Body Fluids, RBC Enzymes and Serum Proteins of Forensic Significance

The four sub-topics in this bullet (body-fluid detection, ABO grouping, RBC enzymes, serum proteins) read like a checklist because that is exactly what they were. An Indian state SFSL serologist working a sexual-assault or homicide case in the 1980s would screen the swab for acid phosphatase, confirm with microscopy or p30, type the ABO from the secretor's stain, then run a starch-gel electrophoresis to phenotype PGM, EsD and Hp. Each step shrinks the donor pool. Stacked together, the combined discriminating power of a full ABO plus six-marker enzyme and protein panel could reach roughly 1 in 1,000 to 1 in 10,000, which was respectable before STR multiplexes.

The three concept clusters to keep separate in your head:

1. Detection of the fluid(is this semen, saliva, blood, urine?). Presumptive crystal and colour tests, then confirmatory immunoassay or microscopy.
2. ABO grouping of the fluid(whose ABO type secreted into this stain?). Lewis-blood-group-system secretor / non-secretor split.
3. Individualisation by polymorphic markers(which donor inside that ABO group?). RBC isoenzymes and serum proteins, typed by electrophoresis and immunoelectrophoresis.

STR DNA typing has supplanted enzyme and protein phenotyping for individualisation wherever DNA is recoverable. The fluid-identification screens (Florence, Barberio, AP, Phadebas, RSID, ABAcard) remain the first step at every CFSL and state SFSL serology section: DNA typing is only applied to stains already confirmed as biological.

Semen identification follows a three-tier workflow used at Indian SFSLs.

Tier 1: Presumptive screening.

• Acid phosphatase (AP) test. Brentamine Fast Blue B salt plus sodium alpha-naphthyl phosphate. Purple colour in 30 to 60 seconds is presumptive positive. Sensitivity is high; specificity is moderate (vaginal fluid, some plant material can give weaker positives, so timing matters).
• Florence test. A drop of Lugol-style iodine and potassium iodide on the suspected stain extract throws dark-brown rhombic or needle-shaped choline iodide crystals under the microscope. Choline is abundant in seminal fluid.
• Barberio test. Saturated aqueous or alcoholic picric acid reacts with spermine to form yellow rhombic or needle-shaped spermine picrate crystals.

Tier 2: Confirmatory.

• Microscopy for spermatozoa. Christmas-tree stain (nuclear fast red + picroindigocarmine) colours sperm heads red and tails green. Intact sperm with head and tail is the gold standard, but azoospermic, oligospermic and vasectomised donors give no sperm at all, so a negative microscopy never excludes semen.
• p30 / PSA immunoassay. RSID-Semen and ABAcard p30 are lateral-flow immunochromatographic strips. They work even on azoospermic donors because PSA is a prostatic protein independent of sperm production. These are now the standard confirmatory at every Indian CFSL serology section. The underlying principle of the strip is covered in the immunoassays topic.

Tier 3: Individualisation. STR DNA typing on the sperm fraction (differential extraction separates sperm-cell DNA from epithelial DNA in a mixed vaginal swab). This is the workflow used in every BNSS Section 176(3) sexual-assault investigation today.

For saliva specifically, the Phadebas reagent(a dye-linked starch tablet that releases blue dye when alpha-amylase cleaves the starch) is the test most commonly cited in forensic practice MCQs. The starch-iodine alternative gives a colourless zone where amylase has digested the starch in an otherwise blue iodine-stained background. Both rely on the fact that salivary alpha-amylase is 700 to 2000 times more concentrated than the same enzyme in serum.

The ABO antigens (A, B, H) exist in two forms. Membrane-bound glycolipids are present on red cells in every ABO-typed person. Water-soluble glycoproteins are secreted into saliva, semen, sweat and other body fluids only in people who carry at least one functional Se (FUT2)allele. These people are secretors and they make up roughly 80% of the Indian population. The remaining 20% are non-secretors(genotype sese) and their body-fluid stains cannot be ABO-typed by the classical absorption-inhibition or absorption-elution methods.

Practical consequences for the serologist:

• Saliva on a cigarette butt or envelope can be ABO-typed in 80% of cases. Absorption-inhibition is the textbook method: known anti-A and anti-B sera are pre-incubated with the saliva extract, and the residual titre against test red cells indicates which antigen was present in the saliva.
• Semen from a secretor donor carries A, B or H substance in seminal plasma. From a non-secretor donor it carries none, and ABO from semen alone fails.
• Urine, sweat, vaginal fluid all follow the same secretor / non-secretor rule.

