Why am I not better after a transfusion?

I have been asked about why blood transfusions do not make you feel better immediately. The answer lies in the Rapoport-Luebering Shuttle. For those who would like to read the long-winded textbook explanation try this link but for others, here is an explanation.

The main purpose of red cells is to take oxygen from the lungs and deliver it to the tissues. For this purpose they contain hemoglobin. Hemoglobin has a high affinity for oxygen, but at some stage it has to let go of its oxygen so that it can be taken up by the tissues. How it does this is complicated but it is very dependent on the acidity of the tissues, but also on the concentration of a molecule called 2,3-DPG.

The change in oxygen affinity with acidity is known as the Bohr effect. Because the tissues are relatively rich in carbon dioxide and the red cell enzyme carbonic anhydrase readily converts carbon dioxide to carbonic acid, the acidity is greater there than in arterial blood; therefore, the Bohr effect helps the transfer of oxygen to tissues.

The other important factor affecting the oxygen affinity of hemoglobin is the concentration of 2,3-DPG. This molecule can insert into the pocket between β globin subunits in the 4-chain hemoglobin molecule and reduces oxygen affinity by displacing oxygen.

2,3-DPG is synthesized from intermediate molecules on the metabolism of glucose by means of a pathway known as the Rapoport-Luebering shuttle. The means by which glucose is broken down to produce energy for the red cell is known as the Embden Meyerhof pathway which is pictured at the beginning of this article. This second drawing is a blow up of the important area. Normally 1,3-DPG is turned into 3-PG by PG kinase (there is a rare type of hereditary anemia that lacks this enzyme) and this generates energy in the form of ATP. The Rapoport-Luebering shuttle is an alternative by-pass that generates more 2,3-DPG. Just how much glycolysis takes place by the main highway and how much takes the by-pass is a complex issue. It depends on the rate of production of its precursor 1,3-DPG, the level of acidity in the cell, how much ATP there is relative to ADP, the ratio of NAD to NADH, and the concentration of inorganic phosphorus.

The most important function of 2,3-DPG is its effect on the oxygen affinity of hemoglobin. In the deoxygenated state, each hemoglobin molecule can bind one molecule of 2,3-DPG, a reaction leading to reduced oxygen affinity and improved oxygen delivery to tissues. The increased oxygen affinity of stored blood is accounted for by reduced levels of 2,3-DPG. Transfusion of such blood results in an increase in oxygen affinity of circulating blood so that less oxygen is delivered to the tissues. It starts to return toward normal in 7 to 12 hours as the function of the pathway breaking down glucose is restored. A noticeable clinical impairment in oxygen delivery due to low 2,3-DPG levels remains disputed in some quarters, but it would be expected to have its greatest impact when large transfusions are required in critically ill individuals.

Weatherall's Lasker

I was very pleased to hear that Sir David Weatherall has won the Lasker Prize for his work in Thalassemia.

I have known him (though not well) for the best part of thirty years. Many years ago I wrote a paper with him, having discovered a patient with beta thal trait and swiss type hereditary persistence of HbF, each inherited from a different parent.

He was quite excited by the discovery and sent his research fellow, Bill Wood, down to Bournemouth to do the necessary confirmatory tests on our primitive equipment. The paper was published as Heterocellular hereditary persistence of fetal haemoglobin (heterocellular HPFH) and its interaction with B thalassaemia. WG Wood, DG Weatherall, JB Clegg, TJ Hamblin, JH Edmonds, AM Barlow. British Journal of Haematology 1977 36: 461-473. and has been cited 35 times.

New on MDS

Although most people associate me with CLL, I spent almost as much of my career on myelodysplastic syndrome (MDS). Even as a young man I was fascinated by this pre-leukemic condition and in my early twenties encountered three patients with what we would now call refractory anemia with sideroblasts who had chromosomal abnormalities (MM, AW and PS). In those days before chromosome banding, we could not identify individual chromosomes and just called them 'C' group chromosomes. In retrospect, chromosomes 7 and 8 were involved.

As a registrar and senior registrar I accumulated a series of unexplained cases, but it wasn't until Dr Mufti came to work for me as a fellow, having previously worked with the late, great, David Galton at the Hammersmith, that we set about identifying what was going on. We wrote an important paper that established that MDS was much more common than people had thought hitherto, and that most of it went undiagnosed. Myelodysplastic syndromes: a scoring system with prognostic significance
G. J. Mufti, J. R. Stevens, D. G. Oscier, T. J. Hamblin*. British Journal of Haematology 1985 59,425–433. We discovered that our hospital 76 cases had been misdiagnosed in the past 5 years. This was the first paper to offer a prognosis based on the initial diagnostic picture and it has since been cited in the medical literature 423 times. Later on we added our data to those of the French, Japanese, Spanish, Germans and a small American series to produce the International scoring system for evaluating prognosis in myelodysplastic syndromes P Greenberg, C Cox, MM LeBeau, P Fenaux, P Morel, G Sanz, M Sanz, T Vallespi, T Hamblin, D Oscier, K Ohyashiki, K Toyama, C Aul, G Mufti and J Bennett Blood 1997, 89, 2079-2088. which is one of the most cited papers in Blood with 1990 citations.

My main interest in MDS has been in diagnosing the very early forms of the disease. Barn door cases need to be distinguished from acute leukemia, but accurate diagnosis of the early cases depends on viewing very good blood films with an expert eye. Abnormalities of nuclear shape and structure, various cytoplasmic inclusions and granulation and overall cellularity are crucial. More can be done diagnostically by looking and the histology of bone marrow trephines and the disposition of the reactive tissue. Unlike CLL, you can't simply stick a sample on a flow cytometer and hope to get an answer. Though you may get clues.

There are certainly similarities between MDS and aplastic anemia, and one of the features of aplastic anemia is the presence of a clone of cells derived from this strange condition, paroxysmal nocturnal hemoglobinuria (PNH), in which the red cells are particularly susceptible to lysis by endogenous complement. It seems that cells lack GPI connected molecules on their surfaces that inhibit complement activity, particularly CD55 and CD59 and they can be tested for by flow cytometry.

