69 PNH). Discriminant diagnostic features Establish whether immune or non-immune \200\224check DAT ?Immune if DAT +ve check IgG and C3 specific reagents \200\224suggest warm and cold antibo dy respectively. Screen serum for red cell alloantibodies. ?Cold antibody present \200\224examine blood movie 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 confirmation. Ch eck Mycoplasma IgM and EBV serology, and for presence of Donath Landsteiner antibody (cold reacting IgG antibody with anti-P specificity). ?Warm antibody present \200\224IgG +ve DAT only suggestive \200\224examine movie for sphe rocytes (usually prominent), lymphocytosis or abnormal lymphs to suggest LPD. Examine patie nt 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 films for parasites. ?Renal/liver abnormality \200\224examine for hepatomegaly, splenomegaly, LFTs and U&E. ?Low platelets \200\224consider TTP/HUS. ?Haemoglobinopathy \200\224check Hb electrophoresis. ?Red cell membrane abnormality \200\224check family history and perform red cell fragility test. ?Red cell enzyme disorder \200\224check family history and do G6PD and PK assay. Note: enz ymes may be falsely normal if reticulocytosis. ?PNH \200\224check immunophenotyping for CD55 + CD59 (Ham \200\231s acid lysis test now la rgely obsolete). Treatment Treat underlying disorder. Give folic acid and iron supplements if low. Gehrs, B.C. & Friedberg, R.C. (2002) Autoimmune hemolytic anemia. Am J Hematol, 69, 258 \200\223271. 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 70 Genetic control of hemoglobin production Hb comprises 4 protein subunits (e.g. adult Hb = 2 a + 2 b chains, a2b2) each linked to a haem group. Production of different globin chains varies from embryo7adult to meet the particular environment at each stage. Globin genes are located on chromosomes 11 and 16. A ll globins related to a globin are located on chromosome 16; all those related to b globin are on chromosome 11. The sequence in which they are produced during development reflects t heir physical order on chromosomes such that z is the first a-like globin to be produced in life. After z expression stops, a production occurs (z7a switch). On chromosome 11 the arr angement of b-like globin genes follows the order (from left7right) e7g7d7b mirroring the b -like globin chains produced during devel- opment. As embryo develops into fetus, z product ion stops and a is pro- duced. The a globin combines with g chains and produces a2g2 (fetal Hb, HbF). After birth g production 5 and d and b chains are produced. Adults have predomin antly HbA (a2b2) although small amounts of HbA2 (a2d2) and HbF are produced. 70 Hb switching is physiological but the mechanism is unclear. HbF (a2g2) binds O2 more tightl y than adult hemoglobin, ensuring adequate O2 delivery to the fetus which must extract its O2 from mother \200\231s circulation. After birth the lungs expand and the O2 is derived fr om the air, with b production replacing that of g, leading to an increase in adult hemoglob in (a2b2). Hemoglobin Globin chains Amount Embryo Hb Gower 1 Hb Gower 2 Hb Portland *by 5th week z2e2 a2e2 z2g2 42%* 24%* Fetus HbF HbA a2g2 a2b2 85% 5 \200\22310% Adult HbA HbA2 HbF a2b2 a2d2 a2g2 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 (bS). Second group of Hb dis- orders results from imba lanced globin chain production \200\224globins pro- 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 71 Red cell disorders duced are structurally normal but their relative amounts are incorrect and lead to the thal assaemias. 71 Haemoglobinopathies result in significant morbidity and mortality on a world-wide scale. Pa tients with these disorders are also seen in Northern Europe and the UK, especially in area s with significant Greek, Italian, Afro- Caribbean and Asian populations. a-like genes on chromosome 16 z yz ya2 (cid:2)(cid:3)1 a2 a1 (cid:4)-like genes on chromosome 11 e Gg Ag yb d b Arrangement of a-like and b-like globin genes (y indicates pseudogene) a globin switching z yz ya2 ya1 a2 a1 b globin switching e Gg Ag yb d b Globin gene switching during development 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 72 Sickling disorders Sickle cell anemia (homozygous SS, bSbS), HbSC (bSbC), HbS/b+ or b thalassaemia, and HbSD (bSbD) all produce significant symptoms but homozygous sickle cell anemia is generally the most severe. The gene has remained at high frequency due to conferred resistance to malari a in het- erozygotes. Inheritance is autosomal recessive. 