scene.

2009 Elsevier Ireland Ltd. All rights reserved.Review

Analysis of body uids for forensic purposes: From laboratory testingto non-destructive rapid conrmatory identication at a crime scene

Kelly Virkler, Igor K. Lednev *

Department of Chemistry, University at Albany, SUNY, 1400 Washington Avenue, Albany, NY 12222, United States

A R T I C L E I N F O A B S T R A C T

journa l homepage: www.e lsevier .com/ locate / forsc i in tdoi:10.1016/j.forsciint.2009.02.013

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destructive methods at the crime scene. The most important and it concludes with the discussion of new biospectroscopy

K. Virkler, I.K. Lednev / Forensic Science International 188 (2009) 1172reason for these tests to be non-destructive is the preservation ofDNA evidence. Body uids such as blood, semen, saliva, vaginaluid, urine, and sweat all contain DNA evidence so it is imperativeto develop identication tests that will protect this valuable data[1]. Another disadvantage of most of these current methods is thatthey are designed to detect a specic body uid, so the investigatorneeds to decide which test to perform based on the uid that ismost likely present. There is a need for a universal conrmatorytest that can be applied to an unknown stain which will be able toidentify any of the body uids that might be present.

Manyof thecommontechniquesused to identifyparticularuidshave been around for decades. Some of these techniques havechanged very little such as the luminol [2] and crystal tests [3] forblood and the microscopic identication of spermatozoa to conrmthe presence of semen [4]. Others such as the presumptive tests toidentify heme in blood, acid phosphatase in semen, and amylase in

methods under development that offer non-destructive conrma-tory identication of body uid traces immediately at the crimescene [15,16]. It is important to emphasize that these newbiospectroscopy techniques are still being developed and are notyet available. We believe that with further testing, these novelmethods will be able to deliver results in a simple and automaticfashion at a crime scene that will be acceptable for court testimony.For specic and in-depth details about a particular test, includingformer tests, tests currently in use, and newly developed tests, it isbest to consult the individual articles and books referencedthroughout the review.

The review is organized as follows:

A description of the composition of each body uid and how thedifferent biological components found in each uid inuencecurrent identication methods.6. Vaginal uid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.1. Current techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.2. Emerging techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7. Urine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .



8. Sweat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .



9. Non-destructive conrmatory identication of body uids. . . . . . .

9.1. Fluorescence spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . .

9.2. Raman spectroscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10. Future developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction

The detection and identication of body uids at a crime sceneare very important aspects of forensic science. Determiningwhether or not there is a body uid present and subsequentlyidentifying it allows the sample to undergo further laboratorytesting including DNA analysis which is a very crucial step in awide range of investigations. Sometimes just knowing the identityof a uid can be enough to inuence the outcome of a case. This isnot always an easy task, however, since many body uid stains areeither invisible to the naked eye or similar in appearance to otheruids or substances. Even when the identity of a stain may seemobvious to a forensic investigator, absolute conrmation isnecessary in order for the evidence to be used in court to eitherprove or disprove a fact in a case. This is especially important withthe possible occurrence of mixtures. A stain could containmultiplebody uids frommore than one donor. Physical tests performed onthese questioned stains allow crime scene investigators andlaboratory technicians to identify a uid or to conrm the absenceof one which can be of equal value in a case. The most commonbody uids found at crime scenes are blood, semen, and saliva, butothers such as vaginal uid, urine, and sweat can also playimportant roles including the contribution of valuable DNAevidence. Each of these uids has one or more screening teststhat are presumptive in nature, and some of them haveconrmatory tests that will conclusively identify their presence.There are also some tests which can identify the species of aparticular uid, and these are also considered to be conrmatory.

The main problem with these tests is the destruction of thesample. Sometimes a case can be broken with just the smallestamount of biological evidence, so it is crucial that these smallquantities are examined as efciently as possible by non-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

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saliva have evolved over the years due to advances in technology,better understanding of the nature of the uids, or even to preventexposure to hazardous chemicals. A few new methods have beendiscovered, and the majority of these involve the detection ofspecic messenger ribonucleic acid (mRNA) markers to identifydifferent bodyuids [57].Over time theywill possibly beexpandedupon and become more accepted by the forensic community.

An extensive and thorough book that describes the knownidentication tests for body uids up to the year 1983 is Sourcebookin Forensic Serology, Immunology, and Biochemistry by R.E. Gaensslen[8]. It is a very in-depth analysis of thevariousmethods studiedup tothat point in time. In the years sinceGaensslens publication, severalother book chapters have summarized the identication of bodyuids. These include Spaldings [9] and Greenelds [10] chapters inForensic Science: An Introduction to Scientic and Investigative

Techniques in 2003, Shalers [11] and Jones [12] work in ForensicScience Handbook Vol. II (2002) and Vol. III (2005), respectively,Watsons 2004 chapter [13] in Crime Scene to Court; The Essentials ofForensic Science, and most recently portions of Lis book ForensicBiology [14]whichwas released in2008. Thesebooks summarize thewell accepted techniques, and Li mentions some of the new mRNAmethods in his chapters. The above mentioned books and chaptersdescribe in detail the presumptive and conrmatory methods ofidentication that are currently being used in forensic laboratories,with the exception of Lis book, which also discusses the morerecently developed mRNA techniques.

The following review briey summarizes all current and formermethods of body uid identication and focuses on the newdevelopments in forensic science during the last 56 years. Itdiscusses both signicant improvements in conventional bioana-lytical methods and developments of novel approaches. The reviewevaluates the advantages and disadvantages eachmethod presents,. . .

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Summary of current and previous techniques used either at thecrime scene or in the forensic laboratory. The body uidsdiscussed will include blood, semen, saliva, vaginal uid, urine,and sweat. Each uid will be reviewed individually in that order,and the tests for that uid will be broken down into presumptiveand conrmatory tests when applicable.

Discussion of new methods of identication that have beenpublished in the last 6 years. These methods have not beensubstantially reviewed by other sources. Again, each uid will becovered individually, and this section on new techniques will fallat the end of each individual uid section.

Introduction of new non-destructive, conrmatory techniquesbased on uorescence and Raman spectroscopies, which areapplicable to all body uids and their dry traces. The potential ofthese spectroscopic methods for forensic applications, speci-

attention to a latent stain at a crime scene, and then furtherpresumptive tests can be utilized to form more conclusions aboutany body uids that are present. A versatile light source productknown as Polilight1 contains a range of wavelengths and can evenreveal stains covered by paint [19]. These light sources must beused with caution, however, since certain ultraviolet wavelengthscan damage the DNA evidence in a sample. One study found thatexposure of 30 s ormore to 255 nm light damaged the DNA enoughthat none was detected during polymerase chain reaction shorttandem repeat (PCR-STR) quantication and amplication [20].Another experiment found that restriction fragment lengthpolymorphism (RFLP) patterns only weakened and were notfalsiedwith exposure to UV light up to 5 days, but thewavelengthused was not specied [21].

The luminol test is one of the rst presumptive blood tests that

agin

-H Acid

-F Lacti

-E Citri

-A Urea

Vagi

Glyc

Acet

Pyrid

Squa

Imm

K. Virkler, I.K. Lednev / Forensic Science International 188 (2009) 117 3mmunoglobulins -Semenogelin -Potassium -

-Zinc -Bicarbonate -

-Citric acid -Phosphorus -

-Lactic acid -Glucose -

-Fructose -Immunoglobulins -

-Urea

-Ascorbic acid

-Immunoglobulins-Glu

-Iemoglobin -Acid phosphatase -Amylase -

ibrinogen -Prostate-specic antigen -Lysozyme -

rythrocytes -Spermatozoa -Mucin -

lbumin -Choline -Buccal epithelial cells -

cose -Spermine -Thiocyanate -Blocally for identication of body uid traces on-eld at a crimescene, will be discussed.

2. Composition of body uids

Each body uid has a unique composition, and the presence ofspecic components in one uid versus another is the basis of itsidentication. Table 1 shows the major components of blood,semen, saliva, vaginal uid, urine, and sweat [8,10,14,17,18]. Thereare several components that are common among more than oneuid, but it is the difference in relative contribution which makestests for these components effective. One example is the largeamount of amylase in saliva compared to the smaller amounts insemen and vaginal uid. Another example is the ratio of citrate tolactate when comparing semen and vaginal uid. Urea is acomponent in urine, semen, and sweat, but it is used as an indicatorof urine based on the much higher concentration in that uid.

3. Blood

3.1. Current techniques

Blood is the most common body uid encountered at crimescenes. There are several presumptive tests to identify blood aswell as conrmatory tests. The following two sections explaintechniques that are well known in the forensic community as wellas some variations to thesemethodswhich are not currently in use.Table 2 summarizes all of the techniques for blood and the otherbody uids.

