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Lymphocyte Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Lymphocyte and links to relevant pages within the course.

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Blood Banking Question Bank - Review Mode (no CE)
Which of the following is responsible for causing graft-versus-host reactions:View Page
Gamma irradiation of cellular blood components is required in which of the following situations:View Page
HLA-A and HLA-B antigens can be detected using which of the following techniques?View Page

Body Fluid Differential Tutorial
Lymphocytes vs. Monocytes

Lymphocytes, for the most part, have a more regular cytoplasmic border without the cytoplasmic blebbing and pseudopods that are present in monocytes.Observe the differences in character of the cytoplasm between the two cell types. Both cells have blue cytoplasm, however the monocyte (red arrow) has a grainy, gritty texture that is absent in the lymphocytes. In addition, there are fine red cytoplasmic granules present in the monocyte that are not apparent in the lymphocytes.Notice the relatively regular nuclear shape of the lymphocytes (blue arrow) versus the more complex nuclear shape of the larger monocyte. There is also a difference in the texture of the chromatin between the two cell types. The lymphocyte chromatin is more dense and clumped.

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Lymphocytes

The image in this slide depicts slightly activated normal lymphocytes (see arrows).Observing the cytoplasm, it is apparent that the cell it is pastel blue and non-granular; identical to what one would expect to see in a slightly viral peripheral lymphocyte.It is also important to note the chromatin texture and staining. While the chromatin is a bit more loose than a normal lymphocyte observed on a peripheral blood smear, it still has course, smudge-like, clumped chromatin of a mature lymphocyte.

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Lymphocytes and Atypical Lymphocytes

Similar to peripheral blood, lymphocytes in fluids come in all sizes. A larger lymphocyte is not necessarily an abnormal or atypical lymphocyte, however. In the image shown here there is one atypical lymphocyte (blue arrow) adjacent to the monocyte at the top of the smear. The atypical lymphocyte is almost as big as the monocyte, however the nucleus is more regular. The amount of cytoplasm is similar between the two cells, but the atypical lymphocyte has a deeper blue shading at the edge of the cytoplasm. Though there are nucleoli present in this atypical lymphocyte, it does not necessarily mean that this is a malignant cell. Cyto-spinning can expose nucleoli in cells that would not normally appear to have nucleoli in the peripheral blood. The balanced amount of cytoplasm in comparison to the nucleus and the overall size of the cell are consistent with the range of variation found with atypical lymphocytes.In this smear, there is also a plasmatoid lymph in the lower left of the cluster (green arrow) and a basophil in the lower right (orange arrow).

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Viral Lymphocytes

The smear in this slide came from a patient suffering from viral meningitis. Notice the absence of neutrophils and the large numbers of lymphocytes, most of which are normal. There is much greater amount of cytoplasm in the three atypical lymphocytes grouped in the center of the image (see arrows). These atypical lymphocytes have a chromatin pattern similar to a mature lymphocyte, even though the cells have increased size.The more activated a lymphocyte becomes in response to a viral infection, the more likely it is to see nucleoli on a cytospin, especially in the pediatric population.Nucleoli alone does not make an atypical lymph malignant or leukemic. Chromatin textures and cytoplasmic volumes will be altered as well in leukemia and lymphoma.

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A patient with an infectious mononucleosis infection presents in the emergency room. Physicians order a spinal tap which is immediately sent to the laboratory for review. Please identify the cell in the image below from this patient's cerebrospinal fluid sample.View Page
Monocytes

Monocytes (red arrows) are larger than lymphocytes (blue arrows). They tend to have a more complex nuclear shape, and cytoplasm that is more grainy than lymphocytes. Their chromatin is softer, finer, and more "lacy" when compared to lymphocyte chromatin.Notice the greater irregularity of the cytoplasmic membranes in the monocytes compared to the lymphocytes.

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Bone Marrow Aspiration Part I: Normal Hematopoiesis and Basic Interpretive Procedures
Hemophagocytosis

One of the normal roles of the marrow macrophage is to remove cellular debris. In a normal bone marrow, this includes the engulfment of extruded RBC nuclei and old, non-nucleated RBCs at the end of their lifespan. In some patients, macrophages lose their ability to distinguish self from non-self (i.e., invader/ pathogen) and good cells from old/senescent cells. This can happen because of an inborn error in the macrophages or by an infection-mediated transformation. When this change occurs, any cell in the vicinity of a defective macrophage become a target for engulfment. This term is called hemophagocytosis.The top image on the right shows two macrophages that have ingested several different cell types (see red arrows). There is the normally ingested non-nucleated RBC, but also the abnormally ingested segmented neutrophil and at least one early nucleated RBC precursor. Viable bone marrow precursors are not the usual diet of macrophages.The lower image on the right shows an even more impressive macrophage with at least a dozen or more ingested RBCs as well as three segmented neutrophils and a lymphocyte.

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Lymphocyte

Lymphocytes mature in the lymph nodes rather than in the bone marrow and therefore are not routinely assessed when deciding if a marrow has "trilinear" (myeloid, erythroid, megkaryocytic) maturation. However, they are normally present in the bone marrow and, when clustered in a lymphoid follicle, can be very prominent. Since lymphocytes mature in the lymph nodes, they will appear identical to peripheral blood lymphocytes when viewed in the bone marrow. They will have the same range of variation in size and cytoplasm and will demonstrate the same types of viral transformations noted in the peripheral blood. Viral/atypical lymphocytes are combined together with normal lymphocytes in a bone marrow differential count and not placed into their own category, as they are in a peripheral blood differential. However, the hematopathologist may include this information in the interpretation, if these changes are noted.Lymphocytes can be found scattered throughout the bone marrow and must be distinguished from early erythroid precursors, which they can closely resemble. Lymphocytes are frequently found in and around early NRBC clusters. In the top image on the right, notice the medium-sized lymphocyte (red arrow) next to the two basophilic normoblasts (blue arrow). The color and texture of the scant lymphoid cytoplasm is almost identical to the NRBC, which can be a bit confusing. However, observe the differences in the nuclei between the two cell types. The lymphocyte has a less distinct chromatin clumping pattern than the basophilic normoblasts and the lymphocyte does not have any "nuclear pores." Also, the lymphocyte has an irregularly-shaped nucleus that is hugging the cytoplasmic border, while the NRBC has a round and regular, centrally-placed nucleus. Identify the three lymphocytes circling the NRBCs in the second image (see red arrows). Notice the chromatin of the lymphocytes; the lymphoid smudgy/clumpy pattern is certainly not as dense and clumped as what is noted in the NRBCs. This nuclear difference becomes more pronounced as the erythroids mature. The cytoplasmic differences should be more apparent as well, since lymphocytes will never produce hemoglobin.

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Hematogone

Hematogone is a term applied to a subset of early B-lymphocytes, found in normal bone marrow, whose morphology greatly resembles that of leukemic lymphoblasts. These cells are larger than the average mature lymphocyte, have scant cytoplasm, and a fine, soft chromatin texture; however, they are not quite as immature in appearance as a true leukemic lymphoblast. Hematogones are more common in younger children but can be found in bone marrow samples of patients at any age. They tend to be found in increased numbers within the bone marrows of patients recovering from bone marrow suppression. Common causes of increased concentrations of hematogones include: viral illness, chemotherapy recovery, and immune mediated cytopenias, such as idiopathic thrombocytpenic purpura (ITP). Hematogones are also common in patients with neuroblastoma.With experience, knowledge of the patients underlying clinical condition, and the ability to review a patient's bone marrow, it is possible to distinguish hematogones from blasts. When necessary, a hematogone flow cytometry panel can be obtained to distinguish these benign cells from lymphoblasts.Notice the size of these blast-like hematogones (see red arrows). They are larger than the few background lymphocytes present in these images. Notice the fine chromatin and scant cytoplasm. They are usually found mixed in with the full range of bone marrow cellular lineages, but can cluster with other lymphocytes within the spicules.

