Anemias - Revisão da Classificação e Análise Diagnóstica

Artigo de revisão Anemias para atividade substitutiva da aula 1 Revisão da classificação das anemias faça um resumo explicando em que se baseiam as classificações das anemias conforme artigo anexo Págs 264265 2 Utilizando o mesmo artigo associe duas anemias com as alterações das fotos das lâminas Ex Anemias hemolíticas Anemia falciforme Foto 6 Envie sua resposta até dia 08082023 Esta atividade terá um valor de 05 na nota da prova prática Anemia Evaluation and Diagnostic Tests Michael J Cascio MDa Thomas G DeLoughery MD MACP FAWMbc KEYWORDS Red cell indices Schistocytes Microcytic Macrocytic Cytogenetics Anemia Diagnostic testing KEY POINTS Both the red cell indices and blood smear can offer clues to diagnosis and help to guide laboratory testing Classification of anemia by either size of the red cell or mechanism decreased production or increased loss can narrow down the differential diagnosis New molecular technologies may offer improved diagnostic sensitivity and specificity ANEMIA DEFINITION Although anemia is common the exact cutoff to establish a diagnosis can be elusive The standard definition is populationbased and varies by gender and race Current hemoglobin cutoff recommendations range from 13 to 142 gdL in men and 116 to 123 gdL in women1 Data from large population studies suggests that hemoglobin levels for African Americans tend to be 08 to 07 gdL lower perhaps owing to the high frequency of alphathalassemia in this population2 Another important factor is the trend of hemoglobin For example a patient with previous hemoglobin values at the higher end of the normal range who now presents with a hemoglobin concentration at the lower end of the normal range can now be considered anemic SYMPTOMS AND SIGNS OF ANEMIA In general the signs and symptoms of anemia are unreliable in predicting the degree of anemia Several factors determine the symptomatology of anemia with time of The authors report no conflict of interest a Department of Pathology Oregon Health and Science University 3181 SW Sam Jackson Park Road MC L471 Portland OR 97239 USA b Division of HematologyMedical Oncology Department of Medicine Knight Cancer Institute Oregon Health and Science University MC L586 3181 Southwest Sam Jackson Park Road Portland OR 97239 USA c Division of Laboratory Medicine Department of Pathology Oregon Health and Science University Hematology L586 3181 Southwest Sam Jackson Park Road Portland OR 972013098 USA Corresponding author Division of HematologyMedical Oncology Department of Medicine Knight Cancer Institute Oregon Health and Science University MC L586 3181 Southwest Sam Jackson Park Road Portland OR 97239 USA Email address deloughtohsuedu Med Clin N Am 101 2017 263284 httpdxdoiorg101016jmcna201609003 medicaltheclinicscom 0025712517 2016 Elsevier Inc All rights reserved onset and overall baseline health of the patient being the most important Patients who gradually develop anemia over a period of months can tolerate lower hemoglobin owing to the use of compensatory mechanisms An example would be a patient with sickle cell disease who can tolerate a chronic hemoglobin concentration of 7 gdL Because blood delivers oxygen many of the signs are related to lack of oxygen deliv ery chiefly fatigue and shortness of breath On physical examination anemia is man ifested by paleness of the mucous membranes and resting tachycardia One should look for other physical examination clues to a possible source of anemia such as splenomegaly guaiacpositive stools or oral telangiectasia COMPENSATION FOR ANEMIA There are 3 physiologic compensatory mechanisms for anemia The first is by increasing cardiac output Because oxygen delivery is cardiac output times hemo globin patients with decreased hemoglobin can maintain the same level of oxygen delivery by increasing cardiac output Therefore patients with limited cardiac reserve heart failure coronary artery disease will have symptoms at higher hemo globin concentrations than those with normal cardiac function Increasing plasma volume is the second compensatory mechanism This allows the remaining red cells to move around more efficiently owing to decreased viscosity The increased plasma volume also increases cardiac output and helps to maintain blood pressure Finally red cell 23diphosphoglycerate increases which decreases oxygen affinity for hemoglobin This results in more oxygen delivery to tissues The high ambient oxygen tension in the alveoli leads to full oxygenation of hemoglobin despite its decreased oxygen affinity but at the tissue level this results in more delivery of oxygen CLASSIFICATION There are 2 classification systems for anemia Box 1 The first is based on Wintrobe observations that red cell size can differentiate potential etiologies of anemia This led to the concepts of microcytic normocytic and macrocytic anemia3 Microcytic anemias are those with a mean corpuscular volume MCV less than normal 80 fL Microcytic anemias reflect defects