155Definition

Examination of the peripheral blood smear should be con-sidered, along with review of the results of peripheral bloodcounts and red blood cell indices, an essential componentof the initial evaluation of all patients with hematologic dis-orders. The examination of blood films stained with Wright'sstain frequently provides important clues in the diagnosisof anemias and various disorders of leukocytes and platelets .

Normal human red blood cells are biconcave disks (disko-cytes) with a mean diameter of about 7 .5 µm. Erythrocytesare slightly smaller than small lymphocytes . The hemoglo-bin of red cells is located peripherally, leaving an area ofcentral pallor equal to approximately 30 to 45% of the di-ameter of the cells. Cells of normal size and hemoglobincontent (color) are termed normocytic and normochromic .Larger than normal erythrocytes are macrocytes (diametergreater than 9 µm); small red cells are microcytes (diameterless than 6 µm) ; and those with central pallor greater than50% of the diameter are hypochromic . Abnormal variabilityin size is termed anisocytosis; unusual variation in shape iscalled poikilocytosis; and significant differences among eryth-rocytes in the amount of central pallor is referred to asanisochromia. Polychromatophilia means the erythrocytes havea blue-gray hue to the color of their cytoplasm .

From a diagnostic standpoint, poikilocytosis has no spec-ificity, but the recognition of specific forms of poikilocytes(irregularly shaped cells) often points to specific disorders .Spherocytes are round, densely staining red cells that lackcentral pallor and have a smaller than normal diameter . Instomatocytes, the area of central pallor is elliptical rather thanround, giving the cell the appearance of the opening of amouth (stoma) . Target cells (codocytes) have a centrally lo-cated disk of hemoglobin surrounded by an area of pallorwith an outer rim of hemoglobin adjacent to the cell mem-brane giving the cell the appearance of a target . Leptocytes(or wafer cells) are thin, flat cells with the hemoglobin atthe periphery of the cell . Sickle cells (drepanocytes) are elon-gated, sometimes crescent-shaped, erythrocytes with pointedends . Elliptocytes (ovalocytes) range from slightly oval toelongated cigar-shaped forms . Teardrop erythrocytes (dacry-ocytes) are red cells with one end round and the other endmore pointed . Acanthocytes have several (usually 3 to 7) ir-regularly spaced blunted projections from the margin ofthe cells . Echinocytes are also cells with cytoplasmic projec-tions, but in contrast to acanthocytes, the projections aretypically evenly spaced on the cell surface, more numerous(often 10 to 15), and frequently have sharper points . Schi-zocytes (schistocytes) are fragmented erythrocytes appearingin a variety of morphologic forms such as small triangularerythrocytes, helmet cells, and normal-size erythrocytes with2 to 3 pointed surface projections (keratocytes, or "horncells"). Round erythrocytes with a single, elliptical or roundsurface defect are termed bite cells . Rouleaux formation is a

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phrase denoting the stacking of erythrocytes, generally ina curving pattern .

Morphologic identification of inclusion bodies withinerythrocytes can be helpful clinically . Howell-Jolly bodiesare purple spheres, usually about 0 .5 µm in diameter, pre-senting singly, or rarely multiply, in the cytoplasm . Baso-philic stippling of erythrocytes refers to numerous very smallcoarse or fine blue granules within the cytoplasm. Whenthe stippled particles are due to iron granules (demonstrableby the Prussian blue stain), they are termed Pappenheimerbodies. Malaria parasites may appear as cytoplasmic inclusionbodies within erythrocytes . Platelets overlying erythrocytesmay be mistaken for erythrocyte inclusions .

There are a number of important morphologic abnor-malities of mature granulocytes . Cytoplasmic vacuoles maybe recognized. Toxic granulation refers to small, dark blue-staining granules . Dohle bodies are light blue cytoplasmicinclusions, 1 to 2 µm in diameter . The Pelger-Hues anom-aly, a disorder characterized by impaired nuclear seg-mentation of mature neutrophilic granulocytes, appearsmorphologically as cells with bilobed nuclei (dumbbell oreyeglass shapes) or with round or oval nuclei (Stodtmeistercells) . Hypersegmented neutrophils are cells in which thereare six or more nuclear lobes .