72 Sickle cell anemia (SCA, HbSS) Pathogenesis Widespread throughout Africa, Middle East, part s of India and Mediterranean. Single base change in b globin gene, amino acid 6 (glu7val). In UK Afro-Caribbean population gene is found in 1:10. RBCs containing HbS deform (elongat e) under conditions of reduced oxy- genation, and form characteristic sickle cells \200 \224do not flow well through small vessels, and are more adherent than normal to vascular e ndothe- lium, leading to vascular occlusion and sickle cell crises. Patients with SCA are t he offspring of parents both of whom are carriers of the bS gene, i.e. they both have sickl e cell trait, and homozygotes for the abnormal bS gene demonstrate features of chronic red cell haemolysis and tissue infarction. Clinical features Highly variable. Many have few symptoms whilst others have severe and fre quent crises, marked haemolytic anemia and chronic organ damage. HbF level plays role in am eliorating symptoms (4 HbF7fewer and milder crises). History may reveal a +ve family histor y or past history of crises. 2 Infancy \200\224newborns have higher HbF level than normal adult, pro- tected during first 8 \200\22320 weeks of life. Symptoms start when HbF level falls. SCA of ten diagnosed <1 year. 2 Infection \200\224high morbidity and mortality due to bacterial and viral infection. Pneumococcal septicaemia (Streptococcus pneumoniae) well recognized. Other infec ting organisms: meningococcus (Neisseria menin- gitidis), Escherichia coli and Haemophilus influenzae (hyposplenic). 2 Anemia \200\224children and adults often severely anemic (Hb 6.0 \200\2239.0 g/dL). Ane mia is chronic and patients generally well-adapted until episode of decompensation (e.g. se vere infection) occurs. Sickle crises 2 Vaso-occlusive \200\224dactylitis, chest syndrome and girdle syndrome. Patients complain of severe bone, joint and abdominal pain. Bone pain affects long bones an d spine, and is due to occlusion of small vessels. Triggers: infection, dehydration, alcoho l, menstruation, cold and tem- perature changes \200\223 often no cause found. 2 Dactylitis \200\224mainly children. Metacarpals, metatarsals, backs of hands and feet sw ollen and tender (small vessel occlusion and infarction). Recurrent, can result in permanen t radiological abnormalities in bones of the hands and feet (rare). 2 Acute chest syndrome \200\224common cause of death. Chest wall pain, sometimes with pleu risy, fever and SOB. Resembles infection, infarc- tion or embolism. Requires prompt and vig orous treatment. Transfer to ITU if pO2 cannot be kept >70 mmHg on air. 10% mortality. Trea t infection vigorously, often due to S pneumoniae, H influenzae, Mycoplasma, Legionella. 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 73 Red cell disorders 2 Aplastic crises \200\224sudden 5 in marrow production (esp. red cells). Parvovirus B19 infection is cause (invades developing RBCs). Mostly self-limiting and after 1 \200\2232 weeks the marrow begins to function nor- mally. Top-up transfusion may be need ed. 73 2 Haemolytic crises \200\224uncommon; markedly reduced red cell lifespan. May be drug-induced, 2 to infection (e.g. malaria) or associated G6PD deficiency. 2 Sequestration crises \200\224mainly children (30%). Pooling of large volumes of blood in spleen and/or liver. Severe hypotension and pro- found anemia may resul t in death. 2 Other problems \200\223 Growth retardation: common in children, but adult may have normal height (weight tends to be lower than normal). Sexual mat- uration delayed. \200\223 Locomotor: Avascular necrosis of the head of the femur or humerus, arthritis and osteomyelitis (Salmonella infection). Chronic leg ulceration is comp lication of many haemoglobinopathies including sickle cell anemia. Ischaemia is main cause. \200\223 Genitourinary: Renal papillary necrosis7haematuria and renal tubular defects. Ina bility to concentrate urine. Priapism in 60% males. Less common if HbF4. Frequent UTIs in women, CRF in adults. \200\223 Spleen: Severe pain (infarction of splenic vessels). Spleen may enlarge in early life but after repeated infarcts diminishes in size (7hyposplenism by 9 \200\22312 months of age). Splenic function is impaired. \200\223 Gastrointestinal: Gallstones common (2 to chronic haemolysis). Derangement of LFTs (multifactorial). \200\223 CVS: Murmurs (anemia), tachycardia. \200 \223 Eye: Proliferative retinopathy (in 30%), blindness (esp. HbSC), retinal artery occlusion, retinal detachment. \200\223 CNS: Convulsions, TIAs or strokes, sensory hearing loss (usually temporary). \200\223 Psychosocial: Depression, socially withdrawn. Laboratory features Anemia usual (Hb 6.0 \200\2239.0 g/dL in HbSS although may be much low er; HbSC have higher Hb). Reticulocytes may be 4 (to 10 \200\22320%) reflecting intense bo ne marrow production of RBCs. Anemic symptoms usually mild since HbS has reduced O2 affinit y. MCV and MCH are normal, unless also thalassaemia trait (25% cases). Blood movie shows ma rked variation in red cell size with prominent sickle cells and target cells; basophilic st ip- pling, Howell \200\223Jolly bodies and P appenheimer bodies (hyposplenic fea- tures aft er infancy). Sickle cell test (e.g. sodium dithionate) will be positive. Does not discrimin ate between sickle cell trait and homozygous disease. Serum bilirubin often 4 (due to exces s red cell breakdown). 