in hemoglobin synthesis Lack of iron either owing to deficiency or sequestration anemia of inflammation thalassemia or sideroblastic anemias defect of heme synthesis all can lead to microcytosis There are 2 general etiologies of macrocytic anemias MCV 100 fL4red cell membrane defects and DNA synthesis defects Red cell membrane defects can occur in the setting of liver disease or hypothyroidism Macrocytic red blood cells RBCs in this setting tend to be round on review of the peripheral smear In contrast defects in DNA synthesis such as those seen with megaloblastic anemia or chemotherapy show a prominent oval macrocytosis One of the most common causes of macrocytic anemia is the presence of a reticulocytosis The average size of the reticulocyte 160 fL can yield an high MCV in the setting of hemolysis The difficulty in using red cell size as a means of distinguishing potential etiologies for anemia is that in many cases the red cells demonstrate a normal size normo cytic anemia MCV 80100 fL This may occur during early stages of a process such as iron deficiency or when multiple processes occur simultaneously concur rent iron deficiency and liver disease and lead to a red cell size within the normal range The other classification schema uses the underlying mechanism of anemia increase in RBC loss or decrease in RBC production The first branch point is if red cell Cascio DeLoughery 264 Box 1 Classification of anemia Size Microcytic Iron deficiency Thalassemia Sideroblastic anemia Anemia of inflammation Macrocytic Round Aplastic anemia Hypothyroidism Liver disease Renal disease Reticulocytosis Thyroid disease Oval Vitamin B12 and folate deficiency Chemotherapy Myelodysplastic syndrome Normocytic Anemia of inflammation Acute onset hemolysis or blood loss Renal disease Mechanism Increased loss Hemorrhage Hemolysis immune microangiopathic intrinsic red blood cell defects Decreased production Stem cell myelodysplastic syndrome acute leukemia Nutritional iron vitamin B12 folate copper Toxindrug Lack of growth factors renal disease anemia of chronic disease Myelophthisic process metastatic cancer infection fibrosis production is increased or decreased as determined by the reticulocyte count If increased then hemolysis and blood loss are primary considerations If red cell production seems to be decreased then basic causes of impaired marrow production should be considered Nutritional iron vitamin B12 folate copper deficiency Marrow failure aplastic anemia pure red cell aplasia myelodysplasia leukemia Lack of growth factors lack of erythropoietin EPO owing to chronic renal disease Myelophthisic process cancer infection DIAGNOSTIC TESTS Diagnostic testing should focus on 1 determining whether an anemia is present and 2 identifying the underlying etiology Basic assays complete blood count reticulocyte count and blood smear will be reviewed first with more specific assays covered elsewhere in this article Complete Blood Count The complete blood count is probably one of the most widely performed tests in the world The complete blood count was originally performed by manual methods but currently there are 2 main forms of automated technology that are used Each auto mated method directly measures the number of cells the volume of individual cells and the hemoglobin concentration The first automated method was devised by Wallace Coulter in the 1950s and relies on electrical impedance When RBCs flow through an aperture in a currentconducting solution the nonconducting RBCs induce a momentary alteration in current imped ance between the sensing electrodes Each impedance pulse correlates with an indi vidual RBC passing through an aperture whereas the magnitude of the impulse is directly proportional to cell size A newer method uses laser light scatter properties In this assay RBCs are hydro dynamically focused in a flow cell and a beam of laser light is applied A photodetector then captures the light scatter emitted from each cell The photodetector then con verts this light scatter to electrical impulses the number of which is proportional to the number of RBCs passing through the laser Similar to the impedance method the amplitude of the light scatter pulse is proportional to RBC size Hemoglobin concentration is determined by photospectometry after erythrocyte lysis Although red cell number size and hemoglobin concentration are directly measured the hematocrit is a calculated value and can be less reliable when there is inaccuracy in the measurements of the other red cell indices Peripheral Blood Smear Assessment The blood smear can offer valuable diagnostic clues to the etiology of anemia Table 1 The clinician can request a formal blood smear review by the laboratory technologist or pathologist to assess red cell white cell and platelet morphology When initiating such requests it is important to convey relevant clinical and laboratory findings that may not be available to the pathologist Assessment of red cell size is an important part of the peripheral