71 THALASSAEMIAS 72 CHAPTER 2 Red cell disorders \235\233\202 thalassaemia Two globin genes on each chro mosome 16, with total of 4 globin genes per cell (normal person is designated / ) making tha- lassaemia more heterogeneous than thalassaemia. Like sickle cell anemia, patients can either have mild thalassaemia ( thalassaemia trait, \210\222 \210\222/ or \210\222 / \210\222 or \210\222 / ) where 1 or 2 globin genes are aff e cted or they may have severe thalassaemia if 3 or 4 of the genes are aff ected. thala ssaemia is generally the result of large deletions within globin complex. High prevalenc e of thalassaemia in Africa, Afro-Caribbeans, South and SE Asia ( \210\222 / \210\222 or \210\222 / ). o thalas- saemia (i.e. \210\222 \210\222/ ) found most commo nly in E Mediterranean and SE Asian ethnic groups. thalassaemia occurs from loss of link ed globin genes on 1 chromosome (i.e. \210\222 \210\222/ ). Deletions in gene H S40 region (upstream regulatory region) account for most o thalassaemia mutations. + re sults from deletion of 1 of the linked genes ( \210\222 / ) or inactivation due to p oint mutation ( T / ). \235\233\202 thalassaemia trait ( \210\222 \235\233\202/ \235 \233\202 \235\233\202, or \235\233\202 \235\233\202/ \210\222 \210\222 or \210\222 \235 \233\202/ \210\222 \235\233\202) 1 gene deleted. Asymptomatic. d MCV and MCH in minority. \235\233\202 thalassaemia trait ( \235\233\202 \235\233\202/ \210\222 \210\222 or \210 \222 \235\233\202/ \210\222 \235\233\202) 2 common + deletions are \210\222 3.7 and \210\222 4.2. The common 0 deletions are ( \210\222 \210\222 /MED), ( \210\222 /(20.5) ), and ( \210\222 \210\222 /SEA), ( \210\222 \210\222 /THAI), ( \210\222 \210\222 /FIL). Requires PCR for diagnosis. Will not be picked up on HPLC or electrophoresis. Asymptomatic carrier \200\224recognized once other causes of microcytic anemia are excluded (e.g. Fe de fi ciency). Hb may be n or minimally d. MCV and MCH are d. Absence of splenomegaly or other clinical fi ndings. Requires no therapy. Hemoglobin H disease ( \210\222 \210\222/ \210 \222 \235\233\202) Three genes deleted; only 1 functioning copy of the globin gene/ c ell. Clinical features variable. May be moderate anemia with Hb 8.0 \200\223 9.0g/dL. MCV a nd MCH are d. Hepatosplenomegaly, chronic leg ulcer- ation, and jaundice (refl ecting under lying haemolysis). Infection, drug treatment, and pregnancy may worsen anemia. Blood fi lm shows hypochromia, target cells, NRBC and i reticulocytes. Brilliant cresyl blu e stain will show HbH inclusions (tetramers of globin, 4, that have polymerized due to lack of chains). Hb pattern consists of 2 \200\22340% HbH ( 4) with some HbA, A2 and F . Treatment Not usually required but prompt treatment of infection advisable. Give regular fo lic acid especially when pregnant. Splenectomy of value in some patients with HbH disease. Needs monitoring and may require blood transfusion. 73 THALASSAEMIA Hemoglobin Bart \200\231s hydrops fetalis ( \210\222 \210\222 / \210\222 \210\222) Common cause of stillbirth in South East Asia. All 4 globin genes aff ected. chains form te tramers (HbBart \200\231s, 4) which bind oxygen very tightly, with resultant poor tissue oxygenation. Fetus is either stillborn (at 34 \200\22340 weeks \200\231 gestation) or dies soon after birth. They are pale, dis- tended, jaundiced, and have marked hepatosplenomegaly and ascites. Hemoglobin is 76.0g/dL and the fi lm shows hypochromic red cells, target cell s, i reticulocytes and nucleated red cells. Hemoglobin analysis shows mainly HbBart \200 \231s ( 4) with a small amount of HbH ( 4); HbA, A2, and F are absent. In utero transfusi ons can aid survival. Long-term transfusions will be required. 74 CHAPTER 2 Red cell disorders \235\233\203 thalassaemia There are only 2 copies of gl obin gene per cell. Abnormality in 1 globin gene results in thalassaemia trait; if bo th globin genes are aff ected the patient has thalassaemia major or thalassaemia i ntermedia. Prevalent in the Mediterranean region, Middle East, India, Pakistan, and SE Asia ; less common in Africa (apart from Liberia and some regions of N. Africa). Unlike thala ssaemia, most thalassaemias are due to single point mutations (>200 identifi ed to date; rarely due to deletions). Results in reduced globin synthesis ( +) or absent globin production ( ). In thalassaemia major, patients have severe anemia requiring lifelon g support with blood transfusion (with resultant Fe overload). There is ineff ective erythr opoiesis. The thalassaemia phenotype is heterogeneous since several factors infl uence t he disease. Recently identifi ed globin stabilizing protein binds free chains, blocki ng production of reactive oxygen species thereby modulating the clinical picture of thal assaemia in mice. Not yet confi rmed in humans. Not obvious at