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 significant red blood cell (RBC) findings. 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 hemolysis—and in hemolysis, RBC findings can suggest specific 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 definitive diagnosis.
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 flow cytometry and point of care testing and molecular analysis, review of a patient's 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 confirm the CBC findings. It is unusual for laboratory error to affect any of the measurements in the CBC, but spurious findings may include the following [1, 2]:
- low counts due to faulty aspiration of whole blood by the automated counter;
- macrocytosis due to RBC agglutination or rouleaux, hyperleukocytosis, or severe hyperglycemia;
- microcytosis due to the blood counter's misidentification of giant platelets as RBCs.
- To review relevant white blood cell (WBC) and platelet (PLT) findings. For example, a high platelet count is expected in anemia due to iron deficiency and a low platelet count is expected in anemia due to microangiopathic hemolysis.
- To review RBC morphology. There are five important aspects:
- Shape. What is/are the dominant poikilocyte(s)?
- Size. Is there anisocytosis or a dual population?
- Color. Is there hypo- or hyperchromasia? Is there anisochromia or polychromasia?
- Inclusions. Are there Howell–Jolly bodies, malaria parasites, nucleated RBCs, etc.?
- Arrangement. Is there agglutination or rouleaux?
A list of RBC morphologies, their definitions, and their associated clinical states is shown in Table 1 . Poikilocytosis must be interpreted in its appropriate context: finding a rare poikilocyte in an otherwise normal smear is likely clinically insignificant, while finding extensive poikilocytosis in a normocytic anemia may indicate specific 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 nonspecific poikilocytosis, and only the finding of a dominant or extensive poikilocytosis in combination with anemia is likely clinically relevant.
|RBC morphology||Morphological definition||Clinical associations|
|Acanthocyte (spur cell)||RBC has irregularly distributed, variably sized, pointy projections off its surface||Advanced liver disease, hyposplenism, some dyslipidemias, pyruvate kinase deficiency, McLeod phenotype|
|Anisochromia||Variation in the amount of central pallor among a population of RBCs||Iron deficiency, myelodysplasia, hypochromic anemia post transfusion|
|Anisocytosis||Variation in size among a population of RBCs||Common nonspecific finding. Seen in iron deficiency, moderate or severe thalassemia, megaloblastic anemia, partially treated anemia of several causes, post transfusion|
|Basophilic stippling: coarse||RBC has variably sized (up to large) basophilic ‘granular’ discolorations across its entire cytoplasm, on a Wright-stained film||Thalassemia, lead poisoning, myelodysplasia, pyrimidine 5′ nucleotidase deficiency, post chemotherapy|
|Basophilic stippling: fine||RBC has small, uniform, punctate basophilic dots across its entire cytoplasm, on a Wright-stained film||Reticulocytosis, normal finding|
|Bite cell/blister cell||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)||Oxidative hemolysis|
|Dimorphism||Two distinct populations of RBC are present, for example, microcytic and normocytic, or hypochromic and normochromic||Myelodysplasia, post transfusion, partially treated iron deficiency|
|Echinocyte (burr cell)||RBC has regularly distributed, equally sized, rounded projections off its surface||Artifact, renal failure, post transfusion, phosphate deficiency, burns|
|Elliptocyte||RBC is oval shaped||Iron deficiency, megaloblastic anemia, hereditary elliptocytosis, post chemotherapy|
|Heinz body||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 film||Oxidative hemolysis, hyposplenism|
|Howell–Jolly body||Solitary round mass, relatively large (e.g., approximately 10–20% of the diameter of the RBC), within the hemoglobinized portion of the RBC. Appears dark blue or purple on a Wright-stained film||Hyposplenism, erythroblastosis, myelodysplasia, megaloblastic anemia, post chemotherapy|
|Hypochromia||The zone of central pallor is > 1/3 the diameter of the RBC||Iron deficiency, thalassemia, anemia of chronic disease|
|Irregularly contracted cell||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’||Nonspecific finding seen in a variety of conditions including G6PD deficiency, hemoglobinopathies, and normal neonates|
|Pappenheimer body||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||Iron overload, hyposplenism, myelodysplasia|
|Polychromasia||RBCs show color variability as a population: some (usually the majority) are the usual red color, while others are bluish||Reticulocytosis, normal neonate|
|RBC agglutination||Some RBCs aggregate into multicellular masses resembling a bunch of grapes||Cold agglutinin, cold autoimmune hemolytic anemia|
|Rouleaux||Some RBCs aggregate into linear patterns, said to resemble a stack of coins||Normal finding in the thick part of the blood smear, hypergammaglobulinemia (monoclonal or polyclonal)|
|Schistocyte||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||RBC fragmentation syndromes, for example, microangiopathic hemolytic anemia and hemolysis secondary to cardiac valve|
|Sickle cell||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)||Severe sickling syndrome, for example, SS, SC and SD|
|Spherocyte||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)||Autoimmune hemolytic anemia, alloimmune hemolytic anemia (e.g., hemolytic disease of the newborn), hereditary spherocytosis|
|Stomatocyte||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||Artifact, obstructive liver disease, hereditary stomatocytosis, South East Asian ovalocytosis, Rh null syndrome|
|Target cell||The RBC has a central red area within the zone of central pallor||Thalassemia, liver disease, hyposplenism, Hgb C disease or SC disease, hereditary xerocytosis. May be seen in iron deficiency|
|Teardrop cell||The RBC is tapered to a point at one end, resembling the classic artist's rendition of a drop of water||Nonspecific finding seen in several conditions including myelofibrosis|
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 five main causes of microcytic anemia, which together form the easily remembered acronym TAILS:
T = Thalassemia.
