i reticulocytes Not seen in normals (removed by spleen), small numbers seen post-splenectomy, oxidant drugs , G6PD defi ciency, sulfonamides, unstable Hb (Hb Zurich, K ln) Heinz bodies Made of DNA, generally removed by the spleen, dyserythropoietic states, e.g. B12 defi cienc y, MDS, post- splenectomy, hyposplenism HbH inclusions, denatured HbH ( 4 tetramer), stain with methylene blue, seen in HbH disease ( \210\222 \210\222/ \210\222 ), less prominen t in thalassaemia trait, not present in normals Howell \200\223Jolly bodies, target cell s, occasional nucleated RBCs, lymphocytosis, macrocytosis, acanthocytes Howell \200\223Jolly bodies H bodies Hyposplenic blood \201 lm 34 CHAPTER 2 Red cell disorders Anemia in renal disease Anemia is consistently found in the presence of chronic renal failu re since the kidneys are the main source of erythropoietin. Severity gen- erally relates to the degree of renal impairment although anemia may occur even if modest renal impairment. The dominant mechanism is inadequate production of erythropoietin (EPO). Other contributory factors include (i) suppressive eff ects of uraemia and (ii) d in RBC sur- vival. Uraemia impairs platelet function l blood loss and Fe defi ciency. Small amounts of blood are inevi tably left in the tubing following dialysis so that blood loss and Fe defi ciency are furth er contributory factors in dialysis patients. Folate is lost in dialysis and supplementatio n is required to avoid defi ciency. Aluminum toxicity (from trace amounts in dialysis fl ui ds) and osteitis fi brosa from hyperparathyroidism are rare contribu- tory factors. Laboratory features \200 Hb typically 5.0 \200\22310.0g/dL. \200 MCV n. \200 Blood fi lm \200\224mostly normochromic RBCs; schistocytes and acanthocytes present. No specifi c abnormalities in WBC or platelets (see Fig 2.1). Microangiopathic haemolytic changes present in vasculitic collagen disorders with renal failure and classically in HUS and TTP. Management \200 Short-term treatment with RBC transfusion, based on symptoms (not Hb concentration). Correction of Fe and folic acid defi ciencies. \200 EPO will correct anemia in most patie nts. \200 Start at 50 \200\223100 units/kg SC \227 3/week. Give IV Fe at same time. Response apparent <10 weeks; reduced doses required as maintenance. Renal Association guide lines have been produced for application and monitoring of EPO therapy. Although expensive it improves quality of life and avoids transfusion dependency and Fe overload. Side eff ects of EPO \200 i BP. \200 Pure red cell aplasia. \200 Thrombotic tendency. 35 anemia IN RENAL DISEASE Fig. 2.1 Blood fi lm: chronic renal failure with burr (irregular shaped) cells (E see color plate section). Further reading Eschbach, J.W. et al. (1987). Correction of the anemia of end-stage renal d isease with recombinant human erythropoietin. Results of a combined phase I and II clinical trial. N Engl J Med, 316, 73 \200\2238. Levin, N. et al. (1997). National Kidney Foundatio n: Dialysis Outcome Quality Initiative \200\223 development of methodology for clinical pra ctice guidelines. Nephrol Dial Transplant, 12, 2060 \200\2233. 36 CHAPTER 2 Red cell disorders Anemia in endocrine disease Anemia and other hematological eff ects occur in various endocr ine disorders. The abnormalities will usually correct as the endocrine abnor- mality is cor rected. Pituitary disorders De \201 ciency/hypopituitarism is associated with normochromic, normoc ytic anemia; associated leucopenia may also occur. Abnormalities correct as normal function is restored, by replacement therapy. Thyroid disorders Hypothyroidism may produce a mild degree of anemia; MCV usually i but may be normal. Corrects on restoration of normal thyroid function. Menorrhagia occurs in hypot hyroidism and can result in associated Fe defi ciency. B12 levels should be checked because of the association with other autoimmune disorders (e.g. pernicious