smear evaluation Normocytic red cells are approximately the same size as the nucleus of a small resting lymphocyte Fig 1 Red cells that are larger than the lymphocyte nucleus are consid ered macrocytic and those that are smaller are microcytic As noted when a macro cytic anemia is present it is important to distinguish round and oval macrocytosis owing to differing underlying etiologies Microcytosis may be seen in the setting of iron deficiency hemoglobin H disease thalassemia minor sideroblastic anemias and in some cases of anemia of chronic disease When evaluating a microcytosis assessment for hypochromia can be helpful in determining whether the changes may be owing to or in part reflect iron deficiency In normal red cells central pallor occupies approximately onethird of the RBC diam eter An increase in the central pallor is indicative of hypochromia which generally oc curs at hemoglobin concentrations less than 10 gdL and the degree of which correlates with the severity of the anemia Figs 2 and 3 The differential diagnosis for microcytic hypochromic anemia includes thalassemia iron deficiency sideroblas tic anemias and anemia of chronic disease Anisocytosis and poikilocytosis refer to the variation of red cell size and shape respectively Although a mild increase in anisopoikilocytosis lacks diagnostic speci ficity it is important to note whether the RBC abnormality observed is a high frequency or low frequency abnormality High frequency abnormalities as the name suggests are RBC types that occur more often in normal blood films Examples include Cascio DeLoughery 266 acanthocytes echinocytes target cells and ovalocytes In contrast low frequency abnormalities are rare or seldom seen in blood of healthy individuals These include schistocytes tear drop cells and blister cells among others The severity of the poi kilocytosis can be estimated based on the relative percent of each abnormal RBC type observed which are commonly reported by the laboratory in a tiered grading scheme Schistocytes or helmet cells are red cell fragments that contain 2 or 3 pointed ends Fig 4 The presence of an increased number of schistocytes implies intravascular damage to red cells The mechanism of schistocyte formation involves mechanical shearing of the red cell membrane by intravascular deposits such as fibrin strands or platelet aggregates Schistocytes can be increased in the setting of disseminated intravascular coagulation thrombotic thrombocytopenia purpura hypertension pre eclampsia mechanical heart valves and a ventricular assist device Spherocytes appear as round slightly smaller red cells that lack central pallor and have a more deeply red appearance than other red cells Spherocytes are formed through loss of red cell membrane this converts the biconcave disk most surface area for a given volume to a sphere which is the least amount of surface area An increased number of spherocytes is seen in hereditary spherocytosis hemolytic ane mias mediated by immune mechanisms and in microangiopathic hemolytic anemias see Fig 4 Table 1 RBC morphologic findings RBC Finding Mechanism Clinical Setting Coarse basophilic stippling Remnant of ribosomes Lead poisoning marrow stress thalassemia Bite cells Hemoglobin denaturation and removal by RES Oxidative hemolytic anemia Burr cells Membrane alteration related to increased lipids calcium Artifact liver disease uremia hyperlipidemia Acanthocytes Increased cholesterol in RBC membrane Liver disease abetalipoproteinemia McLeod syndrome Elliptocytes RBC cytoskeletal defects Heredity elliptocytosis iron deficiency HowellJolly bodies RNA remnants Splenectomy or functional hyposplenism Nucleated red cells Retention of nucleus Splenectomy marrow stress myelopthisis Schistocytes Mechanical RBC damage Microangiopathic hemolytic anemia Reticulocytosis shift cells High RNA content Hemolysis marrow stress Spherocytosis Loss of RBC membrane cytoskeletal defect Hereditary spherocytosis autoimmune hemolytic anemia Target cells Excess RBC membrane cholesterol decreased hemoglobin Thalassemia hemoglobin C and E disease obstructive liver disease Tear drop cells Extension of RBC cytoplasm Myelopthisis fibrosis extramedullary hematopoiesis Abbreviations RBC red blood cells RES reticuloendothelial system Anemia 267 Fig 1 Normal peripheral blood smear original magnification 400 Red blood cells RBCs show a normal central pallor less than onethird of the diameter of the RBC and have a size that is roughly equal to the size of the nucleus of a small lymphocyte red arrows Normal neutrophils black arrows with segmented nuclei and pink cytoplasm are shown Platelets with normal size and bluepurple alpha granules are easily identified in the background black arrowheads Fig 2 Microcytic hypochromic anemia original magnification 630 This peripheral blood film was from a patient with iron deficiency anemia Note the dimorphic red cell populations One population black arrowheads seems to be smaller than the nucleus of a small lymphocyte black