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Plasma Cell

Plasma cells are terminally differentiated B-lymphocytes that have developed a characteristic morphology while actively producing and releasing immunoglobulins. While plasma cells have their origins in the bone marrow as B-cells, they usually leave the bone marrow to develop and mature in the lymph nodes or spleen. Plasma cells begin to produce immunoglobulins after being stimulated by T-cells and exposed to processed antigens.Under normal circumstances, plasma cells are not a large percentage of the lymphoid cells found in a marrow. They are usually placed in a separate category in the differential, unlike viral/atypical lymphs. There can be a relative increase in plasma cells in reactive marrows, and both plasma cells and their early precursors will be markedly increased in plasma cell disorders.While mature plasma cells somewhat resemble lymphocytes, there are a few important differences. The size of the cell is usually larger with more abundant cytoplasm. The nucleus is eccentrically placed and the overall shape of the cell generally resembles a wedge or comet with the nucleus leading the cytoplasm. The chromatin is just as thick and clumpy as a lymphocyte's but is aligned in a more "spokey" or "clockwork" pattern. The cytoplasm is usually more basophilic than the cytoplasm of a normal lymphocyte and will have a well-defined perinuclear halo or noticeable clearing in the golgi area. Vacuoles may or may not be present.Notice the size of the single plasma cell in the top image (see red arrow). It is larger than the neutrophil precursors surrounding it and is almost rectangular in shape. Observe that the nucleus leads the cytoplasm, causing the wedge or comet shape. Notice the prominent perinuclear halo. Find the two plasma cells in the upper left corner of the second image. There is much more cytoplasm in these plasma cells compared to the occasional lymphocyte present in the field. Notice the eccentric nuclear placement as well as the characteristic clearing in the golgi area.

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What are the cells that are indicated by the red arrows in the image on the right?View Page

Cardiac Biomarkers (retired 12/6/2013)
Atherosclerosis

Atherosclerosis is one of the leading causes of heart disease and its presence is an important risk factor for events leading to acute myocardial infarction (AMI). In the past, atherosclerosis was described as a cholesterol and lipid storage event. Now we know it is a chronic inflammatory disorder of the arterial vessels with lipid components. Atherosclerosis begins with damage to the cells that line the blood vessels. Some possible causes of this cell injury are bacterial infection, hyperlipidemia, hypertension, glycosylated products of diabetes, cytokines from adipose tissue, or exposure to toxins such as pollution and second-hand smoke. Monocytes and lymphocytes adhere to the injured site; macrophages enter and ingest proteins and, along with modified lipoproteins, create foam cells. An inflammatory milieu results as cytokines and other inflammatory molecules become involved; foam cells and white blood cells begin secreting cytokines and metalloproteinases. Myeloperoxidase is also released by degranulated white blood cells and macrophages. As inflammation and accumulation of these products continues, fatty dots and streaks are formed on the vessel lining and the formation of plaque begins. As the atherosclerotic process continues, involved cells proliferate forming a complex extracellular matrix and a fibrous cap. If development continues, possibly over decades, the plaque formations are distributed throughout various vessels, become calcified or collagenized and make the vessel walls rigid. The risk to patients with significant atherosclerosis is that eventually a narrowing of the artery (stenosis) can cause a reduction in oxygen delivery to tissues and plaque rupture can lead to an acute coronary event.

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Case Studies in Clinical Microbiology
Case History

A 63-year-old man was seen in the emergency room with the complaints of sudden onset of fever, chills, and abdominal pain, accompanied by mild diarrhea. The blood pressure was 140/84, the pulse rate 82/minute, and the body temperature 39.8C. A blood sample was drawn for a complete blood count, and a blood culture. A second blood culture was drawn from the opposite arm, with 10 mL of blood being placed into each an aerobic and an anaerobic bottle, following customary practice. The complete blood count revealed a hemoglobin of 15.8 mg/dL, a hematocrit of 45%, and a white blood count of 4.2/L. The neutrophils were 39%, lymphocytes 45%, monocytes 10%, eosinophils 4% and basophils 2%. The platelet count was 255/L. The patient was admitted to the hospital for further work-up and empiric antibiotic therapy. Within 24 hours after admission, the body temperature had decreased to 38.2C, although the mild diarrhea persisted. A stool toxin test for Clostridium difficile was negative and neither enteric pathogens nor Campylobacter species were recovered in stool culture after 24 hours incubation. Fecal neutrophils were not seen on direct examination. The anaerobic blood culture became positive 36 hours after inoculation.

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Cerebrospinal Fluid (retired 7/17/2012)
Lymphocyte pleocytosis refers to a decreased number of lymphocytes in a CSF when compared to a normal sample.View Page
What types of cells are present in this field of a cytocentrifuged CSF sample?View Page
Cells Present in Normal CSF

In addition to chemical components, a few cells are also found in normal CSF. In an adult, 0 - 5 WBC/µl is considered normal. Children will have slightly higher cell counts. Up to 30 WBC/µl is within normal limits for newborns. Lymphocytes account for 60 - 100% of these cells.

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How many cells may be seen in a 1 µl spinal fluid sample in a normal adult?View Page
Examining CSF with the Hemacytometer (continued)

White cells are less refractile and appear somewhat granular in appearance. In general, white cells will be larger than red cells. The segmented nucleus in neutrophils can be seen on high power. Lymphocytes and monocytes may be more difficult to differentiate in an undiluted, unstained specimen. If cells are counted in the four corner squares and the center square on both sides of the hemacytometer, the number of cells counted equals the number of cells/mm3 which is the equivalent of cells/µL. The ruled area of one side of a hemacytometer is shown on the right, marked with routine counting squares for red and white cell counts. Each large square is 1 mm2 by 0.1 mm in depth. If the four corner squares that are marked "W" in the image and the entire center square are counted on both sides of the hemacytometer, the area is equal to 10 square millimeters (10 mm2) and the total volume is 1 mm3 (10 mm2 x .1 mm = 1 mm3).

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Cytospin Technique

The cytospin technique uses a high speed centrifuge to concentrate the cells on a slide in a uniform monolayer 6 mm in diameter. The monolayer distribution enhances the morphological appearance of the cells present.Allow the slides to dry in air for several minutes and then stain them with Wright-Giemsa stain. Cytospin slides may be placed in an automatic stainer or stained manually.Perform a 100 or 200 cell differential and record the number of neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages, and blasts cells.Pathologists must review any slide which has tumor cells, unidentified cells, or immature stages of cells, such as blasts.Since criteria for review may vary from one laboratory to another, be sure to check the requirements in your laboratory before reporting the differential.

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What type is the indicated cell?View Page
Mature Peripheral Blood Cells

In normal spinal fluid from an adult, 60% of cells are lymphocytes and up to 30% are monocytes. Up to 2% neutrophils is also considered within normal limits when a cytospin smear is used for the differential. In children, normal CSF cells are 70% monocytes, up to 20% lymphocytes and up to 4% neutrophils. When any of these normal cell abundances are increased, the term pleocytosis is used. Neutrophil pleocytosis is an increase in neutrophils and usually indicates the presence of a bacterial infection.

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Match the condition on the left with associated CSF cells on the right.View Page
Mature Lymphocytes

Four small mature lymphocytes are seen in this picture. Sixty percent of the cells found in normal adult spinal fluid are lymphs.

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Neutrophil and Lymphocyte

Two segmented neutrophils and a lymphocyte are present in this field. (The arrow indicates one of the neutrophils.) Occasional neutrophils are considered a normal finding in cytospin smears.

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More Neutrophils and Lymphocytes

Two segmented neutrophils and a lymphocyte (indicated by an arrow) are in the center of this picture. Notice the mature chromatin structure in the nucleus of the lymphocyte. Three mature red cells are present around the lymphocyte. Two macrophages are also present in this picture.

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Red Cells and Lymphocytes

Many red blood cells and a small, mature lymphocyte are present in this picture. This is typical of fields seen in samples resulting from a bloody tap.

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Lymphocytes

Many lymphocytes are present in this field. Two larger, reactive lymphocytes with intact cytoplasm and slightly indented nuclei are indicated by the blue arrows. Two other large cells with irregular, trailing cytoplasm are macrophages (histiocytes). These are indicated by the red arrows. Increased lymphocytes may be seen in viral meningoencephalitis, partially treated bacterial meningitis, multiple sclerosis, Guillian-Barre's syndrome, or polyneuritis.

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Chemical Screening of Urine by Reagent Strip (retired March 2012)
Which of the following white blood cells would NOT produce a positive leukocyte esterase test?View Page
Leukocyte Esterase Dipstick Test

If leukocyte esterase is detected, a color change occurs on the reagent pad after the strip is dipped in the urine sample. Be sure to follow the manufacturer's directions for read-time and test interpretation. A positive leukocyte esterase test indicates the presence of granulocytic white blood cells. Lymphocytes do not contain granules, and would not produce a positive leukocyte esterase test. Positive results should be confirmed by performing a microscopic examination on the sediment; being aware that white blood cells may be absent if they are lysed, yet releasing their esterases into the specimen. Positive results may occasionally be found in random specimens from females due to contamination of the specimen by vaginal discharge.

