Introduction and Blood Flashcards
“Formed elements”
Non-plasma component of blood
Includes nucleated cells, erythrocytes, and platelets
Resolution of a standard light microscope
0.2 μm
Haemotoxylin
Hematoxylin (the blue dye) is a basic dye which will stain acidic structures a bluish color. Structures that contain DNA or RNA have affinity for this dye (i.e. nucleus), as DNA and RNA are acidic. Structures that react with hematoxylin are referred to as basophilic (base loving), as they bind to the basic dye.
Eosin
Eosin (the pink dye) is an acidic dye that will stain basic components of the cell pink. Since cytoplasmic proteins are typically basic, the cytoplasm is usually stained pink. Components of the cell that react with eosin are referred to as acidophilic or eosinophilic.
Wright-Giemsa
The Wright-Giemsa stain (sometimes just referred to as Giemsa) is frequently used to stain individual cells (as opposed to tissues). It is particularly helpful for staining blood (most of the images you will see in the reading specific to the blood session that accompanies this are actually Wright-Giemsa stains, and not H&E stains) and bone marrow. However, the general “nucleus blue, cytoplasm pink” rule still applies
Trichrome Stain
The Trichrome stain is a variant of H&E that adds a third dye that specifically stains collagen a blue-green color, and is useful for visualizing connective tissue. A similar stain can be used to visualize elastin fibers in elastic connective tissue.
transmission electron microscopy
The electron beam passes through the section. This method is useful for visualizing cells at the ultrastructural level (i.e., organelles and other subcellular structures below the limit of resolution of light microscopy).
scanning electron microscopy
Uses a moving electron source to “scan” the surface of the specimen, which is coated with an electron dense material (gold is commonly used). The electrons bounce off of the surface of the sample, and will be collected by a detection system, which generates a 3D image of the sample being studied. This technique is useful for studying whole cells and tissues
Relative sizes of bacteria, erythrocytes, and megakaryocytes.
Relative size of erythrocyte and virus
Relative size of virus and protein
Myeloperoxidase staining
Immunohistochemistry
In immunohistochemistry, a section of tissue is placed on a slide, and an enzyme-linked antibody is placed on the tissue. The antibody will bind to its specific antigen. Excess, unbound antibody is washed away. The position of the antibody is then visualized using a substrate for the antibody that generates a visible product.
It is sometimes called immunoperoxidase staining (“impox” for short), because the enzyme linked to the antibody is usually horseradish peroxidase.
Immunofluorescence
very similar in concept to immunohistochemistry, but the antibody is linked to a fluorescent molecule rather than an enzyme, and is activated when it is exposed to light of a particular wavelength
in situ hybridization
We take advantage of the principle of sequence complementarity: nucleic acid “probes” with sequence complementary to the region of interest are used to identify a particular DNA or RNA sequence target.
The probe is typically labeled with a visible signal (fluorophore or enzyme) that allows it to be detected. When the probe is fluorescent, the method is referred to as fluorescence in situ hybridization, or FISH.
next-generation sequencing
instead of sequencing one long sequence, many short pieces of DNA (“reads”) are sequenced in parallel on a chip, then tiled together to get the full sequence. This allows sequencing to be performed much more cheaply and efficiently than using older methods.
“shotgun”
Plasma
Unclotted liquid phase of blood
Serum
post-clot liquid phase of blood
lacks fibrinogen and clotting factors
Albumin
The most abundant plasma protein (it makes up 55% of circulating protein all by itself). It has a vital role in maintaining plasma oncotic pressure – without it, fluid would leak into the tissue.
Gamma globulin
antibodies (all, not just IgGs)
Blood electrolyte concentrations
Thrombocyte
another name for platelet
Number of erythrocytes in 1 microliter of adult blood
5x106 per µl
“central pallor”
Indent in erythrocytes
Erythrocyte half-life
half-life in circulation of ~120 days
hemostasis
blood clotting
A Neutrophil
B Eosinophil
C Basophil
D Monocyte
E T Lymphocyte
F Plasma Cell
Neutrophils
The most abundant of the leukocytes and usually comprise 50-70% of white blood cells (even higher during an acute inflammatory response). Neutrophils are the first responders to tissue injury and are the major mediators of the acute inflammatory response.
Eosinophils
comprise up to 3% of circulating leukocytes in a normal adult. They are about 15 μm in diameter, same as neutrophils, but can be distinguished by their bilobed nuclei and bright pink (eosinophilic) granules. Eosinophils function in defense against helminthic infections (parasitic worms), and play an important role in the pathology of allergy.