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 74 74 Blood movie in homozygous sickle cell disease. Note the elongated (sickled) red cells. Confirmatory tests Hemoglobin electrophoresis shows 80 \200\22399% HbS with no normal HbA. HbF may be elevated to about 15%. Parents will have features of sickle cell trait. Screening In at-risk groups pregnant woman should be screened early in pregnancy. If both p arents of fetus are carriers offer prenatal/neonatal diagnosis. Affected babies should be g iven penicillin daily and be immunized against S. pneumoniae, H influenzae type b, and Neis seria meningitidis. Prenatal diagnosis May be carried out from first trimester (chorionic villus sampling from 10 weeks gestation) or second trimester (fetal blood sampling from umbilical cord or tropho blast DNA from amniotic fluid). DNA may be analyzed using restriction enzyme digestion with Mst II and Southern blotting, RFLP analysis assessing both parental and fetal DNA haplotyp es, oligonucleotide probes specific for sickle globin point mutation, or PCR amplification fol- lowed by restriction enzyme digestion of amplified DNA. ARMS (amplifi- cation refracto ry mutation system) PCR is useful in ambiguous cases. In late pregnancy fetal blood samplin g may be used to confirm diagnosis. Management General Lifelong prophylactic penicillin 250mg bd PO with folate replacement. Pneumovax II vaccination advisable. Management during pregnancy and anesthesia Anesthesia should be carried out by experienced anesthetist who is aware of complications of SCA. If the patient is unwell consider transfu - sion to Hb of 10g/dL, but generally transfusion not necessary. Management of crises (cid:2)(cid:2) (cid:9) Hematological Emergencies: Sickle Crisis p532. (cid:27) www.bcshguidelines.com/pdf/SICKLE.V4_0802.pdf 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 75 Red cell disorders 75 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 76 HbS \200\224new therapies Agents that elevate HbF levels It has been recognized for some time that 4 HbF levels ameli orate b tha- lassaemia and sickle cell disease. HbF reduces HbS polymerisation and hence si ckling. HbF level of >10% reduces episodes of aseptic necrosis; levels >20% HbF are associa ted with fewer painful crises. 76 Hydroxyurea \200\224several studies have shown that baboons treated with cytosine arabinos ide showed 4 HbF. Similar results obtained with hydroxyurea which has advantages over other cytotoxics e.g. low risk of secondary malignancy with prolonged use. Hydroxyurea has been evaluated in a large number of clinical trials. Effects are dose-dependent and the highest elevation of HbF is seen at myelosuppressive doses. Erythropoietin \200\224leads to 4 HbF but not widely used in the management of haemoglobinopathies. Evidence suggests that rHuEPO provides an additive effect when alternated with hydroxyurea. Dose required is high (1000 \200\2233000iu/kg 3d/week) with co-administration of Fe supplements. 5-azacytidine \200\224inhibitor of methyltransferase, enzyme responsible for methylation o f newly incorporated cytosines in DNA. Preventing methylation of the g globin gene leads to 4 HbF. (cid:2)(cid:2) Risk of developing 2 malignancy. Short chain fatty acids \200\224butyrate analogs are potent inducers of haematopoietic dif ferentiation. Elevated concentrations of butyrate and other fatty acids in diabetic mothers is responsible for the persistently elevated HbF in the neonates born to such mothers. Ini tial studies involving the use of butyrate to increase HbF levels in patients with sickle c ell anemia appeared promising but subsequent studies have been disappointing. Bone marrow transplantation \200\224sibling donor transplants for sickle cell disease have been carried out in a number of centers. Since the mortality from sickle cell disease has dropped over recent years from 15%71%, and with the advent of hydroxyurea therapy, there is a less compelling argument for BMT in sickle cell disease. Gene therapy \200\224potentially curative but experimental. Globin gene transfer has been attempted with variable results. Expression of exogenous gene has been at levels too low to be of benefit. Charache, S. et al (1995) Effect of hydroxyurea on the frequency of painful crises in sickl e cell anemia. Investigators of the Multicenter Study of Hydroxyurea in Sickle Cell Anemia. N Engl J Med, 332, 1317 \200\2231322; Platt, O.S. et al. (1994) Mortality in sickle cell d isease. Life expectancy and risk factors for early death. N Engl J Med, 330, 1639 \200\2231 644. 