WK 2A Red blood cells detailed

irst part on red cells, second part on anemia, and then after that we've got a case study session to start applying some of the theory we've covered in the last couple of weeks. Okay, so I'm going to be here, I'm around and about, but bear with as I'm ducking in and out, but Richard knows the drill, don't you?

Right, let me click, so now your pointer and everything. Okay, well good morning everybody, excuse me, thank you Natasha, thank you for the welcome, I hope the lecture justifies it.

So I am Richard, as Natasha said, can you hear me at the back by the way, is everything okay? It's all working, good.

I spent 39 years in the NHS in my working career, I am now, I have to say, happily retired, it's the only benefit of being old. Most of that time I spent in hematology at the Royal Berkshire Hospital, just down the road, and for the last portion of my career I was a hematology and blood transfusion manager there. So today we're going to be talking about red blood cells.

This is a huge topic, and there's a lot of information, but hopefully some of it will stick and you'll find it interesting. I'll do my best to make it interesting. So the format of this morning's talk is this, we'll talk about what red cells actually do and what their structure is and how the structure supports the function. We'll talk about how they're produced and how they're destroyed, and we're going to talk in a bit more detail about hemoglobin, and we will then relate the structure of hemoglobin to its function, then a little bit of an aside about synthetic red blood cells, and then we'll have a break, 10 minutes, 15 minutes or so, and then we'll go on to the enormous topic of anemia and hemoglobinopathy, and as Sydna Thachta suggested, the talk is about an hour either side of the break.

So some of this I'm sure is going to be revision for some of you, and I make no apology for that really, but just to make sure we all know where we're starting from. So red blood cells, here they are, we call them erythrocytes if you want the snappy name, and if we look at them, and these are red blood cells on the picture, they look like simple packets of hemoglobin, don't they? There's not much to say really, they're kind of round, got a pale bit in the middle, they're kind of pinky, reddy sort of color because we've stained them, and those of us that have studied them in more detail will know that they are actually a biconcave disc, that is they're thinner in the middle than they are at the sides. They've got a mean diameter of about seven microns, and their volume, depending on how you measure it because you get different results from different equipment, is about 78 to 101 femtolitres.

We can see from this picture, as I'm sure most of you know, that they have no nucleus in the mature form, which is unique amongst mammalian cells, but we'll come on to that later. And in mature red cells that there are also no cytoplasmic organelles to speak of, there's no endoplasmic reticulum for instance or anything like that.

And not surprising really, red cells contain hemoglobin as their major component, it's about 25% by volume and 33% by weight. So what are red cells for?

Well we know that they are the sort of the packaging for hemoglobin, if you like, because we require hemoglobin for the carriage of oxygen, and as we'll found later for carriage of carbon dioxide as well.

And hemoglobin also helps to maintain the pH of our plasma, which is maintained within very strict limits to avoid a nasty condition called acidosis, which we'll come on to a bit later.

So I've mentioned that red cells are a biconcave disc, and the reason for this is, there's a couple of reasons really, and the first is that red cells, as we've said, are about seven microns in diameter, but your smallest capillaries are smaller than that, so red cells have to be very deformable to pass through small capillaries, and the biconcave disc shape makes them very pliable.

And the biconcave disc shape also, as I guess most of you know, gives a much larger surface area for gaseous exchange, because we've said we need to get oxygen in and out, and we've also implied that we need to do the same with carbon dioxide.

So the biconcave disc gives us a larger surface area for gaseous exchange, compared to, for instance, a sphere, but we'll come on to the problem of red cells and spheres later in the second half.

So red cells, as I've said, have no nucleus or cytoplasmic organelles really, that when you've got a cell that needs to be deformable and needs to carry oxygen, you don't really want a big lumpy nucleus if it's going to get in the way of the cell moving through capillaries, and the big lumpy nucleus is going to take up space that could be occupied by hemoglobin.

So evolution, in mammals at least, has removed the red cell nucleus.

Bird erythrocytes, and I think reptiles still have a nucleus, but they haven't evolved the same as us. And we think that each red cell, assuming that it lives its normal lifespan, travels about 300 miles and makes 170,000 circuits through the heart in its lifespan, and the lifespan is approximately 120 days usually, but not always. So let's talk a bit about red cell structure. So we've seen them on the first picture, and they look really quite simple, don't they? But unfortunately, as is often the case with biology, the more you look at them, the more complicated they become. Well, obviously, they must have a membrane.

All cells have a membrane, and I think most cells have a membrane which looks something like this. We call it a lipid bilayer.

This is the outside of the cell.

This is the inside of the cell. The outer and the inner surfaces are hydrophilic so that they can interact with their aqueous environment for the transfer of nutrients, gases, anions, et cetera.

And the membrane itself is made up of about 40 percent lipids, 52 percent protein, and 8 percent carbohydrate.

And we can see this structure with fatty acids and cholesterol as per the diagram.

So we call it a lipid bilayer.

So this is a lower resolution, if you like, picture of the lipid bilayer. So this here is the lipid bilayer that we've just been talking about.

But you can't just leave a membrane as a lipid bilayer. It needs some sort of structure to give it the right shape.

And red cells have what we call a cytoskeleton. And this cytoskeleton is made up of a number of different proteins. And the most important of these are proteins called spectra.

And there are two forms of this, alpha spectrum and beta spectrum. And these are sort of fibrous proteins, if you like, which make a sort of a hoop around the inside of the red cell to give it its characteristic sort of disc shape. Spectrum is actually a tetromeric molecule.

So there are two alphas and two betas in each molecular spectrum.

And the spectrum hoop, if you like, is linked to the lipid bilayer by a couple of entities. There's this entity here, the actin complex, and this entity here, and chyron. And chyron, as you can see, is linked to another protein called BAM3, and I'll come on to that later. And the actin complex is linked to glycol 4 and C. So there are quite a few membrane proteins.

I've listed a few here, because they all do something.

They're there for a reason.

So BAM3 protein, that's these, is there for an iron transport, particularly chloride and bicarbonate. Glycol 4 and A helps to maintain the negative charge of the cell. And that's important. The cells are all negatively charged to stop them aggregating. If they weren't charged, they would tend to clump together. And obviously, you don't want that to happen in small capillaries. So you've heard of the expression that light charges repel.

So that's what's happening here, the red cells all negatively charged. So they tend to put each other away. This charge has, if you read some of the literature, has a fairly antique name. We call it the zeta potential, but I don't think anyone uses the term anymore.

As long as you know that the red cells are all negatively charged to keep themselves apart, then that's as much as we need to as we expect. Glycol 4 and C, as we can see, helps to regulate cell shape, because it's one of the entities by which the actin, sorry, the spectrons are linked to the lipid bilayer membrane. It's also involved in membrane deformability.

And chyrin, that's this lot here, as we've said, links the lipid bilayer to spectrin.

Spectrin itself I've mentioned helps to maintain the biconcave disk. And the actin complex, as I've said, links the lipid bilayer, that's here, links the lipid bilayer to spectrin.

So what we've got then is a floppy lipid bilayer, if you like, with a more rigid sort of protein hoop underneath it. And the lipid bilayer is linked to the protein hoop by these proteins, which we will group together as transmembrane proteins and by these sort of anchoring entities.

So red cells have a structure then. There is a further complexity. Red cells also are quite metabolically active.

And there are a couple of reasons for this really. The first is, as I'm sure you all know, iron, if you leave it in an aqueous environment, tends to oxidize.

As I'm sure you all know, hemoglobin contains iron. And the iron has to be maintained in the reduced state. That's Fe2 plus. If it oxidizes to Fe3 plus, it is no longer useful as an oxygen carrying medium.

It's bought a one-way ticket, essentially, to the iron scrap paper. It is no use if it oxidizes. So one of the prime functions of red cell metabolism is to maintain the heme iron in a reduced state.

And it does that via NADPH reducing agent, which it gets from the pentose phosphate pathway, which I'll show you in a minute. If the iron in hemoglobin does oxidize, we say that the hemoglobin has changed to methemoglobin, which as I've got here is inoperative as an oxygen carrier.

The other forms of hemoglobin, of course, are oxyhemoglobin and deoxyhemoglobin. We'll come on to that later. Another point of having the red cell metabolism is to produce ATP as a source of energy because the cell has to work quite hard to maintain membrane deformability and to regulate iron and water exchange. So ATP is necessary for that as a source of energy.

And if ATP is not available, fairly disastrous consequences ensue. And I've got some pictures of that right at the end of the second part.

I didn't mention just now that the metabolic activity is anaerobic glycolysis. This is important.

We've got a cell whose prime function is to carry oxygen. What we don't want is for that cell to be chomping through its own oxygen.

So the red cell metabolism is anaerobic. And the anaerobic pathway results in the production of a metabolite, which we call 2,3-DPG. It's also called 2,3-BPG.

And we'll mention this again later, but it helps to regulate the way that the hemoglobin molecule takes up and releases oxygen. So this is the red cell metabolic pathway, the anaerobic penthouse phosphate pathway.

I am not a biochemist. I have rather little interest in biochemistry, so I won't be going through this in detail. I'll be pleased to know. I just want you to be aware of some important things, which you just mentioned.

So this is an enzyme called G6PD.

And this is important because it gives rise to NADPH, which is used to maintain the iron in its reduced state.