arrow and shows an increase in central pallor The second population shows a more normal size and lacks central pallor less than onethird of the red blood cell diameter red arrows Fig 3 Hypochromic microcytic anemia original magnification 630 In contrast with Fig 2 the red blood cells RBCs on this smear show a relatively uniform appearance with normal appearing RBCs absent Fig 4 Microangiopathic hemolytic anemias are characterized by an increase in spherocytes blue arrows schistocytes red arrows nonspecific red cell fragments black arrowheads and polychromatophilic cells black arrows original magnification 630 Fig 5 Elliptocytes are increased in a variety of conditions Here a relatively uniform population of elliptocytes is noted incidentally on peripheral smear review during a bleeding diathesis work up The patient had a positive family history of hereditary elliptocytosis original magnification 400 are microcytic and more elongate and are termed pencil cells A dramatic increase in anisopoikilocytosis with hypochromic microcytes can help to support a diagnosis of iron deficiency Ovalocytes can be seen in the setting of hereditary conditions such as southeast Asian ovalocytosis or in acquired cases such as myelodysplastic syndrome MDS Southeast Asian ovalocytosis results from mutations in SLC4A1 and occurs in a geographic distribution that parallels that of malaria endemic regions The presence of this mutation confers resistance to Plasmodium falciparum and Plasmodium vivax Drepanocytes sickle cells are bipolar red cells with characteristic points at each end Fig 6 These cells are characteristic of the sickling syndromes such as Sickle cell disease or trait and hemoglobin SC disease Quantitative assessment of sickle cells on peripheral smear review is of dubious clinical value with no correlation between the degree of disease severity and the number of drepanocytes observed Target cells are formed owing to excess red cell membrane Fig 7 On blood smear review they have a characteristic bulls eye appearance owing to hemoglobin occupying the central portion of the cell Target cells occur when there is excess red cell membrane compared with hemoglobin level In liver disease target cell formation is owing to altered lipid metabolism whereas in the setting of setting of thalassemia the abnormality is owing to lack of hemoglobin production Clinical findings MCV and red cell count can help to differentiate between the two Teardrop cells dacrocytes have an extended cytoplasmic projection resembling a tail Increased numbers of tear drop cells are most often seen in the setting of marrow myelophthisis such as metastatic carcinoma marrow fibrosis and extramedullary hematopoiesis but also can be found in cases of severe iron deficiency Spur cells acanthocytes have a few irregularly spaced and variably sized projections on their surface Fig 8 Additionally the central pallor of the RBC is absent and the red cell has a contracted or dense appearance Acanthocyte formation is related Fig 6 Drepanocytes sickle cells are denoted by black arrows original magnification 630 Fig 7 Innumerable target cells are present in this patient with hemoglobin C disease original magnification 630 Fig 8 Acanthocytes black arrows and Burr cells red arrows in a patient with endstage liver disease owing to hepatitis C virus infection original magnification 630 Note the irregularly spaced sharp projections and loss of central of the acanthocyte versus the more blunt and regularly distributed projections and retained central pallor of the Burr cell Fig 9 Blister cells are seen in settings of drug or medication induced oxidative injury usually in patients with glucose6phosphatedehydrogenase deficiency Note the presence of numerous blister cells red arrows as characterized by the puddling of hemoglobin at 1 aspect of the cell with the other aspect having a clearedout look surrounded by a thin rim of red membrane original magnification 600 A few polychromatophilic cells are present black arrow Fig 10 This peripheral blood smear demonstrates numerous nucleated red blood cells black arrows polychromatophilic cells red arrows and target cells black arrowheads original magnification 630 The patient had beta thalassemiahemoglobin E disease Fig 11 Rouleaux is an in vitro phenomenon characterized by stacking of greater than 3 red blood cells and is mainly seen in the setting of increased serum protein levels typically in patients with a severe infectiousinflammatory process or a monoclonal protein spike This peripheral blood smear demonstrates an increase in Rouleaux formation in a patient with plasma cell myeloma original magnification 50 Rare circulating plasma cells are identified black arrows will be removed from the circulation each day To maintain steady state the marrow needs to constantly produce new RBCs Reticulocytes are immature nonnucleated RBCs that circulate in the peripheral blood for 1 day before losing their RNA and becoming mature RBCs The traditional method of measuring