Unit 1 Flashcards
What is hematology?
The study of the blood and blood forming tissues
What is the composition of blood?
55% plasma
45% formed elements
What are “formed elements”?
Include erythrocytes, leukocytes, and thrombocytes
Describe erythrocytes
- normal range 4.5-.5.9 million per mm3 in adults
- biconcave shape
- diameter 7 microns
- cells for transport of O2 and CO2
- life span is 120 days
Describe leukocytes
- normal range 4.4-11.3 thousand per mm3 in adults
- five types
- size 8-20 microns
- involved in fighting infection, combating allergic reactions, and immune responses
- except for lymphocytes white cells life span is approximately 10 days
Describe thrombocytes
- smallest cells in the blood
- normal range 150,000-400,000
- active role in coagulation and hemostasis
Describe the cellular membrane
- semipermeable separation between the cellular components, the organelles and the surrounding environment
- made up of: proteins, phospholipids, cholesterol, traces of polysaccharide
Describe the Fluid Mosaic Model
- dynamic fluid structure with globular proteins floating in lipids
Describe lipids of the cellular membrane
- as phosopholipids
- arranged in two layers
—> polar (charged) phophates ends are toward inner and outer surfaces with the fatty acid ends toward the interior of membrane
—> protein molecule may be incorporated into the lipid bilayer or associated with either the outer or inner surface membranes
—> polysaccharides (plycoproteins or glycolipids) are attached to the membrane
What is the function of lipid bilayer of the cellular membrane?
- Impermeability of membrane
What is the function of the protein in the cellular membrane?
- acts as transport molecule, for the rapid penetration of polar and non-lipid soluble substances.
-Determines and protects shape and structure of membrane (attached to microtubules and micro filaments)
What is the function of carbohydrates (polysaccharides) of the cellular membrane?
- surface antigen - functions in recognition of and interaction between cells
What are the extrinsic proteins in human blood cells?
Spectrin
Actin
Describe the cytoplasmic membrane, as a unit
- maintain cellular integrity of the interior of the cell by controlling and influencing the passage of materials in and out of the cell.
- This function is accomplished through the major membrane processes of osmosis, diffusion, active transport and endocytosis
Describe Osmosis
- net movement of water moelcules through a semipermeable membrane
- normally water molecule move in and out of cell membrane at an equal rate producing no net movement
- if concentration gradient exists, movement of water moelcules will be greater from areas of low solute to areas of higher solute concentration
What is the basic principle underlying the erythrocyte fragility test?
Alterations int he erythrocyte membrane can be observed by placing RBC’s in varying concentrations of Nacl
What is osmostic pressure?
Pressure exterted by water molecules inside membrane at equilibrium
Describe low sodium concentration during osmosis
The net movement of water is into the cell which will cause the cell to lyse
Describe high sodium concentration of osmosis
The net movement of water is out of the cell which will cause cell creanation
Describe diffusion
- Passive process through a semipermeable membrane (also referred to as dialysis), where substance move down a concentration gradient from areas of high solute concentration to areas of low solute concentration by dissolving in the lipid portion of the membrane
- requires no energy
What is diffusion dependent on?
1) solubility of molecules in liquids
2) temperature
3) concentration gradient via hydrophilic regions (where proteins create a pore like opening)
4) calcium ions affect permeability of membranes. Increase in Calcium in fluid around cell of accumulation of calcium in cytoplasm of cell can decrease permeability
Describe active transport
- sodium-potassium pump, fundamental transport system. Na ions pumped out of cell into extracellular fluid- where concentration of Na is higher- need ATPs
- movement of molecules from area of lower concentration against a concentration gradient to an area of higher concentration
- requires energy
Describe endocytosis
- process of engulfing particles or molecules with formation of membrane-bound vacuoles in the cytoplasm
What are two types of endocytosis?
Pinocytosis
Phagocytosis
Describe pinocytosis
- the uptake (engulfment) of liquids by the formation of tiny vesicles from the cellular membrane and their subsequent pinching off.
- literally “ cell drinking”
Describe phagocytosis
- engulfment and destruction of particles- the vacuole containing the particle is fused with lysosomes and digestive enzymes are emptied into the vacuole for it destruction
- cellular eating - cell ingests solid particles and digests them
Briefly describe reactive and neoplastic growth
- size and shape of cells constant but features can vary because of infectious diseases or malignancy
What is anaplasia?
Bizarre cytologic features associated with poorly differentiated malignant tumors
What is atrophy?
- decrease in number or size of cells - leads to decrease in size of organ or tissue mass
What is dysplasia?
Abnormal cytologic features - often is a premalignant change
What is hyperplasia?
Increase in number of cells in a tissue
What is hypertrophy?
Increase in size of cells - leads to increase in organs size
What is metaplasia?
Change from one adult type to another
What is necrosis?
The death of groups of tissue cells caused by environmental factors such as lack of oxygen
What are cytoplasmic organelles?
Functional units of a cell that can only be viewed with an electron microscope.
- staining with Wrights stain aids in differentiating features of the cells
Describe cytoplasm
- 70-90% water
- contains proteins, lipids, carbohydrates, minerals, salt and water
- background for all chemical reactions that takes place in the cell
- embedded in cytoplasm are the organelles
What are the organelles of the cell?
- centrioles
- endoplasmic reticulum
- Golgi apparatus
- lysosomes
- microbodies
- microfilaments
- microtubules
- mitochondria
- ribosomes
Describe centrioles
- involved in cell division
- two cylindrical organelles found near the nucleus in a tiny round body called centrosome
- they serve as points of insertion of the spindle fibers during cell division
- has 9 (triplet) groups of microtubules
What happens to the centrioles during mitosis?
- separate from each other
- form thin cytoplasmic spindle fibers that connect to each individual chromosome
- pull apart to help equal distribution of chromosomes into two daughter cells
Describe the endoplasmic reticulum
- lace like network, two kinds:
—> rough
—> smooth - network of tubular structures crisscrossing across the cell
- some connect the nuclear membrane to the cell membrane
- channel for transport
- acts as a storage area
Describe rough endroplasmic reticulum
- containing ribosomes - protein production
Describe smooth endoplasmic reticulum
- no ribosomes present - site of lipid synthesis (cholesterol) and breakdown of fats into smaller molecules that can be used for energy
Describe the Golgi Apparatus
- appears unstained area next to nucleus
- arranged in layers of membranes resembling stacks of pancakes
- part of the Golgi apparatus and adjacent portions of the ER produce lysosomes
- products of Golgi apparatus are exported from cell when a vesicle fuses with the plasma membrane
- horseshoe-shaped or hook-shaped organelle with associated stack of vesicles
What are the functions of Golgi Apparatus?
1) it is the site for concentrating secretions of granules, packing and segregating carbohydrate components of certain secretions
2) synthesizes carbohydrates and combines them with proteins
3) abundant in gastric glands, salivary glands, and pancreatic glands
Describe lysosomes
- contain hydrolytic enzymes for digestion of phagocytized products, waste management of the cell
- digest protein molecules
- helps to digest old, worn-out cells, bacteria and foreign matter
- If they rupture, the lysosome will digest the cell’s proteins causing it to die
- “suicide bags”
- three types: primary, secondary, and tertiary
Describe microbodies
- small intracytoplasmic organelles
- contain oxidase enzymes that produce H202 (hydrogen peroxide)
- oxidative activity is important aspect of phagocytosis
Describe microfilaments
- consists of protein actin and larger myosin filaments
- AIDS in amoeboid movement of phagocytic cells
- smallest components of cytoskeleton.
Describe microtubules
- hollow protein fibers— formation is rapid, reversible self assembly of filaments function:
—> cell shape- cytoskeleton
—> organelle movement
—> passive role in intracellular diffusion - mitotic spindle is composed of microtubules
Describe the mitochondria
- rod-shaped, energy producing organelles
- makes energy in the cell
- cells that have the most mitochondria have a greater need for energy
- contain enzymes that break down glucose to ATP (adenosine triphosphate) the energy molecule
- “powerhouse”
- not found in mature erythrocytes
Describe ribosomes
- small dense granules that contain RNA and protein
- exist singly or in clusters in cytoplasm or on ER (making it rough ER)
- site of protein synthesis (with mRNA)
- show lack of membranes
- presences of many ribosomes produces cytoplasmic basophilia (blue color) when stained with Wright stain
Describe glycogen
- storage form of carbohydrate
- can be seen with special stain periodic acid-Schaffer (PAS)
- increased glycogen concentrations in neutrophils related to need for high energy to carry out of body defenses
Describe Ferritin
- storage form of iron
- found in telolysosomes (iron-rich dense bodies)
- siderosomes is term used to refer to iron-saturated hemosiderin
- found in macrophages of spleen and bone marrow
- plays role in recycling and storage of iron for hemoglobin synthesis
- smaller than glycogen
Describe nucleus
- 10-15 um
- control center of cell
- membrane has inner and outer layer, outer layer continuous with ER
- contains one or more nucleoli which contains RNA and is site of synthesis and process of the different types of RNA
- contains chromatin- uncoiled DNA
Describe Chormatin
- genetic material composed of nucleic acids and proteins
- DNA has two functions
- proteins associated with nucleic acids
—> histones
—> nonhistones
—>together = nucleosome - demonstrates characteristic patters when stained - distinctive feature of cell types
- divide into two types
—> heterochromatin
—> euchromatin
DNA + protein = ?
Chromatin
What are the functions of DNA?
1) dictate nature of proteins that can be synthesized- controlling cell
2) transmit information for cellular control from one generation to next
Describe histones associated with with nucleic acid
- positively charged
- essential to structural integrity to chromatin
Describe nonhistones associated with nucleic acid
- Less positively charged
- play role in genetic regulation and organization of DNA and histones
Describe heterochromatin
- condensed, dark staining area
- genetically inactive
- associated with nucleolus
- more restricted function of a cell the more heterochromatin present
Describe euchromatin
- uncoiled, pale-staining area
- genetically active
Describe chromosomes
- genetic material that exists as diffuse elongated chromatin fibers prior to mitosis
- during cellular division that stands condense into short visible structures
- number is constant within each species —humans have 46 chromosomes arranged into 23 pairs, one member of each pair inherited from the father and the other inherited from the mother
- 22 pairs are autosomes (body chromosomes) and pair represents the sex chromosomes, either XX or XY
- chromosomes can be grown, harvested, stained, and banded to produce a Karotype
What are 3 chromosomal alterations?
1) deletion
2) translocation
3) trisomy
Describe deletion of chromosomes
Loss of a segment of a chromosome
Describe translocation of chromosomes
Segment of one chromosome breaks from its normal location and attaches to another chromosomes during meiosis
Describe trisomy
- One of the homologous chromosomes fails to separate from its sister chromatid and leads or a set of three chromosomes instead of a pair
- trisomy 21 = Down syndrome
Describe mitosis
1) interphase: DNA replication
2) prophase: chromatin coils: nuclear membrane disintegrates, centrioles move to opposite ends of the cell
3) metaphase: chromatids move to equatorial plate
4) anaphase: chromatids separate and move to opposite poles
5) telophase: two daughter cells form and nucleolus and nuclear membrane reappear
Describe meiosis
1) cell division unique to gametes
2) gametes have only one homologous of the 23 pair (haploid number 1n)
3) phases differ from mitosis- two cell divisions
4) during phase I of meiosis— homologous sister chromatids undergo process of synapsis (lining up end to end) to allow for crossing over- an exchanging of genetic information
What are two metabolites important to hematologists?
