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Hemoglobinopathies and Thalassemias The ABCs of Lab Evaluation By Shirley L. Welch, PhD http://www.aacc.org/publications/cln/2009/october/Pages/Series1009.aspx# Idividuals with one copy of HbS, called HbS trait, usually are asymptomatic and have normal red blood cell indices and morphology. Typical laboratory findings for these patients are 35 to 45% HbS. In simple cases of HbS trait, the percentage of HbS is always greater than the percentage of HbA. For individuals with HbS levels <30%, labs should consider concurrent iron deficiency or α-thalassemia trait. Individuals with two copies of HbS develop sickle cell disease. Symptoms include hemolytic anemia, with Hb levels of 6 to 10 g/dL. In addition, peripheral-blood smears show sickle cells, target cells, and HowellJolly bodies. Their electrophoretic patterns and HPLC results typically show 90 to 95% HbS, no HbA, and often slightly elevated HbF in the 5 to 10% range. It is important to know a patient’s transfusion history in cases of sickle cell disease, as small amounts of HbA post-transfusion can produce misleading test results. In neonates, blood counts are usually normal, but as HbS levels increase and HbF levels decrease, hematologic abnormalities appear. Individuals can also have HbS in combination with other variants and/or mutations. For example, a person may co-inherit a β−chain variant such as HbC, an α-chain variant such as HbG-Philadelphia, or have concurrent β-thalassemia and/or α-thalassemia. (See HbC section below.) A diagnosis of concurrent β+-thalassemia is likely when test results show HbS >50% and HbA <50% with increased HbA 2 and HbF. If HbA is not present and HbA2 and HbF are elevated, concurrent β0 thalassemia should be considered. Patients with S/β0-thalassemia have a clinical course similar to sickle cell disease, but patients with S/β+thalassemia generally have milder disease due to the presence of some HbA. α-Thalassemias Thalassemias are caused either by mutations that reduce the rate of synthesis of a globin chain or by deletion of one or more of the globin genes. α-Thalassemias usually are caused by deletions of one or more of the four α-globin genes. These deletions decrease the synthesis of the protein, thereby creating an overabundance of γ-chains in a fetus or β-chains after HbF disappears. These γ- or β- chains can aggregate and form HbBart’s or HbH, respectively. For patients whose Hb electrophoresis or HPLC analyses and iron studies are normal and the MCV is low, α-thalassemia trait should be considered. However, DNA studies are required for a definitive diagnosis. Table 3 summarizes the effects of α-gene deletions. Table 3 Common α-Thalassemias 1 Genotype Genes Deleted (-α/αα) 1 Silent Carrier None (-α/-α) 2 Homozygous α2Thalassemia Microcytosis (--/αα) 2 α-thalassemia 1 trait +/- anemia (--/-α) 3 HbH Disease Chronic hemolytic anemia (--/--) 4 Hydrops fetalis Lethal Phenotype Clinical Findings β-Thalassemias At least 150 mutations are known to cause β-thalassemia. The condition occurs mainly in people from the Mediterranean region, the Middle East, India, and Southeast Asia. These mutations have been divided into two categories: β0-thalassemias, which involve complete absence of β-chain production; and β+ thalassemias, which result in reduced synthesis of the β-chain. The severity of the disorder varies widely depending on the amount of β-globin produced. Laboratory findings for individuals with β-thalassemia trait include microcytosis, hypochromia, no or mild anemia, and normal or slightly increased RBCs. HbA 2 levels are elevated in these individuals, and HbF may be normal or increased. Inheritance of two β-thalassemia genes causes more severe disease ranging from β-thalassemia intermedia to Cooley’s anemia or β-thalassemia major. Quantitation of HbA2 and HbF Diagnosis of β-thalassemia requires quantitation of HbA2. In general, labs use anion-exchange column chromatography, HPLC or CE for this analysis. Both HPLC and CE provide acceptable quantitation of HbA 2 and HbF in the presence of many Hb variants; however, anion exchange chromatography only measures HbA2 quantitatively. Therefore, labs should use another method in conjunction with anion exchange chromatography to identify Hb variants. Labs can also measure HbF by alkali denaturation if the levels are >50%. Labs should confirm by a second method levels of HbF >10% detected by HPLC. Sickle Solubility Test Another test used by labs specifically for screening or confirming the presence of HbS is the sickling solubility test. Several commercial kits are available that can detect HbS down to a level of 20%. Solubility testing as a screen is not indicated in infants under 6 months of age, as there is a high potential for false-negative results. Significant haemoglobinopathies: guidelines for screening and 2 diagnosis Kate Ryan,1 Barbara J. Bain,2 David Worthington,3 Jacky James,4 Dianne Plews,5 Anthony Mason,6 David Roper,7 David C. Rees,8 Barbara de la Salle9 and Allison Streetly10 Writing group: On behalf of the British Committee for Standards in Haematology http://www.bcshguidelines.com/documents/sign_haemoglo_bjh_042010.pdf Cellulose acetate electrophoresis Haemoglobin electrophoresis at pH 8Æ4–8Æ6 using a cellulose acetate membrane is simple, reliable