Peripheral blood smear Information and Courses from MediaLab, Inc.

Platelet satellitism and platelet clumping can cause pseudo-thrombocytopenia. Platelet satellitism was first reported in the early 1960's. It is a rare condition that occurs when an IgG antibody forms in the presence of EDTA, the anticoagulant that is used for the collection of hematology blood specimens. The IgG antibody is directed against the glycoprotein IIb/IIIa complex on the platelet membrane. As the antibody coats the platelets, the platelets rosette around segmented neutrophils, bands, and sometimes around monocytes. Antibody-coated platelets that are huddled around white cells will not be counted as platelets by automated equipment and the platelet count will be falsely decreased. If a peripheral blood smear is reviewed, platelets will be observed attached to white blood cells. The image on the right illustrates platelet satellitism with platelets adhering to a neutrophil. Platelet clumping can also occur in the presence of EDTA and the platelet count again will be falsely decreased. The count will probably be flagged by the analyzer for platelet clumps or giant platelets. If either platelet satellitism or platelet clumps are observed on the peripheral smear, the sample could be recollected using sodium citrate as the anticoagulant. Platelets can then be counted using the automated method. The platelet count from a tube that contains liquid sodium citrate will need to be corrected for the dilutional effect of the citrate; this can be accomplished by multiplying the platelet count that is obtained from the automated analyzer by 1.1.

These laboratory findings are associated with TTP and HUS: Thrombocytopenia -- Platelet count is often less than 20 x 109/L in TTP, but may not be as low in HUS. Schistocytes (red blood cell fragments, as indicated by the arrows in the image to the right) may be observed on the peripheral blood smear. Schistocytes are the result of erythrocytic membrane damage caused by sheering of red blood cells as they pass through a fibrin mesh of clot formation occurring in the blood vessels. LDH, serum bilirubin, and reticulocyte counts are elevated. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are usually normal. Proteinuria and hematuria may be present.

Most technologists working in the clinical hematology setting are familiar with the morphology of blood cells found in peripheral blood smears. Many of the same blood cells found in the peripheral blood are also found on cytospin preparations of body fluids. While the morphologies are similar between the two sources, there are changes to the "comfortable/familiar" peripheral blood morphology that are introduced by the cytospin technique. It is paramount to fully conceptualize the appearance of both normal and abnormal blood cell morphologies in body fluids in order to accurately perform fluid differentials.The cytopsin process works by wicking fluid into a filter while fluids samples are spun into a central column and deposited in a mono-layer onto a defined area of a slide. This allows the cells to be concentrated for appropriate identification. This technique can cause pronounced changes to morphology and staining characteristics, as well as cellular destruction if the cytospin malfunctions.The cytospin technique is known to stretch and distort cellular and nuclear morphology and allow nucleoli to appear more prominent than what one would normally see in peripheral smears. Cytospinning, however, does not change nuclear:cytoplasmic ratios nor does it alter relative chromatin textures or clumping patterns. Though this technique can make cells appear larger that on the peripheral blood smear, it can not change cytoplasmic textures and granulation. Focusing on these steadfast features can make cytospin morphology less intimidating.

The image shown to the right depicts a cluster of cells containing both normal and atypical (reactive) lymphocytes. The variations in size, depth of color, and cytoplasmic volume similar to what one would expect to observe on a peripheral blood smear. There is a difference in the density of the chromatin in the lymphocytes as well as the uniformly regular nuclear shape. The atypical lymphocytes on the left (green arrow) are larger than the other lymphocytes while exhibiting a larger amount of generally non-granular cytoplasm. One of the atypical lymphocytes has a few azurophilic granules in the Golgi area.The single cell at the bottom right with the finer, less dense chromatin and irregular nucleus is a monocyte (blue arrow).

Bone marrow smears can be very cellular and it can be difficult to keep track of where you are on the smear while keeping your correct hand position on the keyboard . Having a good strategy to use when counting cells and performing differentials can make this less difficult. On peripheral blood differentials, it is easy to observe and count each cell individually as the stage is moved to bring the next field into view. However, with bone marrows, the total number of keys that need to be used on the differential counter is greater than the number that need to be used with a peripheral blood smear and the number of cells per field is also increased dramatically, making it easy to lose track of the cells on the smear or one's hand/keyboard placement. It can be simpler and less stressful to work on the quadrant system. There are two different ways to do this: Divide the field into quadrants. Count the individual cells in each quadrant separately. This decreases the number of cells into more manageable bites. However, you still have the increased number of cell types to deal with and possible keyboard frame-shifts. Divide your keyboard into quadrants. Search your field for a limited number of cell types and tally all you see before moving on to the next grouping of cell types. Once you tally all your groups then move on to the next field (e.g., lymphocytes, monocytes, macrophages, eosinophils, basophils, plasma cells, erythroids, segmented neutrophils, bands, etc). You can make these small groupings for any cells as long as you cover the entire list of cell types that your laboratory reports in its bone marrow differential protocol. Remember that blasts are identified by cell type and there will usually be a separate key for pronormoblasts, myeloblasts, lymphoblasts, and possibly monoblasts and plasmablasts. It is possible to combine both methods, using the keyboard quadrant technique with a restricted portion of the total microscope field. This is useful when you are getting close to your total tally and do not want to alter the balance by only counting one cell type for the last few cells.