The sorts of patients I am talking about are those that have a degree of isolated and unexplained thrombocytopenia, neutropenia or anemia (particularly with a raised MCV). The degrees of myelodysplastic features are too small to label the condition MDS (although Alan Jacobs of Cardiff used to call such cases NQMDS or NYMDS - not quite- or not yet- MDS). The international community has labelled such cases idiopathic cytopenia of undetermined significance (ICUS).

In this month's BJ Haem there is a letter from Japan (Ando et al 2010, 150: 705-707) reporting on PNH cells in ICUS. It is brief and probably premature, but it picks out 2 of the 11 ICUS cases that they studied and suggest that these who had small populations of PNH cells also had very low numbers of megakaryocytes - perhaps making them aplastic anemia forme fruste rather than MDS. However the numbers are too small to be statistically significant.

People complain that hematologists are poor at diagnosing MDS. True, but that underestimates the difficulty.

Anemia: the thalassemias.

In many parts of the world in the battle between man and the mosquito, the moggies would have won, were it not for the remarkable way that humans have altered their red blood cells. We have already mentioned sickling and G-6PD deficiency and today I am going to tackle the thalassemias. The thalassemias are a group of condition is which there is a defect in the production of the globin part of hemoglobin.


The body has quite a complicated way of making globin. ordinary hemoglobin is made from two alfa chains and two beta chains. Alfa chains are coded on chromosome 16. There are two alfa genes which are clustered with a gene for the zeta chain, an alfa chain equivalent produced in early embryonic life when blood is made in yolk sac. Both alfa chain genes are active in fetal and adult life and necessary to produce enough alfa chains.

Beta chains are coded on chromosome 11 along with a cluster of beta-like genes, including epsilon, gamma and delta chain genes. Epsilon genes are used by the embryo during yolk sac hemopoiesis. Embryonic hemopoiesis produces Hb Gower 1 and Gower 2 as well as Hb Portland. (Gower 1 is zeta 2, epsilon 2; Gower 2 is alfa 2, epsilon 2; Portland is zeta 2, gamma 2) The fetus uses the gamma chain gene to make HbF (alfa 2, gamma 2) and the adult uses mainly the beta chain gene, with small amounts of delta and gamma chains (HbA is alfa 2, beta 2; HbA2 is alfa 2, delta 2).

This is all very wonderfully designed with the appropriate hemoglobin being made at the different stages of development. For example, baby red blood cells need to snatch oxygen molecules from the mother's blood while the baby is in the womb. It so happens that HbF has a higher affinity for oxygen than HbA, and this is how the baby does it.

For the most part, individuals carrying thalassemia genes suffer no ill effects, but they are protected against malaria. You can usually tell that the individual has it because the red cells are smaller than usual. When we were dealing with iron deficiency we said that a red cell consists mainly of hemoglobin, so the amount of hemoglobin controls the size of the red cell. To little iron means not enough heme and therefore small red cells. In the thalassemias, not enough globin is made and therefore not enough hemoglobin and therefore small red cells result. These individuals are said to have thalassemia trait, meaning that they have inherited a thalassemia gene from only one parent. They are not anemic but they have microcytosis (small red cells).

If you inherit thalassemia genes from both parents then you are probably in real trouble with what is usually a severe illness, but it does depend on whether you inherit the same abnormal gene or a different one. Sometimes inheriting different thalassemia genes can actually make the condition less severe, but the various combinations give a wide variety of conditions from no illness to a fatal one.

There are four clinical states of thalassemia. The most severe is death in the womb. This is called hydrops fetalis - the fetus gets severe heart failure because it isn't making any blood and dies. The second most severe is thalassemia major, in which anemia develops at the age of about 6 months. The body strains to make blood, but hardly succeeds. In the normal adult blood making takes place in the flat bones of the body only - the skull, ribs, sternum, pelvis and spine, but in children it is also made in the bones of the arms and legs. In the fetus it is in addition made in the liver and spleen. In thalassemia major, blood is made in all these places and even in lymph nodes. So, big livers and spleens are the order of the day and the bone marrow so expands that the walls of the bones become very thin. The shape of the face is distorted with prominence of the frontal and maxillary bones, and fractures of the other bones are common.
X-ray of the skull demonstrates the expansion of the medulla and thinning of the cortex and the picture is often referred to a the hair on end appearance. (Picture from medispot123.blogspot.com).

These children have to be treated by blood transfusion. In fact they are heavily transfused to try and suppress the growth of the bone marrow. All this transfusion overloads them with iron which has to be removed or else it gets stuck in various organs, causing staining of the skin, arthritis, and damage to the liver and heart, as well as failure of a host of endocrine organs including the pancreas causing diabetes, the thyroid causing myxedema, the parathyroids causing a low calcium, the testes causing failure of puberty and the pituitary causing small stature.

Removal of iron used to involve nightly subcutaneous infusions of desferrioaxamine which itself was unpleasant and cause cataracts and damage to the retina as well as increasing susceptibility to infections, particularly with salmonella and yersinia. Happily we now have XJade an oral and more efficient chelator.

The least severe form of thalassemia, thalassemia minor, is totally asymptomatic with only a funny blood count to let you know it is there. Between major and minor, there is thalassemia intermedia in which there is anemia and a big spleen, but usually transfusions are not necessary.

Those are the clinical conditions; what are the genetic lesions that cause them?

Hydrops fetalis is caused by a complete absence of alfa chains - they are needed for both HbA and HbF. You have four genes coding for alfa chains, two from each parent. For Hydrops all four have to be missing. If it is recognized early by genetic testing, the baby's life can be saved by intra-uterine transfusion, but the baby will never make its own blood and it will be totally reliant on transfusions unless it could have a stem cell transplant. This sequence of treatments is seldom undertaken and termination of pregnancy is a more likely outcome.

Thalassemia major is usually beta thalassemia major, where a point mutation on both maternal and paternal chromosomes has prevented the production of beta chains. Since you don't need HbA until after birth it does not cause hydrops, but it gradually develops in the first six months of life. Free alfa chains are toxic to the red cell, shortening its survival. Production of other chromosome 16 globin chains, gamma and delta, means that there are increased amounts of HbF and HbA2, which help to soak up the excess alfa chains. If there is a degree of alfa thalassemia, this can lessen the severity of the condition. Rare individuals posess three copies of the alfa chain on chromosome 11 and this makes the condition worse. In some forms of beta thalassemia, small amounts of beta chains are produced which help to ease the condition slightly. Although beta thalassemia major is mainly a sign of two defective beta chains, sometimes the patients are double heterozygotes for beta thalassemia and another hemoglobinopathy.