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 77 Red cell disorders 77 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 78 Sickle cell trait (HbAS) Asymptomatic carriers have one abnormal bS gene and one normal b gene (with 30 million carr iers worldwide). Clinical features 2 Carriers are not anemic and have no abnormal clinical features. 2 Sickl ing rare unless O2 saturation falls <40%. Crises have been 78 reported with severe hypoxia (anesthesia, unpressurised aircraft). 2 Occasional renal papil lary necrosis, haematuria and inability to concen- trate the urine in adults. Laboratory features 2 Hb, MCV, MCH and MCHC normal (unless also a thalassaemia trait). 2 Hb S level 40 \200\22355% (if <40% then also a thalassaemia trait). 2 Movie may be normal or s how microcytes and target cells. 2 Sickle cell test will be +ve (HbSS and HbAS). Carrier detection Neither FBC nor movie can be used for diagnostic purposes. Detection of t he carrier state relies on hemoglobin electrophoresis (HbA 50%; HbS 50%). (cid:2) Care needed during anesthesia (avoid hypoxia). 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 79 Red cell disorders 79 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 80 Other sickling disorders HbSC Milder than sickle cell anemia but resembles it. Patients have fewer and milder crises . Retinal damage (microvascular, proliferative retinopathy) and blindness are major complic ations (30 \200\22335%). Arrange regular oph- thalmological review by specialist. Aseptic n ecrosis of femoral head and recurrent haematuria are common. Increased risk of splenic infa rcts and abscesses. (cid:2) Beware thrombosis and PE especially in pregnancy. 80 Clinical Mild anemia (Hb 8 \200\22314g/dL) and splenomegaly common. Less haemolysis, fewer painful crises, fewer infections and less vaso-occlusive disease than SCA. Growth and devel opment normal. Lifespan normal. (cid:2) 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 (b121 re sults in reduced polymerisation. HbDPunjab glu7lys) cause little disease on their own altho ugh there may be mild haemolysis in the homozygote. These haemoglobins cause sickle cell di sease when present with HbS. (b121 glu7gln) and HbOArab HbSaa thalassaemia Common in Black individuals. Lessens severity of SCA by reducing the con centration of Hb in red cells. HbSbb thalassaemia Caused by inheritance of bS from one parent and b thalassaemia from the other. Sickle/b thalassaemia is severe since no normal b globin chains are produced. Sick le/b+ thalassaemia is much milder having b globin in 5 \200\22315% of their Hb. Microcytosi s and splenomegaly are characteristic. Family screening will confirm microcytosis and 4 HbA 2 in one of the parents. Management Essentially as for HbSS with prompt treatment of crises (see above). 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 81 Red cell disorders 81 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 82 Other haemoglobinopathies HbC disease (b6 glu7lys) West Africa. Patients have benign compensated haemolysis. Developm ent is normal, splenomegaly is common. Gallstones are recognized complica- tion. The Hb may be mildly 5. MCV and MCH 5 and reticulocytes 4. Blood movie shows prominent target cells a nd occasional HbC crystals. Hb elec- trophoresis shows mainly HbC with some HbF. HbA is abs ent. Red cells said to be \200\230stiff \200\231. Care with anesthesia. HbC trait (b6 glu7 lys) Asymptomatic. Hb is 6. Movie may be normal or show presence of target cells. HbC 30 \200\22340%. HbD disease (e.g. DPunjab b121 glu 7gln) Found in North West India, Pakistan a nd Iran. Movie shows target cells. HbD trait (e.g. DPunjab b121 glu7gln) Of little conseque nce other than interaction with HbS. Hb and MCV 6. Movie normal or shows target cells. HbE disease (b26 glu7lys) South East Asia (commonest Hb variant), India, Burma and Thailand. Th is Hb is moderately unstable when exposed to oxidants. May produce tha- lassaemic syndrome when mRNA splice mutants. There is mild anemia, MCV and MCH 5, reticulocytes 6. Movie shows target cells, hypochromic and microcytic red cells. There are few symptoms; underlying com pen- sated haemolysis, mild jaundice. Liver and spleen size are normal. Treatment is not us ually required. HbE trait (b26 glu7lys) Asymptomatic. Indices similar to b thalassaemia tra it. Hb usually 6. 