Glycogen
Ferritin
What is epigenetic?
- refers to stable changes in gene function that are transmitted from from one cell to its progeny
- epigenetic changes play important role in normal cellular development and differentiation
What is genome?
Total genetic material stored in an organisms chromosomes
Describe cytogenetics
Contributes to understanding inborn or acquired genetic problems by providing providing a low-power screening method for detecting isolated or missing chunks of chromosomes
What is used today to identify changes ranging from a single chromosome disorder to alterations involving the interchange of DNA between chromosomes?
Molecular methods
What can molecular methods detect in chromosomes?
Abnormalities in:
- erythrocytes
- leukocytes
- coagulation factors
What are disorders that are caused by erythrocyte abnormalties?
- sickle cell disease
- Alpha- and Beta- thalassemias
What are disorders caused by leukocyte abnormalties ?
- acute myelogenous leukemia (AML)
- acute lymphoblastic leukemia (ALL)
- chronic myelogenous leukemia (CML)
- lymphoma
What disorders are caused by coagulation factor abnormalties?
- hemophilia A
- hemophilia B
- factor V Leiden defect
What is a karyotype?
- chromosomes pairs are numbered according to relative size and position of their centromeres and placed in groups according to letters
What chromosome banding technique is NOT obsolete?
- digestion of chromosomes with the enzyme trypsin, followed by Giemsa staining, that produces G bands
What is the Philadelphia chromosome?
- First chromosomal abnormality discovered in a malignant disorder
- example of translocation from chromosome 22 to 9
What is the period of time between mitoses?
Interphase
What is the phase of actual cell division in mitosis?
M phase
What are the three subphases of interphase?
- G1
- S phase
- G2
Describe G1 phase
- lasts 6-8 hours
- nucleolus (nucleoli) becomes visible
- chromosomes are extended and active metabolically
- cell synthesizes RNA and protein in preparation got cell division
- as stage comes to an end, cellular metabolic activity slows
Describe the S phase
- last 6 hours
- time of DNA replication
- metabolic and growth activities to a minimum
- shorter chromosomes are replicated first
- protein portion is also duplicated
- by end of S phase, each chromosome homologue has doubled but is held together by a single centromere
Describe G2 phase
- relatively short, 4-5 hours
- second period of growth
- DNA can function again to maximum in preparation for cell division
By end of G2 phase, proteins have been constructed and both the DNA and RNA are doubled. Centrioles ave divided, forming a pair of new centrioles at right angles to each other.
Describe prophase
1) replicated strands of chromatin become tightly coiled
2) the identical halves, chromatids, are joined at the centromere
3) the nucleolus and nuclear envelope disintegrates, the fragments scattering in the cytoplasm
4) the centrioles (composed of microtubules) separate and migrate to the opposite poles of the cell.
5) microtubules aggregate to form mitotic spindle that is attached to centrioles
Describe Methphase
1) identical sister chromatids move to the center of the spindle
2) each chromatid pair is attached to a spindle fiber and aligned along the equator of the cell.
3) point of attachment is the centromere
Describe anaphase
- begins as soon as chromatids pull apart and lasts until newly formed chromosomes reach the opposite poles of the spindle
- chromatid pains are separated, with one half of each pair being pulled at their centromere by the spindle fibers toward each pole.
Describe Telophase
1) the chromosomes arrive at opposite poles of the cell early in phase. One of each kind of chromosomes arrives at each of the poles of the cell.
2) the nucleolus and nuclear membrane reappear and spindle fibers disappear during this phase
3) because the chromosomes uncoil and become lo nger and thinner, the chromosome structural formation disappears
4) the DNA and proteins (nucleoproteins) now assume their distinctive chromatin arrangement
What is cytokinesis?
Division of cytoplasm
Describe cytokinesis
- follows telophase
1) cytoplasm around the two new nuclei becomes furrowed and the cytoplasmic membrane pinches in.
2) pinching in is accomplished by the contraction of a ring of microfilaments that forms at the furrow.
3) at completion, two new and identical daughter cells have been formed
Describe the G0 phase
Some cells continue through the mitotic cycle repeatedly, but others lose their mitotic ability and enter a protracted state of mitotic inactivity
What is apoptosis?
Cell death
What are the types of apoptosis?
- Active (programmed cell death and necrosis)
- passive (cell death without known regulatory mechanisms
What are some characteristics of apoptosis?
- chromatin condensation and fragmentation
- cell shrinkage
- elimination of dead cells by phagocytosis
Describe physiological cell death
Usually occurs by apoptosis
Describe pathological cell death
Results from acute cellular injury, which produces rapid cell swelling and lysis
How do cells prevent apoptosis?
With specific growth factor:
- one mechanism is increased synthesis of anti-apoptotic proteins
What is apoptosis caused by?
Activation of intracellular proteases, known as caspases.
What are the two pathways of apoptosis?
- intrinsic
- extrinsic
Describe intrinsic pathway of apoptosis
- focuses on mitochondria as initiators of cell death
Describe extrinsic pathway of apoptosis
Relies on TNF family death receptors for triggering apoptosis
What disorders are associated with decreased apoptosis?
- leukemias
—> chronic lymphocytic leukemia
—> SLE
—> follicular lymphomas
—> cancers with p53 mutations
What disorders are associated with increased apoptosis?
- AIDS
- aplastic anemia
What are gametes?
Ova or sperm
What is synapsis of meiosis I?
- Sister chromatids lining up end to end
- allows for easy exchange of genetic material through crossing over.
What is genomics?
The study of the entire genome of an organism
Describe functional genomics
The study of actual gene expression or gene profile of a specific cell at an exact gene expression or gene profile of a specific cell at an exact stage of cellular differentiation or functional activity
Describe proteomics
- the study of the composition, structure, function, and interaction of proteins produced by a cell
Describe genotype
- the specfic genetic makeup of an individual, usually in the form of DNA. it codes for the phenotype of that individual.
Describe phenotype
- an individual organisms EXPRESSED total physical appearance and constitution or a specific manifestation of a trait, such as a size or eye color, that varies between individuals
How are phenotypes determined?
- by genotype, or by the identity of the alleles that an individual carries at one or more positions on the chromosomes
- by multiple genes and influenced by environmental factors
Describe dominant gene
- refers to allele that causes a phenotype that is seen in a heterozygous genotype (ex: brown eye color = Bb)
- a person only needs to inherit one copy of the gene for the trait to be expressed
Describe recessive gene
- an allele that causes a phenotype (visible or detectable characteristic) that is only seen in a homozygous genotype (ex. Blue eye color = bb)
- a person must inherit two copies of this gene for the trait to be expressed
- thus both parents have to carriers of the recessive trait in order for a child to express that trait
- if both parents are carriers, there is a 25% chance with each child to show the recessive trait
What is a gene?
- A segment of DNA that is arranged along the chromosome at a specific position called a locus
- the functional unit of a chromosome
- responsible for determining the structure of a single protein or polypeptide.
What are alleles?
- Genes at specific locus that differ in nucleotide sequence
What are exons?
Coding sequences
What are introns?
Intervening sequences
What is polymorphism?
- the change in the DNA sequence that does not result in a functional abnormality
What is a single nucleotide polymorphism (SNP)?
A region of DNA that differs in only a single DNA nucleotide
Describe sickle cell disease
- hemoglobin S has a difference in one amino acid on the beta Chain.
- individuals heterozygous for this trait
Describe hemoglobin C disorder
A substitution of lysine for glutamic acid in beta chain occurs
What procedures allow determination of the exact sequence of amino acids on each of these two chains?
Electrophoresis
Chromatography
Describe oncogenes
- genetic alteration that causes cancers including leukemias and lymphomas, along with tumor suppressor genes and microRNA genes in affected cells.
- usually somatic cell events but germ-line mutations can predispose a person to inherited or familial cancer
- ex: Burkitt’s lymphoma
Describe protooncogenes
- viral oncogenes have normal counterparts in human genome
- ID of these established the fact that the human genome carries gene with the potential to dramatically alter cell growth and to cause malignancy when altered or activated to an oncogene
What are the functions of protooncogenes?
1) growth factors
2) growth factor receptors
3) signal transducers
4) transcription factors
What does activation of protooncogenes result from?
- mutations
- gene rearrangement or gene amplification
What is a hematopoietic neoplasm?
When a normal hematopoietic precursor cell, stem cell or more differentiated progenitor cell, acquires a cancer-inducing mutation
Describe tumor protein, p53
- specific tumor suppressing genes (p53 genes) inhibit cell growth in normal cells
- transcription factor encoded by TP53 gene
- guardian of the genome because it conserves stability by preventing genome mutations
What are some hemolytic malignancies demonstrating specific genetic alteration of p53?
- acute myeloid leukemia
- chronic lymphocytic leukemia
- chronic myelogenous leukemia
- myelodysplastic syndrome
- non-Hodgkin’s lymphoma
Describe Minimal residual disease (MRD)
- low level of disease cells
- molecular techniques are more sensitive to low number of cells. Can permit for early detection which means earlier intervention
What is apoptosis stimuli?
- deprivation of survival factors such as growth factor or loss of extra cellular matrix
- signals from death cytokines such as TNF
- cell damaging stress
What is the stimuli for necrosis?
- toxins
- massive injury
- severe hypoxia
What is a main characteristic of necrosis?
Conditions of ATP depletion
Describe overall cell size that is undergoing necrosis
Enlarged due to swelling
Describe overall cell size of cell undergoing apoptosis
Reduced by shrinkage
Describe the plasma membrane of a cell undergoing apoptosis
Intact but lost phospholipid asymmetry
Describe the plasma membrane of a cell undergoing necrosis
Disrupted with the loss of integrity
Between apoptosis and necrosis, which one is associated with inflammation?
Necrosis
What are cells?
- smallest organized units of living tissues, have the ability to individually perform all the functions essential for life processes
What is the largest organelle?
Nucleus
What is hematopoiesis?
- Process of blood cell production, differentiation and development
What are the hematopoietic organs?
- bone marrow
- liver
- spleen
- lymph nodes
- thymus
Describe fetal hematopoiesis
Also takes place in yolk sac, aorta-gonad-mesopnephrons (AGM) regions fetal liver and bone marrow
What are the 3 types of stem cells?
1) Totipotential stem cells
2) Pluripotential stem cells
3) multipotential stem cells
Describe totipotential stem cells
- present in the first few hours after ovum is fertilized
- most versatile type of stem cell
- can develop into any human cell type, including development from embryo into fetus
Describe pluripotential stem cells
- present several days after fertilization
- can develop into any cell type, except they cannot develop into a fetus
Describe multipotential stem cells
- derived from pluripotential stem cells
- can be found in adults but are limited to specific types of cells to form tissues. For example, bone marrow stem cells can produce all types of blood cells, bone cartilage and adipose cells
What are the phases of hematopoiesis?