anemia). Thyrotoxicosis may be associated with mild degrees of normochromic anemia in 20% of cases w hich corrects as function is normalized. Erythroid activity is i but a disproportionate inc rease in plasma volume means either no change in Hb concentration or mild anemia. Haematini c defi ciencies occur and should be excluded. Adrenal disorders Hypoadrenalism results in normochromic, normocytic anemia; the plasma vol ume is d which masks the true degree of associated anemia. The abnormalities are corrected by replacement mineralocorticoids. Hyperadrenalism (Cushing \200\231s) results in erythrocytosis with a typical net increase i n Hb (by 1 \200\2232g/dL). Occurs whether Cushing \200\231s is 1 or iatro- genic. Mechani sm is unclear. Parathyroid disorders \200\224hyperparathyroidism may be associated with anemia from impair ment of EPO production, or in some cases from 2 marrow sclerosis. Sex hormones \200\224androgens stimulate erythropoiesis and are occasionally used to stimul ate red cell production in aplastic anemia. The infl uence of androgens explains the higher Hb in adult \231\202 compared with \231\200. Diabetes mellitus when poorly controlled ma y be associated with anemia; however, the majority of hematological abnormalities in dia- b etes mellitus result from 2 disease-related complications, e.g. renal failure. Further reading Spivak, J.L. (2000). The blood in systemic disorders. Lancet, 355, 1707 \200\22312. 37 anemia IN ENDOCRINE DISEASE 38 CHAPTER 2 Red cell disorders Anemia in joint disease Rheumatoid arthritis, psoriatic arthropathy, and osteoarthritis may be com- plicated by anemia. Various factors contribute to anemia; commonly more than one i s present, especially in rheumatoid arthritis. Some of the mechanisms that give rise to ane mia in rheumatoid also apply in other con- nective tissue disease, e.g. SLE, polyarteritis nodosa, etc. (see Table 2.2) Anemia of chronic disorders (ACD) ACD is a cytokine-driven suppression of red cell producti on. The clinical problem is being able to recognize the presence of other contributory fact ors in the pathogenesis of the anemia. BM macrophages fail to pass their stored Fe to devel oping RBCs and a lower than expected rise in EPO suggests some inhibition in its pathway. M arrow also appears less responsive to EPO. i IL-1 has been identifi ed. Detailed studies su ggest a synergistic eff ect of IL-1 with T-cells to produce IFN- which can sup- press ery throid activity. May also be an i level of TNF- which inhibits erythropoiesis through rel ease of IFN- from marrow stromal cells. Typical features of ACD \200 Hb range 7.0 \200\22311.0g/dL. \200 MCV is usually n but w hen longstanding the MCV is moderately d (may look like Fe defi ciency). \200 Ferritin usually n but may be i. \200 Serum Fe n o r d, TIBC n or d. (Note: Fe and TIBC tests now obsolete). Serum transferrin receptor levels normal. \200 i zinc protoporphyrin level. \200 BM Fe stores plentiful. Table 2.2 Additional mechanisms of anemia in rheumatoid disease Warm antibody AIHA in association with rheumatoid and other collagen disorders; fi lm will show reticulocytosis and +ve DAT Autoimmune phenomena Red cell aplasia Chronic blood loss (caused by medication) Drug-related problems Drug side eff ects e.g. macrocytosis from antimetabolite immunosuppressives, e.g. azathiopr ine and methotrexate, oxidative haemolysis 2 to dapsone or sulfasalazine (occurs in norma l individuals as well as those with G6PD defi ciency) Anemia 2 to gold therapy for rheumatoid arthritis Idiosyncratic reactions, unexplained or unforeseeable reactions such as marrow aplasia Rare autoimmune haemolysis due to mefenamic acid, diclofenac, or ibuprofen Hypersplenism, Felty \200\231s syndrome in rheumatoid, renal failure in SLE or polyarteriti s 2 to other organ problems 39 anemia IN JOINT DISEASE Management Supportive transfusion in symptomatic patients; coexistent Fe defi ciency should be excluded and treated. Minority may be suitable for/respon- sive to EPO therapy. Bleeding and Fe de \201 ciency Usually 2 to use of NSAIDs \200\224consider and exclude o ther causes of blood loss which may occur in this patient group. Further reading Bron, D. et al. (2001). Biological basis of anemia. Semin Oncol, 28, 1 \200 \2236. Spivak, J.L. (2000) The blood in systemic disorders. Lancet, 355, 1707 \200\22312. 