If there is not enough G6PD for a genetic reason, for instance, the iron oxidizes, and that gives us a clinical problem, which we'll talk about later on in the talk.

This is where the metabolite 2,3-BPG or 2,3-DPG is produced. And so this is one of the ways in which hemoglobin can adjust its oxygen affinity. And then an enzyme here, PK, called pyruvate kinase, is essential for the production of ATP, which I just said is important for iron exchange and particularly for water take up and loss.

Let's talk a bit about red cell production.

Red cell production, as I'm sure you know, is part of a process called hemopoiesis. Let me call it erythropoiesis.

In adults, it's usually confined to the marrow spaces in the long bones and the big bones, so things like the femur, sternum, and pelvis. And in some disease states, as what I want to talk about later, erythropoiesis can occur in all sorts of other places.

And you're making and destroying a truly astronomic number of red cells.

You make and destroy about 10 to the 11 red cells every day. That's about a million every second, which is, I think, pretty extraordinary.

But even more extraordinary is that if you need to, you can uprate that figure by about 10 times. So this is a throwback to when we were hunter-gatherers hunting lions, I guess. If you have the misfortune to be mauled by something large and toothy and you lose a lot of blood, you can make that use. Evolution has given you the means to try to make that blood up more quickly than you would usually do.

And just to put into context, a cubic millimeter of blood, which is a three and a half to five and a half million red blood cells. So they're very small, and you're making them in enormous quantities.

And when red blood cells are made, they actually do have a nucleus. We've seen from the first picture in the beginning of the talk that they don't have a nucleus in a mature form. But you can't make a cell, obviously, if it doesn't have a nucleus, because you've got no means of producing any protein, for instance, or cell division.

So red cells are made with a nucleus, and this here is a early red cell, a nucleated red cell, which has another name called norm at last, which is kind of falling out of favor. This is a stain's preparation. So we've stained it with methylene blue, and eosin methylene blue stains the nucleus blue, and eosin stains the red cells, this sort of orangey color. Methylene blue also stains the cytoplasmic organelles, the endoplasmic reticulum, all this kind of stuff, which is why the cytoplasm looks kind of speckly and sort of a muddy sort of purple color. But as we'll see later, the nucleus is removed from the red cell before the red cell leaves the bone marrow. Usually, it's gobbled up by macrophages in a particular endothelial system.

And then the cell emerges from the bone marrow as a reticular site.

So this is a stain using new methylene blue, and it's a vital stain, which is our expression to denote that it's actually done on live cells. And what we can see here is this is a reticular site, and it's got these clumps in it, these stained clumps.

And these are clumps of cytoplasmic organelles, which are no longer useful, and eventually they will be removed by the spleen.

But they've stained with new methylene blue. And if we see lots of these cells in the blood, then it tells us that there are more immature red cells than perhaps we might expect normally, and we can go on and look and see why that might be. So why she drives erythropoiesis?

Well, I'm sure you all know that it's driven by a cytokine called erythropoietin, usually shortened to EPO.

And this acts on committed stem cell precursors in the bone marrow, committed erythropoiesis stem cell precursors in the bone marrow, which increases the rate of cell division and hence increases the number of red cells some way down the line.

Erythropoietin is produced in the kidneys, as long as you've got working kidneys, in response to hypoxia that the kidneys can detect, hypoxia being the oxygen tension. So the kidneys say, oh, there's not much oxygen in your peripheral blood. We better increase the amount of erythropoietin, and that acts on the committed stem cell precursors, and you produce more red cells to hopefully rectify the reason for the hypoxia. And as I've said already, red cell nuclei, which the cells are produced with, are extruded and then phagocytosed by macrophages before the cell is released in the bone marrow. The cytoplasmic organelles that we've seen in that particular site on the last slide having no problem.

The cytoplasmic organelles in a particular site do continue to produce a bit of hemoglobin once the cells are released into the circulation, but that's only for a day or two, and after that the cytoplasmic organelles are removed by the macrophages.

And what we get is the sort of red cell that we know and love, which has no nucleus and no cytoplasmic organelles that we can see. So this is a kind of schematic diagram of erythropoiesis. Mostly, this is all in the bone marrow.

This is the point here at which the cells are usually released from the bone marrow.

So this is more rather archaic terminology, I'm afraid, but this is a nucleotide red cell that we've just seen, and this is a reticular site. So these are the earliest stem cells, and as you know stem cells, some of them are in a resting pool, some are just ticking over very, very slowly, and some of them will become committed red cell precursors, and that's what's happening as we work our way from the left across to the right. And they become more numerous as we go, and they become more active in terms of the number of cells in cell cycle. And the cells that are going to become red cells obviously are going to need to produce hemoglobin, amongst other things.

So to do that, they express a number of what we call CD markers, plus the differentiation.

They're kind of maturity markers really. CD34 is a stem cell marker, and you can see that as we work our way from left to right, generally speaking, that reduces as the cells become more erythroid.

CD71 is a receptor for transferion, which the red cells need to recruit iron into the developing red cell. So not surprisingly, as we work our way from left to right, the transferon receptor is much more strongly expressed at a time when the cell is beginning to synthesize large amounts of hemoglobin. The erythropoietin receptor, it becomes more strongly expressed as well, because this is the area on which erythropoietin acts to drive these cells into more active division. Glycoforin A is one of the membrane proteins that we'll miss out, and erythropoietin mRNA, that's just involved in cell division. So not surprisingly, as we're busy, the cell's busy dividing, and it's busy synthesizing hemoglobin, it's going to express more globin messenger RNA. And hemoglobin is finally expressed in very high levels from nucleated red cell through the tick and onwards.

So the stage from being what we might call a very committed red cell to actually being released takes about a week, 108, 188 hours, when the cell is released into the bone, into the peripheral blood to circulate, hopefully for 120 days. So how do we monitor and how do we destroy red blood cells? As I've said, the lifespan of a red cell is about 120 days. Not always. There are some clinical conditions, and we'll talk about these in the second half, where the red cells don't exist, called hemolysis.

And they potentially have a hemolytic anemia, but they don't actually need to be anemic, because as we've said, you can uprate your red cell production enormously.

So even though you might be destroying red cells too quickly, you've got massive reserves to replace them more quickly as well.

So hemolytic anemia doesn't necessarily follow from just hemolysis. But we would find, if the red cell lifespan is shortened, we would find an increased reticular site count, because this tells us that the rate of red cell production is higher than it might normally be. As a rough guide, and is rough, in an adult, we wouldn't expect more than about a half to one and a half percent of circulating red cells to be reticular sites. But in hemolytic anemia, where the process of destruction and red cell production is quicker, that figure can be much higher.

So how are red cells sort of monitored and destroyed? If you're lucky enough to have one, and most of us do, the red cells are destroyed by your spleen, once they reach the end of their normal working life.

So your spleen is a complex organ that you mark out in your studies, but the spleen is a two-fold organ. It's actually a primary organ involved in the maturation of lymphocytes, for instance, and the presentation of antibodies, antigens rather.

But it's also an organ, if you like, of red cell quality control. And I've got a picture to show you this, and I think on the next slide. But what happens is there are macrophages in the spleen which sense particles, the nucleus, the cytoplasmic organelles, in circulating red cells, and they pluck them out.

And they also pluck out bits of damaged red cell membrane.

It might have antibodies stuck to it or whatever. Or it may be the red cell has just become old, and once the red cell becomes old, its metabolism slows down. I can equate with that, that's how I feel. Its metabolism slows down, and it becomes less bendy. I can equate with that as well. So once the red cells are less bendy than normal, they don't deform as they go through the spleen properly, and the spleen says, oh, that's an old one, I'll have that out, and it destroys it. And the spleen is really a very discriminating filter. And this is a rather crude diagram of how the spleen works in terms of red cells. I use this diagram in every talk because I quite like it in some ways, but it's got a really, really stupid mistake on it. I quipped it from Wikipedia years ago, but the spleen is divided into two halves of a set as a lymphoid side, which is white pulp, not right pulp, white pulp and red pulp. We're not going to deal with the white pulp in this talk, we're going to deal with the red pulp, which is the erythroid side. So the blood flow through the spleen is this way. So what we've got are these things called splenic cords, and these are blind sacs and the sacs are surrounded by venous sinuses, which are sort of blood spaces, if you like. And there are very small windows, only about three microns across between the spenic cords and the venous sinuses, and the red cells have to pass through these tiny windows. And if they can't do that, they are destroyed. And the reason that they wouldn't do that is they've lost pliability, or they've got a large particle, and the particle in that case would be plucked out, as I've said.

So it's quite a clever system. Your spleen has a very large blood supply, and if any of you have been unlucky enough to have your spleen removed following trauma, you're quite lucky to have got away with it, really, because splenic rupture can lead to death really quite quickly through blood loss.

So I've said some of this already, but we'll hasten the destruction of the red cell by the macrophages in the spleen, as will things like antibodies stuck to the red cell membrane, and we'll talk a bit more about that later.

So you've got red cells, the splenic macrophages are breaking them all up. What happens to the contents?

Well, the iron, we're pleased to know, is recycled.

The body works very hard to retain the iron that you have on board, and in the absence of any iron loss, and menstruation is an example of iron loss, for instance, but in the absence of any iron loss, you can survive for a number of years on a completely iron-free diet, just on the iron that you've got in your body, and because the recycling mechanism is usually so effective. So the iron is recycled by the splenic macrophages.