3.1.1. Presumptive tests

The simplest test that crime scene investigators use to detectbloodstains that are not clearly visible is an alternate light source(ALS) such as ultraviolet light. This method is especially helpfulwhen the stain is on a dark background [11]. An ALS can direct

Table 1Composition of body uids.

od [17] Semen [17] Saliva [17] Vinvestigators often use at a crime scene, and it has been around forover 40 years [22]. It is based on the ability of hemoglobin andderivatives in blood to enhance the oxidation of luminol in thepresence of an alkaline solution and involves spraying a suspectedarea with an aqueous solution of luminol and an oxidant [8,9]. It isknown to be the most sensitive of the current presumptive testsbeing used [23], and there are also several formulations availablethat have advantages and disadvantages regarding sensitivity,intensity and duration of illumination, and effect on subsequentDNA analysis [24,25]. It can even be used on an area that has beencleaned by a suspect [11]. One study found that a certain popularform of the luminol test known as the Grodsky formulation canhave detrimental effects on subsequent DNA analysis whencompared to the Weber, Weber II, and Bluestar1 alternatives[24]. The luminol test remains popular due to the lack of falsepositives and false negatives in comparison with other screeningtests as well as the fact that luminol is not as hazardous as otherreagents [26]. However, it is limited to use in dark environments[14]. A similar, less popular uorescence technique involvinguorescein depends on heme accelerating the oxidation ofuorescin to uorescein in hydrogen peroxide [8,9]. Studies haveshown that it is just as effective as luminol as a presumptive test forblood and also will not damage potential DNA evidence [27].However, unlike luminol which will emit light on its own,uorescein-sprayed stains need to be exposed to an ALS with awavelength range of 425485 nm [14]. Another technique basedon chemiluminescence that gives positive results without dama-ging the DNA in a sample is Bluestar1 [28], and studies have evenshown it to be more sensitive and stable when compared toluminol [25].

There are several different catalytic tests commonly used toidentify presumptively blood based on the peroxidase-like activityof the heme group [9]. The most utilized of these tests used to bethe benzidine test. A positive result yields a blue color when bloodreacts with the ethanol/acetic acid solution [8,9]. There are several

al uid [14,18] Urine [10,17] Sweat [8,17]

phosphatase -Urea -Urea

c acid -Creatinine -Lactic acid

c acid -Uric acid -Chloride

-Chlorine -Sodium

nal peptidase -Tamm-Horsfall glycoprotein -Potassium

ogenated epithelial cells -Immunoglobulins

ic acid

ine

lene

unoglobulins

cent

hic

l

l

l

l

cent

s

hic

K. Virkler, I.K. Lednev / Forensic Science International 188 (2009) 1174Table 2Summary of all current or older testsa.

Body uid Component Classication

Blood Whole uid ALSHemoglobin Chemilumines

Chemical

Crystal test

SpectroscopicChromatograp

Elements SpectroscopicIsozymes Immunologica

Antibodies Immunologica

Immunologica

Semen Whole uid ALSEnzymes Chemical

Immunologica

Choline Crystal test

Chemical

Chemilumines

Electrophoresi

Chromatograp

Spermine Electophoresisfalse positives for this test such as chemical oxidants and fruit/vegetable peroxidases [8,29], but its main disadvantage is thatbenzidine is a known carcinogen, and this has led to itsreplacement as a presumptive reagent in the forensic laboratory[9,11,29].

A very popular presumptive catalytic method is the phe-nolphthalein test which is also known as the Kastle-Meyer test.Phenolphthalein will cause an alkaline solution to turn pink afterits oxidation by peroxidewhen blood is present [9]. It can also havefalse positives similar to those of benzidine, but tests on other bodyuids do not yield a positive result [8], and it is not carcinogenic.Although it is not as sensitive as luminol [27], it will still detectblood as dilute as 1 part in 10,000 [30].

Two derivatives of benzidine, ortho-toludine and tetramethyl-benzidine (TMB), were developed as catalytic presumptive tests toreplace the hazardous benzidine reagent. Both of these tests areconducted under acidic conditions that involve color changes thatare blue or blue-green, respectively [9]. After more consideration,however, it was found that ortho-toludinewas carcinogenic in rats,and themore sensitive TMB slowly replaced it in the laboratory [8].

Crystal test

Elements Chemical

SpectroscopicSpermatozoa Microscopic

Antigens Immunological

19-OH F1a/F2aAntibodies

Isozymes

Saliva Whole uid ALSAmylase Chemical

Antibodies Immunological

Elements Spectroscopic

Vaginal uid Gly. epithelial cells Chemical

Vaginal peptidase Electrophoresis

Antibodies Immunological

Lactate/citrate Electrophoresis

Urine Whole uid ALSEpithelial cells MicroscopicUrea Chemical

Creatinine

THP Immunological

UA/UN Chemical

Elements Spectroscopic17-Ketosteroid conj. Chromatographic

Sweat Elements Microscopic

Codes: grey: presumptive; dark grey: conrmatory; italic: non-destructive; and bold: aa It is most desirable for a test to be conrmatory, non-destructive, and applicable tMethod(s)

Polilight1

Luminol, Fluorescein, Bluestar1

Benzidine, Kastle-Meyer, O-toludine, TMB/Hemastix1, LMG

Teichman, Takayama

UVvis, Fluorescence (hematoporphyrin)

PC, TLC

SEMEDX

LDH

HemeSelectTM, ABAcard1, HemaTrace1, Hexagon OBTI

ELISA

Woods Lamp, Bluemaxx BM500, Polilight1

SAP, LAP, GDA, CAP, g-GTPGGT ELISA

Florence test

Choline oxidase

Choline oxidase/Luminol solution

Isotachophoresis

HPLC, PC, TLC

Capillary tube electrophoresisThe TMB reagent remains one of the more popular used inpresumptive tests for blood and is the main constituent of theHemastix1 eld test [9,23]. Although Hemastix can be used in theeld, it tends to show even more false positives than the otherpopular presumptive tests [11].

One of the other most popular presumptive tests for blood usedin forensics today is leucomalachite green (LMG) [11]. It is alsoperformed under acidic conditions and involves a heme-catalyzedreaction with a resulting green color [9]. Similar to phenolphtha-lein, it has a sensitivity of 1 part in 10,000 [30]. Like all otherpresumptive tests for blood, it is not species specic and will notindicate whether suspected blood is human or not [13].Leucocrystal violet is another form that can be used but is notas common in forensic investigations [8,11].

Additional non-catalytic presumptive tests have also becomeavailable to be used in conjunction with the previously mentionedscreening tests. Some of these are Heme SelectTM, ABAcard1

HemaTrace1, and Hexagon OBTI which all use immunologicalmethods to identify primate blood. The latter has been recentlycompared to other catalytic tests and was found to be inferior as a

Barberio test, Puanens test

Zinc paperstrip test

SEMEDX

Christmas tree stain

PSA, SVSA

Radioimmunoassay

1E5 ELISA, SAP ELISA

LDH

UV light, High intensity quartz are tubes

Starchiodine, Phadebas1, Amylose Azure, Rapignost1-Amylase

ELISA, Immunodiffusion

SEMEDX

PAS reagent

Starch gel/L-valyl-L-leucine

Oestrogen receptors

Capillary isotachophoresis

UV light

Visualize cells (no stains)

Nesslers, DMAC, Urease/bromothymol blue

Jaffe test, Salkowski test

Sandwich ELISA, SP radioimmunoassay

TZ-UA One/Urea NB-test

SEMEDX

HPLC, ESI-LCMS, PC, TLC

SEMEDX

pplicable to multiple uids.

o multiple uids, but this table contains no methods which t this description.

K. Virkler, I.K. Lednev / Forensic Science International 188 (2009) 117 5presumptive test for blood but rather a good method tosupplement the results of the more sensitive catalytic tests dueto its specicity. Hexagon OBTI works well on aged or degradedmaterial [9], however it does produce false negatives [30].

3.1.2. Conrmatory tests

Once a positive result is obtained from a presumptive bloodtest, several conrmatory tests are available to identify absolutelyan unknown stain to be blood. These tests can be categorized asmicroscope tests, crystal tests, spectroscopic methods, immuno-logical tests, and chromatographic methods.

The simplest of microscope tests involves the identication ofblood cells by directly visualizing them in liquid blood [11]. It isbelieved that visually identifying the red andwhiteblood cells alongwith brin is denite proof that blood is present. Numerous stainshave been developed to aid this process [8], but other more popularconrmatory tests have replaced cell identication as a technique[11]. An expansion on this method involves the use of a scanningelectron microscope (SEM) which allows scientists to study themorphology of an unknown stain and to analyze its chemicalcomposition using an energy dispersive X-ray (EDX) analyzer. Verysmall or dilute stains can be detected with this method [31].