40 CHAPTER 2 Red cell disorders Anemia in gastrointestinal disease Anemia occurs in GIT disorders through mechanisms of blo od loss, ACD-specifi c disease-related complications, or drug side eff ects/idio- syncrasy occurring singly or in various combinations (see Table 2.3). Table 2.3 Blood loss in gastrointestinal disease Immediately following acute hemorrhage \200\224RBC indices usually n Acute Normochromic anemia RBC indices show low normal or marginally d, especially MCV Movie shows mixture of normochr omic and hypochromic RBCs ( \200\230dimorphic \200\231) Acute on chronic RBC indices show established chronic Fe defi ciency features d MCV, MCH, platelets often i Chronic Anemia in GIT disorders can be simply considered against some of the commoner problems aris ing through the GIT Oesophageal \200\224bleeding from peptic oesophagitis, association of o esoph- ageal web and chronic Fe defi ciency. Gastric \200\224pernicious anemia and B12 defi ciency, late eff ects of partial or total gastrectomy producing B12 and/or Fe defi ciency. Microangiopathic haemolytic anemia from metastatic adenocarcinoma. Small bowel \200\224malabsorption states, e.g. Fe and/or folate defi ciency 2 to coeliac disease, malabsorption from other problems including infl amma- tory bowel disease; hypospl enism 2 to coeliac with or without d plate- lets. Large bowel \200\224blood loss anemia from infl ammatory bowel disorders. Note: these may a lso be associated with ACD. Rare occurrence of auto- immune haemolysis associated with ulce rative colitis. Pancreas \200\224ACD associated with carcinoma or chronic pancreatitis, DIC associated with acute pancreatitis. Liver \200\224E see Anemia in liver disease, p.41. Drug-related anemia arises through: \200 Upper GIT irritation causing blood loss \200\224 aspirin, NSAIDs, corticosteroids. Bleeding due to specifi c drugs, e.g. warfarin and heparin. \200 Drug-induced haemolysis, e.g. oxidative (Heinz body) haemolysis due to sulfasalazine or dapsone. Production impairment, e.g. aplasia 2 to mesalazine. 41 anemia IN LIVER DISEASE Anemia in liver disease Anemia is common in chronic liver disorders. There are several pos- sible causes including: \200 ACD \200\224part of marrow response to chronic infl ammator y processes. \200 Macrocytosis anemia: specifi c eff ects on membrane lipids cause iMCV. Alcohol \200\224direct suppressive eff ect on erythropoiesis with iMCV. \200 Folate defi ciency: seen in alcoholic liver disease l nutritional defi ciency and/or direct eff ect of alcohol on folate metabolism. \200 Blood loss from o esophageal varices l acute or chronic anemia. \200 Hypersplenism \200\224portal hypertens ion can produce marked splenic enlargement leading to hypersplenism. Haemolytic anaemias, e.g.: Autoimmune haemolytic anemia in association with chronic active hepatitis. Zieve \200\231s syndrome (hypertriglyceridaemia + self-limiting haemolysis due to acute alcohol excess). Viral hepatitis may provoke oxidative haemolysis in those with G6PD defi ciency. Acute liver failure \200\224DIC and MAHA may occur. \200 Acanthocytosis: acute haemolytic anemia with acanthocytosis (spur cell anemia). Rare. Usually late stage liver disease, wit h poor prognosis. Alcohol Even if no liver disease, chronic alcohol consumption may lead to iMCV (100 \200 \223110fL). Anemia may be associated with poor diet, folate defi - ciency, GI bleeding and Fe defi ciency. Alcohol has direct toxic eff ects on the marrow with reversible suppression of haemopoiesis; dHb and d platelets are most common. Further reading Savage, D., et al. (1986). Anemia in alcoholics. Medicine (Baltimore), 65, 322 \200\22338. Wu, A., et al. (1974). Macrocytosis of chronic alcoholism. Lancet, 1, 829 \200\22331. 