Non Haemolytic Anaemias

1. Non Haemolytic Anaemias Dr Marion Wood Mary Ruth Colchester General Hospital

2. Learning Objectives Identify major features of disease Discuss the aetiology and molecular basis of disease Discuss the prognosis and and diagnosis of disease Understand and evaluate the multidisciplinary approach to diagnosis, monitoring and treatment Define major associated disease processes Evaluate Haematology�s specific role in clinical pathway Discuss key haematological techniques in differential diagnosis Be aware of new diagnostic technologies

3. Non Haemolytic Anaemias How can we define them? An anaemia in which reduction of the red cell lifespan is not the main underlying pathology Truth is slightly stretched, as most of these demonstrate a mildly reduced lifespanTruth is slightly stretched, as most of these demonstrate a mildly reduced lifespan

4. So, if it�s not red cell breakdown- what is the cause??? Impaired production: - marrow failure Infiltration - marrow Deficiency � nutritional Blood loss Physiological/dilutional Impaired production: - marrow failure Infiltration - marrow Deficiency � nutritional Blood loss Physiological/dilutional

5. Non Haemolytic Anaemias- a Clinician�s approach Based primarily on the MCV � Macrocytic Microcytic Normocytic

6. Non Haemolytic Anaemias- a Clinician�s approach (2) After the MCV � what else might help? Is the rest of the count normal? Especially WBC and platelets What about the patient history � importance of clinical details in helping laboratory/clinicians interpret the resultsIs the rest of the count normal? Especially WBC and platelets What about the patient history � importance of clinical details in helping laboratory/clinicians interpret the results

7. Case Study # 1 A 70 yr old retired barber with fatigue, lethargy and breathlessness on walking. Symptoms had developed over last few months. Use study from BMJ - request form (mock up) What might they do next ? (film??, reflex test haematinics or suggest GP does these with rpt FBC) Give some film findings � what do they think now? Give clinical history � does this help? Give results of additional tests - B12/Folate, LFTs and TFTs normal, raised ferritin These are some of the issues we will cover as the lecture progressesUse study from BMJ - request form (mock up) What might they do next ? (film??, reflex test haematinics or suggest GP does these with rpt FBC) Give some film findings � what do they think now? Give clinical history � does this help? Give results of additional tests - B12/Folate, LFTs and TFTs normal, raised ferritin These are some of the issues we will cover as the lecture progresses

8. Non Haemolytic Anaemias Megaloblastic anaemias Iron deficiency anaemia Anaemia of chronic disorders Sideroblastic anaemias

9. Megaloblastosis Defined by defective DNA synthesis Causes include: Reduced availability of deoxyribonucleoside(s)(dNTPs) Asynchronous synthesis of dNTPs Have used the term megaloblastosis rather than megaloblastic anaemia as some patients may not actually appear anaemic, but still have a significantly raised MCV and other features of megaloblastic anaemia Have used the term megaloblastosis rather than megaloblastic anaemia as some patients may not actually appear anaemic, but still have a significantly raised MCV and other features of megaloblastic anaemia

10. Mechanism of defect dNTPs are needed for DNA replication during S phase B12/folate deficiency thought to inhibit conversion of dUMP to dTMP Reduced availability of dTTP May inhibit elongation of short strands decrease in DNA synthesis and cell death and ineffective erythropoiesis Purines - dATP, adenine dGTP, guanine and pyrimidines dTTP,thymine dCTP cytosine dUMP (deoxyuridine monophosphate , dTMP thiamine monophosphate Lack of other dNTPs may be cause/contributory factor of non B12/folate deficiency megaloblastic anaemias Diagram clearly shows the close relationship between B12 and folate and the influence that a lack of one can have on the action of the other Purines - dATP, adenine dGTP, guanine and pyrimidines dTTP,thymine dCTP cytosine dUMP (deoxyuridine monophosphate , dTMP thiamine monophosphate Lack of other dNTPs may be cause/contributory factor of non B12/folate deficiency megaloblastic anaemias Diagram clearly shows the close relationship between B12 and folate and the influence that a lack of one can have on the action of the other

11. General features Major effects are on fast turnover tissues Glossitis, beefy tongue, angular cheilosis Cells enlarged, occ multinucleate with cell death Mild icterus Peripheral neuropathy (SACD)(B12) Loss of cognitive function Risk of neural tube defects in pregnancy (folate) Icterus � due to ineffective haemopoiesis � primrose yellow patients !! Neural tube defects � also occur with B12 defic, but less likely to occur than folate defic in young pt. May be due to buildup of metabolic products Icterus � due to ineffective haemopoiesis � primrose yellow patients !! Neural tube defects � also occur with B12 defic, but less likely to occur than folate defic in young pt. May be due to buildup of metabolic products

12. General laboratory features Peripheral blood Variable anaemia and variably raised MCV Oval macrocytes Hypersegmented neutrophils Leucopenia/thrombocytopenia Circulating megaloblasts Bone marrow Variable erythroid hyperplasia Giant metamyelocytes Megaloblasts Ineffective Haemopoiesis raised unconjugated bilirubin, urinary urobilinogen and LDH due to ineffective haemopoiesis ie features of �haemolysis� but mostly occuring within the marrow !! Decreased haptoglobins � again indicating that there is haemolysis as part of the pathophysiologyraised unconjugated bilirubin, urinary urobilinogen and LDH due to ineffective haemopoiesis ie features of �haemolysis� but mostly occuring within the marrow !! Decreased haptoglobins � again indicating that there is haemolysis as part of the pathophysiology