Crystal tests are the most common conrmatory tests for bloodthat are used, and the Teichman and Takayama crystal tests are thetwo most popular [9,11]. The Teichman test is based on theformation of hematin by heating a dried stain in the presence of ahalide and glacial acetic acid [8,11]. This test forms brown, rhombiccrystals and is very sensitive to under- and over-heating [9]. TheTakayama test is based on the formation of hemochromogen byheating a dried stain in the presence of pyridine and glucose underalkaline conditions [9], although acidic conditions have also beenused [8]. A positive result yields needle-shaped crystals, and thistest is preferred due to advantages such as less heat sensitivity [8],ease of use, and a wider variety of stain compatibility [11]. Both ofthese crystal tests were improved upon in another experiment sothat they became easier to use and more sensitive [32].

Spectroscopic methods, such as UVvis absorption, are con-sidered highly reliable in conrming the presence of blood in a stain[8]. Many different derivatives of hemoglobin have a characteristicstrong absorbance band around 400 nm that is called the Soret Band[11]. Many of these tests can distinguish between differenthemoglobin derivatives and also show a positive result for olderstains that have a negative result with other presumptive tests andcrystal tests [11]. However, there are many conditions which caninterfere with the spectral results such as water submersion,sunlight exposure, heating, and rust [8]. Amicrospectrophotometercan be used to measure an absorption spectrum after a sample hasbeen treatedwith theTakayama reagent [33]. Another spectroscopictest is based on the uorescence of hematoporphyrin fromexcitation with ultraviolet light. This method has been found tobe especially successful when dealing with bloodstains on oxidizedmetals, bloodstains up to 10 years old, and bloodstains exposed toconditions not suitable for UVvis absorption such as heat, sunlight,and humidity. Best results can be obtained by analyzing a salineextract of the bloodstain, and it is important to keep in mind thatthere are possible false positives since porphyrin compounds occurfrequently in biological systems [8]. These spectroscopic tests havenot been used inmany years for the investigation of crimes, but it isimportant to include them in this discussion in order to help tounderstand the evolution of body uid testing.

Amodern and currentlyused conrmatory test forblood that canalso distinguish species involves the cross reaction of thehemoglobin inabloodstainwithanti-humanhemoglobin. Precipitinlines observed after immunodiffusion conrmed the presence of notonly hemoglobin, but specically human hemoglobin. This methodhas been expanded upon to detect numerous different species ofblood [8]. Isozyme analysis is another technique that can conrmthe presence of blood by comparing the differences in lactatedehydrogenase (LDH) isozyme patterns of different uids [29,34].Enzyme-linked immunosorbent assay (ELISA) is another methodthat has been used to identify blood [35] as well as identify bloodgroups using different antibodies [36].

Finally, chromatographic methods can be used to conrm theidentity of blood. Paper chromatography was the rst techniqueand involved the separation of hemoglobin and its derivatives. Analcoholic benzidine spray reagent in conjunction with a hydrogenperoxide spray was used to detect spots, and the hematoporphyrincomponent was visualized due to uorescence without spraying.The main disadvantage of this technique was a lengthy develop-ment time and the necessity of pre-saturation. Thin-layerchromatography has also been utilized with a similar developingtechnique [8]. These tests are also not currently used in forensiclaboratories.

3.2. Emerging techniques

The following section will describe new and emerging techni-ques in the area of forensic blood detection. These techniques arealmost all conrmatory in nature, and most of them involveimmunological markers and can simultaneously identify thespecies of the unknown blood sample. Table 3 summarizes thenew techniques that have emerged in the last 56 years for bloodand the other body uids.

The majority of new techniques being investigated for theidentication of blood found at a crime scene involve RNA with afocus on mRNA. This form of RNA carries information aboutproteins from the DNA to the ribosomes in cells where proteins aresynthesized [37]. Some of these new techniques have beenmentioned in a review on the applications of RNA in forensicscience [38]. In 2004, Alvarez et al. revealed a method to isolatesimultaneously RNA and DNA from the same stain extract.Performing an mRNA expression analysis on the RNA extractswill yield information regarding the identity of the stain, and theDNA analysis will reveal the donors identity. The marker beingused to identify blood in this case was b-spectrin (SPTB). Theability to analyze both of these characteristics at the same timewillhelp to identify better the number of different uids or donors in amixture [39].

In 2005, a multiplex reverse transcription-polymerase chainreaction (RT-PCR)methodwas proposed by Juusola and Ballantyneto identify different body uids including blood [42]. This was anexpansion on their previous work that concluded that RNA couldbe retrieved from a blood stain and be analyzed for identicationpurposes [40]. The RT-PCR process involved the detection ofspecic mRNA markers for gene expression analysis, namely theSPTB and porphobilinogen deaminase (PBGD) genes for blood. Apatent was developed for the same technique [41]. Sensitivity wasin the range of 6 ng of blood for a positive result for both genes.Specicity was provenwhen these genes were only detected in theblood samples, andmenstrual bloodwas even exclusively detectedby the presence of the matrix metalloproteinase-7 (MMP-7)enzyme [42]. ThisMMP-7 enzymehad previously been studied anddetermined to be a conclusive marker for menstrual blood since itwas not detected in vascular blood or other uids [43]. Bauer andPatzelt have also explored this menstrual blood enzyme and theimportance of a negative result that shows a stain is not menstrualblood [44]. A similar study was conducted in 2008 [6] using thesame SPTB and PBGD blood genes that Juusola and Ballantyneworked with, and it was found that positive results could beobtained on stains that were up to 15 months old.

Another RT-PCR approach was developed by Nussbaumer et al.in 2006 and focused on the detection of the alpha locus 1 (HBA)

RNAmarker in blood [7]. In 2007 Juusola and Ballantyne continuedto make alterations and improvements to their technique with the

One more new technique for conrmatory identication ofhuman blood has recently been developed which uses a lateral

Table 3Summary of techniques recently developeda.

Body uid Component Classication Method(s)

Blood Whole uid ALS IR lightHemoglobin Spectroscopic XRF (iron)Antibodies Immunological Lateral ow test strip Antiglycophorin A

mRNA SPTB/PBGD, HBA, ALAS2/SPTB (RT-PCR)

Semen Whole uid ALS Lumatec Superlight 400Elements Spectroscopic XRF (zinc)mRNA Immunological PRM1, PRM1/PRM2, KLK (RT-PCR)Semenogelin RSID-Semen Test, Nanotrap Sg

Saliva Whole uid ALS Lumatec Superlight 400Amylase Chemical SALIgAE1

Spectroscopic Fluorescence spectroscopy (swabbed from skin)

Antibodies Immunological RSID-Saliva Test

Immunological Amylase ELISA, Lateral ow test strips

mRNA HTN3, STATH/HTN3 (RT-PCR)

Vaginal uid Antibodies Immunological E2-17b ELISAmRNA Immunological MUC4, HBD-1/MUC4 (RT-PCR)

Sweat Antibodies Immunological G-81 ELISA

Codes: grey: presumptive; dark grey: conrmatory; italic: non-destructive; and bold: applicable to multiple uids.a It is most desirable for a test to be conrmatory, non-destructive, and applicable to multiple uids, but this table contains no methods which t this description.

K. Virkler, I.K. Lednev / Forensic Science International 188 (2009) 1176introduction of a triplex system that detects the blood-specicgenes erythroid d-aminolevulinate synthase (ALAS2) and SPTBalong with the housekeeper gene GAPDH. This housekeeping genenormalizes the expression of the blood-specic genes to maintainhigh specicity [5].

In 2008, Zubakov et al. comprehensively analyzed the whole-genome gene expression on aged bloodstains in order to generate aset of stable RNA markers. After an initial selection of about 1000possible markers for blood, the options were narrowed bycomparing them to the GNF SymAtlas tissue database. Genemarkers were chosen based on high expression in blood and lowexpression in other uids. In the end, nine were chosen based onthese parameters and the production of positive results on stains asold as 180 days [45].Fig. 1. A non-destructive XRF spectrum of the elemental composition of gun shot residueplanetary exploration. Preliminary results have been obtained for blood and semen base

that does not damage the sample but is not conrmatory. (Reprinted from [47] with pow test strip containing an antiglycophorin A antibody that willreact with an aqueous blood sample when contact is made. This isthe rst test which detects this specic sialoglycoprotein, and thetest claims to overcome the problem of the high dose Hook effectencountered by other similar methods. The basic theory uses adetection antibody which binds to human blood, then migrates upthe membrane and forms a visible complex with an immobilizedcapture antibody [46].