the reticulocyte count is a manual method that uses supravital stains such as methylene blue to highlight the reticulum RNA network of this immature red cell fraction The technologist then enumerates the num ber of reticulocytes and expresses this value as a percent of total RBCs The main dif ficulty with this test is that it depends on the hematocrit Thus a reticulocyte count of 1 in a patient with a hematocrit of 45 would increase to 2 if the hematocrit decreased to 225 Therefore the raw number needs to be corrected for the hemato crit by multiplying the value by patients hematocrit divided by 45 corrected reticulo cyte count 5 retic Hct45 So in the above example the corrected reticulocyte count would be 2 times 22545 equals 1 The number of reticulocytes can be measured directly by most automated analyzers by staining the remnant RNA with a fluorescent dye This is known as the absolute reticulocyte count Advantages of automated methods include improved turnaround time measurement precision and lack of need to adjust for hematocrit The usefulness of the reticulocyte count is in assessing the marrow response to ane mia If the reticulocyte count is elevated then either blood loss or hemolysis is sug gested Normal reticulocyte counts are indicative of production causes of anemia Very low reticulocyte counts 01 or 10000mL are seen in aplastic anemia or pure red cell aplasia RED CELL INDICES OLD AND NEW Originally 3 of the red cell indices were derived or calculated Table 2 1 MCV 2 mean corpuscular hemoglobin concentration MCHC and 3 mean corpuscular he moglobin MCH On most new analyzers MCV hemoglobin and red cell count are directly measured and MCHC and MCH are calculated Because the MCHC and MCH tend to trend with the MCV these indices are rarely used anymore With more widespread use of new automated complete blood count analyzers there are now a wider array of newer indices available6 With the ability to rapidly identify reticulocytes these machines can determine both reticulocyte MCV and retic ulocyte MCHC and from this derive an MCH CHrRetHe Most studies of these newer indices have been in patients receiving EPO who develop functional iron defi ciency where the delivery of iron to the developing red cell cannot keep up with de mand The CHrRetHe falls with the onset of iron deficiency and is the first to increase with iron supplementation However the CHrRetHe also can be reduced in thalassemia therefore hemoglobinopathies should be excluded before it is used to assess iron status Table 2 RBC indices Index Normal Values Comment MCV 80100 fL RBC size Mean corpuscular hemoglobin concentration 338342 mdL Increased in spherocytosis Mean corpuscular hemoglobin 285323 pg Trends with MCV Abbreviations MCV mean corpuscular volume RBC red blood cell Anemia 275 Fig 12 Normal bone marrow aspirate original magnification 400 This aspirate shows the presence of a mixture of maturing granulocytic and erythroid precursors Well prepared aspirate smears with adequate spicules should be obtained when considering a diagnosis of myelodysplastic syndrome Fig 13 A Normal bone marrow core biopsy stain hematoxylin and eosin original magnification 25 The normal adult marrow shows a cellularity of 30 to 70 which is estimated by comparing the proportion of cells to adipocytes The core biopsy is important for determining marrow cellularity and the presence of any focal infiltrates such as granulomas lymphoma or metastatic cancer B Bone marrow core biopsy demonstrating cellularity of greater than 95 in a patient with myelodysplastic syndrome stain hematoxylin and eosin original magnification 25 Box 2 Testing for specific causes of anemia Renal disease Creatineblood urea nitrogen Erythropoietin level Anemia of chronic disease Erythropoietin Ferritin Nutritional deficiencies Iron ferritin Vitamin B12 methylmalonic acid Folate homocysteine Copper serum copper ceruloplasmin Thalassemia Hemoglobin electrophoresis DNA sequencing Sickle cell disease Sickle solubility test Hemoglobin electrophoresis Hemolysis general screening Haptoglobin Lactate dehydrogenase Indirect bilirubin Reticulocyte count Myeloma Serum protein electrophoresis and immunofixation Serum free light chain analysis future measure of hepcidin levelsthe molecular mediator of anemia of chronic dis easewill offer direct testing Endocrine Disease Several endocrine diseases can lead to anemia with the etiology related to decreased red cell production Hypogonadism is an important cause of anemia in men Testos terone sensitizes erythroid precursors to the effects of EPO This provides the ratio nale for why postpubertal males have a 10 to 15 greater hemoglobin concentration hematocrit and RBC counts than women7 Hypothyroidism can lead to a normocytic or macrocytic anemia Iron Deficiency Over time a negative iron balance owing to blood loss or increased demand will lead to a reduction in total body iron stores in the reticuloendothelial system and the bone marrow Three stages in this process have been