The other protein components of hemoglobin, for instance, goes to the liver to make bilirubin, but that's a topic for another day. And as I've said, the spleen can remove particles, so nuclear material.

So even if the nucleus has been removed, there can be some other chunks of nuclear protein, and we call these things how jolly bodies, after Mrs. Howell and jolly who identified them, they're just little bits of spleen, and they are little sort of perfectly round, dark blue staining bodies, about a tenth the diameter of a red cell, and if we see these, it suggests to us that either patient doesn't have a spleen, or if they've got one, that it's not working properly. As I've said, the spleen can also remove cytoplasmic organelles.

It can remove chunks of sort of iron and associated protein, which we call cidorotic granules, another name Papenheimer bodies, left over from the red cell production.

And the spleen can also remove chunks of oxidized hemoglobin, which we've mentioned earlier, called metzhemoglobin, and the chunks of oxidized hemoglobin, if we see them stain, we call them Heinz bodies.

It's suggested that reticular sites, the early red cells with the cytopasma organelles, are actually sort of sticky in some way, which slows their passage through the time to pick out the cytoplasmic organelles that were involved in the maturation of the cell. I'm not sure we actually know that. I think it's just suggested that they might well be. Okay, that's all I wanted to say about red cells per se.

Got somewhere to go to the break yet, I'm afraid, because we've got another big topic called hemoglobin. But anyone got any questions about red cells, any burning issues?

Either shout them out or come and see me in the break. Everyone good so far?

Okay, let's talk about hemoglobin. So the functions of hemoglobin, as I'm sure this is revision for most of us, transportation of oxygen from the lungs to respiring tissue. That much is pretty common knowledge.

And to do that, oxygen is reversibly bound to what we call a heme group. And I've got a picture of this in a minute, which is an ion atom in a porphyrin ring, in a protein ring.

And it's really important to realize that this is a physical interaction.

It's not a chemical process.

A chemical process of bonding iron to oxygen would be oxidation. And as I've said, we don't want that to happen. So this is just a physical association where the oxygen is physically associated but not actually chemically attached to the iron in the hemoglobin molecule.

So as I've said, the iron atom must stay in its reduced form. Fe3 plus is non-functional for oxygen transport.

And if hemoglobin has oxygen on board, as I'm sure you know from watching too much casualty or whatever I'm going to tell you, oxyhemoglobin is a bright red color.

And deoxyhemoglobin, venous hemoglobin, is deoxygenated, and that is a dark red color. So hemoglobin is involved in the transport of oxygen, that much we knew already. But rather less well known is the fact that it's also involved in the transport of carbon dioxide.

And red blood cells are actually associated with the transport of about 95 percent of the carbon dioxide that comes from respiring tissues before it gets to the lungs.

About a quarter of that is directly bound to part of the hemoglobin molecule in terminal valine of the alpha chains.

So that leaves us with 75 percent to do something else with. And red cells are instrumental in transporting that by using a red cell membrane enzyme called carbonic anhydrase.

This, I believe, is the fastest-acting enzyme in biology. And it catalyzes the transformation of carbon dioxide and water to carbonic acid, which fairly rapidly dissociates to hydrogen ions and bicarbonate in the plasma. And so the bicarbonate diffuses back into the plasma off the red cell surface, and that gives us about 70 percent of our carbon dioxide transport. And the bicarbonate circulates around the body, gets to the alveoli, where it dissociates back into carbon dioxide and water, and you exhale the carbon dioxide and all as well.

There is, however, a potential problem, and this is another function of hemoglobin, and that relates to this iodine here. I'm sure you are aware from your chemistry studies that free hydrogen ions would contribute to the formation of an acid, and we don't want that to happen, because if you've got acidic plasma, you have a nasty problem called acidosis.

The pH of your blood is maintained within very tight limits between 7.35 and 7.45, any less than that, and you have acidosis, which gives rise to, well, initially headaches and organ damage and ultimately death. So we don't want that to happen.

So hemoglobin has a function that the ions, the hydrogen ions, left from the dissociation of carbon dioxide to bicarbonate, actually bind to the globin chains of hemoglobin.

So hemoglobin acts as a buffer. So it's got three functions then, really, power to oxygen, the transport of carbon dioxide, and acting as a buffer to keep your plasma pH where it should be. I've kind of labored this point already, I think, but this highlights the difference between oxygenation, which is what we want to happen for hemoglobin and oxygen, and oxidation, which is what we don't want to happen.

So the ion, Fe2 plus, stays as Fe2 plus, and is bound to oxygen physically, and we say that that is the changes from hemoglobin to oxyhemoglobin.

If the ion oxidizes Fe3 plus, then we say that is methemoglobin irreversible change and is no longer useful for oxygen transport. So what is the structure of hemoglobin?

It's quite a large molecule. Molecular weight is about 68 kilodaltons, and it is made up of four globin chains of two different types in each hemoglobin molecule. And as I mentioned earlier with biology, the problem is the more you look at something, the more complicated it becomes.

So it, unfortunately, becomes obvious that there is more than one sort of hemoglobin. Most of us here, probably all of us here, have as our major hemoglobin component hemoglobin A.

This is adult hemoglobin, and it's made up of two alpha chains and two beta chains. We will all mostly have a very minor component, which we call hemoglobin A2, which is made up of two alpha chains and two gamma chains. No, two delta chains. Always getting that one, not two delta chains.

And then babies in utero before they're born have a different hemoglobin.

This is important because they need to get oxygen off mum's hemoglobin, because that's the only way they can get oxygen.

So babies have a hemoglobin with a higher oxygen affinity than mum's hemoglobin, so that the baby's hemoglobin, hemoglobin F, can take oxygen from mum's hemoglobin, hemoglobin A. So babies have what we call a high affinity hemoglobin, hemoglobin F, and that is alpha two gamma two.

So we've identified three different sort of molecular structures of hemoglobin, if you like. But they are all similar in as much as each globin chain, so each alpha and each beta chain, in this case, or each alpha or delta, et cetera.

Each globin chain surrounds a heme group. You'll see in literature, sometimes it's referred to as a prosthetic group. It's another archaic term, I'm afraid. We don't use it much more. And the heme group is made up of an atom of iron and a porphyrin ring. That's the chemical symbol for it, if you want it.

And so there are four heme groups in each hemoglobin molecule.

And it looks a bit like this. It looks like a plate of tagliatelle. So what we've got, we can envisage that these sort of olive green ones are the alpha chains. They're kind of each of the colored ones are the beta globin chains.

And these entities here are the porphyrin rings with an iron atom in the middle of each porphyrin ring.

So it's quite an elaborate structure.

And it is like that because hemoglobin needs to do something really quite cunning in terms of its oxygen affinity.

Oxygen affinity is the relationship, as I've got here, between oxygen saturation in the red cells environment and the oxygen saturation of the hemoglobin and the partial pressure of oxygen in the red cells environment.

What we really want is something which seems impossible, which is we want a variable oxygen affinity. What we want the red cells to do is when they are in an environment with a lot of oxygen, the alveoli, for instance, we want them to take up oxygen and bind it strongly. But then what we want them to do is when they're in an environment where their partial pressure of oxygen is lower in respiring organs, muscles, et cetera, we want the red cells to release the oxygen to have a lower affinity, as is to bind it less strongly. And this change in affinity is achieved, at least in part, by this structure, this tetrameric structure with the globin chains. So this diagram describes, if you like, the oxygen affinity.

So let's say that we are a red cell. And at this point, where our partial pressure of oxygen is quite high, we are in the lungs. And we're in the circulation, and we move along, and the partial pressure of oxygen begins to drop as we move away from the lungs.

But you'll notice that actually on the oxygen saturation graph of the y-axis, the oxygen saturation remains the same, even though the partial pressure of oxygen is reduced. So what's happening is there isn't as much oxygen in the red cells environment, but it hasn't at the moment lost any of its oxygen. And we don't really want it to lose oxygen in or near the lungs, do we?

Because there's plenty of oxygen there. What we want to do is to carry the oxygen to somewhere where there is a rather little oxygen. So as we move away from the lungs, you'll see that the curve begins to fall off quite quickly.

And what's happening here is that the first and the second oxygens are lost really quite quickly with relatively small drop in the partial pressure of oxygen.

And the last oxygen is held on too much more strongly, presumably to keep it until the very last moment at the most hypoxic sites that the red cell encounters.

So what we're saying is really in the tissues you need a relatively large drop in the partial pressure of oxygen to dislodge the first oxygen, but the second and third are given up more easily and says the fourth.

The fourth it hangs on to quite avidly. Okay, I'll set that as well.

So there are a number of ways in which the structure of hemoglobin gives rise to this alterable affinity.

And one way is actually a physical conformational change as the cell becomes oxygenated and deoxygenated.

And this diagram shows you that this is the porphyrin ring here, and this is the iron atom. And the whole lot kind of moves around to shield or expose the iron binding site in the porphyrin ring as a way of tailoring oxygen affinity. And that's this point here, conformational change adjusts the oxygen affinity by varying oxygen access to or from the heme groups.

And then there's another way in which oxygen affinity can be changed.