Reactive lymphocytes are usually larger than small lym-phocytes, may have cytoplasmic vacuolization, sometimeshave deep blue staining of the periphery of the cytoplasm,and contain nuclei that may be kidney bean or monocytoidin shape .

Most platelets in the peripheral blood have diametersbetween I and 3 µm . Platelets greater than 3 µm in diameterare "large" (megathrombocytes) . In a normal person usuallyless than 5% of the platelets appear large .

Figure 155.1 shows examples of morphologically normaland abnormal erythrocytes .

Technique

Morphologic abnormalities of peripheral blood cells are dis-covered by microscopic examination with the oil immersionlens of well-prepared films of peripheral blood stained withWright's stain. For appropriate interpretation of the mor-phology of erythrocytes, one concentrates on areas of theslide where the red cells appear singly and have centralpallor. Examination of erythrocytes far out on the featherededge discloses erythrocytes lacking central pallor, whereasin thick areas of the slide the morphology of the erythro-cytes is distorted by contact between cells .

Artifactual changes of erythrocytes occur commonly onperipheral blood films . Cytoplasmic vacuolization of redcells is an artifact. Echinocytes (crenated red cells) are fre-quently caused by hypertonicity or alkalinity of the stainingsolution. Stomatocytes may form when the staining solution

Figure 155.1Morphologically abnormal erythrocytes .

is too acidic. When target cells appear in one area of theslide and not in another, they are artifacts because naturallyoccurring target cells will be distributed evenly throughoutthe slide .

Neutrophils and monocytes often congregate at thefeathered end and at the edges of the blood film . Granu-locytes of blood anticoagulated with EDTA may acquirecytoplasmic vacuoles . Leukocytes may rupture during thepreparation of the blood film, leaving amorphous nuclearmaterial ("basket" cells) . The lymphocytes of chronic lym-phocytic leukemia are particularly prone to rupture .

Although platelets usually occur singly on the blood film,in some cases there is considerable aggregation of platelets,making estimation of the number of platelets more difficult .

Basic Science

Morphologic abnormalities of erythrocytes may be due eitherto production of abnormal erythrocytes in the bone marrowor to pathologic processes to which the erythrocytes areexposed in the circulation . Macrocytes usually reflect ab-normal erythropoiesis in which there is a reduced numberof cell divisions during maturation of erythroid precursors .Hypochromia generally arises because of impaired hemo-globinization of erythroid cells in the marrow . Spherocytescan be due to an inherited membrane abnormality of eryth-rocytes (hereditary spherocytosis) or can result from theaction of phagocytes on erythrocytes sensitized with anti-bodies wherein the phagocytes remove portions of the redcell membrane, creating spherocytes . In addition, bothspherocytes and schizocytes result from the action of ab-

I55 . PERIPHERAL BLOOD SMEAR

normal physical forces in the circulation (particularly shearstress) that cause fragmentation of normal erythrocytes . Sto-matocytes are overhydrated cells, which in three-dimen-sional views are bowl shaped ; they may appear as anintermediate form in the transformation of diskocytes tospherocytes . The target cell is a bell-shaped cell with a rel-ative excess of membrane; in patients with obstructive liverdisease a significant increase in the total membrane contentof cholesterol leads to the increase in cell surface area . Thespur cells (acanthocytes) of chronic alcoholic liver diseasehave increased cholesterol but, in contrast to target cells,normal content of phospholipids . The echinocytes of py-ruvate kinase deficiency form because of decreased ATPgeneration resulting in loss of water and potassium fromthe red cells. Sickle cells result from aggregation in thedeoxygenated state of molecules of hemoglobin S, whichhave the substitution of valine for glutamic acid of the sixthamino acid position of the beta chain of hemoglobin . Inhereditary elliptocytosis, the elliptical shape of the cell isdue to membrane protein abnormalities . Infiltrative dis-orders of the bone marrow, with disruption of the vascu-lature of the marrow, are associated with the formation ofelliptocytes and teardrop erythrocytes . Bite cells apparentlyarise when a phagocyte removes a portion of the red cellalong with a Heinz body .