A completely different form of presumptive identication ofblood that is non-destructive has been suggested by Trombka et al.Themethod involves a unique NASA technology involving portableX-ray uorescence (XRF) that was originally designed forelemental analysis during planetary exploration. It can detectthe abundance of iron present in the hemoglobin of blood, and itspatteredwith blood. A portable device was usedwhichwas developed for NASA for

d on the presence of iron and zinc, respectively. This is a good screening technique

ermission from Elsevier.)

K. Virkler, I.K. Lednev / Forensic Science International 188 (2009) 117 7has potential to be a valuable aid in the identication of blood at acrime scene since the device is portable [47]. Fig. 1 shows an XRFspectrum displaying a peak for iron. The fact that it is non-destructive is unique compared to the other known techniques andis very helpful in preserving DNA evidence.

Another non-destructive screening test for bloodwas suggestedin 2007 by Chun-Yen Lin et al. and involves the use of infrared (IR)light as a means to identify latent blood traces. This method issimilar to the more familiar techniques involving ultraviolet light.Bloodstains as dilute as 1:8 were detected on black fabrics when adigital camera with an IR sensitive CCD captured pictures of thestains illuminated under IR light. Although this technique is not assensitive as other presumptive tests and does not work well onsome fabrics, it can be helpful due to its non-destructive naturewhich allows preservation of DNA evidence while quicklysearching a crime scene for bloodstains [48].

4. Semen


Semen is one of the other most commonly encountered bodyuids at crime scenes. There are also several presumptive tests toidentify semen as well as conrmatory tests. The followingsections explain techniques that are either well known in theforensic community or have been summarized in previousliterature.

4.1.1. Presumptive tests

Like with blood, semen can also be detected using an ALS suchas ultraviolet light. It is routine procedure to search a crime scenefor semen and other uids using this simple and non-destructivemethod [29]. TheWoods Lamp (WL) is a specic device that emitswavelengths from about 320400 nm, and it is small, inexpensive,safe, and easy to use. However when the WL was put to the testagainst other uids, it was not very specic and sometimes did noteven detect semen stains, and it gave false positive results forointments and creams [49]. Another commercial ALS, the Blue-maxxTM BM500, was tested in a similar way and was 100%sensitive to semen stains. Also, physicians using the ALS were ableto distinguish semen from other products 83% of the time afterreceiving training on how to use the device [50]. Another ALS thathas been used on several uids including semen is Polilight1

which has a wavelength range of 415650 nm as well as white andultraviolet light [19].

The most popular and accepted presumptive test for thepresence of semen is the test for seminal acid phosphatase (SAP).This enzyme has the ability to catalyze the hydrolysis of organicphosphateswhich forms a product thatwill reactwith a diazoniumsalt chromogen to cause a color change [10]. This is the basicprinciple behind all the variations of the SAP test. One popularsubstrate/color developer combination is alpha-naphthyl phos-phate and Brentamine Fast Blue. Other combinations that havebeen successful are beta-naphthol with Fast Garnet B, and alpha-naphthol with Fast Red AL [8]. Additional reagents used have beensodium thymolphthalein monophosphate due to its high selectiv-ity, stability, and low hazardous risk [51], and a combination of p-nitroaniline, NaNO3, a-naphthyl phosphoric acid, and aqueousmagnesium chloride [52]. Of course there are false positives suchas some plant materials and even vaginal acid phosphatase (VAP),so this technique cannot be considered conrmatory [8]. One wayto avoid a potential false positive for VAP is to observe a colorchange that only occurs between 5 and 30 s since VAP has nevergiven a positive result that quickly [12]. Other methods that havebeen developed to distinguish between SAP and VAP involve theseparation of the two acid phosphatases using isoelectric focusing[53], and by using acrylamide gel electrophoresis [54]. Furtherdisadvantages of SAP tests are that the enzyme can degrade whenexposed to heat, mold, putrefaction, or chemicals [12].

There are similar presumptive tests for semen based on thepresence of other enzymes, but these tests are not as popular. In acomparison study with the SAP test, the leucine aminopeptidase(LAP) test had fewer false positives with other human body uids,fruits, and vegetables, and only showed negative results withsemen from other species. However it was less sensitive to highdilutions [55]. Another enzyme that has been tested is glycylpro-line dipeptidyl aminopeptidase (GDA). Results have shown thatstains as old as 24 years can give a positive reaction; false positivesinclude vaginal uid, feces, strawberries, broad beans, and onions.The sensitivity was only tested as low as a 1:4 dilution in which allwere positive [56]. Another enzyme-based test relies on thedetection of cystine aminopeptidase (CAP). It is about 100 timesmore prevalent in semen than other uids. When tested on otherbody uids including vaginal uid and feces, there were no falsepositive results as with some of the previously mentioned enzymetests. There were also only negative results for several fruits andvegetables, including strawberries [57]. A test for the enzyme g-glutamyltransferase (g-GTP) using Fast Garnet GBC salt and a-naphthylamine as indicators showed positive results on stains asold as 23 years, but there were several false positives includingbreast milk, vaginal uid, green peas, broad beans, onions,strawberries, apples, and plums [58]. Finally, the test for seminalzinc has been studied and proposed to be a better and lessdegradable marker than SAP. This study reported no false positiveresults on other body uids, fruits, or vegetables, and it detectedsemen on stains that were 25 years old [59]. A zinc test paperstripmethod was compared to an SAP paperstrip test, and in the endboth tests appeared to be similar in both sensitivity and specicity[60].

Another presumptive test for semen that has been around for along time but no longer regularly used is the test for the presence ofcholine. One test for choline is the Florence test which involvesplacing an extract of a questioned stain on a microscope slide,treating it with a solution of iodine and potassium iodide, andobserving the brown needle-like crystals that form [61]. Thepossibility of false negatives is great due to low sensitivity [61], andthis test is negative for other body uids including vaginal uid aswell as semen from other species [8]. Additional methodsdeveloped for the detection of choline are based on a reactionwith choline oxidase [62] including a chemiluminescent testinvolving a choline oxidase/luminol solution [63]. A morecomplicated method to detect choline known as isotachophoresiswas found to have no false positives with other body uids, fruits,or vegetables. Stains up to 10 years old still showed some positiveresults, and the test showed positive results for semen in vaginaluid swabs taken from deceased females [64].

Onemore presumptive test that has been studied in the past butis not currently in use is the detection of the seminal polyamineknown as spermine (SPM) which has the highest concentration insemen among all the different forms of polyamines. High-performance liquid chromatography (HPLC) combined with asimple extraction method has been found to be a simple andsensitive way to detect SPM in comparison to paper chromato-graphy, TLC [65], capillary tube isotachophoresis [66], and anenzyme method [67]. The most prevalent false positive came fromsoy sauce, and no spermine was measured in body uids includingurine, blood, sweat, breast milk, and saliva. Fruit and vegetablejuices also showed no positive results [68]. Another test forspermine that has been used in the past is called the Barberio testand involves the microscopic conrmation of yellow crystals thatform when semen is exposed to an aqueous solution of picric acid.This test is considered more reliable than the Florence test for

K. Virkler, I.K. Lednev / Forensic Science International 188 (2009) 1178choline, and it still gave positive results on stains as old as 3 yearsand ones that had been heated to 150 8C [8]. A similar methodknown as Puanens test is another crystal test that uses NaphtholYellow S as a reagent and forms orange crystals in the presence ofsemen [8].

Finally, a completely unrelated method to the ones previouslymentioned has also been used to identify semen stains but is not aspopular when compared to SAP tests. It applies scanning electronmicroscopy (SEM) and energy dispersive X-ray microanalysis tothe detection of sodium, phosphorus, sulfur, chlorine, potassium,calcium, and other metal trace elements. These elements occur invarying proportions among different body uids, and identifyingan unknown stains element ratio will distinguish it from otheruids. Chlorine was the largest peak detected in the semensamples, but calcium can be used as an identication marker. Themethod was awed, however, since the substrate spectrum willdominate the uid spectrum, and subtraction of the substrate willalso remove peaks from the uid that are present in both. In theend, this method will work best to identify one uid stain as beingidentical to another [69]. This technique can only be classied aspresumptive due to the interference of the substrate.