And I mentioned this metabolite earlier, 2,3-DPG equals 2,3-BPG. And this metabolite binds preferentially to deoxyhemoglobin, and it reduces its oxygen affinity further.

So it promotes further deoxygenation possibly of that last oxygen. With the intention, obviously, of giving up the oxygen to hypoxic metabolically busy sites. Meta- increasing metabolism gives rise to falling pH, as we said, because of the hydrogen ions. And that also reduces the oxygen affinity, which tends to increase the oxygen supply. So metabolite piaptium areas are going to get more oxygen for that reason as well. I've mentioned that fetal hemoglobin has an increased oxygen affinity compared to adult hemoglobin.

And that is because the gamma chains that fetal hemoglobin has don't bind to 3-DPG as well.

So it becomes a high affinity hemoglobin. And as I said, the point of that is so that baby's hemoglobin can take oxygen from mom's hemoglobin across the placenta.

Okay, slight digression. Those of you who are blood donors or have had a blood transfusion will be aware of some of the issues of transfusing human blood. This is not electron blood transfusion.

Somebody else does that. But I want to talk a little bit about artificial red blood cells because I think they're quite interesting.

We've seen that, actually, red cells are much more sophisticated than one might at first have thought.

And artificial red cells extraordinarily difficult.

But if we could do it, there would be some big advantages over transfused human cells, which, as I'm sure you're aware, have the potential to carry disease, have a finite lifespan, you're dependent on donor pool, some religions prefer not to have transfused blood, et cetera.

So if we could make artificial red blood cells to mimic the functions that we've been talking about, we could get over a lot of the problems of having to transfuse proper human ones.

We get over storage temperature. We could get over the problem of blood groups. You probably know about blood groups, but I am blood group A.

If you're blood group O and you have your transfused the literature of my blood, well, if your transfused five mils of my blood, it could kill you. We obviously want to try to avoid that.

But if we could make blood cells ourselves, we could arrange perhaps for there not to be any blood groups to complicate issues. If you are transfused to a unit of blood, your hemoglobin concentration and your plasma goes up, that's good.

But there is a nasty problem, which is actually that because of the storage issues in the blood that you've been transfused, there is rather little 2,3-DPG.

And what this means is that the unit that you're transfused, initially at least, has really quite a high oxygen affinity. That is, it's good at taking up oxygen, but it's not good at giving it up because there isn't enough 2,3-DPG in the stored unit, in the stored unit and the stored red cells. So once your hemoglobin might go up, actually, it's not as useful as you might think in the early stages. After a little while, the 2,3-DPG permeates the red blood cells and all as well.

But initially, at least, there is a problem that the transfusion isn't as effective as you think it might be. If we could make artificial cells, then we could get rid of the risk of disease transmission, which wouldn't be risking transmission of syphilis, HIV, glandular fever, malaria, a raft of others, and we could get rid of that. We wouldn't be dependent on donors. And as I've said, we could avoid religious issues as well.

The problem is that making red blood cells is extraordinarily difficult.

We can't just purify hemoglobin and transfuse that. The free globin chains are toxic to the kidneys.

The free globin chains scavenge nitrous oxide from the circulation leading to vasoconstriction, i.e., your capillaries or restrict. That's the last thing you want.

And they have a very unfavorable oxygen affinity. And even though they're toxic to the kidneys, assuming your kidneys carry on working, free hemoglobin is removed with a half-life of about 30 minutes.

And even though a lot of effort over the decade has been put, particularly by the military, as you say, has been put into the production of artificial red blood cells, there are currently no hemoglobin-based products licensed for clinical use in Europe or the US. And there is no alternative to donor blood in general use. People have tried all sorts of things, remanufacturing hemoglobin to sort of ... In South Africa, back in the early stages of the 2000s, they had a very big problem with HIV, as you're sure you're aware.

And Hemopure, which was a remanufactured hemoglobin, was licensed in South Africa, but only in South Africa, and I don't think it's licensed anymore. You can conjuate hemoglobin to something to give it some stability, polyethylene glycol is one, and hemospan was the product, but that's not licensed.

People have tried to make other cells, like yeast or E.

coli, into what you might call neohemocytes by genetically engineering them to make hemoglobin. And they do, but they don't make anything like enough to be useful as oxygen-carrying cells.

There is, however, some optimism on the horizon. And that is a trial called RESTORE, which is actually based on some UK work between, I think, the University of Bristol and the NHS blood transfusion service.

And this is a trial to investigate the effectiveness and the possibility, if you like, of growing stem cells into red blood cells in vitro. And the trial started in November 2022 with a transfusion of five mils of synthetic, if you like, farmed red blood cells into donors.

The trial is still ongoing. It's expected to report in 2025. But at the moment, that looks as if it might be the only possibility of getting artificial red blood cells.

The problem really is, as I've said, you're making and destroying such huge numbers of red cells for a blood transfusion to be effective. We don't need five mils. We need 10 times that.

So to make all those red blood cells might be just too difficult, expensive, time-consuming, et cetera.

But the trial to establish the possibility and the clinical effectiveness reports quite soon.

OK. That's a lot of information, I'm aware. I'm happy to take any questions now or come and see me in the break. But we'll have a short break. Is it 10 or 15 minutes, Natasha? What do you say? 10-2.

Yeah, 11 o'clock by the clock then. So you've got about 11 minutes to go and put your brains back into order. Be careful if you can put chairs to try and let them go through. So just be careful if you come and go and trip up. Hopefully someone will be out looking at you. Let me pause the recording. On the record, click on the screen and it should.

Lovely. We'll carry on there for the second half. OK.

Thank you, Natasha. Welcome back. So in the second half, we're going to talk about anemia. This is another pretty vast topic.

It's quite clinical. I've got quite a lot of pictures of red cells in sort of various disease states, which hopefully you'll find interesting. But we can define anemia really very simply as a reduced concentration of hemoglobin in the blood. And the symptoms of anemia generally start to appear when the hemoglobin falls below about 90 to 100 grams per liter of hemoglobin.

The normal range, depending on age and gender, is something like 125 to 150 for women, about 130 to about 180 grams per liter for men. And in general, if the hemoglobin falls slowly, then the symptoms are much more subtle than if the hemoglobin falls quickly.

And it's been reported that hemoglobin can fall as low as 60 grams per liter in an otherwise well young patient. And there's a story which may well be apocryphal of someone who goes to see their GP. They felt a little bit tired and a bit breathless, but not too much.

And he takes one look at her and he says, gosh, I better check your hemoglobin. So he does his own fairly crude hemoglobin check in the surgery. And his correctly, your hemoglobin is only 30.

You better go to hospital straight away. And she said, I can't possibly. I've got a game of squash booked in an hour.

It's fairly well documented. And even if it's not true, it kind of highlights the issue that some people can tolerate a low hemoglobin really remarkably well.

If the hemoglobin has fallen quickly, as we'll see later, if the hemoglobin falls, sorry, they're sorry.

Well, if it falls slowly, if the hemoglobin falls quickly, then the situation is much more worrying.

Another symptom of hemoglobin, then of anemia, rather shortness of breath, you become weak, pale, particularly mucous membrane.

So if you pull your pinky color looks pale, you feel lethargic palpitations, you know, those big sort of sudden heartbeats get headaches and heart failure and confusion, especially in older patients. And the clinical signs of anemia, I said, paloremepous membranes, and also the nail beds then start to look pale. And then some other things happen, depending on the reason for the anemia.

You can find concave nails, mainly in iron deficiency, for instance, jaundice, if you're destroying red cells more quickly than they ought to be destroyed, leg ulcers, particularly in older patients with iron deficiency, bone deformities in some of the hemoglobin office that we're going to talk about later.

There's an old wives tale that if you're anemic, you're going to get lots of infections and this sort of thing. Generally speaking, that isn't true. It's an old granny's myth. But the only reason that you would get lots of infections, generally speaking, with anemia are something like bone marrow, folia or leukemia.

Anemia is not generally speaking connected with susceptibility to infection. So once again, things become more complicated the more we look at them. We have to classify anemia because there are quite a few different types. And the way we classify them is based essentially on two red cell parameters. So we look at the red cells in terms of their size. We looked at that picture of red cells on the first diagram that I showed you in the first part. And we could see that the red cells are discs and I said they're about seven microns across.

And they have a volume usually depending on how you measure it of something like 78 to 100 centimeters. So if we look at the red cells, we find that they are small.

That is generally speaking, they have a small diameter and a small volume. We would say that they are macrocytic, small cells, kind of obvious really.

Are they of normal size, normalcytic or are they large macrocytic?

So that's one part of the classification of anemia.

The other part is about their hemoglobin concentration. We say do they have a reduced hemoglobin concentration that is?

Do they essentially, are they very pale when we look at them? Is there a lot of wasted space inside the red cell? So if there is, then we would say that they are hypochromic, which literally means I suppose less color. Or is the hemoglobin concentration normal?

That is they have a normal concentration of hemoglobin. We say they are normochromic.

Or is the hemoglobin concentration actually high? This is rather unusual. We would say they are hyperchromic but there's only one condition, I'll show you that later, in which that happens.

So we classify anemia based on the red cell size and the red cell hemoglobin concentration.

So to do that we have to use some measurements that we've made.