42 CHAPTER 2 Red cell disorders Iron (Fe) de \201 ciency anemia Microcytic anemia is common and the commonest cause is chr onic Fe defi ciency. Fe physiology and metabolism Normal (Western) diet provides 715mg of Fe/d, of which 5 \200 \22310% is absorbed in duodenum and upper jejunum. Ferrous (Fe2+) Fe is better absorbed tha n ferric (Fe3+) Fe. Total body Fe store 74g. Around 1mg of Fe/d lost in urine, feces, sweat , and cells shed from the skin and GIT. Fe defi ciency is commoner in \231\200 of reproduc tive age since menstrual losses account for 720mg Fe/month and in pregnancy an additional 5 00 \200\2231000mg Fe may be lost (transferred from mother l fetus) (see Table 2.4). Table 2.4 Causes of Fe defi ciency Menorrhagia Reproductive system Oesophagitis, oesophageal varices, hiatus hernia (ulcerated; simple hiatus hernia would not cause Fe defi ciency), peptic ulcer, infl ammatory bowel disease, hemorrhoids, carcinoma: stomach, colorectal, (rarely angiodysplasia, hereditary haemorrhagic telangiectasia) GIT Coeliac disease, atrophic gastritis (Note: may also result from Fe defi ciency), gastrectom y Malabsorption Growth spurts, pregnancy Physiological Vegans, elderly Dietary Haematuria (uncommon cause) Genitourinary system PNH, frequent venesection, e.g. blood donation Others Commonest cause is hookworm infestation Worldwide Assessment Clinical history \200\224review potential sources of blood loss, especially GIT loss. Menstrual loss \200\224quantitation may be diffi cult; ask about number of tampons us ed per day, how often these require changing, and duration. Other sources of blood loss, e.g. haematuria and haemoptysis (these are not common causes o f Fe defi ciency). Ask patient if he/she has been a blood donor \200\224regular blood donat ion over many years may cause chronic Fe store depletion. Drug therapy, e.g. NSAIDs and corticosteroids may cause GI irritation and blood loss. Past medical history, e.g. previous gastric surgery (l malabsorption). Ask about previous episod es of anemia and treatments with Fe. 43 IRON (Fe) DEFICIENCY anemia In patients with Fe defi ciency assume underlying cause is blood loss until proved otherwis e. In developed countries pure dietary Fe lack causing Fe defi ciency is almost unknown. Examination \200 General examination including assessment of mucous membranes (e.g. hereditary haemorrhagic telangiectasia). Seek possible sources of blood loss. \200 Abdominal examination, rectal examination, and sigmoidoscopy mandatory. Gynecological examination also required. Laboratory tests \200 d Hb. \200 d MCV (<76fL) and d MCHC (Note: d MCV in thalassaemia and ACD). Red cell distribution width (RDW): i in Fe defi ciency states with a greater frequency than in ACD or thalassaemia trait (see Fig. 2.2). Serum ferritin (measurement of Fe/TIBC generally unhelpful). Ferritin assay preferred \200\224low serum ferritin identifi es the presence of Fe defi cie ncy but as an acute phase protein it can be i, masking Fe defi ciency. d Fe and i TIBC indi cates Fe defi ciency (though tests are obsolete). The soluble transferrin assay (sTfR) is useful in cases where i ESR. sTfR is i in Fe defi ciency but n in anemia in presence of i ESR (e.g. rheumatoid, other in fl ammatory states). This assay is not universally available at present. % hypochromic RBCs \200\224some modern analysers provide this parameter. i % hypo RBCs are seen in Fe defi ciency but also thalassaemia, CRF on EPO where insuffi cient Fe given. Zinc protoporphyrin (ZPP) \200\224in the absence of Fe, zinc is incorporated into protoporp hyrin. i ZPP in Fe defi ciency is a non-specifi c marker since i ZPP is seen in any disorde r that restricts Fe availability to