Thalassemia trait is the non-significant form of thalassemia, only picked up on the blood count.
The blood picture is of a microcytosis with prominent target cells. With beta-thalassemia trait the diagnosis can be confrimed by Hb electrophoresis, though not with alfa thalassemia trait.




Here is is necesary to look for HbH inclusions by incubating the blood with brilliant cresyl blue. If only one alfa gene is missing then the incidence of the 'golf ball' inclusions may be as small as 1 cell in 50,000.though with two genes missing they are a bit commoner.

Thalassemia intermedia may have many causes. The patients are moderately anemic and have large spleens but they don't require regular transfusions. It may be caused by homozygous beta thalassemia - if the gene inherited from both parents allows for the production of some beta chains, or if alfa thalassemia is also inherited, or if a condition called hereditary persistence of fetal hemoglobin (which allows the gamma chains to soak up the excess alfa chains) coexists. Or it might be caused by the asociation of beta thalassemia trait and three (rather than 2) alfa chains on each chromosome 11. Or it could be HbH disease, where three of the four alfa chains have gone missing. Or it could be delta beta thalassemia (where no HbA2 is made). Or it could be Hb Lepore. This is a strange molecule made from the fusion of parts of the beta genes and the delta genes. Homozygotes show intermedia and heterozygotes the trait.

Most forms of alfa thalassemia are caused by deletion of one or more alfa chain gene. Hb Constant Spring is an alfa chain varient in which a mutation affects the termination code so that an elongated chain is produced. this fails to function as a proper alfa chain and effectively produces one of the forms of alfa thalassemia, though these non-deletional forms of alfa thalassemia (and there others with strange names like Hb Quong Sze) tend to be more severe than the deletional forms.

Hemoglobin E is now the commonest form of thalassemia in North America. Newer migrants from South-East Asia have carried the gene. HbE is caused by a substitution of glutamic acid by lysine at codon 26 of the beta chain. This mutation activates a cryptic mRNA splice site which results in reduced synthesis of the mutant beta chain, leading to a thalassemic phenotype. It also interferes with the interface with the alfa chain, leading to increased susceptibility to oxidative stress. Although individuals with homozygous HbE may be similar to Thlassemia trait patients, the condition is variable. Double heterozygotes with HbE and beta thalassemia tend to have thalasemia intermedia with hemolytic crises, though some have a thalassemia major picture. Some patients have benefited from hydroxycarbamide treatment which raises HbF levels.

The thalassemic syndromes are very common throughout the Mediterranean world and in South East Asia. With the migration consequent on war we are seeing and understanding more about these strange conditions.

Sickle cell anemia

I have committed to teaching the juniors next week on sickle cell disease. In my 35 years at Bournemouth I had only one patient plus some visiting students who had this disease, so I can hardly pose as an expert.

But it is one of the most common genetic diseases worldwide and in America occurs in 1 in 2400 live births. Among African Americans the incidence is 1 in 400. In some places in Britain everybody has a screening test for it at birth, but Bournemouth is not one of them, malaria being quite rare here.

The abnormality is due to the substitution of valine for glutamic acid at position 6 in the beta chain of hemoglobin. Hb S is insoluble and forms crystals when exposed to low oxygen tension. It then polymerizes into long fibers, each consisting of seven intertwined double strands with cross linking.

It is only when the sickle gene is inherited from both parents that the full picture emerges. The features are of a severe hemolytic anemia punctuated by crises. Hb S releases oxygen to tissues more easily than Hb A so the anemia is often more severe than its symptoms. Interestingly some patients live fairly normal lives without incident.

The crises may be painful, visceral, aplastic or hemolytic.

Most common are painful crises caused by the occlusion of small blood vessels. They are precipitated by such factors as infection, acidosis, dehydration or any condition causing low oxygenation (altitude, anesthetic, obstetric delivery or, violent exercise), exposure to cold or vascular stasis. The occlusions may occur in bones (hips, shoulders and vertebrae are most common), the lungs and spleen. The most serious events occur in the brain. In children the hand-foot syndrome is common when occlusions occur in the small bones. It may lead to digits of varying length.

Visceral crises are due to sickling within organs. The most common occurs in the lungs with infiltrates in the lungs causing severe respiratory distress and is the commonest cause of death. Sickling in the liver and spleen can also cause severe illness.

Aplastic crises occur either because parvovirus puts a halt to red cell production or because the body runs out of folic acid.

Hemolytic crises often accompany other types of crisis and are associated with a fall in Hb with a reticulocytosis.

Other clinical features include splenomegaly in infancy, but splenic infarction means that most adults have signs of hyposplenism. Leg ulcers are common.

Treatment is to avoid things that precipitate crises, especially dehydration, anoxia, infections, stasis of the circulation and cooling of the skin surface. Patients should receive folic acid 5mg a day as prophylaxis, they should be vaccinated against pneumococcus as children and adults should receive prophylactic penicillin like other people without a spleen. Good hygiene and nutrition should be practised.

Crises are treated with rehydration, antibiotics, if appropriate, and bicarbonate if there is acidosis. Strong pain relief is often necessary, but some patients do become dependent on opiates. Blood transfusion is only needed for severe symptomatic anemia (normal oxygenation is achieved with hemoglobin lower than in patients with Hb A). Exchange transfusion may be needed if there is neurological damage or visceral crisis, or if painful crises are common. The aim should be to achieve a Hb S level of less than 30%.

Transfusion is often necessary during pregnancy and a Hb S level of less than 30% should be achieved by delivery. Careful anesthetic technique is required to avoid hypoxemia or acidosis

Iron overload may become a problem in patients requiring frequent transfusions.

In carefully selected patients, stem cell transplant may be indicated.

Sicklers in Saudi Arabia often have high Hb F levels and this seems to prevent the worst complications. Drugs have been used to raise Hb F levels, particularly hydroxyurea (called hydroxycarbamide more recently).