birth due to presence of HbF ( 2 2) but as chain production diminishes and globin production increases eff ects of the mutation become obvious. Children fail to thrive, and development is aff ected. Hep atosplenomegaly (due to production and destruction of red cells by these organs) is typical . Children also develop facial abnormalities as the fl at bones of the skull and other bone s attempt to produce red cells to overcome the genetic defect. Skull radiographs show \200 \230hair on end \200\231 appear- ances refl ecting the intense marrow activity in the skull bones. Investigation and management thalassaemia trait \200 Carrier state. \200 Hb may be d but is not usually <10.0g/dL. \200 MCV d to 763 \200\22377fL. \200 Blood fi lm: microc ytic, hypochromic RBCs; target cells often present. Basophilic stippling especially in Mediterraneans (see Fig 2.8). RCC i. \200 HbA2 ( 2 2) i \200\224provides useful diagnostic test for thalassaemia t rait. \200 Occasionally confused with Fe defi ciency anemia, however, in thalassaemia trait the serum Fe and ferritin are n (or i) whereas in IDA they are d. BM shows 6 \227 the number of erythroid precursors as n though this is not required to make the diagnosis of thalassaemia major. Treatment Not usually required. Usually detected antenatally or on routine FBC preop. th alassaemia intermedia (non-transfusion dependent phenotype) \200 Regular blood transfusio n not required; more severe than thalassaemia trait but milder than thalassaemia majo r. May arise through several mechanisms, e.g: Inheritance of mild thalassaemia mutations (e.g. homozygous + thalassaemia alleles, co mpound heterozygote for 2 mild + thalassaemia alleles, compound heterozygotes for mild pl us severe + thalassaemia alleles). 75 THALASSAEMIA Fig. 2.8 Blood fi lm in thalassaemia trait. Elevation of HbF. \200 Coinheritance of thalassaemia. \200 Coinheritance of thala ssaemia trait with, e.g. HbLepore. \200 Severe thalassaemia trait. Clinical \200 Variable presentation with only moderate degree of anemia. \200 Hepatospl enomegaly. \200 Fe overload is a feature. \200 Some patients are severely anemic (Hb 76 g/dL) although not requiring regular blood transfusion, have impaired growth and developmen t, skeletal deformities and chronic leg ulceration. \200 Others have higher Hb (e.g. 10 \200\22312g/dL) with few symptoms. Management Depends on severity. May require intermittent blood transfusion (especially duri ng growth spurts and pregnanacy), Fe chelation, folic acid supplementation, prompt treatmen t of infection, as for thalas- saemia major. Hydroxycarbamide sometimes used to increase HbF. Occasionanally splenectomy can be helpful to maintain higher Hb. 76 CHAPTER 2 Red cell disorders thalassaemia major (Cooley \200\231s anemia) Patients have abnormalities of both glob in genes. Presents in childhood with anemia and failure to thrive. Untreated children have extramed- ullary haemopoiesis with hepatosplenomegaly and skeletal deformities. Clinical \200 Moderate/severe anemia (Hb 73.0 \200\2239.0g/dL). \200 d MCV and MCHC. \200 i Reticulocytes. \200 Blood fi lm: marked anisopoikilocytosis, target cells, and nu cleated red cells (see Fig 2.9). Methyl violet stain shows RBC inclusions containing precipitated globin. \200 Hb elect rophoresis or HPLC shows mainly HbF ( 2 2). In some thalassaemias there may be a littl e HbA ( 2 2) if some globin is produced. HbA2 may be n or mildly elevated. Fig. 2.9 thalassaemia major. Note: bizarre red cells with marked anisopoikilocytosis. Management \200 Regular lifelong blood transfusion (every 2 \200\2234 weeks) to maintain pre- transfusion Hb 9.5 \200\22310g/dL and suppress ineff ective erythropoiesis allowing no rmal growth and development in childhood. This will help to prevent hepatosplenomegaly and facial bone changes. 77 THALASSAEMIA Fe overload (transfusion haemosiderosis) is major problem \200\224 damages liver, heart, endocrine glands including pancreas. Compliance with iron chelation t herapy determines prognosis, and can be challenging to manage especially in younger patient s. Splenectomy is rarely required with timely transfusion therapy but can be of value (e.g. if massive splenomegaly or increasing transfusion requirements) but b est avoided until after the age of 5 years due to i risk of infection. BM transplantation has been carried out using sibling donor HLA-matched transplants with go od results in young patients with thalassaemia major. The procedure carries a signifi ca nt procedure-related morbidity and mortality, along with GvHD (E BMT section pp.424 \200 \223431). i HbF levels using 5-azacytidine or hydroxycarbamide should ameliorate thalassaemia. Fe chelation therapies \200 Choice of iron chelation therapy (mono or combination therapy ) should be determined by pattern, extent of Fe loading, patient