This is why the classical workflow always paired the fluid-detection step with the secretor-status step. The companion bullet on blood group systems (ABO, Rh, MNS)covers the antigen genetics in depth; the blood-grouping techniques from bloodstains bulletcovers absorption-elution and Lattes crust in detail.

Red blood cells carry a set of enzymes that are polymorphic in the population, meaning two or more allelic forms circulate at frequencies high enough that the phenotype of one stain donor differs from another with useful probability. Before STR DNA, these were the forensic individualisation markers. You separate them by electrophoresis of red-cell haemolysate on starch gel, polyacrylamide gel or by isoelectric focusing (IEF)then stain for enzyme activity in situ to visualise the band pattern.

A stain typed as PGM 2-1, EsD 1, AK 1, GLO 2-1 belongs to a much smaller fraction of the population than a single ABO group narrows down. Stacked across four polymorphic enzymes plus ABO, the discriminating power was historically respectable. The classical Indian SFSL panel was PGM + EsD + AK; some labs added GLO and ADA.

STR multiplexes have replaced this workflow for individualisation. The RBC enzyme panel is now of historical and educational interest at most operational labs; the underlying chemistry and inheritance patterns remain important reference knowledge for forensic serologists.

Plasma carries a set of polymorphic proteins separable by starch-gel, polyacrylamide-gel or isoelectric-focusing electrophoresis. The forensic three are haptoglobin, group-specific component and transferrin.

• Haptoglobin (Hp). Binds free haemoglobin released from lysed red cells; the Hp-Hb complex is cleared by liver Kupffer cells. The Hp1 and Hp2 alleles give three common phenotypes:Hp 1-1(single fast band),Hp 2-1(multiple intermediate bands),Hp 2-2(slower band pattern). Detect by haemoglobin staining of the gel (benzidine, o-tolidine) after running plasma against a known Hb standard.
• Gc (group-specific component, vitamin D binding protein). Three classical alleles:Gc1FGc1SGc2. IEF separates them sharply. Some Indian population groups show distinct allele frequencies that improved discriminating power locally.
• Transferrin (Tf). Iron transport protein. The common variant is TfC with TfB and TfD subvariants found at low frequency in specific populations. Typed by starch-gel or PAGE with iron-saturation.

Two methodological points:

1. Isoelectric focusing (IEF)is the high-resolution technique that separates phenotypes which conventional starch-gel cannot resolve. Carrier ampholytes generate a pH gradient in the gel; each protein migrates to the pH equal to its isoelectric point and stops. This is the same separation principle used for PGM subtyping. The detailed treatment is in the electrophoresis and immunoelectrophoresis bullet.
2. Modern role. STR DNA typing has replaced these markers for forensic individualisation almost everywhere. Hp, Gc and Tf phenotyping survives mainly in transfusion-medicine and paternity-exclusion contexts in India, not in operational stain casework.

The Indian operational picture in 2026:

• CFSL Hyderabad houses the country's reference serology and DNA division under DFSS. Its serology section still runs presumptive AP and crystal screens on every sexual-assault case before differential extraction and STR typing.
• State SFSLs(Maharashtra, Tamil Nadu, Karnataka, West Bengal, Kerala, UP, Punjab and others) run serology sections at every regional FSL. Most have transitioned ABO and isoenzyme work onto a "screen-only" footing and route confirmatory grouping into the DNA queue.
• AIIMS forensic medicine and major medical-college departments do the post-mortem collection of body-fluid samples that the SFSL serology section then tests, with a clean documented chain of custodyjoining the two.
• BNSS Section 176(3)since 1 July 2024, makes forensic examination mandatory at any crime-scene where the offence is punishable with seven years or more of imprisonment. For a sexual-assault investigation this means presumptive AP and PSA screens, plus DNA collection, are now legally required, not optional. The applicable evidentiary regime in court is the Bharatiya Sakshya Adhiniyam 2023.
• Method validation and proficiency testing under ISO/IEC 17025 and NABL accreditationkeep the screening tests defensible at trial even when the chemistry is a century old.

CFSL Hyderabad serves as the central DNA and serology reference lab; BNSS 176(3) drives the operational workflow; and the historical isoenzyme panel was the direct predecessor of the STR-based practice that supplanted it.