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False Positive Leukocyte Esterase Test

A false positive result may occur in the presence of strong oxidizing agents in the collection container. In random urine specimens from women, a positive result for leukocyte esterase may be due to a source external to the urinary tract. Other urine sediment findings such as bacteria, squamous or renal epithelial cells, lymphocytes or red blood cells do not contain esterases, and would not produce a positive leukocyte esterase test.

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A positive leukocyte esterase test indicates the presence in a urine specimen of which of the following?View Page

Chemistry / Urinalysis Question Bank - Review Mode (no CE)
Which of the following statement about synovial fluid is true:View Page
Match the tissues on the left with the corresponding LDH isoenzyme peak on the right.View Page

General Laboratory Question Bank - Review Mode (no CE)
A hapten is only antigenic when it is coupled with which of the following:View Page
Which of the following is responsible for humoral response:View Page
A patient with atypical (reactive) lymphocytes in his peripheral blood smear should be tested for:View Page

Hematology / Hemostasis Question Bank - Review Mode (no CE)
Identify the cell in this illustration indicated by the arrow:View Page
Identify the cell in this illustration indicated by the arrow:View Page
Identify the cell in this illustration indicated by the arrow:View Page
What is the cell indicated by the arrow in this illustration:View Page
Found frequently in a newborn's blood the cells indicated by arrow in this illustration are:View Page
Identify the cell in this illustration indicated by the arrow:View Page
Identify the cell in this illustration indicated by the arrow:View Page
Which of the two WBCs indicated by the arrows on this illustration is normally the most numerous in peripheral blood and what is its name:View Page
Identify the leukocyte seen in this illustration:View Page
Identify the cell in this illustration indicated by the arrow:View Page
Erythropoietin is produced in:View Page
Which of the following major cellular elements does not develop solely in the bone marrow:View Page
What is another name used to designate a fully committed B-lymphocyte:View Page
Which of the following is not primarily a hemolytic process?View Page
Which blood cell is found in the largest numbers in the peripheral blood of a normal adult:View Page
Which of the following cells are capable of producing antibodies and lymphokines:View Page
Phagocytosis is a function of which of the following types of cells:View Page
If greater than 50% lymphocytes were found on the peripheral blood smear of a 5 month old child you would suspect which of the following conditions:View Page
Which of the following is least likely to stimulate the production of reactive lymphocytes:View Page
Which of these is arranged from least mature to most mature:View Page

HIV Safety for Florida
Introduction

Acquired Immunodeficiency syndrome (AIDS) is caused by the Human Immunodeficiency virus (HIV). When HIV enters a person's bloodstream, it attacks and kills the T-helper lymphocytes, which are essential to the body in fighting off infections. As these cells are lost, so is the body's ability to fight infection. Possibly months after the initial infecting episode, an infected person develops a mononucleosis-like illness lasting a week or two. A person may then be free of symptoms for years. But as the T-helper cells die, the person becomes vulnerable to many serious infections. The expected mortality is 100%, and there is no vaccine available to develop specific immunity.

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Introduction to Bone Marrow
Which of the following statements are true for the hematopoietic cords?View Page
Lymphocytes

Lymphocytes are often located in nodules and these nodules are unevenly distributed throughout the marrow so the lymphocyte count may vary in bone marrow samples from different sites. Plasma cells are often found clustered around blood vessels. Monocytes seem to congregate about arterioles in the center of the cord.

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Normal M:E Ratio

The normal M:E ratio in adults varies from 1.2:1 to 5:1 myeloid cells to nucleated erythroid cells. An increased M:E ratio (6:1) may be seen in infection, chronic myelogenous leukemia or erythroid hypoplasia. A decreased M:E ratio (<1.2-1) may mean a decrease in granulocytes or an increase in erythroid cells. M:E ratios are somewhat higher in newborns and infancy than in later childhood and in adults. It is important to note that lymphocytes, monocytes and plasma cells are not included in the M:E ratio.

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High Power Examination

High power (40x objective) examination can be used to estimate the myeloid-to-erythroid ratio. The erythrocytes are nucleated, immature erythrocytes. Under high power, nucleated red cells appear to have a dark purple nucleus as opposed to the lighter staining nucleus of the myeloid or granulocyte series. Lymphocytes also have a dark staining nucleus and some may be erroneously included in the erythroid estimate. In the normal marrow these numbers are insignificant.

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Evaluating M:E Ratio in a Patient with Chronic Lymphocytic Leukemia.

A thin area of a slide taken from a patient who has chronic lymphocytic leukemia, which is characterized by an increased number of small lymphocytes in the bone marrow. At this power, numerous small dark cells similar in appearance to immature red cells are seen, but can be quickly confirmed as lymphocytes when viewed under oil. The actual M:E ratio is normal, since lymphocytes are not included in the final ratio. The arrows show several cell most likely representing small lymphocytes. Some small lymphocytes are normal in the bone marrow.

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Match the following.View Page

Introduction to Flow Cytometry: Blood Cell Identification
Gating: Selecting the Population of Interest

Gating is a method that is used in flow cytometry to isolate cells, thereby selecting a cell population of interest. An electronic "gate" is placed around the cells that have the same light scatter or fluorescence properties. The white oval in the cytogram below is drawn around the lymphocyte population in the tested sample. This selection of a specific cell population is very important because the extrinsic/fluorescent CD marker data (shown in the histogram on the right) directly reflects the population "gated" in the cytogram.

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Case One

A 50-year old woman presented to her physician reporting increasing fatigue over the past several weeks. Routine blood tests were ordered. Her white blood cell count was elevated with a slightly abnormal absolute lymphocyte count. To evaluate the patient's lymphocytosis, a peripheral blood sample was submitted for flow cytometry.The following cytograms and histograms represent data from the blood sample that was analyzed using flow cytometry.

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If 100% of the cells in the gated lymphocyte population are positive for CD45 (i.e., they are leukocytes) and 7% are B cells (represented by CD19 and CD20 positivity), approximately what percentage of T cells would be expected?View Page
T Cell Markers

Until now, the interpretation has been single color analysis. Ruling out a monoclonal B cell population does not guarantee that this is a normal peripheral blood. The T-cell lymphocyte population must also be evaluated. In order to do so, it is important to not only determine how many T cells are present, but also what subsets they represent. Dual color analysis can be used to distinguish C4 from C8 subsets in the C3 population.C2= 91%CD5= 90%CD7= 91%Image #1 CD3 is plotted with CD4. CD3/CD4 dual positive (+) cells = 55% Add Q1 and Q2 percentages together to get total CD3 = 88%Image #2 CD8 plotted with CD3. CD8/CD3 dual + cells = 33%Add Q1 and Q2 percentages together to get total CD3 = 88%Since this is the same sample and the same gated population, the total CD3 values should be very close from image to image. In the two images on the right, the total CD3 values are within one-tenth of each other (both round to 88%). C2= 91% CD5= 90% CD7= 91% CD3= 88% CD4= 55% CD8= 33%

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Final Interpretation of Case #1

The cell representations are listed below. B cellsCD19= 7%CD20= 7%CD10= 0%HLA-DR= 10%CD19/CD5 dual + = 0%Kappa=4%Lambda=3%Interpretation of the above data (refer to the decision tree to assist with interpretation of the case): There is a good mix of kappa and lambda; neither one is predominant. The B cells do not express CD5 (a B cell that expresses CD5 is abnormal because CD5 is normally a T cell marker) CD10 is not present on the B cells. Conclusion: B cells are NORMALT cellsCD2= 91%CD5= 90%CD7= 91%CD3= 88% The pan T cell markers all are within a few percentage points of one another, which translates to the sample having all markers that should be there and no values are skewed toward one marker. CD4 plus CD8 equals the total CD3 (55 + 33 = 88), therefore, there is good representation of both T helper and cytotoxic T cells (a neoplastic process could have predominantly one or the other or dual positive CD4 and CD8). Conclude: T cells are NORMALFinal conclusion: this is a normal peripheral blood lymphocyte population and there is no indication of a neoplastic process. The B cells that are present, though a small percentage of the total lymphocytes, have no abnormal markers. Most of the lymphocytes in this sample are T cells, yet they are normal as well. The CD markers that should be there are present and there is a good representation of both helper and cytotoxic cells.