characteristics, and patie nt choice. Available chelators \200 Desferrioxamine: parenteral, painful, compliance problems but go od effi cacy. Should be given IV continuosly in decompensated organ dysfunction. Deferiprone: oral, given in 3 divided doses. Side eff ects include: arthralgia, nausea, other GI symptoms, LFT disturbance, agranulocytosis (weekly FBC recomme nded), Zn2+ defi ciency. Particularly good for cardiac iron loading. Deferasirox: new oral chelator, once daily (suspension), similar effi cacy to desferrioxamine. Side eff ects include GI upset, i creatinine, cytopenias. Newer agents undergoing clinical trials. Monitoring \200 Ferritin 3-monthly. Annual assessment \200\224CardiacT2* MRI, liver R2 or T2* MRI, endocrine tests. Audiology/ophthalmology (chelation side eff ects). Acute complications \200 Cardiac damage \200\224can present with cardiac arrhythmia and c ardiac failure, older patients prone to AF even when myocardial T2* satisfactory. Need urgent ECG, ECHO, CXR, cardiac monitoring and management on coronary care ward. Acute severe sepsis \200\224i risk of overwhelming bacterial sepsis (i further if splenectomy). Beware central venous catheter infection. Particularly Klebsiel la, Yersinia enterolitica, encapsulated organisms. Give broad-spectrum IV antibiotics, cons ider HDU input. \200 Liver failure \200\224cirrhosis, portal hypertension, acute hepatic decompensation. Early liver unit input, careful fl uid balance. \200 Endocrine dysfunctio n \200\224hypocalcaemia with tetany due to hypoparathyroidism. Need cardiac monitoring, IV calcium. Diabetic complications. 78 CHAPTER 2 Red cell disorders Screening Screen mothers at fi rst antenatal visit. If mother is thalassaemic carrier, scre en father. If both carriers for severe thalassaemia off er prenatal diag- nostic testing. F etal blood sampling can be carried out at 18 weeks \200\231 gesta- tion and globin chain sy nthesis analyzed. Chorionic villus sampling at 10+ weeks \200\231 gestation provides a sour ce of fetal DNA that can be analyzed in a variety of methods: Southern blotting, oligonucle otide probes, or RFLP analysis may determine genotype of fetus. Moving towards PCR- based t echniques; likely to improve carrier detection. Preimplantation diagnosis is performed usin g embryonic biopsies. Further reading Dzik, W.H. (2002). Leukoreduction of blood components. Curr Opin Hematol, 9 , 521 \200\2236. Hoff brand, A.V., et al. (2003). Role of deferiprone in chelation therapy for transfusional iron overload. Blood, 102, 17 \200\22324. Olivieri, N.F. (1999). The beta -thalassemias. N Engl J Med, 341, 99 \200\223109. Rund, D., et al. (2005). Beta-thalassemia . N Engl J Med, 353, 1135 \200\22346. Shalev, O., et al. (1995). Deferiprone (L1) chelates pathologic iron deposits from membranes of intact thalassemic and sickle red blood cells bo th in vitro and in vivo. Blood, 86, 2008 \200\22313. United Kingdom Thalassaemia Society (2 008). Standards for the clinical care of children and adults with thalasaemia in the UK. M www.ukts.org/pdfs/awareness/standards2008.pdf Weatherall, D.J. and Provan, A.B. (2000). Red cells I: inherited anaemias. Lancet, 355, 1169 \200\22375. 79 OTHER THALASSAEMIAS Other thalassaemias Heterozygous \235\233\205 \235\233\203 thalassaemia Produces a picture similar to thalassaemia trait with i HbF (5 \200\22320%) and microcytic RBCs; HbA2 is n or d. Homozygous \235\233\205 \235\233\203 thalassaemia Homozygous condition is uncommon. There is failure of production of both and globins. Milder than thalassaemia majo r, i.e. thalas- saemia intermedia. Represents a form of thalassaemia intermedia. Hb 8 \200\22311g/dL. Absence of HbA and HbA2; only HbF is present (100%). Heterozygous \235\233 \203 thalassaemia/ \235\233\205 \235\233\203 thalassaemia Similar to thalassaemia major (but less severe). Hb produced is mainly HbF with small amount of HbA2. \235\233\204 \235 \233\205 \235\233\203 thalassaemia Homozygote is not viable. Heterozygous condition is asso ciated with haemolysis in neonatal period and thalassaemia trait in adults with n HbF and H bA2. HbLepore This abnormal Hb is the result of unequal crossing over of chromo- somes. Aff ects and globin genes with generation of a chimeric globin with sequences at NH2 termi nal and globin at COOH terminal. Production of globin is ineffi cient; there is abs ence of normal and globins. The phenotype of the heterozygote is thalassaemia trait; the homozygote picture is thalassaemia intermedia. HbE/ thalassaemia Compound heterzygote (coinheritance of HbE and thalassaemia mutation). Varied clinical course (mostly thalas saemia intermedia phe- notype) with mild-moderate anemia, splenomegaly. Some patients requi re regular transfusion. Further reading BCSH haemoglobinopathy diagnosis guidelines: M www.bcshguidelines.com/pdf/b jh809.pdf 80 