Acquired \200\224non-immune \200 MAHA. \200 TTP/HUS. \200 Hypersplenism. \200 Prost hetic heart valves. \200 March haemoglobinuria. \200 Sepsis. \200 Malaria. \200 Par oxysmal nocturnal haemoglobinuria. Clinical features Symptoms of anemia, e.g. breathlessness, fatigue. Urinary changes, e.g. r ed or dark brown of haemoglobinuria. Symptoms of underlying disorder. 53 HAEMOLYTIC SYNDROMES Con \201 rm haemolysis is occurring \200 Check FBC. \200 Peripheral blood fi lm \200 \224polychromasia, spherocytosis, fragmentation (schistocytes), helmet cells, echinocytes. i reticulocytes. \200 i serum bilirubin (unconjugated). \200 i LDH. \200 Low/absent s erum haptoglobin (bind free Hb). \200 Schumm \200\231s test (for intravascular haemolysis ). \200 Urinary haemosiderin (implies chronic intravascular haemolysis, e.g. PNH). Discriminant diagnostic features Establish whether immune or non-immune \200\224check DAT. ?Immune \200\224if DAT +ve check IgG and C3 specifi c reagents \200\224suggest warm and col d antibody respectively. Screen serum for red cell alloantibodies. ?Cold antibody present \200\224examine blood fi lm for agglutination, check MCV on initial FBC sample and again after incubation at 37 C for 2h. High MCV at room temperature due to agglutinates falls to normal at 37 C. Check anti-I and anti-i titres for confi rmation. Ch eck Mycoplasma IgM and EBV serology, and for presence of Donath \200\223Landsteiner anti- b ody (cold reacting IgG antibody with anti-P specifi city). ?Warm antibody present \200\224IgG +ve DAT only suggestive \200\224examine fi lm for sphero cytes (usually prominent), lymphocytosis or abnormal lymphs to suggest LPD. Examine patient for nodes. ?Intravascular haemolysis \200\224check for urinary haemosiderin, Schumm \200\231s test. ?Sepsis \200\224check blood cultures. ?Malaria \200\224examine thick and thin blood fi lms for parasites. ?Renal/liver abnormality \200\224examine for hepatomegaly, splenomegaly, LFT s and U&E. ?Low platelets \200\224consider TTP/HUS. ?Haemoglobinopathy \200\224check Hb electrophoresi s. ?Red cell membrane abnormality \200\224check family history and perform red cell fragili ty test. ?Red cell enzyme disorder \200\224check family history and do G6PD and PK assay. Note: enzy mes may be falsely normal if reticulocytosis since increased levels are present in young RB Cs. ?PNH \200\224check immunophenotyping for CD55 + CD59 (Ham \200\231s acid lysis test now lar gely obsolete). Treatment Treat underlying disorder. Give folic acid and Fe supplements if low. Further reading Gehrs, B.C. and Friedberg, R.C. (2002). Autoimmune hemolytic anemia. Am J H ematol, 69, 258 \200\22371. 54 CHAPTER 2 Red cell disorders Genetic control of hemoglobin production Hb comprises 4 protein subunits (e.g. adult Hb = 2 \227 + 2 \227 chains, 2 2) each linked to a haem group. Production of diff eren t globin chains varies from embryo l adult to meet the particular environment at each stage . Globin genes are located on chromosomes 11 and 16. All globins related to globin are l ocated on chromosome 16; all those related to globin are on chromosome 11. The sequence in which they are pro- duced during development refl ects their physical order on chromosom es such that is the fi rst -like globin to be produced in life. After expres- sion stops, production occurs ( l switch). On chromosome 11 the arrangement of -like g lobin genes follows the order (from left l right) l l l mirroring the -like g lobin chains produced during develop- ment. As embryo develops into fetus, production st ops and is pro- duced. The globin combines with chains and produces 2 2 (fetal Hb, HbF). After birth production d and and chains are produced. Adults have predom inantly HbA ( 2 2) although small amounts of HbA2 ( 2 2) and HbF are produced (see Tabl e 2.8). Hb switching is physiological but the mechanism is unclear. HbF ( 2 2) binds O2 m ore tightly than adult hemoglobin (higher oxygen affi nity), ensuring adequate O2 delivery to the fetus which must extract its O2 from mother \200\231s circulation. After birth the l ungs expand and the O2 is derived from the air, with production replacing that of , le ading to an increase in adult hemoglobin ( 2 2). (See Figs. 2.5 and 2.6.) Table 2.8 Structure and quantity of normal haemoglobins Hemoglobin Globin chains Amount Embryo Hb Gower 1 Hb Gower 2 Hb Portland aby 5th week 2 2 2 2 2 2 42%a 24%a Fetus HbF HbA 2 2 2 2 85% 5 \200\22310% Adult HbA HbA2 HbF 2 2 2 2 2 2 97% 2.5% 0.5% Hemoglobin abnormalities Fall into 2 major groups: structural abnormalities of Hb due to al terations in DNA coding for the globin protein leading to an abnormal amino acid in the glo bin molecule, e.g. sickle hemoglobin ( S). 2nd group of Hb dis- orders results from imbala nced globin chain production \200\224globins produced 55 GENETIC CONTROL OF hemoglobin PRODUCTION are structurally normal but their relative amounts are incorrect and lead to the thalassaem ias. Haemoglobinopathies result in signifi cant morbidity and mortality on a worldwide scal e. Patients with these disorders are also seen in Northern Europe and the UK, especially in areas with signifi cant Greek, Italian, Afro-Caribbean and Asian populations. -like genes on chromosome 16 \210 \210 2 \210 1 2 1 -like genes on chromosome 11 G A \210 Fig. 2.5 Arrangement of -like and -like globin genes ( \210 indicates pseudogene). globin switching \210 \210 2 \210 1 2 1 globin switching G A \210 Fig. 2.6 Globin gene switching during development. Further reading Rund, D., et al. (2001). Pathophysiology of alpha- and beta-thalassemia: th erapeutic implications. Semin Hematol, 38, 343 \200\2239. 56 CHAPTER 2 Red cell disorders Sickling disorders Sickle cell anemia refers to homozygous SS, S S, but compound HbSC ( S C), HbS/ + or thalassaemia, and HbSD ( S D) are all sickling disorders. Clinica l symptoms can be quite variable between individuals but HbSS is generally the most severe. The gene has remained at high frequency due to conferred resistance to malaria in heterozy gotes. Inheritance is autosomal recessive. Sickle cell anemia (SCA, HbSS) Pathogenesis Widespread throughout Africa, Middle East, part s of India and Mediter- ranean. Single base change in globin gene, amino acid 6 (glu l v al). Heterozygosity rates: West Africa 10 \200\22330%, African Americans and UK AfroCaribbe ans 10%). Patients with SCA are the off spring of parents both of whom are carriers of the S gene, i.e. they both have sickle cell trait, and homozygotes for the abnormal S gene demonstrate features of chronic red cell haemolysis and tissue infarction. RBCs containing HbS deform (elongate) under conditions of reduced oxygenation, and form characteristic sick le cells \200\224do not fl ow well through small vessels, and are more adherent than normal to vascular endothelium, leading to vascular occlusion and sickle cell crises. The vaso-oc clusive process is complex and, in addition to the polymerization of globin, there is inter action of sickle cells with other types of cell and proteins. Sickle cells are more adheren t to vascular endothelium than normal red cells. During vascular crises there is endothelia l damage with exposure of molecules such as throm- bospondin, laminin, and fi bronectin. Th ere is activation of blood coagula- tion with enhanced thrombin generation and hyperreactiv ity of platelets. Chronic haemolysis depletes the body of nitric oxide (NO) which is needed for vasoregulation resulting in chronic complications such as pul- monary hypertension and leg ulceration. Clinical features \200 Highly variable. Many have few symptoms whilst others have severe and frequent crises, marked haemolytic anemia, and chronic organ damage. HbF level plays ro le in ameliorating symptoms (iHbF l fewer and milder crises). Likely impact of inherited an d environmental