Chapter 18: Blood Group Typing and Protein Profiling

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Welcome back to the Deep Dive.

So imagine this, it's way before modern DNA testing.

How did forensic scientists actually link someone to a crime using biological clues?

Yeah, it's easy to forget there was a whole science before DNA.

Exactly.

So today we're kind of time traveling.

We're going deep into that pre -DNA world.

We're looking at the absolute bedrock blood group typing and protein profiling.

This is really where it all started.

That's right.

And if you really want to get a handle on how investigators work back then, this Deep Dive is definitely for you.

We're aiming to quickly get you up to speed on the core ideas, the key techniques, things like glutination, electrophoresis, and you know, the molecules involved.

And you really have to start with one person, Karl Landsteiner.

His discovery, the ABO system, early 1900s.

I mean, it wasn't just about making blood transfusions safe.

No, it completely changed that.

It won him a Nobel Prize in 1930.

And basically it launched the whole field of identifying people using biology.

Right.

It was the first proof really that biologically, we weren't all the same, a massive realization for forensics.

Okay, let's dive into the science then.

When we talk about a blood group, what are we actually measuring?

Is it just A, B, A, B, O,

or is there more to it?

Well, fundamentally, yes, it's about those types.

But what we're really looking at are specific molecules,

antigens on the surface of red blood cells.

Think of them as tiny biological flags.

Antigen polymorphisms, right?

Exactly.

And while the official count is like nearly 30 different blood group systems recognized now.

Wow, 30.

Yeah.

But forensically, the big ones were always ABO.

And then sometimes others like RH, MNS, KEL, Duffy, KID got used to.

Okay.

And here's maybe the key thing for crime scenes, right?

These flags, these antigens, they aren't only in the blood.

Precisely.

That's crucial.

The AB, you know, antigens, you find them in saliva, semen, other bodily fluids, even in organs like the kidney and liver.

So a blood type test wasn't just for blood stains.

Not at all.

It meant you could potentially type evidence from a sexual assault or maybe saliva from a bite mark.

Big deal.

Definitely expands the possibilities.

Yeah.

But, okay, why do we have these different types?

It comes down to just one biological pathway.

Pretty much, yeah.

Think of it like a little molecular assembly line.

Everyone starts with the same basic structure.

It's called the O antigen, or technically the H antigen.

It's a sugar structure and an enzyme called phecociltransferase builds it.

So typo blood just

stops there.

Exactly.

That's the foundation.

If you're typo, that's all you have.

Okay.

So how do you get A or B then?

Where's the split?

Ah, that depends on another enzyme, a transferase.

And which transferase you have is determined by the ABO gene, which is on chromosome nine.

Okay.

So if you inherit the A version, the A allele, your enzyme adds a specific sugar, it's N -acetylglycosamine onto that O foundation.

Boom, you're type A.

And the B version?

The B foundation plus galactose equals type B.

What about O then?

You said it stops early.

Right.

The O allele has a tiny mutation, a small dilution actually.

And that mutation completely knocks out the enzyme's activity.

It just can't add any extra sugar.

So you're left with just the basic O antigen.

Wow.

So it's literally just one sugar molecule difference between A and B.

Essentially, yes.

And the difference between the A and B enzymes themselves, it boils down to just four amino acid changes.

Positions 266 and 268 seem to be the really critical ones for determining which sugar gets added.

Tiny difference, big outcome.

Okay.

Knowing these antigens can be in saliva and semen leads us straight to something called secretor status.

This sounds like it could cause some real headaches for forensic work.

Oh, it absolutely did.

A secretor is someone, and this is about 80 % of Caucasians, for example, whose body fluids like saliva and semen actually contain the A, B or O antigens corresponding to their blood type.

So their secretions match their blood.

What about the other 20 %?

The non -secretors.

If someone's blood type is A, but they're a non -secretor, does their semen still show up as type A?

No, it doesn't.

And that's the huge complication.

There are actually two related genes involved here, the FUT genes.

FUT1 is mainly responsible for putting the O antigen foundation onto red blood cells.

But FUT2 is the gene that directs the synthesis of that same O foundation in secretory glands, the ones making saliva, semen, et cetera.

