Peripheral Blood Smear Review

An automated complete blood count (CBC) with differential white blood cell count can determine 32 different hematological parameters. Automated cell counters incorporate a method for flagging white blood cell (WBC), red blood cell (RBC) and platelet abnormalities. The flagged abnormalities prompt a medical scientist to perform a manual peripheral smear review and differential to verify the abnormality flagged as well as allow identification of additional morphological abnormalities.

The initial microscopic evaluation of all flagged specimens is performed by a clinical laboratory scientist. Their review serves several important functions including verification of automated results; identification of abnormal, immature and atypical cells; and recognition of clinically significant morphological abnormalities. Blood smear abnormalities identified on new patients or infrequent outpatient visitors are then submitted to a clinical pathologist for confirmation and further evaluation. Any comments and findings identified by the clinical pathologist are reported as free text within the finalized report. 

In rare circumstances, a peripheral blood specimen may not generate any abnormal flags by the instrument. These mostly include qualitative abnormalities such as hematological malignancies (e.g. chronic and acute leukemias, myelo-proliferative neoplasm, and myelodysplasia), hereditary hemolytic disorders (hereditary spherocytosis), and presence of infectious agents (malaria and ehrlichia). A separate request for peripheral blood smear review by a clinical pathologist should be restricted to these disorders, if clinical suspicion is high.

Different zones of the peripheral smear need to be examined. The lateral edges contain large cells such as monocytes, immature cells, and blasts. The feathered edge will contain platelet clumps, malignant cell clumps and microorganisms. Differential counts and morphology examination should be performed in the thin area where cells are nicely separated. 

Red Blood Cells

The following abnormal red blood cell abnormalities may be observed:

• Elliptocytes & ovalocytes - hereditary elliptocytosis, iron deficiency, RBC enzymopathies
• Pencil cells – iron deficiency
• Microcytes – iron deficiency, thalassemia
• Oval macrocytes - megaloblastic anemia & dyserythropoiesis
• Round macrocytes - liver disease
• Spherocytes - hereditary spherocytosis, WAIHA and DHTR
• Echinocytes (burr cells) have evenly spaced projections- storage artifact, uremia, HUS, severe burns, CABG, RBC enzymopathies
• Acanthocytes (spur cells) have irregular spaced spikes - abetalipoproteinemia, malnutrition, end stage liver disease, splenectomy, Vitamin E deficiency, McLeod syndrome
• Stomatocytes - alcohol abuse & liver disease, hydroxyurea Rx, hereditary stomatocytosis
• Target cells (codocytes) – liver disease, Hgb C, D & O, SCD, beta thalassemia, splenectomy
• Teardrop cells (Dacrocyte) - megaloblastic anemia, myelofibrosis, myelodysplasia
• Bite cells (Blister cells) - G6PD deficiency and dapsone Rx, unstable hemoglobins
• Helmet cells – TMA, mechanical valves, pulmonary emboli
• Schistocytes (fragments) - TMA, DIC, prosthetic heart valve 
• Keratocyte - DIC, burns, vascular prosthesis
• Sickle cells (Drepanocytes) – Sickle cell disease, sickle cell beta thalassemia
• Rouleaux – myeloma, chronic liver disease, chronic inflammation

RBC Inclusions

Red blood cells may contain different kinds of inclusions.

• Basophilic stippling - megaloblastic anemias, sideroblastic anemias, lead poisoning
• Cabot rings - hemolytic anemia leukemia, megaloblastic anemia
• Heinz bodies - G6PD deficiency, oxidizing chemical exposure, unstable hemoglobin disorder
• Hemoglobin C crystals - splenectomy in homozygous hemoglobin C disorder
• Hemoglobin H inclusuions - alpha thalassemias
• Howell-Jolly bodies - acute hemolytic anemias, post-splenectomy, megaloblastic anemias
• Pappenheimer bodies - sideroblastic anemias, megaloblastic anemias, post-splenectomy
• Parasites - babesiosis, malaria 

Platelets

Platelet counts my be decreased (thrombocytopenia) or increased (thrombocytosis). A low platelet count may be due decreased production, increased destruction, or platelet clumping.