Hydroxyurea is a type of chemotherapy used in polycythemia and before Gleevec for CML. It is regarded as very safe, though it can suppress the platelets, Hb and neutrophils, and therefore has to be regularly monitored with a CBC. There has been a randomized clinical trial in patients with sickle cell disease and this showed a 44% reduction in pain episodes, a longer time to the first painful crisis, fewer episodes of acute chest syndrome and fewer transfusions or admission to hospital. In long term follow up, mortality rates were 40% lower.

There have been several less convincing studies which have suggested that Hb F levels can be raised to 20% without significant toxicity, that neurologic function and splenic function improves, and that hydroxyurea does not lead to a higher incidence of second malignancies. This has been a worry since there has been a suggestion of a higher incidence on acute leukemia in patients with polycythemia who take hydroxyurea. It is not clear however whether it is safe to take during pregnancy, and there have been reports of reduced sperm production in men on the drug.

There are a number of experimental treatment being tested. I noticed viagra on the list.

If the sickle cell gene is inherited from only one parent it is completely benign, unless it occurs in association with another hemoglobinopathy. The picture is then that of sickle cell disease, but patients have large spleens. Patients with HbSC disease are particularly prone to thrombosis and pulmonary emboli - especially in pregnancy. and they have a high incidence of retinal problems.

Homozygous Hb C disease, which is common in West Africa, is due to the substitution of lysine for glutamic acid ant position 6 on the beta chain. It tends to form rhomboidal crystals in red cells. It causes a mild hemolytic anemia with many target cells. Splenomegaly is prominent.

Homozygous Hb D disease is a mild hemolytic anemia. Homozygous Hb E disease is the commonest hemoglobinopathy in South East Asia and resembles a mild thalassemia.

Hemolytic anemias: classification.

I divide up the haemolytic anemias like this:

A] Hemolytic anemias due to intrinsic properties of the red cell.

A red cell consists of three things: hemoglobin, membrane and a few enzymes. Things can go wrong with any of these to cause hemolysis. Mostly they are congenital conditions.

1 Hemoglobin problems. The most important is sickle cell disease, but there are many other polymorphisms that shorten red cell survival.
2 Membrane problems. Hereditary spherocytosis; hereditary elliptocytosis and the one acquired problem, paraoxysmal nocturnal hemoglobinuria.
3 Enzyme pathway problems. Red cells gain their energy by breaking down glucose (glycolysis). They lack the citric acid cycle and have to make do with the Embden-Meyerhof pathway that turns glucose into pyruvate. There are many enzymes on this pathway but only two important ones as far as hemolytic anemia is concerned, and both relate to little by-passes. About 10% of the traffic travels on the first by-pass, which has the purpose of generating hydrogen ions. It is known alternatively as the Hexose Monophosphate Shunt or the Pentose Phosphate Pathway.

The chief enzyme along it is Glucose 6-phosphate dehydrogenase (G-6PD). Around 250 million people in the world have a defect in their G-6PD. Hydrogen ions are needed by the cell to turn NADP to NADPH and thus to reduce glutathione. The normal activity of the cell leaves it susceptible to oxidation and the reduced glutathione prevents this. It is seldom that there is no G-6PD, but commonly there is no enough. Certain environmental agents increase the oxidative stress on a cell. Chief among these are anti-malarials and suphonamides. Dapsone can cause a hemolytic anemia in mormal people, such is the oxidative stress that it produces. In Mediterranean types of G-6PD deficiency Fava beans can cause the oxidative stress (I'm not sure about a good Chianti, though).

The second by-pass is called the Rappaport-Lubering Shuttle, which has the purpose of producing a chemical called 2,3,DPG. This is necessary for hemoglobin to be able to release oxygen to the tissues. The more 2,3,DPG, the easier it is to do this. The important enzyme here is pyruvate kinase. If it is absent then there is more 2,3,DPG and it is easier to get oxygen to muscles. There are only about 200 people in the world with this deficiency.

All the other enzyme deficiencies are so rare that you have to be a super-specialist even to have heard of them. The next commonest has 6 people affected in the world.

B] Hemolytic anemias due to extrinsic factors.

The most important of these are antibodies which come in warm and cold types. I shall discuss these at length at a later time.

Otherwise I tend to think of them under the headings of animal, vegetable and mineral.

There are few animals that cause hemolytic anemia and they are mostly very small, namely the malarial parasite and the bacteria Bartonella spp and Clostridium Welchii. Otherwise there are a few poisonous animals including snakes and spiders.

Vegetables include fava beans that we have mentioned and the poison ricin from the Castor Oil plant.

The mineral causes are mainly drugs, which again I will deal with separately. A special mention goes to water. I remember seeing a man with very severe hemolysis who had been swept through the drainage system of a local lake. He never recovered from his fresh-water drowning. Salt water would not have done the same thing. Finally we have hot and cold to remind us of burns and teh cold antibody symdromes.

Another way of classifying hemolytic anemia is to think about intravascular and extravascular hemolysis. Extravascular hemolysis releases free hemoglobin into the circualtion and causes renal failure accompanied by severe symptoms. The cause are incopatible blood transfusion, paroxysmal cold hemoglobinuria and paroxysmal nocturnal hemoglobinuria, microangiopathic hemolytic anemias, fresh water drowning, some toxins, severe G-6PD deficiency especially after some drugs or beans, severe AIHA, some forms of unstable hemoglobinopathies, and march hemoglobinuria. This last is caused by the squashing of red blood cells in the small vessels of the feet as they slap against the tarmac. It is a problem in long distance runners and schizophrenics who repeatedly slap their heads with the palms of their hands! All the rest are intravascular hemolytic anemias.

Yet another way of classifying hemolytic anemias is by what you see down the microscope. Spherocytes are formed when bits of membrane are chewed off. it happens mostly in the spleen to red cells coated with antibody, or with a membrane lacking the protein spectrin or to cells whose membrane has been damaged by the enzyme lecithinase, produced by the bacterium Clostridium Welchii. The cell assumes the shape with the largest volume and the smallest surface area, namely a sphere. The exit doors from the spleen are only two microns wide, but a sphere cannot squeeze itself through so small a gap, the way that a biconcave disc can, so spherocytes are doomed to a splenic death.