factors. Spectrum of haemolytic and vasoocclusive phenotype. Newborns \200\224have higher HbF level than normal adult, protected during fi rst 8 \200\22320 weeks of life. Symptoms start when HbF level falls. \200 Infection \200\224high morbidity and mortality due to bacterial and viral infection due to functional hyposplenism. Pneumococcal septicaemia (Streptococcus pneumonia e) well recognized. Other infecting organisms: meningococcus (Neisseria meningitidis), Esch erichia coli, and Haemophilus infl uenzae (hyposplenic). i malaria risk with higher complic ation rate (prophylaxis encouraged) Steady state anemia \200\224children and adults often severely anemic (Hb 76.0 \200\2239.0 g/dL). The steady state Hb of each patient should be noted at the annu al review. Anemia is chronic (haemolytic) and patients generally well-adapted until episode of decompensation (e.g. severe infection) occurs. Causes of actue-on-chronic anemia includ e 57 SICKLING DISORDERS i haemolysis (usually 1 \200\2232g/dL) due to infection including malaria, vaso-occlusive c risis, transfusion reaction. G6PD defi ciency can contribute to i haemolysis. Sequestration syndromes including acute chest syndrome, splenic and hepatic sequestration and acute apla sia usually due to parvovirus B19 infection. Apart from aplastic crisis all others should b e accompanied by i reticulocyte count. In some cases transfusion therapy can be life saving but decision to transfuse should be made following discussion with an experienced clinicia n. Acute and chronic sickle complications \200 Vaso-occlusive crisis: presents with severe b one, joint, and muscle pain. Bone pain aff ects long bones and spine, and is due to occlusi on of small vessels. Triggers: infection, dehydration, alcohol, menstruation, cold, and tem perature changes \200\224often identifi able precipitant found. Dactylitis is a subform mai nly children age <6 years. Metacarpals, metatarsals, backs of hands and feet become swollen and tender (small vessel occlusion and infarction). Recurrent, can result in permanent rad iological abnormalities in bones of the hands and feet (rare) with digit shortening. Sepsis: fever without source, commence broad-spectrum antibiotics without delay and organize septic screen. Pulmonary complications: acute chest syndrome \200\224common cause of death, seen in 40% of patients, children > adults (more severe in adults). Chest wall pain, sometimes with pleur isy, fever, and SOB. Resembles infection, infarction, or embolism. May follow simple pain c risis and common postoperatively. Early recognition important. Requires prompt and vigorous treatment. Transfer to ITU if pO2 cannot be kept >70mmHg on air. Consider CPAP. Treat infe ction vigorously, cover for S. pneumoniae, H. infl uenzae, Mycoplasma, Legionella. Exchange transfusion can be life saving. Pulmonary hypertension recognized chronic complication. Sc reening ECHO recommended. TRV max. jet velocity >2.5m/s associated with i risk of death. Ch ronic lung disease also seen, restrictive defect on PFT. Sequestration crises \200\224mainly children (30%). Pooling of large volumes of blood in spleen and/or liver. Severe hypotension and profound anemia may result in death. Splenic sequestration is seen predominantly in children <6 years of age; often oc curs following a viral infection. Abdominal pain and hepatobilary complications: gallstones common due to chronic haemolysis. RUQ pain may represent biliary colic, cholangitis, sickle chronic hepatopathy. Laparoscopi c cholecystectomy may help prevent recurrent acute biliary complications. Neurological complications: silent infarcts common due to small vessel vasculopathy. i risk of ischaemic stroke (young children > adults but increases in older adults). Haemorrhagic stroke more common in young adults. Signifi cant cause of death. Cognitive defi cits recogn ized in children and adults. TCD screening recommended for children from age 2 with transfu sion therapy for 1 stroke prevention. Acute stroke should be managed with exchange transf usion. 