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All helper cells are T cells. Consequently, in a normal lymphocyte population, it is reasonable to expect all CD4-positive cells to be CD3-positive as well. True or False?View Page
Calculating Absolute Cell Counts

Lymphocytes are a specific type of white blood cell. Lymphocytes can either be T cells or B cells. In the mature T cell population, the T cells can either be helper T cells or suppressor/cytotoxic T cells.Understanding the principles behind the identification of these T cell populations is important. To begin, CD3 marks all mature T cells. Then: CD4 marks T-helper cells CD8 marks cytotoxic T cells Therefore, in any given lymphocyte population, the CD4+ cells plus the CD8+ cells should equal the CD3+ cells. This is because the CD4 cells and the CD8 cells will also mark with CD3 since they are both mature T cells.Provided the total white blood cell (WBC) count and the percentage of lymphocytes from a complete cell count/differential, one can calculate various values. These values include: absolute CD3 counts, CD4 counts, CD8 counts, and CD4:CD8 ratios.The following results represent a patient sample which is used to calculate the values above:WBC count= 2.5 cells/uL (2500 cells/L), % Lymphs= 30%Using the following calculation: Absolute (Abs) lymphs= WBC count x 1000 x percent lymphs (expressed as a decimal) we can determine the absolute lymphocyte count per liter.2.5 x1000 x 0.30 = 750 lymphs/L

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Calculating Absolute Cell Counts- continued

Now that the absolute lymphocyte value has been calculated, the results from the flow cytometric analysis can be incorporated as follows:Values from the flow cytometer analysis%CD3= 60% (CD3 positivity reflects total T cell percentage)%CD4= 40% (CD4 positivity reflects T-helper cell percentage)%CD8= 20% (CD8 positivity reflects cytotoxic T cell percentage)*Note that 60% CD3 (total mature T cells) is further broken down into 40% CD4 (T-helper cells) + 20 % CD8 (cytotoxic T cells)By using the following calculation, the absolute (Abs) cell CD marker populations = Abs lymphocytes x % CD (as decimal)For Example:AbsCD3= 750 x 0.60 = 450 cells/LAbsCD4= 750 x 0.40 = 300 cells/LAbsCD8= 750 x 0.20 = 150 cells/LCD4:CD8 ratio= 40/20 = 2.0

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Case Two

A 60-year-old man presented to the emergency department with complaints of fatigue and weakness which began during the previous week. He also stated that he had been experiencing shortness of breath without chest pain, cough, or dizziness. Upon physical examination, he had normal vital signs. However, his spleen was mildly enlarged. Laboratory tests were ordered. The white blood count (WBC) and absolute lymphocyte counts were both elevated:WBC= 28.5 x 109/L (normal 4 - 10) Lymphocyte count= 20.0 x 109/L (normal 1.2 - 4.0)A manual differential confirmed the elevated lymphocyte count. Smudge cells were also noted, as shown in the image.To evaluate the patient's lymphocytosis, a blood sample was submitted for flow cytometry analysis.

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T Cell Analysis

Though the percentages of gated lymphocyte populations (T or B cells) are important, it is still necessary to determine if the cells present have a normal or abnormal CD marker representation. A population will also need to be scrutinized to evaluate whether it is 10% or 99% of the total lymphocytes.Remember that lymphocytes will either be T cells or B cells and mature T cells will either be helper cells or suppressor/cytotoxic cells. With this in mind, normal mature T cells should express CD2, CD3, CD5, CD7, and either CD4 or CD8. CD4 marks T-helper cells CD8 marks cytotoxic T cells The CD markers present in the peripheral blood sample in Case Two are: CD2 = 17% CD5 = 97% CD7 = 17% CD3 = 18% CD4 = 10% CD8 = 8% Note the markedly increased CD5 percentage in relation to the other T cell markers. If this were a normal T cell population, it would have CD5 at roughly 17%. In order to determine if the elevated CD5 percentage is due to abnormal T cells or to abnormal B cells that are marking with CD5, analyze a sample that has been stained with both CD5 and CD19 monoclonal antibodies. Two areas of interest would be CD5+ only and whether or not there is a CD19+/CD5+ dual marking population. Note that on the image shown on the right, CD5+ only is 16.5% (rounds to 17%) and is in alignment with a normal T cell population in this sample.T-cell analysis conclusion: T-cells are normal and the CD5+ cells that are also positive for CD19 represent abnormal B cells that express the T-cell marker, CD5.

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B Cell Analysis and Final Interpretation of Case Two

The B cell analysis produced the following results (abnormal findings are highlighted in yellow):CD19 = 81% CD20 = 82% HLA-DR = 83%Kappa = 80% Lambda = 1%CD19+/CD5+ = 81% CD10 = negative CD45 = 99%Two apparent abnormalities are present; the B cells are clonal because they express primarily Kappa light chains and the B cells are positive for CD5, which is not normally present on B cells.If Kappa or Lambda is predominant and CD5 is co-expressed with CD19 (CD19/CD5 dual positive lymphocyte population), B-cell chronic lymphocytic leukemia (B-CLL) is a possible diagnosis. In order to confirm this finding, it is important to determine whether or not CD23 is present. The flow cytometry pattern can look identical for B-CLL and Mantle Cell Lymphoma (MCL), except CD23 is generally positive in B-CLL and negative in MCL, as shown in the decision tree on the right. This particular case does have CD23 = 82% Therefore, the final interpretation of Case Two is B-CLL.

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Laws and Rules of the Florida Board of Clinical Laboratory Personnel (retired 9/1/2010)
Description of Specialties (4)

Specialists in cytogenetics detect chromosome abnormalities and genetic disorders. Cytogenetics counseling may only be performed by an individual licenses in the cytogenetics specialty at the director level. Specialists in molecular genetics analyze DNA and RNA to find disease-related genotypes, mutations, and phenotypes in order to detect or predict disease and identify carriers. Specialists in histocompatibility test to determine tissue compatibility, prevent infections, and investigate and post-transplant problems. Techniques include blood typing, HLA typing, HLA antibody screening, disease markers, flow cytometry, crossmatching, HLA antibody identification, lymphocyte immunophenotyping, immunosuppressive drug assays, allogenic, isogeneic and autologous bone marrow processing and storage, mixed lymphocyte culture, stem cell culture, cell mediated assays, and assays for the presence of cytokines. Specialists in andrology and embryology examine gametes and embryos, including production, morphology, number, and motility, to address issues of fertility and infertility.

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Microbiology / Serology Question Bank - Review Mode (no CE)
A decrease in which of the following in an AIDS patient are associated with increased susceptibility to infection:View Page

Normal Peripheral Blood Cells
Match the following cells with their corresponding characteristics:View Page
Appearance of the Erythrocyte

Erythrocytes are non-nucleated, round, biconcave, disc-shaped cells They are 6.7 to 7.7μ in diameter, 2μ thick, and have an average volume (Mean Corpuscular Volume, MCV) of 80-100μ3. In stained blood films, only the flattened surfaces of the RBC's are seen. Therefore, they appear circular with an area of central pallor corresponding to the indented area. The central pallor occupies about 1/3 of the diameter of the cell. The overall red blood cell diameter is slightly less than that of the nucleus of the small lymphocyte. The cytoplasm stains pink to brick-red, and no nucleus is present.

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Glossary of Terms N through Z.

N:C Ratio - Nuclear: cytoplasmic Ratio - The ratio of nuclear volume to cytoplasmic volume within any one cell.Neoplasm - Any new and abnormal growth, such as a tumor.Neutrophilic Granules - Specific granules present in the cytoplasm of neutrophils. These granules resemble pencil stippling and stain a lilac color due to their affinity for both basic and acid dyes.Phagocyte - Any cell that ingests microorganisms or other cells and foreign particles.Phagocytosis - The ingestion and destruction of microorganisms or other foreign particles.Plasma - The fluid portion of blood in which the various blood cells are suspended.PF3 (platelet Factor 3) - A lipoprotein component of the platelet membrane; functions as a surface catalyst during blood coagulation.Pseudopod - A temporary protrusion of the cytoplasm of a cell.Refractile - Capable of refracting or changing the direction of light.Senescence - The process or condition of growing old.Serotonin - A constituent of blood platelets and other cells and organs; induces constriction of the blood vessels.Specific Granules - Granules found in cells of the more mature stages of the granulocytic series. They have distinct staining reactions which differ with each type of granulocyte.T-cell - Thymus derived lymphocyte which mediates cellular immunity.Thrombocyte (Platelet) - A circular or oval disk found in the blood; concerned with hemostasis.Thymus - A ductless gland-like body situated in the anterior mediastinal cavity; reaches its maximum development during the early years of childhood.Vacuole - Any small space or cavity formed in the cytotoplasm of a cell.