and rapid. It enables the provisional identification of haemoglobins A, F, S/G/D, C/E/ O-Arab, H and a number of less common variant haemoglobins. Differentiation between haemoglobins migrating to a similar position can be obtained by using electrophoresis on acid (agarose) gels, HPLC or IEF. The provisional identification of any variant haemoglobin should be supported by at least one further unrelated method. Application of an alternative technique will exclude the possibility that a single band in either the S or C position represents a compound heterozygous state such as SD or SG and CE or CO-Arab respectively. If a patient has microcytosis the possibility that a single band represents compound heterozygosity for a variant haemoglobin and b0 thalassaemia must also be considered. Sickle solubility test The kits for sickle cell solubility tests that are predominantly used in the UK will detect haemoglobin S down to a concentration of 20% (and sometimes below; in some cases as low as 8%) (BCSH 1998). The method of Lewis et al (2006), although less sensitive than some commercial kits, can detect Hb S down to a concentration of 20%. The methods are therefore capable of detecting all cases of sickle cell trait beyond the period of infancy, even when there is coexisting a thalassaemia trait (but possibly not when there is coexisting Hb H disease). False positives have been described in patients with high plasma protein levels (Canning & Huntsman, 1970) and in anaemic patients when double the volume of blood is used in the test (Arras & Perry, 1972; Lilleyman et al, 1972). The latter problem can be avoided, however, by using a more concentrated sample of blood or washing the red cells. All positive and equivocal sickle solubility tests should be confirmed by HPLC or an alternative technique both for confirmation of the presence of Hb S and to distinguish sickle cell trait from sickle cell anaemia and from compound heterozygous states. In an emergency, e.g. preanaesthesia, this distinction can be made with reasonable accuracy with a sickle solubility test combined with a blood film and a blood count. It is also recommended that all negative sickle solubility tests be confirmed by HPLC or an alternative technique. 3 Conversely, sickle solubility testing should be employed whenever an unknown haemoglobin is encountered that elutes in the position of the ‘Hb S Window’ by HPLC or is in the position of Hb S on CAE or IEF. In general, a sickle solubility test is not indicated in an infant before the age of 6 months because a negative result may be misleading. However, a sickle solubility test can sensibly be performed in an emergency, prior to anaesthesia, as if it is negative it is unlikely that anaesthesia will cause any clinical problems because the Hb S % will be too low. The wording of the report on such a test must state that a negative test does not exclude the presence of a low percentage of haemoglobin S and that further testing is necessary and will follow. If the patient is anemic, it is necessary to correct the hematocrit to 0.5 in order to avoid false negative results. All negative or equivocal sickle solubility tests be centrifuged before reading in order to increase sensitivity and reliability. It is important not to omit this step if samples have low percentage of Hb S. All sickle solubility tests, whether, positive, negative or equivocal, should be confirmed by Hb electrophoresis or HPLC If rapid results are required, such as before emergency anaesthesia, the distinction between sickle cell trait and sickle cell anaemia or compound heterozugous states can be made with reasonable accuracy by combining a sickle solubility test with a blood film and a blood count. Significant haemoglobinopathies: guidelines for screening and diagnosis Kate Ryan,1 Barbara J. Bain,2 David Worthington,3 Jacky James,4 Dianne Plews,5 Anthony Mason,6 David Roper,7 David C. Rees,8 Barbara de la Salle9 and Allison Streetly10 Writing group: On behalf of the British Committee for Standards in Haematology http://www.bcshguidelines.com/documents/sign_haemoglo_bjh_042010.pdf Pre-operative/pre-anaesthesia It is important to detect SCD prior to anaesthesia because its presence will influence clinical management. Testing should be initiated by clinical staff on the basis of a clinical history and assessment of family origin. All patients from groups with a high prevalence of Hb S (Table I) should be offered testing as some cases of milder disease may be unrecognized and the presence of Hb S heterozygosity may also influence perioperative techniques. Haemoglobin S African including North Africans, African-Caribbeans, African-Americans, Black British and any other African ethnicity (e.g. Central and South Americans of partly African ethnicity), Greeks, Southern Italians including Sicilians, Turks, 4 Arabs, Indians α thalassaemia Chinese, Taiwanese, South-East Asian (Thai, Laotian, Cambodian, Vietnamese, Burmese, Malaysian, Singaporean, Indonesian, Philippino), Cypriot, Greek, Turkish and Sardinian β thalassaemia All ethnic groups other than Northern Europeans Table I. Ethnic groups with a clinically significant prevalence of haemoglobin S and α0 and β thalassaemia. 