CHAPTER 2 Red cell disorders Hereditary persistence of fetal hemoglobin Heterogeneous groups of rare disorders caused by deletions or crosso- vers involving and chain production, or non-deletional forms du e to point mutations upstream of the globin gene, with high levels of HbF production in adult life. There is d and chain production with enhanced chain production. Globin chain imbalance is much less marked than in thalassaemia, resulting in milder disorder. There are few clinical eff ects even when 100% Hb is HbF. May be pancellular (very high levels of HbF hemoglobin synthesis with uniform distribution in RBCs) or heterocellular (i numbers of F cells). Ethnic diff erences \200 Black individuals with heterozygous pancellular HPFH have HbF le vels of 15 \200\22330%. Greek individuals with pancellular HPFH have HbF 710 \200\22320% (most HbF is A ). Mechanism Like thalassaemia, HPFH frequently arises from deletions of DNA, which remov e or inactivate the globin gene (note: heterocellular HPFH may be result of mutations ou tside the globin gene). There are also many transcription factors and epigenetic regulat ors that can account for the variation in HbF in adults. Transcription factors identifi ed include BCL 11A, KLF1, MYB and chromatin modifi ers (histone deacetylases, DNA methyltransf erases, and nuclear receptors TR2/TR4). Heterozygous HPFH Anemia may be mild or absent. Hematological indices are normal. There is balanced /non- globin chain synthesis. HbF level 725%. 81 HB PATTERNS IN hemoglobin DISORDERS Hb patterns in hemoglobin disorders See Table 2.10. Table 2.10 Hb patterns in hemoglobin disorders % Hemoglobin A F A2 S Other Normal thalassaemia trait thalassaemia intermedia thalassaemia major 97 <1 2 \200\2233 80 \200\22395 30 \200\22350 1 \200\2235 50 \200\22370 3 \200\2237 0 \200\2235 0 \200\22320 80 \200\223100 0 \200\22313 60 \200\22385 15 \200\22335 1 \200\2233 HPFH (black heterozygote) HPFH (black homozygote) thalassaemia trait 100 85 \200\22395 Bart \200\231s 0 \200\22310% at birth H 5 \200\22330% Bart \200\231s 20 \200\22330% at birt h Bart \200\231s 80 \200\22390% 60 \200\22395 HbH disease HbBart \200\231s hydrops HbE trait HbE disease HbE/ thalassaemia HbE/ thalassaemia HbD trait HbD disease HbD/ thalassaemia HbC trait HbC disease 60 \200\22365 0 0 13 1 \200\2232 5 \200\22310 30 \200\22340 2 \200\2233 5 \200\223 E 30 \200\22335 E 95 E 60 \200\22370 E 80 50 \200\22365 1 \200\2235 0 \200\2237 1 \200\2235 1 \200\2233 1 \200\2237 1 \200\2233 D 45 \200\22350 D 90 \200\22395 D 80 \200\22390 60 \200\22370 C 30 \200\22340 C 95 slight i 3 3 \200\223 30 \200\22345 90 60 \200\22385 Sickle trait Sickle cell anemia Sickle/ + thalassaemia Sickle/ o thalassaemia Sickle/D Si ckle/C 1 7 5 \200\22315 55 \200\22370 0 5 \200\22330 4 \200\2238 70 \200\22390 5 \200\22330 0 50 50 \200\22365 D 50% C 50% 1 \200\2235 1 0 0 2 \200\2233 HbLepore trait 80 \200\22390 1 \200\2233 2.0 \200\2232.5 Lepore 9 \200\22311% Lepore 8 \200\22330% Lepore 5 \200\22315% HbLepore disease 0 70 \200\22390 0 HbLepore/ thalassaemia 70 \200\22390 2.5 82 CHAPTER 2 Red cell disorders Non-immune haemolysis 4 major groups \200 Infections. \200 Vascular (mechanical damage). \200 Chemical dama ge. \200 Physical damage. Infection \200 Malaria \200\224especially falciparum. Causes anemia through marrow suppre ssion, hypersplenism, and RBC sequestration. In addition there is haemolysis due to destruc tion of parasitized RBCs by RES and intravascular haemolysis when sporozoites released from infected RBCs. Blackwater fever refers to severe acute intravascular haemolysis with haemo globinaemia, dHb, haemoglobinuria, and ARF. Babesiosis \200\224Babesia (RBC protozoan). Rapid onset of vomiting, diarrhea, rigors, jaundice, iT . Haemoglobinaemia, haemoglobinuria, ARF and deat h. Clostridium perfringens \200\224septicaemia and acute intravascular haemolysis. Viral \200\224especially viral haemorrhagic fevers, e.g. dengue, yellow fever. Mechanical \200 Cardiac \200\224turbulence and shear stress following mechanical valve re placement. General features of haemolysis: i reticulocytes, i LDH, i plasma Hb, with d hapt oglobins d platelets. Urinary haemosiderin +ve. MAHA \200\224E see Microangiopathic haemolytic anemia, p.96. \200 HUS/TTP \200\224E see T hrombotic thrombocytopenic purpura, pp.538 \200\223540. March haemoglobinuria \200\224with severe strenuous exercise, e.g. running. Destruction of RBCs in soles of feet. Worse with hard soles and uneven hard ground. Mild an emia. No specifi c features on fi lm. May be associated GIT bleeding and d ferritin (lost i n sweat). Chemical and physical \200 Oxidative haemolysis \200\224chronic Heinz body intravascular haemolysis with dapsone or sulafasalazine in G6PD defi cient people or unstable Hb (and nor mals if dose high enough). Movie: bite cells (RBC). Heinz