Okay.

So non -secretors have an issue with FUT2.

Exactly.

They have a specific mutation, a nonsense mutation in both copies of their FUT2 gene.

This basically shuts down the enzyme production in secretory tissues.

So let me get this straight.

A type A non -secretor has a working A enzyme.

So their blood cells are type A because FUT1 made the O foundation there.

Correct.

But because their FUT2 is inactive, their saliva and semen lack that O foundation.

So the A enzyme has nothing to stick the A sugar onto in those fluids.

You've got it.

Precisely.

So imagine a sexual assault case.

The suspect's blood is tested type A, but the semen evidence from the victim comes back negative for A antigen.

It's like an exclusion.

It looks like it.

Yeah.

But if the suspect is a type A non -secretor, that semen should be negative for A antigen.

You have to be incredibly careful interpreting that kind of result.

That is a major forensic pitfall.

Now, historically, ABO types follow Mendelian genetics, right?

A and B are dominant over O.

Yep.

Simple dominance.

That's why blood typing was used early on in basic paternity testing.

You could rule fathers out sometimes.

But what was the biggest limitation, forensically speaking,

for ABO by itself?

Just the sheer lack of power to tell people apart.

I mean, think about it.

In Caucasian populations, something like 42 % are type A, 47 % are type O.

Wow.

Yeah.

So if your crime scene stain is type O, well, you basically just implicated almost half the population.

It's not very discriminatory.

That's not great for narrowing down suspects.

Not at all.

That huge probability of a random match meant labs absolutely could not rely on just ABO.

They had to start combining it with other blood group systems and, crucially, move on to protein profiling to get better odds.

Right.

Okay, so let's talk techniques.

The crime scene reality isn't usually fresh liquid blood.

It's often a dried stain, maybe degraded.

How did they even get results from that early on?

Well, they started with Landsteiner's basic principle, agglutination.

You know, the clumping of red blood cells when they meet the right antibody.

The classic original method was called the lattes crust assay.

Lattes crust.

I'm picturing someone carefully scraping a tiny bit of dried blood off, say, a shirt.

How did they make the agglutination work with a crust?

They use the antibodies that are naturally in the blood serum.

So say the dried blood stain was type A.

That means the dried serum in the crust contains anti -B antibodies.

So they take a tiny bit of that crust, expose it to known type B indicator red cells if those B cells clumped up.

Ah, then the crust must have contained anti -B, meaning the original blood was type A.

Exactly.

It sounds pretty straightforward and it was relatively quick.

I'm guessing there's a catch.

Tried blood cells probably aren't intact, right?

They break open.

That's the big limitation.

Lysis.

When blood dries, the red cells tend to burst.

So the lattes assay needed relatively intact antibodies in the serum crust and it just wasn't very sensitive.

For old stains or really tiny ones, it often just failed, didn't work.

Okay, so they needed something better, especially those challenging dried stains.

This leads us to the more sensitive method.

Yes, the absorption elution assay.

This was developed specifically for dried stains and it's actually quite clever because it detects the antigen itself indirectly using temperature changes.

Absorption elution.

How did that work step by step?

Okay, so first you take your sample, the bit of dried stain on say a thread.

You basically immobilize the antigens that are hopefully there.

Right.

Second, you add known antibodies.

Let's say you suspect it's type A, so you add anti -A antibodies.

You do this at a low temperature, like in a fridge, which encourages the antibodies to bind to any A antigens present in the stain.

That's the absorption part.

Okay, antibodies stick to the stain if the antigen is there.

Then what?

Third, you wash away all the antibodies that didn't bind.

So only the ones stuck to the antigen remain.

Got it.

Cleaning up the unbound stuff.

Fourth, and this is the elution part, you raise the temperature maybe to around 56 degrees Celsius.

This heat causes the bound antibodies to detach, to elute from the antigen, and go back into a clean solution.

Ah, so you're collecting just the antibodies that were specifically stuck to the stain.

Exactly.

Fifth, you take that solution containing the eluted antibodies and you pest it against known type A indicator cells.

If those cells clump up, then you know the eluted liquid contained anti -A antibodies, which means must have had A antigens.