0 Table 2. Geographic distribution of ethnic populations at increased risk for thalassemia or sickle cell disorders Regions of Origin Africa Mediterranean region e.g., Sardinia, Corsica, Sicily, Italy, Spain, Portugal, Greece, Cyprus, Turkey, Egypt, Algeria, Libya, Tunisia, Morocco, Malta Middle East e.g., Iran, Iraq, Syria, Jordan, Saudi Arabia and other Arabian peninsula countries, Qatar, Lebanon, Palestine, Israel (both Arabs and Sephardic Jews affected), Kuwait South East Asia e.g., India, Afghanistan, Pakistan, Indonesia, Bangladesh, Thailand, Myanmar Western Pacific region e.g., China, Vietnam, Philippines, Malaysia, Cambodia, Laos Caribbean countries South American countries Thalassemia Sickle Cell Disease ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑in parts of India ↑ _ ↑ ↑ ↑ ↑ For routine operations, FBC and haemoglobin analysis using HPLC or a suitable alternative diagnostic method should be performed at the pre-assessment visit. In an emergency, an FBC and a sickle solubility test should be performed. Results in this situation should be evaluated clinically and must be followed by definitive testing (see below). Investigation of microcytosis outside the antenatal situation Recommendation Preoperative testing should be carried out in patients from ethnic groups in which there is a significant prevalence of sickle cell haemoglobin. Emergency screening with a sickle solubility test and full blood count must always be followed by definitive analysis. The need to investigate for thalassaemia and haemoglobinopathies should be considered in patients with unexplained microcytosis. 5 Haemoglobin electrophoresis at pH 8.4 8.6 using a cellulose acetate membrane is simple, reliable and rapid. It enables the provisional identification of haemoglobins A, F, S/G/D, C/E/O-Arab, H and a number of less common variant haemoglobins. Differentiation between haemoglobins migrating to a similar position can be obtained by using electrophoresis on acid (agarose) gels, HPLC or IEF. The provisional identification of any variant haemoglobin should be supported by at least one further unrelated method. Application of an alternative technique will exclude the possibility that a single band in either the S or C position represents a compound heterozygous state such as SD or SG and CE or CO-Arab respectively. If a patient has microcytosis the possibility that a single band represents compound heterozygosity for a variant haemoglobin and β0 thalassaemia must also be considered. Hydropic fetus Neonate or infant with anaemia and either haemoglobin F only or unexpectedly low percentage of haemoglobin A Haemoglobin Bart’s hydrops fetalis β thalassaemia major Unexplained anaemia and splenomegaly β thalassaemia major or intermedia, haemoglobin H disease, unstable haemoglobin Thalassaemias including haemoglobin H disease Haemoglobin S and interacting haemoglobins (C, D-Punjab, O-Arab) or β thalassaemia Haemoglobin H disease, unstable haemoglobin Thalassaemia, variant haemoglobin Suspected thalassaemia or unexplained microcytosis Clinical and haematological features suggestive of sickle cell disease Unexplained haemolysis Unexplained target cells Unexplained irregularly contracted cells Unexplained polycythaemia Unexplained cyanosis with normal oxygen saturation Variant haemoglobin, particularly haemoglobin C or an unstable haemoglobin High affinity haemoglobin Haemoglobin M Table 2. Conditions for haemoglobinopathy investigations in investigation of clinical disorders. Variant haemoglobins, such as Hb S can be quantified by scanning densitometry after electrophoresis/staining; however quantification of haemoglobin A2 by this method is not recommended as the precision is not good enough for the diagnosis of b thalassaemia trait (BCSH 1998). High-performance liquid chromatography (HPLC) High-performance liquid chromatography can be used for the quantification of haemoglobins S, A2 and F and for the detection, provisional identification and quantification of many 6 variant haemoglobins. HPLC usually provides accurate quantification of Hb A2 and is therefore suitable for the diagnosis of b thalassaemia trait. High-performance liquid chromatography usually separates haemoglobins A, A2, F, S, C, DPunjab and G-Philadelphia from each other. However, both Hb E and Hb Lepore often co-elute with A2 (as other haemoglobins co-elute with A, S and F) but may be recognized by alternative techniques. HPLC has the disadvantage that it also separates glycosylated and other derivative forms of haemoglobin, which can make interpretation more difficult. For example, derivatives of haemoglobin S co-elute with haemoglobin A2, rendering its quantification inaccurate. Careful examination of every chromatogram is essential. As with every method of haemoglobin analysis, controls should be run with every batch. Identification of variants is only provisional, and unrelated second-line methods should be used for confirmation. If HPLC is used as the screening technique, it is essential to check and maintain the positions of the windows, which are used as the first stage identification of any variants found. This is generally done by adjusting the column temperature or the flow rate so that the Hb A2 peak appears at a standard time. This is just as important as the calibration of the Hb A2 and Hb F levels and should be checked daily. Appropriate controls should be included wherever possible. Sickle solubility test The