bodies not prominent if intact sp leen. Haemolysis well compensated. MetHb \200\224E see Methaemoglobinaemia, p.95. \200 Lead poisoning \200\224moderate dRBC lifespan. Anemia mainly due to block in haem synthesis although lead also inhibits 5 \200 nucleotidase (NT). Basophilic stippling on fi lm. Ring sideroblasts in BM. O2 \200\224haemolysis in patients treated with hyperbaric O2. \200 Insect bites \200\224e .g. spider, bee-sting (not common with snake bites). \200 Heat \200\224e.g. burns l sever e haemolysis due to direct RBC damage. \200 Liver disease \200\224reduced RBC lifespan in acute hepatitis, cirrhosis, Zieve \200\231s syndrome is an uncommon form of haemolysis \200\224intravascular associated with acute abdominal pain (E see Anemia in liver disease, p.41). 83 NON-IMMUNE HAEMOLYSIS Wilson \200\231s disease \200\224autosomally inherited disorder of copper metabolism, with hepatolenticular, hepatocerebral degeneration. PNH \200\224E see Paroxysmal nocturnal haemoglobinuria, p.104. \200 Hereditary acanthocyt osis \200\224a- -lipoproteinaemia. Rare, inherited. Associated with retinitis pigmentosa, steatorrhoea, ataxia, and mental retardation. 84 CHAPTER 2 Red cell disorders Hereditary spherocytosis Most common inherited RBC membrane defect characterized by vari- a ble degrees of haemolysis, spherocytic RBCs with i osmotic fragility. Pathophysiology Abnormal RBC cytoskeleton: partial defi ciency of spectrin, ankyrin, band 3 or protein 4.2 (leads to d binding to band 4.1 protein and ankyrin). Loss of lipid from RB C membrane l spherical (cf. biconcave) RBCs with reduced surface area l get trapped in sple nic cords and have reduced lifespan. RBCs use more energy than normal in attempt to maintai n cell shape. RBC membrane has i Na+ permeability (loses intracellular Na+) and energy requ ired to restore Na+ balance. Red cells are less deformable than normal. Epidemiology In Northern Europeans 1:5000 people are aff ected. In most cases inher- itance is autosomal dominant although autosomal recessive inheritance has been reported. Clinical features Presents at any age. Highly variable clinical expression from asympto- ma tic to severely anemic, but usually there are few symptoms. Note: phenotype is fairly unifo rm within a family. Well-compensated haemol- ysis; other features of haemolytic anemia may be present, e.g. spleno- megaly, gallstones, mild jaundice. Occasional aplastic crises occu r, e.g. with parvovirus B19 infection. Diagnosis \200 +ve family history of HS in many cases. \200 Blood fi lm shows ii sphero cytic RBCs (see Fig 2.10). \200 Anemia, ireticulocytes, iLDH, unconjugated bilirubin, uri nary urobilinogen with d haptoglobins. DAT \210\222ve. \200 If typical family history, clinical and laboratory fi ndings, additi onal tests may not be necessary If additional tests needed, screening tests recommended are EMA binding and cryohaemolysis test (good predictive value). Gel electrophoresis of red cell me mbrane for atypical cases. Osmotic fragility is supportive. Eosin-5-maleimide binding test (EMA binding) \200\224uses fl ow cytometry. EMA is bound to some transmembrane proteins and is lost if there is defi ciency of certain proteins. Better specifi city and sensitivity than osmotic fragility. Osmotic fragility test \200\224RBCs incubated in saline at various concentra- tions. Result s in cell expansion and eventually rupture. Normal RBCs can withstand greater volume increa ses than spherocytic RBCs. +ve result (i.e. confi rms HS) when RBCs lyse in saline at near to isotonic concentration, i.e. 0.6 \200\2230.8g/dL (whereas normal RBCs will simply show s welling with little lysis). Osmotic fragility more marked in patients who have not undergon e splenectomy, and if the RBCs are incubated at 37 C 85 HEREDITARY SPHEROCYTOSIS for 24h before performing the test (see Fig 2.11). Note: a normal result does not exclude H S and may occur in 10 \200\22320% cases. Not routine for diagnosis. Autohaemolysis test \200\224since spherocytic RBCs use more glucose than normal RBCs (to ma intain normal shape) red cells incubated in buff er or serum for 48h show lysis and release of Hb into solution, which can be measured. In HS RBCs release greater amounts of Hb cf. n ormal RBCs (3% vs 1% in normal). Cryohaemolysis test, osmotic gradient, ektacytometry and EMA (eosin- 5-maleimide) tests hav e higher predictive value but tests are not specifi c for HS (e.g. +ve result seen in SE As ian ovalocytosis). SDS-PAGE form of gel electrophoresis that can be used to detect defi - ciency in RBC membra ne proteins. Should be considered when clinical features worse than predicted. Genetic analyzes \200\224e.g. DNA-based assays to detect mutations within genes for RBC mem brane proteins \200\224not routinely available. Complications \200 Aplastic crisis (e.g. parvovirus B19 infection, but