1) embryonic phase
2) fetal hepatic phase
3) medullary ( or myeloid) phase
Describe the embryonic phase of hematopoiesis
- primitive hematopoiesis takes place in the yolk sac in the blood islands
- begins day ~19 post conception and continues until week 8 of gestation
- cells produced: erythrocytes, macrophages, and platelets
- erythrocytes contain Gower and Portland hemoglobin
Sites: from the yolk-sac to the hepatic (liver) phase to the bone marrow
Describe Fetal hepatic phase of hematopoiesis
- begins at 5-7 weeks post conception and continues until approximately 24 weeks of gestation (6th month)
—> peak hematopoiesis is ~3 months of gestation - cluster of erythroblasts, granulocytes, and monocytes colonize the fetal liver to eventually mobilize to the bone marrow
- microenvironment conducive to hematopoiesis is created by different cell populations that produce cytokines and chemoattractants to the liver
- spleen, thymus, and lymph nodes contribute to hematopoiesis during this phase
Describe the medullary phase
- bones are large enough to have marrow cavities
- 5th month, the bone marrow takes on its role and the chief organ of hematopoiesis in the adult with the liver and spleen acting as supplementary organs of hematopoiesis (EPO), granulocyte colony-stimulating factor (G-CSF), and granulocyte-monocyte colony-stimulating factor (GM-CSF) are present
- M:E ratio is 3:1 at this stage
- hemoglobin F and Hemoglobin A are present at this stage
Describe definitive erythropoietin
- begins 1-2 days later than primitive hematopoiesis
- process starts with with the formation of self-renewing HSCs in AGM
What is self-renewal?
HSCs have the ability to proliferate without differentiation
What is the first fully developed organ in the fetus?
Thymus
What are important characteristics of hematopoietic organs?
1) the anatomic structure consists of different tissue types and component cells
2) the stroma consists of various cells and extracellular macromolecules that occupy the hematopoietic tissue with hematopoietic cells. Stroma is the microenvironment where hematopoietic progenitor cells (HPCs) grow and differentiate
3) The HPCs are the HSCs and their offspring that become blood cells of multiple specific lineages
Describe yellow marrow
- adipose tissue - inactive
- replaces red bone marrow in many bones
Describe red marrow
- cellular - active
- active production of of most type of leukocytes, erythrocytes, and thrombocytes
- in children, red bone marrow is located throughout the skeletal system
- activity occurs in central portion of skeleton
What does bone marrow consist of?
- hematopoietic cells
- fat tissue
- osteoblasts
- osteoclasts
- stroma
What do hematopoietic cells consist of?
- erythroid
- myeloid
- lymphoid
- megakarocyte
- marrow stromal cells
- mast cells
- macrophages
- bone cells
What is hepatosplenomegaly?
Enlargement of the spleen and liver
Describe bone marrow
- ## found within central cavities of axial and long bones. It consists of hematopoietic tissue islands and adipose cells surrounded by vascular sinuses interspersed with a meshwork of trabecular bone,
Describe hematopoiesis
- takes place in marrow consisting of stromal cells and extracellular matrix
- must be supported by microenvironment that is able to to recognize and retain hematopoietic stem cells and provide factors required to support proliferation, differentiation, and maturation of stem cells along committed lineages
- stimulated by erythropoietin
What does the hematopoietic microenvironment consist of?
- adventitial reticular cells
- endothelial cells
- macrophages
- adipocytes
- bone lining cells (osteoblasts)
- elements of the extracellular matrix
Describe erythropoiesis
- occurs in distinct anatomical sites called erythropoietin islands, specialized niches in which erythroid precursors proliferate, differentiate, and enucleate
- each island consists of an iron-laden macrophage surrounded by a cluster of erytrhoblasts
- within each island, cell-cell and cell-extracellular matrix adhesion, positive and negative regulatory feedback, and central macrophage function occur
Describe granulopoiesis
- myeloid cells account for 23-85% of the nucleated cells in normal bone marrow
- early cells are located in the cords and around bone trabeculae
- neutrophils in the BM reside in the proliferating pool and the maturation storage pool
- need 3 to 6 days in the proliferating pool to mature
- if needed, the cells from the storage pool can enter peripheral circulation and have life span of 6-10 hours
Describe megakaryopoiesis
- take places adjacent to the sinus endothelium
- protrude through the vascular wall as small cytoplasmic processes to deliver platelets into blood
- take approximately 5 days to develop
What primarily produces erythropoietin (EPO)?
- kidney
- minor amounts from liver
Describe the 4 phases of hematopoietic cells
- according to maturity
1) primitive - multipotential cells. The most immature group of capable of self-renewal and differentiation into all blood cell lines
2) intermediate cells (committed progenitors). This group consists of committed progenitor cells destined to develop into distinct cell lines
3) precursors: give rise to mature cells; morphologically distinguishable in bone marrow and peripheral circulation
4) mature cells. The most developed group with specific physiologic functions
Describe Hematopoietic progenitor cells (HPCs)
- exists in the bone marrow in a highly organized, 3-D microenvironment composed of an diverse population of stromal cells and extracellular matrix rich in fibronectin, collagens, and various proteoglycans
What are the 5 growth factors for blood development?
1) G-CSF (granulocyte - colony stimulating factors)
2) GM-CSF (granulocyte-macrophage colony stimulating factors)
3) M-CSF (monocyte colony stimulating factors)
4) erythropoietin
5) IL-3
Describe lymphopoiesis
- unlike other cell lines, lymphocytes and plasma cells are produced in lymphoid follicles
- plasma cells are located along the vascular wall
- lymphoid cells typically account for 1-5% of the nucleated cells in the normal BM
Describe marrow stromal cells of the bone marrow
The meshwork of stromal cells is composed of reticulum cells, histiocytes, adipose cells, and endothelial cells
Describe mast cells of the bone marrow
- tissue mast cells, a connective tissue cell of mesenchymal origin, are normally observed in bone marrow
- form from HPCs in response to stem cell factor (KIT ligand, which is ligand for CD117)
- major effector cells of allergic reactions that are activated by exposure to antigen that cross-links allergen-specific IgE
- contain heparin, histamine, serotonin and proteolytic enzymes.
Describe macrophages of the bone marrow
Also called reticulum cells or histiocytes, appear as large cells in the bone marrow
- these cells constitute a physiological system, mononuclear phagocyte system
- macrophages and monocytes migrate freely into the tissues from the blood to replenish and reinforce the macrophage population
- most important step in the maturation of macrophages is cytokine-driven conversion of the normal resting macrophages to the activated macrophages.
- can be activated by IFN-gamma and G-CSF
Describe bone cells of the bone marrow
- osteoblasts are bone-matrix-synthesizing cells that resemble plasma cells and are usually observed in groups
Describe the thymus
- populated by primitive lymphoid cells from the yolk sac and liver.
- lymphoid cells physically push the epithelial cells apart
- T-cell progenitors migrate to the thymus from the bone marrow for further differentiation
- CD34+ progenitor cells develop in the thymic cortex
- lymphocytes in the thymus = thymocytes
- lymphocyte precursors differentiation is under influence of thymosin
- also regulates immune function by secretion of multiple soluble hormones
Describe cortex of thymus
- characterized by a blood supply system; unique because it only consists of capillaries.
- function is to act as a densely populated “waiting zone” of progenitor T cells
- before cells enter the cortex,the lack CD4 and CD8 surface markers. As they get closer to the medulla, the cells will progress to mature T cells that express their CD4 and CD8
Describe cytokines
- Proteins produced by many different type of cells
- bind to their receptors on the surface of a target cell
- induce intracellular signaling to the target cell for survival, proliferation, or differentiation responses to initiate a signaling pathway
- includes: interleukins, colony-stimulating factors, and interferons
What are the secondary lymphoid tissues?
- lymph nodes
- spleen
- gut-associated lymphoid tissue (GALT)
- thoracic duct
- bronchus-associated lymphoid tissue (BALT)
- skin-associated lymphoid tissue and blood,
- lymphocytes circulate in the peripheral blood and lymphatic tissues and through secondary lymphoid organs
Describe the spleen
- highly vascular
- major function of removing aging and damaged blood cells and particles, such as antigen-antibody complexes and opsonized microbes from the circulation and initiate adaptive (antibody) immune response to blood-borne antigens
- also stores platelets and participates in immune defense. About 1/3 of body pool of platelets are here
- contains largest amount of lymphocytes and macrophages int he body
- has red pulp, white pulp, and the marginal zone
- only lymphoid organ that has majority B lymphocytes instead of T lymphocytes
Describe red pulp of spleen
- sinusoids of the red pulp are lined by discontinuous epithelium, allowing passage of cells between the cords and the sinuses
- sinuses lined with macrophages which create filter that blood can seep through
- these sinuses trap red cell inclusions, older RBCs (older than 120 days) for recycling, and platelets
- the reservoir for platelets
What is “pitting”?
- The spleens ability to pluck out particles from intact erythrocytes without destroying them.
- blood cells coated with antibody are susceptible to pitting by splenic macrophages
- reduces the surface to volume ratio and results in formation of abnormally shaped spherocytes
Describe white pulp of the spleen
- initiating immune reactions involving both cellular and humoral immunity
- lymphoid cells form a cylindrical cuff around splenic arterioles, and these are mainly T cells.
Describe splenomegaly
- also called hypersplenism
- enlargement of the overall size of the spleen. Causes some degree of extreme reduction of erythrocytes, leukocytes, and platelets in circulating blood
- can result in pooling of 80-90% of platelets and produces peripheral blood thrombocytopenia
What are some disorders of the spleen?
- thrombocytopenia
- Gauchers’s disorder (macrophage accumulate large quantities of indigestible substances)
- Neoplasm (malignant cells occupy much of the splenic volume
- hepatosplenomegaly
- tumor cells - will show hypersplenism which is complication of sickle cell anemia
—> functional splenectomy or autosplenectomy
Describe the lymph nodes
- located all along the lymphatic vessels, and lymph fluid circulates through the nodes as it progresses through the lymphatic system
- lymphocytes circulate between the blood, organs and lymphatic tissues
What are the 3 main functions of the lymph nodes?
1) sites of lymphocyte proliferation in the germinal center
2) filter particulate matter, bacteria, or debris that enters the node via the lymph
3) participate in the immune response to foreign antigens
What is immunocompetent?
Able to function in the immune response
What are disorders of the lymph nodes?
- Adenitis ( increased number of microorganisms flow through the lymph nodes and overwhelm macrophages and infection of node can start
- lymphadenopathy (enlargement of lymph nodes)
- cancel cells can break lose and enter the lymph nodes. Malignant ones have potential of growing and metastasizing to other nearby nodes
Describe blood (as an organ)
- immunologic tissue effector
- most frequent sampled lymphoid organ
Describe the extrinsic regulation theory
- each HSC or progenitor cell has the unlimited and equivalent capacity to differentiate into many potential progeny, and specific signals from the cells environment determine the pathway of an individual cell.
Describe the intrinsic regulatory theory
Supports the proposition that the cellular environment makes no difference
Describe progenitor blood cells
- foundation of two major ancestral cell lines: lymphocytic (lymphoid) and nonlymphocytic (myeloid) cells
Describe the lymphoid stem cells (lymphocytic)
- precursor of either mature T cells or B cells/plasma cells
Describe nonlymphocytic stem cells
- progresses to the progenitor colony-forming unit, granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM)
What is the acronym for CFU used for?