Howell-Jolly bodies are remnants of DNA ; they are or-dinarily removed from red cells by the spleen . Basophilicstippling occurs in conditions in which the biosynthesis ofhemoglobin is impaired ; the stippled particles are aggre-gates of ribosomes or, in the case of Pappenheimer bodies,aggregates of ferritin, lysosomes, ribosomes, and degen-erating mitochondria.

Pelger-Huet cells occur in a hereditary disorder in whichthe granulocytes function normally and no hematologic ill-ness exists . As an acquired disorder, Pelger-Hiiet cells gen-erally reflect neutrophilic granulocytic dysplasia .

Clinical Significance

Two abnormalities of erythrocytes can be recognized by low-power microscopic examination of the blood. Rouleaux oferythrocytes is related to very high serum protein concen-trations, generally due to multiple myeloma or to macro-globulinemia. Agglutination of red cells on the slide is usuallydue to cold agglutinins .

Macrocytes (frequently oval) in substantial numbers areobserved in patients with megaloblastic anemias (vitaminB12 or folic acid deficiency) often with considerable aniso-cytosis (with some microcytes present as well) . In addition,macrocytes may be prominent in individuals with erythro-leukemia, myelodysplastic disorders, acquired sideroblasticanemia, and with antimetabolite or androgen drug therapy .A lesser degree of macrocytosis is seen commonly in alco-holic patients . Polychromatophilic macrocytes usually in-dicate a high reticulocyte count.

A predominance of hypochromic microcytic cells is foundin iron deficiency anemia, thalassemia, and hereditary sid-eroblastic anemia, and in some patients with the anemia ofchronic disorders and with lead intoxication . For individualswith mild anemia, the degree of microcytosis is usually sub-stantially greater in patients with thalassemia minor thanthose with iron deficiency . Anisochromia with the presenceof a dimorphic red cell population (hypochromic and nor-mochromic) is observed in acquired sideroblastic anemia,

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patients with thalassemia minor after transfusions, and per-sons with iron deficiency following transfusions or treat-ment with iron .

Examination of peripheral blood films of normal personsreveals small numbers of poikilocytes, usually less than 2% .In the assessment of the significance of poikilocytosis, onemust identify the predominant abnormal morphologic formand exclude artifactual alterations of the red cells .

Spherocytes are the predominant morphologic abnor-mality in patients with hereditary spherocytosis, autoim-mune hemolytic anemia, and hemolytic transfusion reactions,and are common, along with schizocytes, in patients withred cell fragmentation disorders . Spherocytes may also beobserved in less common hemolytic states such as the Heinzbody hemolytic anemias and clostridial sepsis . Stomatocytesare seen in large numbers in alcoholics and in the raredisorder of hereditary stomatocytosis, and in small numbersin normal persons. There are four major circumstancesin which target cells appear as the major morphologicabnormality : thalassemia, hepatic disease with jaundice,hemoglobin C disorders, and the postsplenectomy state .Lesser numbers of target cells are found in sickle cell ane-mia, iron deficiency, and lead intoxication . Leptocytes areseen in thalassemic disorders and with obstructive liver dis-ease. Sickle cells and dense, deformed poikilocytes ("irre-versibly sickled cells") are characteristic of sickle cell anemia,hemoglobin SC disease, hemoglobin S-thalassemia, andhemoglobin C-Harlem, but are not observed in sickle celltrait.

A large number of elliptocytes (25 to 75% of the redcells) usually indicates hereditary elliptocytosis. Moderatenumbers of elliptocytes are seen in thalassemia and mye-lofibrosis, and lesser numbers in iron deficiency andhypersplenic states. Teardrop erythrocytes (usually with el-liptocytes) are particularly prominent in patients with mye-lofibrosis with myeloid metaplasia and occur frequently inpatients with other infiltrative disorders of the bone marrowsuch as leukemia and metastatic carcinoma . Acanthocytesare the principal morphologic abnormality in abetalipopro-teinemia and in the "spur cell anemia" associated with severealcoholic liver disease . Acanthocytes are found along withother poikilocytes after splenectomy . Conditions associatedwith the appearance of echinocytes are pyruvate kinase de-ficiency of erythrocytes, uremia, carcinomas, and immedi-ately after the transfusion of aged or metabolically depletedblood (echinocytes form during storage of the blood) . Cor-rection of the metabolic abnormalities of uremia results indisappearance of echinocytes . Schizocytes are the morpho-logic hallmark of the hemolytic anemias associated with redcell fragmentation (i.e ., the microangiopathic hemolyticanemias and those hemolytic anemias associated with mal-functioning cardiac prostheses) . Schizocytes also may formduring disseminated intravascular coagulation . Bite cells areprincipally related to the various Heinz body hemolytic ane-mias, such as glucose-6-phosphate dehydrogenase defi-ciency .