58 CHAPTER 2 Red cell disorders Eye complications: proliferative retinopathy (in 30%; more common in SC disease, aff ecting 750% adults), blindness (esp. HbSC), retinal artery occlusion, retinal detachment. Refer t o ophthalmology if sudden-onset visual impairment, eye trauma. Renal and genitourinary: haematuria common, likely due to medullary sickling. Frank haematu ria \200\224acute papillary necrosis. Renal reserve is impaired. Risk of acute kidney injur y. Risk factors for chronic sickle nephropathy: hypertension, proteinuria, severe anemia, h aematuria. Avoid NSAIDs, start ACE inhibitor, good BP control. Postrenal transplant exchang e transfusion recommended. Hyposthenuria (inability to concentrate urine) common cause of n octurnal enuresis. Priapism common (40%) men. Form of compartment syndrome so fulminant (>4 h) priapism is medical emergency. Give oral alpha agonist, urgent penile aspiration, washou t, and intracorporal alpha agonist injection (urology). Other problems \200 Growth retardation: common in children, but adult may have normal hei ght (weight tends to be lower than normal). Sexual maturation delayed. Acute fat embolism syndrome: bone marrow infarction and necrosis with embolization of fat d roplets. Triggers acute systemic illness. Progressive anemia, thrombocytopenia, DIC, reticu locytopenia. Can lead to multiorgan failure. Locomotor: avascular necrosis of the head of the femur or humerus, arthritis, and osteomyelitis (Salmonella infection). Chronic leg ulceration is a complication of many haemoglobinopathies including sickle cell anemia. Ischaemia is main cause. Rare in SC disease. CVS: murmurs (anemia), tachycardia. \200 Psychosocial: depression, socially withdrawn. Anesthesia and surgery \200 Data from the TAPS trial demonstrated a benefi t from pre- operative transfusion in patients with HbSS/HbS/ thalassaemia having low/medium-risk proc edures (e.g. adenotonsillectomies, lap chlocystectomies). All patients should have IV hydra tion the evening prior to procedure under GA. Attention should be paid to oxygenation, hydr ation, and temperature control pre/peri- and postoperatively. Early mobilization, incentive spirometry, and VTE prophylaxis should be considered. Obstetric care \200 Pregnant patients should be managed by team of experienced hematologists/obstetricians. Continue penicillin/folic acid. Add aspirin from 12 weeks \200\231 gestation. Stop NSAIDs. Prophylactic LMWH should be considered during antenatal admissions/post delivery. No eviden ce for routine transfusions. 59 SICKLING DISORDERS Laboratory features Anemia usual (Hb 76.0 \200\2239.0g/dL in HbSS although may be much lowe r; HbSC have higher Hb). Reticulocytes may be i (to 710 \200\22320%) refl ecting intense BM production of RBCs. Anemic symptoms usually mild since HbS has reduced O2 affi nity with O 2 dissociation curve shifted to the right. MCV and MCH are normal, unless also thalassaemia trait (25% cases). Blood fi lm shows marked variation in red cell size with prominent sick le cells and target cells; basophilic stippling, Howell \200\223Jolly bodies, and Pappenhei mer bodies (hyposplenic features after infancy) (see Fig 2.7). Sickle solubility test (e.g. sodium dithionate) will be positive. Does not discriminate between sickle cell trait and h omozygous disease. Serum bilirubin often i (due to excess red cell breakdown). Fig. 2.7 Blood fi lm in homozygous sickle cell disease. Note the elongated (sickled) red ce lls. Con \201 rmatory tests Hb electrophoresis or HPLC shows 80 \200\22399% HbS with no normal HbA. HbF may be elevated to about 15%. Parents will have features of sickle cell trait. Laboratory screening tests Acceptable tests include high-performance liquid chromatography (HPLC), generally used as 1st-line screening as less labor