Bingo.

Much more sensitive than lattes for old, dried material.

It became the workhorse for typing stains.

Okay, so absorption elution helped with ABO on tricky samples.

But even with that, the discrimination power wasn't fantastic.

They needed more, right?

Definitely.

To get that probability of a random match down further, say from one in two down to maybe one in several hundred, they had to look beyond just blood groups.

That's where protein profiling came in.

Why protein?

Because a surprising number of our proteins, maybe 20 to 30 percent, are polymorphic, meaning their amino acid sequences vary slightly between different people.

These tiny variations could be detected.

And how do they actually separate and see these different protein versions in a tiny blood or semen sample?

This brings us to electrophoresis, doesn't it?

Electrophoresis, yeah.

That was the core technology.

Basically, you use an electric field to pull molecules through a gel matrix, separating them based on, well, usually two main things, their electrical charge and their size, or molecular weight, MR, we call it.

Okay, the source material mentions two main ways electrophoresis was used for proteins.

Let's start with the one that separates based on size,

denaturing electrophoresis.

Right.

So if you want to separate proteins only by size, you first have to kind of neutralize the other factor, which is their natural electrical charge.

How do you do that?

You treat the sample with chemicals.

First, reducing agents break down some of the protein's

structure, unfold it a bit.

Then you add a strong detergent, usually one called SDS sodium dodecyl sulfate.

SDS.

What does that do?

SDS molecules basically coat the protein, giving it a strong, uniform negative charge.

It essentially masks the protein's own natural charge.

Wait, so if everything's coated in negative charge from the SDS, how does the electric field separate them?

Isn't everything just moving towards the positive pole?

It is, but now, because the charge to mass ratio is pretty much the same for all the coated proteins, the only thing left determining how fast they move through the gel is their size.

Like a molecular sieve.

Smaller proteins wiggle through the gel faster, larger ones get tangled up more and move slower.

Exactly.

And you'd run known marker proteins alongside your sample, proteins with known molecular weights.

By comparing how far your unknown protein traveled relative to markers, you could estimate its size.

Okay, that makes sense for size.

But then there's the other technique, isoelectric focusing, IEF.

This sounds more complex.

It focuses on charge.

Yeah, IEF is really powerful.

It doesn't really care about size.

It separates proteins based purely on their isoelectric point or PI.

Isoelectric point.

What's that exactly?

Every protein has a unique pH value at which its overall net electrical charge is exactly zero.

That specific pH is its PI.

Okay.

So how does IEF use that?

Well, you create a stable pH gradient across the electrophoresis gel.

Maybe it goes from pH 3 at one end to pH 10 at the other.

You apply your protein mixture.

When you turn on the electric current, a protein will move through the gel.

If it's in a pH region below its PI, it'll have a net positive charge and move towards the negative electrode.

If it's above its PI, it'll be negative and move towards the positive electrode.

So it migrates until?

Until it reaches the exact point in the pH gradient that matches its own unique PI.

At that point, its net charge becomes zero and it just stops moving.

It focuses into a sharp band right there.

Wow.

So different proteins stop at different places based on their specific PI.

Precisely.

And because PI is very sensitive to even single amino acid changes, IEF can separate proteins that differ only very slightly.

It has much higher resolving power than just separating by size.

Okay.

So they had these powerful separation techniques.

What specific protein variations, these polymorphisms, do they actually target for forensic work?

They started looking heavily at enzymes found inside red blood cells, erythrocyte isoenzymes.

Isoenzymes just means different forms of an enzyme that do the same job but have slightly different structures and therefore potentially different charges or sizes.

And the first really big success story there was?

Fosiglucomutase, or PGM.

Detecting different variants of PGM was a major breakthrough.

Why PGM specifically?

Was it just common?

It was common, yes, but the really crucial thing was where you find it.

PGM is in red blood cells, sure, but it's also present in detectable amounts in semen.

Ah.

So PGM typing suddenly became really valuable for sexual assault cases.

Hugely valuable.

It added significant discriminating power, way beyond what ABO alone could offer in those cases.