4.1.2. Conrmatory tests

The most reliable and widely accepted conrmatory techniquefor the detection of semen is the microscopic identication ofsperm cells. Semen is the only body uid which possesses spermcells, and the large amount of DNA in the heads can be treatedwitha stain tomake the sperm visible [13]. Themost popular stain usedis the Christmas tree stain, and it is known for its characteristiccolors which stain the heads red and the tails green. There is anadditional technique using a solution of proteinase K that willdenature epithelial cells and make the unaffected sperm headsmore visible [10]. Other stains that are less effective than theChristmas tree stain that have been tested are Gram modiedChristmas tree, hematoxylin and eosin, Baecchis, Papanicolaous,and Wrights [12]. Of course the largest disadvantage of thismicroscope technique is if the semen donor is azoospermic due tonatural causes or by vasectomy; for this reason, other chemicaltests have been developed.

The most popular conrmatory test for semen beyond lookingfor sperm cells is the test for prostate-specic antigen (PSA). Thesemen from azoospermic males will still contain this antigenwhich is present in the seminal plasma; other body uids contain avery low level of PSA and do not interfere [10]. These low levelsrequire that PSA tests are not too sensitive so that there are no falsepositives. An important aspect of detecting PSA involves the abilityto detect it on contaminated or scarce samples including launderedfabrics and decomposed cadavers [70]. The original methodsinvolved immunoelectrophoresis or ELISA [71], and some specictechniques involved a dot blot immunoassay with a radiolabeledProtein A [72] as well as a dot-immunobinding method called themembrane aspiration test (MAT) [73]. Another used thin-layerimmunoassay (TIA) and showed no false positive results withblood, saliva, urine, sweat, and tears [74]. Now several test kitshave emerged that depend on antibodyantigen reactions and aremuch quicker and easier to use [12]. One commercially availablekit is the Biosign1 PSA test, and it was also found to be cheaper tooperate than the traditional ELISA method [75]. The OneStepABAcard1 is another commercial test kit. It also relies on thetechnique of amobilemonoclonal anti-human PSA antibodywhichbinds to human PSA and migrates along the strip to theimmobilized polyclonal anti-human PSA antibody and forms avisible line [76]. PSA in semen diluted 106 times is able to bedetected with this test, and only male urine samples gave a falsepositive result [77]. Other commercial tests that have beendeveloped include Chembio, Medpro, Onco-screen [70], PSA-check-1, and Seratec1 PSA Semiquant [76]. The Seratec1 test wasfound to show false positives with semen-free vaginal samples aswell as some readily available contraceptive foam [78]. Acomparison study among the PSA Rapid Test Kit, Rapid PSA, andSMITEST determined that all three had equal specicity, butSMITEST [79] had the greatest sensitivity [80]. An automated testkit that allows simultaneous analyses is the Hybritech Tandem-EPSA Immunoenzymetric Assay and uses less labor and funds thantraditional methods [81].

MHS-5, also known as immunoglobulin G1 and seminal vessel-specic antigen (SVSA), is an antigen that will only react with theepithelium in human seminal vesicles [82]. The major gel-formingproteins in human semen, semenogelin I and semenogelin II, bothcontain SVSA and are recognized by amonoclonalMHS-5 antibody.Sema1 is an ELISA kit designed to detect the presence of semenbased on the reaction between the MHS-5 antibody and SVSA. Thismethod is sensitive for semen samples diluted as many as 106

times, but not nearly as specic as typical PSA tests and is no longerused [12]. Other immunological methods that have been devel-oped to detect semen involve the presence of semenogelin [83],semenogelin II [84], 19-OH F1a/F2a prostaglandin [85], and amonoclonal antibody called 1E5 [86]. Finally, an ELISA techniquefor the detection of a monoclonal antibody to SAP has beendeveloped which showed no false positives with other body uidsand is sensitive with semen dilutions up to 1:100,000 [87].

As mentioned earlier in the discussion about conrming thepresence of blood, an LDH isozyme can also play a role inconrming the presence of semen. It was discovered to haveproperties in between LDH-3 and LDH-4 and was soon namedLDH-X. It is unique to human sperm, however, so like theChristmas tree staining technique, the LDHmethod will only workif the donors semen contains sperm [8,88]. The isozyme isdetected by electrophoresis, and this technique will give positiveresults on stains at least 30 weeks old [88] and on post-coitalvaginal swabs [29]. Sperm can even be detected in a mixture withblood and vaginal uid using this method [89]. This method is notcommonly used in modern forensic laboratories.

Another isozyme that can be used to detect semen isg-glutamyltranspeptidase (GGT). It is much more active in seminal plasmathan other body uids and is believed to be spermic or testicular innature [29]. A few other isozymes have been studied such assperm diaphorase, creatine phosphokinase (CPK), and variousesterases, but these methods have not proved to be as effective asthe more popular PSA tests [29].


The following section will describe new and emergingtechniques in the area of forensic semen detection. Like withblood, these techniques are almost all conrmatory in nature, andmost of them involve immunological markers and can simulta-neously identify the species of the unknown semen sample.

Many of the new methods being developed to identify semenstains involve mRNA markers and are the same methods alreadymentioned for the detection of bloodstains. Bauers review of theuses of RNA in forensic science also covers methods to detectsemen [38]. The same RNA and DNA co-isolationmethod describedby Alvarez et al. can also be applied to semen samples. Protamine 1(PRM1) was the semen-specic marker under investigation in thisstudy [39].

The RT-PCR method proposed by Juusola and Ballantyne in2005 also applies to semen. The process involves the detection ofthe semen-specic genes PRM1 and protamine 2 (PRM2) [42]. Apatent was developed for the same technique [41]. Sensitivity forthe semen samples was the highest among body uids beingdetected with less than 200 pg of input RNA needed for a positive

upe

foun

K. Virkler, I.K. Lednev / Forensic Science International 188 (2009) 117 9result with both genes. Specicity was proven when these geneswere only detected in the semen samples. This octaplex RT-PCR

Fig. 2. A white and black cloth containing semen is illuminated with the Lumatec Sdarkness and daylight. Semen was detectable 100% of the time, and saliva was only

decrease. (Adapted from [92] with permission from the author.)method produced no false positives, no false negatives, and nosingle gene drop outs [42]. The updated version of this experimentthat was presented in 2007 involves a triplex system as previouslymentioned during the bloodstain discussion, and the genesinvolved are PRM1 and PRM2 for semen along with the GAPDHhousekeeping gene [5]. A similar study was conducted in 2008using the same PRM1 and PRM2 semen genes that Juusola andBallantyne worked with, and it was found that positive resultscould be obtained on stains that were up to 15 months old [6].

The RT-PCR approach developed by Nussbaumer et al. in 2006focuses on the kallikrein 3 (KLK) marker for semen (also known asPSA). There was no cross reactivity with samples from other uidssuch as blood, vaginal secretions, and saliva, and none of thesesamples showed positive results for the KLK assay. Also, thisspecic semen mRNA marker showed great stability and wasequally detected after 10 days of room temperature storage withno stabilizing buffer. This technique proved to be more sensitivethan popular protein-based assays for PSA [7].

In 2007 Pang and Cheung [90] compared a new commercialrapid stain identication (RSID)-Semen Test to the previouslymentioned ABAcard1 PSA test for the detection of semen stains.The RSID-Semen Test is based on the detection of semenogelin (Sg)usingmonoclonal anti-human Sg antibodies. Both tests involve thesame immunochromatographic membrane assay technology. TheRSID-Semen Test was found to bemore sensitive by detecting Sg ata semen dilution of 1:100,000. The several different species ofsemen that tested positive for spermatozoa all showed negativeresults for Sg. This test also showed no false positives with otherbody uids, and the analysis takes only 10 min [90]. Anothercommercial test kit that detects Sg is called Nanotrap Sg. It has asimilar sensitivity as the RSID-Semen Test, but more than threerepeats of freezing and thawing semen samples caused thesensitivity of the results to drop. This test was able to detectsemen in 67% of samples that contained no spermatozoa. The

rlight 400 at 550 nm. The device has a range from 320 to 700 nm and will work in

d 60% of the time. Dark fabrics absorbed uorescence and made the stain visibilityability for this method to nd male DNA in samples showing nospermatozoa makes it valuable for subsequent DNA analysis [91].

As with blood, Trombka et al. have introduced a non-destructive method of presumptive semen identication. Themethod which involves a unique NASA technology involvingportable XRF can detect the abundance of zinc present in thesemen stains, and it also has potential to be a valuable aid in theidentication of semen at a crime scene since the device isportable. The process only takes about a minute, and it has aprecision of better than 10% in measuring 1 ml of semendistributed over a 40 cm2 area [47]. The non-destructive natureof the method will be very helpful in sexual assault cases that relyon DNA evidence not being destroyed by early screening tests.