13. Megaloblastic anaemia

14. Features of Megaloblastosis Slide 1 one early normoblast (purple arrow) and two intermediate normoblasts (red arrows). Stages of erythroblast maturation can be distinguished by the decreasing size, decreasing cytoplasmic basophilia, increasing chromatin clumping and increased haemoglobinization as maturation proceeds. Slide 2 . The megaloblast is larger and shows finer reticular nuclear structure than a normoblast at the same stage of haemoglobinization. Purple arrow - haemoglobinization just starting Red arrow - haemoglobinization almost complete but nucleus is still finely reticular, instead of being darkly staining and amorphous; this is also referred to as nucleocytoplasmic asynchrony Slide 1 one early normoblast (purple arrow) and two intermediate normoblasts (red arrows). Stages of erythroblast maturation can be distinguished by the decreasing size, decreasing cytoplasmic basophilia, increasing chromatin clumping and increased haemoglobinization as maturation proceeds. Slide 2 . The megaloblast is larger and shows finer reticular nuclear structure than a normoblast at the same stage of haemoglobinization. Purple arrow - haemoglobinization just starting Red arrow - haemoglobinization almost complete but nucleus is still finely reticular, instead of being darkly staining and amorphous; this is also referred to as nucleocytoplasmic asynchrony

15. Causes of Megaloblastosis B12 deficiency/metabolic defect Folate deficiency/metabolic defect Folate antagonists Lesch-Nyhan syndrome Orotic aciduria Drugs affecting DNA synthesis Chemotherapy, anticonvulsants, azathioprine and anti-retroviral treatments Some cases of AML and MDS/other BM infiltration Folate antagonists eg methotrexate Orotic aciduria � rare defect responds to uridine, Lesch Nyhan � very rare may respond to adenine�may be examples of a lack/defect in other dNTPs Hydroxyurea occasionally used to treat non-haem conditions (eg some types of chr. liver or skin disease) Potentially very misleading if requester does not include this information and patient records are not available. Folate antagonists eg methotrexate Orotic aciduria � rare defect responds to uridine, Lesch Nyhan � very rare may respond to adenine�may be examples of a lack/defect in other dNTPs Hydroxyurea occasionally used to treat non-haem conditions (eg some types of chr. liver or skin disease) Potentially very misleading if requester does not include this information and patient records are not available.

16. NB diagram uses terminology FH4 � usually termed TH4 MSR � methionine synthase reductase B12 is a co factor for the conversion of homocysteine to methionine Folate acts as a methyl donorNB diagram uses terminology FH4 � usually termed TH4 MSR � methionine synthase reductase B12 is a co factor for the conversion of homocysteine to methionine Folate acts as a methyl donor

17. Role of Cobalamins Act as co-factor for 2 enzymes Methionine synthase, which converts homocysteine to methionine and requires methylcobalamin (with 5-TH4 as a methyl donor) Methylmalonyl CoA mutase which converts methylmalonyl-CoA to succinyl-CoA and utilises the 5� deoxyadenosyl form. In adenosylobalamin deficiency. Succinyl � CoA cannot be generated, leading to an accumulation of methylmalonic acid (MMA). MMA levels can thus be used as a functional measure of B12 status Also needed to maintain the folate cycle In adenosylobalamin deficiency. Succinyl � CoA cannot be generated, leading to an accumulation of methylmalonic acid (MMA). MMA levels can thus be used as a functional measure of B12 status Also needed to maintain the folate cycle

18. Vitamin B12 Natural cobalamins synthesised by bacteria in human food � animal proteins only Metabolically active forms eg methylcobalamin (methyl ligand) and adenosine (adenosyl), Synthetic forms eg cyanocobalamin (cyanide) or hydroxycobalamin(hydroxyl) � IF cannot bind these Recommended daily amount about 2?g, easily obtained from a balanced Western diet. Dietary defic alone rare except strict vegans. Good reserves (about 2.5mg) particularly in liver. Takes 3-5 years to deplete body stores Cobalamins are complex molecules and are characterised by a single central cobalt atom, with variable ligands . Found in meat, fish, and other animal derived products. Also added to breakfast cereals. Synthetic forms eg in treatment readily convert to metabolically active forms Average Western diet contains up to 30ug per dayCobalamins are complex molecules and are characterised by a single central cobalt atom, with variable ligands . Found in meat, fish, and other animal derived products. Also added to breakfast cereals. Synthetic forms eg in treatment readily convert to metabolically active forms Average Western diet contains up to 30ug per day

19. B12 absorption Cobalamin (B12) cleaved from food by pepsin/ HCl in stomach, then binds to R binder protein In duodenum, B12 is cleaved off R binder - binds to IF IF complex binds to cubulin in distal ileum, endocytoses into cells - IF cleaved off. B12 enters portal blood IF also binds B12 in portal circulation: �enterohepatic� cycle B12 binds to transcobalamin (TC), complex carried to tissues or BM TC/B12 binds to cellular receptors promoting cell entry TC is cleaved off, making B12 available for use Approx 1% absorbed passively - not enough to avoid deficiency developing in affected people. Pepsin produced by the gastric chief cells, HCl is produced by gastric parietal cells R binder is produced by gastric parietal cells and salivary gland epithelial cells Cleavage of cobalamin from R binder by action of pancreatic proteases trypsin Intrinsic factor is a glycoprotein produced by gastric parietal cells. Binding is promoted by more alkaline pH in small gut. cubulin is a surface receptor protein located on ileal mucosal cells Transcobalamin (formerly known as TCII) is a transport protein Passive absorption occurs in the duodenum and ileum. Can also occur through other mucous membranes. Oral, sublingual and nasal sprays have been used to give therapeutic B12Pepsin produced by the gastric chief cells, HCl is produced by gastric parietal cells R binder is produced by gastric parietal cells and salivary gland epithelial cells Cleavage of cobalamin from R binder by action of pancreatic proteases trypsin Intrinsic factor is a glycoprotein produced by gastric parietal cells. Binding is promoted by more alkaline pH in small gut. cubulin is a surface receptor protein located on ileal mucosal cells Transcobalamin (formerly known as TCII) is a transport protein Passive absorption occurs in the duodenum and ileum. Can also occur through other mucous membranes. Oral, sublingual and nasal sprays have been used to give therapeutic B12