82 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 83 Red cell disorders 83 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 84 Unstable haemoglobins Congenital Heinz body haemolytic anemia caused by point mutations in globin genes. Hb preci pitates in red blood cells7Heinz bodies. In normal Hb there are non-covalent bonds maintain ing the Hb structure; loss of bonds leads to Hb denaturation and precipitation. Production of Heinz bodies leads to less deformable red cells with reduced lifespan. 84 Predominantly autosomal dominant; most patients are heterozygotes. Mainly affects b globin chain e.g. HbHammersmith (mutation involves amino acid in contact with haem pocket); HbBris tol (replacement of non- polar by polar amino acid with distortion of protein). Clinical features 2 Well compensated haemolysis. 2 Hb may be 6 if unstable Hb has high O2 a ffinity. 2 Haemolysis exacerbated by infection and oxidant drugs. 2 Jaundice and splenomega ly are common. 2 Some Hbs are unstable in vitro but show little haemolysis in vivo. Investigation 2 Hb 6 or 5. 2 MCV often 5. 2 Movie shows hypochromic RBCs, polychromatic RBC s, basophilic stip- pling. 2 Heinz bodies seen post-splenectomy. 2 Reticulocytes are 4. 2 Demonstrate unstable Hb usin g e.g. heat or isopropanol stability tests. 2 Brilliant cresyl blue will stain Heinz bodies . 2 Estimation of P50 may be helpful. 2 DNA analysis of value in some cases. Management Most cases run benign course. Treatment seldom required. Gallstones common. Recommend regul ar folic acid supplementation. Splenectomy of value in some patients. Avoidance of precipit ants of haemolysis advised. 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 85 Red cell disorders 85 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 86 Thalassaemias Arise as a result of diminished or absent production of one or more globin chains. Net resu lt is imbalanced globin chain production. Globin chains in excess precipitate within RBCs l eading to chronic haemolysis in bone marrow and peripheral blood. Occur at high frequency i n parts of Africa, the Mediterranean, Middle East, India and Asia. Found in high frequency in areas where malaria is endemic and thalassaemia trait probably offers some protection. 86 Named after affected gene e.g. in a thalassaemia the a globin gene is altered in such a way that either a globin synthesis is reduced (a+) or abol- ished (a ) from RBCs. Severity va ries depending on type of mutation or deletion of the a or b globin gene. aa thalassaemia Two a globin genes on each chromosome 16, with total of 4 a globin genes per cell (normal p erson is designated aa/aa). Like sickle cell anemia, patients can either have mild a thalas saemia (a thalassaemia trait) where one or two a globin genes are affected or may have seve re a tha- lassaemia if three or four of the genes are affected. a thalassaemia is gen- eral ly the result of large deletions within a globin complex. Silent aa thalassaemia ( \200\223 a/aa) One gene deleted. Asymptomatic. 5 MCV and MCH in mi nority. a thalassaemia trait (aa/ \200\223 \200\223 or \200\223 a/ \200\223 a) Asymptomatic carri er \200\224recognized once other causes of microcytic anemia are excluded (e.g. iron defici ency). Hb may be 6 or minimally 5. MCV and MCH are 5. Absence of splenomegaly or other clin ical findings. Requires no therapy. Hemoglobin H disease ( \200\223 \200\223/ \200\223 a) Three a genes deleted; only one func tioning copy of the a globin gene/cell. Clinical features variable. May be moderate anemia with Hb 8.0 \200\2239.0g/dL. MCV and MCH are 5. Hepatosplenomegaly, chronic leg ulceration and jaundice (reflecting underlying haemolysis). Infection, drug treatment and pregnancy ma y worsen anemia. Blood movie shows hypochromia, target cells, NRBC and increased reticulo- cytes. Brilliant cresyl blue stain will show HbH inclusions (tetramers of b globin, b4, that have polymerise d due to lack of a chains). Hb pattern con- sists of 2 \200\22340% HbH (b4) 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. 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 87 Red cell disorders Hemoglobin Bart \200\231s hydrops fetalis ( \200\223 \200\223 / \200\223 \200\223) Common cause of stillbirth in South East Asia. All 4 a globin genes affected. g chains form tetra mers (HbBart \200\231s, g4) which bind oxygen very tightly, with resultant poor tissue oxyg enation. Fetus is either stillborn (at 34 \200\22340 weeks gestation) or dies soon after bi rth. They are pale, distended, ascites. jaundiced Hemoglobin is 6.0g/dL and the movie show s hypochromic red cells, target cells, increased reticulocytes and nucleated red cells. Hem oglobin analysis shows mainly HbBart \200\231s (g4) with a small amount of HbH (b4); HbA, A 2 and F are absent. 