An overview of presumptive and confirmatory testing in the forensic sciences

The Amanda Knox/Raffaele Sollecito case introduced some observers to the uses and misuses of presumptive and confirmatory blood tests. The use of two stages of testing is encountered in at least two forensic disciplines:  testing for body fluids (blood, saliva, and semen), and testing for illicit drugs.  Some clinical testing also employs two-stage testing.  Focusing on blood among body fluids and cocaine among illicit drugs, this article will cover the basic theory of testing and introduce some problems in the criminal justice system surrounding presumptive and confirmatory testing.


Sensitivity and specificity in clinical or forensic tests

Altman and Bland defined sensitivity as “the proportion of true positives that are correctly identified by the test,” and they defined specificity as “the proportion of true negatives that are correctly identified by the test.”  A web site at Emery University states, “A sensitive test helps rule out disease (when the result is negative)…A very specific test rules in disease with a high degree of confidence.”


Lalkhen and McCluskey define sensitivity as the ratio of true positives to the sum of true positives plus false negatives.  They define specificity as the ratio of true negatives to the sum of true negatives plus false positives.  Lalkhen and McCluskey further state, “Although the ideal (but unrealistic) situation is for a 100% accurate test, a good alternative is to subject patients who are initially positive to a test with high sensitivity/low specificity, to a second test with low sensitivity/high specificity. In this way, nearly all of the false positives may be correctly identified as disease negative.”


Harvey Motulsky wrote, “Sensitivity measures how well the test identifies those with the disease…Specificity measures how well the test excludes those who don’t have the disease…”  He continued, “Consider a screening test for a disease that is fatal if untreated but completely treatable.  If the screen test is positive, it is followed by a more expensive test that is completely accurate and without risk.  For this screening test you want to set the sensitivity very high, even at expense of a low specificity.  This ensures that you will have few false negatives but many false positives.  That’s OK.  False positives aren’t so bad, they just result in a need for a more expensive and more accurate (but safe) test.”


Presumptive tests must generally be sensitive and confirmatory tests must generally be specific.  Kobilinsky, Liotti, and Oeser-Sweat wrote, “Initially a presumptive screening test is used to identify the evidence.  Presumptive tests are usually sensitive but not specific, and thus small amounts of the substance can be detected.  False-negative results are very uncommon unless the amount of specimen is so small as to go undetected.  However, occasionally, false-positive results that could potentially be misleading may be observed.  Presumptive tests are useful as preliminary screening procedures that reduce the number of items that would otherwise have to be analyzed.  Substances that provide negative presumptive results are not tested further.  Presumptive tests that are positive should always be followed by confirmatory tests.  The latter are less sensitive but more specific and therefore results are more reliable.” (p. 34)


It is possible to use receiver-operating characteristic curves to visualize the tradeoff between sensitivity and selectivity, but to the best of the author’s knowledge, this has not been done in forensic testing of drugs or body fluids (see below).  Sensitivity and specificity should be distinguished from positive predictive value and negative predictive value.  The former two depend only upon the test, but latter two depend on the test and also the prevalence of the condition in the population.  The definition of the word “specificity” in forensic or clinical chemistry is not necessarily the same as the statistical definition presented above; moreover, it has been the subject of debate (Pardue).  The sensitivities of presumptive tests for blood are usually given in terms of the dilution at which blood is still detectable; in the author’s view this definition is close to what is meant by the limit of detection (Armbruster and Pry).


Presumptive and confirmatory tests in body fluid analysis

In a 2009 review article Virkler and Lednev wrote, “Each of these fluids has one or more screening tests that are presumptive in nature, and some of them have confirmatory tests that will conclusively identify their presence.  There are also some tests which can identify the species of a particular fluid, and these are also considered to be confirmatory.”  They continued, “Once a positive result is obtained from a presumptive blood test, several confirmatory tests are available to identify absolutely an unknown stain to be blood.”  The use of the word screening suggests the dual function of presumptive tests, to identify some stains for further analysis and to identify others as being of no further interest for the questioned substance.  The same logic that applies to blood applies to other body fluids.  In 2014 Tobe, Vennemann and their collaborators wrote, “Such testing strategies [for blood and semen] provide easy-to-use, low-cost and time efficient tools for forensic science. However, detailed knowledge of specificity and sensitivity of presumptive tests is crucial for correct interpretation and inclusion into forensic investigations.”


A positive result from a presumptive test for blood indicates the possibility that blood is present.  A positive result from a confirmatory test allows one to conclude that blood is present.  The Amanda Knox/Raffaele Sollecito case illustrated the problem of relying upon one positive presumptive test for blood (luminol) while ignoring negative results from another presumptive test, tetramethylbenzidine.  The Gregory Taylor case illustrated the situation that arises when a presumptive test for blood is positive, the confirmatory test is negative, and the laboratory chooses to obfuscate.  In the Lindy and Michael Chamberlain case, negative results from a presumptive test were ignored, and positive results were claimed to indicate that blood was present.  Furthermore some positive results from the presumptive test came from an inaccessible location in the Chamberlain’s car, a result that should have caused the forensic workers to reassess their conclusions but did not.  Based upon these cases, one might imagine that the limitations of presumptive testing for blood are well understood.  However not every laboratory performs confirmatory testing for blood; instead, some move straight into DNA profiling, as noted by Castro and Coyle.