intensive and cost eff ective. A lso quantitates HbS %. Isoelectric focusing (IEF), capil- lary electrophoresis (CE), and ce llulose acetate electrophoresis (CAE) 60 CHAPTER 2 Red cell disorders at alkaline pH can be used for screening or confi rmation. Sickle solubility can be used as confi rmation. Also examine the blood fi lm. Antenatal screening Pregnant women should be off ered screening as part of their antenatal care in early pregnancy. The family origins questionnaire should be used for low-prevalence regions. If both parents of fetus are carriers off er prenatal/neonatal diagnosis. Prenatal diagnosis May be carried out from 1st trimester (chorionic villus sampling from 10 weeks \200\231 gestation) or 2nd trimester (fetal blood sampling from umbilical cord or tr ophoblast DNA from amniotic fl uid). DNA may be analyzed using restriction enzyme digestion with Mst II and Southern blotting, RFLP analysis assessing both parental and fetal DNA hap lotypes, oligonucleotide probes specifi c for sickle globin point mutation, or PCR amplifi cation fol- lowed by restriction enzyme digestion of amplifi ed DNA. ARMS (ampli- fi cation refractory mutation system) PCR is useful in ambiguous cases. In late pregnancy fetal bloo d sampling may be used to confi rm diagnosis. Newborn screening program Detects infants in the neonatal period to allow early interventio n with penicillin prophylaxis, initiation of parental education and awareness. Dry blood sp ots used with HPLC or IEF. As well as the routine childhood vac- cinations additional pneum ococcal vaccination (prevenar + pneumovax) recommended and timely hepatitis B and annual in fl uenza vaccination. Management \200\224general \200 Health maintenance: avoid dehydration, extremes of temper ature, over exertion. Compliance with penicillin prophylaxis (current UK guidelines recomme nd lifelong), folate replacement. Pneumococcal vaccination, screening for complications \200\224transcranial Doppler (TCD) age 2 \200\22316, urinalysis for proteinuria, ECHO for p ulmonary hypertension (TRV jet velocity >2.5m/s). Education around analgesic use, life styl e adaptation. At least annual review in specialist clinic. \200 Acute and chronic pain ma nagement: most patients with sickle cell disease suff er painful crises. Chronic pain increases with age. May be clear precipitant o f acute pain \200\224infection, temperature changes (hot or cold), or stress. Chronic pain requires multidisciplinary team input (pain specialists, psychology). Fluid replacement \200\224good daily hydration. During acute crisis IV maybe required. Hyperhydration should be avoided. Management of pain crisis 3 E see Hematological emergencies: sickle pain crisis p. 666. Management of painful crises \200 Regular parenteral opiates are useful initially but sho uld be tapered and replaced with oral analgesia. Red cell transfusion is generally not required although is indicated in sudden anemia in ch ildren (e.g. splenic sequestration), parvovirus B19 infection (with associated transient re d cell aplasia), or acute chest syndrome (hypoxia). 61 SICKLING DISORDERS Further reading Bonaventura, C. et al. (2002). Heme redox properties of S-nitrosated hemogl obin A0 and hemoglobin S: implications for interactions of nitric oxide with normal and sic kle red blood cells. J Biol Chem, 277, 14557 \200\22363. Howard, J. et al. (2013). The Tran sfusion Alternatives Preoperatively in Sickle Cell Disease (TAPS) study: a randomized, cont rolled, multicentre clinical trial. Lancet, 381, 930 \200\2238. Lottenberg, R. et al. (2005 ). An evidence-based approach to the treatment of adults with sickle cell disease. Hematolo gy Am Soc Hematol Educ Program, 2005, 58 \200\22365. Rees, D.C. et al. (2003). Guidelines f or the management of the acute painful crisis in sickle cell disease. Br J Haematol, 120, 7 44 \200\22352. Royal College of Obstetricians and Gynecologists (2011). Management of Sickl e Cell Disease in Pregnancy. London: RCOG. Steinberg, M.H. (1999). Management of sickle cel l disease. N Engl J Med, 340, 1021 \200\22330. 