87 and have marked hepatosplenomegaly and 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 88 bb thalassaemia There are only 2 copies of b globin gene per cell. Abnormality in one b if both b globin ge nes are globin gene results in b thalassaemia trait; affected the patient has b thalassaemi a major or b thalassaemia intermedia. Unlike a thalassaemia, most b thalassaemias are due t o single point muta- tions. Results in reduced b globin synthesis (b+) or absent b globin p ro- duction (b ). In b thalassaemia major, patients have severe anemia requiring lifelong support with blood transfusion (with resultant iron over- load). There is ineffective eryth ropoiesis. Not obvious at birth due to presence of HbF (a2g2) but as g chain production dim inishes and b globin production increases effects of the mutation become obvious. Children fail to thrive, and development is affected. Hepatosplenomegaly (due to production and dest ruction of red cells by these organs) is typical. Children also develop facial abnormalitie s as the flat bones of the skull and other bones attempt to produce red cells to overcome t he genetic defect. Skull radiographs show \200\230hair on end \200\231 appearances reflect ing the intense marrow activity in the skull bones. 88 Investigation and management bb thalassaemia trait 2 Carrier state. 2 Hb may be 5 but is not usually <10.0g/dL. 2 MCV 5 to 63 \200\22377fL. 2 Blood movie: microcytic, hypochromic RBCs; target cells often presen t. Basophilic stippling especially in Mediterraneans. 2 RCC 4. 2 HbA2 (a2d2) 4 \200\224provides useful diagnostic test for b thalassaemia trait. 2 Occasionally confused with iron deficiency anemia, however, in thalas- saemia trait the s erum iron and ferritin are normal (or 4) whereas in IDA they are 5. Blood movie in thalassaemia trait Treatment Not usually required. Usually detected antenatally or on routine FBC pre- op. 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 89 Red cell disorders b thalassaemia intermedia 2 Denotes thalassaemia major not requiring regular blood transfus ion; more severe than b thalassaemia trait but milder than b thalassaemia major. 89 2 May arise through several mechanisms e.g. \200\223 Inheritance of mild b thalassaemia mutations (e.g. homozygous b+ tha- lassaemia a lleles, compound heterozygote for two mild b+ thalas- saemia alleles, compound heterozygote s for mild plus severe b+ thalassaemia alleles). \200\223 Elevation of HbF. \200\223 Coinheritance of a thalassaemia. \200\223 Coinherita nce of b thalassaemia trait with e.g. HbLepore. \200\223 Severe b thalassaemia trait. Clinical 2 Present with symptoms similar to b thalassaemia major but with only moderate degree of anemia. 2 Hepatosplenomegaly. 2 Iron overload is a feature. 2 Some patients are severely anemic (Hb 6g/dL) although not requiring regular blood transfusion, have impaired growth and devel- opment, skeletal defor mities and chronic leg ulceration. 2 Others have higher Hb (e.g. 10 \200\22312g/dL) with few symptoms. Management Depends on severity. May require intermittent blood transfusion, iron chelation, folic acid supplementation, prompt treatment of infection, as for b thalassaemia major. b thalassaemia major (Cooley \200\231s anemia) Patients have abnormalities of both b globin genes. Presents in childhood with anemia and recurrent bacterial infection. There is extra medullary haemopoiesis with hepatosplenomegaly and skeletal deformities. Clinical 2 Moderate/severe anemia (Hb 3.0 \200\2239.0g/dL). 2 MCV and MCHC 5. 2 Reticulocy tes 4. 2 Blood movie: marked anisopoikilocytosis, target cells and nucleated red cells. 2 Methyl violet stain shows RBC inclusions containing precipitated a globin. 2 Hb electrophoresis shows mainly HbF (a2g2). In some b thalassaemias there may be a little HbA (a2b2) if some b globin is produced. 2 HbA2 may be 6 or mildly elevated. 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 90 90 b thalassaemia major: note bizarre red cells with marked anisopoikilocytosis Management 2 Regular lifelong blood transfusion (every 2 \200\2234 weeks) to suppress inef- fective erythropoiesis and allow normal growth and development in childhood. 2 Iron overload (transfusion haemosiderosis) is major problem \200\224damages heart, endoc rine glands, pancreas and liver. Desferrioxamine reduces iron overload (by promoting iron e xcretion in the urine and stool), and is given for 8 \200\22312h per day SC for 5 days/week . Compliance may be dif- ficult, especially in younger patients. Complications of desferrio xamine include retinal damage, cataract and infection with Yersinia spp. 2 Splenectomy may be of value (e.g. if massive splenomegaly or increasing transfusion requirements) but best avoided until after the age of 5 years due to 4 risk of infection. Infective episodes should be treated promptly with intravenous antibi otics. 