20. Causes of B12 deficiency (1) Likely to cause anaemia Pernicious anaemia/other gastric defect Total/partial gastrectomy Malabsorption/intestinal problem Dietary deficiency Transport problem Fish tapeworm Gastric causes - PA , congenital absence of IF Intestinal eg Crohns, esp if terminal ileum severely affected or resected because of disease. blind loop syndrome- rare, post surgery, tropical sprue Transport � transcobalamin deficiency Fish tapeworm diphyllobothrium latumGastric causes - PA , congenital absence of IF Intestinal eg Crohns, esp if terminal ileum severely affected or resected because of disease. blind loop syndrome- rare, post surgery, tropical sprue Transport � transcobalamin deficiency Fish tapeworm diphyllobothrium latum

21. Causes of B12 deficiency (2) Some malabsorption but anaemia unlikely Alcohol, HIV, GvHD, severe pancreatitis, gastric bypass surgery etc etc Megaloblastosis but normal B12 (and folate) � rare TC deficiency � this is the essential transport protein but majority of plasma B12 is bound to haptocorrin (TCI) NB: TC is synthesised by granulocytes, can bind B12 analogues. MPDs may give rise to very high B12 levels In TC deficiency, B!2 cannot be delivered to tissues and serum B!2 is actually normal. Most B12 is bound to haptocorrin (TCI) cannot be utilisedIn TC deficiency, B!2 cannot be delivered to tissues and serum B!2 is actually normal. Most B12 is bound to haptocorrin (TCI) cannot be utilised

22. History of PA Late 1890s German Dr Biermer described a fatal anaemia which he called PA Ehrlich noted very large abnormal early erythroblasts in PB. Some clinicians suggested dietary deficiency cause, others noted PM stomach changes 1920s US doctors Minot and Murphy awarded Nobel prize for discovery that a diet high in raw beef liver could improve condition. (YUK) Finally in 1940s missing vitamin found to be B12 �Pernicious Anaemia � because it was refractory to all known treatments He called these large erythroblasts megaloblasts�Pernicious Anaemia � because it was refractory to all known treatments He called these large erythroblasts megaloblasts

23. Pathophysiology of PA Gastric parietal cells produce IF, R binder and HCl Chr inflammation of gastric mucosa + autoantibody damages/destroys cells. Progression to gastric atrophy reduction/absence IF incidence in those with other autoimmune problems eg thyroid disease or IDDM. incidence in group A, vitiligo, premature grey hair and blue eyes. Frequent glossitis, beefy tongue and mild jaundice. May get peripheral neuropathy or SACD, loss of balance, loss of memory and cognitive function Parietal cells located in gastric fundus - upper part of stomach Progression to atrophy very slow process Neuropathy � cause unclear � possibly related to build up of intermediate products of metabolismParietal cells located in gastric fundus - upper part of stomach Progression to atrophy very slow process Neuropathy � cause unclear � possibly related to build up of intermediate products of metabolism

24. Role of folate compounds Act as coenzymes in transfer of single carbon units Required for purine and pyrimidine synthesis 5- methyl tetrahydrofolate (5 methyl TH4) required as methyl donor in conversion of homocysteine to methionine using B12 as co enzyme Resulting tetrahydrofolate (TH4) can be converted to other intracellular polyglutamate co enzymes by the action of folate polyglutamate synthase Illustrates the highly interactive process; because 5 methyl TH4 can only convert to TH4 and on into a polyglutamate such as 5,10-methylene THF, which is needed for thymidylate synthesis in presence of B12. Equally, folate cycle can only continue if B12 is available. Illustrates the highly interactive process; because 5 methyl TH4 can only convert to TH4 and on into a polyglutamate such as 5,10-methylene THF, which is needed for thymidylate synthesis in presence of B12. Equally, folate cycle can only continue if B12 is available.

25. Folate Large group of compounds with variable glutamate residues Found in most food sources concentration in yeast, greens, nuts and liver. Normal diet contains about 250ug/daily, adult requirement approx 100ug/daily Easily destroyed by heating/boiling Total stores about 10mg, some loss in urine, etc Stores sufficient for up to 6 months � risk of rapid depletion Especially spinach Also added to cereals Especially spinach Also added to cereals

26. Folate absorption Rapid and efficient absorption of monoglutamates from upper GI tract � probable active process Polyglutamates hydrolysed to mono forms ? In gut, ? In mucosa Absorption with higher no of glutamate residues ? Affects transfer, ? Affects hydrolysis Conversion to 5-methyltetrahydrofolate (5-methyl TH4) in mucosa � entry into portal circulation. Transported bound to albumin or free in plasma Active entry into cells Folate is absorbed from duodenum and upper jejeunum Polyglutamates are hydrolysed by pteroylpolyglutamate hydrolaseFolate is absorbed from duodenum and upper jejeunum Polyglutamates are hydrolysed by pteroylpolyglutamate hydrolase