No one can be certain of the future directions that confirmatory testing for body fluids may take.  Virkler and Lednev suggested that quantitation of messenger ribonucleic acid, mRNA, may be used.  In this technique RNA is reverse-transcribed into DNA, and the DNA is amplified and quantitated by the polymerase chain reaction.  Because RNA is generally more prone to degradation and because there is an extra step, relative to the quantitation of DNA, this technique requires carefully executed controls, or the experiment will provide misleading information.  The laboratory which undertook the RNA testing in the Mark Lundy retrial in New Zealand may have detected DNA, which is not useful in identifying a particular body fluid.



Presumptive and confirmatory tests in drug analysis

On page 223 of Forensic Chemistry Suzanne Bell wrote, “Drug analysis, and to a lesser extent, toxicology utilize traditional color-based presumptive testing, targeting both drugs and diluents.14  In a recent survey, 86% of responding laboratories reported using spot testing (another term used to describe presumptive testing) for drug analysis”  After visual screening and presumptive testing, is “a definitive identification of the controlled substance or a determination that the sample does not contain one at detectable levels.”


Changes of color are part of many presumptive tests in drug analysis, and the competent analyst must be keenly aware of the details of the protocol.  On pages 225-226 Suzanne Bell sounded an important note of caution:  “The time of contact is an important consideration in color testing in general; leaving samples in the reagents, many of which contain strong acids, can lead to colors unrelated to the presence of the target analyte.”  The Kastle-Meyer presumptive test for hemoglobin will give a false positive for blood if the incubation time is too long.  Another problem with color-change tests is that there is often some subjectivity in reading it.  Alan Harris wrote, “Two major concerns with the tests themselves are a high risk of false positives and a high risk of human error in administration.”  Harris continued, “Given that these are ‘field tests,’ it is entirely likely, and possibly common, that they would be done under poor visibility conditions—such as at night under streetlights or near police cruisers with their lights on.”


Some presumptive tests for cocaine use cobalt thiocyanate; one such test is the Scott test (Bell).  John Kelly was highly critical of U.S. District Court Judge William Alsup for declaring that the cobalt thiocyanate test is a confirmatory test, and rightly so.  This test produces false positives both in the laboratory and in the field.  John Kelly quoted a United States Army forensic chemist who stated in 1982 that, “As a footnote, twenty to thirty percent of all substances initially field tested positive for a drug and subsequently submitted to this laboratory for analysis are devoid of any drugs or contain a drug different than the one indicated by the field test.”  David Lohr mentioned a Nevada study that indicated that thirty-three percent of field tests gave false positives, although it is unclear from his report whether this refers to tests for cocaine or includes other illicit drugs.


Kelly sketched the case of Janet Lee, in which flour gave a false positive.  She spent three weeks in jail before further testing showed that the substance was indeed flour.  David Lohr offered the case of truck drivers Gale Griffin and her husband Wendell Harvey, in which baking soda gave a false positive.  Ms. Griffin and Mr. Harvey spent eight weeks in jail; their truck was damaged; and their security clearances have yet to be reinstated.  In some instances it is well understood why a given chemical would give a false positive in the Scott test.  A number of compounds produce the same blue color at the end of th test (Tsumura).  Yet sodium bicarbonate does not resemble these compounds in the slightest.  Flour is a complex mixture, and one can only speculate as to what compound might have given rise to this false positive.


In some ways Ms. Lee and Ms. Griffin were lucky.  Ryan Gabrielson and Topher Sanders recounted the story of Amy Albritton, who was arrested in Harris County, which includes the city of Houston, Texas.  Her lawyer seemed to think that a presumptive test proved the presence of cocaine. According to Barry Scheck field testing was intended to minimize testing backlogs at crime laboratories.  Such a policy assumes that innocent people would not plead guilty after a false positive field test.  As Mr. Scheck pointed out however, empirical evidence from many cases in Harris County in Texas refuted this belief, and indeed it is unreasonable to expect the average person to be aware of the difference between a presumptive test and a confirmatory one.  Upon being apprised of the problem of guilty pleas based on incorrect presumptive test results, DA Devon Anderson then implemented a policy that did not allow for guilty plea deals unless a positive confirmatory result was also observed.  This is a sensible reform.


