Structure, function, production and fate of red blood cells

This chapter does not refer to any Section of the 2017 CICM Primary Syllabus, because nowhere in the Syllabus Document is there anything about erythrocytes spelled out specifically, except as vague waving gesture in the direction of "the physiological production of blood and its constituents". That would be Section Q1(i). Not so, in the actual exam, where erythrocytes have appeared multiple times:

• Question 14 from the second paper of 2016
• Question 11 from the first paper of 2014
• Question 15 from the first paper of 2012

These were largely solid SAQs calling for a detailed understanding of what erythrocytes are, how they are made and what makes them awesome. Judging by the pass rates of 19% and 33%, most of the candidates were not prepared for this. 

As far as a single best peer-reviewed resource, the paper by Gordon-Smith (2013) would be ideal because it is densely packed with the right answers, but unfortunately it happens to paywalled. Instead, three free papers can be suggested. Karen Brown (1996) has you covered for erythrocyte metabolism, Dzierzak & Philipsen (2013) for erythropoiesis, and Kuhn et al (2017) for red cell structure and function.

Structure of red blood cells

From an evolutionary perspective, we have had these things since the lamprey bodyplan was a revolutionary new concept. Though erythrocytes appear to be an essential part of a vertebrate circulatory system, there's a whole wide range of different possible engineering answers to the same problem (Nikinmaa, 1990). They vary considerably in size, shape,  nucleation, mitochondrial content, and deformability.  Here, an excellent and  underrecognised contribution from George Gulliver (1875) is presented to illustrate the breadth of this diversity:

Human erythrocytes are typically only 6-8μm in diameter, and as you can see they are on the lower end of the size spectrum.  For contrast,  Amphiuma, a genus of aquatic salamander, has massive nucleated red cells (48-78 μm), which is thought to be an adaptation to the need for big wide low-resistance systemic capillaries (Snyder & Sheafor, 1999)- as the amphibians have a single ventricle circulation, they really can't afford to have one high-resistance circuit and another low-resistance circuit, so both their systemic and pulmonary circulation basically need to be pressurised to roughly the same level.  What is the point of this digression? They don't have to be biconcave disks. 

Biconcave disk shape of red blood cells

But why are they biconcave in humans, when clearly there are other options? We speculate; but broadly, the benefits of being biconcave are:

• Greater surface area: though, admittedly, this is not much, but still the surface-to-mass ratio of a biconcave disk is better than that of a sphere. To say that "this particular profile maximizes the surface to volume ratio" is probably only true because surface area is not the only engineering specification for an erythrocyte, and - by compromise with all the other requirements - this shape is the best we could do. Consider that an echinocyte would have much more surface area, but terrible rheology in narrow tubes, reduced deformability, and so forth. 
• Maximal diffusion of gases: the radial distance from the centre of a disk is always going to be smaller than the radial distance from the centre of a sphere with the same surface area, and this matters for very simple Fickian reasons. You would want to maximise the diffusion of oxygen from the haemoglobin molecule which happens to be sitting right in the middle of the erythrocyte.
• Enhanced deformability is essential, as red cells need to proceed through thin capillaries, often narrower than the actual diameter of the cell itself. In particular, the way you deform seems important. Here, in a landmark paper from 1963, Guest et al produced stills from a high-speed video of capillary blood flow, illustrating the change in shape which takes place 
  
  In support of biconcavity, Guest et al opined that "it may be that this shape is particularly
  adapted to paraboloidal transformation", but why a parabolid is required at all remains unsettled.
• Maximised laminar flow: Uzoigwe (2006) suggested that "the biconcave profile of the discocyte means that much of the mass is distributed in the periphery", which makes it less prone to rotation during flow in the large vessels, which therefore promotes laminar flow and discourages atherogenic platelet scattering. This is only a hypothesis, eg. it was published in Medical Hypotheses, but with plausible reasoning.