Fragmented cells are seen in anemias where the red cells are mechanically damaged. this can be from fibrin strands in the circulation, such as occur in disseminated intravacular coagulation and microangiopathic hemolytic anemias (thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, malignant hypertension, toxemia of pregnancy, meningococcal septicemia) or by mechanical heart valves or arterial grafts. The anemia can be acute (and usually intravascular) or chronic (also intravascular but without the symptoms) and likely to result in iron deficiency with stainable iron in the urine.

How to detect hemolytic anemia.

A hemolytic anemia is an anemia in which the red cells are being destroyed more rapidly than they can be replenished. Red cells normally last about 120 days in the circulation after which they become rather tatty and are destroyed, mainly in the spleen. However, the bone marrow can make new ones at up to eight times its normal rate, so hemolysis has to be quite severe before the patient becomes anemic.

The symptoms and signs of hemolytic anemia are similar to those of any other anemia except in two circumstances. If the liver is immature and cannot cope with all the red cell breakdown products, the individual may become severely jaundiced; and if the hemolysis is mainly inside the blood vessels when certain severe systemic effects may occur with back pain, shivering attacks, fever, dark urine, collapse and possibly kidney failure.

Otherwise special tests may be necessary to recognise hemolysis. The blood film may show spherocytes (the red cells no longer have a pale central area and a flat shape, but become like little balls with dark centers), elliptocytes (elliptical cells rather than round ones) or fragmented cells. Staining with certain 'supravital' stains will reveal increased numbers of reticulocytes (large young cells with a network of RNA strands showing up) and perhaps 'Heinz' bodies (tiny dark faceted lumps on the surface membrane of the cells). Reticulocytes are a sign of the bone marrow working overtime to replenish the cells that have been destroyed, and are a usual finding in hemolytic anemia unless the marrow has failed (which it sometimes does in a condition known as paroxysmal nocturnal hemoglobinuria) or run out of folate or is stuffed with things that shouldn't be there not allowing room for the red cell precursors to expand (the best example of this is CLL)

Special tests will also reveal signs of increased red cell destruction. When heme is destroyed it gets changed to bilirubin. This is fat soluble, but it gets changed by the liver to a water soluble form and is excreted in the bile. An immature liver cannot do this and so fat soluble bilirubin accumulates in the blood (sometimes called 'indirect bilirubin') causing jaundice. If it gets too much it dissolves in the white matter of the brain thereby damaging it and causing kernicterus or brain damage. This is really only a problem in babies and was once an important disease (hemolytic disease of the newborn) but with the introduction of anti-D prophylaxis for Rhesus negative mothers, it is now seldom seen.

Another heme breakdown product, urobilinogen is excreted in the urine, so a good test for hemolysis is to test for increased urobilinogen in the urine. This a 'sticks' test but if it is raised it should be confirmed by the biochemistry laboratory. Urobilinogen causes dark urine when it is left to stand. When hemoglobin is first released from the red cells it binds to a protein called haptoglobin and the bound hemoglobin is rapidly cleared from the blood, so a low level of haptoglobin is a sensitive test for hemolysis. Hemolysis taking place in the blood vessels overwhelms the haptoglobin and free hemoglobin then binds to albumin as methemalbumin. This is tested for by the Schumm's test. This is a good test for intravascular hemolysis, but the lab doesn't like doing it because it releases a nasty 'bad eggs' smell. Next free hemoglobin can be detected in the plasma and possibly in the urine, but in the kidney tubules it gets destroyed leaving a deposit of iron in the urine which can be tested for with Perl's stain (hemosiderosis).

Detecting hemolytic anemia in CLL is strewn with hazards and it is often missed. The Coombs test is a good indication that the body is thinking about destroying red cells, but often it stops there. You can't rely on finding reticulocytes because the marrow often has no space to make abundant erythrocytes. Nor is measuring bilirubin a good indication of red cell breakdown since any increase may be very short-lived as the liver metabolises the indirect bilirubin. Reduction in haptoglobin levels is the most useful test along with urinary urobilinogen levels. In teh days when hematologists looked at blood films they would often find spherocytes to clinch the diagnosis. Alas radioactive Chromium-labelled red cell survival studies are no longer done except in specialized laboratories, and sometimes the diagnosis depends on a therapeutic trial of steroids.

Anemia with large red cells: 3 Non-megaloblastic causes

This will be final essay on anemias with large red cells. To complete the two previous essays, I should add that it is dangerous to treat B12 deficiency with folic acid, since although the anemia will improve, the neurological problems will not.

But today I want to deal with macrocytosis that is not due to megaloblastic anemia since megaloblastic anemia accounts for less than 10% of all cases of macrocytosis seen in a hematology lab.

Large red cells are not necessarily associated with anemia and the best example of this is in the newborn where the cells are large and the hemoglobin is high. Why this is so is not clear, but in view of what I shall say later, it should be noted that baby red cells have fetal (HbF) rather than adult (HbA) hemoglobin in them.

When I say baby red cells I mean the red cells of babies rather than freshly produced red cells, although these too are larger than normal. Freshly produced red cells (or reticulocytes) are larger than normal too, probably because they contain extra things like strands of RNA and some nuclear remnants. These are removed as the red cell goes through the spleen so patients who have had a splenectomy also have large red cells. The commonest cause for an increase in reticulocytes is hemolytic anemia, but that is a subject for another day. Response to hematinics or hemorrhage can also cause a reticulocytosis.

In many populations the commonest cause of a raised MCV is alcohol (in Finland it is the cause in 65% of cases!). Alcohol causes a macrocytosis in a number of ways. It can be malnutrition and therefore folate deficiency, but this is relatively rare. Also rare is a severe hemolytic anemia. Most cases are believed to be caused by a direct toxic effect on the bone marrow. In acute alcoholic poisoning the bone marrow shows a characteristic pattern of abnormalities. In terminal alcoholic abuse the red cells are large because of liver failure. A confirmatory test of alcoholism is the serum gamma-GT.