Basophils
The least numerous of the leukocytes, comprising about 0.5% (or roughly 1 in 200 cells). They are about the same size as neutrophils and eosinophils, but distinguished by their dark blue (basophilic) granules that typically obscure the nucleus from view.
Basophils release acute inflammatory mediators, including histamine and serotonin, during acute hypersensitivity reactions. If you see more than one or two basophils on a blood smear, something is most likely wrong with the patient. In particular, basophilia (too many basophils – see note on terminology) should make you suspect a particular hematologic malignancy, chronic myelogenous leukemia (CML)
Lymphocytes
Typically comprise 10-20% of circulating leukocytes and include B, T, and NK cells (these cannot be distinguished morphologically – you need to use methods such as flow cytometry that assess cell surface marker expression to tell them apart). They are 7-8 μm in diameter. As a general rule, the nucleus of a resting lymphocyte should be about the same size as a red cell.
A resting lymphocyte should have a round nucleus, condensed chromatin (why?), and very little cytoplasm (again, why?).
Monocytes
10-20% of circulating leukocytes, similar to lymphocytes. They are the largest circulating cells in normal blood, about 25 μm in diameter
In addition to their size, they can be identified by their folded (often bean-shaped) nuclei and bluish-gray cytoplasm without granules. When attracted to tissue by molecular signals (chemokines) during chronic inflammation, monocytes migrate out of the blood and into tissue, where they become macrophages and play an important role in “cleaning up” tissue damage and killing pathogens.
–cytosis
too many of a cell type
–penia or -cytopenia
too few of a cell type
Anemia
too few red cells
–philia
often used in place of –cytosis to indicate too many of a cell type.
hematocrit
Historically, red cell mass was assessed using the hematocrit. To measure hematocrit, whole blood is placed in a very thin glass tube and spun down in a centrifuge. The ratio of the height of the red cell pellet to the total height of the column is the hematocrit (normal is around 36-45%; higher in men, lower in women). This method is quick, cheap, and easy – all you need is a tube, a centrifuge, and a ruler – and is still used in resource-poor settings
Most hospital labs that use automated instrumentation instead measure the concentration of hemoglobin in lysed blood as a surrogate for red cell mass (normal is 12-15 g/dl). The hematocrit is then calculated based on the hemoglobin concentration.
hematopoiesis
The process of formation of the formed elements in the blood
Nearly all hematopoiesis in adults occurs in the bone marrow. (Interestingly, it occurs in the spleen and liver in the fetus, and migrates to the marrow during the last few months of gestation.) The bone marrow contains the precursors of all the formed elements of the blood: myeloid precursors (give rise to granulocytes, lymphocytes, and monocytes), erythroid precursors, and megakaryocytes, the precursors of platelets
Turnover of erythrocytes, platelets, neutrophils
dynamic flexibility of bone marrow
the ability to increase production of blood cells in response to stress
hematopoietic stem cell differentiation pathways
“Left Shift”
When blood progenitor cells exit the blood marrow early in response to stress and finish proliferating and differentiating in the bloodstream
erythropoietin
Production of erythrocytes, or erythropoiesis, is regulated by the hormone erythropoietin
EPO is a small (166 amino acid) glycoprotein hormone produced by specialized cells in the kidney (the juxtaglomerular cells)
Production of EPO (and by extension, of erythrocytes) occurs in response to hypoxia, which triggers a signaling cascade involving the transcription factor HIF1α. Once in the nucleus, HIF1α drives transcription of EPO
Erythropoetin in action
Once released into the blood, EPO acts on the earliest erythrocyte precursors in the bone marrow (BFU-E and CFU-E; see diagram on page 19) to drive them to proliferate and prevent them from undergoing apoptosis, resulting in increased production of mature erythrocytes. Once the red cell mass increases, and the hypoxic state is resolved, then HIF1α will be inactivated, and EPO production will decline. This is a classic example of a negative feedback loop.
thrombopoietin
Production of platelets, or thrombocytes, is regulated by the hormone thrombopoietin (TPO).
TPO is produced at a relatively constant rate by the liver.
Both megakaryocytes (platelet precursors) and platelets themselves express the receptor for TPO. However, only megakaryocytes have the ability to respond to TPO and make platelets. The mature platelets therefore act as a “sink” for TPO. If there is a decline in the number of platelets, more TPO will bind to receptors on megakaryocytes, resulting in an increase in platelet production. These additional platelets will restore the balance of TPO bound to platelets vs. megakaryocytes, and production will return to normal.
Leukocyte hematopoiesis
Mononuclear Cells
Lymphocyte, monocyte, or macrophage
Quantitative erythrocyte, platelet, and leukocyte measurement by light scattering
Spherocytosis
lack of erythrocyte pallor
spherical erythrocytes