62 CHAPTER 2 Red cell disorders HbS \200\224sickle-modifying therapies Standard therapies Hydroxycarbamide \200\224it has been recognized for some time that i HbF levels ameliorate sickle cell disease as HbF reduces HbS polymeriza- tion and hence sickli ng. Hydroxycarbamide has been evaluated in a large number of clinical trials. Mechanism of action: initially believed to ameliorate crises through elevation of HbF levels but impact on infl am- mation/endothelial dysfunction may also be contributory. Clinical indi- cations include recurrent pain, acute chest syndrome, frequent hospital admissions. Could be consi dered for chronic organ dysfunction. Regular monitoring required. Transfusion \200\224can be life saving for acute complications (acute chest syndrome, acute ischaemic stroke, sequestration, aplastic crisis). All patients should have extended red c ell phenotype to minimize alloim- munization. Chronic transfusion program (exchange preferr ed) for 1 stroke prevention (high-risk TCD), 2 stroke prevention. Considered in some pa tients for chronic organ dysfunction, e.g. renal impairment. Erythropoietin \200\224leads t o iHbF and HbS. Not widely used in the manage- ment of haemoglobinopathies as EPO levels no rmally not low. Evidence suggests that rHuEPO provides an additive eff ect when alternated with hydroxcarbamide (especially if renal impairment). Dose required is high (1000 \200\223 3000IU/kg \227 3d/week) with co-administration of Fe supplements. Newer therapies Aimed at ameliorating or preventing clinical complications of SCD. Modulators of HbF can ac t by inhibiting DNA methylation (decitabine, azacytidine) or histone deacetylation (dimethy butyrate, vorinostat and panobinostat) that work by changing chromatin structure and enhanc e globin gene expression and also haematopoietic diff erentiation. Membrane-active drugs \200\224these help reverse cellular dehydration (sickle Hb polymeriza tion is concentration-dependent). Some drugs block cation transportation channels. Includes clotrimazole and magnesium salts. Anti-sickling agents \200\224act by increasing oxygen affi nity (Aes-103), pre- venting Hb S polymerization (PEG cardroxyhaemoglobin), or blocking adenosine signaling (adenosine 2B r eceptor antagonists). Modulators of endothelial dysfunction (selectin inhibitors), platelet aggregation (prasugre l,eptifi batide), infl ammation (regadenoson,statins), oxidant damage (glutamine). Curative interventions BM transplantation \200\224sibling donor transplants for sickle cell disease have been carried out in a number of centers. Mostly restricted to children and co nsidered for severe complications and/or requirement for trans- fusion program, e.g. cerebr ovascular disease. Recent data using RIC transplants in adults promising. 63 HBS \200\224SICKLE-MODIFYING THERAPIES Gene therapy \200\224potentially curative but experimental. Globin gene transfer has been a ttempted with variable results. Expression of exog- enous gene has been at levels too low t o be of benefi t. Further reading Brugnara, C. et al. (1993). Inhibition of Ca(2+)-dependent K+ transportatio n and cell dehydration in sickle erythrocytes by clotrimazole and other imidazole derivativ es. J Clin Invest, 92, 520 \200\2236. Charache, S. et al. (1995). Eff ect of hydroxyurea on the frequency of painful crises in sickle cell anemia. Investigators of the Multicenter St udy of Hydroxyurea in Sickle Cell Anemia. N Engl J Med, 332, 1317 \200\22322. Charache, S. et al. (1996). Hydroxyurea and sickle cell anemia. Clinical utility of a myelosuppressive \200\234switching \200\235 agent. The Multicenter Study of Hydroxyurea in Sickle Cell Anemi a. Medicine (Baltimore), 75, 300 \200\22326. Platt, O.S. et al. (1994). Mortality in sickle cell disease. Life expectancy and risk factors for early death. N Engl J Med, 330, 1639 \200\22344. 