International Journal of Laboratory Hematology

The Ofcial journal of the International Society for Laboratory Hematology

REVIEW

INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY

Red blood cell morphology

J. FORD

Division of Hematopathology, BC Childrens Hospital, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada Correspondence: Jason Ford, BC Childrens Hospital, 4500 Oak St, Vancouver V6H 3N1, BC, Canada. Tel.:604-875-2044; Fax: 604-875-2815; E-mail: jford@cw.bc.ca
doi:10.1111/ijlh.12082

S U M M A RY

Received 31 December 2012; accepted for publication 21 January 2013 Keywords Anemia, morphology, red blood cell, poikilocytosis, peripheral smear

The foundation of laboratory hematologic diagnosis is the complete blood count and review of the peripheral smear. In patients with anemia, the peripheral smear permits interpretation of diagnostically signicant red blood cell (RBC) ndings. These include assessment of RBC shape, size, color, inclusions, and arrangement. Abnormalities of RBC shape and other RBC features can provide key information in establishing a differential diagnosis. In patients with microcytic anemia, RBC morphology can increase or decrease the diagnostic likelihood of thalassemia. In normocytic anemias, morphology can assist in differentiating among blood loss, marrow failure, and hemolysisand in hemolysis, RBC ndings can suggest specic etiologies. In macrocytic anemias, RBC morphology can help guide the diagnostic considerations to either megaloblastic or nonmegaloblastic causes. Like all laboratory tests, RBC morphologies must be interpreted with caution, particularly in infants and children. When used properly, RBC morphology can be a key tool for laboratory hematology professionals to recommend appropriate clinical and laboratory follow-up and to select the best tests for denitive diagnosis.

INTRODUCTION
Medical school educators around the world emphasize the importance of teaching future physicians the correct approach to the history and physical examination. These basic skills are widely understood to be the foundation of medical practice, even in the face of technological change. For laboratory hematology professionals, the complete blood count (CBC) and the peripheral smear are, respectively, our history and physical examination. Despite quantum leaps in technological development in the clinical laboratory, with evolutions and revolutions in ow cytometry and point of care testing and

molecular analysis, review of a patients CBC and peripheral smear morphology is still the mainstay of hematologic diagnosis. For patients with anemia, the peripheral smear morphology provides key information to create the differential diagnosis. Review of the peripheral smear has three main components: To conrm the CBC ndings. It is unusual for laboratory error to affect any of the measurements in the CBC, but spurious ndings may include the following [1, 2]: (i) low counts due to faulty aspiration of whole blood by the automated counter;

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(ii) macrocytosis due to RBC agglutination or rouleaux, hyperleukocytosis, or severe hyperglycemia; (iii) microcytosis due to the blood counters misidentication of giant platelets as RBCs. To review relevant white blood cell (WBC) and platelet (PLT) ndings. For example, a high platelet count is expected in anemia due to iron deciency and a low platelet count is expected in anemia due to microangiopathic hemolysis. To review RBC morphology. There are ve important aspects: (i) Shape. What is/are the dominant poikilocyte(s)? (ii) Size. Is there anisocytosis or a dual population? (iii) Color. Is there hypo- or hyperchromasia? Is there anisochromia or polychromasia? (iv) Inclusions. Are there HowellJolly bodies, malaria parasites, nucleated RBCs, etc.? (v) Arrangement. Is there agglutination or rouleaux? A list of RBC morphologies, their denitions, and their associated clinical states is shown in Table 1 [3]. Poikilocytosis must be interpreted in its appropriate context: nding a rare poikilocyte in an otherwise normal smear is likely clinically insignicant, while nding extensive poikilocytosis in a normocytic anemia may indicate specic causes of hemolysis. In the neonatal period and in patients on chemotherapy, poikilocytosis must be interpreted with special caution: these patients may be expected to have a background level of mild or moderate nonspecic poikilocytosis, and only the nding of a dominant or extensive poikilocytosis in combination with anemia is likely clinically relevant. Most clinicians and laboratory professionals use an approach to anemia centering on the mean cell volume (MCV). This review of RBC morphology will follow the MCV approach. Morphology in the assessment of microcytic anemia Medical students often learn that there are ve main causes of microcytic anemia, which together form the easily remembered acronym TAILS: T = Thalassemia. A = Anemia of chronic disease.