Chromatography and microcrystalline tests

Chromatography is a family of techniques that separates substances based upon their chemical or physical characteristics.  The characteristics might be boiling point, polarity, charge, size, or other chemical or physical properties.  The distance a compound move or the time it takes the compound to move a defined distance is often measured as part of a chromatographic experiment.  Types of chromatography include gas chromatography, liquid chromatography, and thin-layer chromatography.  Chromatography may be performed as part of either a presumptive or confirmatory test.  On their own chromatography tests may be employed as screening tests for drugs.  This time or distance of migration of a compound has some limited value in identifying the substance, but it is prone to misinterpretation.  In the Patricia Stallings case chromatography results misidentified propionic acid as ethylene glycol and nearly put an innocent woman away for life.  A single chromatographic experiment is insufficient to identify a compound with a high degree of certainty.  Likewise, the morphology of a small crystal (microcrystalline tests) should not be used as confirmatory test.


Conclusions and suggestions for reforms

Some high profile cases, such as those of Lindy and Michael Chamberlain, Amanda Knox and Raffaele Sollecito, and Gregory Taylor have illustrated the pitfalls of incorrectly interpreted presumptive tests for blood.  Yet the lessons of these cases are not being entirely heeded.  Moreover in terms of quantity, the far more significant problem is probably overinterpretation of presumptive tests for illicit drugs, as pointed out by Ryan Gabrielson and Topher Sanders.


With respect to presumptive testing for illicit drugs, one might specify a maximum time that a person could be held on the basis of a positive result, such as seventy-two hours.  If a confirmatory test were not run or if it were negative, then the person would have to be released.  Alternatively, one might mandate that a positive result would not be grounds for arrest but only for taking a sample for confirmatory testing.  This might alleviate one problem, that of incorrectly performed field tests, a problem noted by Gabrielson and Sanders.  One could exclude the results of presumptive drug or body fluid tests in criminal trials unless they are supported by confirmatory tests; some states do this with respect to testing for blood (Harris).  However, this reform would not be of any use in cases that are decided by plea bargain.  The Harris County policy of not accepting plea deals without a confirmatory test (see above) could be made mandatory across the country.


Web Resources

A discussion of positive and negative predictive value can be found here.

This site (from the North Carolina Indigent Defense Services) has information on a variety of forensic sciences and also has a terminology section.

The author’s website has several discussions of presumptive and confirmatory testing for blood and a treatment of the Patricia Stallings case.



Douglas G. Altman and J. Martin Bland, “Diagnostic Tests 1:  sensitivity and specificity” 1994 British Medical Journal 308 1552-1553.


David A. Armbruster and Terry Pry “Limit of Blank, Limit of Detection and Limit of Quantitation” 2008 Clinical Biochemical Review 29 S49-52.


Suzanne Bell Forensic Chemistry, 2nd ed. 2013, Pearson.


Diane Castro and Heather Coyle, “Review: Biological Evidence Collection and Forensic Blood Identification” unpublished manuscript.


Ryan Gabrielson and Topher Sanders, “Busted” The New York Times Magazine, 7 July 2016.


Alan Harris, “A Test of a Different Color:  The Limited Value of Presumptive Field Drug Tests and Why That Value Demands Their Exclusion from Trial” 2011 Southwestern Law Review 40 531-550.


John Kelly, “False Positives Equal False Justice” California Attorneys for Criminal Justice (2008).


Wendy Koen and C. Michael Bowers, eds., Forensic Science Reform 2017 Elsevier.


Abdul Ghaaliq Lalkhen and Anthony McCluskey,“Clinical tests: sensitivity and specificity” Continuing Education in Anaesthesia, Critical Care & Pain 2008 8(6) 221-223.


David Lohr, “Unreliable Field Drug Tests Result In Innocent People Pleading Guilty” The Huffington Post 18 November 2016.


Harvey Motulsky Intuitive Biostatistics, 3rd ed. 2013, Oxford University Press.


Lawrence Kobilinsky, Thomas Liotti, Jamel L. Oeser-Sweat, and James D. Watson, DNA Forensic and Legal Applications, 2004, Wiley-Interscience.


Harry L. Pardue “Counterpoint The inseparable triad: analytical sensitivity, measurement uncertainty, and quantitative resolution” Clinical Chemistry 1997 43(10) 1831-1837.


Richard Saferstein, Criminalistics:  An introduction to forensic science, 9th ed. Pearson Prentice Hall (2009)


Barry Scheck, “Unreliable Field Drug Tests Result In Innocent People Pleading Guilty” The Huffington Post, 15 July 2016.


Yukari Tsumura et al., “False positives and false negatives with a cocaine-specific field test and modification of test protocol to reduce false decision” 2005 Forensic Science International 155 158-164.


Marielle Venneman, Georgina Scott, Lynn Curran, Felix Bittner, and Shanan S. Tobe “Sensivity and specificity of presumptive tests for blood, saliva, and semen” (2014) Forensic Science Med Pathol 10 69-75.


Kelly Virkler and Igor K. Lednev, “Analysis of body fluids for forensic purposes:  From laboratory to non-destructive rapid confirmatory identification at a crime scene” 2009 Forensic Science International 188, 1-17.

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