Ultrastructural features of red blood cells

All of these features have some unique (or at least notable) quality which needs to be mentioned for completeness:

• Extremely negative surface charge. The erythrocyte cell membrane, itself a fairly unremarkable phospholipid/cholesterol bilayer, is on the surface severely greebled with glycosylated protein residues. These - part from being physiolgically important transmembrane proteins (for example, the proteins that make up the blood grouping antigen system) - also contribute to making the outside of the membrane extremely negative. This has massive benefits. Most significantly, it prevents the cells from blobbing together into a lump (the professional term is probably agglutination) because, as the result of the repulsive force of negative charge, two red cells cannot approach each other any closer than about 50-100 Å (van Oss & Absolom, 1983)
• Dynamic cytoskeleton which is basically a scaffold formed by Band 3, a protein which sticks out of the inner membrane like a tentpole, and long bands of other proteins (spectin, actin, etc) which connect these tentpoles to one another and maintain tension on the inner surface of the membrane. The image below is a  concatenation of a three-dimensional model by a Li et al (2018) and a TEM image of the erythrocyte membrane magnified 200,000 times from Byers & Branton (1985). Looking at these, one might come to the conclusion that this cytoskeleton takes on an orderly mesh-like form, but when David Stokes and colleagues imaged it in 3D, they found it to be a "densely packed, heterogeneous network of filaments", as seen in the lowermost image.
  
  This loose weave of apparently random threads remodels itself dynamically, allowing the red cell to squeeze through tiny capillaries (perhaps a quarter of its diameter) and then to return to its original shape, repeatedly many thousands of times over its expected 120-day lifespan.
• Lack of organelles is not really a structural feature so much as it is a lack of such features, but there was nowhere else to put this point. RBCs have no nuclei or mitochondria, which has several obvious benefits. Firstly, it maximises the space for haemoglobin (which is a fair and reasonable move, as a nucleus could be large), and two, it supposedly increases the deformability of the cell. The latter point seems odd, as one may come away from highschool biology with the impression that the cytosol and organelles have the consistency of custard, but in fact the nucleus is surprisingly rigid (5-10 times more rigid than the rest of the cytoskeleton) and its diameter is the main limiting for for getting sucked up into a micropipette of a given caliber, for example. In short, if your function depends on squeezing effortlessly through tight capillaries, consider not having a nucleus. 

Metabolism in red blood cells 

As mentioned above, the normal adult RBC has no organelles, which also means no mitochondria. That should raise the question: how, precisely, does it drive energy-dependent cellular machinery? There is one main pathway which splits three ways, and from past examiner remarks, it appears that "a complete answer" would mention these shunts. However, its is highly unlikely that the trainee would ever be expected to know these pathways in enough detail to draw them, or name individual enzymes. Still, in case somebody requires a huge flowchart of the Embden-Meyerhof-Parnas pathway, here is that eyesore, dug out from (of all places) the marketing section of the Sigma-Aldrich website.

In summary, short enough to be memorised five minutes before the exam:

• The glycolytic (Embden-Meyerhof) pathway is the main mechanism by which a red cell gets its ATP.  The pathway takes one molecule of glucose and converts it into two molecules of pyruvate. This is entirely anaerobic, i.e. the red cell uses none of its oxygen itself. As the result, ATP and NADH are produced. 
• Methaemoglobin reductase pathway borrows this NADH to reduce methaemoglobin back to haemoglobin (returning NAD to the pathway). If this step is taken, the borrowed NADH cannot be used later in the pathway, where lactate dehydrogenase uses it to convert pyruvate into lactate. 
• The Luebering-Rapaport shunt produces 2,3-diphosphoglycerate (2,3-DPG) by borrowing 1,3 diphosphoglycerate from the pathway before it can be converted to pyruvate. If this step is taken, the borrowed metabolite obviously won't continue down the pathway to turn into pyruvate or lactate. However, 2,3-DPG can eventually be converted back into 3-phosphoglycerate, and therefore could be viewed as a form of energy storage.
• The hexose monophosphate shunt produces NADPH, which then converts oxidised glutathione to reduced glutathione, and this is used as an antioxidant to protect the RBC membrane and enzymes.

Notable functional features of red blood cells

In the college answer to Question 11 from the first paper of 2014, CICM examiners complained that the candidates

"failed to mention, and describe, the RBC’s role in acid base buffering and HCO3- production". There is, in fact, much more:

• Oxygen transport:  yes, the red cells are full of haemoglobin. Some might say that oxygen carriage is actually a function of haemoglobin, but in actual fact haemoglobin in the circulation on its own would generally be quite useless, as it tends to fall apart into dimers which are incapable of normal oxygen transport. Moreover, if haemoglobin were free in the circulation, it would have a host of toxic effects.   There would be no nitric oxide, for one; as haemoglobin would very effectively bind every last molecule of it with very high affinity, resulting in catastrophic widespread vasoconstriction. In other words, it is not as if red cells are completely superfluous, and haemoglobin would be fine all on its own.  
• Protein buffering is mainly due to the presence of abundant haemoglobin inside the cell, which acts as an important buffer because of its multiple histidine residues. Authoritative sources state that 50-60% of all buffering in the bloodstream is due to haemoglobin.
• Bicarbonate buffering: red blood cells contain a large amount of carbonic anhydrase which catalyses the reaction between CO₂ and water to produce bicarbonate.
• Mitigation of pH change in the peripheral circulation: pH of the peripheral blood would change significantly more if deoxygenated RBCs were not there to buffer the acid and sequester the chloride. Fortunately, the bicarbonate produced in the red cells is quickly exported into the circulation, and chloride is imported into the cells by the Band 3 protein in order to maintain electroneutrality, in a process usually referred to as the Hamburger effect. 
• Protecting everything from haemoglobin. If haemoglobin were free in the circulation, it would have a host of toxic effects, and it would  There would be no nitric oxide, for one; as haemoglobin would very effectively bind every last molecule of it with very high affinity. 
• Keeping complement under control. Erythrocytes have two proteins protruding from their membranes (decay accelerating factor CD55 and membrane inhibitor of reactive lysis CD58) which inactivate complement. A deficiency of these proteins results in paroxysmal nocturnal haemoglobinuria (Gordon-Smith, 2013).
• Regional blood flow autoregulation: Apart from the nitric oxide scavenging by haemoglobin, red cells also release other vasoactive mediators (eg. ATP) which contribute to the regulation of regional blood flow, for example producing vasodilation in hypoxic tissue regions (Kuhn et al, 2017)

Haematogeny: production of red blood cells

The three main steps along the pathway from stem cell to red cell are:

• Myeloid progenitors
• Pro-erythroblasts
• Reticulocytes

Myeloid progenitors are rather boring pluripotent myeloid stem cells, the products of the even less differentiated haematopoietic stem cells.  They then undergo erythroid lineage commitment and become pro-erythroblasts (where they could have also chosen to become granulocytes, monocytes or megakaryocytes). The switch to erythroid lineage is not visually spectacular, and mainly characterised by the expression of different surface CD markers.

Pro-erythroblasts  are relatively large cells with a big nucleus . They reproduce helishly fast; the rate of cell cycle turnover is 6-7 hours, which is something you would otherwise only ever see in embryonic tissue (Vandekerckhove et al, 2009). These are also the cells that produce all that haemoglobin, and in the course of becoming filled with it, they progress through stages:

• basophilic erythroblasts (i.e. mainly filled with ribosomes and other cellular machinery,
• polychromatophilic erythroblasts whigh have enough hamoglobin to be multicoloured, and
• orthochromatophilic erythroblasts, which have the "right" colour (i.e. red)

The additional cellular machinery and nucleus contribute to their size significantly; they are usually about ten times the size of a mature RBC. As they mature, the size of the nucleus decreases. The orthochromaic stage eventually expels its nucleus, along with all their other organelles, to become reticulocytes. This "enucleation" is a super weird process which is basically mitosis, except one cell ends up without any reproductive machinery, and the other cell ends up a "pyrenocyte", which is all reproductive machinery, just nucleus modestly draped in some minimum figleaf of a cell membrane. Carrying on the lewd metaphor, these pyrenocytes present an "eat me" signal to the macrophages of the erythroblastic island by everting their cell membranes and revealing the seductive  phosphatidylserine from its inner surface. Yoshida et al (2005) were able to explain this much better, and without giggling like schoolchildren. 

Reticulocytes are the enucleated product of orthochromatophilic erythroblasts. They are sufficiently ready to exchange gas and do other red cell things, and so they are released into the circulation, where they continue to mature over the subsequent 24-48 hours.  During that first couple of days they decrease in surface area by about 20-30% and lose about 10-15% in volume (Waugh et al, 1997). The thing that makes them reticulocytes, the mesh-like mess of ribosomal RNA strands in their cytoplasm, is quickly jetissoned by exosome exocytosis; vesicle-like blebs of membrane are pinched off and sent as flotsam into the bloodstream, to be picked up by the reticuloendothelial system (Blanc et al, 2005). These exosomes are massively numerous, the bloodstream is literally lousy with them, and the only reason you don't typically order an "exosome count" on an FBC is that they are too small to count optically, being about 60nm in diameter, and they seem to be completely physiologically inert, in the sense that we have not discovered any important roles for them. This is remarkable, as apparently, about 10¹⁴ of them are released into the bloodstream every day, and they are covered in/full of working enzymes such as acetylcholinesterase. Anyway: when reticulocytes lose their ribosomes, they are considered mature red cells. At about this time they also acquire their final biconcave form.