Erythrocyte inclusions are elements that may be present in red blood cells (RBCs). The appearance, composition, and associated physiology of the inclusions are specific for each type of inclusion. Identification and reporting of these inclusions are important because their presence may indicate diseases or disorders.Many erythrocyte inclusions can be visualized on a Wright-stained smear. However, some erythrocyte inclusions can only be observed by using a special stain. For example, to confirm the presence of Heinz bodies, hemoglobin H bodies, or reticulocytes, smears must be prepared after staining an aliquot of fresh whole blood with a supravital stain such as new methylene blue or brilliant cresyl blue. The image on the right is a peripheral blood smear prepared after staining the blood sample with a supravital stain; Heinz bodies, which are clumps of precipitated hemoglobin, are indicated by the arrows. To detect siderosomes, a stain must be used that is specific for iron, such as Prussian blue.

Howell-Jolly bodies are round, smooth, almost pyknotic, dark-purple bodies ranging in size from 0.5 to 1.0 micron in diameter. These RBC inclusions contain DNA. Howell-Jolly bodies are thought to be nuclear remnants or aggregates of chromosomes that have separated from the mitotic spindle and remain behind after the remainder of the RBC nucleus is expelled. The spleen normally removes these bodies from the RBCs. However, Howell-Jolly bodies can be observed on a Wright-stained peripheral blood smear post-splenectomy or when the spleen is not functioning properly. Howell-Jolly bodies may also be seen in hemolytic anemias such as sickle cell anemia.When Howell-Jolly bodies are present, they usually occur singly, as shown in the image on the right. It is possible, though, that two or more may be seen in a single RBC, usually occurring in the presence of megaloblastic anemia or abnormal erythropoiesis.

Pappenheimer bodies may be seen in the cytoplasm of mature and immature erythrocytes on a Wright-stained peripheral blood smear. They appear as small dark purple granular bodies of varying size, frequently clustered in groups of two, three, or more near the edge of the cell. They are composed of degenerating cellular remnants that contain iron. Pappenheimer bodies are most likely caused by accelerated red cell division or impaired hemoglobin synthesis.

Codocytes, also referred to as target cells can be observed on the peripheral blood smear from a patient with sickle cell trait (HbSA), as indicated by the arrows in the image on the right. Codocytes are cells that can be seen in hemoglobinopathies, thalassemia, iron deficiency, and other anemias where there is a decrease in the mean corpuscular hemoglobin concentration (MCHC).Sickle cell trait will not usually show completely sickled cells because of the HbA that is present in each cell. HbA usually comprises greater than 60% of the hemoglobin in HbSA.However, rare drepanocytes (sickle cells) and occlusive crisis may be found during times of extreme exercise and fluid restriction.

CBC results for patients with sickle cell disease (HbSS) can reflect a slight, moderate, or severe anemia. The CBC and peripheral blood smear shown below are those of an African-American 12-year-old male patient with HbSS.Reference intervals may vary between facilities and are dependent on patient age and gender. The reference intervals that are shown below are specific to this case. Parameter Patient Result Reference Interval White blood cell count (WBC) 18.9 3.2 - 9.8 x 109/L Red blood cell count (RBC) 3.88 4.50 - 5.70 x 1012/L Hemoglobin 10.0 13.8 - 17.2 g/dL Hematocrit 32 41 - 50% MCV 82 80 - 110 fL RDW 22.7 11 - 14.5 Platelet count 458 140 - 440 x 109/L

CBC results for patients with double heterozygous condition of Sickle Cell with Beta thalassemia (HbS/Bthal) can be asymptomatic or demonstrate a slight to moderate anemia. The CBC and peripheral blood smear for a patient with HbS/Bthal, which appear below, reflect results more consistent with thalassemia than hemoglobinopathy. The hemoglobin electrophoresis results on the following page revealed this condition.Reference intervals may vary between facilities and are dependent on patient age and gender. The reference intervals that are shown below are specific to this case. Parameter Patient Result Reference Interval White blood cell count (WBC) 11.4 4.0 - 11.0 x 109/L Red blood cell count (RBC) 6.37 4.5 - 5.9 x 1012/L Hemoglobin 13.6 13.8 - 17.2 g/dL Hematocrit 38.1 41 - 53% MCV 59.9 80 - 100 fL RDW 18.9 12 - 14.6 Platelet count 150 - 440 x 109/L