I = Iron deciency. L = Lead poisoning. S = congenital Sideroblastic anemia. Only three of these are common in most parts of the world, namely iron deciency, anemia of chronic disease (ACD), and thalassemia. Lead poisoning is not usually considered a common cause of anemia, but it may be seen in pediatrics particularly in areas where paint, toys, or jewelry containing lead can be eaten by small children. Lead can also be consumed by infants in formula made with contaminated water [4] and may rarely cause anemia in adults with extensive industrial exposure. Congenital sideroblastic anemia is vanishingly rare. In classic cases, the morphological differentiation of the three common microcytic anemias is straightforward. The classic morphology in ACD is of unremarkable RBCs, while iron deciency shows anisocytosis, anisochromia, and elliptocytosis, and thalassemia trait demonstrates target cells and coarse basophilic stippling. Regrettably, these so-called classic presentations are unreliable in practice. Elliptocytes and anisocytosis are often seen in thalassemia, target cells may occur in iron deciency, and both iron deciency and thalassemia may appear as unremarkable as ACD. The red blood cell distribution width (RDW), classically taught as a key differentiator of iron deciency from thalassemia, is also unreliable [5]; far better than the RDW is the RBC count [5, 6], although even a high RBC count is not proof of thalassemia. The only reliable classic morphologic nding that can separate these three conditions is the presence of coarse basophilic stippling. Coarse stippling is seen in some cases of thalassemia and is never seen in uncomplicated iron deciency or ACD. A microcytic patient with coarse basophilic stippling likely has thalassemiaalthough the patient should be in an ethnically at-risk group, and there should not be another reasonably likely cause of basophilic stippling. Even a likely diagnosis of thalassemia must still be conrmed by hemoglobin HPLC, H body staining, molecular testing, or some other reliable method. Morphology is essentially never diagnostic of thalassemia: it can only suggest whether thalassemia is more or less likely.

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Table 1. Common RBC morphological ndings RBC morphology Acanthocyte (spur cell) Anisochromia Anisocytosis Morphological denition RBC has irregularly distributed, variably sized, pointy projections off its surface Variation in the amount of central pallor among a population of RBCs Variation in size among a population of RBCs Clinical associations Advanced liver disease, hyposplenism, some dyslipidemias, pyruvate kinase deciency, McLeod phenotype Iron deciency, myelodysplasia, hypochromic anemia post transfusion Common nonspecic nding. Seen in iron deciency, moderate or severe thalassemia, megaloblastic anemia, partially treated anemia of several causes, post transfusion Thalassemia, lead poisoning, myelodysplasia, pyrimidine 5 nucleotidase deciency, post chemotherapy Reticulocytosis, normal nding

Basophilic stippling: coarse

Basophilic stippling: ne Bite cell/blister cell

Dimorphism

Echinocyte (burr cell) Elliptocyte Heinz body

RBC has variably sized (up to large) basophilic granular discolorations across its entire cytoplasm, on a Wright-stained lm RBC has small, uniform, punctate basophilic dots across its entire cytoplasm, on a Wright-stained lm RBC has a semi-circular indentation in its outer cytoplasmic border. There may be a roof to this indentation (blister cell) or no roof (bite cell) Two distinct populations of RBC are present, for example, microcytic and normocytic, or hypochromic and normochromic RBC has regularly distributed, equally sized, rounded projections off its surface RBC is oval shaped RBC has a submembranous or epimembranous small round mass, which can only be seen by supravital or specialized Heinz body stains. This body is not visible on a routine Wright-stained lm Solitary round mass, relatively large (e.g., approximately 1020% of the diameter of the RBC), within the hemoglobinized portion of the RBC. Appears dark blue or purple on a Wright-stained lm The zone of central pallor is > 1/3 the diameter of the RBC The RBC is small, dark, and lacks a zone of central pallor. Its outer margin is not spherical: it may appear dented, compressed, or otherwise contracted Usually multiple small dark blue or purple granular inclusions, all within the hemoglobinized portion of the RBC. These occupy only one portion or region of the RBC, unlike basophilic stippling which is more global throughout the entire RBC

Oxidative hemolysis

Myelodysplasia, post transfusion, partially treated iron deciency

Artifact, renal failure, post transfusion, phosphate deciency, burns Iron deciency, megaloblastic anemia, hereditary elliptocytosis, post chemotherapy Oxidative hemolysis, hyposplenism