may be any virus); see temporary dd reticulocytes, Hb and Hct. Megaloblastic changes in folate defi ciency. \200 i haemolysis during intercurrent illnes s, e.g. infections. \200 Gallstones (in 50% patients; occur even in mild disease). \200 Leg ulceration. \200 Extramedullary haemopoiesis. \200 Fe overload if multiply transfu sed. Exclude Other causes of haemolytic anemia, e.g. immune-mediated, unstable Hbs and MAHA, whi ch can give rise to spherocytic RBCs. CDA type II should be excluded particularly is splene ctomy considered. Treatment Supportive treatment is usually all that is required, e.g. folic acid (5mg/d) (mo derate and severe cases). In parvovirus crisis Hb drops signifi cantly and blood transfusio n may be required. Splenectomy is \200\230curative \200\231 but is reserved for patients w ho are severely anemic or who have sympto- matic moderate anemia. Best avoided in patients <6 years old due to risk of i fatal infection post-splenectomy. Those who do need splenec- tomy should be considered for cholecystectomy too. Further reading Bolton-Maggs, P.H., et al. (2011) Guidelines for the diagnosis and manageme nt of hereditary spherocytosis. Br J Haematol, 126, 455 \200\22374. 86 CHAPTER 2 Red cell disorders Fig. 2.10 Blood fi lm in hereditary spherocytosis. Note: large numbers of dark spherical re d cells (E see color plate section). 100 Patient Control Normal range 75 % RBC lysis 50 25 0 0 0.2 0.4 0.6 0.8 10 % Saline Fig. 2.11 Osmotic fragility assay: note control red cells (red) lyse at lower % saline sinc e they are able to take up more water than spherocytic red cells before lysis occurs. 87 HEREDITARY ELLIPTOCYTOSIS Hereditary elliptocytosis Heterogeneous group of disorders with elliptical RBCs. 3 major groups \200 Hereditary elliptocytosis. \200 Spherocytic HE. \200 South East A sian ovalocytosis. Pathophysiology Mutations in or spectrin. There may be partial, complete defi ciency, or structural abnormality of protein 4.1, or absence of glycophorin C. Epidemiology In Northern Europeans 1:2500 are aff ected. Inheritance is autosomal dominant. More common in areas where malaria is endemic. Clinical features Most are asymptomatic. Well-compensated haemolysis. A few patients have c hronic symptomatic anemia. Homozygote more severely aff ected. Diagnosis \200 May have +ve family history. \200 Blood fi lm shows ii elliptical or ova l RBCs. \200 Anemia, i reticulocytes, i LDH, unconjugated bilirubin, urinary urobilinogen with d haptoglobins. DAT is \210\222ve. \200 Osmotic fragility usually n (unless spherocytic HE). \200 Tran sient increase in haemolysis if intercurrent infection. Complications Usual complications of haemolytic anemia, e.g. gallstones, folate defi - cien cy, etc. Treatment Supportive care: folic acid (5mg/d). Most patients require no treatment. In more severe cases consider splenectomy. Spherocytic HE Elliptical and spherical in peripheral blood. Haemolysis and i osmotic fragi lity distinguish it from common heredi- tary elliptocytosis. Molecular basis is unknown. \200\230sphero-ovalocytes \200\231 Southeast Asian ovalocytosis Caused by abnormal band 3 protein. RBCs are oval with 1 \200 \2232 trans- verse ridges. Cells have i rigidity and d osmotic fragility. RBCs are more res istant to malaria than normal RBCs. 88 CHAPTER 2 Red cell disorders Glucose-6-phosphate dehydrogenase (G6PD) de \201 ciency G6PD is involved in pentose phosph ate shunt l generates NADP, NADPH, and glutathione (for maintenance of Hb and RBC membrane integrity, and reverse oxidant damage to RBC membrane and RBC com- ponents). G6PD defi cien cy is X-linked and clinically important cause of oxidant haemolysis. Aff ects \231\202 pre dominantly; \231\200 carriers have 50% normal G6PD activity. Occurs in West Africa, Southe rn Europe, Middle East, and South East Asia. >300 variants identifi ed. Features \200 Haemolysis after exposure to oxidants or infection. \200 Chronic non-sphe rocytic haemolytic anemia. \200 Acute episodes of haemolysis with fava beans (termed favi sm). \200 Methaemoglobinaemia. \200 Neonatal jaundice. 3 main forms of the disease, those associated with: 1. Acute intermittent haemolytic anemia . 2. Chronic haemolytic anemia. 3. No risk of haemolytic anemia. Mechanism Oxidants l denatured Hb l methaemoglobin l Heinz bodies l RBC less deformable l d estroyed by spleen. 2 main forms of the enzyme \200 Normal enzyme is G6PD-B, most prevalent form worldwide. \200 20% of Africans are type A. \200 A and B diff er by 1 amino acid. \200 Mutant enz yme with normal activity = G6PD A(+), fi nd only in black individuals. G6PD A( \210\222) is main defect in African origin; d stability of enzyme in vivo; 5 \200\22315% normal activity. 