64 CHAPTER 2 Red cell disorders Sickle cell trait (HbAS) Asymptomatic carriers have one abnormal S gene and one normal gene (with 30 million carriers worldwide). Clinical features \200 Carriers are not anemic and have no abnormal clinical features. \200 Sickling rare unless O2 saturation falls <40%. Crises have been reported with severe hypoxia (anesthesia, unpressurized aircraft). Associated with i risk of VTE. \200 i exercise-associated sudden death. \200 Renal comp lications (haematuria, renal medullary carcinoma, may exacerbate other conditions that cause ESRF). Laboratory features \200 Hb, MCV, MCH, and MCHC n (unless also thalassaemia trait). \200 HbS level 40 \200\22355% (if <40% then also thalassaemia trait). \200 Movie may be n or show microcytes and target cells. \200 Sickle cell test will be +ve (HbSS and HbA S). Carrier detection Neither FBC nor fi lm can be used for diagnostic purposes. Detection of t he carrier state relies on HbS quantitation (HPLC): <50%, HbA> 50%, in contrast to HbS/ + thalassaemia (HbS >50%, HbA <50%). 3 Care needed during anesthesia (avoid hypoxia). 65 OTHER SICKLING DISORDERS Other sickling disorders HbSC Milder than SCA but resembles it. Patients have fewer and milder crises. Retinal damag e (microvascular, proliferative retinopathy) and blindness are major complications (30 \200 \22335%). Arrange regular ophthalmological review by specialist. Aseptic necrosis of femora l head and recurrent haematuria are common. i risk of splenic infarcts and abscesses. 3 Bew are thrombosis and PE especially in pregnancy. Clinical Mild anemia (Hb 8 \200\22314g/dL) and splenomegaly common. Less hae- molysis, fewe r painful crises, fewer infections, and less vaso-occlusive disease than SCA. Growth and de velopment normal. Lifespan normal. Pregnancy may be hazardous. Movie Prominent target cells with fewer NRBC than seen in SCA. Howell \200\223Jolly and Pap penheimer bodies (hyposplenism). Occasional C crystals may be seen. Diagnosis Hb electrophoresis and family studies. MCV and MCH are much lower than in HbSS. HbSD, HbSOArab Milder than HbSS. Both rare. Interactions of these globins with HbS ( 121 r esults in reduced polymerization. HbDPunjab glullys) cause little disease on their own alth ough there may be mild hae- molysis in the homozygote. These haemoglobins cause sickle cell disease when present with HbS. HbS/ \235\233\203 thalassaemia Caused by inheritance of S from one parent and thalassaemia from the other. Sickle/ thalassaemia is severe sin ce no normal globin chains are produced. Sickle/ + thalassaemia is much milder having globin in 5 \200\22315% of their Hb. Microcytosis and splenomegaly are charac- teristic. Family screening will confi rm microcytosis and i HbA2 in one of the parents. See UK Standa rds. ( 121 glulgln) and HbOArab Management Essentially as for HbSS with prompt treatment of crises (E Sickle crisis, p.666) . Further reading http://www.sicklecellsociety.org/app/webroot/fi les/fi les/CareBook.pdf 66 CHAPTER 2 Red cell disorders Other haemoglobinopathies HbC disease ( \235\233\2036 glullys) West Africa. Patients have b enign compensated haemolysis. Development is normal, splenomegaly is common. Gallstones are recognized compli- cation. The Hb may be mildly d. MCV and MCH d and reticulocytes i. Bloo d fi lm shows prominent target cells and occasional HbC crystals. Hb electrophoresis shows mainly HbC with some HbF. HbA is absent. Red cells said to be \200\230stiff \200\231. Car e with anesthesia. HbC trait ( \235\233\2036 glullys) Asymptomatic. Hb is n. Movie may be n or show presence of target cells. HbC 30 \200\22340%. HbD disease (e.g. DPunjab \235\233 \203121 glullys) Found in North West India, Pakistan, and Iran. Movie shows target cells. H bD trait (e.g. DPunjab \235\233\203121 glulgln) Of little consequence other than