kits for sickle cell solubility tests that are predominantly used in the UK will detect haemoglobin S down to a concentration of 20% (and sometimes below; in some cases as low as 8%) (BCSH 1998). The methods are therefore capable of detecting all cases of sickle cell trait beyond the period of infancy, even when there is coexisting a thalassaemia trait (but possibly not when there is coexisting Hb H disease). False positives have been described in patients with high plasma protein levels (Canning & Huntsman, 1970) and in anaemic patients when double the volume of blood is used in the test (Arras & Perry, 1972; Lilleyman et al, 1972). The latter problem can be avoided, however, by using a more concentrated sample of blood or washing the red cells. All positive and equivocal sickle solubility tests should be confirmed by HPLC or an alternative technique both for confirmation of the presence of Hb S and to distinguish sickle cell trait from sickle cell anaemia and from compound heterozygous states. In an emergency, e.g. preanaesthesia, this distinction can be made with reasonable accuracy with a sickle solubility test combined with a blood film and a blood count. It is also recommended that all negative sickle solubility tests be confirmed by HPLC or an alternative technique. Conversely, sickle solubility testing should be employed whenever an unknown haemoglobin is encountered that elutes in the position of the ‘Hb S Window’ by HPLC or is in the position of Hb S on CAE or IEF. In general, a sickle solubility test is not indicated in an infant before the age of 6 months because a negative result may be misleading. However, a sickle solubility test can sensibly be performed 7 in an emergency, prior to anaesthesia, as if it is negative it is unlikely that anaesthesia will cause any clinical problems because the Hb S % will be too low. The wording of the report on such a test must state that a negative test does not exclude the presence of a low percentage of haemoglobin S and that further testing is necessary and will follow. The following techniques can be used for haemoglobin variants: • High performance liquid chromatography (HPLC) • Isoelectric focusing (IEF) • Cellulose acetate electrophoresis (CAE) Abnormal results should be confirmed by a different technique that is appropriate for the likely variant. Another technique that can be used for confirmation, besides those listed above, is acid agar or acid agarose electrophoresis although this is not suitable as a screening technique. Sickle solubility testing can be used as confirmation of an initial screen that suggests the presence of sickle haemoglobin. Screening for thalassaemia. Methods used are red cell indices in conjunction with measurement of Hb A2 levels. Routine measurement of blood indices includes measurements of MCH and mean cell volume (MCV); it is recommended that MCH is used to screen for thalassaemia as this parameter is more stable than MCV. Hb A2 is quantified by HPLC or microcolumn chromatography. HPLC system A national recommended cut-off Hb A2 of 3.5% or above has been set as the action point in the diagnosis of carriers of b thalassaemia. A value of 5.0% for Hb F has been set for the investigation of a raised fetal haemoglobin in pregnancy. In a patient with an MCH below the cut-off point (<27 pg), further investigation will be required if the total Hb A2 is above 3.5%. The a thalassaemia risk needs to be considered in the light of the family origin of the patient. The major risk is for b thalassaemia, but the risk of Hb Bart’s hydrops fetalis should not be overlooked. Hb A2 values >4.0% with normal indices may indicate b thalassaemia trait with or without coexisting a thalassaemia. 8 In this case: • Re-analyse FBC • Repeat Hb A2 • Consider B12/folate deficiency, drugs, liver disease/alcohol or HIV infection Hb A2 values ≤4.0% with normal red cell indices and a normal Hb F level can usually be regarded as normal, although some mild b thalassaemia alleles (mainly in subjects of Mediterranean origin) are associated with an A2 of 3.5–4.0%. Interpretation of results in the presence of iron deficiency Severe iron deficiency anaemia(Hb<80 g/l)can reduce theHbA2 level slightly (by up to 0.5%). Outside of pregnancy, anaemia should be treated and the haemoglobin analysis repeated when the patient is iron replete. In pregnant women there is no justification for delaying the investigation for haemoglobinopathies whilst treating iron deficiency presumptively, as this will delay the process of identifying at-risk carrier couples, who could be offered prenatal diagnosis. Neonatal samples Neonatal samples are typically composed of mostly Hb F (approximately 75%) with approximately 25% Hb A and small quantities of acetylated Hb F and sometimes Hb Bart’s. It is also important to realize that occasionally the presumptive identification of a haemoglobin variant using screening methods is incorrect, because some variants give exactly the same results using current screening techniques. The sensitivity and specificity are approximately 99% for the methods used. Unequivocal identification of haemoglobin variants can only be achieved by either protein analysis (e.g. mass spectrometry) or DNA analysis. Recommendation Abnormal laboratory screening results should be confirmed by a different technique that