2 Bone marrow transplantation has been carried out using sibling donor HLA-matched transpla nts with good results in young patients with b thalassaemia major. The procedure carries a significant procedure- related morbidity and mortality, along with GvHD ((cid:9) BMT sectio n p324 \200\223326). Screening Screen mothers at first antenatal visit. If mother is thalassaemic carrier, scree n father. If both carriers for severe thalassaemia offer prenatal diag- nostic testing. Fet al blood sampling can be carried out at 18 weeks gesta- tion and globin chain synthesis ana lyzed. Chorionic villus sampling at 10+ weeks gestation provides a source of fetal DNA that can be analyzed in a variety of methods: Southern blotting, oligonucleotide probes or RFLP analysis may determine genotype of fetus. Moving towards PCR based techniques; likely to i mprove carrier detection. Weatherall, D.J. & Provan, A.B. (2000) Red cells I: inherited anaemias. Lancet, 355, 1169 \200\2231175. 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 91 Red cell disorders 91 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 92 Other thalassaemias Heterozygous db thalassaemia Produces a picture similar to b thalassaemia trait with 4 HbF (5 \200\22320%) and microcytic RBCs; HbA2 is 6 or 5. 92 Homozygous db thalassaemia Homozygous condition is uncommon. There is failure of production of both d and b globins. Milder than b thalassaemia major, i.e. b thalassaemia intermedia. Represents a form of thalassaemia intermedia. Hb 8 \200\22311g/dL. Absence of HbA and HbA2 ; only HbF is present (100%). Heterozygous b thalassaemia/db thalassaemia Similar to b thalassaemia major (but less sever e). Hb produced is mainly HbF with small amount of HbA2. gdb thalassaemia Homozygote is not viable. Heterozygous condition is associated with haemol ysis in neonatal period and thalassaemia trait in adults with 6 HbF and HbA2. HbLepore This abnormal Hb is the result of unequal crossing over of chromosomes. Affects b and d globin genes with generation of a chimeric globin with d sequences at NH2 terminal an d b globin at COOH terminal. Production of db globin is inefficient; there is absence of no rmal d and b globins. The phenotype of the heterozygote is thalassaemia trait; the homozygo te picture is thalassaemia intermedia. BCSH haemoglobinopathy diagnosis guidelines (cid:27) www.bcshguidelines.com/pdf/bjh809.pdf 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 93 Red cell disorders Hereditary persistence of fetal hemoglobin 93 Heterogeneous groups of disorders caused by deletions or cross-overs involving b and g chai n production, or non-deletional forms due to point mutations upstream of the g globin gene, with high levels of HbF produc- tion in adult life. There is 5 d and b chain production wi th enhanced g chain production. Globin chain imbalance is much less marked than in b thalas saemia, resulting in milder disorder. There are few clinical effects. May be pancellular (very high levels of HbF hemoglobin synthesis with uniform distribution in RBCs) or heterocellular (increased numbers of F cells). Mechanism Like db thalassaemia, HPFH frequently arises from deletions of DNA, which remove or inactivate the b globin gene (note: heterocellular HPFH may be result of mutations outsi de the b globin gene). Heterozygous HPFH Anemia may be mild or absent. Hematological indices are normal. There is balanced a/non-a globin chain synthesis. HbF level 25%. 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 94 Hb patterns in hemoglobin disorders % Hemoglobin A F A2 S Other Normal 97 <1 2 \200\2233 b thalassaemia trait 80 \200\22395 b thalassaemia intermedia 30 \200\22350 b thalassaemia m ajor 0 \200\22320 1 \200\2235 50 \200\22370 80 \200\223100 3 \200\2237 0 \200\2235 0 \200\22313 94 HPFH (Black heterozygote) 60 \200\22385 HPFH (Black homozygote) 15 \200\22335 100 1 \200\2233 a thalassaemia trait HbH disease 85 \200\22395 60 \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% HbBart \200\231s hydrops HbE trait HbE disease HbE/b thalassaemia HbE/a thalassaemia 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 HbD trait HbD disease HbD/b thalassaemia 50 \200\22365 1-5 0-7 1-5 1-3 1-7 1 \200\2233 D 45 \200\22350 D 90 \200\22395 D 80 \200\22390 HbC trait HbC disease 60 \200\22370 C 30 \200\22340 C 95 slight 4 Sickle trait Sickle cell anemia Sickle/b+ thalassaemia Sickle/bo thalassaemia Sickle/D Sick le/C 55 \200\22370 0 5 \200\22330 0 0 0 1 7 5 \200\22315 5 \200\22330 1 \200\2235 1 3 3 \200\223 4 \200\2238 30 \200\22345 90 60 \200\22385 70 \200\22390 50 50 \200\22365 D 50% C 50% HbLepore trait 80 \200\22390 HbLepore disease HbLepore/b thalassaemia 1 \200\2233 0 2 \200\2232.5 70 \200\22390 70 \200\22390 Lepore 9 \200\22311% Lepore 8 \200\22330% Lepore 5 \200\22315% 0 2.5 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 95 Red cell disorders 95 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 96 Non-immune haemolysis 4 major groups 2 Infections. 