Finally, a newly developed presumptive test for semen stains isa UVvis light source called Lumatec Superlight 400. It emits lightfrom 320 to 700 nm and was tested on human and boar semensamples on different types and colors of fabrics. Fig. 2 shows theresults of illuminating a stain on light and dark fabricwith differentlters. The Superlight was able to detect stains both in darknessand in the presence of daylight; storage times of 3 and 5 weeksshowed no difference in results. Semen was best detected using arange of 415490 nmwith orange or red goggles. Poor results wereobtained on dark fabrics and on fabrics that had been washed, butdifferent types of fabrics showed similar results [92].

5. Saliva


In addition to blood and semen, saliva is another commonlyencountered body uid at crime scenes. There are a few wellknown and accepted presumptive tests for saliva, but there are no

K. Virkler, I.K. Lednev / Forensic Science International 188 (2009) 11710currently used conrmatory tests that are specic to saliva. Thefollowing section explains presumptive techniques that are eitherwell known in the forensic community or have been summarizedin previous literature.

Like blood and semen, saliva can also be located using an ALS.Saliva stains will appear bluish-white when being viewed underan ultraviolet light, though this will not distinguish it fromanother body uid stain [8]. Saliva is also harder to detect thansemen due to the lack of solid particles in the saliva sample [12].One study that compared two different argon laser light sourcesto a high intensity quartz arc tube found the quartz arc tube to besuperior based on portability, cost, sensitivity, and power output.The lifetime of the quartz tubes was found to be the largestdisadvantage, though they were cheaper to replace than the laserlight sources [93].

The most popular and widely accepted technique to testpresumptively for saliva is based on the activity of amylase. Twodifferent forms are found in the human body. Amylase found insaliva, breast milk, and perspiration is coded by the AMY1 locus onchromosome 1, while amylase found in the pancreas, semen, andvaginal secretions is coded by the AMY2 locus [10,29]. AlthoughAMY1 is found more in saliva than any other uid, it can still onlygive presumptive information since it is not exclusive to saliva[10]. A radial diffusion assay has been used to distinguish sourcesof AMY1 and AMY2 [94]. The starchiodine test is based on the factthat starch appears blue when in the presence of iodine, andsalivary amylase will break down the starch to cause a colorchange. However, competing proteins such as albumin andgamma-globulin in blood and semen will also break down iodineto form a false positive result. The Phadebas1 test reagent, whichincludes procion red amylopectin, has been applied in tube testsand press tests which both can detect saliva diluted up to 1:128[10]. A study testing the Phadebas1 reagent for false positiveresults found that hand cream, face lotion, washing powders, urine,and feces tested positive using Red-Starch paper [95]. Anothermethod utilizing an insoluble amylase/dye complex calledAmylose Azure as a substrate will yield a blue color uponhydrolysis in amylase. This technique is more sensitive since it candetect saliva dilutions as high as 1:1000, it requires a shorterincubation time, and it will detect saliva present in body uidmixtures. A commercial test strip known as Rapignost1-Amylaseused to detect amylase in urine has also been applied to salivasamples. The method is simple and requires only 30 min ofreaction time [96].

There have been some immunological methods presented forthe identication of saliva, though they are still not completelyexclusive or widely used. An ELISA method using a horseradishperoxidase conjugate combined with monoclonal antibodies hasbeen used to detect a-amylase activity in saliva stains [36]. Rabbitantisera against a-amylase have been used in conjunction with a-amylase puried from human submaxillary glands in a traditionalimmunodiffusion experiment. This experiment did not test otherbody uids, so it can only be used as a species indicator once thesample has already been identied as saliva [97]. Many otherexperiments have been conducted involving immunoelectrophor-esis and saliva antigens, but there were too many cross reactionswith other uids such as serum to lead to a reliable technique thatwas conclusive for saliva [8].

There are also some techniques that have dealt with micro-scopy. Like with semen, the use of SEM coupled with EDX canidentify the relative concentrations of sodium, phosphorus, sulfur,chlorine, potassium, calcium, and othermetal trace elements in thequestioned sample. In the saliva samples tests, potassium was thelargest peak and can be used as a basis of identication. Aspreviously mentioned, this technique can only be used to screen asample and determine if it is identical to another. The dominanceof the background spectrum and subsequent subtraction will losevaluable data about the uid [69].


The following section will describe new and emergingtechniques in the area of forensic saliva detection. Like with bloodand semen, most of these methods involve mRNAmarkers and canpotentially be considered conrmatory in nature. The applicationof some of these techniques to forensic casework could besubstantially helpful.

Many of the same methods already discussed for blood andsemen can also be applied to saliva. Bauers review of the uses ofRNA in forensic science also covers methods to detect saliva [38].The same RNA and DNA co-isolation method described by Alvarezet al. can also be applied to saliva samples, and for this uid histatin3 (HTN3) is being detected [39].

The RT-PCR method proposed by Juusola and Ballantyne in2005 also applies to saliva. The saliva-specic genes statherin(STATH) and HTN3 were the ones under investigation in this study[42]. A patent was developed for the same technique [41].Sensitivity was similar to that of blood with only 9 ng of inputRNA needed to detect both genes. Specicity was slightly less thanthat of blood and semen. Although the main components STATHand HTN3 were only detected in saliva, the larger and less specichistatin 1 (HTN1)was detected using the HTN3 primer and showedsome slight false positive results in semen. Likewise, a smallerversion of STATH was minutely detected in menstrual blood [42].The updated version of this experiment that was presented in 2007involves a triplex system as previously mentioned during thebloodstain and semen discussions, and the genes involved areagain STATH and HTN3 for saliva along with the GAPDHhousekeeping gene [5]. A similar study was conducted in 2008using the same STATH and HTN3 semen genes that Juusola andBallantyne worked with, and it was found that positive resultscould be obtained on stains that were up to 15 months old withmuch better sensitivity than tests for amylase [6].

In 2008, Zubakov et al. comprehensively analyzed the whole-genome gene expression on aged saliva stains in order to generatea set of stable RNA markers. After an initial selection of about 500possible markers for saliva, the options were narrowed bycomparing them to the GNF SymAtlas tissue database andtargeting genes for tissues like the salivary gland, tongue, trachea,and tonsils. Genemarkerswere chosen based on high expression inblood and low expression in other uids. In the end, ve werechosen based on these parameters and the production of positiveresults on stains as old as 180 days [45].

An ELISAmethod developed byQuarino et al. uses amonoclonalanti-human salivary amylase antibody to detect saliva stains, andit shows no cross reactivity with other forms of amylase such aspancreatic or bacterial. The salivary amylase can be quantitativelydetected by absorption at 405 nm directly from the sample welland there is a direct relationship between absorption and amylaseactivity. The results showed that 100% of the saliva samples andonly 13% of other body uids showed absorption. However, thefalse positive absorption results were ten times weaker than thelowest saliva result [98].

In 2007 Karl Reich presented a lateral ow test strip as amethodto conrm the presence of saliva rapidly, accurately, and with highsensitivity. The technique uses nine antibodies against humansalivary amylase which can be monoclonal, polyclonal, orrecumbent antibodies. This test is species specic and can beapplied to many different types of samples. The test strip isimmunochromatographic in nature and uses a mobile andstationary antibody. Positive results were obtained from samplestaken from buccal swabs, plastic bottles, plastic mugs, ceramic

K. Virkler, I.K. Lednev / Forensic Science International 188 (2009) 117 11mugs, cigarette butts, and soda cans. There was only some crossreactivity with feces and breast milk, and only human salivayielded positive results [99].

Another recently released colorimetric assay test kit for salivathat is presumptive in nature is called SALIgAE1 and is alsoavailable as a spray [14]. Myers and Adkins performed a studythat compared this test to the Phadebas1 and starchiodinemini-centrifuge test. They found that SALIgAE1 is much lesssensitive than the other two, and it even had detection limits thatwere higher than the average a-amylase concentrations inhuman saliva. It could only detect dilutions of neat saliva as lowas 1:10. Waiting longer than 10 min sometimes revealed positiveresults that were not formerly visible [100]. Another studycompared SALIgAE1 to an immunochromatographic test knownas the RSID-saliva test and the Phadebas1 test [101]. The RSID-saliva test uses a mobile and stationary monoclonal anti-humansalivary a-amylase antibody that forms a visible pink line in thepresence of antigen [14]. The results of the experiment weredifferent than that of Myers and Adkins. The RSID-saliva testcould detect saliva diluted up to 1:10,000, SALIgAE1 up to1:2000, and the Phadebas1 test only up to 1:100. Speciesspecicity was also highest with the RSID-saliva test with onlyrat saliva testing positive [101].