developing RBCs, e.g. infection, infl ammation, cancer, etc. Reticulocyte Hb concentration (CHr) appears to be a sensitive method for detecting early Fe defi ciency. Examination of BM aspirate (Fe stain) is occasionally useful. \200 Theoretically FOB test ing may be of value in Fe defi ciency but results can be misleading. False +ve results seen in high dietary meat intake. Treatment of Fe de \201 ciency Simplest, safest and cheapest treatment is oral ferrous sal ts, e.g. FeSO4 (Fe gluconate and fumarate equally acceptable). Provide an oral dose of elem ental Fe of 150 \200\223200mg/d. Side eff ects in 10 \200\22320% patients (e.g. abdominal d istension, constipation and/or diarrhea) \200\224try d the daily dose to bd or od. Liquid F e occasionally necessary, e.g. children or adults with swallowing diffi culties. Increasing dietary Fe intake has no routine place in the management of Fe defi ciency except where in take is grossly defi cient. 44 CHAPTER 2 Red cell disorders Fig. 2.2 Blood fi lm in Fe defi ciency anemia: note pale red cells with pencil cell (top le ft). Response to replacement A rise of Hb of 2.0g/dL over 3 weeks is expected. MCV will i concom i- tantly with Hb. Reticulocytes may i in response to Fe therapy but is not a reliable indi cator of response. Duration of treatment Generally 76 months. After Hb and MCV are normal continue Fe for at l east 3 months to replenish Fe stores. Failure of response \200 Is the diagnosis of Fe defi ciency correct? \200 Consider ACD or thalassaemia trait. \200 Is there an additional complicating illness? Chronic infection, collagen disorder, or neoplasm. \200 Is the patient complying with pre scribed medication? \200 Is the preparation of Fe adequate in dosage and/or formulation? \200 Is the patient continuing to bleed excessively? \200 Is there malabsorption? \200 Are there other haematinic defi ciencies (e.g. B12 or folate) present? \200 Reassess pa tient: ?evidence of continued blood loss or malabsorption. 45 IRON (Fe) DEFICIENCY anemia Parenteral Fe Occasionally of value in genuine Fe intolerance, if compliance is a problem, or if need to replace stores rapidly, e.g. in pregnancy or prior to major surgery. Note: Hb will rise no faster than with oral Fe. Intravenous Fe Fe may be administered IV as Fe hydroxide sucrose (ferric hydroxide with suc rose) or Fe dextran (ferric hydroxide with dextran). Facilities for cardiopulmonary resusci tation should be available though serious adverse events are uncommon. Further reading Andrews, N.C. (1999). Disorders of iron metabolism. N Engl J Med, 341, 1986 \200\22395. Brugnara, C. (2000) Reticulocyte cellular indices: a new approach in the diagn osis of anemias and monitoring of erythropoietic function. Crit Rev Clin Lab Sci, 37, 93 \200\223130. Kuhn, L.C. and Hentze, M.W. (1992). Coordination of cellular iron metabolism b y post- transcriptional gene regulation. J Inorg Biochem, 47, 183 \200\22395. Labbe, R.F. e t al. (1999) Zinc protoporphyrin: A metabolite with a mission. Clin Chem, 45, 2060 \200\223 72. Rettmer, R.L. et al. (1999) Zinc protoporphyrin/heme ratio for diagnosis of preanemic i ron defi ciency. Pediatrics, 104, e37. Tapiero, H. et al. (2001). Iron: defi ciencies and r equirements. Biomed Pharmacother, 55, 324 \200\22332. 46 CHAPTER 2 Red cell disorders Vitamin B12 de \201 ciency B12 defi ciency presents with macrocytic, megaloblastic anemia ranging from mild to severe (Hb <6.0g/dL). Symptoms are those of chronic anemia: fatigue, d yspnoea on eff ort, etc. Neurological symptoms may also be present \200\224classically peri pheral paraesthesiae and disturbances of position and vibration sense (see Table 2.5). Occa sionally neurological symptoms occur with no/minimal hematological upset. If uncorrected, t he patient may develop subacute combined degeneration of the spinal cord l permanently atax ic. Pathophysiology B12 (along with folic acid) is required for DNA synthesis; B12 