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Large Lymphocytes and Reactive Lymphocytes

Large lymphocytes have abundant pale blue transparent cytoplasm. If you imagine putting a printed page behind the cell, the cytoplasm looks as though you could see through it to read the words. Although there are usually no cytoplasmic granules present, a few large well-defined azurophilic granules (lysozomes) can occasionally be seen. In this case, the cells would be called large granular lymphocytes. A large lymphocyte can be found in the upper image to the right.Reactive, or atypical, lymphocytes are relatively fragile cells, and as a result can be squeezed out of shape by surrounding cells, giving them a scalloped appearance instead of a smooth cytoplasmic edge. The nucleus of the reactive lymphocyte is larger than that of the small lymphocyte, and is more irregular in shape. Sometimes it is rounded, oval or indented with a typical "stretched" appearance. A reactive lymphocyte can be found in the lower image to the right.

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The nucleus of a small lymphocyte is about the same size as a:View Page
Which of the following cells is characterized by a thin rim of cytoplasm around the nucleus?View Page
Monocyte Appearance

Monocytes are the largest of the normal peripheral blood cells, ranging from 14-20µm in diameter with an N:C ratio of approximately 3:1. Monocytes have abundant blue-gray cytoplasm containing many fine lilac granules. These give the cytoplasm a "ground glass" appearance. However, these granules may be difficult to see if the blood film is poorly stained. Frequently, cytoplasmic vacuoles are present. These vacuoles appear as unstained areas or "holes" in the cytoplasm; an example of which can be found in the lower image to the right.Because monocytes are extremely motile cells, blunt pseudopods may be seen. These should not be confused with the apparent cytoplasmic projections produced when large lymphocytes are indented by surrounding cells. Monocytes have generally lighter staining nuclei than do other leukocytes. The nucleus stains a pale bluish-violet, and the chromatin is fine. Overall, the nucleus has a soft, spongy, three-dimensional appearance, in contrast to the hard, flat nucleus of the large lymphocyte and the densely clumped nucleus of the band. The nucleus may be round, kidney-bean shaped, folded, indented, or horseshoe, and may show "brain-like" convolutions.

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Differentiating Monocytes from Large Lymphocytes

At times it can be very difficult to differentiate monocytes from large lymphocytes. Monocytes may be mistaken for large lymphs when their cytoplasm stains too lightly, when the characteristic granules are indistinct, or when the nucleus is rounded or only slightly indented. Sometimes a cell will have the nucleus of a lymphocyte and the cytoplasm of a monocyte, or some other confusing combination of characteristics.In order to properly identify the cell, it is necessary to weigh all of the characteristics together to determine which cell type it most resembles. Even then it is occasionally necessary to judge the cell on the basis of the company it keeps. For instance, if there are many monocytes, but few large lymphocytes around, the confusing cell is probably a monocyte.

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Identify the nucleated blood cell that is indicated by the arrow:View Page
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Normal Peripheral Blood Cells (retired 6/20/2012)
Match the descriptions with the cells.View Page
Match the functions below with the corresponding cell type.View Page
Identify each of the cells shown in the image on the rightView Page
The white blood cell that can vary most in appearance on a Wright-stained smear is:View Page
Please identify the illustrated leukocyte.View Page
Please identify the illustrated leukocyte.View Page
Please identify the illustrated leukocyte.View Page
Please identify the illustrated leukocyte.View Page
Please identify the illustrated leukocyte.View Page
Cellular Immunity

Cellular immunity includes delayed hypersentivity reactions, graft rejection, graft-versus-host reactions, defense against intracellular organisms, and probably defense against neoplasms.Cellular immunity is mediated by lymphocytes which we call T-cells.T-cells are so named because they are dependent on the thymus for their production and development.The majority of T-cells are long-lived with an average lifespan of 4.4 years, but it is known that some survive for as long as 20 years or more.T-cells are capable of leaving and re-entering the circulation many times during their long life.T and B cells cannot be differentiated when viewing blood films.They are identified through the use of immunologic cell markers.

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Null Cells

A third class of lymphocytes are called null cells. Null cells are not dependent on the thymus and they can attack certain types of specific cells without prior sensitization. This category includes killer (K) cells, which aid in the destruction of antibody-coated targets, and natural killer (NK) cells, which can lyse targeted cells.

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When Lymphocytes Transform

Lymphocytes "transform" in response to antigenic stimuli.Their nuclei becomes larger with more open chromatin and a greater degree of nuclear folding.The cytoplasm becomes abundant, the number of azurophilic granules may be increased and vacuoles may be present.The cytoplasmic membrane may be easily indented by surrounding red blood cells, resulting in a scalloped appearance of the cell's outer edge.These lymphocytes may also be referred to as reactive, activated or stimulated.

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What is the Function of Lymphocytes?

Lymphocytes are primarily involved in the body's immune response mechanism. This involves complex phenomena which end in the development of humoral and cellular immunity.

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Humoral Immunity

Humoral immunity involves the production of antibodies (immunoglobulins), and is brought about by lymphocytes which we call B-cells. B-cells are bone-marrow derived lymphocytes. After B-cells are stimulated by an antigen, they proliferate and transform into plasma cells which produce specific antibodies.

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T lymphocytes are larger and have more vacuoles than B lymphocytes.View Page
Monocytes

Monocytes are phagocytes which remove injured and dead cells, cell fragments, microorganisms and insoluble particles from the blood and body tissues.Monocytes also secrete substances that affect the function of other cells, especially lymphocytes.They are produced in the bone marrow, and when mature are released into the peripheral blood. Although they do serve a phagocytic role in the blood, their main site of action is the body tissues.The half-life for monocytes in the peripheral blood is approximately 8 hours. Monocytes migrate into the tissues, often to sites of inflammation, where they serve their primary purpose.Here they transform into fixed or free macrophages, and continue their function as avid phagocytes.When activated, macrophages may enlarge and have enhanced metabolism.

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Cell Diameter

The cell diameter is slightly less than that of the nucleus of the small lymphocyte. The cytoplasm stains pink to brick-red, and no nucleus is present.

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Glossary of Terms A through M.

Antibody - A modified type of serum globulin synthesized by lymphoid tissue in response to antigenic stimulus. By virtue of specific combining sites each antibody reacts with only one antigen. Anucleate - Having no nucleus. Azurophilic granules - The well-defined large reddish granules (lysosomes) which may be present in large lymphocytes. They are called "azurophilic granules" because they stain blue with the azure stains which were originally used. Basophilic granules - Specific granules present in the cytoplasm of basophils. These granules are large and stain purple-black due to their strong affinity for basic stain. B-cell - Bone marrow derived lymphocytes which produce humoral antibodies. Biconcave - Having two concave surfaces. Cellular Immunity - The capacity of a small proportion of lymphoid population to exhibit response to a specific antigen. Chromomere - The centrally located granular portion of the platelet. Clone - A population of cells descended from a single cell. Delayed Hypersensitivity - (part of cellular immunity) that develops slowly over a period of 24-72 hours after an antigenic stimulus. It consists of an accumulation of cells around small vessels and/or nerves. Example: Tuberculin skin test reaction. Digestive Enzyme - A substance that catalyzes or accelerates the process of digestion. Eosinophilic Granules - Specific granules present in the cytoplasm of eosinophils. These granules are large, refractile spheres which stain reddish-orange due to their strong affinity for acid stain. Erythrocyte (red blood cell, RBC) - One of the elements found in peripheral blood. Normally the mature form is a non-nucleated, circular, biconcave disk adapted to transport respiratory gases. Fixed Macrophage - A phagocyte that is non-motile. Free Macrophage - An ameboid phagocyte present at the site of inflammation. Graft Rejection - A transplanted tissue that is rejected by the body's antibodies. Graft vs. Host Reaction - A complication that occurs when an implanted piece of tissue, which contains antibodies, rejects the host's tissue. Granulocyte - A leukocyte which contains granules in its cytoplasm, i.e., neutrophilic, eosinophilic, or basophilic granules. Half-life - is the length of time it takes for half of the cells circulating at a given time to leave the blood for the tissues. Hemocyte - Any blood cell or formed element of the blood. Hemostasis - A mechanism of the vascular system to arrest an escape of blood. It involves an interaction between blood vessels, platelets, and coagulation. Heparin - A mucopolysaccharide acid which, when present in sufficient amounts, functions as an anticoagulant by inhibiting thrombin. Histamine - A powerful dilator of capillaries and a stimulator of gastric secretions. Humoral Immunity - Acquired immunity produced after response to an antigenic stimulus in which B cells produce circulating antibodies. Hyalomere - the clear, blue non-granular zone surrounding the chromomere of a platelet. Immune Response - The interaction of a cell and an antigen that results in a proliferation of the cell and a capacity to produce antibodies. Isotonic Fluid - A fluid whose elements have an equal osmotic pressure. Leukocyte (white blood cell, WBC) - One of the formed elements of the blood; involved primarily with the body's defense. Lysosome - A microscopic body within cell cytoplasm; contains various enzymes, mainly hydrolytic, which are released upon injury to the cell. Megakaryocyte - A giant cell of the bone marrow from which platelets are derived. Mononuclear - A cell having a single nucleus.