is appropriate for the likely abnormality. Quantification of Hb A2 by CAE plus scanning densitometry 9 is not recommended. A sickle cell solubility test is not generally indicated in infants below the age of 6 months and is not recommended as a primary screening tool except in an emergency situation. All sickle solubility tests should be confirmed by HPLC or an alternative method. Assessment of iron status may be useful in the interpretation of laboratory tests since in severe iron deficiency anaemia(Hb<80 g/l)can reduce the HbA2 level slightly (by up to 0.5%). Examination for Hb H bodies cannot reliably distinguish between a thalassaemia traits and should not be used for screening. Laboratories should be aware of the effect of blood transfusion on the interpretation of results. Haemoglobin interpretation is misleading after a recent blood transfusion and necessitates repeat testing after 4 months if a pre-transfusion sample has not been analysed. DNA testing for the sickle gene is now recommended for transfused neonates to avoid the need for a repeat specimen at 4 months posttransfusion, although a pre-transfusion specimen is still the preferred specimen. No technique can identify all abnormalities but the combined sensitivity/specificity of the HPLC and IEF techniques for haemoglobins present at the time of screening is approximately 99%. The pattern of haemoglobin variants is not unique however and whilst some will be clarified by using the second technique, unequivocal identification can usually only be made by DNA analysis or mass spectrometry. The diagnosis of a thalassaemia is more complicated because DNA analysis is the only accurate way to distinguish between a+ and a0 thalassaemia. However it is not practical to seek to confirm all potential cases of a thalassaemia by DNA analysis because the a+ form is too common and not usually clinically important; it is not cost-effective for DNA laboratories to perform analysis on all such cases. Furthermore, non-deletional forms of a+ thalassaemia are more common than was thought and rapid methods for their detection are not available. If a blood transfusion has been received within 4 months, misleading data and conclusions may result. Analytical fact should be separated from interpretative opinion. The factual results should be given first and should 10 be followed by a clear conclusion, which may include recommendations. If information from the blood count is used in coming to a conclusion about the significance of the analytical data (as in probable a thalassaemia) then those aspects of the blood count used (such as haemoglobin concentration, red cell count, MCH, MCV) must be included in the haemoglobinopathy report. Similarly, if information on ethnicity/family origin is used, it should be stated in the report. Results of the sickle solubility test, in the absence of resultsfrom an unrelated confirmatory method, should only be reported as an ‘interim’ report. The final report with information from the blood film, HPLC and/or electrophoresis and any other appropriate tests should follow as soon as possible. As it improves clarity, the conclusion should always be given both in full text and in standard abbreviation form in parentheses. For example: Sickle Cell Carrier (AS) or Sickle Cell Anaemia (SS). Laboratory Investigation of Hemoglobinopathies and Thalassemias: Review and Update 1. 2. Gwendolyn M. Clarke1 and Trefor N. Higginsa,1 + Author Affiliations 1. 1. Dynacare Kasper Medical Laboratories, 14940 123rd Ave., Edmonton, Alberta T5V 1B4, Canada. 1 ↵aAuthor for correspondence. Fax 780-452-8488; e-mail [email protected]. http://www.clinchem.org/content/46/8/1284.full cbc Structural hemoglobinopathies may have an impact on the red cell indices, and red cell indices are critical to the diagnosis of thalassemias. The key components of the CBC include: Hb, red blood cell (RBC) number, mean corpuscular volume (MCV), and red cell distribution width (RDW). The thalassemias generally are classified as hypochromic and microcytic anemias. Hence the MCV is a key diagnostic indicator. Virtually all automated hematology analyzers now provide a measurement of MCV that is both precise and accurate. This cell volume, reported in femtoliters, in most adult populations ranges from ∼80 to 100 fL. Thalassemic individuals have a reduced MCV, and one study has suggested that an MCV of 72 fL is maximally sensitive and specific for presumptive diagnosis of thalassemia syndromes (13). 11 The RDW is a measure of the degree of variation in red cell size. Some causes of microcytic anemia, most notably iron deficiency, are characterized by an increase in RDW. The thalassemias, in contrast, tend to produce a uniform microcytic red cell population without a concomitant increase in RDW. This observation is variable among the thalassemia syndromes, however, with notable increases in RDW in the setting of Hb H disease and δ β-thalassemia minor (1). Therefore, the RDW may provide information useful as an adjunct to diagnosis but is not useful as a lone indicator. The RBC count is also useful as a diagnostic adjunct because the thalassemias produce a microcytic anemia with an associated increase in the RBC number. Other causes of microcytic anemia, including iron deficiency and anemia of chronic disease, are more typically associated