Smokers as well as drinkers may also have a macrocytosis. I used to think that this was because most smokers are also drinkers or perhaps a toxic effect of cigarette smoke, but I now know that it is because smokers frequently have chronic obstructive pulmonary disease (COPD). As many as 50% of patients with COPD have a raised MCV. It has been suggested that this is related to the frequent finding that such patients also have numbers of HbF cells.

Liver disease per se causes a raised MCV, again, often with a normal hemoglobin. It is thought to be caused by increased cholesterol in the cell membrane, a feature of a disordered fat metabolism. The same cause has been attributed to the macrocytosis of hypothyroidism. Hypercholesterolemia is a feature of this and the red cells on the blood film have characteristically wavy edges.

In my practice one of the commonest causes of macrocytosis was myelodysplastic syndrome (MDS). All types of MDS are associated with macrocytosis including sideroblastic anemia, which in the older text-books was said to be a cause of small red cells. This is not completely untrue, since the very rare sex-linked congenital sideroblastic anemia does have small cells, and the MDS-type of sideroblastic anemia does have some small red cells on the blood film, even though the MCV is usually raised.

Anything that is in the bone marrow that shouldn't be there can cause a macrocytosis, be it lymphoma, myeloma, myelofibrosis or secondary cancer. We should not forget drugs as a cause. We have mentioned cytotoxic drugs like hydroxyurea (hydroxycarbamide), 6-mercaptopurine and azathiaprine that produce a pseudo-megaloblastic picture or interfere with folate metabolism like methotrexate, 5-fluorouracil and phenytoin, or its absorption, like metformin and cholestyramine, but we should also mention the anti-AIDS medications, stavudine, lamivudine and zidovudine.

Megaloblastic anemia is one of the less common causes of macrocytosis, so a full history needs to be taken to exclude the other causes. Paradoxically, a history of hypothyroidism should alert the physician to pernicious anemia, since both are autoimmune diseases and there is a considerable overlap between the two.

The order of investigation should be blood film first, followed by a reticulocyte count. The blood film should pick up most cases of megaloblastic anemia and a serum B12 should follow if they are there. A low B12 should send you on a hunt for the cause, but PA is the most likely. Borderline B12 levels are best incestigated by methylmalonic acid and homocysteine levels if the lab does them, as well as a red cell folate level. Serum folates should no longer be done. The main purpose of a bone marrow is to diagnose primary or secondary malignancy (including MDS).

Anemia with large red cells 2 Folic acid

I hesitate to put up a diagram of folate metabolism because it is so complicated. The correct name for folic acid is pteroylglutamic acid. Humans cannot synthesize it so we must take it in as a vitamin. Bacteria do synthesize it from pteridine, p-amino benzoic acid and glutamic acid, but this is inhibited by suphonamides which are therefore toxic to bacteria but harmless to humans.

I don't know whether the second diagram is more understandable, but if you focus on the top right hand corner, this is the bit that is relevant for megaloblastic anemia. The rest of the diagram is all about how various forms of chemotherapy interact with folate metabolism and we may come back to that. The part I am referring to is the change from dUMP to dTMP.

DNA and RNA differ in a number of ways. DNA is where the information for the genes is stored. Its structure is well known by now; the famous double helix where a chain of sugar molecules forms the banisters and interlocking purine and pyrimidine bases form the steps of the spiral staircase.
The bases are Guanine that always mates with Cytosine and Adenine that always mates with Thymine. The order of the bases is the basis of the code. RNA is single stranded and acts as both the messenger and the template for protein production; three bases representing a single amino acid. But the bases for RNA are different: instead of Thymine we have Uracil.


These molecules are very similar but thymine has an extra methyl (CH3) group hanging on one of the benzene rings. In Man thymine can only be made from uracil and there is a special enzyme that does it called thymidilate synthetase. One form of folic acid, 5, 10-methylene tetrahydrofolate, acts as the donor of the CH3 group to make thymine.

So although a patient without enough folic acid or who can't get it into the right form because of an absence of B12, although such a patient can make RNA, he can't make DNA properly. I was once buttonholed by a cytologist who told me that she had just diagnosed pernicious anemia from a cervical smear; a timely reminder that the megaloblastic process is not confined to blood cells, but is found in any tissue that is making new cells and therefore new DNA. Again it should be emphasized that B12 deficiency and folate deficiency both end up with identical blood pictures - megaloblastic anemia.

Diagnosis of folate deficiency is made by measuring the red cell folate, having first ascertained that the serum B12 is normal (red cell folate may be low in B12 deficiency). The serum folate is not a useful investigation - it merely tells you what sort of diet the patient has been having recently.

How can you get folate deficient? Folic acid is present in green vegetables so it is possible for those who live on tea and jam and bread to become deficient because of a poor diet, but it is unusual. Alcoholics are among those most likely to have a poor diet. More commonly there is an overuse of folic acid leading to a relative shortage. Typically, any condition where there is increased cell turnover puts the patient at risk. Pregnancy used to be one of the commonest causes, but most pregnant mums get folate supplements. Hemolytic anemia, any severe skin condition like eczema or psoriasis and any rapidly growing cancer or leukemia can cause a shortage of folate. Patients in intensive care may also develop folate deficiency.

Then there is malabsorption. Folate is absorbed from the small bowel and the condition most commonly associated with folate deficiency is celiac disease. Tropical sprue may be a problem in the Tropics (its why the ex-pats used to take Marmite with them), and Crohns disease, sclerodema of the gut and surgical resections may all be a cause. Some drugs interfere with folate absortion, notably phenytoin and sulphasalazine.

Folate deficiency is not associated with neurological syndromes apart from neural tube defeccts for which pregnant women should receive 400 micrograms a day as prophylaxis. This is thought to act through the methionine to homocysteine interaction. Eaised levels of homocytsteine are also associated with thrombotic lesions, for which folic acid is also a remedy.

Megaloblastic anemia generally has a low red cell count and a high red cell distribution width (RDW). Thrombocytopenia and neutropenia are also common and sometimes the presentation is of severe pancytopenia.

There are rare enzyme deficiencies like orotic aciuria and Lesch-Nyan syndrome that cause megaloblastic anemia, and certain drugs have an antifolate activity like methotrexate. Other drugs that interfere with DNA synthesis can cause megloblastosis with normal B12 and folate levels incude hydroxocarbamide, 5-fluorouracil, cytosine arabinoside, 6-mercaptopurine, azathiaprine, and anti-retroviral drugs. Megaloblastic changes may also be seen in some myelodysplastic syndromes.