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Glossary of Terms N through Z.

N:C Ratio - Nuclear: cytoplasmic Ratio - The ratio of nuclear volume to cytoplasmic volume within any one cell.Neoplasm - Any new and abnormal growth, such as a tumor.Neutrophilic Granules - Specific granules present in the cytoplasm of neutrophils. These granules resemble pencil stippling and stain a lilac color due to their affinity for both basic and acid dyes.Phagocyte - Any cell that ingests microorganisms or other cells and foreign particles.Phagocytosis - The ingestion and destruction of microorganisms or other foreign particles.Plasma - The fluid portion of blood in which the various blood cells are suspended.PF3 (platelet Factor 3) - A lipoprotein component of the platelet membrane; functions as a surface catalyst during blood coagulation.Pseudopod - A temporary protrusion of the cytoplasm of a cell.Refractile - Capable of refracting or changing the direction of light.Senescence - The process or condition of growing old.Serotonin - A constituent of blood platelets and other cells and organs; induces constriction of the blood vessels.Specific Granules - Granules found in cells of the more mature stages of the granulocytic series. They have distinct staining reactions which differ with each type of granulocyte.T-cell - Thymus derived lymphocyte which mediates cellular immunity.Thrombocyte (Platelet) - A circular or oval disk found in the blood; concerned with hemostasis.Thymus - A ductless gland-like body situated in the anterior mediastinal cavity; reaches its maximum development during the early years of childhood.Vacuole - Any small space or cavity formed in the cytotoplasm of a cell.

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Mononuclear Cells: Lymphocytes and Monocytes.

The mononuclear leukocytes consist of two cell types: lymphocytes and monocytes. In contrast to the granulocytes, these cells have rounded nuclei, some with indentations or folds. Granules are not prominent.

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Identify the nucleated blood cell:View Page
Large Lymphocytes

Large lymphocytes have abundant pale blue transparent cytoplasm.If you imagine putting a printed page behind the cell, the cytoplasm looks as though you could see through it to read the words.Although there are usually no cytoplasmic granules present, a few large well-defined azurophilic granules (lysozomes) can occasionally be seen.

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Large Lymphocytes are Fragile Cells

Large lymphocytes are relatively fragile cells, and as a result are frequently squeezed out of shape by surrounding cells, giving them a scalloped appearance.

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Large Lymphocyte Nuclei

The nucleus of the large lymphocyte is larger than that of the small lymphocyte, and is more irregular in shape. Sometimes it is rounded, oval or indented.

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Chromatin Pattern of Large Lymphocytes

The chromatin pattern of the large lymph, seen in the upper image, is not as dense as that of the small lymphocyte, seen in the lower image, but even so the nucleus appears hard and flat.

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"Stretching" of Large Lymphocyte Nuclei

At other times the nucleus appears to be stretched across the cell, attached to the cell membrane at each end. A nucleus like this seems to have visible "stretch lines" through it.

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Lymphocyte or Lymph

Lymphocytes are a heterogeneous group of cells that have different origins, lifespans and functions, and vary markedly in size. Some have a diameter of approximately 7µ, while others are as large as 18µ. The variations in size are mainly due to different amounts of cytoplasm. Therefore, the N:C ratio may range from 5:1 in some lymphocytes to 1:2 in others.

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Small Lymphocytes

Small lymphocytes have only a thin rim of clear, homogenous, moderate blue cytoplasm around the nucleus.

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Nucleus of Lymphocyte

The nucleus is slightly larger than a normal RBC. It is usually round or oval in shape, but may be slightly indented. The chromatin is very dense and clumped.

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Overall Appearance of the Nucleus

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Differentiating Monocytes from Large Lymphocytes: Table.

The table below lists some characteristics that help to distinguish large lymphocytes from monocytes. CellImageNucleusCytoplasmLarge lymphocyteOval, round, indented, "stretched Deep purplish-blue Dense Sky-blue Clear, transparent No granules or azurophilic (reddish) granules Infrequent vacuoles Cytoplasm may be indented by surrounding cells MonocyteRound, oval, indented, convoluted Pale purplish- blue Fine, lacy, spongy Blue-gray Cloudy, opaque, "ground-glass" appearance Fine granules Frequent vacuoles Cytoplasm may have pseudopods

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The nucleus of a small lymphocyte is about the same size as a:View Page
Which of the following cells is characterized by a thin rim of cytoplasm around the nucleus?View Page
Identify the nucleated blood cell:View Page
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Monocytes are Extremely Motile Cells

Because monocytes are extremely motile cells, blunt pseudopods may be seen. These should not be confused with the apparent cytoplasmic projections produced when large lymphocytes are indented by surrounding cells.

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Differentiating Monocytes from Large Lymphocytes

At times it can be very difficult to differentiate monocytes from large lymphocytes.Monocytes may be mistaken for large lymphs when their cytoplasm stains too lightly, when the characteristic granules are indistinct, or when the nucleus is rounded or only slightly indented.Sometimes a cell will have the nucleus of a lymphocyte and the cytoplasm of a monocyte, or some other confusing combination of characteristics.In order to properly identify the cell, it is necessary to weigh all of the characteristics together to determine which cell type it most resembles.Even then it is occasionally necessary to judge the cell on the basis of the company it keeps.For instance, if there are many monocytes, but few large lymphocytes around, the confusing cell is probably a mono.

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Match the characteristics with the cell type.View Page
Identify the nucleated blood cell:View Page
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Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
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Identify the nucleated blood cell:View Page

OSHA Bloodborne Pathogens
What Causes HIV Infection?

HIV infection is caused by the human immunodeficiency virus. The infection occurs when HIV enters a person's bloodstream, where it attacks and kills the helper T-cells. Helper T-cells are part of a group of white blood cells known as lymphocytes that are essential for fighting off infections.As the numbers of these cells decreases, so does the body's ability to fight infection.

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OSHA Bloodborne Pathogens (retired)
What causes HIV?

HIV is caused by the Human Immunodeficiency virus.When HIV enters a person's bloodstream, it attacks and kills the T-helper cells. These cells are part of a group of white blood cells known as lymphocytes, which are essential to the body in fighting off infections.As these cells are lost, so is the body's ability to fight infection.

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Phlebotomy
Yellow top tubes

Contain either acid citrate dextrose (ACD), which maintains RBC viability and may be used for HLA phenotyping, DNA, paternity testing, or lymphocyte surface markers, or: Sodium polyanetholesulfonate (SPS) which is sometimes used to collect blood culture specimens.

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Red Cell Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
A blood smear represented by the photograph was submitted for hematologic review. Based on the erythrocyte morphology and the accompanying histogram, which of the following choices is the most likely situation or condition?View Page
The complete blood count was obtained from a patient recently admitted to the emergency room. The red blood cell indices obtained revealed an MCV of 115 femtoliters (fL) (normal range 80 - 90 fL). The patient met the criteria for a peripheral blood smear examination. A representative field is shown on the right.Which of the following conditions may be indicated by the results seen on this peripheral blood smear?View Page
Anisocytosis Reference Table

Cell TypeImageCellular DescriptionAssociated Diseases and ConditionsNormocyteMCV 80-100 fL (approximately the same size as the nucleus of a small lymphocyte) MCHC 32-36 g/dL or 32%-36%N/A; cell is normalMicrocyteRed blood cells are smaller than normalMCV < 80 fLIron deficiency anemiaSideroblastic anemia Certain thalassemias Lead poisoning Certain hemoglobinopathiesMacrocyteRed blood cells are larger than normalMCV > 100 fLMegaloblastic Anemia (Vitamin B12/Folate deficiency)MyelodysplasiaLiver disease

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Red Cell Morphology
Using the lymphocyte as a guideline, the cells that are indicated by the arrows would be described as what type of cells?View Page
Normal Red Cells

The normal red blood cell (RBC) is often described as a biconcave disc. The center of the red cell is thinner than the edge. When a normal RBC is flattened on a microscope slide and stained, it has a diameter of 6-9 microns and a pale center area surrounded by a buff- or pink-stained rim. The pale center area, called the central pallor, normally occupies about one-third of the cell diameter.