described The iron depletion stage shows a decrease in iron stores without a decrease in serum iron levels or hemoglobin concentration outside of the normal range Serum ferritin is low in this stage The sec ond stage is characterized by the detection of abnormal iron serologies such as reduced transferrin saturation increased total ironbinding capacity and increased zinc protoporphyrin The third and final stage demonstrates a hemoglobin concentra tion of less than the lower limit of the normal range Although there are a variety of tests to assess iron stores studies have shown the most efficient test is the serum ferritin A serum ferritin level greater than 100 ngmL essentially rules out iron deficiency8 Other classic tests such as serum iron levels and transferrin saturation have low predictive value Increased total iron binding ca pacity is specific but not sensitive for early stages of iron deficiency Iron assessment can also be performed on bone marrow aspirate material Adequate bone marrow spicules must be present to assess for storage iron which is contained in macrophages Some investigators suggest a minimum of 7 particles be present to establish the absence of storage iron8 In addition to assessment of iron stores evaluation for increased ring sideroblasts erythroid precursors containing iron particles that encircle greater than twothirds of the diameter of the nucleus can also be performed An increase in ring sideroblasts can signify a number of different pathologies such as drugstoxins alcohol heavy metal poisoning lead and MDSs Other Nutritional Deficiencies Vitamin B12 deficiency Vitamin B12 deficiency produces a megaloblastoid macrocytic anemia which mani fests in the peripheral blood with hypersegmented neutrophils and in the marrow with megaloblastoid maturation of granulocytic and erythroid lineages There is increasing recognition of the difficulty in using serum vitamin B12 levels to determine tissue deficiency Currently measurement of methylmalonic acid is recommended Excess methylmalonic acid is produced in the absence of vitamin B12 and is more sen sitive and specific for vitamin B12 deficiency than direct measurements of serum vitamin B124 Folate deficiency Similar to vitamin B12 deficiency folate deficiency manifests as a megaloblastoid macrocytic anemia and shows identical morphologic features Both serum and red cell folate concentration lack diagnostic specificity and like vitamin B12 deficiency the use of metabolite assays are more accurate4 Lack of folate leads to elevation in Anemia 279 serum homocysteine levels Because vitamin B12 deficiency can also lead to elevated homocysteine methylmalonic acid levels should be incorporated into the workup for folate deficiency Copper Determining copper status is relatively straightforward Measurement of serum copper and its carrying protein ceruloplasmin can aid in determining whether a copper defi ciency is present In most laboratories the ceruloplasmin assay has a faster turn around time and may be more reflective of copper deficits9 Thalassemia Thalassemias are inherited genetic disorders that lead to impaired production of he moglobin They can range in severity from just a decreased MCV to very severe ane mia Most common types are alpha and beta thalassemia The hallmark of all thalassemia is a microcytosis owing to decreased production of hemoglobin with a concurrent increase in RBC count There are many prediction rules based on MCV and red cell count with the most popular being the Mentzer index MCVRBC count A Mentzer index of less than 13 is thought to favor a diagnosis of thalassemia over iron deficiency but this tends to lack sensitivity and specificity More complex algorithms have also been derived and improve accuracy The diagnosis of beta thalassemia trait is made by hemoglobin electrophoresis In this assay hemoglobin is separated by size and charge to assess the presence and quantity of the different hemoglobin species In betathalassemia the levels of a minor hemoglobin componenthemoglobin A2is increased In contrast with hemoglobin A which is composed of 2 alpha chains and 2 beta chains hemoglobin A2 is composed of 2 alpha chains and 2 delta chains Increased hemoglobin A2 is a compensatory process owing to decreased production of beta chains Alpha thalassemia trait cannot be diagnosis by electrophoresis because there is no increase in hemoglobin A2 Often it is a diagnosis of exclusion in a patient with micro cytosis who has normal iron stores and normal hemoglobin electrophoresis To estab lish the diagnosis DNA testing can be performed to determine the presence or absence of the alpha chain gene deletions Sickle Cell Disease and Other Hemoglobinopathies A rapid screening test for sickle cell is the sickle solubility test In this assay whole blood is lysed with a reducing agent If a sickling hemoglobin is present an insoluble precipitate is produced Because this is a screening one cannot differentiate between sickle cell disease sickle cell trait or one of the