27. Causes of folate deficiency Dietary Poor diet or cooking Malabsorption Coeliac disease, tropical sprue, surgery, Crohns Increased utilisation Pregnancy, prem babies, Haem disorders, Inflammatory Disease Folate antagonist drugs Anticonvulsants � mechanism unclear Dihydrofolate reductase inhibitors eg methotrexate ? alcohol Diet Elderly, dementia, alcoholism, infancy When dihydrofolate is reduced back to the TH4 form, process is catalysed by dihydrofolate reductase. Enzyme is inhibited by drugs eg methotrexate. Can also occur with related Drugs eg pyrimethamine (daraprim) used for malaria and toxoplasmosisDiet Elderly, dementia, alcoholism, infancy When dihydrofolate is reduced back to the TH4 form, process is catalysed by dihydrofolate reductase. Enzyme is inhibited by drugs eg methotrexate. Can also occur with related Drugs eg pyrimethamine (daraprim) used for malaria and toxoplasmosis

28. Investigation of Megaloblastic Anaemia Remember non pathologic causes � pregnancy 3 stages a. is it simply macrocytic or truly megaloblastic. b. confirmed megaloblastic � is it B12 or folate (or rarely something else) c. What is the underlying cause of the deficiency of B12/folate Which tests and in which order � likely to be guided by initial film and history Clinical details (if available) PB film, ? Retics; other things that may be pointers eg HJ bodies & possible Coeliac; acute onset or concomitant Fe defic. May give dimorphic picture; also expect hypersegmented neuts and ?low WBC/plts May occ. Have wk +ve DAGT(complement only) Serum B12 and folate; TFTs and LFTs, red cell folate BM � occ misdiagnosis with leukaemia � care required. IF and PCA antibodies Investigation of GI tract/absorption (Schilling test) Clinical details � if available FBC and film ? retics Serum B12 and folate, LFT and TFT Red cell folate Bone marrow IF and PCA antibodies Investigation of GI tract/absorption (Schilling test) IF and PCA antibodies Homocysteine levels Methylmalonate levels MTHR Tests for congenital deficiency/altered metabolism Remember non pathologic causes � pregnancy 3 stages a. is it simply macrocytic or truly megaloblastic. b. confirmed megaloblastic � is it B12 or folate (or rarely something else) c. What is the underlying cause of the deficiency of B12/folate Which tests and in which order � likely to be guided by initial film and history Clinical details (if available) PB film, ? Retics; other things that may be pointers eg HJ bodies & possible Coeliac; acute onset or concomitant Fe defic. May give dimorphic picture; also expect hypersegmented neuts and ?low WBC/plts May occ. Have wk +ve DAGT(complement only) Serum B12 and folate; TFTs and LFTs, red cell folate BM � occ misdiagnosis with leukaemia � care required. IF and PCA antibodies Investigation of GI tract/absorption (Schilling test) Clinical details � if available FBC and film ? retics Serum B12 and folate, LFT and TFT Red cell folate Bone marrow IF and PCA antibodies Investigation of GI tract/absorption (Schilling test) IF and PCA antibodies Homocysteine levels Methylmalonate levels MTHR Tests for congenital deficiency/altered metabolism

29. Auto Antibodies in PA Parietal Antibodies - found in 90% patients but common in older people (16% normal 60+ females) IF antibodies Type 1 � inhibits B12 binding 50% Type 2 � inhibits ileal binding site 35% IF antibodies are therefore highly specific but not v sensitive Mechanism also related to cell mediated immunity � seen on biopsy (& as mentioned earlier) May be found in gastric secretions as well as plasma/serum Childhood PA � congenital lack or abnormality of IF (so no Abs, normal stomach and acid). Usually presents at about 2 yrs old, when maternal transferred B12 has been used upIF antibodies are therefore highly specific but not v sensitive Mechanism also related to cell mediated immunity � seen on biopsy (& as mentioned earlier) May be found in gastric secretions as well as plasma/serum Childhood PA � congenital lack or abnormality of IF (so no Abs, normal stomach and acid). Usually presents at about 2 yrs old, when maternal transferred B12 has been used up

30. Investigation (2) Deoxyuridine suppression test Schilling test Endoscopy and gastric/jejeunal/duodenal Bx ? Imaging of small intestine In practice � therapeutic trial is frequent choice Homocysteine levels Urinary Methylmalonate excretion MTHR Tests for congenital deficiency/altered metabolism . DU suppression test � in vitro measure of uptake of radioactive thymidine into marrow cell DNA � result is abnormal in both B12 and folate deficiency; can use correction tests to distinguish which it is. Result will be normal in megaloblastic anaemia not due to B12/folate deficiency or other problems with thymidylate synthesis. Schilling test specific for B12 absorption � give 2 forms of radioactive cyanocobalamin, one on it�s own, the other with active IF; measure output of B12 in urine. Must have replenished B12 stores first, so give IM B12 Homocysteine levels � can be used as a reflection of folate status, but also influenced by B12, B6 and B2 levels. Methylmalonate � accumulates when conversion of methymalonyl-CoA to succinyl-CoA by Methylmalonyl CoA mutase is inhibited by lack of B12. Both tests not readily available and lack specificity MTHR catalyses conversion of 5,10 methylene TH4 to 5-methyl TH4 . Deficiency can cause severe developmental delay. Etc. DU suppression test � in vitro measure of uptake of radioactive thymidine into marrow cell DNA � result is abnormal in both B12 and folate deficiency; can use correction tests to distinguish which it is. Result will be normal in megaloblastic anaemia not due to B12/folate deficiency or other problems with thymidylate synthesis. Schilling test specific for B12 absorption � give 2 forms of radioactive cyanocobalamin, one on it�s own, the other with active IF; measure output of B12 in urine. Must have replenished B12 stores first, so give IM B12 Homocysteine levels � can be used as a reflection of folate status, but also influenced by B12, B6 and B2 levels. Methylmalonate � accumulates when conversion of methymalonyl-CoA to succinyl-CoA by Methylmalonyl CoA mutase is inhibited by lack of B12. Both tests not readily available and lack specificity MTHR catalyses conversion of 5,10 methylene TH4 to 5-methyl TH4 . Deficiency can cause severe developmental delay. Etc