A = Anemia of chronic disease.
I = Iron deficiency.
L = Lead poisoning.
S = congenital Sideroblastic anemia.
Only three of these are common in most parts of the world, namely iron deficiency, 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  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 deficiency 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 deficiency, and both iron deficiency and thalassemia may appear as ‘unremarkable’ as ACD. The red blood cell distribution width (RDW), classically taught as a key differentiator of iron deficiency from thalassemia, is also unreliable ; 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 finding 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 deficiency or ACD. A microcytic patient with coarse basophilic stippling likely has thalassemia—although 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 confirmed 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.
The ethnicities that are not at high risk of thalassemia include northern Europeans, American Indians, Canadian First Nations, Inuit, and patients from Japan . 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 α- from β-thalassemia, as it may be seen in either condition. It must not be confused with fine basophilic stippling, which is a normal finding.
The morphology of H bodies , which are consistent with (if not pathognomonic for) α-thalassemia, is well known: using supravital stains, these precipitates of β-globin tetramers appear as innumerable dark spots distributed in a geometric fashion across the entire cytoplasm of the RBC like the pits on the surface of a golf ball. Patients with a single or double α-gene deletion may show a single H body RBC in many high-power fields, while patients with hemoglobin H disease (α-/-) 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 α-deletions  and the laboratory's technical expertise. H bodies also usually require the presence of exclusively normal β-globin chains (i.e., βA-chains): if a patient has both α-thalassemia and a simultaneous β-variant, such as hemoglobin E, it may be much more difficult to find H bodies. This variable sensitivity means that although the presence of H bodies can indicate α-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 α-thalassemia. However, even in this context, H bodies are not ‘proof’ of α-thalassemia: H-like bodies can be formed by other unstable hemoglobins besides β4, such as Hemoglobin J-New York.
The analogous RBC inclusion in β-thalassemia, consisting of precipitates of α4, may be designated ‘Fessas bodies’ . These are solitary large round deposits within the cytoplasm of an RBC: like the surrounding soluble hemoglobin, the precipitated α-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 deficiency. 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 ‘fish cells’ described by Barbara Bain [Bain, personal communication]. These are generally not seen in patients with iron deficiency or ACD. Fish cells resemble teardrop cells, with one rounded end and one tapered end: unlike teardrops, the tapered end flares out into two buds resembling a fish's tail. One fish cell is seen at the center of Figure 1. This 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 first 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 significant.
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 patient's anemia.
The most common role of RBC morphology in patients with normocytic anemia is in the assessment of patients with hemolysis. Poikilocytosis will often suggest a specific cause or mechanism of hemolysis (Table 1):
- Bite and blister cells, as well as irregularly contracted cells, are the classic findings in oxidative hemolysis: for example, because of G6PD deficiency. Oxidative hemolysis may also lead to (less prominent) schistocytosis and spherocytosis.
- Acanthocytes are rarely the dominant finding in a hemolytic patient, but may suggest pyruvate kinase deficiency (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 Sβ0, 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 Howell–Jolly bodies. Patients with SC disease and any of the sickle thalassemia compound disorders (including Sβ0 and SS-α thalassemia) may have considerably more target cells than patients with uncomplicated SS. Patients with SC disease may also demonstrate C crystals in some RBCs . 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 flow cytometry ) 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 deficiency.
- Elliptocytosis is most commonly due to iron deficiency or hereditary elliptocytosis (HE). Although there are several other causes of elliptocytosis, as a practical matter if iron deficiency 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) . 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 .
- Schistocytes generally reflect 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 Kasabach–Merritt 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 deficiency, 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 benefit 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 ‘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 deficiency, 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 findings 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., Diamond–Blackfan anemia, idiopathic aplastic anemia, etc.) may produce morphologically unremarkable RBCs.
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 definitive diagnosis.