is also requ ired for neurological functioning. B12 is absorbed in terminal ileum after binding to intri nsic factor produced by gastric parietal cells. Body stores of B12 are 2 \200\2233mg (suffi cient for 3 years). B12 is found in meats, fi sh, eggs, and dairy produce. Strictly vegeta rian (vegan) diets are low in B12 although not all vegans develop clinical evidence of defi ciency. Presenting hematological abnormalities \200 Macrocytic anemia (MCV usually >110fL). In ex treme cases RBC anisopoikilocytosis can result in MCV values just within normal range. RBC changes include oval macrocytosis, poikilocytosis, basophilic stippling, Howell \200\223Jolly bodies, circulating megaloblasts. Hypersegmented neutrophils. \200 Leucopenia and thrombocytopenia common. \200 BM shows megaloblastic change; marked erythroid hyperplasia with predominance of early erythroid pre cursors, open atypical nuclear chromatin patterns, mitotic fi gures and \200\230giant \200 \231 metamyelocytes Fe stores usually i. \200 Serum B12 d. \200 Homocysteine levels are i in both B12 and f olate defi ciency. \200 Serum/red cell folate usually n or i. \200 LDH levels markedly i refl ecting ineff ective erythropoiesis (RBC destruction within the BM and d RBC lifespan. Autoantibody screen in pernicious anemia: 80 \200\22390% show circulating gastric parietal cell antibodies, 55% have circulating intrinsic factor antibodies. Note: parietal cell anti bodies are not diagnostic since found in normals; IFA is only found in 50% of patients with PA but is diagnostic. Management of B12 de \201 ciency \200 Identify and correct cause if possible. \200 Inv estigations are undertaken and a test of B12 absorption is carried out (e.g. Schilling test). Urinary excretion of a test dose of B12 labeled with trace amoun ts of radioactive cobalt is compared with excretion of B12 bound to intrinsic factor; the t est is done in two parts. B12 malabsorption corrected by intrinsic factor is diagnostic of pernicious anemia (in absence of previous gastric surgery). This test is now largely obsole te since the reagents are no longer available. \200 Management \200\224hydroxocobalamin 1 mg IM and folic acid PO should be given immediately. 47 VITAMIN B12 DEFICIENCY Table 2.5 Causes of B12 defi ciency Commonest, due to autoimmune gastric atrophy resulting in loss of intrinsic factor producti on required for absorption of B12. Incidence i >40 years and often associated with other au toimmune problems, e.g. hypothyroidism. Pernicious anemia May develop after major partial gastrectomy. Following total gastrectomy Resection of ileum, Crohn \200\231s disease. Ileal disease E.g. diverticulae or localized infl ammatory bowel changes allowing bacterial overgrowth wh ich then competes for available B12 Diphyllobothrium latum. Blind loop syndromes Fish tapeworm Malabsorptive disorders Tropical sprue, coeliac disease. E.g. vegans Dietary de \201 ciency Supportive measures \200\224bed rest, O2, and diuretics may be needed while awaiting respon se. Transfusion is best avoided but 2 units of concentrated RBCs may be used for patients s everely compromised by anemia (risk of precipitating cardiac failure); hypokalaemia is occa sionally observed during the immediate response to B12 and serum [K+] should be monitored. Response apparent in 3 \200\2235d with reticulocyte response of >10%; normoblastic conversi on of marrow erythropoiesis in 12 \200\22324h. Patients frequently describe a subjective im provement within 24h. \200 B12 replacement therapy \200\224initially hydroxocobalamin 5 \227 1mg IM should be given during the fi rst 2 weeks, thereafter maintenance injections ar e needed 3-monthly. If dietary defi ciency seems likely and B12 defi ciency mild, worth trying oral B12 (cyanoc obalamin 50 \200\223150mcg or more, daily between meals). Long-term follow-up depends on the 1 cause. Pernicious anemia patients require lifelong t reatment and