less common sickling hemoglobin species Definitive diagnosis is made by hemoglobin electrophoresis Hemoglobin electrophoresis is the standard screening test for hemoglobinopathies Clues to the presence of hemoglobinopathies are hemolysis cyanosis or erythrocytosis Increas ingly DNA sequencing to find specific mutations is being used to make a precise diagnosis10 Hemolysis The diagnosis of hemolysis is a 2step process The first is to assess for the presence of a hemolytic anemia and the second it to establish the etiology Screening for hemo lysis uses a variety of tests which often must be interpreted in unison and with knowl edge of the patients medical history11 Haptoglobin is a protein that acts as a scavenger of free hemoglobin thereby pro tecting the body from its toxic effects12 Low haptoglobin levels are sensitive for Cascio DeLoughery 280 hemolysis but are not specific because severe liver disease transfusions and regular exercise can lead to low levels An additional cause of absent haptoglobin levels is ahaptoglobinemia This is an inherited condition that results in total lack of haptoglobin production This is a relatively common condition that is found in 11000 whites and up 4 of African Americans13 Lactate dehydrogenase is an intracellular enzyme found in abundance in the red cell Although lactate dehydrogenase is sensitive for hemolysis an increased level can be found in many other disorders especially liver disease Indirect bilirubin increases as the breakdown of heme overwhelms the livers ability to form conjugated bilirubin and excrete it into the bile Liver disease can also increase levels and an increased level of indirect bilirubin can only be interpreted to imply he molysis in the setting of a normal direct bilirubin level Outside of fulminate hemolysis it is rare to see an indirect bilirubin greater than 4 mgdL As noted the reticulocyte count will be increased in hemolysis Sensitivity may be an issue because some patients will have normal reticulocyte counts owing to nutri tional deficiencies lack of EPO or destruction of red cell precursors14 Once the presence of hemolysis is established the next step is to determine the cause Acquired causes of hemolysis are most often owing to autoimmune disease mechanical destruction toxins or paroxysmal nocturnal hemoglobinuria The direct antibody test Coombs test helps to determine the presence of an antibody mediated cause such as might be seen in the setting of autoimmune causes Inherited etiologies for hemolysis may involve defects in the RBC membranecytoskeletal pro teins hemoglobin or intracellular enzymes Often the blood smear provides clues to the presence of membrane or cytoskeletal defects such as those seen in hereditary spherocytosis or elliptocytosis Hemoglobin electrophoresis is useful for identifying he moglobinopathies whereas specific assays are needed to identify enzymatic defects Plasma Cell Myeloma A rare but important cause of anemia especially in older patients is plasma cell myeloma An high index of suspicion for this possibility should be had when evaluating anemic patients with back pain or renal disease Serum protein electrophoresis with immunofixation and serum free light chain analysis should be obtained when evalu ating for myeloma Assessing for light chain disease is important because 10 of my elomas only excrete light chains Myelodysplastic Syndrome MDS is a group of clonal bone marrow failure disorders that result in anemia and vary ing degrees of leukopenia and thrombocytopenia MDS occurs in predominantly older patients but may occur in younger individuals exposed to cytotoxic chemotherapy for childhood malignancies Laboratory clues for MDS include a macrocytic anemia with nonelevated reticulocyte count hypoproliferative anemia Other cytopenias may be present A morphologic review of the peripheral blood smear may reveal an oval macrocytosis without an increase in polychromatophilic cells Neutrophils may show dysplastic features including nuclear hypolobation unilobed or pseudoPelger Huet nuclei or hypogranular cytoplasm Fig 14 Platelets may also show dyspoeitic features including the presence of large or giant forms or those with absent granules Bone marrow examination is a requisite in the workup of a patient with suspected MDS Adequate and wellprepared aspirate smears are of paramount importance because this allows the pathologist to assess for diagnostic dyspoietic changes in the granulocytic erythroid and megakaryocytic lineages and enumerate blasts see Fig 13 Figs 15 and 16 Cytogenetic studies including FISH for MDSrelated Anemia 281 Fig 14 Dysplastic neutrophils in the setting of acute myeloid leukemia with myelodysplasiarelated changes black arrows original magnification 630 These polymorphonuclear leukocytes PMNs show bilobed nuclei with water clear hypogranular cytoplasm This is contrast with the pink cytoplasm seen in normalappearing PMNs red arrow Identification of such features on peripheral blood film can help to direct the workup