Intracellular Information and Courses from MediaLab, Inc.

This is a CSF sample from a patient diagnosed with bacterial meningitis.Note the intracellular and extracellular bacteria present on this smear (see arrows).Notice the white blood cells clumps around the bacteria. This is not simply an artifact of the preparation, but rather a response to the presence of the actual bacteria. WBC clumping can be a great clue to the presence of bacteria, so laboratorians should always scan any clumps present for infectious organisms.

When intracellular bacteria are present in a body fluid cytospin, they classically appear within neutrophils (see arrows). When the bacterial burden is high, they may also be found in monocytes/histiocytes. It is important to note that monocytes are the less effective phagocytes of these two cell types, so they will never contain intracellular organisms if the neutrophils do not do so as well. If you think you are seeing bacteria in a monocyte/macrophage, and the neutrophils do not display intracellular bacteria, you may actually be observing granules of hemosiderin within the macrophages. Hemosiderin usually stains more green-blue or black than bacteria, and the granules are less regular in size than ingested bacteria. Comparing the Wright stain to the Gram stain, and performing an iron stain, will help with this distinction.

Cardiac biomarkers are important in heart disease where there is damage to the myocardium and myocytes. Interrupted or blocked bloodflow and decreased oxygen cause injury to the myocytes and cell death. As the myocytes become necrotic, their membranes are disrupted and intracellular biomolecules diffuse into cardiac microvasculature and then to lymphatics. From there, these compounds, many of which are biomarkers, enter the peripheral circulation.Cardiac biomarkers are relied upon for the detection and monitoring of two categories of cardiovascular disease: Acute Ischemic Disease -- an AMI is the most serious disease in this category Heart Failure

Venous BloodVenous blood is deoxygenated blood that flows from tiny capillary blood vessels within the tissues into progressively larger veins to the right side of the heart. Venous blood is the specimen of choice for most routine laboratory tests. The blood is obtained by direct puncture to a vein, most often located in the antecubital area of the arm or the back (top) of the hand. At times, venous blood may be obtained using a vascular access device (VAD) such as a central venous pressure line or Hickmann Catheter or an IV start. Most laboratory reference ranges for blood analytes are based on venous blood.Arterial BloodDeoxygenated blood is pumped from the right side of the heart to the lungs where it takes up oxygen. The now oxygenated blood is pumped through the left side of the heart via arteries.The most common reason for collection of arterial blood is the evaluation of arterial blood gases. Arterial blood may be obtained directly from the artery (most commonly, the radial artery) by personnel who are trained to perform this procedure and are knowledgeable about the complications that could occur as a result of this procedure. Arterial blood may also be obtained from a vascular access device (VAD) inserted in an artery such as a femoral arterial line or Swan-Gantz catheter. Capillary BloodCapillary blood is obtained from capillary beds that consist of the smallest veins (venules) and arteries (arterioles) of the circulatory system. The venules and arterioles join together in capillary beds forming a mixture of venous and arterial blood. The specimen from a dermal puncture will therefore be a mixture of arterial and venous blood along with interstitial and intracellular fluids.Capillary blood is often the specimen of choice for infants, very young children, elderly patients with fragile veins, and severely burned patients. Point-of-care testing is often performed using a capillary blood specimen. Specimen Type Method of Collection Common Use Venous Direct puncture of vein by venipuncture; vascular access device Routine laboratory tests Arterial Direct puncture of artery; vascular access device Arterial blood gases Capillary Dermal puncture of fingertip or heel Infants and young children Elderly patients with fragile veins Severly burned patients Point-of-care testing

Cells containing hemoglobin S have a decreased capacity to maintain normal levels of potassium (K+). As K+ leaves the cell, water follows. Two mechanisms are responsible for maintaining intracellular ion levels, the Gardos channel and the K+-Cl- channel. Both channels are abnormally activated in patients with sickle cell disease. The resulting loss of water from the cell increases the hemoglobin concentration and the chances for sickling.

Several mutations of the HFE gene have been described. The most common mutation in patients with hereditary hemochromatosis is the C282Y mutation. In the C282Y mutation, a base substitution leads to a change in the amino acid in position 282 from cysteine (C) to tyrosine (Y). The loss of the sulfhydryl-containing amino acid disrupts the tertiary structure of HFE so that it no longer binds to beta-2 microglobulin. Beta-2 microglobulin appears to act along with other proteins to chaperone the newly synthesized HFE out of the Golgi apparatus and to the cell surface where it can then bind to TfR. In the C282Y mutation, HFE remains in the Golgi, never making it to the cell surface. The result is that transferrin binding to TfR is enhanced and excessive amounts of iron enter the cells of the small intestine, liver, and other tissues. A second mutation, H63D, causes a histidine (H) residue in position 63 to be replaced by aspartic acid (D). The mechanism by which this mutation leads to increased iron uptake is less well understood when compared to the C282Y mutation. Unlike the C282Y mutation, the H63D mutation does not seem to affect the binding of beta-2 microglobulin and intracellular movement, since detectable concentrations of the mutated protein are found on cell membranes. Some researchers speculate that the H63D mutation affects the binding of proteins involved in iron regulation and uptake at the cell surface.A third mutation, S65C, leads to a serine-to-cysteine substitution in its associated protein. This mutation has been been found in some compound heterozygotes for C282Y or H63D, but is rarely associated with iron overload in HH.Additional mutations of HFE have been identified, but their clinical significance is unclear. Most laboratories performing molecular assays test for only the C282Y, H63D, and S65C mutations.