The anemia of chronic disorders

Although iron deficiency anemia can be easily recognized by a low hemoglobin and a low MCV, there are other causes of this picture and other tests for confirming iron deficiency. Looking at a stained blood film reveals red blood cells that are paler than usual. They are paler because they are thinner and thin cells let more light through. You can also measure the amount of iron in the blood: the serum iron level. Iron is carried in the blood on a carrier protein known as transferrin. Normally, the whole body plasma transferrin contains about 3mg of iron and it functions as a transit compartment. About 20mg of iron flows through it in a normal day. In iron deficiency there is spare carrying capacity and the serum transferrin is raised. Elsewhere in the body iron is bound to a storage protein called ferritin, so in iron deficiency the serum ferritin level is low. Finally, you can normally see bits of iron in macrophages in the bone marrow (they stain blue with a special stain called Perl’s stain). In iron deficiency the macrophages are empty.

However, in some apparent iron deficient anemias, although the serum iron is low the transferrin is also low and the ferritin high and there is plenty of stainable iron in the bone marrow macrophages. The red cells on the blood film are still pale and indeed the blood film is indistinguishable from any other case of iron deficiency. These cases often have chronic inflammatory conditions like rheumatoid arthritis, ulcerative colitis or Crohn’s disease, or chronic infections like TB or perhaps certain types of cancer. Sometimes the condition comes on very quickly, especially in severe acute infections. We call these anemias, the anemias of chronic disorders or sometimes the anemias of inflammation. We used to say that something was preventing the release of iron from the macrophages. We now know what that ‘something’ is, and as we might have expected it is more complicated than that.

The ‘something’ is now known to be Hepcidin (pronounced ‘hep – side – in’; ‘hep’ comes from the Greek for liver – ‘hepar’; ‘sidero’ is the Greek for iron). Hepcidin is a small peptide consisting of 25 amino-acids. As you might expect from its name, it is made mainly in the liver, though it can be made by both granulocytes and macrophages. A knockout mouse has been produced that lacks hepcidin and from studying this we know that hepcidin controls intestinal iron uptake and the retention of iron in macrophages. If you inject hepcidin it produces a 75% reduction of serum iron levels within an hour and the effect persists for two days. A diet laden with iron produces an increase in the production of hepcidin, and anemia or a shortage of oxygen reduces hepcidin production.

During inflammation, inflammatory cytokines are produced and one of the most important of these is interleukin-6 (IL-6) which is an important inducer of hepcidin production. Other cytokines including IL-1 and TGF-beta are also involved in hepcidin regulation.

There are several mechanisms for the absorption of iron from the diet. Dietary iron is either in the ferric form (Fe+++) or as heme (myoglobin, the respiratory pigment of muscle [otherwise known as meat] contains heme). Fe+++ must be reduced to the ferrous form (Fe++) for absorption and this is done with a ferric reductase enzyme. Fe++ is absorbed using DMT1 (a silly name that just stands for divalent metal transporter 1) and heme absorption uses the equally obviously named heme carrier protein 1 (HCP1). Both these mechanisms only take the iron as far as the lining cells (or enterocytes) of the duodenum. From here they have to pass into the plasma and thence to macrophages in the bone marrow and elsewhere for the manufacture of hemoglobin, myoglobin and other iron-dependent proteins. The protein responsible for getting iron out of cells into the plasma is called ferroportin (Latin this time meaning iron-door). Ferroportin is equally important for enterocytes and macrophages. Without it iron would be stuck in the enterocytes and lost when they migrate up the duodenal villi and are shed into the intestinal lumen, and iron that had entered the macrophages would be trapped there and never get to the developing red blood cells.

Hepcidin binds to ferroportin in the cell membrane and causes it to be internalized and degraded (to put it another way, hepcidin locks the iron door). So hepcidin acts as a regulator of iron absorption and usage. In anemia, when there is a shortage of iron, or when the patient is short of oxygen, hepcidin production is suppressed so that more iron is absorbed and more iron is released from macrophages. Of course, if the anemia is not caused by a shortage of iron there can be inappropriately increased absorption of iron and this occurs in some types of thalassaemia.

Hepcidin is produced when iron absorption is sufficient, when there is no anemia or hypoxia, so as to stop excessive iron absorption. Hepcidin is also produced in response to some inflammatory cytokines, particularly IL-6; hence in the anemia of chronic disorders iron gets trapped in macrophages and not released to the developing red cells. Thus in the anemia of chronic disorders, because the iron-door is bolted, the anemia has the appearance of iron deficiency.

Added later. I need to say that there is no point in trying to treat the anemia of chronic disorders with either oral or intravenous iron. It just won't work.

Anemia with small red cells.

I prefer to classify anemias according to the size of the red blood cells and for this reason I regard the mean cell volume (MCV) as the most important of the red cell indices. An MCV of less than 80 fl indicates a microcytic anemia.

Since red cell consists of mainly hemoglobin, a microcytic anemia occurs when the body can't make enough hemoglobin. Either it can't make globin or it can't make heme. Inability to make globin is almost always a congenital condition, and collectively these are known as the thalassemias. I shall write about these on another day.

The rate-limiting step in heme production is the availability of iron.

We need to obtain iron from our diet because we are constantly losing it. Everybody loses about 0.5 mg a day, mainly from bowel cells shed in the motions, though shed skin cells are also involved. Women lose on average 0.5 mg a day from menstruation (equivalent to 80ml of blood loss a month). Therefore women need about twice as much iron every day in their diet as men. During pregnancy the average extra requirement is about another 1mg spread out over the whole 9 months, though at certain times they need as much as 6mg a day. The extra iron is required to provide for the baby's blood and muscle, the extra blood in the mother's circulation and the increased muscle in the uterus.

In the diet, iron is available in a usable form in sufficient quantities, only in meat. There is iron in vegetables but in such small quantities and so poorly absorbed that it can be discounted. This is particularly true of spinach. I will write that in capitals. SPINACH IS A VERY POOR SOURCE OF IRON. A German chemist put the decimal point in the wrong place when making the calculation.