The Kleihauer-Betke test is performed to quantitate the number of fetal cells present in the maternal circulation. Once the size of the feto-maternal hemorrhage (FMH) is determined, the appropriate RhIg dose can be calculated and administered to prevent the mother from making anti-D.The test is based on the principle that red cells containing fetal hemoglobin (HbF) are less susceptible to acid elution than cells containing adult hemoglobin (HbA).General descriptionA peripheral blood smear is made from the maternal postpartum sample and treated with acid. Fetal cells remain intact because of high concentrations of HbF, while HbA is eluted from the maternal cells.After acid treatment the slides are washed, stained, and examined microscopically.The number of fetal cells (which take up the stain) are counted per number of maternal cells (which appear as ghost cells) to give % fetal cells.The volume of fetal bleed is then calculated to determine how much additional RhIg is required.See Kleihauer-Betke graphic (Source: ENet Answers) The top image on the right illustrates a negative Kleihauer-Betke test. The blue arrows in the bottom image point to fetal cells that have taken up the stain. The red arrows indicate maternal cells, which appear as ghost cells.These images were provided courtesy of Mount Sinai Blood Transfusion Laboratory, Toronto, Ontario and can be found on Canada's Transfusion Safety Officer's Website. Available at: http://www.transfusionsafety.ca/library/kb-ros.html. Accessed September 26, 2011.Limitations: Despite its widespread use, the Kleihauer-Betke test has significant limitations, including Low sensitivityPoor reproducibility.

The first group is composed of erythrocytes or red blood cells (RBC's). The main function of the erythrocytes is the transport of oxygen from the lungs to the body tissues. All of the cells in this Wright's stained peripheral blood smear are red blood cells. The image to the right shows the normal morphology of red blood cells.

Glassy, Eric F.,(Ed). Color Atlas of Hematology: An Illustrated Field Guide Based on Proficiency Testing. 1998. College of American Pathologists Hematology and Cliical Microbiology Research Committee. College of American Pathologists, Northfield, IL 60093-2750.Hookey,L., Dexter, D., Lee,D. H. The Use and Interpretation Of Quantative Terminology In Reporting Red Blood Cell Morphology. Laboratory Hematology 7:85-88, 2001.Peterson P, Blomberg DJ, Rabinovitch A, Cornbleet PJ. Physician Review of the Peripheral Blood Smear: When and Why. For the Hematology and Clinical Microscopy Resource Committee of the College of American Pathologists. Laboratory Hematology 7:175-179, 2001

Homozygous Hb E is common in Southeast Asia and presents with very mild anemia and seldom requires transfusion. Over 30 million people in the world are HbE carriers, making this abnormal hemoglobin almost as common as HbS. Hb E is uncommon in North America and in Europe, but with changing immigration patterns, Hb E and related diseases cannot be ignored. Peripheral blood smear findings of target cells, microspherocytes, red cell hypochromia, red blood cell fragments, and nucleated red blood cells may be noted. Evidence from hemoglobin electrophoresis is required to establish a diagnosis.Clinically, a very important and severe disease is Hb E/beta thalassemia in which there is hemolysis requiring repeated transfusions. Severe anemia, low MCV, and elevated RBC are characteristic of Hb E/beta thalassemia.

Ovalocytes/elliptocytes are oval or elliptical red blood cells that range in shape from slightly egg-shaped to rod or pencil forms. They have normal central pallor with the hemoglobin concentrated at the ends of the elongated cells. The ends of the cells are blunt and not sharp like sickle cells.A rare ovalocyte/elliptocyte (less than 1%) may be found on almost any peripheral blood smear. However, when they comprise more than 25% of the red blood cells on the blood smear, hereditary elliptocytosis (HE) is probable. In most cases, patients are asymptomatic while having normal red blood cell life spans, although a mild anemia may occur. Resistance to malarial infection may be a beneficial attribute of HE.

When an Initial analysis of red blood cells (RBCs) from an automated instrument are found to be abnormal, many laboratories will microscopically evaluate the peripheral blood morphology of the RBCs. This important step can help to establish which, if any, abnormalities are present as well as correlate possible disease states or conditions associated with the findings. Most laboratories will employ guidelines for review of the peripheral blood smear for RBC morphology. Though each laboratory will create their own guidelines, the following are a few examples that could trigger a manual, microscopic peripheral blood smear review:Hemoglobin: < 8 or >18 g/dL (<10 or > 21g/dL in a newborn)Hematocrit: <20% or > 60% in adults (<40% or >65% in a newborn)MCHC: <29 g/dLMCV: <69 femtoliters (fl) or >110flFlags generated by the hematology analyzer that indicate possible red cell abnormalities or spurious results In most laboratories, when these findings are noted, they should be followed up with a peripheral blood smear review for RBC morphology.

