Scope and History of Forensic Serology

The word serology comes from serum, the fluid fraction of blood that carries antibodies, and historically the discipline was heavily focused on blood-group antigens and antibodies. The forensic version widened that focus to all biological fluids that appear at crime scenes and that carry markers capable of being characterised. In practice this means blood (liquid, dry, or degraded), semen, saliva, vaginal secretions, urine, faeces, and, in some laboratories, perspiration and breast milk.

The discipline is best understood by its position in a two-stage workflow. Serology identifies what a stain is and eliminates impossible sources before DNA analysis attempts to identify who. That sequencing matters for two reasons: first, it avoids wasting DNA extraction on a stain that turns out to be canine blood or rust; second, it preserves the stain as much as possible by starting with non-destructive or minimally destructive tests.

Landsteiner's 1901 paper in Wiener Klinische Wochenschrift described the haemagglutination pattern, the way certain blood samples clump together and others do not. He sorted the pattern into three groups by 1901 and a fourth (AB) was identified in 1902 by two of his colleagues, Alfred von Decastello and Adriano Sturli. Within a decade the system was being applied in paternity disputes in Germany and Austria, and by the 1920s forensic scientists in Europe and Japan had begun testing dried bloodstains for ABO type.

The most influential early forensic application came through the work of Leone Lattes in Italy. His 1916 method for recovering ABO antigens from dried bloodstains made it possible, for the first time, to type a stain found at a scene and compare it to a suspect's blood. The Lattes crust test used an elution approach, washing antigens off dried red cells, and it remained in use in European laboratories for decades.

The ABO system's forensic utility is statistical: group O occurs in roughly 44% of Northern Europeans, A in about 42%, B in 10%, and AB in 4%. A stain typed as group B eliminates all group A, O, and AB individuals, which in many suspect comparisons is a meaningful exclusion. Millions of people share any given ABO group, so typing could exclude but could rarely individualise. That limitation drove the search for more polymorphic markers through the mid-twentieth century, and the discrimination power that Alec Jeffreys' 1984 DNA profiling delivered resolved it decisively.

The precipitin reaction was described by Rudolf Kraus in 1897, but it was Paul Uhlenhuth, a bacteriologist working at the Imperial Health Office in Berlin, who in 1901 turned it into a practical species test. Uhlenhuth immunised rabbits with human serum, producing antisera that precipitated human protein but not animal protein when mixed with a test extract. His first forensic application was the Tessnow case, where he tested stains on a carpenter's clothing suspected in the murders of two children in Rugen in 1901 and confirmed them as human blood.

The precipitin test was adopted across European police laboratories within a decade and remained the primary species-confirmation test for most of the twentieth century. Modern lateral-flow immunoassay cards work on the same antibody-antigen principle but deliver a result in two to five minutes rather than requiring overnight incubation. Cards validated for human haemoglobin, human IgG, or prostate-specific antigen (PSA) are now standard in many jurisdictions.

After the ABO system, researchers discovered additional red-cell antigen systems with forensic relevance. The MN system was described by Landsteiner and Philip Levine in 1927; the S antigen, which extended it to MNSs, was not discovered until 1947 by Walsh and Montgomery. The Rhesus (Rh) system was identified in 1940 by Landsteiner and Alexander Wiener. Combined typing using ABO, Rh, MNSs, and other systems reduced the frequency of any given combination but still could not approach the discrimination power that DNA would later deliver.

A parallel development was the systematic study of secretors by Wilhelm Schiff and others in the 1920s and 1930s. Roughly 80% of the population secrete blood-group antigens into body fluids: saliva, semen, sweat, and vaginal secretions. This meant that a swab from a bite mark or a semen stain could, in many cases, yield ABO grouping information without requiring a blood sample. Secretor analysis became routine in sexual assault casework through the 1950s to 1980s.

By the 1970s, a well-equipped serology laboratory could type a stain at five or six genetic marker systems and produce combined frequencies of one in several hundred or one in a few thousand. This was useful for including or excluding suspects. But it still left thousands of possible contributors in any large city, and it required a relatively well-preserved stain. Degraded samples often yielded only ABO, if anything at all.

In September 1984, Alec Jeffreys at the University of Leicester produced the first DNA fingerprint, using restriction fragment length polymorphism (RFLP) analysis. The first forensic use came in 1986, in the investigation of two murders in Leicestershire. Jeffreys' technique produced discrimination powers that made all previous serological markers look coarse. By the early 1990s, PCR-based STR profiling had made DNA analysis faster, more sensitive to small or degraded samples, and amenable to database matching.

The consequence for serology was a reshaping of purpose rather than an elimination. Serology became the gateway: identify the fluid, confirm it is human, and direct the most informative sample to the DNA laboratory. Where once a serologist might spend a day typing a stain through a battery of blood-group markers, the modern workflow often terminates serological testing after a confirmatory species test and routes the sample onward. Serology expanded rather than contracted: lateral-flow PSA tests for semen, amylase tests for saliva, and vaginal epithelial cell protocols were refined through the 1990s and 2000s to give DNA analysts a clean characterisation of what they are extracting from.

Today's forensic serology laboratory uses a tiered toolkit. Presumptive tests for blood include catalytic colour tests: the leucomalachite green (LMG) test, Kastle-Meyer (KM) phenolphthalein test, and luminol or Bluestar for large-area scenes with low-visibility stains. All exploit the peroxidase-like activity of haemoglobin. None is specific to blood, and all can give false positives from plant peroxidases, bleach, or certain metallic salts. They are fast screening tools, not conclusions.

Confirmatory identification of human blood is achieved with lateral-flow haemoglobin cards validated to a defined sensitivity threshold. Semen is confirmed either by the Christmas tree stain (Kernechtrot-Picroindigocarmine) identifying spermatozoa under microscopy, or by PSA (p30) lateral-flow cards when azoospermic donors are possible. Saliva is confirmed by amylase testing, with alpha-amylase activity measured enzymatically, though saliva tests are still regarded as presumptive by many laboratories given the ubiquity of salivary amylase in the environment.

• Leucomalachite green and Kastle-Meyer tests: rapid colorimetric presumptive tests for blood, exploiting haemoglobin peroxidase activity.
• Luminol and Bluestar: chemiluminescent presumptive tests for blood on large surfaces or after cleaning attempts, visible in low light.
• Haemoglobin lateral-flow cards: confirmatory human-blood test; species-specific antibody produces a visible line within minutes.
• PSA (p30) cards and Christmas tree stain: confirmatory semen identification by prostate-specific antigen or spermatozoa morphology.
• Alpha-amylase assay: presumptive-to-confirmatory test for saliva; high activity levels indicate salivary origin.

Accreditation under ISO/IEC 17025 now requires that each test used in casework has a defined validation record covering sensitivity, specificity, and interference substances. This is not merely bureaucratic: a defence expert who demonstrates that a confirmatory test cross-reacts with a common household substance under the conditions found in the case can undermine the entire downstream DNA result. Rigorous serology underpins the reliability of the DNA result that follows.