Structure and Function of RBC - Strom 03.16.2015 Flashcards
Objective 1
Differential Diagnosis 101: Be able to provide at least one possible reactive (non-neoplastic) etiology for an increased number of red cells, platelets, or any type of leukocyte.
Red cells:
Platelets:
Leukocytes:
Objective 2
Predict the cellular and physiologic effects of reduced production of ATP, NADH, or NADPH in the red cell.
- Reduced ATP: Na+/K+ pump fails to maintain electrolyte balance, leading to excess water entry into cell. Lysis/burst
- NADPH: required for the anti-oxidant system; used along with glutathione to eliminate peroxide; requires energy;
- NADH: required for anti-oxidant system; used along with cytochrome b5 reductase to reduce methemoglobin back to hemoglobin; requires energy
- Lacking NAD(P)H or the energy needed to run the system can result in clumps of oxidized hemoglobin and excess red cell lysis, termed hemolytic anemia
EDTA and wright-giemsa stain
EDTA is an anti-coagulant used when blood samples are collected. You don’t want the blood to clot before you can get it on a slide, so you use an EDTA tube. A wright-giemsa stain is used when examining a blood stain.
Eosin-Y
A stain that is –> negatively charged and aromatic –> soluble in ethanol, not water It stains hydrophobic, basic macromolecules, such as heme, which is an aromatic molecule with a positive charge in its center (thus you can see why the stain sticks to the heme). Used to stain red cells and eosinophils. Eosinophils have cytoplasmic granules that just LOVE to take up this stain.
Appearance of normal RBC stained with eosin -Y
Bi-concave discs, uniform in size, with an area of central pallor that occupies 1/3 of the diameter of the RBC.
Eosinophil
These are rare in blood, except in cases where the patient is having an allergic reaction (drug or otherwise) or is infected with parasites. This fact can help with your differential diagnosis! As with all other cells, an increase in eosinophils might also indicate the stirrings of a neoplastic process.
Reactive vs. neoplastic
This is the starting point for diagnosing ANY increase in the number of ANY type of blood cell.
Methylene Blue
A stain that –> is soluble in water and methanol –> stains hydrophobic, acidic macromolecules such as proteins and nucleic acids. –>is flat and positively charged Used to stain basophils
Basophils
- Rare in the blood - Increase in neoplastic conditions - Related to mast cells/degranulate in allergic reactions
Monocyte
3%-8% of leukocytes
The intellgence gathering arm of the immune system; spend a lot of time sharing information with lymphocytes in the lymphatic tissues in the way that they are phagocytes that present foreign antigens via MHC Class II molecules
Most macrophages are derived from these cells
Monocytosis is VERY NON-SPECIFIC, as levels can increase in a variety of illnesses, so you wouldn’t use monocytosis to help you with your differential.
Appearance: The nucleus looks like a horse shoe (proper way to say = amoeboid or S-shaped). Use the appearance of the nuclues to differentiate between the monocyte and a reactive lymphocte.
Lymphocyte
20-30% of blood leukocytes
Can increase in viral syndromes or as a neoplastic process
Can live days to years
Mostly T-cell, then B-cells and NK cells.
Reactive Lymphocyte
Nucleus will be round/oval, which is how one can distinguish the reactive lymphocyte from a monocyte. If a pathologist is still unsure of which type of cell he’s seeing, he will call it a mononuclear cell.
Might have more cytoplasm and prominent nucleoli.
Increased levels of reactive lymphocytes indicates viral infection.
Large granular lymphocyte
NK cells and CTLs sometimes show small numbers of basophilic cytoplasmic granules, so we call them “large granular lymphocytes”
Is knowing the neutrophil count essential for evaluating any infectious disease?
Yes
Neutrophils: Facts and Appearance
Think hand-to-hand combat: they eat bacteria (phagocytosis) and secrete enzymes to kill bacteria (degranulation). Though they live for only one day, their post-suicidal chromatin can form NETs (neutrophil extracellular traps), which help control sepsis.
40-70% of leukocytes
Their numbers increase 10-fold or more in response to bacterial infection.
Appearance: Segmented nuclei; cytoplasmic granules that stain light pink and do not attract much eosin-Y or methylene blue stain.
Neutrophils: Tactics
Migrate in response to chemokines
Recognize and swarm enemy
Degrade and immobilize enemy
Minimize collateral damage
Neutrophils: Tools (receptors and the like)
Consequences of Bad Tools
TOOLS
IL-8 receptor (AKA: CXCR-2)
Integrins –> CD11a/CD18 complex, a “grab and hold” device
Very active cytoskeletons
CONSEQUENCES
Congenital CD18 defect –> leukocyte adhesion defect (recall delayed sloughing of umbilical cord as clue!)
Wiskott-Aldrich Syndrome (WAS) –> failure of the cytoskeleton to respond during adhesion; these individuals have trouble with the mechanism that initiates cytoskeletal reorganization in response to signals from the cell surface (for example, signal can come from binding of integrins to their receptors)
Recognition Tools of Neutrophils
Neutrophils express receptors for many bacterial and fungal constituents
- Toll-like receptors, which recognize microbial constituents (LPS, dsDNA, flagellin, peptidoglycans, etc) and mediate signaling that initates cytokine production; These are considered PRRs
- Complement receptors (which are not PRRs)
- Fc receptors, which bind to antibodies that are attached to infected cells or invading pathogens. Their activity stimulates neutrophils to destroy microbes by antibody-mediated phagocytosis
Chemokines associated with neutrophils
CXCL2: secreted by monocytes and macrophages; a chemotactic for neutrophils
IL-8 (CXCR-2): produced by macrophages and endothelial cells to recruit neutrophils
TNF: produced mostly by macrophages, but neutrophils as well; induces fever, apoptosis, and cachexia, among other terrible things.
Excess chemokine production is associated with excess inflammation, which can sometimes lead to death.
How Neutrophils Kill
What happens when they can’t kill good
KILL
Phagocytose the enemy
Produce a lysozyme to chew through polysaccharide cell wall
Produce proteases to attack enemy’s proteins
Use bleach (hypochlorite) to kill the enemy. To make the bleach requires myeloperoxidase; can use a stain with Ab for myeloperoxidase in order to identify neutrophils
Apoptose in order to 1) limit the amount of nasty enzyme released so that surrounding tissues are not damaged; and 2) release chromatin-derived NETs
CAN’T KILL GOOD
Chronic Granulomatous Disease: a condition in which neutrophils fail to make hypochlorite once they have migrated to and recognized an enemy. Clusters of ineffectual and dying neutrophils result; we call these clusters “granulomas”
Appearance of neutrophils during bacterial infection
As a whole, immature forms of neutrophils seen during bacterial infection is called “left shift”
- Non-segmented nuclei that are horseshoe-shaped (these neutrophils are called bands)
- Less mature neutrophils called metamyelocytes that have bean-shaped nuclei
- Neutrophils called **myelocytes **that have round nuclei
Neutrophils will also have increased cytoplasmic granules (called “toxic granulation”)
- Primary granules (blue in color) will be present; indicates immaturity because primary granules should only be seen in myeloid precursors in bone marrow
- Secondary granules (pink in color) will also be present
- If your patient’s lab report reads “toxic granulation”, then the patient’s cells looked as described above.