31. Treatment and monitoring Depends on underlying cause !! ?? Stop drugs/ alcohol abstention ? Correct the diet Folate (oral; rarely IV folinic acid) or B12 (usually IM) replacement Alcohol � v common cause of raised MCV; however may also have direct toxic effect on marrow and be associated with poor diet � folate deficiency Liver disease of any cause � will require further investigations Also remember physiological macrocytosis � pregnancy, neonates, reticulocytosis for other reason � usually not oval macrocytes and no associated WBC changes � ie normoblastic not megaloblastic MDS and myeloma may only have mild anaemia and no other abnormalities on FBC, need considerationAlcohol � v common cause of raised MCV; however may also have direct toxic effect on marrow and be associated with poor diet � folate deficiency Liver disease of any cause � will require further investigations Also remember physiological macrocytosis � pregnancy, neonates, reticulocytosis for other reason � usually not oval macrocytes and no associated WBC changes � ie normoblastic not megaloblastic MDS and myeloma may only have mild anaemia and no other abnormalities on FBC, need consideration

32. Iron Earliest Iron Bridge 1779 CoalbrookdaleEarliest Iron Bridge 1779 Coalbrookdale

33. Distribution of iron Majority of iron is contained within Rbcs and is salvaged from senescent cells and re-utilised. Dietary absorption and excretion should be just about steady state in a normal patient. Therefore unusual to have pure dietary deficiency and takes long time Absorbed and salvaged is transported by transferrin back to BM for re-use. Some iron is stored in macrophages and in liver stores. Iron is also found in myoglobin and as a component of enzymes such as catalase, cytochromes and succinic dehydrogenaseMajority of iron is contained within Rbcs and is salvaged from senescent cells and re-utilised. Dietary absorption and excretion should be just about steady state in a normal patient. Therefore unusual to have pure dietary deficiency and takes long time Absorbed and salvaged is transported by transferrin back to BM for re-use. Some iron is stored in macrophages and in liver stores. Iron is also found in myoglobin and as a component of enzymes such as catalase, cytochromes and succinic dehydrogenase

34. Iron absorption Ingested iron mostly absorbed into duodenal enterocytes Haem binds to specific receptor HCP-1 Dietary iron must be in ferrous (Fe2+) form to be absorbed. Dcytb reduces ferric Fe3+ to Fe2+. DMT1 transports iron across cell membrane into cell Iron oxidised back to Fe3+ by action of haephastin then transferred across basolateral membrane by ferroportin Some iron may be retained and is stored as ferritin Iron levels controlled by regulation of each step. eg, cells can produce more Dcytb, DMT1 and ferroportin in response to iron deficiency anaemia Dcytb � ferric reductase enzyme located on brush border of enterocytes DMT1 is divalent metal transporter 1 not specific to iron active in transport of all types of divalent metals ferroportin, a transmembrane iron export protein. abundantly expressed on cell surface of RE macrophages and on the basolateral membrane of duodenal enterocytes, under control of hepcidin Stored iron is shed into faeces Dcytb � ferric reductase enzyme located on brush border of enterocytes DMT1 is divalent metal transporter 1 not specific to iron active in transport of all types of divalent metals ferroportin, a transmembrane iron export protein. abundantly expressed on cell surface of RE macrophages and on the basolateral membrane of duodenal enterocytes, under control of hepcidin Stored iron is shed into faeces

35. Factors affecting absorption Acid environment Solubilising agents eg sugars Ferrous and haem iron Pregnancy Iron deficiency Ineffective erythropoiesis Hereditary Haemochromatosis Increased expression of DMT-1 and ferroportin Decreased hepcidin Alkali environment Precipitating agents eg phytates Tea Ferric and inorganic iron �Iron excess� Decreased erythropoiesis Infection Decreased expression of DMT-1 and ferroportin Increased hepcidin Acid vs alkaline eg pt with achlorhydria or taking antacids. Might occur in conjunction with megaloblastic Acid vs alkaline eg pt with achlorhydria or taking antacids. Might occur in conjunction with megaloblastic

36. Iron transport Absorbed and recycled Fe is carried by transferrin Polypeptide synthesised in liver - inversely proportional to iron stores Binds 2 atoms of ferric iron Binds to transferrin receptors on erythroblasts etc. Iron released and transferrin re-used Variable concentration of transferrin receptors dependent on iron stores Serum levels of transferrin receptors can also be determinedSerum levels of transferrin receptors can also be determined

37. Regulation of iron storage Facilitated by binding of IRPs to IREs on transferrin receptor and ferritin mRNA Location of binding site either promotes or decreases protein mRNA translation Binding at 5� end reduces and 3� end increases translation In iron deficiency, ferritin levels will drop and synthesis of transferrin receptor 1 (TfR1)increases DMT-1 synthesis may also be controlled in this way IRP iron regulatory protein IRE iron regulatory elementsIRP iron regulatory protein IRE iron regulatory elements