So we'll come on later in tomorrow's talk to talk about how we actually measure some of these things. But we can measure the volume of red blood cells. That gives us the MCV. And the normal range is about 80 to 101 femtoliters. That's 10 to the minus 15 of a liter. But it does change the normal range varies depending on what sort of machine you've measured it on. Then the way we assess hemoglobin concentration is this parameter called mean corpuscular hemoglobin concentration.

It's a bit of a mouthful. We abbreviate it to NCHC. And it's a calculator parameter with the hemoglobin the total blood hemoglobin divided by this number, hematocrit, which is it's the ratio of red cells to plasma.

More on that later.

And the normal range is 300 to 350 grams per liter. And we can also look at the mean cell hemoglobin. That is the amount of hemoglobin in each red cell.

And usually that's between about 27 and 34. But obviously if a cell is small and it's got hemoglobin at a low concentration, it's not going to have much hemoglobin in it in absolute terms.

So the MCH will be there. So let's talk about normal cystic normal chroma anemia.

Well, as we said, the red cells in this condition then are of normal volume. The MCV is normal.

The hemoglobin is at a normal concentration. The MCHC is normal.

So the red cells contain a normal amount of hemoglobin.

So the MCH is normal. The problem is just that there aren't enough red cells.

The count is reduced. Anybody wants to suggest a reason why we might get a reduced red blood cell count?

Go on. Be brave. Blood loss. Absolutely. I mentioned before about losing your spleen or rupturing your spleen.

So it could be an acute bleed.

Other reasons for getting a bone marrow failure.

So for whatever reason, there's not enough erythropoiesis going on. It could be hemolysis that we're destroying the red cells too quickly or renal failure.

As I mentioned earlier, you need working kidneys to make erythropoietin to drive the red cell precursors into proliferation and to make more and more red cells. If you haven't got working kidneys, then that doesn't happen. At least not adequately. So those are all examples of normal cystic normal chromachromachinemia.

Red cells are normal just that there aren't enough.

Okay. Next one. Microcytic hypochromachinemia.

So the red cells are reduced in volume.

They're small. So their diameter is going to be small as well. They have less hemoglobin and it's at a lower concentration than normal.

So the NCV, the NCH, and the NCHC are all reduced. Anybody want to be brave and give us an example of a microcytic hypochromachinemia?

Commonest reason, iron deficiency. But there are others.

Fallicemia is what we call a hemoglobinopathy and we'll come onto that at the end of this talk. And there's another one called anemia of chronic disorder.

Some chronic disorders such as, I don't know, rheumatoid arthritis, Crohn's, that sort of thing, tend to give you a disorder of iron metabolism.

So you have often mild iron deficient looking picture. The patient isn't actually iron deficient though, but it looks like they are. But the commonest reason for a microcytic hypochromachinemia usually is iron deficiency.

Next one then. So we've dealt with normal cells, we've dealt with little cells, and now we've got big cells, macrocytic normal chromachinemia.

So this is where the red cells are increased in volume. They have hemoglobin at a normal concentration, but the red cell count is low. So in terms of the numbers that we can produce on our machines, the mean cell volume is increased.

The MCHC, the concentration of hemoglobin, is normal. So the MCH is increased.

They are big cells. The hemoglobin concentration is normal, so the amount of hemoglobin in each cell is higher than normal. So macrocytic normal chromachinemia. A common reason for that would be B12 or folate deficiency.

B12 comes, I'm sure you're aware, from some meat product, dairy, eggs.

It's the thing that vegans and vegetarians can become proficient in.

Folate is found in dark green vegetables particularly, but it's very thermolabel, so if you boil it, it gets destroyed. I'm not a biochemist, as I've said before. One is a cofactor for the other, but you need them both together to make protein, essentially. So if you are a B12 or folate deficient, you're likely to become anemic, possibly severely so, but you also find problems with some of the other areas where cell division is usually rapid. So for instance, patients who are B12 or folate deficient get sore mouth.

They get disruption of their gastrointestinal tract, because usually you're sloughing off cells in your mouth and your gastrointestinal tract, and they're replaced. But if you can't make the cells because you're B12 or folate deficient, then you get typically sore mouth.

Men become sterile.

It gives rise to nervous complaints as well.

So B12 or folate deficiency affects quite a lot processing in the body.

Hematologically, it also gives rise to a very low platelet count potentially, and a low white cell count, particularly neutrophils, for the same reason that you can't make the protein that these cells are made of if you're B12 or folate deficient.

So we're going to look at some examples of anemias now, and this is a kind of a list of them, which I won't talk through because there's slides for each. Okay, it's a bit of a big area, but let's talk about the first sort of anemia, then anemia due to acute blood loss.

So as we've said, there's nothing wrong with your red cells. It's just that you've left half of them in the road having been knocked off your bike.

So the anemia is normocytic, normal.

Well, those of you who are blood donors will know that you can tolerate some blood loss without too much trouble. If you give blood in, you lose about 500 mils, but you can lose more than that 10 to 20 percent of your blood volume with little or no effect, generally.

If you are unlucky enough to lose more than that between the lecture and 1500 mils, then you'll be okay if you're lying down or sitting.

But don't try and stand up.

If you lose more than that, 1500 to two liters, you're going to be drifting in and out of consciousness, short of breath and sweating, and a blood loss of greater than two liters, severe shock.

This is not shock as in a nasty surprise.

This is shock as in poor perfusion of essential organs so that they suffer hypoxia and irreversible organ damage and death.

There is a problem with looking at bleeding patients, bleeding in terms of blood loss, which is that obviously if you've been unlucky enough to have a cycling accident, then you've bled all over the road.

The blood that you've got in your vessel, in your blood vessel still, is much the same as before you were knocked off your bike.

You've taken the body two to three days to make that volume up, so you can lose a lot of blood without actually dropping your hemoglobin much, unless of course you're put on a drip.

Hypovolemia is something to be aware of.

Hypovolemia obviously is a lack of blood volume, but not necessarily anemia. Following anemia due to acute blood loss, we expect a ridiculous response. As I said, reticular sites are those early red blood cells, or immature red blood cells rather, and we expect to see a reticular site response in between three and five days, which is the time it takes for the kidneys to generate erythropoietin and for that to have its influence on the stem cells and for the maturation process to actually produce viable red cells at the end of it.

Okay, let's talk about hemotenic deficiency.

In this case, iron deficiency anemia.

So we've said, I'm sure you'll know, that iron deficiency anemia is going to cause anemia because you can't have hemoglobin without iron.

So what we find in iron deficiency anemia is, as I suggested, is it's a necrocytic hypochromechanemia, so the cells are small.

You have to take my word for that, but they look quite small, and they have a very large area of central pallor. That is, the middle of the cells is very pale. There are also quite a lot of sort of misshapen ones, which happens because the cells, because they haven't got enough hemoglobin in, they change their shape either in the circulation or when we spread them on a glass slide.

So that's iron deficiency anemia caused by either blood loss, as we've said, or well chronic blood loss, that's important, so that you actually lose iron over a long period of time, or poor diet or malabsorption. That is, there's iron in your diet, but you're not absorbing it.

So what would he do about iron deficient patients? Well, the first thing is to replace the iron stores. Two ways of doing that, you can have iron tablets, or you can have the iron injected. And the tablets, obviously, is the easiest way of doing it, but they're not without their consequences. They cause severe constipation, I gamma.

The hemosologic response in terms of the increasing hemoglobin is no different, irrespective of which of these routes that you use, oral or parenteral.

But you would use parenteral injected iron if you want to boost the iron stores more quickly, so for instance, in late pregnancy, or if the patient's got an absorption problem.

And once you've increased the boot of the patient's iron stores, the hemoglobin should rise, assuming that the kidneys are working properly and the bone marrow is working properly, should rise by about 10 grams per liter per week.

In severe cases, obviously, we can give a blood transfusion that has two important benefits. One is clearly that you're increasing the amount of hemoglobin. The other is that you're also giving a massive amount of iron. But the problems are it's expensive, donor units are scarce, there is a risk of transfusion related complications due to blood group mismatching or disease or whatever, so best not to transfuse unless you really have to. But it can be used in severe cases. But an important thing about iron deficiency is to rectify the underlying course. So if it's a little old lady who's living on tea and that needs to be investigated and rectified if possible, and the same with bleeding. Okay, so this is a different hematonic deficiency.

So I should have said hematinics are iron B12 and folate, and they are the things, the sort of dietary things that you need to make hemoglobin. So this is another hematonic deficiency, vitamin B12 or folate deficiency. So as I've said before, this is a macrocytic, normal chronic anemia. The cells are big.

You can see that they look pretty well hemoglobinized. There's a few which are relatively pale in the middle, but mostly they're pretty well stuffed.

Some of them are oval, and this is an indication to us when we look at patients' blood cells that they might be B12 or folate deficient.

Oval macrocytes are found in B12 or folate deficiency, as are these things.

To pre-empt a little bit of what I'm going to say tomorrow about neutrophils, this neutrophil is what we describe as being hypersegmented. So neutrophils are a white blood cell.

They're one of the phagocytes, and they have this nucleus, which has this rather wibbly-wobbly sort of structure. We've made up of lots of lobes. Should be between three and five lobes, but this one's got, probably a dozen. And in patients who are B12 or folate deficient, we find oval macrocytes and hypersegmented neutrophils as a result of the interruptions to cell division caused by poor availability of protein.