Thrombocytopenia

Thrombocytopenia has many etiologies:

• EDTA induced platelet clumping
• Incidental thrombocytopenia of pregnancy
• Thrombotic thrombocytopenic purpura (TTP)
• Hemolytic uremic syndrome (HUS)
• Disseminated intravascular coagulation (DIC)
• Drug induced
• Hypersplenism
• Idiopathic thrombocytopenic purpura (ITP)
• Heparin induced thrombocytopenia (HIT)
• Hereditary thrombocytopenia - May-Hegglin, gray platelet syndrome, Bernard Soulier, X-linked, and Wiskott Aldrich
• Posttransfusion purpura

Platelet clumps are often observed at the feathered edge of the blood smear. In vitro platelet clumping can occur in EDTA anticoagulated blood resulting in falsely low platelet count. Platelet clumps may be counted as WBC, resulting in a falsely elevated WBC count. Platelet clumps may be broken up by vortexing the sample. If clumping persists, blood should be recollected in a blue top tube containing sodium citrate. If platelet clumping cannot be resolved, a platelet estimate can be reported. Eight to 25 platelets per immersion oil field correlates with a normal platelet count. 

Thrombocytosis

Thrombocytosis can be primary or reactive. Platelet count is a poor discriminator between them.

• Primary - often accompanied by increased Hgb, MCV or WBC                                                                                                  
• Reactive - often accompanied by iron deficiency microcytic anemia. Platelet count can be more than 1 million.

White Blood Cells

White blood cell counts can be decreased (leukopenia) or increased (leukocytosis). Decreased white blood cell counts are usually due to a decrease in either neutrophils (neutropenia) or lymphocytes (lymphopenia). There are many etiologies for both. 

Neutropenia

• Mild neutropenia: 1000-1500 cells/uL (1.0-1.5 x 10³/uL)
• Moderate neutropenia: 500-999 cells/uL (0.5 - 0.99 x 10³/uL)
• Severe neutropenia: < 500 cells/uL (<0.5 x 10³/uL)  
• Chronic benign – resolves in 5 to 15 months
• Benign ethnic – Duffy antigen negative
• Drug induced - antibiotics
• Sepsis
• Immune
• Large granular lymphocyte leukemia

Lymphopenia

• Corticosteroids
• Anti-thymocyte globulin
• Viral infection such as HIV
• Critical illness such as sepsis, autoimmune disease, sarcoidosis, chronic renal failure, and alcohol abuse

Leukocytosis

Elevated white blood cell counts (leukocytosis) can be classified as granulocytosis, monocytosis, or lymphocytosis. Each can be reactive or neoplastic. 

Reactive Leukocytosis

Reactive morphologic changes include toxic granulation, vacuolization and the presence of Dohle bodies in segmented and band neutrophils & Howell Jolly bodies.

• Toxic granulation is the presence of large purple or dark blue primary granules in the cytoplasm of neutrophils, bands, and metamyelocytes
• Dohle bodies are single or multiple pale blue cytoplasmic inclusions near the cellular membrane of neutrophils, bands, or metamyelocytes. They are remnants of the endoplasmic reticulum.
• Vacuoles are variable in size and may coalesce. They are considered toxic only if accompanied by other toxic changes. 
• Blue-green crystals are indicative of liver failure and imminent death.
• Growth factor therapy can cause retention of primary granules in cells with mature nuclei.

Neoplastic Leukocytosis

• Myeloproliferative disorder 
• Acute leukemia
• Chronic leukemia

Monocytosis

• Monocytosis can be reactive or neoplastic.Reactive monocytosis includes inflammation, infection, autoimmune disorders, corticosteroids & growth factors
• Relative monocytosis accompanies recovery from chemotherapy or drug induced neutropenia.
• Neoplastic causes include CMML, AML with monocytic differentiation, CML with p190 fusion & myeloid neoplasm with rearrangements of PDGFRA, PDGFrB, FGFR1 or PCM1-JAK2

Eosinophilia

Eosinophilia can be primary or secondary. 

• Primary eosinophilia is a bone marrow disorder
• Secondary eosinophilia accompanies parasitic infestation, drug reactions, asthma and other allergic reactions, vasculitides and metastatic cancer