38. Iron storage Vast majority in Rbcs. Also in myoglobin and enzymes � of remainder�.. Majority stored as Ferritin in hepatocytes water soluble protein: iron complex of approx 20% iron Stored in ferric form Haemosiderin � mostly in macrophages Insoluble complex - about 40% iron Stains with Perls Prussian Blue Stored in ferric form Small amount in macrophages. Ferriitn comprises an outer shell of apoferritin with an inner iron complex. Not visible by light microscopy Small amount in macrophages. Ferriitn comprises an outer shell of apoferritin with an inner iron complex. Not visible by light microscopy

39. Role of hepcidin Synthesis in response to iron and inflammation in response to active erythropoiesis. Regulates iron metabolism by interaction with Ferroportin. Ferroportin normally facilitates transmembrane transfer of Fe Hepcidin inhibits Fe release by binding to surface ferroportin. Hepcidin can therefore be considered as a negative regulator of iron absorption and recycling. Hepatic derived peptide hormone secreted into the circulation Ferroportin is a hepcidin receptor Hepcidin bound ferroportin is internalised and degradedHepatic derived peptide hormone secreted into the circulation Ferroportin is a hepcidin receptor Hepcidin bound ferroportin is internalised and degraded

40. Control of Hepcidin Induction Hepcidin synthesis in the liver is promoted by the presence of iron and infection/inflammation Is inhibited by active erythropoiesis and hypoxia Hepatic synthesis also under control of: Hfe/Tfr1 (haemochromatosis gene/transferrin receptor1), Tfr2 � Transferrin receptor2 found in erythroid precursors, hepatic cells and duodenal crypt cells Hjv � haemojuvelin, Smad4 cell signalling molecule, BMPs � bone morphogenetic protein may be ligand for Hjv Hepcidin is also induced in other tissues by action of IL6 during inflammatory processes Hepcidin is secreted in to the circulation, where it regulates systemic iron metabolism by interacting with its receptor, ferroportin, a transmembrane iron export protein. Ferroportin is abundantly expressed on the cell surface of RE macrophages and on the basolateral membrane of duodenal enterocytes. These 2 cell types are the main suppliers of iron to the plasma. By recycling iron from senescent erythrocytes, RE macrophages release 20�25 mg of iron into the plasma per day. Enterocytes add another;1�2 mg of iron to the plasma through the absorption of dietary iron. Hepcidin inhibits iron release at both of these sites by binding to cell-surface ferroportin and causing its internalization and subsequent degradation. Hepcidin can therefore be considered as a negative regulator of iron absorption and recycling. Hepcidin synthesis in the liver is promoted by the presence of iron and infection/inflammation Is inhibited by active erythropoiesis and hypoxia Hepatic synthesis also under control of: Hfe/Tfr1 (haemochromatosis gene/transferrin receptor1), Tfr2 � Transferrin receptor2 found in erythroid precursors, hepatic cells and duodenal crypt cells Hjv � haemojuvelin, Smad4 cell signalling molecule, BMPs � bone morphogenetic protein may be ligand for Hjv Hepcidin is also induced in other tissues by action of IL6 during inflammatory processes Hepcidin is secreted in to the circulation, where it regulates systemic iron metabolism by interacting with its receptor, ferroportin, a transmembrane iron export protein. Ferroportin is abundantly expressed on the cell surface of RE macrophages and on the basolateral membrane of duodenal enterocytes. These 2 cell types are the main suppliers of iron to the plasma. By recycling iron from senescent erythrocytes, RE macrophages release 20�25 mg of iron into the plasma per day. Enterocytes add another;1�2 mg of iron to the plasma through the absorption of dietary iron. Hepcidin inhibits iron release at both of these sites by binding to cell-surface ferroportin and causing its internalization and subsequent degradation. Hepcidin can therefore be considered as a negative regulator of iron absorption and recycling.

41. Iron deficiency Increased iron loss Nutritional deficiency Increased demand for iron Impaired absorption. Occure because losses> than intake, normally a delicate balance is maintained� 1mg/day in men and non menstruating women, double or more needed in menstuating F Only 5-10% dietary Fe usually absorbed; av. UK diet 14mg = 1.4mg/day Ok for men!!! Increased loss usually blood loss eg GI loss, menorrhagia, is there a co-existent bleeding tendency (Hereditary Taelangiectasia, vWD, angiodysplasia of bowel) Nutritional lack � rare in UK as isolated cause Can be iron poor diet, diet in which absorption is decreased Increased demand � eg pregnancy, adds requirement of 500mg for inc. RBC mass, 250-300mg for foetus, potential delivery loss of 500ml ~ 300mg Breast feeding � important source of iron for infant and further demand for maternal iron stores. Impaired absorption � usually related to intestinal damage eg duodenal surgery or Crohns and coeliac disease Acid suppression also causes reduced absorption because of reduced ability to convert ferric to ferrous iron � so found in association with PA (gastric achlorhydria) and in some patients on log term acid suppresssion � failure to recognise this may lead to extensive investigation and accusations that patient is not taking their iron tablets�.Occure because losses> than intake, normally a delicate balance is maintained� 1mg/day in men and non menstruating women, double or more needed in menstuating F Only 5-10% dietary Fe usually absorbed; av. UK diet 14mg = 1.4mg/day Ok for men!!! Increased loss usually blood loss eg GI loss, menorrhagia, is there a co-existent bleeding tendency (Hereditary Taelangiectasia, vWD, angiodysplasia of bowel) Nutritional lack � rare in UK as isolated cause Can be iron poor diet, diet in which absorption is decreased Increased demand � eg pregnancy, adds requirement of 500mg for inc. RBC mass, 250-300mg for foetus, potential delivery loss of 500ml ~ 300mg Breast feeding � important source of iron for infant and further demand for maternal iron stores. Impaired absorption � usually related to intestinal damage eg duodenal surgery or Crohns and coeliac disease Acid suppression also causes reduced absorption because of reduced ability to convert ferric to ferrous iron � so found in association with PA (gastric achlorhydria) and in some patients on log term acid suppresssion � failure to recognise this may lead to extensive investigation and accusations that patient is not taking their iron tablets�.