400+ variants but only 2 are relevant clinically: Type A( \210\222) = Africans (10% enzyme activity). \200 Mediterranean (with 1 \200\2233% activity). Drug-induced haemolysis in G6PD de \201 ciency \200 Begins 1 \200\2233d after ingestion of drug. \200 Anemia most severe 7 \200\22310d after ingestion. \200 Associated with lo w back and abdominal pain. \200 Urine becomes dark (black sometimes). \200 Red cells de velop Heinz body inclusions (cleared later by spleen). \200 Haemolysis is typically self- limiting. \200 Implicated drugs shown in Table 2.11. \200 But heterogeneous; variable s ensitivity to drugs. \200 Risk and severity are dose related. 89 GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY Haemolysis due to infection and fever \200 1 \200\2232d after onset of fever. \200 Mild anemia develops. \200 Commonly seen in pneumonic illnesses. Favism \200 Hours/days after ingestion of fava beans (broad beans). \200 Beans contain oxidants vicine and convicine l free radicals l oxidize glutathione. Urine becomes red or very dark. \200 Shock may develop \200\224may be fatal. Table 2.11 Risk of haemolysis in G6PD-defi cient individuals De \201 nite risk Possible risk Aspirin (1g/d acceptable in most cases) Chloroquine Probenecid Quinine and quinidine (accep table in acute malaria) Antmalarial drugs Primaquine Pamaquine (not available in UK) Analgesic drugs Aspirin Phenacetin Others Dapsone Methylthioninium chloride (methylene blue) Nitrofurantoin 4-quinolones (e.g. ciprofl oxacin, nalidixic acid) Sulfonamides (e.g. co-trimoxazole) Neonatal jaundice \200 May develop kernicterus (possible permanent brain damage). \200 Rare in A( \210\222) variants. \200 More common in Mediterranean and Chinese variants. Laboratory investigation \200 In steady state (i.e. no haemolysis) the RBCs appear normal . \200 Heinz bodies in drug-induced haemolysis (methyl violet stain). \200 Spherocytes and RBC fragments on blood fi lm if severe haemolysis. \200 i reticulocytes. \200 i unc onjugated bilirubin, LDH, and urinary urobilinogen. \200 d haptoglobins. \200 DAT \210 \222ve. 90 CHAPTER 2 Red cell disorders Diagnosis Demonstrate enzyme defi ciency. In suspected RBC enzymopathy, assay G6PD and PK f i rst, then look for unstable Hb. Diagnosis is diffi cult during haemolytic episode since r eticulocytes have iilevels of enzyme and may get erroneously normal result; wait until stea dy state (76 weeks after episode of haemolysis). Family studies are helpful (see Fig 2.12). Management \200 Avoid oxidant drugs \200\224see BNF1. \200 Transfuse in severe haemolys is or symptomatic anemia. \200 IV fl uids to maintain good urine output. \200 exchan ge transfusion in infants. \200 Splenectomy may be of value in severe recurrent haemolysi s. \200 Folic acid supplements (?proven value). \200 Avoid Fe unless defi nite Fe defi ciency. GLUCOSE ATP Glucose 6-phosphate dehydrogenase ADP Glucose 6-phosphogluconate Glucose 6-phosphate NADPH NADP Hexose monophosphate shunt Fructose 6-phosphate ATP ADP GSSG G-SH Fructose 1,6-diphosphate Ribulose 5-phosphate Glyceraldehyde 3-phosphate NAD GLYCOLYSIS NADH 1,3-diphosphoglycerate Mutase ADP 2,3-diphosphoglycerate ATP 3-phosphoglycerate Phosphatase Luebering-Rappaport Shunt 2-phosphoglycerate Phosphoenolpyruvate ADP Pyruvate kinase ATP Pyruvate Fig. 2.12 Glycolytic pathway showing key enzymes in red. Reference 1. British National Formulary. M www.bnf.org Further reading Mason, P.J. et al. (2007). G6PD defi ciency: the genotype-phenotype associa tion. Blood Rev 21, 267 \200\22383. 91 PYRUVATE KINASE DEFICIENCY Pyruvate kinase de \201 ciency Congenital non-spherocytic haemolytic anemia, caused by def i ciency of PK enzyme (involved in glycolytic pathway), leading to unstable enzyme with red uction in ATP generation in RBCs. O2 curve is shifted to the right due to i 2,3-DPG product ion. Epidemiology Autosomal recessive. Aff ected persons are homozygous or double het- erozygote s. Clinical features Highly variable, with chronic haemolytic syndrome. May be apparent in neo nate (if severe) or may present in later life. Diagnosis \200 Variable anemia. \200 Reticulocytes ii. \200 DAT \210\222ve. \200 L DH i. \200 Serum haptoglobin d. \200 Defi nitive diagnosis requires assay of PK level. Complications Aplastic crisis may be seen in viral infection (e.g. parvovirus B19). Treatment Dependent on severity. General supportive measures include daily folic acid (5mg/ d). Transfusion may be required. Splenectomy may be of value if high transfusion requiremen ts. In aplastic crisis (e.g. viral infec- tion) support measures should be used. Further reading Prchal, J.T. et al. (2005). Red cell enzymes. Hematology Am Soc Hematol Edu c Program, 19 \200\22323. Zanella, A. and Bianchi, P. (2000). Red cell pyruvate kinase defi ciency: from genetics to clinical manifestations. Baillieres Best Pract Res Clin Haematol, 13, 57 \200\22381. 