HowellJolly body

Hyposplenism, erythroblastosis, myelodysplasia, megaloblastic anemia, post chemotherapy

Hypochromia Irregularly contracted cell

Iron deciency, thalassemia, anemia of chronic disease Nonspecic nding seen in a variety of conditions including G6PD deciency, hemoglobinopathies, and normal neonates

Pappenheimer body

Iron overload, hyposplenism, myelodysplasia

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Table 1. (Continued) RBC morphology Polychromasia Morphological denition RBCs show color variability as a population: some (usually the majority) are the usual red color, while others are bluish Some RBCs aggregate into multicellular masses resembling a bunch of grapes Some RBCs aggregate into linear patterns, said to resemble a stack of coins The RBC appears to have been fragmented: it lacks the usual circular shape, instead showing a triangular or other angulated morphology. The zone of central pallor is often missing There are several sickle RBC morphologies, including the classic sickle (crescentic with two sharply pointed ends) or boats (linear with two tapered if somewhat rounded ends) The RBC is smaller and darker than normal. There is no zone of central pallor. The outer edge must be almost perfectly round (to differentiate this cell from irregularly contracted cells) The zone of central pallor is linear, rather than circular. Usually the line of pallor runs parallel to the long axis of the RBC, if the latter is ovoid, but in certain variants (e.g., South East Asian ovalocytosis), the line may run across the long axis or may be nonlinear, for example, bifurcated or trifurcated The RBC has a central red area within the zone of central pallor The RBC is tapered to a point at one end, resembling the classic artists rendition of a drop of water Clinical associations Reticulocytosis, normal neonate

RBC agglutination Rouleaux

Cold agglutinin, cold autoimmune hemolytic anemia Normal nding in the thick part of the blood smear, hypergammaglobulinemia (monoclonal or polyclonal) RBC fragmentation syndromes, for example, microangiopathic hemolytic anemia and hemolysis secondary to cardiac valve

Schistocyte

Sickle cell

Severe sickling syndrome, for example, SS, SC and SD

Spherocyte

Autoimmune hemolytic anemia, alloimmune hemolytic anemia (e.g., hemolytic disease of the newborn), hereditary spherocytosis

Stomatocyte

Artifact, obstructive liver disease, hereditary stomatocytosis, South East Asian ovalocytosis, Rh null syndrome

Target cell

Teardrop cell

Thalassemia, liver disease, hyposplenism, Hgb C disease or SC disease, hereditary xerocytosis. May be seen in iron deciency Nonspecic nding seen in several conditions including myelobrosis

The ethnicities that are not at high risk of thalassemia include northern Europeans, American Indians, Canadian First Nations, Inuit, and patients from Japan [7]. Everyone else should be considered at risk. Coarse basophilic stippling is not pathognomonic for thalassemia: it can also be seen in lead poisoning, myelodysplastic syndrome, post chemotherapy, and in rare other conditions (see Table 1). Coarse stippling does not help differentiate a- from b-thalassemia, as it