42. Laboratory features of iron deficiency 3 stage process, low stores - increasingly microcytic and hypochromic picture, variable polychromasia � increasing anaemia must differentiate from thalassaemia (high rbcs, RDW lower), Increasing anaemia, if bleeding may see high retics, high RDW May see thrombocytosis, May see rouleaux and increased ESR dependent on underlying cause Ferritin low, serum iron low, TIBC typically high, serum transferrin receptor high BM iron stores decreased /absent and erythroblast iron the same � unlikely to do a BM !! Beware -May be complicated by anaemia of chr disorders, megaloblastic anaemia etc 3 stage process, low stores - increasingly microcytic and hypochromic picture, variable polychromasia � increasing anaemia must differentiate from thalassaemia (high rbcs, RDW lower), Increasing anaemia, if bleeding may see high retics, high RDW May see thrombocytosis, May see rouleaux and increased ESR dependent on underlying cause Ferritin low, serum iron low, TIBC typically high, serum transferrin receptor high BM iron stores decreased /absent and erythroblast iron the same � unlikely to do a BM !! Beware -May be complicated by anaemia of chr disorders, megaloblastic anaemia etc

43. Clinical features of iron deficiency Pallor, fatigue, exertional breathlessness, exacerbation of underlying ischaemia Angular cheilosis Tachycardia In prolonged or severe cases �Koilonychia� May have features of underlying disease Remember skin rash with CoeliacRemember skin rash with Coeliac

45. Case study #2 RH age 17 yrs, college student Pale and tired, some exertional dyspnoea Recent (past 3 months) recurrent flu-like illness No abnormal bleeding or bruising. Hb 6.7g/dl MCV 87, WBC 5.8, Plts 138 RDW 22.6Recent (past 3 months) recurrent flu-like illness No abnormal bleeding or bruising. Hb 6.7g/dl MCV 87, WBC 5.8, Plts 138 RDW 22.6

46. Investigation of Fe Deficiency Start with the history Evidence of blood loss G I Tract Menstrual Urine Good dietary history essential Simple obvious bleeding � eg: haemorrhoids, nose bleeds (consider underlying bleeding disorder), assessment of nenstrual losses �Occult bleeding� � GI tract- �FOB� � potential for false +ves if not done with careful dietary preparation, still a useful screen however. Ferritin � if low, Fe defic. Unequivocal; normal result does not exclude � May need Fe/TIBC, especially if raised ESR/CRP or ass. with chr. Disease. Drugs � Aspirin, steroids, acid suppressionGood dietary history essential Simple obvious bleeding � eg: haemorrhoids, nose bleeds (consider underlying bleeding disorder), assessment of nenstrual losses �Occult bleeding� � GI tract- �FOB� � potential for false +ves if not done with careful dietary preparation, still a useful screen however. Ferritin � if low, Fe defic. Unequivocal; normal result does not exclude � May need Fe/TIBC, especially if raised ESR/CRP or ass. with chr. Disease. Drugs � Aspirin, steroids, acid suppression

47. Treating Iron Deficiency Depends on the cause !! Stop/prevent bleeding Oral iron Parenteral iron Gluten free diet Worldwide � parasitic infections are main cause. Maternal Fe deficiency leads to neonatal deficiency Oral iron � important to replenish stores and not simply correct anaemia Parenteral can be IM � numerous injections � may be painful and stain tissuesWorldwide � parasitic infections are main cause. Maternal Fe deficiency leads to neonatal deficiency Oral iron � important to replenish stores and not simply correct anaemia Parenteral can be IM � numerous injections � may be painful and stain tissues

48. Anaemia of Chronic Disease/Inflammation Chronic infections TB, SBE, osteomyelitis etc Malignancies Lymphoma, metastatic carcinoma Chronic inflammatory disease PMR, RA, SLE, IBS Associated with a �hypoproliferative marrow, low retics, low Epo levels and poor/no response to iron Bleeding, poor nutrition, drug related effects may also impact Tends to occur in patients with systemic disease Associated with a �hypoproliferative marrow, low retics, low Epo levels and poor/no response to iron Bleeding, poor nutrition, drug related effects may also impact Tends to occur in patients with systemic disease

49. Pathophysiology Presence of inflammatory cytokines, mainly IL-6 also IL-1, interferons and TNF alpha Liver produces increased amounts of hepcidin. Hepcidin stops ferroportin from releasing iron stores. Decreases ferroportin expression Probably affects bone marrow response to Epo. May inhibit release of Epo from kidney Rbc survival mildly decreased Bacterial lactoferrin may compete with transferrin