Now that you have reviewed the red blood cell morphology tables, we will incorporate disease state correlations with the help of case studies and questions with answers. The following case studies and questions will be presented in groups of related morphologic findings on the peripheral blood smear. The case studies and questions will contain multiple choice and true/false questions for you to answer. There will be helpful information regarding the morphologies noted and disease states present displayed in the feedback sections for each question- so be sure to review the explanations after answering the questions. Enjoy!

The Kleihauer-Betke test is performed to quantitate the number of fetal cells present in the maternal circulation. Once the size of the FMH is determined, the appropriate RhIg dose can be calculated and administered to prevent the mother from making anti-D.The test is based on the principle that red cells containing fetal hemoglobin (HbF) are less susceptible to acid elution than cells containing adult hemoglobin (HbA).General description A peripheral blood smear is made from the maternal postpartum sample and treated with acid. Fetal cells remain intact because of high concentrations of HbF, while HbA is eluted from the maternal cells. After acid treatment the slides are washed, stained, and examined microscopically. The number of fetal cells (which take up the stain) are counted per number of maternal cells (which appear as ghost cells) to give % fetal cells. The volume of fetal bleed is then calculated to determine how much additional RhIg is required. See Kleihauer-Betke graphic (Source: ENet Answers) Limitations: Despite its widespread use, the Kleihauer-Betke test has significant limitations, including Low sensitivity; Poor reproducibility.

Many variations in morphology may be seen when examining Wright stained peripheral blood smears. One method of classifying these variations in white cell morphology is based on the way the body responds to a stimulus, deficiency, or the presence of an inherited defect. This classification falls into three groups:Pathological: Cells may show abnormalities in appearance and/or function. The body is responding abnormally to a stimulus or inherited defect, resulting in physiological impairment in the patient. Nonpathological: Cells may show variation in morphology but their function is normal. Their presence does not cause physiological impairment. Reactive: Cells show variation in morphology but are functioning normally in response to a specific stimulus, such as a virus or bacteria. There is a disease process in progress to which the cells are responding. Although the morphology has varied from normal and their presence is significant, the body is responding normally to a stimulus.

The findings in the image to the right (peripheral blood smear) would elicit a report comment of "increased platelets" of a high magnitude, such as "marked" or "4+." Estimates of platelet counts from review of a peripheral blood should be made on each smear examined. This provides a simple estimate of "high", "low", or "normal" which usually corroborates the value generated from an automated cell counter. A formula for estimating platelet counts must be established for each laboratory. One guideline for the estimation of platelets is as follows: Count platelets on 5 fields using 1000X magnification (care should be taken to ensure the fields used for counting are not too thick or too thin) Average the platelet counts obtained Multiply by 15 X 109/L to obtain estimated platelet count (some laboratories prefer a 20 X 109 multiplier in this step if capillary blood is used)Such a counting scheme for platelets when clustered, as in the image, is probably not needed, as there are more than 100 platelets in the field. This translates into a platelet count of 1500 X 109/L or more.

The blood count alone might be interpreted as reflecting infection, possibly supporting a diagnosis of acute appendicitis. However, the technologist performing the differential noted that more than 70% of the segmented neutrophils had bi-lobed or mono-lobed nuclei, strongly suggesting Pelger-Huet anomaly. Since the peripheral blood smear did not support the diagnosis of appendicitis in this patient, and since abdominal pain localized to the right lower quadrant never developed, the boy was hydrated with intravenous fluid and observed. After hydration, his constitutional symptoms improved and the abdominal pain subsided. People entering high altitude where the humidity may be very low are susceptible to dehydration and may experience symptoms related to mountain sickness.

Plasma cells are uncommonly observed in the peripheral blood smear. They are normal constituents of lymph nodes, spleen, connective tissue and bone marrow. The presence of plasma cells in the peripheral blood is indicative of a large number of conditions, mostly related to infections , immune disorders, malignancies, toxic exposures, hypersensitivity reactions and their responses.Although mature plasma cells have a distinct appearance, they still may be confused morphologically with immature plasma cells and other cells with inclusions, reactive changes or nucleated red bloods cell with altered identities. In the image to the right, a plasma cell is present. The plasma cell has an eccentric immature nucleus with a muddy chromatin pattern. Note also clumping and stacking of the erythrocytes, typical of rouleaux formation, implicating an increase in plasma gamma globulin. Further studies are in order, including a bone marrow examination, where at least 30% of bone marrow cells should be variations of mature and immature plasma cells. Serum protein electrophoresis will reveal a monoclonal globulin spike, and light chains in excess of 1.0 gm/24 hours may be seen in the urine. The presence of lytic bone lesions is a convincing clinical clue. With these findings in combination, a diagnosis of myeloma can be made with assurance.