Other common enzyme markers they used were things like erythrocyte acid phosphatase, often called ECP or ENT, and esterase D or ESD.

Beyond enzymes, hemoglobin variants also turned out to be important.

Absolutely.

Two in particular gave really useful investigative information.

Which were?

First, fetal hemoglobin, HBF.

This is the main oxygen carrier in fetuses and newborns.

It has a different structure, gamma chains instead of the adult beta chains.

And it disappears after birth, right?

It gets replaced by adult hemoglobin, HBA, usually by about six months of age.

So finding HBF in a blood stain at a crime scene, that's very strong specific evidence, often pointing towards tragic cases like infanticide or perhaps a concealed birth.

A very specific and powerful marker.

And the second key hemoglobin variant?

Hemoglobin S, HBS.

That's the variant responsible for sickle cell disease.

Okay, how is that useful forensically?

Well, HBS is found much more frequently in certain populations, particularly people with African or Hispanic ancestry.

So detecting HBS in a sample could provide investigators with an early, potentially valuable lead about the possible ethnic origin of the person the sample came from.

And that difference, HBS versus normal HBA, is it a big structural change?

It's incredibly small, molecularly speaking.

It's just a single amino acid substitution.

At position six on the beta chain, a glutamic acid residue is replaced by a valine.

That tiny change causes all the problems associated with sickle cell disease, but it also made it detectable by electrophoresis.

Amazing.

Okay, so we have red cell enzymes, hemoglobin variants.

What about proteins just floating in the blood plasma or serum?

Yep, they looked at serum protein polymorphisms too.

The most widely used one was probably HP.

Its job is to bind onto free hemoglobin that might escape from red cells.

It has common variants that could be typed.

They also looked at variations in immunoglobulins, antibodies,

specifically markers on the gamma chains, GM markers, and kappa light chains, kilamin markers.

And there are others too, like group -specific component, GC, and transferrin, TF, which transports iron.

So it wasn't just one test.

They were layering all these different systems together, ABO, RH, then PGM, maybe HP, perhaps HP variants.

Exactly.

You had to combine the results from multiple independent systems.

Each test added a bit more discriminating power, and when you put them all together, you could finally start getting results that were statistically meaningful, reducing that chance of a random match down to something much more useful for an investigation.

Okay, let's try and pull this all together then.

What were the main pillars of this pre -DNA forensic biology?

Well, if we synthesize it, you basically had two major categories we've discussed.

First, blood group typing, that's ABO, RH, and the others, plus dealing with the whole secret torn on secretor issue, using techniques like the basic lattes assay, but more importantly, the sensitive absorption elution method for stains.

Right, that was pillar one.

And pillar two was protein profiling.

This meant using electrophoresis, both the size -based SDS method, and especially the high -resolution isoelectric focusing to detect variations in proteins.

Key targets there were enzymes like PGM found in red cells and semen, and significant hemoglobin variants like HBF and HBS, plus various serum proteins like haptoglobin.

It's easy to dismiss these methods now because we have DNA, but back then, this was the cutting edge.

Combining all these markers was what allowed labs to take that random match probability from

50 -50 down to maybe one in several hundred, or even better sometimes.

Absolutely.

It was painstaking work requiring real skill.

And understanding this history, I think, is really vital.

It shows you the journey, the complexity of analyzing biological evidence, and how far we've actually come.

Yeah, and maybe the key takeaway, the final thought for you is this.

All these older techniques relied on detecting the products, the antigens on the cell surface, the slightly different protein structures like PGM variants or HBS.

They were looking at the physical result of genetic variation.

Right, detecting the protein itself.

Modern forensics just took the next logical step.

We figured out how to bypass the product altogether and go straight to the source.

The underlying genetic code, the DNA sequence in genes like ABO or FUT or the ones for PGM and hemoglobin, that actually dictates what those products will be.

That shift of analyzing the protein manifestation to analyzing the genetic blueprint, well, that changed absolutely everything.

A fascinating look back at the foundations truly shows the ingenuity involved even before the DNA revolution.

Thanks for walking us through that complex history.

My pleasure.

It's important stuff to remember.

Indeed.

We'll catch you on the next Deep Dive.