50. Laboratory findings Typically mild to moderate anaemia, often non progressive Normocytic, normochromic or mildly microcytic and hypochromic. Low reticulocyte count Often show neutrophil leucocytosis, thrombocytosis and rouleaux with Increased ESR Ferritin normal or high,, serum iron low, TIBC typically low or normal BM iron often increased in macrophages, but erythroblast iron is decreased CRP increased Beware -May be complicated by true iron deficiency Typically mild to moderate anaemia, often non progressive Normocytic, normochromic or mildly microcytic and hypochromic. Low reticulocyte count Often show neutrophil leucocytosis, thrombocytosis and rouleaux with Increased ESR Ferritin normal or high,, serum iron low, TIBC typically low or normal BM iron often increased in macrophages, but erythroblast iron is decreased CRP increased Beware -May be complicated by true iron deficiency

51. Erythropoietin in AI Best approach: treat the underlying condition Epo &ESAs: Initial dramatic improvements in Hb and QoL But tempered with poorer overall survival or shorter progression free survival In renal failure � adverse effect on BP if excessive rise in Hb Improved response if also give parenteral iron � esp in renal failure Much pressure to prescribe Epo or ESAs for patients with cancer and low Hb - linear relationship between rise in Hb &improved QoL up to 12g/dl. Also benefit (financial) based on reduction in transfusion requirementsMuch pressure to prescribe Epo or ESAs for patients with cancer and low Hb - linear relationship between rise in Hb &improved QoL up to 12g/dl. Also benefit (financial) based on reduction in transfusion requirements

52. Disorders of Haem synthesis Sideroblastic anaemia Lead poisoning (Porphyrias) Porphyrias included for completeness, but have little haematology impactPorphyrias included for completeness, but have little haematology impact

53. Sideroblastic anaemias Refractory anaemias Typically dimorphic with variable hypochromic population Ring sideroblasts in BM Excess iron in BM

54. Inherited Sideroblastic Anaemia Rare disorders Often X linked and presenting in childhood Mostly linked to mutations of ALA synthase May respond to pyridoxine but most are transfusion dependent and need iron chelation ALA synthase � delta aminolaevulinic acid synthase, required for first stage of haem synthesis where succinyl Co A and glycine convert to d-aminolaevulinic acid using pyridoxal 6 phosphate as co-factor Often present with markedly microcytic and hypochromic picture and severe anaemia. Pyridoxal 6 phosphate is a co-factor for ALA synthaseALA synthase � delta aminolaevulinic acid synthase, required for first stage of haem synthesis where succinyl Co A and glycine convert to d-aminolaevulinic acid using pyridoxal 6 phosphate as co-factor Often present with markedly microcytic and hypochromic picture and severe anaemia. Pyridoxal 6 phosphate is a co-factor for ALA synthase

55. Acquired Sideroblastic Anaemias Primary MDS FAB classification RARA Characterised by erythroid dysplasia and >15% ring sideroblasts in BM Approx 10% of all MDS, rarely show cytogenetic abnormalities May be Tx dependent Majority of MDS pts show occ ring sideroblasts in BM, but WHO classification subdivides another group with >15% ring sideroblasts and bi or trilineage dysplasiaMajority of MDS pts show occ ring sideroblasts in BM, but WHO classification subdivides another group with >15% ring sideroblasts and bi or trilineage dysplasia

56. Secondary Sideroblastic Anaemias Alcohol usually in conjunction with folate deficiency/ poor nutrition Lead poisoning Mainly inhibits Haem synthetic enzymes Drugs Chloramphenicol � inhibits mitochondrial mRNA translation Anti TB drugs eg izoniazid � pyridoxine antagonist Deficiencies/changes in pyridoxine metabolism Rarely in coeliac, SCD, pregnancy Alcohol may be due to defective pyridoxine metabolism or haem synthesis Lead poisoning also inhibits pyrimidine 5� nucleotidase leads to accumulation of denatured RNA ie basophilic stipplingAlcohol may be due to defective pyridoxine metabolism or haem synthesis Lead poisoning also inhibits pyrimidine 5� nucleotidase leads to accumulation of denatured RNA ie basophilic stippling

57. Diagnosis and Investigation History Laboratory investigations History � Age of onset, Drugs & alcohol (isoniazid and pyrazinamide) FBC, presence of microcytic, hypochromic picture, basophilic stippling, dimorphism,etc BM � erythroid hyperplasia, presence of ringed sideroblasts Haematinics � is ferritin raised ? ? Measure levels of enzymes involved in Haem synthesisHistory � Age of onset, Drugs & alcohol (isoniazid and pyrazinamide) FBC, presence of microcytic, hypochromic picture, basophilic stippling, dimorphism,etc BM � erythroid hyperplasia, presence of ringed sideroblasts Haematinics � is ferritin raised ? ? Measure levels of enzymes involved in Haem synthesis

58. Sideroblastic anaemias - treatment Withdrawal of potential causative agent Piridoxine � high dose +/- folic acid May be transfusion dependent(will develop iron overload if not chelated) RARS ~ 10% risk of leukaemic transformation

59. Porphyrias Rare conditions caused by deficiency of haem synthetic enzymes used in conversion of porphyrins to Haem Deficiency causes reduction in synthesis Causes accumulation of unused precursors and intermediate metabolites, Intermediates become deposited in tissues. Can cause photosensitivity, mania, severe abdo pain Some intermediates can be detected in the urine or faeces Little impact in Haem lab � need to know of their existenceLittle impact in Haem lab � need to know of their existence

60. Further Reading Postgraduate Haematology � Hoffbrand, Catovsky and Tuddenham Essential Haematology - Hoffbrand, Moss &Pettitt Current journals and scientific papers