Okay, different sort of anemia now. The red cell cytoskeletal disorder.

So the cytoskeleton, to recap from early this morning, the cytoskeleton is the protein structure that gives the red cell membrane its shape. So it doesn't take a genius to recognize that these red cells look a rather strange shape. They're all this kind of elliptical shape.

They should be discs, but they're not. And the reason for this is that the patient has either a spectrum, a glycoformin C or a protein 1 disorder.

4. So the red cell membrane is not adequately anchored to an effective red cell cytoskeleton, and it causes the cells to become this shape. It can cause a hemolytic anemia.

It's very variable. Some patients actually become transfusion dependent. Other patients will go through their whole life and not realize they've got it.

If it causes severe anemia, then following on from what I said a little while ago, one way of treating it is to remove the patient's spleen, because the spleen looks at these cells and it says, oh, these are a strange shape and they're not adequately deformable. I'll pick those off so it destroys them. So you get a hemolytic anemia, which is, as I said, a shortening of red cell lifespan.

So removing the patient's spleen, whilst it's a fairly extreme measure, is better than a patient who could be transfusion dependent. It's reasonably common in this country, one in three to four thousand, and it is a normocytic, normal chroma canemia. This is another cytoskeletal disorder, hereditary spherocytosis.

So if you look at these red cells, you can see that they all look sort of spherical. Well, they look round, and mostly, excuse me, red cells don't have an area of central pallor, and that's because they become spheres. Well, the reason they become spheres is pretty much like the last slide, that the lipid bile membrane is not properly connected to an effective red cell cytoskeleton. In this case, almost certainly what happens is the red cells are made as a normal cell, as a normal biconcoped disc.

But probably what happens is the red cell membrane sort of begins to flop about, and you get little buds on it, and the spleen, the spelic macrophages pick those off.

So each time the red cell goes through the spleen, it loses a bit of membrane, and as you're probably aware, a sphere is the shape with a minimum surface area to volume ratio. So eventually, the cell becomes a sphere.

Many of these are clinically silent, a bit like the elliptic cytosis, also considerable genetic heterogeneity.

There's a rather interesting test you can do to stress the red cells osmotically to see if they burst earlier on endosmosis. It's what we call a function, and I say it's fallen out of favor a bit now because there are other ways of demonstrating spherocytosis.

But as the first with the last slide, treatment might be the next week if the patient is particularly badly effective. Even though the red cells look small, they're not particularly, and it's still a normocytic sort of normocytic normal chromokinemia.

Though actually, the spherocytes in this case we could refer to as being hyperchromic, but that's a difficult parameter to measure, and generally speaking, we don't want to. So this is a repeat of the slide I showed earlier, just to clarify the entities involved in the last two slides, the elliptic cytosis and the spherocytosis. So it's these. There's either the spectrum itself or the entities that anchor the spectrum to the lipid bilayer.

Okay, next sort of anemia. Acquired impairment of erythropoiesis. So for some reason, erythropoiesis has lost its effectiveness. It's either stopped completely or it's not going quickly enough. Why might that be?

Well, number of reasons. Children are susceptible to an infection with a virus called parvovirus. It causes a common condition called slap jig disease, and they get a red face and they recover from it pretty quickly. But it has an interesting effect on the red cells in that it causes what we call transient red cell aplasia, which is, for some reason, I don't think we know why, red cell production stops. Not for very long, but it stops largely.

And I think you can envisage that if you've got a normal red cell lifespan of 120 days, stopping making red cells for a few days isn't going to cause any problems. But if you're a child with spherocytosis or elliptic cytosis, as we've just seen, which has got a shortened red cell lifespan, your hemoglobin, your circulating hemoglobin, is going to fall quite quickly because you're destroying your red cells, but you're not making them.

So just occasionally, these children require a blood transfusion, but it's not common. Another reason for transient erythropoiesis will be drugs. And cytotoxic drugs, you could say, cause this.

Cytotoxic drugs, some of them, target any process involving rapid cell turnover. So what we're looking at in erythropoiesis, obviously, is a process involving rapid cell turnover.

Same is true for hemopoiesis in general. So cytotoxic drugs can cause anemia, they can cause too few neutrophils, called a neutropenia, and thrombocytopenia, too few platelets. Generally speaking, hemopoiesis will start up again once the drug has been metabolized. So hopefully, the problem isn't too severe. But patients on cytotoxic drugs need continual monitoring to find out what's happening to their hemopoiesis.

And then another category of acquired impairment erythropoiesis could be bone marrow infiltration.

So your bone marrow, as I've said earlier, is generally speaking, as an adult, in your large bones and pelvis, pelvis and sternum.

But some tumors, unfortunately, have the property to spread into these areas to set up what we call metastases.

And the tumors which metastasize, particularly, are CA prostate and breast.

There are others, I think, London as well, but breast and prostate are particularly bad in this case. Leukemia is a cancer of the blood cells.

So that sets up in normal erythropoietic tissue and disrupts erythropoiesis. And there's another condition called malafibrosis, which is actually a platelet disorder. But the bone marrow erythropoietic space fills up with fibrotic tissue. So all of these things have the same effect that at one point in time, your erythropoiesis was normal. But you've acquired a situation which means that it is now not normal. And may well be not adequate to support your needs.

So anemia but a bone marrow infiltration looks a bit like this. What we've got are red cells of all different shapes and sizes. We call these things teardrop follicular sites because they look like teardrops. They are scientifically called dacrocytes, but I don't think anyone uses that term anymore. So if we find nucleic red cells and immature white cells present, we say that the patient has a leuka as a erythropoplastic picture. I've got that written down somewhere in a future slide.

As a result of bone marrow infiltration, probably, which leads to this thing here, bit of a mouthful, extra modullary hemopoiesis. But it's where hemopoiesis starts up in other parts of the body, typically in the liver. So the red cells look like this, these teardrop follicular sites, because they are made either in a bone marrow where they haven't got the space to grow properly or in some other hemopoiesic environment like the liver, which is set up because of the extreme situation.

And that obviously doesn't have the right architecture to cause shapes to be the right shape.

So teardrop follicular sites, if there are a lot of them, are not a good sign. It tends to suggest something rather sinister is going on. Okay, I think I've probably said most of this. Extra modullary hemopoiesis then. Hemopoiesis outside the bone marrow, especially in the liver, yeah, can be associated with severe anemia, like your body is trying everything to make enough red blood cells.

Categorized then by teardrop follicular sites, nucleic red cells, and immature white cells.

And we call it a leukarythroplastic picture, if they are immature granulocytes, principally myocytes, which I'll show you tomorrow if they're present as well. Okay, another acquired impairment of erythropoiesis then. This is a picture of leukemia. It's a particularly graphic picture, really, of chronic granulocytic leukemia. This chap actually came into A&E one night when I was working in the lab many years ago now, and he had a pain in his gut and did a blood count, and his white cell count is about 700. The upper limit of normal is about 10.

And the reason he's got a pain in his gut is that his spleen is probably about as big as a grapefruit, should be as big as an apple, and because it's absolutely infiltrated with all these leukemic cells, which are crowding out his bone marrow. All of these white cell precursors are taking up space that ought to be occupied by, well, some of them ought to be occupied by some white cell precursors, but mostly by red cells. So he has got a severe anemia.

I think his hemoglobin was about seven as a result of his chronic granulocytic leukemia.

You can inherit conditions which cause impaired erythropoiesis, but it's rather rare, but I mention them not because you need to remember them really, but just to make you aware. There's one called fang, cone is anemia. It's rare, but it's strong in some other races.

First reported in 1927, progressive bone marrow failure, giving rise to death from hemorrhage or reinfection. It's a complicated issue, I think, involving chromosomal breakage and the hematological malignancies and all that sort of thing.

Patients tend to get acute myeloid leukemia much more readily than normal, but it's very unusual, so you don't have to worry too much about it. There's another one called diamond black fan.

The incidence is seven in a million. Congenital pure red cell aplasia. I believe it can be treated by steroids, but the median survival is not particularly long, about 38.

The only cure for both actually is either a stem cell or a bone marrow transplant from a matched healthy donor.

Okay, so this is autoimmune hematocinemia, which is an acquired hematocinemia.

Autoimmune hematocinemia then is where you have developed an antibody against your own red blood cells.

The body normally, although the immune system normally goes to considerable length to avoid making what we call autoantibodies, which are antibodies which are active against your own cells, because clearly you don't want to be damaging or destroying your own cells.

But in some cases, these antibodies are made. Some drugs can do it.

Some viruses can do it. Sometimes we don't know why it happens. And the causes of autoimmune hematocinemia, about 50% are idiopathic.

Idiopathic means we don't know why it's happened. It just has a patient who's made an antibody against their own cells. It can happen in lymphoproliferous disorders, like lymphoma or chronic lymphatic leukemia. Mycoplasma, TB by another name, and extract biovirus, glandular fever, they can also cause you to make an antibody against your own cells. I said already, drugs can do it, particularly penicillin. And other autoimmune diseases, for instance rheumatoid arthritis, once again, can cause you to make an antibody against your own cells. But what happens is that the antibody sticks to the cell membrane, red cell membrane, and each time the red cells go through the spleen, the spleen it macrophages, oh, that didn't belong there. That antibody stuck there. I have that off. So it removes that bit of membrane.