Due to their transparent nature, the cellular and intracellular structure of tissue samples can not be microscopically examined until they are colored by dyes. Dyes used to stain tissue samples for microscopic analysis in the clinical histology laboratory are called biological dyes or biological stains. Biological dyes can be grouped into the following two categories:Natural: Dyes that are derived from natural resources. The most important natural dye in the histopathology laboratory is Hematoxylin. Artificial: Dyes that are derived through chemical reactions. Artificial dyes greatly outnumber natural dyes.

Due to their transparent nature, the cellular and intracellular structure of tissue samples cannot be microscopically examined until they are colored by dyes. Dyes used to stain tissue samples for microscopic analysis in the clinical histology laboratory are called biological dyes or biological stains. Biological dyes can be grouped into the following two categories:Natural: Dyes that are derived from natural resources. The most important natural dye in the histopathology laboratory is hematoxylin.Artificial: Dyes that are derived through chemical reactions. Artificial dyes greatly outnumber natural dyes.

When HPV infects host cells, several HPV DNA-coded proteins initiate cellular changes. Two such areas in the genome are the open reading frames E1 to E7 and the late open reading frames L1 and L2. The proteins encoded by E1 to E7 regions of the genome are responsible for HPV-gene regulation and cell transformation. Proteins resulting from L1 and L2 form the viral shell.E6 and E7 encoded proteins are the most important HPV proteins in malignancy transformations. These viral proteins work together to convert normal host cells to malignant cells. E6 proteins interact with intracellular protein p53 and E7 proteins interact with intracellular retinoblastoma (Rb) protein. Intracellular proteins p53 and Rb regulate cellular growth. Both p53 and Rb are tumor suppressor proteins.When chromosomal damage occurs in normal cellular growth, p53 halts cellular growth and allows DNA repair enzymes to repair damage. Rb also halts cellular growth in DNA damage by inducing apoptosis (cellular death). When HPV E6 proteins bind to p53 and HPV E7 to Rb, mutations accumulate, unchecked cellular growth occurs, and a state of chromosomal instability results. This instability and unregulated cellular growth increases the chance of forming malignant cells. Viral E1, E2, and E5 encoded proteins may also damage cellular processes when HPV infects cells and can lead to malignant transformations.

Numerous genetic events occur over a relatively long period of time that lead to the development of cervical carcinoma. The protein products of tumor suppressor genes are the regulators of cell growth as discussed previously. Two intracellular protein products of tumor suppressor genes located within human cells are p53 and Rb. As noted earlier, protein products from HPV genes E6 and E7 bind to p53 and Rb, which results in unregulated cell growth. This unregulated growth prevents normal DNA repair, allowing for mutations to accumulate in the cell. As this process continues, it is postulated that a proto-oncogene becomes mutated, which in turn activates oncogenes.The E2 gene in HPV controls the production of E6 and E7 in the normal viral life cycle. When the viral genome is integrated into host cells, the E2 gene is disturbed and uncontrolled production of E6 and E7 protein products occurs. This leads to a greater interaction and disabling of host cell tumor suppressor gene products. The genes E6 and E7 of HPV Types 16 and 18 have a greater affinity for tumor suppressor gene products than other HPV types. This explains the greater virulence associated with Types 16 and 18 and their association with 70% of cervical cancer.

The role of leptin in obesity and insulin resistance is sometimes confusing. Some authors refer to leptin as a hormone, not an adipokine. Leptin is synthesized and released from adipose cells in response to adipose tissue changes. It reduces intracellular lipid levels in many types of body cells and thus improves insulin sensitivity. It is an appetite suppressant and inhibitor of fatty liver formation.Leptin is referred to as a "starvation signal" and the leptin gene, is sometimes referred to as "the obesity gene". These names refer to leptin's important function as a messenger in energy metabolism. Leptin signals the hypothalamus when there are increases in fat stores. The hypothalamus then restores metabolic balance by decreasing appetite, stimulating physical activity, and burning of excess calories. During fasting, leptin levels decrease rapidly and hypothalamus signaling results in an increase in cortisol and a decrease in thyroid, sex, and growth hormones. These actions work together to restore energy balance. Leptin is usually increased in obesity, however, similar to increased insulin in obesity, leptin resistance develops. In obesity, appetite suppression does not take place and metabolic rates are lowered. Secreted leptin is not able to stimulate energy balance and healthy caloric intake.