with a decrease in the RBC number that is proportional to the degree of decrease in Hb concentration. The Hb concentration typically is decreased in thalassemia. The thalassemia minor conditions produce minimal decrements in the Hb concentration, whereas thalassemia intermedia and thalassemia major may be associated with moderate to severe decreases in Hb concentration. Various indices utilizing these CBC components have been developed with a view to providing a mathematical derivation to reliably differentiate iron deficiency from thalassemia minor. None are useful in all clinical settings, and probably none exceed the value of the MCV alone in selecting cases for subsequent investigations (13). Carrier Screening for Thalassemia and Hemoglobinopathies in Canada 2008 http://www.sogc.org/guidelines/documents/gui218CPG0810.pdf Individuals with two deleted copies of the a-globin gene have α-thalassemia trait. These individuals are either heterozygotes for α0-thalassemia (αα/—) or homozygotes for α+-thalassemia (α-/α-). Both of these types of α-thalassemia trait (i.e. αα/—or α-/α-) are essentially identical clinically and on routine hematology testing. Patients are generally asymptomatic. A CBC will typically show microcytosis (low MCV, e.g., < 80 fL) and hypochromia (low MCH, e.g., < 27 pg); the patient may also be mildly anemic. Despite anemia, the red blood cell count is often mildly elevated. Hb electrophoresis and Hb HPLC are normal in -thalassemia trait after the newborn period, and the HbA2 level is normal, which is not the case in -thalassemia trait (see below for a discussion of HbA2 in -thalassemia). -Thalassemia trait is usually diagnosed by staining a peripheral blood smear with brilliant cresyl blue to detect abnormal red blood cell inclusions called H bodies. 12 Patients with Hb H disease are usually anemic, microcytic, and hypochromic on routine hematologic testing. Hb electrophoresis and HPLC may show an abnormal hemoglobin, Hb H, although in some cases this may be difficult to detect or require special techniques. On H-body staining, almost every red blood cell will show HbHinclusions. Disease α+-thalassemia silent carriers α-thalassemia trait Genotype/ Clinical Findings Ethnic Group at Risk (αα/α-) These individuals are asymptomatic usually found by chance among various ethnic populations, particularly African American Individuals with two deleted copies of the aglobin gene: either heterozygotes for α0thalassemia (αα/—) or homozygotes for α+-thalassemia (α-/α-) Patients are generally asymptomatic. The patient may be mildly anemic This condition is encountered mainly in Southeast Asia, 13 Haematological Findings These generally have normal routine hematologic findings: normal Hb, MCV, and MCH. Rarely the MCV and/or MCH can be low. Hb electrophoresis, Hb HPLC, and H body staining of a peripheral blood smear are usually negative outside the newborn period. In this form, the diagnosis cannot be confirmed based on hemoglobin electrophoresis results, which are usually normal in all α thalassemia trait. Genetic, molecular (DNA) testing testing is necessary to confirm the diagnosis. A CBC will typically show microcytosis (low MCV, e.g., < 80 fL) and hypochromia (low MCH, < 27 pg); Occasional target cells; the red blood cell count is often mildly elevated. Hb electrophoresis and Hb HPLC are normal, and the HbA2 level is normal. It is usually diagnosed by staining a peripheral blood smear with brilliant cresyl blue to detect abnormal red blood cell inclusions H bodies. the finding of H bodies in the right clinical and ethnic context is typically considered diagnostic of α-thalassemia trait. the Indian subcontinent, and some parts of the Middle East. Hb H disease ( α thalassemia intermedia) ( α-/—) three-gene deletion. Patients with Hb H disease have one functioning α globin gene they inherit α0thalassemia (— /) from one parent and α+-thalassemia (α-/) from the other. This condition is in most cases clinically mild: many patients require occasional transfusion of red blood cells, but these patients are usually not considered “transfusion dependent.” However, marked phenotypic variability has been noted, and some patients do require regular transfusions to survive. There are even occasional cases of Hb H disease presenting as hydrops fetalis.Patients with Hb H disease are usually anemic 14 (αα/—) and patients with single deletions on both chromosomes (α-/α-) have essentially the same results in all these tests, and can be differentiated only by molecular methods. Such molecular distinction is crucial for the identification of a couple at risk for having a fetus with four-gene deletion α-thalassemia (hemoglobin Bart’s hydrops fetalis, or Hb Bart’s disease,—/—). Hb Bart’s disease can occur only when the fetus inherits a double deletion from each parent, that is, when both parents are carriers of α0-thalassemia, (αα/—). In such a situation, the couple has a 25% risk of having a fetus with all four α globin genes deleted. A CBC will typically show microcytosis (low MCV, e.g., < 80 fL) and hypochromia (low MCH, e.g., < 27 pg). Hb electrophoresis and HPLC may show an abnormal hemoglobin, When peripheral blood films stained with supravital stain or reticulocyte preparations are examined, unique inclusions in the RBCs (Hemoglobin H bodies) are typically