interacti on with HbS. Hb and MCV n. Movie normal or shows target cells. HbE disease ( \235\233\20326 glullys) South East Asia (commonest Hb variant), India, Burma, and Thailand. This Hb is mo derately unstable when exposed to oxidants. May produce thalassaemic syndrome when mRNA spl ice mutants. There is mild anemia, MCV and MCH d, reticulocytes n. Movie shows target cells , hypochromic and microcytic red cells. There are few symptoms; under- lying compensated ha emolysis, mild jaundice. Liver and spleen size are normal. Treatment is not usually require d. HbE trait ( \235\233\20326 glullys) Asymptomatic. Indices similar to thalassaemia tra it. Hb usually n. HbE/ \235\233\203 thalassaemia Compound heterozygote. Clinical picture va riable from thalassaemia minor to thalassaemia major. Most patients have moderate disease. Hb is lower than HbEE; MCV and MCH are lower than HbE trait. Reticulocytes are raised sligh tly at 4 \200\2236%. 67 OTHER HAEMOGLOBINOPATHIES 68 CHAPTER 2 Red cell disorders Unstable haemoglobins Congenital Heinz body haemolytic anemia caused by point mutations in globin genes. Hb precipitates in RBCs when there is oxidative stress l Heinz bodies. In nor mal Hb there are non-covalent bonds maintaining the Hb structure; loss of bonds leads to Hb denaturation and precipi- tation. Production of Heinz bodies leads to less deformable red cells with reduced lifespan. The degree of haemolysis varies depending on the mutation, but may be more severe if there is fever or if the patient ingests oxidant drugs (see Table 2. 9). Predominantly autosomal dominant; most patients are heterozygotes. Mainly aff ects globi n chain, e.g. Hb Hammersmith (mutation involves amino acid in contact with haem pocket); Hb Bristol (replacement of non-polar by polar amino acid with distortion of protein). Table 2.9 Examples of unstable haemoglobins Hemoglobin Mutation 98 val l met 63 his l arg 30 arg l ser 136 leu l pro 42 phe l ser 67 v al l asp 130 trp l gly Hb Koln Hb Zurich Hb Tacoma Hb Bibba Hb Hammersmith Hb Bristol Hb Poole Clinical features \200 Well compensated haemolysis. \200 Hb may be n if unstable Hb has high O2 affi nity. \200 Haemolysis exacerbated by infection and oxidant drugs. \200 Ja undice and splenomegaly are common. \200 Some Hbs are unstable in vitro but show little h aemolysis in vivo. Investigation \200 Hb n or d. \200 MCV often d. \200 Movie shows hypochromic RBCs, po lychromatic RBCs, basophilic stippling. Heinz bodies seen post-splenectomy. \200 Reticulocytes are i. \200 Demonstrate unstable Hb using, e.g. heat or isopropanol stability tests. \200 Electrophoresis may be normal. \200 Brilliant cresyl blue will stain Heinz bodies \200\224falsely +ve in neonate due to high HbF levels. Estimation of P50 may be helpful. \200 DNA analysis of value in some cases. 69 UNSTABLE HAEMOGLOBINS Management Most cases run benign course. Treatment seldom required. Gallstones common. Reco mmend regular folic acid supplementation. Splenectomy of value in some patients, e.g. sever e haemolysis and splenomegaly. Avoidance of precipitants of haemolysis advised. 70 CHAPTER 2 Red cell disorders Thalassaemias Arise as a result of diminished or absent production of one or more globin ch ains. Net result is imbalanced globin chain production. Globin chains in excess form tetram ers and precipitate within RBCs l chronic apoptosis in BM and chronic haemolysis in the per ipheral blood. Occur at high frequency in parts of Africa, the Mediterranean, Middle East, India, and Asia. Found in high frequency in areas where malaria is endemic and thalassaemia trait off ers some protection. Named after aff ected gene, e.g. in thalassaemia the globin gene is altered in such a way that either globin synthesis is reduced ( +) or ab olished ( ) from RBCs. Severity varies depending on type of muta- tion or deletion of th e or globin gene. 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