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Another View

Another view taken from the same patient's slide. Although no lymphocyte is seen in this field, many of the cells appear quite small with increased areas of central pallor. This patient had iron deficiency anemia.

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Microcyte with Normal Hemoglobin Content

The arrow points to a microcyte with normal hemoglobin content (one-third of central pallor). Since many of the other cells in this field are normal or larger than normal, the mean corpuscular volume (MCV) would be within the normal range although the diameter and volume of this individual cell would be lower than normal. This type of microcyte can be seen in some hemolytic anemias and the rare enzyme deficiency, pyruvate kinase deficiency anemia.

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Pseudomacrocytes

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Microcyte

Microcytes are less than 6.7 µm in diameter and have an MCV of <80 fL. Notice that many of the red cells shown in this field are smaller than the nucleus of the lymphocyte and, in addition, have a greater area of central pallor. Hypochromic microcytic RBCs can be seen in iron deficiency anemia and thalassemia.

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Another Example of Microcytes

Another example of microcytes seen in a slide from a patient with hemolytic anemia. Compare the two microcytes in the center of the field with the lymphocyte to the right. Notice the larger red cell just below the microcytes is about the same size as the lymphocyte. Several other microcytes can also be seen in this field.

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Normal RBC Size

A normal RBC is 6.7-7.8 µm in diameter, approximately the same size as the nucleus of a small lymphocyte, which is often used as a guideline when determining the size of the red cells on a slide. Notice that most of the red cells in this field are about the same size as the nucleus of the lymphocyte shown in the image.The MCV of a normal-sized RBC is 80-100 fL.

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Macrocyte

Macrocytes have a diameter of 9-14 µm (1.5 to 2 times larger than normal red cells) and the MCV is >100 fL. Several macrocytes are indicated by the blue arrows in the top image on the right. By comparison, macrocytes are larger than the nucleus of a normal lymphocyte. Macrocytes are frequently oval; two examples are indicated by the red arrows in the top image on the right.Oval macrocytes are most commonly associated with vitamin B-12 and/or folic acid deficiency. Decreased DNA synthesis causes the nucleus in the developing red cells to mature at a slower than normal rate. Since hemoglobin production is not affected, the mature red cell that is larger than normal is filled with hemoglobin, resulting in cells with little central pallor. Note that polychromatophilic red cells may also be larger than normal RBCs. However, if stained with a supravital stain, it would be obvious that these cells are reticulocytes. From a Wright-stained smear, they would be reported as polychromatophilic RBCs and not macrocytes. Examples of polychromatophilic RBCs are indicated by the arrows in the bottom image on the right. The cells stain gray-blue with Wright stain.

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The Urine Microscopic: Microscopic Analysis of Urine Sediment
Urine Sediment

Urine sediment may also contain white blood cells (WBCs). Most of the WBCs in urine are segmented neutrophils. Since it is possible that lymphocytes, monocytes, and/or eosinophils may be present, the cells in urine can be stained if it is necessary to differentiate them. The segmented neutrophil indicated by the blue arrow shows a distinct nucleus. When viewing urinary sediment under the microscope, the fine focus adjustment must be used to identify white blood cells. White blood cells swell in dilute alkaline urine and the cytoplasmic granules exhibit brownian movement resulting in "glitter cells." These cells lyse rapidly. "Glitter cells" are most easily seen when viewed under phase-contrast microscopy.

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Variations in White Cell Morphology -- Granulocytes
What morphological change is present in this image?View Page
The structure that is indicated by the arrow in this image, along with giant platelets (also present in the image), is characteristic of which of the following conditions?View Page
What morphological change is present in this image?View Page

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Alder Anomaly, continued

This image shows a peripheral smear from another patient with Alder anomaly. Notice that neutrophil seen in the image has granulation which is much heavier than what would be classified as normal. The amount of granulation may vary from cell to cell, with some cells being unaffected. A lymphocyte showing abnormal granules is also present on this slide.

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Chediak-Higashi Anomaly vs. Toxic Granulation

The neutrophils found in Chediak-Higashi must be differentiated from toxic granulation. In conditions causing toxic granulation, the granules are smaller and more numerous. In toxic granulation, neutrophils are usually the only cells affected. In Chediak-Higashi anomaly, eosinophils, basophils, lymphocytes and monocytes are affected. In eosinophils larger than normal eosinophilic granules may be seen, basophils may exhibit larger than normal basophilic granules, lymphocytes, large azurophilic granules. Larger pale granules/bodies may appear in monocytes.

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Chediak-Higashi Syndrome Lymphocyte

A lymphocyte from a patient with Chediak-Higashi syndrome. The azurophilic granules appear much larger than those seen in normal lymphocytes.

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Chediak-Higashi Anomaly

Chediak-Higashi anomaly is a rare autosomal recessive disorder. It results from a mutation of the gene LYST which encodes a protein with multiple phosphorylation sites. This defect causes a cellular abnormality involving the fusion of cytoplasmic granules. Early in neutrophil maturation normal azurophilic granules form, but they fuse together to form megagranules. Later during the myelocyte stage, normal specific granules form. The mature neutrophils contain both normal specific granules and abnormal azurophilic granules. These large abnormal granules can be seen in the cytoplasm of neutrophils, eosinophils, basophils, monocytes and lymphocytes. These abnormal granules are able to kill bacteria in neutrophils and monocytes; however, the process is much less effective than in normal cells in part, because these neutrophils have impaired locomotion. For these reasons, individuals with Chediak-Higashi have recurrent infections. An accelerated lymphoma-like phase occurs, with lymphadenopathy, hepatosplenomegaly, and pancytopenia. Death often occurs at an early age.

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Alder Anomaly (Alder-Reilly Anomaly )

Alder anomaly is a rare autosomal recessive disorder in which the basic defect involves protein-carbohydrate complexes called mucopolysaccharides. The accumulation of partially degraded (broken down) protein-carbohydrate complexes within the lysosomes account for the larger than normal purple-staining inclusions seen in all types of mature white blood cells, and sometimes in earlier cells. The granules may occur in clusters, rather than diffusely, throughout the cytoplasm as in toxic granulation. These inclusions may be seen in the bone marrow more frequently than in peripheral blood. The physical characteristics associated with this disorder include gargoylism and dwarfism. The function of the cells involved is not affected. This morpholical change would be classified as pathological since the body is responding abnormally even though the function is not affected.

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White Cell and Platelet Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
The cells included in the composite image were found in the peripheral blood smear of a patient with the following results:total WBC of 24.5 x 109/L. Differential count:myelocytes 1 metamyelocytes 4 band neutrophils 15 segmented neutrophils 40 monocytes 8 eosinophils 2 basophils 1 lymphocytes 29This hematologic picture is most consistent with:View Page
Match the letter representing the cell type with the condition in which increased numbers of the cell may be found in the peripheral smear.View Page
The white blood cell indicated by the arrow is representative of the atypical white blood cell associated with infectious mononucleosis.View Page
Normal Bone Marrow Cells

A normal bone marrow smear stained with Wright/Giemsa stain is captured in this photograph.Note the normal maturation sequence beginning with myelocytes (the two large cells in the left upper corner)through metamyelocytes, band neutrophils,and multi-lobed segmented neutrophils.The small cells with darkly staining, centrally placed nuclei are normoblasts (three are clustered in the left lower field).Absent in this field are eosinophils, basophils and megakaryocytes.A normal M:E ratio of 2.4:1 is calculated from the twelve myeloid cells and five normoblasts. Two lymphocytes are identified, one left center, the other left upper.