- a prefix to record the number of colony-forming units of different progenitor cells that are identified through in vitro clonal assays
What can CFU-GEMM can lead to formation to?
- CFU-granulocyte-macrophage/monocyte (CFU-GM)
- CFU-eosinophil (CFU-Eo)
- CFU-basophil (CFU-B)
- CFU-megakaryocyte (CFU-Meg)
- in erythropoiesis, differentiates into the burst forming unit, erythroid (BFU-E)
What are conditions associated with increased mast cells?
- chronic lymphoproliferative disorders
- chronic infections
Describe mononuclear phagocyte system
- includes promonocytes and their precursors in bone marrow, monocytes in circulating blood, and macrophages in tissues
- functions: rapid phagocytosis mediated by receptors for IgG and the major fragment of C3
- macrophages, network of reticular cells of the spleen, thymus, and other lymphoid tissues are organized into this system
Describe erythropoietin (hematopoietic growth factor)
- cellular source: Peritubular cells of kidney, and Kupffer’s cells
- progenitor cell target: CFU-E, late BFU-E and CFU-Meg
- mature cell target: none
- gene located on chromosome 7
Describe IL-3 (hematopoietic growth factor)
- cellular source: activated T cells
- progenitor cells Target: CFU blast, CFU-GEMM, CFU-GM, CFU-G, CFU-M, CFU-Eo, CFU-Meg, CFU-Baso, BFU-E
- mature cell target: eosinophils and monocytes
Describe G-CSF (hematopoietic growth factor)
- cellular source: monocytes, fibroblasts, endothelial cells
- progenitor cell target: CFU-G
- mature cell target: granulocytes
- located on chromosome 17
Describe M-CSF (hematopoietic growth factor)
- cellular source: monocytes, fibroblasts, endothelial cells
-progenitor cell target: CFU-M - mature cell target: monocytes
Describe GM-CSF (hematopoietic growth factor)
- cellular source: T-lymphocytes, monocytes, eosinophils, monocytes, fibroblasts, endothelial cells
- progenitor cell target: CFU-blast, CFU-GEMM, CFU-GM, CFU-G, CFU-M, CFU-Eo, CFU-Meg, and BFU-E
- Mature cell target
Describe hematopoietic growth factor
- major role is regulating the proliferation and differentiation of HPCs as well as regulating the survival and function of the mature blood cells.
What does G-CSF and GM-CSF predominately affect?
Myeloid cells
What is T and B lymphocytes stimulated by?
IL-17
What targets NK cells?
IL-12
What are the molecules on HPCs that are important for mobilization?
VCAMs receptors
What does a comprehensive examination of bone marrow involve?
- examination of both bone marrow smears and histologically tissue sections
- it is also important to evaluate morphology in both bone marrow and peripheral blood
What is peripheral blood (PB) consist of?
- formed elements: 45%
—> RBC- erythrocytes
—> WBC - leukocytes
—> platelets - thrombocytes - liquid portion: 55%
—> plasma - liquid portion of unclotted blood - contains fibrinogen
—> serum - liquid portion of clotted blood - contains no fibrinogen
What are general cellular characteristics?
- the ID and stage of maturation of stained blood can be guided by a variety of systemic features
- two important characteristics to observe initially in cell ID are as follows: overall cell size and nuclear-cytoplasmic ratio
What is nuclear-cytoplasmic ratio?
- the amount of space occupied by the nucleus in the relationship to the space occupied by the cytoplasm
- size of nucleus general decreases as the cell matures
Describe the nuclear characteristics
- play an important role in cell ID. Important features of the nucleus include the following:
—> chromatin pattern
—> nuclear shape
—> the presence of nucleoli
Describe chromatin patterns of nuclear characteristics
- lymphocytes exhibit a smooth or homogenous pattern of chromatin throughout development until the mature stage, when clumped heterochromatin is more obvious
- granulocytes progress from having a fine to a highly clumped pattern
- monocytes have a lacy pattern, which becomes finer as the cell matures.
- erythrocytes continue too develop a more clumped pater as maturation progresses, until the extremely dense (pyknotic) nucleus is lost (extruded) from the mature cell.
Describe nuclear shape of nuclear characteristics
- lymphocytes usually continue to have a round or oval nucleus. Some cells may have a small cleft in the nucleus
- monocytes have a kidney bean-shaped nucleus, but folded or horseshoe shapes are common
- mature neutrophils, eosinophils, and basophils have segmented nuclei attached to one another by fine filaments. The number of distinctive lobes ranges from two to five depending on the cell type
Describe presence of nucleoli of nuclear characteristics
- the presence or absence of nucleoli is important in the ID of cells
- the three cell lines of erythrocytes, leukocytes, and megakaryocytes all have nucleoli in the earliest cell stages
- as cells mature, nucleoli are usually not visible. These changes in the appearance of the nucleoli are related to the rate of synthesis of ribosomal RNA
How many nucleoli are found in lymphoblasts?
1-2
How many nucleoli are found in myeloblasts?
1-5
How many nucleoli do monoblasts have?
1-2 but occasionally 3-4
How many nucleoli does erythroblasts have?
0-2, may stain darker than other blast cells
How many nucleoli do megakaryoblasts have?
1-5
What are cytoplasmic characteristics?
- staining color and intensity
- granulation
- shape
- quantity of cytoplasm
- vacuolization
- inclusion bodies
Describe staining color and intensity of cytoplasmic characteristics
- variation among stages of maturation: younger cells tend to be more blue, whereas more mature cells are more pink to neutral colored
Describe granulation of cytoplasmic characteristics
- identify both the presence and granule to identify the cell
Describe shape of cytoplasmic characteristics
- blasts, monocytes, and megakaryocytes are most distinctive
Describe quantity of cytoplasm of cytoplasmic characteristics
- in some cell types, such as megakaryocytes, the quantity of cytoplasm increases with age
Describe vacuolization of cytoplasmic characteristics
- vacuoles can be seen in monocytes at a normal finding
- in other cell types, however, they are a sign of increasing age or abnormal conditions
Describe inclusion bodies of cytoplasmic characteristics
- examples include Auer rods, which can aid in the ID of the cell that it contains it
What is the cell line of a basophil?
- HSC
- myeloid progenitor cell
- myeloblasts
- myelocyte
- basophilic metamyelocyte
- basophilic band cell (released from marrow)
- basophil
- Function: allergic reaction
What is the cell line of a eosinophil?
- HSC
- myeloid progenitor cell
- myeloblast
- myelocyte
- eosinophilic metamyelocyte
- eosinophilic band cell (released from marrow)
- eosinophil
- Function: allergic reactions and anti parasitic reactions
What is the cell line of a neutrophil?
- myeloblast
- Promyelocyte
- myelocyte
- Metamyelocyte
- Band
- Neutrophil
- Function: Phagocytosis and acute inflammation
What is the cell line of megakaryocytes?
- HSC
- myeloid progenitor cell
- megakaryoblast
- promegakaryocyte
- megakaryocyte
- platelets (or thrombocytes)
- Function: coagulation
What is the cell line of erythrocytes?
- Rubriblasts
- Prorubricytes
- Rubricytes
- Metarubricytes
- Reticulocyte
- Mature erythrocyte
What is the cell line of monocyte?
- HSC
- myeloid progenitor cell
- monoblast
- promonocyte
- monocyte
- Function: phagocytosis, and antigen capture
What is the cell line of a plasma cell?
- HSC
- lymphoid progenitor cell
- pre-B cell
- bone marrow
- either stays B cell or differetiates into plasma cell
- both play roles in the humoral immunity
What is the cell line of a T cell?
- HSC
- lymphoid progenitor cell
- pre-T cell
- thymus
- T cell
- plays role in cellular immunity
What are the granular leukocytes?
- eosinophil
- neutrophil
- basophil
What are the agranular leukocytes?
- monocytes
- lymphocytes
Describe segmented neutrophils
- Lobulated nuclear shape average
- very clumped chromatin
- pink cytoplasmic color
- many granules
- pink granules with a few blues ones
- 56%
Describe band from neutrophil
- curved nuclear shape average
- moderately clumped chormatin
- blue/pink cytoplasmic color
- many granules
- pink granules
- 3%
Describe lymphocyte characteristics in the blood
- Round nuclear shape average
- smooth chromatin
- light blue cytoplasmic color
- few or absent granules
- red granules
- 34%
Describe monocyte characteristics
- indented or twisted nuclear shape average
- lacy chromatin
- gray-blue cytoplasmic color
- many granules
- dusty blue granules
- 4%
Describe eosinophil characteristics
- lobulated nuclear shape average
- very clumped chromatin
- granulated cytoplasmic color
- many granules
- orange granules
-2.7%
Describe basophil characteristics
- Lobulated nuclear shape average
- very clumped chromatin
- granulated cytoplasmic color
- many granules
- dark blue granules
- 0.3%
Describe extramedullary hematopoiesis
- occurs when the bone marrow becomes dysfunctional in cases such as aplastic anemia, infiltration by malignant cells, over proliferation of cell OR when the bone marrow is unable to meet the demands places on it, as in the hemolytic anemias
- sites: spleen, liver, and lymph nodes
What are anuclear?
Thrombocytes
Erythrocytes
Describe the complete blood count (CBC)
- foundation procedure performed in hematology laboratory
- consists of quantitative measurements of:
—> hemoglobin
—> hematocrit
—> red and white blood cell counts
—> platelet count
—> leukocyte differential
—> morphological evaluation of a peripheral blood smear
What are some specimen requirements for hematology?
- plasma specimen
- lavender top (EDTA)
-store at room temp or refrigerated
What are reasons to reject a specimen?
- hemolyzed
- clotted specimen
- wrong tube or container
- tube note labeled
- inadequate quantity
- tube mislabeled
What are the 3 phases of laboratory testing?
- pre-analytical - specimen collection, transport, and processing
- analytical - testing
- post analytical - testing results transmission, interpretation, follow-up, retesting
What is phases of testing constitute for 90% of errors?
- pre- analytical
- post analytical
What are some pre-analytic errors?
- patient ID
- phlebotomist technique
- test collection procedures
- specimen transport
- specimen processing
What are some analytic errors?
- test system not calibrated
- results reported when control results are out of range
- improper measurements of specimens and/or reagents
- reagents prepared incorrectly
- reagents stored inappropriately or used beyond expiration date
- instrument maintenance not done
- dilution and pipetting errors
- inaccuracy
- imprecision errors
- insensitivity
- linearity issues
What are some post analytic errors?
- interpretation of results
- critical value not called
- transcription errors in reporting
- report sent to the wrong location
- report illegible
- report not sent
Describe overall quantitative measurements
- erythrocytes, leukocytes, and platelets are measured quantitatively using automated instrumentation. Usually automated but occasionally manual counts may need to be performed due to low counts or quality issues
What can manual counts be used for?
- quality control strategy
- backup method of analysis
Describe RBC indices
- standard part of the CBC, specifically MCV
- used to mathematically define cell size and the concentration of hemoglobin within the cell. They are MCV, MCH, and MCHC
- can also include:
—> reticulocyte information
—> Red cell distribution width (RDW)
—> Blood cell histograms
What does MCV stand for?
Mean corpuscular volume
What does MCH stand for?