92 CHAPTER 2 Red cell disorders Other red cell enzymopathies Glycolytic pathway \200 Hexokinase defi ciency. \200 Glucose phosphate isomerase defi c iency. \200 Phosphofructokinase (PFK) defi ciency. \200 Aldolase defi ciency. \200 Tr iosephosphate isomerase (TPI) defi ciency. \200 Phosphoglycerate kinase (PGK) defi ciency . Epidemiology Incidence <1 in 106. Inheritance is autosomal recessive (most double heterozyg ote) except for phosphoglycerate kinase defi ciency (X-linked recessive). Clinical features Similar to PK defi ciency although most are more severely aff ected for t he degree of anemia (glycolytic block results in d 2,3-DPG and left shift of O2 dissociatio n curve). PFK defi ciency is associated with myo- pathy. TPI and PGK defi ciencies are asso ciated with progressive neuro- logical deterioration. Diagnosis \200 E See Pyruvate kinase defi ciency, p.91. \200 Non-specifi c morphology w ith anisocytosis, macrocytosis, and polychromasia. Defi nitive diagnosis requires assay of defi cient enzyme (lreference lab). Complications \200 E see Pyruvate kinase defi ciency, p.91. Treatment Folic acid (5mg/d). Transfusion may be required (beware Fe overload if high trans fusion requirement). Role of splenectomy controversial. Natural history Similar to PK except TPI and PGK \200\223TPI present in childhood and cause progressive paraparesis, most die <5 years old due to cardiac arrhyth- mias. PGK can cause exertional rhabdomyolysis and consequential renal failure. Those aff ected show progressiv e neurological deterioration. Nucleotide metabolism \200\224pyrimidine 5 \200 nucleotidase de \201 ciency Epidemiology Autosomal recessive. Note: lead poisoning causes acquired pyr imidine 5 \200 nucleotidase defi ciency. 93 OTHER RED CELL ENZYMOPATHIES Clinical features \200 Moderate anemia (Hb 710g/dL). \200 i reticulocytes. \200 i bil irubin. \200 Splenomegaly. Diagnosis \200 RBCs show prominent basophilic stippling. \200 Pyrimidine 5 \200 nucleo tidase assay. Treatment Symptomatic, splenectomy is of limited value. 94 CHAPTER 2 Red cell disorders Drug-induced haemolytic anemia Large number of drugs shown to cause haemolysis of RBCs. Mec hanisms variable. May be immune or non-immune. \200 Some drugs interfere with lipid compo nent of RBC membrane. \200 Oxidation and denaturation of Hb: seen with, e.g. sulfonamides , especially in G6PD-defi cient subjects, but may occur in normal subjects if drugs given i n large doses, e.g: \200 Dapsone. \200 Sulfasalazine. Hapten mechanism describes the interaction between certain drugs and the RBC membrane components generating antigens that stimulate antibody production. DAT +ve. \200 Penicillins. \200 Cephalosporins. \200 Tetracyclines. \200 Tolbutamide. Autoantibody mediated haemolysis is associated with warm antibody mediated AIHA. DAT +ve. \200 Cephalosporins. \200 Mefenamic acid. \200 Methyldopa. \200 Procainamide. \200 Ibuprofen. \200 Diclofenac. \200 Interferon alfa. Innocent bystander mechanism occurs when drugs form immune complexes with antibody (IgM commonest) which then attach to RBC membrane. Complement fi xa tion and RBC destruction occurs. \200 Quinine. \200 Quinidine. \200 Rifampicin. \200 Antihistamines. \200 Chlorpromazine. \200 Melphalan. \200 Tetracycline. \200 Prob enecid. \200 Cefotaxime. Laboratory features As for autoimmune haemolytic anemia, d Hb, i reticulocytes, etc. Diff e rential diagnosis \200 Warm/cold autoimmune haemolytic anemia. \200 Congenital haemolyt ic disorders, e.g. HS, G6PD defi ciency, etc. Treatment \200 Discontinue off ending drug. \200 Choose alternative if necessary. \200 If DAT +ve with methyldopa no need to stop unless haemolysis. \200 Corticosteroids generally unnecessary and of doubtful value. \200 Transfuse in severe or symptomatic cases only. \200 Outlook good with complete reco very usual. 95 METHAEMOGLOBINAEMIA Methaemoglobinaemia The normal O2 dissociation curve requires Fe to be in the ferrous form (i.e. reduced, Fe2+). Hb containing the ferric (oxidized, Fe3+) form is termed methaemoglob in (MetHb). MetHb is unable to bind O2 leading to poor tissue oxygenation. In health metHb should be \211 3% total Hb. May be congenital or acquired. (See Table 2.12.) Table 2.12 Methaemoglobinaemia Methaemoglobinaemia Congenital HbM or globin mutation in vicinity of Fe. Fe becomes stabilized in Fe3+ form. Heterozygote has 25% HbM MetHb reductase def. Due to defi ciency of NADH-cytochrome b5 reductase. Autosomal recessive inheritance; sympto ms mainly in homozygote Cyanosis from infancy. PaO2 is normal. General health is good Clinical features Occurs when RBCs are exposed to oxidizing agents, producing HbM. Implicated agents include: phenacetin, local anesthetics (e.g. lignocaine), inorganic nitrates (NO2). Patients may ex perience severe tissue hypoxia. HbM binds O2 tightly and fails to release to tissues Acquired 22 HbM = 50% requires urgent medical attention.