Techniques for Determining Blood Groups from Bloodstains

Liquid blood is grouped by adding a drop of anti-A and anti-B to two drops of cell suspension and reading the clumping in seconds. None of that works on a dried stain. Five things have changed by the time the stain reaches the lab.

1. Red cells have lysed. The classical agglutinin endpoint needs intact red cells; antigen on stromal fragments does not clump in the textbook way.
2. Antigens have partly denatured. Heat, sunlight and time degrade A, B and H epitopes. ABO is more robust than MN, which is more robust than Rh; Rh becomes almost ungroupable on old stains.
3. Microbial overgrowth. Bacteria carry B-like antigens; a damp stain can falsely test as group B without controls.
4. Substrate interference. Cotton, wool, denim and soil all absorb antibody non-specifically.
5. Sample size. Scene stains are often a few millimetres across, with a fraction of the cell count direct grouping needs.

The classical response was to stop trying to read direct agglutination on the stain and instead use the stain as a passive antigen surface. Absorption-elution and absorption-inhibition both rely on this shift: do not look at the stain's cells, look at what the staindoesto a known antibody. Before grouping is attempted, the standard Indian CFSL workflow first confirms human blood via presumptive and confirmatory blood-stain identificationgrouping never precedes identification.

Leone Lattes, working in Turin in 1915, was the first to show that the ABO group of a dried stain could be read directly. The method bears his name and remains the entry point in every Indian forensic serology lab manual.

Principle. A small crust of the dried stain is teased into a drop of known antiserum on a glass slide. Residual intact red cell stroma carries enough A or B antigen to bind and visibly agglutinate when the matching antibody is present.

Workflow.

1. Scrape a thin flake of the dried crust with a clean scalpel.
2. Place separate flakes on two slides, one with anti-A and one with anti-B (an anti-AB control is good practice).
3. Cover, incubate at room temperature for 5 to 10 minutes.
4. Read under low-power microscopy. Clumping with anti-A only indicates group A; with both, AB; with neither, group O (or a failed test).

Limitations. Needs a relatively fresh stain, a crust thick enough to flake, and gives weak or false results on degraded samples., hold one anchor:Lattes = direct agglutination of the dried crust. The underlying antigens are those covered in the prerequisite reading on the ABO, Rh and MNSs blood group systemsthe same antisera are reused across all classical grouping techniques.

When the stain is too small or degraded for Lattes, the next step up is absorption-inhibition. The logic flips: instead of looking at the stain's cells clumping, you measure how much antibody the stain pulled out of solution.

Principle. Add a known volume of standardised antiserum of known titre to the stain. The stain's surface antigen binds and removes a proportion of the antibody. After incubation, the supernatant is titrated against indicator cells. A drop in residual titre indicates the antigen was present.

Workflow. Cut three small threads of stain (and one of unstained substrate as control). Place each in a separate tube with a fixed volume of anti-A, anti-B and anti-H. Incubate at 4 °C for 1 to 2 hours. Serially dilute the supernatant and add indicator cells (A1 cells for the anti-A tube, B cells for anti-B, O cells for anti-H). Read the end-titre. A two-tube or greater drop from the reagent's starting titre is positive.

Reading the result. Drop in anti-A and not anti-B indicates group A; drops in both indicate AB; only an anti-H titre drop indicates group O (O cells carry abundant H antigen but no A or B).

Limitations. Quantitative but indirect. Needs careful reagent control, and the substrate control is essential because cotton itself can absorb low levels of antibody.

Stuart Kind, working at the British Home Office in 1960, formalised the absorption-elution test that remains the workhorse of classical stain grouping. It is roughly an order of magnitude more sensitive than absorption-inhibition and is the benchmark against which later refinements are measured.

Principle. Three steps. (1) Absorb known antiserum onto the dried stain so any matching antibody binds the antigen. (2) Wash thoroughly so only specifically bound antibody remains. (3) Elute the bound antibody by gentle heat (typically 56 °C for 10 to 15 minutes), then test the eluate against indicator red cells. Agglutination of the indicator cells proves which antibody was bound, and therefore which antigen the stain carried.

Workflow. Cut three small threads of stain plus one substrate control. Treat each with anti-A, anti-B or anti-H at 4 °C overnight. Wash repeatedly in cold saline until the wash is antibody-free. Transfer each thread into fresh saline with the matching indicator cells. Elute at 56 °C in a water bath; the released antibody now agglutinates the indicator cells. Read after centrifugation.

Sensitivity. Reliable on stains as small as a few millimetres, including stains months or years old. The DFSS Quality Manual and most state SFSL serology SOPs list absorption-elution as the technique of choice for dried-stain ABO grouping where DNA is unavailable. The grouping result still has to ride a documented chain of custodyand survive scrutiny under Section 39 of the Bharatiya Sakshya Adhiniyam 2023.

Advantages. Most sensitive of the four classical techniques. Works on old stains. Gives a positive read (agglutination) rather than an inhibition read, which is easier to interpret in court.

When the available material is so small that even absorption-elution would not return enough antibody to assay (a few single fibres, a wood splinter, a fingernail scraping), the mixed agglutination test is the last classical option.

Principle. The same first step as absorption-elution: known antiserum is absorbed onto the stain so matching antibody binds the antigen. Instead of eluting, indicator red cells of the corresponding group are added directly to the fibre. The indicator cells coat onto the bound antibody, producing a rosette around the fibre read under low-power microscopy.

Workflow. Pick one or two fibres of the stained substrate. Soak in anti-A (or anti-B) at 4 °C for 1 to 2 hours. Wash thoroughly in cold saline. Add a drop of indicator A1 cells (or B cells). Mount on a slide and examine at 100x to 400x. A halo of indicator cells coating the fibre indicates the corresponding antigen.

When to use it. Single hairs, a few stained fibres on a button, a thread from inside a wound, fingernail clippings. The technique trades quantitative resolution for the ability to work on samples too small for any other method.

Limitations. Slow, microscope-dependent, easy to call false positives if substrate controls are sloppy. Modern Indian labs prefer to escalate such samples to low-template DNA STR analysis if the budget allows.

Indian casework today treats classical ABO grouping of a stain as a screening or triage step rather than a definitive identification. It still appears in the workflow for three reasons: (1)triage since a quick ABO group can exclude a suspect cheaply before a full STR profile is ordered; (2)old case re-openings where pre-2000 files contain only ABO data and new samples must first be matched at that level; (3)substrate constraints where heavily putrefied tissue or certain dyed textiles defeat DNA extraction but still yield a usable ABO read by absorption-elution.

The institutional frame to memorise: the DFSS(Directorate of Forensic Science Services, 2003, MHA) sets the Quality Manual;seven CFSLs plus the state SFSLs run the actual serology and DNA work;CFSL Hyderabad is the central DNA division. Section 176(3) of the Bharatiya Nagarik Suraksha Sanhita 2023 makes forensic examination mandatory at scenes of offences carrying seven years or more, increasing stain submissions across SFSLs. Section 39 of the Bharatiya Sakshya Adhiniyam 2023 makes the analyst's opinion admissible. Both statutes replaced the colonial-era CrPC and Indian Evidence Act in 2024. ABO grouping by absorption-elution still has a place; STR profiling carries the individualisation ABO cannot. Both have distinct evidentiary roles in modern casework.