Mean corpuscular hemoglobin
What does MCHC stand for?
Mean corpuscular hemoglobin concentration
Briefly describe manual cell counting
- Blood specimens are diluted to an exact ratio with specific diluent
- cells counted in hemacytometer
What is hemacytometer?
Accurately ruled chamber, ruled off in areas of square millimeters
Describe hemoglobin
- an iron-containing pigment of RBCs composed of four amino acid chains (alpha, beta, gamma, and delta) that delivers oxygen from the lungs to the tissues of the body
- Normal values:
—> female = 12-16 g/dL
—> male = 14-18 g/dL
Describe hematocrit
- the percentage of RBCs in whole blood
- also called the PCV (packed cell volume)
- normal values:
—> female: 36-45%
—> male: 42-50%
Describe hemoglobin measurement (HB)
- manual and automated determination is most commonly performed using the cyanmethemoglobin method
Describe cyanmethemoglobin method
- modified Drabkin’s reagent that contains potassium cyanide is mixed with blood specimen, incubate to induce lysing, and measured using a spectrophotometer at 540 nm
- also called hemiglobincyanide method
What are sources of errors in the HB measurement result from?
Problems with specimen integrity
- elevations in HB measurement are seen in lipemic, icteric, or hemolyzed blood samples
- elevations in HB measurement are also seen in specimens with high WBC counts or RBCs containing HB S or C
What are some strategies used to fix errors in Hb measurement?
- saline (plasma) replacement for icteric, lipemic or hemolyzed plasma
- specimen can be centrifuged and clear supernatant be transferred to a cuvette and measured on a spectrophotometer to address high WBC count interference.
- specimens with Hb C or S can be diluted 1:2 with distilled water and run with the result multiplied by the dilution factor
Describe the traditional cyanmethemoglobin method
- usually used in automated methods
- sample is lysed using the detergent-modified Drabkin’s reagent and light absorbance is measured at 540 nm
Describe the hematocrit (packed cell volume) procedure
- the hematocrit (Hct) or packed cell volume is a macroscopic observation of volume of the packed RBCs in a sample of whole blood, if measured manually
- The Hct is the percentage of RBCs in a volume of whole blood. It is expressed as units of percent or as a ration in the SI system
- an automated hematocrit result is obtained when multi-parameter instruments are used
—> the results are computed from individual MCVs and the red cell count and is not affected by the trapped plasma that is left in the RBC column.
What occurs when whole blood is centrifuged?
- the heavier particles fall to the bottom of the tube, and the lighter particles settle on top of the heavier cells
What are sources of errors of manual Hcts?
Include specimen and technical errors
- specimen errors can falsely decrease Hct values due to inadequate filling of EDTA tube, which causes RBC shrinkage
- technical errors include overcentrifugation or improper sealing of the test capillary tube
- patients with clinical RBC disorders such as macrocyclic or sickle cell anemia produce falsely elevated Hct levels
What is hematocrit used for?
Evaluating and classifying the various types of anemias according to red cell indices
Describe Rule of Three
- used to determine accuracy between Hb, Hct, and RBC counts
- the states:
—> the microhematocrit be 3x the value of the hemoglobin (+/- 3%)
—> the hemoglobin should be 3x the value of the RBC count
—> the microhematocrit should be 9x the value of the RBC count - the rule only applies to normocytic normochromic red cells, and discrepant values may be seen in other conditions
Describe blood volume measurement
- in most cases, the total number of RBCs is closely related to the red cell concentration, but in situations such as severe hemorrhage dehydration, or overhydration, plasma volume or red cell mass or volume must be determined
- is injected and the volume distribution is calculated from the degree of dilution of the injected substance over a period of 15-30 minutes
- radiolabeled albumin is most commonly used
- may also be estimated from red cell volume using radioactive labeled RBCs
How is plasma volume measured?
- by dilution methods
Describe Red cell distribution width
- quantitative cellular volume heterogeneity reflecting the range of RBC sizes within a sample
- useful in early classification of anemia as it first starts to become abnormal earlier in nutritional deficiencies such as iron deficiency anemia
- useful in characterizing micro types anemias, allowing to distinguish between iron deficiency anemia and beta thalassemia
- RDW can also be helpful in identifying RBC fragmentation
Briefly describe manual erythrocyte, leukocyte, and platelet counts
- blood specimens are diluted to an exact ratio with specific diluents and the cells are counted in hemacytometer and accurately ruled chamber in areas of square millimeters
- since RBC counts surpass WBC and PLT counts, they need to be eliminated to count WBC and PLT counts accurately
- most of the lysing agents use the principle of osmotic pressure; placing an aliquot of blood in a hypotonic solution will cause the cell to lyse
Describe Manual erythrocyte count
- can be very inaccurate
- in analysis of RBCs an isotonic solution is used as diluent from whole blood.
- because RBCs greatly exceed WBCs, the error introduced by counting red cells and white cells negligible
What is the calculation for a manual RBC cell count?
(average total of RBCs in 5 squares) X (dilution correction factor) X (volume correction factor) = # of RBCs
- the specimen dilution factor is 200
- the volume correction factor is 50
- 5 of 25 squares in the large 1-mm square are counted
Example: if the average # of RBCs counted is 400, what is the total RBC count?
400 X 200 X 50 (or 400 X 1000) = 4.0 x 10^12/L
What are sources of errors in a manual RBC count?
- increased or erratic results may be seen if contaminated diluting fluid, wet or dry pipette, a dirty hemacytometer, or drying of the dilation in the hematocytometer occurs
Describe the manual RBC cell count diluent. What is the microscope objective power used?`
- dilution = 1:100
- diluent = 0.85% NaCl
- Objective = 40x
Describe the manual WBC cell count diluent. What is the microscope objective power?
- dilution = 1:20
- diluent = 3% acetic acid, 1% ammonium oxalate, 1% hydrochloric acid
- objective = 10x
Describe the manual platelet count diluent. What is the microscope objective power?
- dilution = 1:100
- diluent = 1% ammonium oxalate
- objective = 40x
What are the reference values for packed cell volume?
- Men: 41.5–50.4% (0.415–0.504L/L)
- Women: 36–45% (0.36–0.45 L/L)
- at birth: 51%
- age 6-12: 40%
What are the reference values for erythrocyte count?
- Men: 4.5–5.9 x 10^6/uL (4.5–5.9 x 10^12/L)
- Women: 4.5–5.1 x 10^6/uL (4.5–5.1 x 10^12/L)
- at birth: 4.7 x 10^12/L
- age 6-12: 4.6 x 10^12/L
What are the reference values for hemoglobin concentration?
- Men: 14.0–18.0 g/dL (145–180 g/L)
- Women: 12.0–16.0 g/dL (120-160 g/L)
- at birth: 16.5 g/dL
- age 6-12: 13.5 g/dL
What are the reference values for Normal MCV?
- Men: 80–96 (fL)
- Women: 80–96 (fL)
- at birth: 180 fL
- age 6-12: 86 fL
What are the reference values for Normal MCH?
- Men: 27.5–33.2 (pg)
- Women: 27.5–33.2 (pg)
- at birth: 34 pg
- age 6-12: 29 pg
What are the reference values for Normal MCHC?
- Men: 33.4–35.6% (32–36 g/dL)
- Women: 33.4–35.6% (32–36 8/dL)
Describe MCV
- expresses the average size or volume of an erythrocyte
- useful parameter for classification of anemias based on size
- terms associated with MCV evaluation are normocytic, microcytic, and macrocytic
What are elevated MCV values associated with?
- macrocytic Anemia
- pathological megablastic anemias (such as pernicious anemia and folic acid deficiencies
What are low levels of MCV associated with?
- iron deficiency anemia
- heterozygous thalassemias
- anemias of chronic inflammation/anemia of chronic disorders
What are sources of errors for MCV?
- autoagglutination of red cells can cause falsely elevated MCV values
- red clumps are not counted in automated methods which will affect the Hct
- severe hyperglycemia can cause osmotic swelling of red cells which leads to falsely elevated levels
- leukocytes is may also falsely elevate MCV values
What is the MCV formula?
(Patients packed cell volume or Hct (L/L))/Erythrocyte count (x10^12/L))
- example:
—> patient Hct = 35% (0.35 L/L)
—> erythrocyte count = 4.0 x 10^12/L
(0.35 L/L)/(4.0 x 10^12/L) = 8.75 x 10^-15 = 87.5 fL
*one femtoliter (fL) = 10^-15 L = 1 cubic micrometer (um^3)
Describe MCH
- expresses average weight (content) of hemoglobin per red cell. It is directly proportional to the amount of hemoglobin and the size of the erythrocyte
What are clinical conditions of MCH?
- in anemia secondary to impaired hemoglobin synthesis, the hemoglobin mass per red cell decreases, resulting in a lower MCH value
What are sources of errors for MCH?
- may be falsely elevated by hyperlipidemia As increased plasma turbidity will erroneously elevate hemoglobin measurement
Describe MCHC
- expresses the average concentration (weight) of hemoglobin per unit volume of erythrocytes in the sample
- also defined as the ratio of the weight of hemoglobin to the volume of erythrocytes
-reference values: 33-36% (g/dL) - terms associated with MCHC evaluation are normochromic, hypochromic and hyperchromic (which can only be spherocytosis or a problem with the Hgb or Hct values)
What is the formula of MCH?
(Hemoglobin (x10g/dL))/(erythrocyte count (x10^12/L)) = pg
- example
—> hemoglobin = 14 g/dL
—> erythrocyte count = 4 x 10^12/L
(140 g/dL)/(4 x 10^12/L) = 35 x 10^-12 g = 35 pg
- one picogram (pg) = 10^-12 g = 1 micromicrogram (uum)
What is the formula for MCHC?
(Hemoglobin (g/dL))/(Hct (L/L)) x 100= g/dL
- example
—> hemoglobin = 14 g/dL
—>Hct = 45% (0.45 L/L)
(14 g/dL)/(0.45 L/L) = 31 g/dL
What are clinical conditions of MCHC?
- 50% patients with hereditary spherocytosis demonstrate an elevated MCHC
- this make spherocytosis most common condition associated with MCHC
- other conditions can elevate MCHC such as lipemia, active cold agglutinin disease
What are sources of errors for MCHC?
- accuracy is affected by factors that affect measurement of either Hct, such as plasma trapping, or presence of abnormal red cells or hemoglobin such as hyperlipidemia or leukocytosis
- autoagglutination at room temperature will falsely elevate MCV and decrease RBC count which results elevated MCHC
- falsely elevate hemoglobin concentration
- hemolysis
What is the reference range for WBC?
4.5–11.0 x 109/L
What is leukocytosis?
- Values above the reference range
- can be seen in acute bacterial infections, inflammation, malaria, hemorrhage, pregnancy, some anemias, and in some carcinomas, including leukemia
What is leukopenia?
Values below the reference range
What are methods used to count WBC?
- automated: ACT diff 2
- manual:
—> Neubauer hemocytometer
* 3mm x 3mm x 0.1mm = 0.9mm^3
—> Fochs-Rosenthal hemocytometer
* 4mm x 4mm x 0.2 mm = 3.2 mm^3
What reagents are used for a manual WBC count?