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Criteria for Performing a Manual Review of the Smear

Automated cell counters will flag abnormal findings. The following guidelines represent typical review criteria, although specific requirements vary between laboratories. Follow the criteria established by your laboratory's procedure:Total white blood cell count <3.0 X 109/L or >12.0 X 109/LNeutrophils >80% Lymphocytes >45% or <10% Monocytes >15% Eosinophils >10% Basophils >5%A morphology review may also be indicated if the platelet count is <100 X 109/L or >650 X 109/L.

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An automated hematology counter flagged the white blood cell count. Upon review of the peripheral blood smear, the technologist viewed many cells that appeared similar to those in this image. What should the technologist report?View Page
Assume that several other lymphocytes similar to the one in the center of the photograph, are found on review of the peripheral smear. A work up for leukemia should be recommended.View Page
Additional Comments

The following pages in this presentation includes a series of white blood cell and platelet abnormalities (nonneoplastic) that may be identified in a peripheral blood smear. Many cases will simulate the practice of a peripheral smear review by a hematology technologist. He or she must assess what responses in patient care may be triggered by the clinician attempting to interpret the reported findings on a peripheral smear.Observations of white blood cell abnormalities in the peripheral blood smear should be reported in order to direct the physician to an immediate specific diagnosis, such as: Atypical lymphocytes, suggesting infectious mononucleosis rather than leukemia Toxic granules in neutrophils, as found in acute infections, or atypical granules suggesting a genetic disorder An unusual mix of cells, such as too many or too few neutrophils, monocytes, or other myeloid cells The presence of giant platelets, myelocytes, or other cells, suggesting a myelodysplastic syndromeIn summary, laboratory data should be presented to clinicians in a user-friendly fashion to promote effective decision making.

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Evaluation Criteria: White Blood Cells and Platelets

In most clinical hematology laboratories, an initial blood count is performed by an automated cell counting instrument. Additionally, most of these instruments also produce a five-part differential count, indicating the percentage of neutrophils, lymphocytes, monocytes, basophils, and eosinophils. Some instruments can also provide information about cellular immaturity and abnormal cellular morphologies.Occasionally, atypical cells, similar to those shown in the image to the right, would be flagged or counted as mixed cells, at which point a smear review would be required to make an identification. In cases where there are automated instrument differential flags, mixed cell count is high, or there are other indications that atypical cells may be present, a review of the smear is indicated.

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The neutrophils illustrated in this image are representative of those seen in a female patient's peripheral blood smear. The total WBC was 28.5 X 109/L (reference interval = 4.0 - 11.0). Which of the following BEST describes the condition associated with this WBC count and the white blood cells that are present in the image?View Page
Alder Anomaly

Alder anomaly is characterized by large azurophilic granules that stain dark-purple and are seen throughout the leukocyte cytoplasm, even covering the nucleus. The inclusions (granules) are seen in the cytoplasm of almost all mature leukocytes i.e., granulocytes, lymphocytes, and monocytes. This distinguishes Alder anomaly inclusions from toxic granulation, which is only observed in neutrophils. Another feature that distinguishes Alder anomaly from toxic changes is the lack of cytoplasmic vacuoles of toxic origin in the neutrophils of Alder anomaly.The background condition in Alder anomaly is mucopolysaccharidosis, collectively, a group of inherited disorders where a deficiency of lysosomal enzymes are lacking that are needed to degrade mucopolysaccharides. The inclusions observed in the leukocytes represent partially degraded mucopolysaccharides within lysosomes. Accompanying conditions are hepatosplenomegaly, corneal opacities, and mental retardation.

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Basophils

A basophil and a small lymphocyte are compared in the same field in the upper image, while a single basophil is shown in the lower image.The cytoplasmic granules of the basophil are larger than the granules of toxic granulation.They contain chemical mediators of immediate hypersensitivity, and are found in the cytoplasm and overlying the nucleus (better seen in the lower image). Basophilic granules stain metachromatically with toluidine blue indicating the presence of acid mucopolysaccharide or proteoglycans, both thought to be heparin or heparin-like substances.Basophils are related to mast cells (tissue basophils), each involved in hypersensitivity responses and following anaphylactic episodes. Under the stimulation of complement components C3a and C5a, many mediators are released from the basophil granules, including histamine, heparin, and eosinophil chemotactic factors of anaphylaxis, or ECF-A.Basophils are the least common granulocytes in the peripheral blood, comprising 2% or less of the differential count. The presence of large granules of irregular size in basophils and the admixture of eosinophilic granules may indicate dysplastic changes associated with myelodysplastic disorders and leukemia. Basophils may be increased in:Myeloproliferative disordersChronic metabolic conditions Myxedema Diabetes mellitusHypersensitivity responses Tuberculosis

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A peripheral blood smear is observed during a manual differental review. The patient is a 10 year-old boy with symptoms suggesting appendicitis and an appendectomy is being considered. The total WBC is 18.5 X 1000/uL, RBC's = 5.45 X 1M/uL, hemoglobin = 16.0 g/dL, hematocrit 48.2%.WBC differential:Segs = 53%, bands = 42% (two of which are shown in the image) monocytes = 2% lymphocytes= 2% These findings support the diagnosis of appendicitis.View Page
The smudge cells pictured in this image may be found in each of the following situations except:View Page
A peripheral smear was submitted for review due to increased monocytes on the automated differential. The images on the right are representative fields from the Wright-Giemsa stained blood smear (1000X magnification). The increased monocytes and peripheral picture are consistent with each of the following conditions EXCEPT:View Page
Approximately 10% of the circulating white cells were similar to the one seen in this image. The patient was 42 years old and visited his physician because of recent bruising. Note the absence of platelets on the smear. Possible associated conditions include:View Page
The peripheral smear on the right was submitted for morphologic/clinical examination. The predominant cells comprised 70% of the total white blood cells and are consistent with lymphocytes in a 4-month-old infant.View Page
Case History One

A 14-year-old boy came to the physician's office with a sore throat that progressively worsened over a three-day period. His posterior pharynx was swollen, shiny and erythematous. The boy complained of pain on swallowing. His temperature was 98.5°F. A rapid direct streptococcal antigen test was positive. However, his symptoms did not subside over the next two days while on antibiotic therapy. Anorexia and nausea were persistent and compounded by a frontal headache. Cervical lymph nodes became noticeably enlarged. A complete blood count (CBC) was ordered. The results of the CBC were: WBC 11.9 x 109/L ( Reference interval= 3.8 - 9.8 x 109/L) with: 17% segmented neutrophils 5% band neutrophils 72% lymphocytes 6% monocytes All red cell findings were normal. The automated differential flagged for atypical cells, presumptively atypical lymphocytes. A peripheral blood smear was prepared. The image on the right is a representative field from the Wright-Giemsa stained smear (1000X magnification).A rapid qualitative test for infectious mononucleosis was positive. This is a case of group-A streptococcal infection superimposed on infectious mononucleosis. Symptoms subsided in three weeks following completion of the antibiotic therapy.

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The large blue staining cells represented here in the photographs comprise 50% of the total white blood count. This picture is most consistent with which of the following conditions? (choose all that apply)View Page
Hairy cell leukemia

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Case History Two

An 80-year-old man was seen in the emergency room with sudden onset of right-side chest pain accentuated on inspiration. His cough was productive of yellow sputum, and he was short of breath. His temperature was 101.2°F. A chest X-ray revealed right middle lobe pneumonia. A complete blood count (CBC) was ordered. The results were as follows:CBC ParameterPatient ResultReference IntervalWBC33.0 x 109/L4.0 - 11.0 x 109/LRBC4.5 x 1012/L4.5 - 5.9 x 1012/LHemoglobin15.2 g/dL13.5 - 17.5 g/dLHematocrit44%41 - 53%Platelet200 x 109/L150 - 450 x 109/LSegmented neutrophil6540 - 80%Band neutrophil100 - 5%Lymphocyte 525 - 35%Eosinophil 30 - 5%Basophil 20 - 2%Monocyte252 - 10%A peripheral smear was reviewed based on the elevated WBC and increased monocyte count. A representative field from the Wright-Giemsa stained smear (1000X magnification) is shown on the right. The cells indicated by the blue arrows are atypical monocytes. They have abundant cytoplasm that is more blue than the typical gray-blue cytoplasm of normal monoctes. A few scattered vacuoles are also present. The atypical monocytes, in company with toxic neutrophils (indicated by the red arrow), appeared to be a response to infection. The patient had a past history of tuberculosis, which may account for the monocytosis.

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