may be seen in either condition. It must not be confused with ne basophilic stippling, which is a normal nding. The morphology of H bodies [8], which are consistent with (if not pathognomonic for) a-thalassemia, is well known: using supravital stains, these precipitates of b-globin tetramers appear as innumerable dark spots distributed in a geometric fashion across the entire cytoplasm of the RBC like the pits on the
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surface of a golf ball. Patients with a single or double a-gene deletion may show a single H body RBC in many high-power elds, while patients with hemoglobin H disease (a-/-) demonstrate H bodies in the majority of their RBCs. Unfortunately, the sensitivity of H body staining is variable, ranging from approximately 40% up to approximately 90% depending on the pattern of a-deletions [8] and the laboratorys technical expertise. H bodies also usually require the presence of exclusively normal b-globin chains (i.e., bA-chains): if a patient has both a-thalassemia and a simultaneous b-variant, such as hemoglobin E, it may be much more difcult to nd H bodies. This variable sensitivity means that although the presence of H bodies can indicate a-thalassemia, their absence does not rule this diagnosis out. In the right context (e.g., microcytic anemia with a high RBC count, in a patient from a high-risk ethnicity such as South East Asian), H bodies are in general considered diagnostic of a-thalassemia. However, even in this context, H bodies are not proof of a-thalassemia: H-like bodies can be formed by other unstable hemoglobins besides b4, such as Hemoglobin J-New York. The analogous RBC inclusion in b-thalassemia, consisting of precipitates of a4, may be designated Fessas bodies [9]. These are solitary large round deposits within the cytoplasm of an RBC: like the surrounding soluble hemoglobin, the precipitated a-chains are red on a Wright stain, so Fessas bodies are generally not visible in routine peripheral smears. They can sometimes be seen as red cytoplasmic inclusions in polychromatophilic RBCs or in nucleated RBCs in the peripheral blood, and in RBC precursors in marrow aspirate specimens. One helpful morphological clue in microcytic anemias is the broad range of poikilocytosis seen in many cases of thalassemia, compared to iron deciency. In some patients with thalassemia, there are not only target cells but also numerous teardrop cells and schistocytes. Among the poikilocytes seen in thalassemia are the sh cells described by Barbara Bain [Bain, personal communication]. These are generally not seen in patients with iron deciency or ACD. Fish cells resemble teardrop cells, with one rounded end and one tapered end: unlike teardrops, the tapered end ares out into two buds resembling a shs tail. One sh cell is seen at the center of Figure 1. This
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image also shows examples of the teardrops and schistocytes which can be seen in thalassemia trait. Morphology in the assessment of normocytic anemia Most cases of normocytic anemia are caused by blood loss, suppressed production of RBCs, or hemolysis. In hemorrhage the RBC morphology is entirely unremarkable, except for the polychromasia that typically arises after the rst twelve to 24 h. In patients with reduced RBC production, red cell morphology may be normal where the cause is extrinsic to the red cell itself: for example, because of low erythropoietin in a patient with renal failure. But where erythropoiesis is intrinsically disordered (e.g., myelodysplasia) and in cases of hemolysis, RBC morphology may be diagnostically signicant. Patients with disordered RBC production (such as myelodysplastic syndrome, MDS, or congenital dyserythropoietic anemia, CDA) may have a dual population, elliptocytes, teardrop cells, or other poikilocytes. There may also be circulating nucleated RBCs (nRBCs), showing dysplastic features including asymmetric nuclear budding, multinuclearity, megaloblastoid changes, or karyorrhexis. In children, particularly infants, reactive (transient) dysplastic nRBCs are frequently seen in many patients with brisk reticulocytosis following hemorrhage or hemolysis. Reactive dysplasia in children will abate after correction of the patients anemia. The most common role of RBC morphology in patients with normocytic anemia is in the assessment

Figure 1. A sh cell and other poikilocytes in a case of thalassemia trait. Wright stain, 9100.

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of patients with hemolysis. Poikilocytosis will often suggest a specic cause or mechanism of hemolysis (Table 1): Bite and blister cells, as well as irregularly contracted cells, are the classic ndings in oxidative hemolysis: for example, because of G6PD deciency. Oxidative hemolysis may also lead to (less prominent) schistocytosis and spherocytosis. Acanthocytes are rarely the dominant nding in a hemolytic patient, but may suggest pyruvate kinase deciency (where they will be accompanied by irregularly contracted cells) or the McLeod phenotype. Acanthocytes are more commonly observed in patients with hyposplenism, liver disease, a variety of dyslipidemias, and even anorexia nervosa. Sickle cells will suggest a diagnosis of sickle cell anemia or any of the severe sickling syndromes (including Sb0, SD and SO-Arab). In essentially every patient with sickle cell anemia by the age of 2 years, there will also be evidence of hyposplenism including targets, acanthocytes, and HowellJolly bodies. Patients with SC disease and any of the sickle thalassemia compound disorders (including Sb0 and SS-a thalassemia) may have considerably more target cells than patients with uncomplicated SS. Patients with SC disease may also demonstrate C crystals in some RBCs [10]. C crystals and targets by themselves, without sickle cells, of course may suggest homozygosity for hemoglobin C. Spherocytes have two common causes: immune-mediated hemolysis and hereditary spherocytosis (HS). Some patients with HS will demonstrate occasional mushroom cell or pincer cell variants: these cells resemble spherocytes with mirror-image indentations, resulting in an appearance similar to a button mushroom. RBC morphology is not usually very helpful in differentiating immune hemolysis from HS: further testing (such as direct antiglobulin testing and ow cytometry [11]) may be required. It should be noted that spherocytosis may also be seen in neonates with gram-negative sepsis and in patients with thermal burns, as well as in other hemolytic anemias including G6PD deciency. Elliptocytosis is most commonly due to iron deciency or hereditary elliptocytosis (HE). Although there are several other causes of elliptocytosis, as a practical matter if iron deciency is excluded then elliptocytosis is most likely due to HE. Parents with typical HE may