So a little bit like the process that gives rise to spherocytes in hereditary spherocytosis.

What we find in autoimmune hematocinema is that the cells become spheres because they progressively lose red cell membrane as the cells go through the spleen. So you can see that these cells don't really have an area central color that they, if you like, they are hypochromic. They are highly colored. But we also find these cells here, which have a kind of a purple issue, if we were to do the right stain, these would be reticulocytes because they're early red cells. And there are two of them here, which is more than we'd expect. And the body's made these, obviously, because the hypoxia has been sensed.

So the bone marrow kicks out more red cell precursors to try to boost the circulating hemoglobin. Because these are other red cells are going to have a rather short lifespan because the spleen is busy destroying them. And we call these cells sort of progressively lost a lot of membrane, microus spherocytes. They're not necessarily actually small in terms of their volume, but they look small in terms of their diameter because they are much thicker than normal discs.

And it is, in fact, a normal cytic, normal chroma canina. The red cell MCV is not small. So there is a complication here.

If it was small, and you've got lots of new red cells, as you can see here, these new red cells are quite large compared to some of the others. So if you've got lots of new red cells, they're big. If you've got some red cells which have become smaller, the mean of the cellular volumes is going to be the same. So that needs a bit of careful thought sometimes that just because the mean cell volume is normal doesn't tell you that there are no little ones or no big ones.

OK. This is another acquired hemelytic anemia.

Completely different process. So if we look at this picture, we can see that these red cells look really quite unhappy.

These are red cell fragments which have a snappy name schistocyte, which once again we tend not to use. And what's happened here is these cells have been mechanically broken up.

And they've been broken by one of a number of processes. It's commonly associated with a particular sort of replacement heart valve where it could be referred to as cardiac hemolysis.

And this is a type of heart valve which essentially is a plastic ball in a metal cage to replace one of the aortic valves. And the ball moves up and down in the cage to seal or open the port.

And in doing so, it tends to smash the red cells. So it gives rise to this condition.

If you like cardiac hemolysis, but it's a micro, it's a mechanical fragmentation which would give the rather snappy name of micro angiopathic. Because what this means is the cells are being broken down within the circulation as opposed to in the spleen.

So we find it commonly with mechanical heart valves can also occur in two clinical conditions. I will mention, I'm not going to tell you too much about them because of the whole new topic.

DIC is a coagulation disorder called disseminated intravascular coagulation where you deposit fibrin on the insides of your blood vessels. And HUS, hemolytic uremic syndrome stroke thrombotic thrombocytopenic perfura. This results in the deposition of fibrin in the renal circulation and has a similar consequence to the fibrin in DIC.

The fibrin strands cause shear stress which I'll show you in a minute, which breaks the red cells up.

The last identified cause of micro angiopathic hematocinemia, just as a bit of levity really is, you can also find it in what we call March hemoglobinuria. This was described I think in the 19th century where troops on, Napoleonic troops on long marches started to pee blood or at least red urine, hemoglobinuria rather than hemoglobin. So it is hemoglobin in their blood. And the reason for that is that the constant bashing of the soles of their feet breaks the red cells up in the capillaries in soles of your feet. So they get free hemoglobin in their circulation, as we've said it's clear by the kidneys to give you hemoglobinuria.

And I gather that people who play the bongo drums for many hours can have a same problem associated with fragmentation in the capillaries in the palms of their hands.

So this is shear stress. So what's happening is this if you like is fibrin deposited in the walls of a capillary.

So as the red cells approach, the flow speeds up and the flow rate is much faster in the middle than it is at the sides. And this physically tears the red cells apart.

So that's the mechanism of red cell fragmentation in things like DIC, HOS, TGP. Okay.

So this is a congenital hematocinemia caused by a problem with one of the red cell enzymes. And I mentioned right at the beginning of the first half this morning about how red cells need an active metabolism to maintain their shape and their water status.

And I mentioned this enzyme called pyruvate kinase, which is essential for the production of adequate amounts of ATP to give the red cell energy. And this picture, I took this from a patient who is pyruvate kinase deficient. And what's happening is these cells are unable to regulate their water content. So they're losing water through exosmosis.

So I don't know whether you can see it very clearly from where you are, but the cells have taken on a very sort of spiky appearance. And this is associated with their dehydration. And we call them burr cells after those sort of seed heads, which form on top of tweezers because it looks a lot spiky.

Needless to say, the spleen is going to destroy these reasonably quickly.

So that's the problem.

It is another example of a normal cytic and normal chromokinemia. But it's an example of one example, and there are others, we'll show you in a minute, of a congenital hematocinemia caused by a red cell enzymeopathy. So this is another red cell enzymeopathy.

I mentioned G6PD, which is the enzyme necessary to produce NADPH, which is essential to maintain the ion in a reduced state.

If G6PD is deficient, the heme ion oxidizes to metemoglobin, and it forms chunks, which the splenic macrophages pluck out.

And we call them bite cells because quite literally the spleen takes a bite out of a variable hematocinemia.

But it can be made much, much worse if the patient challenges themselves with oxidizing agents.

And oxidizing agents, some of them are dietary. Faber beans, for instance, are a category of bean. Broad beans is one of them. They're present in quite a lot of diets. And they give you an oxidative challenge. So if you have an impaired ability to keep your hemoglobin reduced, then you're going to make more of these metemoglobin chunks, and you will get a hemoglobinous anemia.

Malaria treatment can do the same. The snappy word for bite cells is keratocytes after the Greek for horn, because these aren't supposed to be horns, but I don't get anyone who uses that term anymore. So once again, that is a normocytic, normal chroma anemia. The last category of anemia I want to talk about is hemoglobinopathy.

There are books, a number of inches thick written about hemoglobinopathy, so I will try to summarize it probably rather crudely. But there are two categories of hemoglobinopathy. The first one is what we call structural variations, where the hemoglobin is made in normal amounts, but its structure is normal, so its function is impaired. The other sort is what we call thalassemia syndromes, in which you variably lose the ability to produce a particular type of globin chain, and the ones of particular concern are obviously making alpha or beta chains, because that's what adult hemoglobin is made from.

So let's talk about the structural variations first.

So the structural variations occur when you get an amino acid substitution in, usually, but not always, one of the globin chains, and we demonstrate this by high-performance liquid chromatography.

So the most significant variants are in the beta chain, and the most significant of these by a country mile is this one, sickle cell hemoglobin, which is caused by one amino acid chain, glutamic acid at position six, on what the beta chain is substituted by valine.

And I've got a slide, I think it's probably the next picture, of the very extreme problem that causes other variants, even globin D.

Punger, and you can read them as well as I can. So these are beta chains, and we get alpha chain variants as well, which tend to be less significant. And many years ago, we did actually find hemoglobin reading. So this is the significance of these structural variants.

Hemoglobin S, if you're a heterozygote, usually asymptomatic, usually no problems.

If you're a homozygote, gross life-threatening hemolysis, talk about in a minute.

Hemoglobin D, no symptoms of heterozygote, or homozygote usually.

Hemoglobin C can have a slight microcytic anemia, but usually it's more microcytic than anemic.

But if you combine it with hemoglobin S as a heterozygote, it does tend to exacerbate sickling, and this can be a problem in some anti-natal situations.

Homozygotes for hemoglobin C, just mild hemolysis.

So I mentioned sickle cell, so this is what the blood of a patient in sickle cell crisis looks like. So what happens in sickle cell is that, so there is one amino acid change on the beta chains, and that is enough to give the cell, and I've picked the hemoglobin, a particularly unfortunate property, which is that when you take its oxygen away, it's in the deoxygenated state, it becomes insoluble. And the hemoglobin crystallizes to form the long crystals, which we call tactoids, which are like twisted up electrical cables, and they elongate across the diameter of the cell. And that distorts the cell.

Initially that process is reversible, but unless the cell is fully hemoglobinized quite quickly, it becomes irreversible, and you can envisage that these sickle cells are going to be picked off by the spleen very quickly, and that gives rise to a violent hematic episode, which is called a sickle cell crisis.

It's a very serious condition, very painful, potentially life-threatening, and it can be brought on by a number of things, I think, diet and dehydration, particularly.

The good news is that sickle is an autosomal recessive condition, so the heterozygotes are largely unaffected.

In terms of its classification, it is still a normal cytic, normal chromokinemia.

So sickle cell can have lethal consequences, but it is the commonest inherited blood disease in the world, which is surprising. Even though, left untreated, the lifespan of a homozygote for sickle in sub-Saharan Africa is less than 10 years, and even with modern medicine, it's only about 60 years in the UK. So you would ask yourself, if it causes such widespread death, why is it so common?

And the reason is this issue of being part of a balanced polymorphism, because it turns out that the frequency of the hemoglobin S gene is maintained in malaria areas, despite the severe consequences of homozygosity, because what happens is the heterozygote for hemoglobin S is more fit than either the homozygote for S or the homozygote for A.

The homozygote for A, hemoglobin A, this isn't normal adult hemoglobin, tends to be very prone to the worst sort of malaria infection, plasmodium falciparum, which, untreated, causes widespread death before the reaching childbearing age. Ahemoglobin S, sickle, as we've said, is a life-limiting, a life-limiting hemological disorder. But heterozygotes are protected, to a large extent, from the worst effects of plasmodium falciparum. They tend not to get the infection.