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. Humoral ImmunityHumoral 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. Cellular ImmunityCellular 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.

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.

Cultures often require 24 or more hours before a pathogen can be recovered. A Gram stain can give preliminary information about the type of bacterial and/or fungal organisms that are present. A rapid diagnosis of bacterial meningitis, made after examining a gram-stained smear of the patient's cerebrospinal fluid, allows the physician to begin treatment immediately. Intracellular gram-negative diplococci observed in a male urethral specimen may be confirmatory of the diagnosis of gonorrhea. Cultures may not even be needed unless susceptibilities are required. (In the female genital specimen, the presence of gram-negative diplococci is not specific enough to confirm the diagnosis, and a culture or other confirmatory testing must be performed).

When evaluating Gram stains of clinical samples, keep in mind the source of material from which the smear was made. Bacteria found in cerebrospinal fluid (CSF), blood, tissue and specimens from other sterile sites are always significant. Gram stains of body fluids that are normally sterile must be examined carefully. For every one organism per oil immersion field, there are about 105 organisms per mL present in the sample! Examining stained smears of CSF sediment may assist the clinician in establishing a presumptive diagnosis. The Gram stain result and the results of other special stains could also guide in the selection of culture media. If bacteria are observed in a CSF specimen, it is important to determine and report whether the bacteria are inside or outside of white blood cells (intracellular or extracellular). The quantity of organisms seen and the amount and type of host cells are also important to report. Bacteria observed in specimens from the throat, genital tract and other areas containing normal flora suggest infection only if their composition and type varies significantly from the norm.

The Gram stain reaction, shape, and arrangement of bacteria, and the presence or absence of intracellular organisms must be noted on the worksheet.Examples:Gram positive cocci in chains are present.Gram negative diplococci, intracellular, are present within white blood cells.Quantitate by approximating the average number of each cell type seen in 10 oil immersion fields, and record as:Many = More than 15/fieldModerate = 4-15/fieldFew = 1-3/fieldOccasional = 2-10/10 fieldsRare = 0-2/10 fields

Direct smear results are generally reported in the same way that they are read, except that bacterial cell arrangement (ex: clusters, chains, pairs) may be misleading and is generally not reported except in the case of intracellular diplocci in genital smears.

Important events that occur in an immune-mediated hemolytic transfusion reaction (HTR) include: Antibody Binding to Red Blood Cells Antibodies may be either IgM or IgG class. IgM antibodies activate complement and lead to intravascular hemolysis where free hemoglobin is released into the plasma. IgG antibodies rarely activate complement but they are often involved in effecting phagocytosis. The concentration of the antibody is directly related to the severity of the HTR. Activation of Complement The end result of complement activation is red cell lysis. Activation of Mononuclear Phagocytes and Cytokines Sensitized red cells are removed from circulation by mononuclear phagocytes. Macrophages in the spleen and Kupffner cells in the liver are active in this process. Activation of Coagulation Antibody-antigen complexes may initiate coagulation and cause disseminated intravascular coagulation (DIC). Shock and Renal Failure Hemolysis can be intravascular or extravascular. In intravascular hemolysis, free hemoglobin, RBC stroma, and intracellular enzymes are released into the blood stream. This results in hemoglobulinemia and hemglobinuria which can lead to kidney damage. In extravascular hemolysis, there is no release of free hemoglobin. Sensitized red cells are removed from the circulation by the monocytes and macrophages in the reticuloendothelial system.

Transfusion-associated graft versus host disease (TA-GVHD) is generally unresponsive to medical treatment. Hematopoetic stem cell transplantation has been successful in rare instances. Gamma-irradiation of blood components containing viable lymphocytes is effective in preventing TA-GVHD. Irradiation is recommended for all Whole Blood, Red Blood Cell (RBC), Platelet, and Granulocyte transfusions to patients at risk. Patients at risk include neonates less than four months, patients with an acquired or congenital immunodeficiency, or patients receiving a directed donation from a family member. Irradiation prevents proliferation of donor lymphocytes with a required dose of 25 Gy to the mid plane of the blood container and a minimum of 15 Gy elsewhere. The dosage must not exceed 50 Gy to prevent harm to the patient from irradiation. Irradiation of blood can result in a decreased survival of red cells and a leakage of potassium from intracellular stores. Because of this, red cell units may only be stored for up to 28 days following irradiation. No reduction in storage time is required for platelets. Because Fresh Frozen Plasma (FFP) and Cryoprecipitate do not contain cells, irradiation is not required to prevent TA-GVHD in patients at risk.