Lymphocyte Information and Courses from MediaLab, Inc.

In the cytospin procedure, use 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, such as Hema-Tek, 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.

In normal spinal fluid from an adult, 60% of cells are lymphocytes and up to 30% are monocytes. Neutrophils abundance up to 2% 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.

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

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

The chromatin pattern is not as dense as that of the small lymphocyte, but even so the nucleus appears hard and flat.

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.

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.

Please refer to the table on the right to distinguish lymphocytes and monocytes. But no table will ever completely remove the problem of confusing cells. You will soon discover that no matter how experienced you become, there will always be a cell or two that will cause you to scratch your head with frustration. Perhaps that is what makes hematology so challenging.

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.

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.

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.

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.

Another type of macrocyte can be seen in the center of this slide. Notice it appears larger than the lymphocyte but in contrast to megalocytes has an area of central pallor. These macrocytes are sometimes referred to as "pseudo macrocytes," since their size is the result of exaggerated flattening (leptocyte) and thus the presence of the central pallor. The MCV for this type of macrocyte is within normal range. Pseudomacrocytes can be seen in patients with cirrhosis of the liver, obstructive jaundice, post splenectomy and conditions that affect the red cell membrane.

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.

Macrocytes have a diameter of 9-14 microns (1 1/2 to 2 times larger than normal red cells) and the MCV is 100 cubic microns or more. The macrocytes seen in this slide are referred to as true macrocytes, or megalocytes. Compare the red cells in the field to the nucleus of the lymphocyte in the lower left. Many of the red cells in the field are larger than the lymphocyte and have little or no central pallor. As a point of reference, the cells just below and above the lymphocyte are macrocytes. Megalocytes are frequently oval and several examples of oval macrocytes can be seen in this field. Megalocytes are the result of decreased deoxyribonucleic acid (DNA) synthesis, frequently due to vitamin B12 and/or folic acid deficiencies. 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 is larger than normal is filled with hemoglobin.

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 just above center of the image to the right 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.

This slide is also from a patient who has Alder-Reilly anomaly. Notice that neutrophil seen in this slide has granulation which is much heavier than in the previous slide. The amount of granulation may vary from cell to cell with some cells being unaffected. A lymphocyte showing abnormal granules is also present in this slide.

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

Alder Reilly Anomaly is a rare autosomal recessive hereditary 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 granules seen in the granulocytes, monocytes and/or lymphocytes. 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.

The following are suggested guidelines directed toward white blood cell data necessitating a hematologist's review:Total white blood cell count <3000/cumm or >12,000/cummNeutrophils >85%Lymphocytes >43% or <10%Monocytes >8%Eosinophils >6%Basophils >4%,.Mixed cells >8% on a 3-part automated differentialA morphology review may also be indicated if the platelet count is <100,000/cumm or >650,000/cumm.Thus, if the granulated cells illustrated in the photograph exceed 6% of the total WBC on a five-part differential or, in combination with monoctytes and basophils, exceed 8% of the total WBC on a three-part differential, a flag would alert the operator that a morphology review or manual differential is needed.

In most clinical hematology laboratories, an initial blood count is performed by an electronic instrument. Some of these instruments also produce a differential blood count, and a platelet count. Instruments that provide a 3-part differential indicate the percentage of neutrophils, lymphocytes, and a mixed field group that includes monocytes, eosinophils, basophils, immature and atypical cells. Thus, the atypical cells shown in the photograph would be counted as mixed cells and a smear review would be needed to make an identification. Instruments providing a 5-part differential count include monocytes and eosinophils. In cases where the mixed cell count is high, or there are other indications that atypical cells may be present, a hematologist's review of the smear is indicated.

Large inclusions in leukocyte cytoplasm appear with Alder-Reilly syndrome. Inheritance patterns are not completely clear. The condition is characterized by larger than usual azurophilic and deeply violet staining granules clustered throughout the cytoplasm (even covering the nucleus)in all granulocytes. There are variations in which some lymphocytes and monocytes may be affected. These inclusions represent partially degraded mucopolysaccharides within lysosomes.Alder-Reilly bodies may be found independently of genetic mucopolysaccharidoses as an inherited anomaly (Jordan's anomaly). Cytoplasmic vacuoles of toxic origin are not present in Alder-Reilly cells. The background condition in Alder-Reilly syndrome is mucopolysaccharidosis with various types of bone and cartilage disorders, reported first in gargoylism, then in Hunter and Hurler syndromes. Accompanying conditions are hepatosplenomegaly, corneal opacities, and mental retardation. Reference: Brunning, Richard D. Morphologic Alterations in Nucleated Blood and Marrow Cells in Genetic Disorders. Human Pathol: 99-124, March, 1970

An 80 year old man was seen in the emergency room with sudden onset of right sided chest pain accentuated on inspiration. His cough was productive of yellow sputum, and he was short of breath.His temperature was 101.2F. A chest X-ray revealed right middle lobe pneumonia. His hemoglobin was 15.2 gm/dl, HCT 44%, and RBC 4.5 m/ml. The white blood count was 35,000/cuml, with 45% neutrophils, 20% bands, 5% lymphocytes, 3% eosinophils, 2% basophils, and 25% atypical monocytes as noted in the photograph.The atypical monocytes had abundant blue-grey cytoplasm with a few scattered vacuoles, which, in company with toxic neutrophils appeared to be a response to infection.The patient had a past history of tuberculosis which may account for the monocytosis.