Finally, some new spectroscopic and ALS techniques have beenrecently presented. Soukos et al. developed a rapid and non-destructive method to detect dried saliva swabbed from skin usinguorescence spectroscopy [102]. Emission spectra were measuredfrom solutions containing the dissolved swab contents in KCl in therange of 345355 nm. Compared to a water control, the emissionspectra showed greater intensity. The uorescence spectra ofsaliva were similar to that of pure aqueous amylase andtryptophan [102]. The ALS technique introduced by Fielder et al.in 2008, which was previously mentioned in regards to semenidentication, can also be applied to saliva. The Lumatec Superlight400 emits light from 320 to 700 nm and was tested on humansaliva samples on different types and colors of fabrics. It was able todetect stains both in darkness and in the presence of daylight;storage times of 3 and 5 weeks showed no difference in results.Saliva was also best detected using a range of 415490 nm withorange or red goggles. Saliva was only detected in 60% of the cases,but the rate of positive results was much higher than with otherreported ALS techniques [92].

6. Vaginal uid


Although not as common at crime scenes as blood, semen, andsaliva, vaginal uid evidence can play an important role in sexualassault cases. However, there are not very many tests available totest for the presence of this uid mainly because it is not very welldened. The constituents can change based on themenstrual cycleof the female, and this makes testing for specic components verydifcult [8]. A fewpresumptive tests that have been established arementioned in this section.

One test is based on the detection of glycogenated epithelialcells using a periodic acid-Schiff (PAS) reagent. This reagent willstain glycogen in the cytoplasm a magenta color, and the intensityof the color is rated to determine the concentration of cells.However, since glycogenation varies based on themenstrual cycle,this test is not very reliable. Also, some females will likely show noglycogenated cells if they are pre-pubescent or postmenopausal, sothis technique can easily have false negative results. False positiveresults can emerge from the mouth or urethral tract in males.Finally, the test uses a large amount of sample and will destroyvaluable DNA evidence [10].Another older method involves an enzyme known as vaginalpeptidase that has been found in vaginal uid samples. Thetechnique uses electrophoresis in a starch gel at a pH of 7.4 inwhich the vaginal peptidase hydrolyses the dipeptide substrate L-valyl-L-leucine. It was found that no other body uid showedpositive results using this method, and the vaginal uid samplestested positive in 64% of the cases. Positive results were alsoobtained for mixtures of vaginal uid and semen or blood [103].Other studies have suggested additional possible components forvaginal uid originating from the epithelial linings such asesterase, alkaline phosphatase, b-glucuronidase, and DPNH-diaphorase [8].

Another study investigated whether oestrogen receptors couldbe detected in vaginal uid samples using monoclonal antibodiesvia immunohistochemical techniques. Samples were tested fromliving females and female corpses as well as male prepuces andurethral mucosa. Oestrogen receptors were detected in all vaginalbiopsy samples taken from the living females regardless of age,though the supercial layer showed no positive results. Thepostmortem female samples also showed no positive results evenwith a short postmortem interval of only 8 h. False positive resultswere obtained from the male prepuce and urethral mucosasamples [104].

Finally, the ratio of lactic acid and citric acid present in vaginaluid can be compared to the ratio found in semen to identify thepresence of vaginal uid either by itself or in a mixture. Lactic acidis present in large quantities in vaginal uid when compared tosemen, and semen has larger quantities of citric acid. Capillaryisotachophoresis was used for the assay, and the results showedthat all semen samples had a much higher concentration of citratecompared to lactate, and vaginal uid samples showed theopposite. The levels of citrate present in post-coital vaginal uidsamples decreased with time which shows the diminishingpresence of semen in the samples. Saliva and urine showed smallamounts of both carboxylic acids, but there were not enough tocause confusion among the semen and vaginal uid samples [105].


Almost all of the recently developed techniques for vaginal uidare based on mRNA markers and can also be applied to the otherbody uids already discussed. Juusola and Ballantynes method ofmultiplex mRNA proling using RT-PCR [41] can identify vaginaluid based on the presence of the human beta-defensin 1 (HBD-1)and mucin 4 (MUC4) markers. These genes were also amplied inmenstrual blood, but they were not detected in vascular blood,semen, or saliva. This uid had the least sensitivitywhen comparedto blood, semen, and saliva, requiring 12 ng of input RNA toidentify both genes [42]. Haas et al. used the same two markerswith success on 15 year old stains [6]. The MUC4 gene was alsodetected using the mRNA and DNA co-isolation techniquedeveloped by Alvarez et al. [39] as well as the RT-PCR techniquepresented by Nussbaumer et al. [7].

One additional new technique that has not yet been discussed isthe detection of 17b-estradiol (E2-17b) which has the strongestoestrogenic activity of all the female hormones [106]. It wasstrongly detected in all 6 extracts from vaginal uid stains usingGCMS and only in 1 of 10 semen stains and 1 of 9 female salivastains. A commercial ELISA kit utilizing a monoclonal antibody toE2-17b was also tested and had similar results to the GCMSexperiment. There were some false positive results from semen,female urine, and female saliva, and there were other problemswith the test such as the sample adhering to the glass tube duringpreparation and a prozone phenomenon of the antigenantibodyreaction. This hormone has potential to identify vaginal uid stainsafter further experimentation [106].

K. Virkler, I.K. Lednev / Forensic Science International 188 (2009) 117127. Urine


Like vaginal uid, urine is a difcult body uid to detect due tolow sensitivity of the available tests and false positive results. It canbe useful in sexual assault, harassment, and mischief cases. Thenature of the uid causes it to spread out and become diluted onfabric surfaces, and this makes it hard to nd. Odor can be anindicator, but it will cover an entire item and not localize itself tothe stained area [10]. This section will mention the many differenttechniques that have been established to identify presumptively aurine stain.

As with other uids mentioned, urine will also uoresce whenexposed to ultraviolet light [8]. This is not specic, of course, andurine is especially hard to locate since the stains tend to be moredilute than other body uids and this makes them uoresce muchless [10]. Other basic microscopic methods have been establishedwhich rely on solids in the sample which consist of variouscrystalline materials and epithelial cells characteristic of theurinary tract linings. This only gives useful results in liquid samplesas opposed to stains, however, so there is limited usefulness toforensic cases. Inorganic ions such as chloride, phosphate, andsulfate have also been used to identify urine. These ions are notunique to urine, but phosphate and sulfate are present in muchhigher concentrations than in other body uids [8].

Urea is an organic compound that is found in higherconcentrations in urine than any other body uid. Tests forurea depend on the activity of the enzyme urease which releasesammonia and carbon dioxide upon breaking down urea. Theammonia can then be detected by either using Nesslers reagentas an indicator or p-dimethylaminocinnamaldehyde (DMAC)[10]. Another method that uses a spray reagent containing ureaseand bromothymol blue has been performed to identify urinestains. A positive result will yield a blue spot at the crime scene[107]. Another technique using urease and bromothymol blue isradial gel diffusion. This method is both qualitative andquantitative with the diameter of the reaction circle beingproportional to the square root of the urea concentration. Semenand sweat stains showed slight false positive results due to theirurea content [108].

Creatinine is another organic compound that is present in highconcentrations in urine. This can be detected using a solution ofpicric acid in toluene or benzene on an extract taken from aquestioned stain. The result in the presence of urine is creatininepicrate which is a red product [10]. This is known as the Jaffe test[8,14]. The Salkowski test, which uses sodium nitroprusside, willyield a blue product when the reagent is heated with creatinine.Potassium ferricyanide acts as an oxidizing agent. O-nitrobenzal-dehyde can also be used [8,12].

An immunological method that has been attempted to identifyurine in stains involves the detection of Tamm-Horsfall glycopro-tein (THP). It is specic to urine, but is also present in other animalspecies [10]. One method involves two monoclonal antibodies toTHP called THP-27 and THP-22. A sandwich ELISA technique withTHP-27 as a capture antibody and THP-22 as a detector antibodyshows no false positive results from other body uids [109]. Asimilar ELISA technique using goat anti-human uramucoid alsoshowed promising results even on samples 3months old, and otherthan cross reactivity with some primate species, it was specic tohuman urine [110]. THP has also been detected in urine stainsusing a solid phase radioimmunoassay. This technique usesafnity-puried rabbit anti-human THP, and any positive resultsfromother body uidswere less than half that of a 1:100 dilution ofurine. The crab-eater monkey urine was the only signicantpositive result from other species [111].A different technique is based on the ratio of uric acid (UA) tourea nitrogen (UN) found in urine. Commercial tests kits utilizinguricase-peroxidase and urease-indophenol were used to quantifyUA and UN, respectively. When compared to other body uids andplant juices,most had neither UA nor UN, and none contained both.Other species of urine had ratios that were either much lower thanthe human urine average or they were much higher. The centers ofthe urine stains tested were found to have a much higher UAcontent than the edges, so it is important not to use the peripheralpart of the stain when doing an analysis [112].