2 Vascular (mechanical damage). 2 Chemical damage. 2 Physical damage. 96 Infection Malaria \200\224especially falciparum. Causes anemia through marrow suppression, hypersple nism and RBC sequestration. In addition there is haemolysis due to destruction of parasitis ed RBCs by RES and intravascular haemolysis when sporozoites released from infected RBCs. B lackwater fever refers to severe acute intravascular haemolysis with haemoglobinaemia, 5 Hb , haemoglobinuria and ARF. Babesiosis \200\224Babesia (RBC protozoan). Rapid onset of vomiting, diarrhea, rigors, jau ndice, 4T . Haemoglobinaemia, haemoglobinuria, ARF and death. Clostridium perfringens \200\224septicaemia and acute intravascular haemolysis. Viral \200\224especially viral haemorrhagic fevers e.g. dengue, yellow fever. Mechanical Cardiac \200\224turbulence and shear stress following mechanical valve replacement. Genera l feature of haemolysis: 4 reticulocytes, LDH, plasma Hb, with 5 haptoglobins platelets. Urinary haemosiderin +ve. MAHA \200\224see p112. HUS/TTP \200\224see p530. 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 anemia. No specif ic features on movie. May be associated GIT bleeding and 5 ferritin (lost in sweat). Chemical & physical Oxidative haemolysis \200\224chronic Heinz body intravascular haemolysis with dapsone or s alazopyrine in G6PD deficient people or unstable Hb (and normals if dose high enough). Movi e: bite cells (RBC). Heinz bodies not prominent if intact spleen. Haemolysis well compensat ed. MetHb \200\224see p110. Lead poisoning \200\224moderate 5 RBC lifespan. Anemia mainly due to block in haem synthesis although lead also inhibits 5 nucleotidase (NT). Basophilic st ippling on movie. Ring sideroblasts in BM. 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 97 Red cell disorders O2 \200\224haemolysis in patients treated with hyperbaric O2. Insect bites \200\224e . g . spider, bee-sting (not common with snake bites). Heat \200 \224e . g . burns7severe haemolysis due to direct RBC damage. 97 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 abd ominal pain ((cid:9) p54). Wilson \200\231s disease \200\224autosomally inherited disorder of copper metabolism, with hepatolenticular, hepatocerebral degeneration. PNH \200\224see p124. Hereditary acanthocytosis \200\224a-b-lipoproteinaemia. Rare, inherited. Associated with r etinitis pigmentosa, steatorrhoea, ataxia and mental retardation. 02-red cell disorders.qxd 10.12.2003 04.11 PM Page 98 Hereditary spherocytosis Most common inherited RBC membrane defect characterized by variable degrees of haemolysis, spherocytic RBCs with 4 osmotic fragility. Pathophysiology Abnormal RBC cytoskeleton: partial deficiency of spectrin, ankyrin, band 3 or protein 4.2 (leads to 5 binding to band 4.1 protein and ankyrin). Loss of lipid from RBC membrane7spherical (cf. biconcave) RBCs with reduced surface area7get trapped in splenic c ords and have reduced lifespan. RBCs use more energy than normal in attempt to maintain cel l shape. RBC membrane has 4 Na+ permeability (loses intracellular Na+) and energy required to restore Na+ balance. Red cells are less deformable than normal. 98 Epidemiology In Northern Europeans 1:5000 people are affected. In most cases inheri- tance is autosomal dominant although autosomal recessive inheritance has been reported. Clinical features Presents at any age. Highly variable from asymptomatic to severely anemic , but usually there are few symptoms. Well-compensated haemol- ysis; other features of haem olytic anemia may be present e.g. splenomegaly, gallstones, mild jaundice. Occasional aplas tic crises occur, e.g. with parvovirus B19 infection. Diagnosis 2 Positive family history of HS in many cases. 2 Blood movie shows 44 spherocytic RBCs. 2 Anemia, 4 reticulocytes, 4 LDH, unconjugated bilirubin, urinary urobilinogen with 5 haptoglobins. DAT \200\223ve. Osmotic fragility test \200\224RBCs incubated in saline at various concentrations. Results in cell expansion and eventually rupture. Normal RBCs can withstand greater volume increas es than spherocytic RBCs. Positive result (i.e. confirms HS) when RBCs lyse in saline at ne ar to isotonic concentration, i.e. 0.6 \200\2230.8g/dL (whereas normal RBCs will simply sho w swelling with little lysis). Osmotic fragility more marked in patients who have not under gone splenectomy, and if the RBCs are incubated at 37 C for 24h before performing the test .