should be checked annually with a FBC and thyroid function; the incidence of g astric cancer is twice as high in these patients compared to the normal population. Broad-spectrum antibiotics should be given to suppress bacterial over-growth in blind loop syndrome local surgery if appropriate. Long-term IM B12 may be the pragmatic solution if blind loop cannot be corrected. Further reading Guidelines on the investigation and diagnosis of cobalamin and folate defi ciencies. A publication of the British Committee for Standards in Hematology. BCSH General Hematology Test Force (1994). Clin Lab Haematol, 16, 101 \200\22315. Toh, B.H., van Driel, I.R., and Gleeson, P.A. (1997). Pernicious anemia. N Engl J Med, 337, 1441 \200\2238. 48 CHAPTER 2 Red cell disorders Folate de \201 ciency Folate defi ciency represents the other main defi ciency cause of me galo- blastic anemia; hematological features indistinguishable from those of B12 defi cienc y. Distinction is on basis of demonstration of reduced red cell and serum folate. See Table 2.6 for causes of folate defi ciency. 3 Megaloblastic anemia patients should never receive empirical treatment with folic acid alone. If they lack B12, folic acid is potentially cap able of precipitating subacute combined degeneration of the cord. Pathophysiology Adult body folate stores comprise 10 \200\22315mg; normal daily require- me nts are 0.1 \200\2230.2mg, i.e. suffi cient for 3 \200\2234 months in absence of exog- enou s folate intake. Folate absorption from dietary sources is rapid; proximal jejunum is main site of absorption. Main dietary sources of folate are liver, green vegetables, nuts, and y east. Western diets contain 70.5 \200\2230.7mg folate/d but availability may be lessened as folate is readily destroyed by cooking, especially in large volumes of water. Folate coen- zymes are an essential part of DNA synthesis, hence the occurrence of megaloblastic change in defi ciency. Diagnosis Hematological fi ndings are identical to those seen in B12 defi ciency \200\224 m acrocytic, megaloblastic anemia. Other fi ndings also similar to B12 except parietal cell a nd intrinsic factor autoantibodies usually \210\222ve. Reduced folate levels \200\224serum folate levels refl ect recent intake, red cell folate levels give a more reliable indicati on of folate status (see Figs. 2.3 and 2.4). Table 2.6 Causes of folate defi ciency Poor nutrition, e.g. poverty, old age, \200\230skid row \200\231 alcoholics d intake i requirements/ losses Pregnancy, i cell turnover, e.g. haemolysis, exfoliative dermatitis, renal dialysis Coeliac disease, tropical sprue, Crohn \200\231s and other malabsorptive states Malabsorption Phenytoin, barbiturates, valproate, oral contraceptives, nitrofurantoin may induce folate m alabsorption Drugs Methotrexate, trimethoprim, pentamidine antagonize folate cf. induce defi ciency Antifolate drugs Poor nutrition plus a direct depressant eff ect on folate levels which can precipitate clin ical folate defi ciency Alcohol Management \200 Treatment and support of severe anemia as for B12 defi ciency. \200 Fol ic acid 5mg/d PO (never on its own, see Table 2.6), unless patient known to have normal B12 level. 49 FOLATE DEFICIENCY Treatment of underlying cause, e.g. in coeliac disease folate levels and absorption normalize once patient established on gluten-free diet. Long- term supplementati on advised in chronic haemolysis, e.g. HbSS or HS. Prophylactic folate supplements recommended in pregnancy and other states of i demand, e.g. prematurity. Fig. 2.3 Blood fi lm: normal neutrophil: usually has <5 lobes. This one has 3 lobes (E see color plate section). Fig. 2.4 Hypersegmented neutrophils with 7 \200\2238 lobes: found in B12 or folate defi - c iency. Note: blood fi lms and marrow appearances are identical in B12 and folate defi cienc ies (E see color plate section). 