Although dietary iron deficiency is common in developing countries, it is almost unknown in the West, except in vegetarian women. Iron deficiency is almost always caused by bleeding. I used to call it iron loss anemia rather than iron deficiency anemia.

The commonest cause of excessive iron loss is menorrhagia, but it is difficult to assess. It is not something women compare notes on. Most women think they have normal periods, but a Swedish gynecologist who investigated exactly how much women who thought their periods were normal actually lost per month, found it varied tenfold, from 50 ml to 500 ml, and the person who thought 500 ml was normal was the Swedish gynecologist herself.

The most important source of bleeding is the gastro-intestinal tract. Often overlooked is aspirin. It always causes some bleeding from the stomach. It may be as little as 0.2 ml per tablet or as much as 2 ml per tablet. Occasionally it is responsible for a torrential hemorrhage. Acetaminophen (paracetamol) does not do this, but ibuprofen does.

Peptic ulcer in stomach or duodenum is less common than it used to be because it is now recognized to be a treatable infection. From the large bowel, we are most concerned about colorectal cancer. In people over 60, iron deficiency is caused by occult bleeding from colorectal cancer in 18% of cases. Therefore if no obvious cause of bleeding is apparent, a person with iron deficiency should be investigated by both upper GI endoscopy and colonoscopy. Colonic diverticulae may cause acute bleeds but they are not the cause of chronic blood loss. Angiodysplasia, abnormal blood vessels, in the large bowel can be the source of bleeding, but in my experience this is only the case when there is an associated clotting disorder.

If no obvious source of bleeding is found there are some techniques to employ to look harder. The urine should be examined for blood and iron. Hematruria from bladder or kidney cancer is important to identify, but in some hemolytic anemias iron, rather than blood, is lost from the kidneys. Blood coagulation should be tested. Bleeding disorders such as mild hemophilia or Von Willebrand's disease may be the cause. Sometimes the bleeding is from the small bowel, which cannot be reached by either gastroscope or colonoscope. Special techniques are required for these.

Does malabsorption of iron occur? People with celiac disease malabsorb fats and other vital dietary elements. What about iron? Some people think that they also malabsorb iron, but for me the evidence favors increased loss of iron-laded bowel cells. In celiac disease they are shed 7 times more rapidly. In past-gastrectomy syndromes they may be shed five times as fast as normal and 50% of those who have had a gastrectomy become iron deficient.

There are still a large number of people who have apparent iron deficiency in whom no cause will be found. Some of them have plenty of iron in their body; it's just that they cannot use it. Collectively these are known as the anemias of chronic disorders, and I shall write about these next.

Anemia in cancer patients

Why have a blood transfusion? Is it a] to raise your hemoglobin? b] to improve your color? c] to improve the oxygen carrying capacity of your blood?

The answer, of course, is c] but while it will do both a] and b] it won't do c] immediately. Oxygen is carried by hemoglobin and is released to the tissues where it is needed. It is helped to do this by a chemical called 2,3,DPG. In order to release oxygen, its place on the hemoglobin molecule must be taken by this chemical. 2,3,DPG is manufactured by all cells from the burning of glucose (the process is known as glycolysis and the biochemical pathway is known as the Emden-Myerhoff pathway. The particular trick of producing 2,3,DPG is known as the Rapaport-Leubering Shuttle). If a cell is not metabolising glucose then it won't make any 2,3,DPG, and this is what happens when blood is stored in a fridge. So when you receive blood from the blood bank it has very little 2,3,DPG and therefore the hemoglobin molecules cling on to their oxygen. That's why someone who has been transfused gets no immediate benefit from it; it can't release its oxygen to the tissues until it regenerates 2,3,DPG, which takes a couple of days.

This is one of the reasons that patients are dissatisfied with blood transfusion. That and the risk of transmitting viruses, or prions, or making their cancer worse by suppressing their immunity. In fact blood transfusion is very safe. Blood is screened for HIV, hepatitis B and C and a host of other possible infections. It cannot be screened for prions, but the risk of developing new variant CJD from transfusion is vanishingly small. The greatest risk of a transfusion is that some idiot will give you the wrong blood.

The alternative is EPO. Lots of cancer patients have been given it, but the unfortunate fact is that it shortens life. A recent meta-analysis published in the Lancet looked at 53 separate trials involving 13,933 patients. Although the effect was not great (a hazard ration of 1.06) it was statistically significant. Partly this effect is because EPO may raise the hemoglobin too much and the thrombotic complications of polycythemia come into play, but also there is the worrying fact that EPO is a growth factor for some tumors.

With so much to worry about we need to look at anemia again. In some cases the problem can be sorted with iron therapy. (Oral iron is best; there is no evidence that intravenous or intramuscular iron is more likely have a response or have a quicker response.) But we need to be able to recognise iron deficiency (which is almost always caused by bleeding). The easiest way is to look at the mean cell volume on the blood count. If this is below 80 fl then iron deficiency is the most likely diagnosis. Thalassemia trait can cause a low MCV and this is an important catch for people of Mediterranean background, but the only other cause of a low MCV is the anemia of chronic disorders (ACD).

I have bee surprised recently by how little even hematologists know about ACD. They seem to be unaware that it can cause a low MCV. I can only think that they have never looked at the blood counts of patients with rheumatoid arthritis, ulcerative colitis and Crohn's disease, let alone people with chronic infections or disseminated cancer. The serum iron in ACD can be as low as it is in iron deficiency, as can the MCV. The difference is that the iron binding capacity or the blood (or the serum transferrin) is raised in iron deficiency and lowered in ACD. Serum ferritin is low in iron deficiency and raised in ACD.

What happens in ACD is that the macrophages are over active and snaffle all the iron in the body and won't release it to the newly formed red blood cells. We now know that a chemical called hepcidin, a small peptide produced by the liver that inhibits both iron absorption and release of iron from the macrophages. Patients with excess hepcidin will not respond to more iron, whether oral or intravenous, nor to EPO. Only blood transfusion will raise the hemoglobin.

High hepcidin levels are found in association with high sed rates, high CRPs, high IL-6 levels and other indicators of inflammation.