- LeukoCount Solution
—> Acetic buffer
—> Gentian violet
Describe specimen used in manual WBC count
- EDTA whole blood
- 1:20 dilution for testing
Describe how to read a hemocytometer
- read on 10x
- read 4 sections (in 4 corners)
- count from both sides should be no more than 10 cells
How do you total volume counted for WBC?
- each “section” has an area of 1 mm^2 and a depth of 0.1mm
- volume of 1 “section” : area x depth
—> 1 mm^2 x 0.1 mm = 0.1 mm^3 - count 4 “sections for total volume counted of 4 x 0.1 mm^3 = 0.4 mm^3
How is the correction factor found?
(Volume desired)/(volume used)
(1.0 mm^3)/(0.4 mm^3) = 2.5
- volume desired is always 1 mm^3
- if different # of squares were counted, the correction factor would change.
- example: 5 squares counted
(1 mm^3)/(0.5 mm^3) = 2.0
What is the formula used for manual WBC counts?
Correction factor (CF) x dilution factor (DF) x # of cells counted = WBC count
-example: 90 cells counted
20 x 2.5 x 90 (or 50 x 90) = 4.5 x 10^3/mm^3
- report out —> WBC count: 4.5 x 10^3/mm^3
- 50 is the multiplication factor (CF x DF)
What are sources of errors in manual WBC counts?
- failure to mix blood properly
- dilution error
- failure to wipe off pipette
- failure to mix
- failure to discard first drop of dilution
- improper charging or chamber
- dirty hemocytometer
What are some important things to know about manual WBC count?
- all numbers MUST have units
- count both sides of hemocytometer and average out counts
- count cells ON the left and top lines but not on the bottom and right lines.
When may leukopenia occur?
- certain viral infections with typhoid fever and malaria
- after radiation therapy
- classic pernicious anemias
- after chemotherapy treatments
When may leukocytosis occur?
- many acute infections, especially bacterial infections
- severe malaria
- after hemorrhage
- during pregnancy
- postoperatively
- some forms of anemias
- in some carcinomas
- leukemia
Why would leekocytopenia occur?
- sepsis
- immunosuppressive agents
What tests are performed for the diagnosis for inflammation?
- absolute cell counts
- assessment of eosinophils and basophils
- neutrophilic hypersegmentation index
- neutrophilic function
- leukocyte alkaline phosphatase test
- erythrocyte sedimentation rate (ESR)
Describe absolute cell count
- the absolute number of segmented neutrophils and bands
- less specific for inflammation
Describe assessment of eosinophils and basophils
- examination of a peripheral blood smear normally demonstrates an average of approximately 4% eosinophils
- procedure required only if an extreme increase in eosinophils is demonstrated on a peripheral blood smear
What is the formula for absolute cell counts?
Total leukocyte count x percentage of cell type = absolute cell value = absolute count
Example:
—> total leukocyte count: 15.0 x 10^9/L
—> bands 12%, segmented neutrophils 80% and lymphocytes 8%
15.0 x 10^9/L x 0.80 = 12.0 x 10^9/L segmented neutrophils
* this formula can be used to determine the absolute value of any cell appearing on a leukocyte differential blood smear
Describe neutrophilic hyper segmentation index
- counting the number of lobes can be performed to determine the neutrophilic hypersegmentation index (NHI)
- a right shift or increase in the number of lobes to five or more occurs in various conditions, being chronic nephritis, pernicious anemia and folic acid diagnosis
What are the three methods for calculating NHI?
1) lobe average: determined by counting the number of lobes in a number of neutrophils. Reference value is 2.5–3.3
2) percentage of neutrophils with five or more lobes. Count the number of lobes in randomly selected segmented neutrophils. Add up the total number of lobes for each segmented neutrophil counted, and divide by the total number of cells counted. Reference range is greater than 3%
3) Hypersegmentation index.
(# of neutrophils with 5 or more lobes x 100)/(# of neutrophils with 4 lobes)
—> values greater than 16.9 are considered to indicate hypersegmentation
—> most sensitive method
Describe erythrocyte sedimentation rate (ESR)
- established parameter of inflammation in the modern clinical laboratory
- standard method of choice: Westergren method
- nonspecific indicator of disease
- most commonly performed lab tests
What are the reference ranges for ESR?
- Male, under age 50 = 10 mm/hour
- Female, under age 50 = 13 mm/hour
- Male, age 50+ = 13 mm/hour
- Female, age 50+ = 20 mm/hour
What could lower ESR?
Erythrocytes with abnormal or irregular shape, such as sickle cells or spherocytes, hinder rouleaux formation
- removal or fibrinogen by defibrination
What could increase ESR?
- rouleaux
- increased fibrinogen levels
- relative increase of plasma globulins caused by loss of plasma albumin
- absolute increase of plasma globulins
What are clinical conditions associated with ESR?
- increased ESR levels
—> anemia
—> infections
—> inflammation
—> tissue necrosis (myocardial infarction)
—> pregnancy
—> some types of hemolytic anemia
What are the reference values for RBC counts?
- Male: 4.5–5.9 x 10^6/mm^3 (or uL)
- Female: 4.5–.5.1 x 10^6/mm^3 (or uL)
- infant: 9.0–30.0 x 10^6/mm3 (or uL)
What are 5 factors that affect on RBC count?
1) extreme physcial exercise - increases count
2) severe dehydration - increase count
3) Age
—> newborn has increased counts
—> drops rapidly, reaches lowest point at 2-4 months
—> gradually rises to adult levels by 14 years of age
—> gradually drops in old age
4) sex
5) altitude
—> increased altitude causes increased RBC counts
—> hypoxia (lack of O2 cause increased RBC counts
What is erythrocytosis?
- Elevated RBC counts
- three types:
—> polycythemia Vera
—> secondary polycythemia or absolute
—> relative polycythemia
What is polycythemia Vera?
-type of erythrocytosis
- is a pathological increase in RBCs and other blood cells
What is secondary polycythemia or absolute?
- increase in erythropoietin production
- can occur in:
—> hypoxia
—> certain types of anemia
—> chronic lung disease
—> inappropriate erythropoietin production
—> smokers
Describe relative polycythemia
- caused by decrease in plasma volume
- total RBC mass is not increased it just looks like it due to the loss of plasma volume, dehydration
- replace fluid, RBC count is back to normal
What is oligocythemia?
Deficiency in the total number of RBCs present in the body
- occurs with anemia and leukemia
What methods are used for RBC counts?
- automated
—> ACT diff 2 - manual
—> Neubauer hemocytometer
* 3 mm x 3 mm x 0.1 mm = 0.9 mm^3
What is the diluting fluid for RBC count?
- Hayem’s (HgCl3 + NaCl + NaSO4 + H20)
Describe how to find the counting factors of RBC counts
- dilution for manual RBC counting is 1:200
- count 5 “R” sections of the 25 sections in the middle portion of the hemocytometer
- 1/25 or 0.04 mm^2
- 0.04 mm^2 0.1 mm (depth) = 0.004 mm^3 (this is the volume)
What is the formula for RBC count?
Dilution factor x volume correction factor x cells counted
200 x 50 x # of cells counted = RBC count
Example: 400 RBC counted
200 x 50 x 400 (or 10,000 x 400) = 4,000,000
What is rouleaux?
Overlapping RBCs, often referred to as a “stack of coins” caused by abnormal or increase plasma proteins
What is erythrocyte morphology?
- Can scale the severity of the erythrocyte abnormalities
- semiquantitative
What is anisocytosis?
Variation in normal RBC size
What is poikilocytosis?
Variation in normal RBC shape
How is erythrocyte morphology graded?
0 = normal appearance or slight variation
1+ = only a small population displays a particular abnormality. Terms: “slightly increased” or “few”
2+ = more than occasional numbers of abnormal erythrocytes can be seen in microscope. Terms: “moderately increased”
3+ = severe increase in abnormal erythrocytes in each microscopic field. Terms: “many”
4+ = most severe state of erythrocytic abnormality, with the abnormality prevalent through microscope. Terms: “marked” or “increased marked”
Describe semiquantitative assessment of leukocytes
- semi quantitative estimate of WBCs to qualitative measurements of WBC is good quality control step
Describe total WBC estimate
1) select appropriate area of stained blood smear at 40x magnification
2) count the # of WBCs in each of the ten fields
3) calculate estimate total concentration of WBCs per uL (average # of WBC) x (2,000)
4) compare estimate with the quantitatively determined automated or manual WBC count
Example: 8 WBCs per field
8 x 2,000 = 16,000/uL (or mm^3) or 16.0 x 10^9/L
Describe peripheral blood film evaluation
- microscopic examination of PB smear
- perform a differential on a blood smear from PB
—> wright stain is used to stain smear
—> Normally see only more mature forms
—> performed systematically using the maturational features
Describe the PROCEDURE for peripheral blood film evaluation
1) start with low power (10x)
—> evaluate blood smear quality, color, distribution of cells
—> inspection for rouleaux or agglutination can be observed
—> large immature abnormal cells or disintegrating lymphocytes and smudge cells can be seen
—> platelet clumping or satellism may also be seen.
2) Move to High dry power (40x)
—>WBC estimates can be assessed
—> oil immersion lens (100x)
—> leukocytes differential can be performed here
—> RBC morphology can also be graded and evaluated here
What is the differential count procedure?
1) begin with a correctly prepared and stained smear
2) focus on 10x objective, scan the smear to check for cell distribution, clumping and abnormal cells
3) move to 40x and using the fine adjustment, focus then.
—> add a drop of immersion oil and switch to 100x
—>find suitable are and begin counting the manual leukocyte differential
4) a total of at least 100 leukocytes should be counted. Express the results as a percentage of total leukocytes counted
5) abnormalities of leukocytes, erythrocytes, and platelets should be noted.
What specimens can be used for a differential count?
- peripheral blood
- bone marrow
- body fluid sediments (spinal fluid)
*smears should be made within 1 hour of blood collection from EDTA specimens to avoid distortion of cell morphology
What are some characteristics of segmented neutrophils in PB?
- lobulated nucleus
- very clumped chromatin
- pink cytoplasm
- many granules
- pink granules with few blue
- average percent: 56%
What are some characteristics of band neutrophils in PB?
- curved nucleus
- moderately clumped chromatin
- blue/pink cytoplasmic
- many granules
- pink granules
- average percentage: 3%
What are some characteristics of lymphocytes in PB?
- round nucleus
- smooth chromatin
- light blue cytoplasm
- few or absent granules
- red granules
- average percentage: 34%
What are some characteristics of monocytes in PB?
- indented or twisted nucleus
- lacy chromatin
- gray-blue cyroplasm
- many granules
- busty blue granules
- average percentage: 4%
What are some characteristics of eosinophils in PB?
- lobulated nucleus
- very clumped chromatin
- granulated cytoplasm
- many granules
- orange granules
- average percentage: 2.7%
What are some characteristics of basophils in PB?
- lobulated nucleus
- very clumped chromatin
- granulated cytoplasm
- many granules
- dark blue granules
- average percentage: 0.3%
What are sources of error in differential counts?