Howell-Jolly bodies are found in patients who have hadsplenectomies or are hyposplenic (e.g., sickle cell anemia)and rarely in megaloblastic anemias . The three disordersparticularly associated with coarse basophilic stippling arelead poisoning, sideroblastic anemia, and thalassemia .

Nucleated erythrocytes, usually in small numbers (inadults), may appear in the blood when the marrow is under

Xl . HEMATOPOIETIC SYSTEM

intense stimulation due to severe hemolysis, hemorrhage,or hypoxia . In addition, nucleated red cells and immaturemyeloid cells may be recognized with infiltrative disordersof the marrow such as myelofibrosis, leukemia, and meta-static carcinoma. In patients with megaloblastic anemias thenucleated erythrocytes in the blood have megaloblastic nu-clear features .

Cytoplasmic vacuolization of granulocytes is observed inpatients with bacteremia or other severe infections . Toxicgranulation, a rather nonspecific finding, is found in a va-riety of disorders including infections and metabolic de-rangements . Dohle bodies are seen in patients with infectionsand burns, during pregnancy, after cytotoxic chemotherapy(particularly with cyclophosphamide), and with the May-Heggelin anomaly. Pelger-Huet cells, on an acquired ratherthan hereditary basis, are particularly associated with mye-lodysplastic and myeloproliferative disorders . Hyperseg-mented neutrophils usually are an important clue to thepresence of vitamin B 12 or folic acid deficiency but are oc-casionally found in patients with myelodysplasia or mye-loproliferative disorders .

The diagnosis of leukemia is commonly obvious by rec-ognition of abnormal numbers and stages of developmentof myeloid or lymphoid cells in the blood. Immature mon-ocytes suggest either leukemia or myelodysplasia . A signif-icant increase in the number of basophils usually indicatesa myeloproliferative disorder .

A high percentage of reactive lymphocytes may be seenin viral illnesses such as infectious mononucleosis, viral hep-atitis, cytomegalovirus infection, HIV infection and rubella,or with reactions to drugs such as phenytoin and para-aminosalicylic acid . Lymphocytes with convoluted nuclei maybe found in T cell lymphomas and in the Sezary syndrome .

An increased number of large platelets is observed inthrombocytopenia with immune-mediated hyperdestruc-tion, disseminated intravascular coagulation, myeloprolifer-ative disorders (particularly myelofibrosis), megaloblasticanemias, the Bernard-Soulier syndrome, and the May-Heggelin anomaly . Platelet size is normal in hypersplenicstates . Microthrombocytes are found in the Wiskott-Ald-rich syndrome . Hypogranular platelets are seen in the mye-loproliferative disorders .

References

Bessis M. Red cell shapes. An illustrated classification and its ra-tionale . Nouv Rev Fr Hematol 1972 ;12:721-46 .

Bessis M, Lessin LS, Beutler E. Morphology of the erythron . In :Williams WJ, Bender E, Ersler AJ, Lichtman MA, eds . Hema-tology . 3rd ed. New York: McGraw-Hill, 1983 ;257-79 .

Garg SK, Lackner H, Karpatkin S. The increased percentage ofmegathrombocytes in various clinical disorders . Ann Intern Med1972 ;77:361-69 .

Lessin LS, Klug PP, Jensen WN . Clinical implications of red cellshape. In : Stollerman GH, ed . Advances in internal medicine .Chicago: Year Book Medical Publishers, 1976 ;21 :451-99 .

Schwartz SO, Stansbury F . Significance of nucleated red blood cellsin peripheral blood . Analysis of 1,496 cases. JAMA 1954 ; 154 :1339-40 .

Zeigler Z, Murphy S, Gardner FH . Microscopic platelet size andmorphology in various hematologic disorders. Blood 1978 ;51 :479-86 .