50 CHAPTER 2 Red cell disorders Other causes of megaloblastic anemia Megaloblastic anemia not due to actual defi ciency of either B12 or folate is uncommon, but may occur in the following situations. Congenital \200 Transcobalamin II defi ciency \200\224absence of the key B12 transportati on protein results in severe megaloblastic anemia (corrects with parenteral B12). Congenital intrinsic factor defi ciency \200\224autosomal recessive, results in failure to produce intrinsic factor. Presents as megaloblastic anemia up to age of 2 years and respond s to parenteral B12. Inborn errors of metabolism \200\224errors in folate pathways, also occurs in orotic aciduria and Lesch \200\223Nyhan syndrome. Megaloblastosis commonly present in the congenital dyserythropoietic anaemias (E see Congenital dyserythropoietic anaemias, p.588). Acquired \200 MDS \200\224often present in sideroblastic anemia (RARS). \200 Acute leuk emia \200\224megaloblastic-like erythroid dysplasia in AML M6. \200 Drug induced \200\224 2 to antimetabolite drugs including 6-mercaptopurine, cytosine arabinoside, zidovudine, and hydroxyurea. Anesthetic agents \200\224transient megaloblastic change after nitrous oxide. Alcohol excess \200\224may result in megaloblastic change in absence of measurable folate defi ciency. Vitamin C defi ciency \200\224occasionally results in megaloblastic change. 51 anemia IN OTHER DEFICIENCY STATES Anemia in other de \201 ciency states Fe, folate, or vitamin B12 defi ciencies account for the majority of clinically signifi cant defi ciency syndromes resulting in anemia. Anemia is recog- nized as a complication in other vitamin defi ciencies and in malnutrition. Vitamin A de \201 ciency Produces a chronic disorder like Fe defi ciency anemia with d MCV and MCH. Vitamin B6 (pyridoxine) de \201 ciency Can produce hypochromic microcytic anemia; siderobl astic change may occur. Pyridoxine is given to patients on antituberculous therapy with iso niazid which is known to interfere with vitamin B6 metabolism and cause sideroblastic anemi a. Vitamin C de \201 ciency Occasionally associated with macrocytic anemia ( megaloblastic change in 10%); since the main cause of vitamin C defi ciency is inad- equate diet or nutri tion there may be evidence of other defi ciencies. Vitamin E de \201 ciency Occasionally seen in the neonatal period in low birth weight infa nts \200\224 results in haemolytic anemia with abnormal RBC morphology. Starvation Normochromic anemia leucopenia occurs in anorexia nervosa; features are not a ssociated with any specifi c defi ciency; BM is typically hypocellular. 52 CHAPTER 2 Red cell disorders Haemolytic syndromes De \201 nition Any situation in which there is a reduction in RBC life-span due to iRBC de struction. Failure of compensatory marrow response results in anemia. Predominant site of R BC destruction is red pulp of the spleen. Classi \201 cation \200\2243 major types 1. Hereditary vs acquired. 2. Immune vs non-immun e (see Table 2.7). 3. Extravascular vs intravascular. Hereditary cause suggested if history of anemia refractory to treatment in infancy FH, e.g. other aff ected members, anemia, gallstones, jaun- dice, splenectomy. Acquired haemolytic anemia is suggested by sudden onse t of symptoms/signs in adulthood. Intravascular haemolysis \200\224takes place in periphera l circulation cf. extravascular haemolysis which occurs in RES. Hereditary \200 Red cell membrane disorders, e.g. HS and hereditary elliptocytosis. \200 Red cell enzymopathies, e.g. G6PD and PK defi ciencies. \200 Abnormal Hb, e.g. thalassa emias and sickle cell disease, unstable Hbs. Acquired \200\224immune Table 2.7 Acquired: immune Alloimmune Autoimmune HDN \200 RBC transfusion incompatibility Warm AIHA \200\2231 or 2 to SLE, CLL, drugs \200 Cold \200\223Mycoplasma or EBV infec tion \200 Cold haemagglutinin disease (CHAD) \200 Lymphoproliferative disorders \200 Paroxysmal cold haemoglobinuria (PCH) 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.