and prompt earlier consideration of bone marrow examination Fig 15 Multilineage dysplasia in patient with myelodysplastic syndrome original magnification 630 A small megakaryocyte with a hypolobate nucleus red arrow in a background of granulocytic dysplasia manifest as hypogranular neutrophils black arrows A normal megakaryocyte is shown for comparison inset original magnification 630 Fig 16 At greater magnification megakaryocytes are increased and show prominent dysplastic findings including small size and hypolobate nuclei black arrows Rare megakaryocytes also show separated nuclear lobes red arrow another diagnostic feature of myelodysplasia stain hematoxylin and eosin original magnification 630 Box 3 Rational approach to anemia Basic tests Complete blood count with indices Reticulocyte count Ferritin Methylmalonic acid Homocysteine Creatinineblood urea nitrogen If older or back pain add Serum protein electrophoresis with immunofixation Serumfree light chain analysis If neurologic disease present add Copper A Rational Approach to Anemia By combining clues from the patient history and initial complete blood count one can plot a rational approach to the anemic patient The reticulocyte counts should be ob tained to determine the presence of hemolysis Given how common iron deficiency is and the lack of specificity of the MCV for this diagnosis serum ferritin should be tested in every patient Further specific testing is guided by the results of these initial studies Box 3 REFERENCES 1 Cappellini MD Motta I Anemia in clinical practicedefinition and classification does hemoglobin change with aging Semin Hematol 20155242619 2 Beutler E Waalen J The definition of anemia what is the lower limit of normal of the blood hemoglobin concentration Blood 20061075174750 3 Brugnara C Mohandas 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Int J Lab Hematol 2014361112 11 Ruiz EF Cervantes MA Diagnostic approach to hemolytic anemias in the adult Rev Bras Hematol Hemoter 20153764235 12 Shih AW McFarlane A Verhovsek M Haptoglobin testing in hemolysis measure ment and interpretation Am J Hematol 20148944437 13 Delanghe J Langlois M De BM Congenital anhaptoglobinemia versus acquired hypohaptoglobinemia Blood 19989193524 14 Liesveld JL Rowe JM Lichtman MA Variability of the erythropoietic response in autoimmune hemolytic anemia analysis of 109 cases Blood 19876938206 Cascio DeLoughery 284 1 De acordo com o artigo existem dois tipos de sistemas de classificação para anemias Sendo eles o tamanho dos glóbulos vermelhos e o aumento da perda de hemácias ou diminuição da produção de hemácias O tamanho dos glóbulos vermelhos podem variar de microcítica VCM 80 fL normocítica VCM 80100 fL e macrocítica VCM 100 fL A anemia microcítica ecoa defeitos na síntese de hemoglobina como por exemplo a falta de ferro e talassemia A anemia macrocítica ocorre quando têmse defeitos na membrana das hemácias e defeitos na síntese do DNA A anemia normocítica ocorre normalmente em estágios iniciais de processos ou quando vários processos ocorrem ao mesmo tempo O outro sistema de classificação é o aumento da perda de hemácias ou diminuição da produção da mesma No caso do aumento da perda de hemácias perda de sangue e hemólise são as causas primárias E se for a diminuição pode ter uma causa nutricional insuficiência medular falta de fatores de crescimento e processo mielfísico 2 Figura 2 Anemia microcítica hipocrômica Anemia normocítica normocrômica Figura 8 Anemia hemolítica Anemia de doença crônica Figura 9 Anemia hemolítica hereditária Anemia megaloblástica Figura 11 Anemia por mieloma múltiplo Anemia por processo infecciosoinflamatório grave Figura 15 Anemia aplásica Síndrome mielodisplásica 1 De acordo com o artigo existem dois tipos de sistemas de classificação para anemias Sendo eles o tamanho dos glóbulos vermelhos e o aumento da perda de hemácias ou diminuição da produção de hemácias O tamanho dos glóbulos vermelhos podem variar de microcítica VCM 80 fL normocítica VCM 80100 fL e macrocítica VCM 100 fL A anemia microcítica ecoa defeitos na síntese de hemoglobina como por exemplo a falta de ferro e talassemia A anemia macrocítica ocorre quando têmse defeitos na membrana das hemácias e defeitos na síntese do DNA A anemia normocítica ocorre normalmente em estágios iniciais de processos ou quando vários processos ocorrem ao mesmo tempo O outro sistema de classificação é o aumento da perda de hemácias ou diminuição da produção da mesma No caso do aumento da perda de hemácias perda de sangue e hemólise são as causas primárias E se for a diminuição pode ter uma causa nutricional insuficiência medular falta de fatores de crescimento e processo mielfísico 2 Figura 2 Anemia microcítica hipocrômica Anemia normocítica normocrômica Figura 8 Anemia hemolítica Anemia de doença crônica Figura 9 Anemia hemolítica hereditária Anemia megaloblástica Figura 11 Anemia por mieloma múltiplo Anemia por processo infecciosoinflamatório grave Figura 15 Anemia aplásica Síndrome mielodisplásica