- smears not made within 1 hour of extraction with EDTA blood specimen
- changes due to technical error: alterations of the cellular nucleus, degranulation or cytoplasmic vacuoles
Describe corrections for Nucleated Red Blood Cells (NRBC)
- similar size of lymphocytes
- automated analyzer WBC counts can be falsely elevated due to NRBC’s in the sample
- When completing a differential if there are more than 10 NRBC/100 WBC’s, a correction of total WBC count must be done
What is the formula for correction for NRBCs?
(Uncorrected WBC count x 100)/(100 + # NRBC per 100 WBC differential count)
Describe shift to the left
- when the percentage of band forms and other immature neutrophils like metamyelocyte and myelocyte increases, the condition is sometimes referred to as a left shift
- could be an indicator of infection or malignancy
- some authorities advocate doing away with the identification of band forms on the differential because of individual variability in cell identification and limited usefulness
What is microcytic?
When MCV range is less than 80 fL
What is normocytic?
When MCV range is normal: 80-96 fL
What is macrocytic?
When the range is over 96 fL
What is hypochromic?
When MCHC range falls below 33%
What is normochromic?
when MCHC is at normal range: 33-36%
What is hyperchromic?
When MCHC exceeds 36%
Describe Coulter Principle
- a method of counting and measuring (the size) of particles (or biological cells) suspended in a fluid
- reference method throughout the world for particle size analysis
- method based on measuring changes in electrical resistance when a particle (such as a cell) is suspended in an electrolyte solution
- several thousand particles per second are individually counted and sized with great accuracy
- this method is independent of particle shape, color and density
Describe the PROCEDURE of Coulter principle
1) small opening (aperture) between electrodes is the sensing zone through which suspended particles pass. In the sensing zone each particle displaces its own volume of electrolyte
2) as each cell goes through the aperture, it impedes the current and causes a measurable pulse
3) the number of pulses signals the number of particles
4) the height of each pulse is proportional to the volume of that particle
5) the quantity of suspension drawn through the aperture is precisely for an exact reproducible volume
What are the two classic methods of counting cellular elements of blood?
1) electrical impedance
2) optical detection
Describe electrical impedance
- referred to as the coulter principle
- cell counting and sizing are based on the detection and measurement of changes in electrical impendance (resistance) produced by a particle as it passes through a small aperture
- the number of pulses generated during a specific period is proportional to the number of particles or cells, recorded by a scattergram
- the amplitude (magnitude) of the electrical pulse produced indicates the cells volume
- the output histogram is a display of the distribution of cell volume and frequency
Describe optical detection principle
- in the optical or hydrodynamic focusing method of cell count in and cell sizing, laser light is used.
- a diluted blood specimen passes in a steady stream through which a beam of laser light is focused
- as each cell passes through the sensing zone of the flow cell, it scatters the focused light
- scattered light is detected by a photodetector and converted to an electrical pulse
- the number of pulses generated is directly proportional to the number of cells passing through the sensing zone in a specific period
What information does scattered light provide?
- cell structure
- shape
- reflectivity
Describe Optical light scatter
- light amplification is generated by stimulated emission of radiation. Three independent processes are operational. These are as follows:
1) diffraction and the bending of light around corners with the use of small angles
2) refraction and the bending of light beach of a change in speed with the use of intermediate angles
3) reflection and light rays ruined back by the surface or an obstruction with the use of large angles
Describe Angles of light scatter
- various angles of light scatter can aid in cellular analysis. These are as follows:
1) forward light scatter 0(degrees) —> this is diffracted light, which relates to the volume of the cell
2) forward low-angle light scatter 2-3(degrees) —> can relate to size or volume
3) forward high angle light scatter 5-15(degrees) —> allows or description of the refractive index of cellular components
4) orthogonal light scatter 90(degrees) —> based on reflection and refraction of internal components, which correlates with internal complexity
Describe Radiofrequency (RF)
- in this newer application, high-voltage electromagnetic current is used to detect cell size, based on the cellular density
- The RF pulse is directly proportional to the the nuclear size and density of a cell. RF or conductivity is related to the nuclear: cytoplasmic ratio, nuclear density and cytoplasmic granulation
Describe the Fundamentals of Laser technology
- the electromagnetic spectrum ranges from long radio waves to short, powerful gamma rays
- within this spectrum is a narrow band or visible or white light, which is composed of red, orange, yellow, green, blue and violet light
- light amplified by stimulated emission of radiation (laser) light ranges from the ultraviolet and infrared spectrum through all the colors of the rainbow
Describe Lasers
L —> Light
A —> amplified (by)
S —> stimulated
E —> emission (of)
R —> radiation
- its emission ranges from ultraviolet to infrared through all the color of the rainbow
-in contrast to other diffuse forms of radiation, laser light is:
—> concentrated
—> almost exclusively of one wavelengths or color
—> its parallel waves travel in one direction
What are some types of lasers?
- glass-filled tubes of helium and neon lasers
- the yttrium
- aluminum
- garnet (YAG) type
- imitation diamond
- argon
- krypton
What occurs when an atom extends beyond the orbits of its electrons or when a molecule vibrates or changes shape?
— they instantly snap back and shed energy
The shorter the wavelength of an electromagnetic disturbance, the _____ energy each photon contains
More
Describe fluorophores
- shorter wavelength light is used as the excitation light for fluorophores
- shorter wavelength light absorbed by an electron of the fluorophores and as a result, this higher energy photon excites the fluorophores
- excitation doesn’t last long because fluorophores and as a result, this higher energy photon excites the fluorophore
- excitation doesn’t last long because fluorophores natural state is ground state
Describe photons
- energy packets of an emitted visible-light particle
- basic unit of all radiation
- when photons reaches an atom of the medium, the energy exchange stimulates the emission of another photon in the same wavelength and direction. This process continues until a cascade of growing energy sweeps through the medium
- travel the length of the laser and bounce off mirrors. First, a few and eventually countless photons synchronize themselves, until an avalanche of light streaks between the mirrors
What is the unit of a wavelength?
Nanometer (nm)
What are stroke shifts?
The difference, in nm, between the peak excitation and the peak emission wavelength
What is the power of lasers to pass on energy and information is measure in?
Watts
Describe principle of flow cytometry
- laser light is the most common light source used in flow cytoemeters because of the properties of intensity, stability, and monochromatin
- flow cytometry is defined as the simutaneous measurement of multiple physical characteristics of a single cell, as the cell flows in suspension through a measuring device
- flow cytometry combines that rapidly classify groups of cells with heterogenous mixtures
- principle of flow cytometry is based on the fact that cells are stained in suspension. Flow cytometry has specifically come to denote the use of fluoroscencee measurement, usually with a laser light source
- the cellular light scatter patterns can be used to identify cells
- both intrinsic and extrinsic properties of cells can be analyzed by flow cytometry
What are intrinsic properties of flow cytometry?
- forward- and right-angle light scatter, which correlate with size and granularity of a cell, respectively. This data output does not require addition of cues or stains for detection
What are extrinsic properties of flow cytometry?
- rely binding of various probes to the cell. The scattered light passes through a variety of filters and lenses and it then measured by photomultiplier tubes, which convert the light signals into electronic signals for computer analysis
What are some characteristics of flow cytometry?
- light scattered along the axis of the laser beam is “forward scattering”
- light scattered perpendicular to the axis is side scatter” or orthogonal scatter
- forward scatter is roughly proportional to cell size; side scatter is roughly proportional to cytoplasmic granularity
- granulocyte have a much larger side-scattered light signal that do lymphocytes
- data are plotted on histograms
- populations of similar cells from secrete and characteristics two-dimensional “clusters” of scatter when the forward and side scatters are plotted against each other
What information does the interactions between each cell and the laser beam provide?
- the amount of light scattered by each cell hit by the laser beam
- the intensity of the fluorescence emitted by labeled antibodies bound to antigen on the different types of suspended cells
Describe WBC analysis
- degree of instrument sophistication is frequently described by a the number of parameters that the instrument generates
What is a parameter?
- any numerical value that describes an entire population
What is a sample?
A subset of a population
What is a statistic?
Any numerical value describing a sample
What are recent innovations of hematology instumentation?
- quantitation of nRBC counts
- a channel for enumeration of immature granulocytes (IGs)
- Random access CD4 lymphocyte counting
- analysis of CD34, CD38, and CD61 cell markers
- measurement of reticulocyte hemoglobin
- enumeration of hematopoietic progenitor cells (HPCs)
- counting of IGs
Describe histograms
- are graphic representation of cell frequencies versus sizes
- in a homogenous cell population, the curve assumes a symmetrical bell-shaped or Gaussian distribution. A wide or more flattened curve is seen when the standard deviation from the mean is increased
- not only do they provide information about erythrocyte, leukocyte, and platelet frequency and their distribution about the mean but also depict the presence of subpopulations
- provide a means of comparing he size of a patients cells with normal populations. Shifts in one direction or the other can be of diagnostic importance. The position of the curve on the x-axis reflects cell size. In the coulter system. The size (volume in femtoliters) is represented on the x-axis
What are methods for analysis of instrumental data output?
- the erythrocyte histogram
- quantitative descriptors of erythrocytes
- the leukocyte histogram
- platelet histograms
- derived platelet parameters
What are the 8 parameters that entry-level hematology measure?
- WBC
- RBC
- hemoglobin
- Hct
- MCV
- MCH
- MCHC
- platelets
What is a delta check?
- another quality control method for comparing a patients own leukocytes, hemoglobin, MCV, and platelet values with previous results
Describe erythrocyte histogram
- reflects the native size of erythrocytes or any other particles in the erythrocyte size range
- In the Coulter system, cells are displayed as small as 24 fL, but only those greater than 36 fL are counted as erythrocytes
- the extension of the lower end of the scale from 36 to 24 fL allows for the detection of erythrocyte fragments, leukocyte fragments, and large platelets
What will the erythrocyte histogram look like if the cells are larger than normal? What about if they were smaller than normal?
- larger: curve to the right (megaloblastic anemias)
- smaller: curve to the left (iron deficiency anemias)
Describe quantitative descriptors of erythrocytes
- an expression of erythrocyte size is the RDW in the Coulter series. This term refers to variation in erythrocyte size
- correlations between the RDW and the MCV exist for various types of anemia
Describe Red cell distribution width
- a parameter that expresses the coefficient of variation of the erythrocyte volume distribution.
RDW = (SD)/mean size x 100
Reference range: 11.5-14.5%
Describe the relationship between MCV and RDW when the MCV is high?
High RDW: causes megaloblastic anemia
Normal RDW: Aplastic anemia in adults
Describe the relationship between MCV and RDW when the MCV levels are normal?
- RDW high: normocytic anemias
- RDW normal: reticulocytosis
Describe the relationship MCV and RDW when MCV levels are low
High RDW: iron deficiency anemia
Normal RDW: heterozygous thalassemias, anemias of chronic inflammation or disorders
Describe the leukocyte histograms
- size-referenced leukocyte histograms display the classification of leukocyte according to size following lysis. It does not display the native cell size
- the histogram of leukocyte subpopulations reflects the sorting of these cells by their relative size, which is primarily related to their nuclear size
- as the leukocyte passes through the aperture in the electrical impedance system, they displace their volume in a conductive fluid, which causes a change in electrical resistance as each cell passes through the aperture
- only those greater than 35 fL are counted as leukocytes
What are the three different cells that are identified on an electrical impedance leukocyte histogram?