At least, if they are infected, it tends not to progress in the same way.

So, they don't get the malaria infection, but they don't sickle either. So, the heterozygote A.S., to some of, is protected from malaria and doesn't sickle. So, if you like, A.

nature sacrifices the homozygotes and S. to preserve the heterozygotes. Interesting, I think.

And then there are some more hemovobins, which we call the unstable hemovobins.

They are not massive considerations, but I include them here, really, for the sake of completion. And, for instance, the hemovobin clone and the hemovobin zurich, there's lots of them. They usually cause, I think, a mild hemovitokinemia. Quite often, it's a sort of a spontaneous, arising mutation.

So, the abnormality is in the hem not to be firmly bound. Water gets into the pocket and oxidizes the hemovobin, oxidizes the ions to get the hemovobin.

Also, of course, there's interference in binding of the alpha and the beta chains, as we saw earlier in the first bit of the talk. The way the alpha and the beta chains bind is important because of that conformational change that they undergo for deoxygenation and oxygenation.

And also, it just interferes with the alpha chain structure, let's say, not a major clinical concern.

Unstable hemovobins, you have said this, result in oxidation of heme ion, which can precipitate and damage the cell membrane. We call the precipitates hind bodies.

We can stain for those in pretty much the same way as we stain for reticular sites with either methylene blue or brilliant creosal green. And they stain up as chunks, which the splenic macrophages usually remove. Not surprisingly, then, in doing that, they cause ahemosuchinemia. This is what hind bodies look like.

Chunks of oxidized hemovobin near the edges of the cell. Sorry about the rather blurred picture.

Okay, so that's the structural defects. So, we now come on to the thalassemia syndrome, and this is the last area of the talk. So, the thalassemia syndromes are a group of inherited red cell disorders characterized by a variable inability to make the globin chains. And we're going to think mainly about alpha and beta chains.

Interestingly, malaria is involved here as well, because despite the fact that being a homozygote for a thalassemia, for beta thalassemia particularly, is a severe clinical problem, it's actually quite a common disorder.

And the malaria parasite seems not to like to develop in red cells of a thalassemic patient, possibly because the hemoglobin is a sort of concentration of the normal. And I think probably that deprives the developing parasites of some of their food.

But generally speaking, thalassemias developed or are common in areas where malaria is endemic.

So, warm air is next to the sea. So, we've said that in the adult hemoglobin and hemoglobin A, there are two globin chains, two alpha chains, two beta chains. So, let's now look at what happens if you've got thalassemia for those.

So, alpha thalassemia, so impaired ability to synthesize globin, alpha globin chains.

If you've got excess beta chains, then that's a problem, because normally speaking, of course, the beta chains match up to the alpha chains to make hemoglobin. But if there aren't any alpha chains, the beta chains have got nowhere to go. So, they make what we call beta tetramers, which we call hemoglobin H, or in the fetus, that's excess gamma chains.

We get hemoglobin barks. And the beta tetramers aggregate, and they can be stained by brilliant creasal blue, and they do give rise to a mild microcytic hypochromaginemia.

So, in alpha thalassemia, you've got, essentially, the clinical problem is the deletion of some alpha chains. We normally inherit two alpha chains from each parent. So, we've normally got, sorry, two alpha genes from each parent. So, we've normally got four alpha genes.

And in alpha thalassemia, one or more of them is deleted.

So, if you've got three normal alpha genes, there's almost no effect on hemoglobin production. Everything proceeds normally, and there's no clinical effect.

Two normal genes, you've got what we call alpha thalassemia tray. You've got nearly normal hemoglobin production, but you do get a mild microcytic hypochromic anemia.

If you've only got one functioning alpha gene, you've got what we call hemoglobin H disease, well, you can find those beta tetramers that stain if you do the right stain, causes a microcytic hypochromaginemia.

If you've got no functioning alpha genes, then that's incompatible with life, and the fetus aborts at an early stage. Beta thalassemia, then.

So, this, not surprisingly, is the impaired ability to synthesize beta globin chains.

And it's caused by mutations on this gene, HPB gene, located on chromosome 11, and there are many of them.

And genetically, it's quite heterogeneous.

Some beta mutations, that's the beta naught mutations, allow no beta chain production at all. Beta plus allows some So, if you can't make enough beta chains, you've got lots of alpha chains left over, and these alpha chains bind to the red cell membrane, and they damage the red cell membrane, causing ineffective erythropoiesis.

The cell is actually aborted at an early stage in its erythropoiesis, but also, of course, reduced red cell survival.

Okay. I mentioned earlier on that hemoglobin A2 is a minority component of most adult hemoglobin. And in beta thalassemia, we find that hemoglobin A2 can be quite elevated, because if you can't make hemoglobin A, which is alpha 2, beta 2, you can make hemoglobin A2, which is alpha 2, delta 2. So, there are no beta chains in hemoglobin A2, so you can make that hemoglobin, and patients that have served with beta thalassemia tend to have quite elevated hemoglobin A2.

They also have elevated HPF for the same reason. HPF production is usually switched off at the moment of birth, because the child doesn't need high-affinity hemoglobin anymore, and a high-affinity hemoglobin is actually, it does bring problems.

It causes, variably, a polycythema, that is where the patient has too many red blood cells, because the hemoglobin doesn't give its oxygen up very readily, as readily as hemoglobin A.

You get hypoxia at what would otherwise be normal hemoglobin levels. So, the hemoglobin is increased by the erythropoiesisin process.

So, you get a polycythema in patients with a lot of hemoglobin F. But nevertheless, it's better having some hemoglobin F than no hemoglobin A.

A bit like the alpha thalassemias in some ways, patients who are heterozygous have a mild microcytic hypochromic anemia, but it's not clinically significant, usually, and we say they have carrier status or alpha thalassemia trait.

But patients who are severe anemia, depending on whether they've got beta plus or beta naught thalassemia, and left to its own devices, it's usually fatal in infancy or in childhood without intervention, because they just can't make beta chains.

And what we find is that you get skeletal abnormalities in these patients, because the degree of hypoxia is so great that erythropoiesis sets up in all other sites in the body, apart from just the long bones and sternum, etc.

And it sets up in the skull, for instance. So, if you x-ray the skull of these patients, they exhibit an abnormality called hair on end, which is that their skull is very thick with vertical striations, and it's an abnormality caused by erythropoiesis starting up in the skull.

And these patients, generally speaking, are transfusion dependent, because they can't make hemoglobin A.

This gives rise to more problems, of course. I mentioned earlier that if you give a unit of eryopolite, you're giving a lot of iron, so these patients become iron overloaded, because they will have to be transfused at regular intervals, typically, I think, monthly.

So, they have to be given treatment of some sort, which binds the iron and the cystin is removable from their body.

So, this is not a particularly good quality of life.

But there is some optimism. For a while now, we've had the ability to do bone marrow transplants, which can cure these patients, but that's a very extreme measure to take. Or more recently, we could subject the patient or offer the patient gene therapy to try to give them functioning genes, so they can make their own beta chains.

Come on to that in a minute. There are more complications of thalassemia in that it can combine with hemoglobin S and hemoglobin C.

Okay, so I mentioned about gene therapy. This is a recent development, and it is a clever system by which the patient has their genetic makeup altered, so that they, rather than stopping hemoglobin F production at that moment of birth, hemoglobin F production actually continues, hopefully, for life. And the treatment is this. It's called Cascivi. I don't know how it's pronounced. I guess that's it.

It's a gene therapy in which the production of birth is actually knocked out using this gene editing tool from the patient's stem cells, which have to be harvested.

So what you end up with is a few stem cells from the patient, which have the potential to continue producing the produced red cells, which can continue to produce hemoglobin F, rather than trying to produce hemoglobin A. So these stem cells are grown up into a clone, and they are then reintroduced or transfused into the patient, where they colonize the prepared bone marrow, and I'm not sure what the preparation involves. It might involve ablation that is kind of destroying a lot of the patient's bone marrow content, but I don't know when that's the case.

But the idea of the treatment is to set up a clone to recolonize the patient's bone marrow with stem cells, which produce hemoglobin F. And as I've suggested, really, as hemoglobin F has gamma chains, not beta, it gives the patient some viable red cell function.

And hopefully, this is a lifelong cure. We don't know yet. It's still early days. It's just been approved by the UK medicine regulator at the astonishing cost of $2 million per patient. Again, the NHS has quite a large discount. But the cost of maintaining patients with beta thalassemia in the population at large is very high.

And obviously, the emotional cost, their quality of life. So it has been decided that it's a cost worth paying. And you can use this treatment for sickle as well. I mentioned beta thalassemia, but for sickle as well.

This is a schematic of the treatment that I grabbed off the BBC website a little while ago. It kind of says what I just said, so I won't go over it again. This is a summary of anemia classification, which I also won't go. I left it just so you can get clarification for you. So that's my lot, I think, time-wise.

I'm sorry it's so much information. I hope you found some of it at least interesting and memorable. I'm happy to take questions now, or come and see me. And unless Natasha wants to start the next session straight away, I'll be around for the next few minutes. Yeah, I'll be around for the next few minutes if you want. Want any more information? Thank you for your attention. I'll see you tomorrow for something completely different.

BI2HI1 Week 2(6) - RBCs and Anaemia

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