have newborns with a much more abnormal phenotype, featuring severe microschistocytosis as well as elliptocytosis. These infants may have either hereditary elliptocytosis with infantile poikilocytosis (HEIP) or hereditary pyropoikilocytosis (HPP) [12]. In South East Asian ovalocytosis (SEAO), the elliptocytes show a transverse (as opposed to longitudinal) zone of central pallor, or two zones of pallor separated by a transverse bar of cytoplasm, or even a zone of central pallor divided into two or three spokes like the open spaces on a sleigh bell. SEAO is considered hematologically benign, although there is a suggestion that it may be responsible for transient anemia in the newborn period [13]. Schistocytes generally reect intravascular hemolysis. When seen with thrombocytopenia, schistocytes suggest microangiopathic hemolytic anemia (MAHA), a group of conditions consisting primarily of thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), and disseminated intravascular coagulopathy (DIC). Morphology is not useful in differentiating among these three conditions, nor among their subtypes (such as congenital vs. acquired TTP or typical vs. atypical HUS). Morphology is also unreliable in predicting the severity of a case of MAHA: a patient with more schistocytes is not necessarily more sick than a patient with fewer schistocytes. There are other important causes of schistocytosis, including vasculitis, intracardiac hemolysis (e.g., due to a septal defect or prosthetic cardiac valve), thermal burn, march hemoglobinuria, the HELLP syndrome in pregnancy, and the KasabachMerritt phenomenon in infants. All of these lesions share the common pathogenetic step of extrinsic mechanical injury to the red blood cell. Many hemolytic anemias show multiple poikilocytes: G6PD deciency, for example, often shows not only bite and blister cells but also schistocytes and spherocytes. The RBC morphology may not so much suggest a single diagnosis as several relevant avenues for clinical and laboratory follow-up. A patient with bite cells and spherocytes may benet from G6PD screening and a direct antiglobulin test, for example. This problem is particularly notable in neonates, in whom the usual hemolytic morphologies may not be clearly evident. Neonatal hemolysis may lead to a very broad range of poikilocytosis, without the same

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classic patterns as are relied upon in adults: oxidative hemolysis, for example, may lead to more schistocytosis than bite/blister cells. The morphologic differential diagnosis for hemolysis in a neonate must therefore be broader than in an adult. Morphology in the assessment of macrocytic anemia The usual approach to macrocytosis is to differentiate between megaloblastic and nonmegaloblastic causes: megaloblastosis is seen with B12 and folate deciency, MDS and CDA, HIV infection, and rare inborn errors of metabolism, while nonmegaloblastic causes include liver and thyroid disease, alcohol, Down syndrome, aplastic anemia, and reticulocytosis. Medications can be responsible for both megaloblastic and nonmegaloblastic anemia, while RBC agglutination may lead to spurious macrocytosis. Red blood cell morphology usually plays a small but important role in this differentiation of megaloblastic from nonmegaloblastic causes. Important preliminary ndings include agglutination, polychromasia (reticulocytosis), target cells (liver disease or alcohol), and a dual population (MDS or post transfusion).

Oval macrocytosis and severe macrocytosis (e.g., >115 fL) are both classically found in megaloblastic anemia, while round macrocytosis is seen in nonmegaloblastic anemia. Circulating nRBCs may show dysplastic features suggesting megaloblastic change: that is, large immature nuclei within mature red cytoplasm. In many patients with macrocytic anemia, the RBC morphology is quite bland: for example, marrow failure (e.g., DiamondBlackfan anemia, idiopathic aplastic anemia, etc.) may produce morphologically unremarkable RBCs.

CONCLUSION
The review of red blood cell morphology is a critical step in the evaluation of a patient with anemia. It can be very useful in evaluating microcytic, normocytic, and macrocytic anemias and is especially helpful in the work-up of patients with hemolysis. Assessment of RBC morphology can be the best tool for laboratory hematology professionals to recommend clinical and laboratory follow-up in a patient with anemia and to select the right tests for denitive diagnosis.

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