observed in almost every red blood cell. These inclusions represent β chain tetramers (Hemoglobin H), which are unstable and precipitate in the RBC, giving it the appearance of a golf ball. Hemoglobin Bart’s hydrops ( α Thalassemia Major) (—/—) four-gene deletion Silent Carrier β Thalassemia The mutation that causes the thalassemia is a β+ thalassemia mutation which diminishes but does not eliminate beta globin chain production from one of two beta globin genes. individuals with one normal gene and one affected gene (genotypically represented as β-thalassemia trait or β-thalassemia minor Svere intrauterine anemia resulting in fetal hydrops and, in almost all cases, intrauterine death. There are only rare case reports of infants surviving with this condition. Without any α globin production, these fetuses are unable to make any fetal (HbF) or adult (HbA) hemoglobin. Instead, they can produce only embryonic hemoglobins, which generally cannot support life past the third trimester. Some of these fetuses will also have congenital abnormalities such as terminal limb defects. these patients have minor changes in RBC indices no symptoms often asymptomatic. Patients have mild anemia. CBC will show mild or no anemia, and MCV and MCH are usually low RBC count is often high. The routine diagnostic test is Hb 15 B/B+ or B/B0). The production of β chains from the abnormal allele varies from complete absence to variable degrees of deficiency. β -thalassemia intermedia or β -thalassemia major HbS/ β -thalassemia Two mutated β globin genes are inherited, one from each Parent. Genotypically, intermedia is usually β+/ β+ or possibly β+/ β0 Two mutated β globin genes are inherited, one from each Parent. there is either zero or almost zero β globin chain synthesis in β thalassemia major (β+/ β0 or β0/ β0). The most common variant hemoglobins that may be electrophoresis or Hb HPLC: these will demonstrate increases in HbA2 (i.e., > 3.5% of total hemoglobin) and usually HbF (i.e., >1%). In the right clinical and ethnic context, an elevated HbA2 is considered diagnostic of β-thalassemia trait. This condition results in anemia of intermediate severity, which typically does not require regular blood transfusions This condition is characterized by transfusiondependent anemia, massive splenomegaly, bone deformities, growth retardation, and peculiar facies in untreated individuals, 80% of whom die within the first 5 years of life from complications of anemia. can produce a sickling syndrome of variable severity 16 Peripheral blood film examination usually reveals marked hypochromia and microcytosis (without the anisocytosis usually encountered in iron deficiency anemia), target cells, and faint basophilic stippling. patients will be anemic,microcytic, and hypochromic, and Hb electrophoresis or HPLC will show elevated HbA2. The peripheral blood films reveal hypochromia, microcytosis And anisocytosis By 6 to 12 months of age, patients will be anemic,microcytic, and hypochromic, and Hb electrophoresis or HPLC will show elevated HbA2. The peripheral blood films reveal severe hypochromia and microcytosis, marked anisocytosis, fragmented RBCs, polychromasia, nucleated RBCs, and, on occasion, immature leukocytes. HbE/ β-thalassemia co-inherited with βthalassemia HbE mutation from one parent and a βthalassemia mutation from another. Extremely common in some Southeast Asian populations, reaching a gene frequency of up to 70% in northern Thailand. Hemoglobin C/ β-thalassemia Sickle Cell Anemia (HbSS) Homozygous HbS disease No normal HbA is produced: instead, red cells contain May vary in its clinical severity from as mild as thalassemia intermedia to as severe as β thalassemia major HbE/ β-thalassemia is one of the most important causes of clinically severe thalassemia worldwide. Hemoglobin C/ β-thalassemia is clinically and hematologically very heterogeneous, ranging from very mild to very severe. Individuals with Hb C Beta + thalassemia have a mild anemia, low MCV, and target cells. Individuals with Hb C Beta 0 thalassemia have a moderately severe anemia, splenomegaly, and may have bone changes. If they inherit B+ thalassemia there is 65 - 70% Hb C, 20 - 30% Hb A, and increased Hb F. If they inherit Beta 0 thalassemia on electrophoresis there is no Hb A and increased Hb F with Hb C. In HbSS, sickled RBCs have a considerably shorter life span than normal RBCs (due to extravascular sickled RBCs and target cells. Howell- Jolly bodies seen when there is splenic atrophy. 17 primarily HbS (with small amounts of HbA2 <1% and HbF (5%10%). HbS heterozygotes HbAS SC disease co-inheritance of HbS and HbC Sickle-β+ thalassemia co-inheritance of HbS with Bthalassemia S/B+ or S/Bo β+thal/βs hemolysis) and cause intermittent episodes of vascular occlusion under conditions of decreased oxygen tension. This causes tissue ischemia, and acute and chronic organ dysfunction involving the spleen, brain, lungs, and kidneys. Pain and swelling of hands and feet (hand-foot disease) is a frequent early presentation of this disease in infants and young children. The hemolysis leads to chronic anemia and predisposes the patient to aplastic crises. heterozygous have normal red cell indices on carriers are CBC, with normal RBC asymptomatic. morphology on peripheral blood smear. Both HbSS and HbAS are routinely