As with semen and saliva, the use of SEM coupled with EDX canidentify the relative concentrations of sodium, phosphorus, sulfur,chlorine, potassium, calcium, and othermetal trace elements in theurine sample. Urine stains were made on handkerchiefs for thepurpose of the experiment. Urine showed amuch stronger chlorinepeak than the other uids, and was lacking the calcium peak. Aspreviously mentioned, this technique can only be used to screen asample and determine if it is identical to another. The dominanceof the background spectrum and subsequent subtraction will losevaluable data about the uid [69].

Finally, some other instrumental techniques have been used tostudy urine stains. These methods revolve around identifying 17-ketosteroid conjugates found in urine, and one study used high-performance liquid chromatography (HPLC) [113]. A spectro-photometer at 380 nm measured results, and ve conjugates weredetected in thehumanurine sampleswhile only tracesof someweredetected in animal samples. Electrospray ionization liquid chroma-tographymass spectrometry (ESI-LCMS) was used to conrmthese results [114] with a lower detection limit for each conjugatebeing 10 ng in scan mode [115]. Some older paper chromatographyand thin-layer chromatography methods have also been suggestedto identify components of urine such as urea, creatinine, urochrome,indican, purine and pyrimidine bases, and steroid derivatives [8].


There have been no recent publications on new techniques toidentify urine stains since Nakazono et al. presented their methodof HPLC and ESI-LCMS in 2002 [114], but they did expandupon that project in 2008. The new technique involved the sameHPLC and ESI-LSMS analysis, but it was carried out simulta-neously as DNA extraction on the same stain sample. This allowedless evidence to be destroyed via testing which is a positiveattribute of any forensic analysis. It is noteworthy that about twiceas much sample was needed to identify simultaneouslymale urinealong with DNA analysis as opposed to female urine due to theabundance of cells in female samples [116].

8. Sweat


Sweat is the least common body uid found at crime scenes incomparison to the other ve that have been mentioned, but it cancontain DNA evidence and has been extracted from sweatbands inhats and waistbands in pants [12]. It is similar in composition tourine but contains less urea and creatinine [8].

The only current technique that has been developed topresumptively test for the presence of sweat is the previouslymentioned SEM coupled with EDX that can identify the relativeconcentrations of sodium, phosphorus, sulfur, chlorine, potassium,calcium, and other metal traces. The sweat analysis showed thatchlorine and sodium were the only consistently clear peaksamong the different samples, and potassium was sometimesvisible. The large chlorine peak is used as the basis of comparisonand identication [69].

scene of a crime without needing lengthy laboratory testing. Theremay be a need for duplicate testing in the laboratory in thebeginning stages of the application of these methods to show thatthe results are reliable and suitable for court testimony, buteventually the new methods should be able to produce acceptableresults on site that are automatic and simple for a crime sceneinvestigator or police ofcer to use.

9.1. Fluorescence spectroscopy

Fluorescence spectroscopy is a sensitive technique that relieson the presence of a uorophore, which is a chemical group in ananalyte that absorbs radiation (normally in the ultraviolet region)and emits light with a specic, longer wavelength. This techniqueuses no damaging reagents and does not cause contact with thesample, but its non-destructive nature is questionable due to thepossibility of photodegradation upon exposure to ultraviolet light.A method to detect saliva using uorescence spectroscopy hasalready been discussed [102]. In addition, Powers and Lloyd [16]have demonstrated that ultraviolet uorescence spectroscopy canbe applied to identify multiple body uids. The ability of any singletest to be applied to multiple uids is a valuable characteristic

K. Virkler, I.K. Lednev / Forensic Science International 188 (2009) 117 138.2. Emerging techniques

In 2003 a study was released that involved the production of amonoclonal antibody for a sweat-specic protein [117]. The G-81monoclonal antibody was chosen since it was specic to sweat butnot to other body uids such as serum, saliva, semen, milk, urine,and tears using an ELISA technique. Fig. 3 shows the specicity ofthis technique for the identication of sweat. G-81 only reactedwith sweat glands in the skinwhen an immunohistochemical stainwas used on different tissues. Using a Western blot, the G-81antibody only reacted to the specic peptides in sweat and not theother body uids thatwere examined. An antibiotic peptide knownas dermcidin was found to have an identical 18-amino acidsegment as the N-terminus of a peptide that reacted with G-81.Finally, ELISA analysis was able to detect the G-81 reactive peptidein sweat samples subjected to an 8192-fold dilution, and storage ofsamples at room temperature for 11 weeks still gave positiveresults [117].

9. Non-destructive conrmatory identication of body uids

Table 2 reveals that many of the current methods of body uididentication use presumptive and destructive chemical tests toidentify certain components in each uid. Looking at Table 3, it is

Fig. 3. A sweat-specic protein was discovered, and a monoclonal antibody (G-81)was developed for this protein. The G-81 antibody specically forms a colored

complex with sweat antigens. Absorbance measurements are shown for sweat (1),

plasma (2), semen (3), saliva (4), milk (5), tears (6), and urine (7) at 492 nm after

analysis by ELISA. The G-81-reactive protein was stable for up to 11weeks in stains.

(With kind permission from Springer Science + Business Media [117].)evident that many of the emerging techniques use mRNAmethodswhich are very specic and conrmatory in nature but stilldestructive to the sample. Unless a DNA prole is simultaneouslyanalyzed as proposed by Alvarez et al. [39], additional samplematerial will be needed to perform a second DNA test. Aspreviously mentioned, the ability to identify body uids at a crimescene in a non-destructive manner is imperative in order topreserve the sample and DNA evidence. In addition to being non-destructive, a test that can be performed on site as opposed tobeing limited to the laboratory is very important because itprovides instant feedback. This gives investigators valuableinformation about the unknown stain almost instantly so theycan alter their investigation accordingly. There have been a fewmethods already mentioned that are non-destructive, but twomore in particular show the best ability to conrm the presence ofeach uid. These two new procedures involving uorescencespectroscopy and Raman spectroscopy are still in the experimentalstages of development and are not currently being used for forensictesting. The hope is that these techniques will ultimately beaccepted by the forensic community and will be able to deliverconrmatory, non-destructive identication of body uids at thesince it eliminates the guesswork regarding what test to use basedon what the unknown uid might be. Many components found inbody uids can exhibit uorescence such as nucleic acids, proteinsand lipids, metabolite breakdownproducts in urine, heme in blood,and dried semen as a whole. The individual composition of each ofthe uids (see Table 1) is unique, and their chemical ngerprintwill correspond to characteristic emission spectra. Fig. 4 shows theemission spectra of semen, blood, saliva, and urine measured withan excitation wavelength of 260 nm [16]. The lowest excitationwavelength used was 260 nm, and it is unclear whether thiswavelength will damage DNA evidence similar to the damageobserved in another study using a 255 nm excitation [20]. Thistechnique is ideal since it can quickly scan a large region of stainedmaterial and is very sensitive. The use of multiple excitationwavelengths and the detection of uorescence over awide range ofwavelengths allows for the identication of a particular body uidwithout the overlap of characteristics [16].

Another independent study introduces a handheld uorescenceinstrument that could potentially be used at a crime scene to giveinstant feedback about the identity of body uids [118]. Theinstrument was designed in 2003 to detect microbial contamina-

Fig. 4. The uorescence emission spectra of semen (), skin oil (--), blood (- - -),saliva (--), and urine () excited at 260 nm. This technique uses no destructivereagents and makes no contact with the sample. The emission lines are broad and

the sample must be analyzed at several wavelengths to be distinguishable from

other body uids. No data is available about the possible photodegradation from

prolonged exposure to 260-nm UV light. (Adapted from [16] with permission from

the author.)

tion, but its high sensitivity wouldmake it ideal for the applicationof body uid detection when merged with the right software.

9.2. Raman spectroscopy

Raman spectroscopy is another non-destructive bioanalyticaltechnique which has a great potential for conrmatory identica-tion of body uids at a crime scene [15]. Raman spectroscopy,when compared to uorescence spectroscopy, exhibits muchhigher selectivity and specicity to chemical and biochemicalspecies despite having a lower sensitivity, and it could potentiallybe useful in resolving mixtures of multiple body uids. The theorybehind Raman spectroscopy is based on the inelastic scattering oflow-intensity, non-destructive laser light by a solid, liquid or gassample. Very little or no sample preparation is needed, and therequired amount of material tested with a Raman microscope canbe as low as several picograms or femtoliters. A typical Ramanspectrum consists of several narrow bands and provides a uniquevibrational signature of the material [119]. Unlike IR absorptionspectroscopy, another type of vibrational spectroscopy, Ramanspectroscopy shows very little interference from

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