Rate of haemopoiesis is under complex regulatory control, but for exam purposes one can merely say "erythropoietin". This hormone is one of the endocrine functions of the kidney, and its release is stimulated by hypoxia of the renal medulla and the abundance of angiotensin-II, which is interpreted as haemorrhage. The normal maturation time, from stem cell to red cell, is thought to take about seven days under normal circumstances, and about 1% of the erythrocytes are cleared every day and replaced by new cells. Under extreme stress, this can be markedly increased. A patient with anaemia who is for whatever reason opposed to blood transfusion can have a rate of erythropoiesis which increases their haemoglobin very rapidly, provided the right metabolic substrates are available.

How fast could you erythropoiese, unforgivably verbing that noun, if everything else needed for red cell synthesis was also offered? An excellent example of what is possible can be seen in Myerson  & Carrroll (1955), who reported the case of R.B, a well nourished 34 year old gentleman with hemochromatosis (and therefore abundant iron reserves). Each month they saw him in clinic, and each month they would drain him of up to 2.7 L of blood, with no change in his haemoglobin concentration. At that rate of loss, with a haemoglobin concentration of 120g/L (and thus losing about 324g/month), this man was producing about 10-11g of haemoglobin per day between venesections, which would correspond to an increase of about 2g/L/day. This corresponds nicely to another case report, this time by Bates et al (2008). The authors gave 20,000 units of EPO daily, as well as oral supplemental iron and all the vitamins and proteins you could possibly want. As you can see, the rate of hemoglobin increase was greatest over the first week of therapy, where it incremented by about 3.4g/L/day. 

The fate of red cells

The term "fate" is interesting, as it implies a certain fatalistic inevitability. It is unfortunately disappearing from modern writing on haematology and chemistry, being more popular in the days of Rous & Robertson (1917). As discussed to some extent in the chapter on haemoglobin, there are really only two final destinations for the red cells, one being the reticuloendothelial system and the other being haemolysis in the bloodstream. As the latter is not predictable or controlled, it will suffice to say that probably about 10% of them end that way, and this is considered normal. The rest are consumed by the reticuloendothelial system.

The lifespan of an erythrocyte is said to be about 120 days in humans, 30 days in mice and 60 days in rats. In non-mammalian species the red cells are nucleated and have machinery for aerobic metabolism, which makes the lifespan of erythrocytes a little longer, eg. in the metabolically conservative reptiles it averages 600-800 days. During their lifespan, red cells undergo a variety of changes, well summarised by Lutz & Bogdanova (2013). In short:

• They increase in density and decrease in size.
• Enzyme activity decreases, especially metabolic enzymes such as hexokinase and pyruvate kinase.
• They become covered in antibodies. Specifically, 7 fold higher amounts of autologous IgG were found when labelled cells were administered and then recollected from the circulation three months later by Rettig et al (1999). 
• They contain more denatured and irreversibly oxidised proteins, especially haemoglobin

The reticuloendothelial system, for this purpose mainly represented by the spleen and liver, identifies senescent red cells by these and other criteria. In short:

• Deformability: the ability to penetrate sinusoidal capillaries in the liver and spleen is a characteristic of a young flexible red cell; elderly shrunken cells with a degraded cytoskeleton will get stuck, and end up being phagocytosed.
• Opsonins: Autologous IgG targets clusters of Band 3 protein which form as a part of red cell senescence, and labels them for phagocytosis
• Surface "eat me" signals such as reversal of membrane phosphatidylserine 

The cells identified in this way are phagocytosed by reticuloendothelial macrophages.

• Inside reticuloendothelial macrophages:
  • Globin molecules are catabolised and released as amino acids
  • Haem is stripped of iron and converted into biliverdin, then bilirubin
    • Bilirubin circulated bound to albumin and is conjugated in the liver
    • Conjugated bilirubin is secreted and converted into urobilinogen in the bowel, then into stercobilinogen and then into stercobilin
    • Urobilinogen can also be reabsorbed and excreted in the urine as urobilin
  • Iron is stored in the reticuloendothelial cells as ferritin or exported into the circulation, in which case it circulates as transferrin