diagnosed by Hb electrophoresis or HPLC. Mild anemia, vasoocclusive problems. they have a sickle syndrome which is very similar to sickle cell anemia Mild sickle cell hematological findings are disease comparable to sickle cell anemia (with some differences, such as microcytosis in S/B- thalassemia but not in HbSS) Differentiating S/Bo -thalassemia from HbSS requires not only Hb HPLC but also additional 18 nformation such as CBC results, physical examination findings, and occasionally family or molecular studies. SD disease co-inheritance of HbS with HbD βs/βD Punjab inherit a Hb C gene from each parent As for sickle cell anemia Hemoglobin C carriers inherit Hb C from one parent and Hb A from the other. They have no Usually have target cells on blood anemia but Usually smear and may have a slightly have target cells on low MCV. blood smear and may have a slightly low MCV. There are no other clinical problems. HbSC disease HbA is not present. The RBCs contain 50% HbS and 50% HbC. Anemia is much milder, with Hb levels of 11 g/dL or higher. Hb CC disease They have a mild hemolytic anemia. There may be very occasional episodes of joint and abdominal pain which are attributed to Hb CC disease. Splenomegaly is common. Aplastic crises and gall stones may occur. microcytosis, and target cell formation The peripheral blood smear may have some sickled cells and a high proportion of target cells. In addition, microcytic, dehydrated, dense RBCs are seen. These may contain crystal-like condensations. HbS variants may occur as double heterozygotes with other Hb variants. These include HbD, HbE, and HbO Arab. Double 19 heterozygosity for certain variants (e.g., Hb S/Hb D Los Angeles, Hb Montgomery/ Hb S) that occur with appreciable frequency in the same ethnic populations as Hb S may also produce significant sickling disease decreased (β+ ) or absent (β0) β globin production Recommendations for thalassemia investigation An MCV ≥ 80 fL and an MCH ≥ 27 pg, with a normal electrophoresis or HPLC, requires no further testing. The finding of a normal MCV (i.e.,≥ 80 fL) in combination with a normal MCH (i.e., ≥ 27 pg) would rule out most cases of thalassemia and would require no additional thalassemia testing. Individuals with an MCV < 80 fL or MCH < 27 pg can have α- and/or β-thalassemia and/or iron deficiency anemia. For individuals with MCV < 80 fL or MCH < 27 pg, the next step is hemoglobin electrophoresis or HPLC, quantitation of HbA2 and HbF, and a blood smear stained for H bodies. It is appropriate to recommend that all pregnant women from an ethnic background at increased risk of hemoglobinopathy and/or thalassemia be screened by both CBC, to assess the MCV and MCH, and a hemoglobin electrophoresis or HPLC. In general, β-thalassemia trait can be reliably diagnosed by hemoglobin electrophoresis or HPLC, with HbA2 and HbF quantitation. Patients with β-thalassemia trait have an elevated HbA2, i.e., > 3.5%. 20 In patients with a low MCV, but with a normal Hb electrophoresis/HPLC and HbA2 and HbF quantitation, the differential diagnosis includes iron deficiency anemia and α-thalassemia. A serum ferritin (to exclude iron deficiency anemia) and a smear to screen for the H bodies of α-thalassemia are therefore required. For pregnant patients, these tests (ferritin and H body stain) should be ordered concurrently. In the right clinical context (e.g., microcytic anemia in an “ethnically appropriate” patient), the presence of H bodies can identify the patient as a carrier of α-thalassemia. H body testing is not 100% sensitive (see below), and therefore the absence of H bodies does not completely exclude α-thalassemia carrier status in an ethnically at-risk patient. If iron deficiency is ruled out in a pregnant woman with a negative H body test, testing of the partner remains crucial to determine the risk of having an affected fetus with Hemoglobin Bart’s hydrops fetalis. Molecular studies may also be done to confirm or exclude carrier status for α-thalassemia. Hemoglobin electrophoresis or HPLC will allow identification of Hb variants, such as HbS, C, D, and E. A phenotypic sickle cell preparation (such as a slide or tube sickling test) is not helpful in identifying other types of β globin variants besides HbS, so should not be used as a carrier screen for hemoglobinopathies. The finding of any abnormality (e.g., low MCV or MCH, or abnormal hemoglobin electrophoresis or HPLC) requires screening of the partner, which entails CBC, smear for H bodies, hemoglobin electrophoresis or HPLC, and HbA2 and HbF quantitation. If the patient is pregnant, testing of the partner should be done promptly. B-thalassemia trait or -thalassemia minor. The CBC will show mild or no anemia, and MCV and MCH are usually low. Like -thalassemia trait, the RBC count is often high. Because reduced production of globin means an inability to generate as much normal HbA (22), they compensate by increasing production of other -like chains, namely and , leading to increases in the levels of the minor hemoglobins HbA2 (22) and HbF (22). The routine diagnostic test is Hb electrophoresis or Hb HPLC: these will demonstrate increases in HbA2 (i.e., > 3.5% of total hemoglobin) and usually HbF (i.e., >1%).12 In the right clinical 21 and ethnic context, an elevated HbA2 is considered diagnostic of -thalassemia trait. 22