- lymphocytes —> left side
- mononuclear cells —> middle
—> ex. Blasts and other immature cells such as promyelocytes, myelocytes, and monocytes - granulocytes —> right side
When should a valley or depression be seen on a leukocyte histogram?
- between the lymphocytes and mononuclear cells
- between mononuclear cells and granulocytes
Describe platelet histograms
- platelet counting and sizing in both the electrical impedance and the optical systems reflect the native cell size
- in the electrical impedance method, counting and sizing take place in the RBC aperture
- in the optical system, forward light scatter pattern discrimination between erythrocytes and platelets in the flow cell determines the platelet count and frequency distribution
What is the expected cell coincidence error?
More than one cell passing through the aperture at the same time
Describe derived platelet parameters
- coulter model system yield the additional parameters or MPV and PDW
- derived from platelet histograms
- allow for a size comparison between a patients specimen and the normal populations. Size comparisons are useful as an indicator of certian disorders
Describe Mean platelet volume calculation (MPV)
- a measure of the average volume of platelets in a sample. The MPV is analogous to the erythrocytic MCV
- it is derived from the same data as the platelet count
- in healthy patients, there is an inverse relationship between platelet count and size. For example, the volume increases as the platelet count decreases
What MPV Nomogram used to determine?
Whether a patients MPV is normal.
What are disorders of MPV?
- decreased:
—> aplastic anemia
—> megaloblastic anemia
—> result of chemotherapy
—> hypersplenism - increased:
—> thrombocytopenia
—> myeloproliferative disorders
—> heterozygous thalassemia
Describe platelet distribution width (PDW)
Measure of the uniformity of platelet size in a blood specimen
- serves as a validity check and monitors false results
- normal less than 20%
When will PDW be increased?
- aplastic anemias
- megaloblastic anemias
- in chronic myelogenous leukemia
- anti-leukemic chemotherapy
Describe laser technology
- principle of flow cytometry relies n three distinct steps:
1) cytochemical reactions prepare the blood cells for analysis
2) a cytometer measures specific cell properties
3) algorithms convert these measurements in familiar results for cell classification, cell count, cell size and hemoglobinization - the instruments sampling mechanism divides blood samples into aliquots that are treated in four separate reactions chambers
What are the 4 reaction chambers of laser technology ?
1) hemoglobin
2) RBCs/platelet
3) peroxidase
4) basophil/lobularity
Describe RBCs/platelets laser technology
- the RBC/platelet channel uses a laser-based optical assembly that is shared with the basophil/lobularity channel
- the light scattered at low and high angles simutaneously measures RBC volume (size) and optical density (hemoglobin concentration) of each cell
- additional parameters obtained from the histogram are cell
- additional parameters obtained from the histogram are the MCV and RDW
- the platelet histogram is derived from measuresurments made with the high-angle detector
What are the applications of flow cytometry?
- general properties of flow cytometry
- hemtaological applicatons
- clinical applicatons of flow cytometry
- other cellular applications
Describe be General properties of flow cytometry
- major advances this technology are owing to several factors:
1)the ability to produce monoclonal antibodies resulted in the subsequent development of specific surface markers for various subpopulations of cells
2) the development of new fluorescent probes for DNA, RNA, and other cellular components increased the variety of possible applications at the molecular and cellular levels
3) the expansion of computer applications has improved the instrumentation technology, making it easier to operate and more practical for use in clinical as well as research laboratories
What are the hematological applications?
1) automated differentials can be based on a variety of principles
2) these include determination of cell volume by electrical impedance or forward light scatter, cytochemistry, or peroxidase staining, and VCS technology
3) flow cytometry can be applied practically to several techniques in the clinical hematology laboratory
Describe the peroxidase application for laser technology
- tungsten light- based optics channel, RBCs are lysed and WBCs are fixed and then stained
- thousands of cells are characterized by a combination of their size (scatter) and peroxidase activity (absorbance)
- scatter is plotted on the y-axis and absorbance is plotted on the x-axis
- used to generate the total WBC count and differential count, except for basophils, MPXI
Describe Mean Peroxidase index (MPXI)
- the index of the mean peroxidase activity of neutrophils as measured by their stain intensity
What can increased myeloperoxidase activity be associated with?
- megaloblastic anemia
- hyperproliferative granulopoiesis, or reactive states
Describe the basophil/lobularity (Nuclear) channel application of laser technology
- the nuclear channel is used to measure the conformation of the nucleus of WBCs.
- principle is when WBCs are exposed to a surfactant at a low pH, the membranes and cytoplasm of specific leukocytes, neutrophils, eosinophils, Lymphocytes, and monocytes disintegrate and only the bare nuclei remain
- the nuclear channel cytometer distinguished leukocytes by differences in nuclear shape and counts basophils.
- this laser-based cytometer measures light scattering at two different angles:
—> low (0-5 degrees) measures size
—> high (5-15 degrees) responsive to the lobularity of nuclei
Describe the cytogram of basophil/lobularity channel
- polymorphonuclear neutrophil (PMN) appears on the right and the mononuclear nuclei (MN) appear on the left with a valley between them
Describe the lobularity index
PMN:MN
- an index of the degree of PMN nuclear segmentation: a low value suggests a left shift
Describe automated differentials
- evaluation of internal cellular organelles and nuclear characteristics can be by:
—> 90-degrees laser scatter
—> polarizing laser light
—> RF - separate measurements can be made of individual measurements of volume, conductivity, and light scatter
Describe clinical applications of flow cytometry
- reticulocyte: enumeration of reticulocytes by flow cytometry is more accurate, precise and cost-effective
- platelet counts: can provide an estimate of young,reticulated platelets by counting platelets that stain with an RNA dye
- Immunophenotyping: monoclonal antibodies, identified by a cluster of designation (CD), are used in most flow cytometry immunophenotyping
- hematological malignancy: flow cytometry has become an important tool in the diagnosis and classification of hematologic neoplasia by immunophenotyping. Flow cytometry techniques with bone marrow cells are applicable to DNA cell cycle analysis
What are other cellular applications for flow cytometry?
- solid organ transplantation
- stem cell transplantation
- monitoring monoclonal antibody therapy
- paroxysmal nocturnal hemoginermia testing
- fetal hemoglobin
- blood parasites
- cell functioning analysis
- chromosomal analysis
- cell sorting
What are some commonly used monoclonal antibodies in flow cytometry?
- CD3: target cell: T cells
-CD4: target cell: T cells (helper) and monocytes (dimly expressed) - CD8: target cells: T cells (cytotoxic) and macrophages
- CD19: target cells: B cells
- CD34: target cell: progenitor (hematopoietic stem cells)
Describe the basic lymphocyte screening panel
A basic immune screening panel typically consists of detection and quantitation of CD3, CD4, CD8, CD19, and CD16/CD56
Describe hematological malignancy
- important tool in the diagnosis and classification of hematologic neoplasia by Immunophenotyping
- intracellular staining is most often used to aid in the diagnosis of acute leukemias and lymphomas
Describe measuring T cells for acquire immunodeficiency syndrome analysis
- the quantitation of T and B cells using monoclonal surface markers can be performed using flow cytometry.
- CD4 T cell count is one of the standard measures for diagnosing AIDS and management of HIV
Describe solid organ transplantation flow cytometry
- flow cytometric cross-matching uses fluorochrome-conjugated antihuman IgG to detect the binding of alloantibodies to donor lymphocytes in allogeneic organ transplantation.
- CD3 and CD19 coupled with anti-IgG in a three-color assay can distinguish T-cells and B-cells mismatches.
Describe stem cell transplantation
- flow cytometry is widely used to enumerate the CD34-positive implanted stem cells.
- in some cases CD45 and nucleic acid stains are also detected.
Describe monitoring monoclonal antibody therapy in flow cytometry
- essential for measuring the expression of cell surface and intracellular markers of multiple drug resistance (MDR) in cancer patients
Describe paroxysmal nocturnal hemoglobinemia testing in flow cytometry
- GPI-linked proteins, CD55 and CD59, are examined to determine if a deficiency or absence of these cell markers exists.
Describe fetal hemoglobin in flow cytometry
- detection of fetal hemoglobin and F cells by flow cytometry is becoming common
- assay uses monoclonal antibodies to hemoglobin F.
- allows for detection of sickle cell disease, and fetal maternal hemorrhage
- also allows for quantitation of fetal hemoglobin
Describe blood parasite testing in flow cytometry
- malarial parasites can be screened
- uses a riding orange to erythrocytes, and will detect erythrocytes with DNA (malarial) and will fluoresce
Describe cell functioning analysis of flow cytometry
- every event that occurs during the process of lymphocyte activation can be measured by flow cytometry
Describe chromosomal analysis
- flow cytometry can be used for karyotyping analysis.
- has 7 peaks that represent different groups of chromosomes
Describe cell sorting of flow cytometry
- after quickly making the appropriate measurements, the computer makes the decision to sort or isolate a single cell by applying a charge to the cell just as it leaves the flow cell.
- the cell is electrostatically deflected into a test tube.
Describe digital microscopy
- this new technology, referred to as automated digital cell morphology, provides an unprecedented level of efficiency and consistency
- in its simplest form, automated digital cell morphology is a process where blood cells are automatically located and preclassified into categories of blood cells
- method that combines ANNs, image analysis and slide handling
Describe Artificial Neural Networks (ANN)
- an information-processing model that stimulates the way the human brain processes information.
- emulated the neural structure of the brain which is composed of a lot of neurons that work together to solve specific problems
Describe digital cell morphology
- virtual microscopy
- Instrument: DiffMaster Octavia
- processes 8 slides per batch
- allows for remote review of a smear and storage of up to 20,000 slides with images in a database
What are instruments used in coagulation studies?
- electromechanical
- photo-optical methods
- platelet agglutination
- platelet aggregation
- new automation
- suggested screen panels: thrombotic hemostasis panel and a fibrinolytic hemostasis panel
What are the fibrinolytic hemostasis panel assay?
- alpha-2-antiplasmin
- plasminogen
- plasminogen activator inhibitors
- tissue plasminogen activator
What are the thrombotic hemostasis panel assays?
- antithrombin
- Factor VIII:C
- Heparin
- Lupus anticoagulant
- protein C
- protein S and free protein S
Describe electromechanical methods of coagulation analysis
- earliest instrument to detect blood clotting
- the principle of electromechanical methodology is the measurement of conduction or impedance of an electrical current by the formation of fibrin.
- example: semiautomated instrument is the fibrometer
- system consists of a 37C heat block, an automatic pipette, and a mechanical mixer and timer block
- After the appropriate containers are filled and plasma is added to the substrate to initiate the timing mechanism.
- timing mechanism triggers a digital readout time and the probe unit. Probes contain electrodes which created electrical potential between it and the moving electrode.
- a detection circuit is activated when a fibrin strand is formed between two electrodes
- circuit activation stops time and prevents further movement of electrodes
What assays are used in electromechanical methods?
- activated partial thromboplastin time (APTT)
- prothrombin time (PT)
- factor assays
Describe photo-optical methods
- the principle of this measurement is that a change in light transmission measured as optical density (absorbance) versus tome can be used to quantitatively determine the activity of various coagulation stages or factors
- red LED light is passed through cuvette where it is altered by fibrin clot formation
- light is converted into electrical signal and this amplified signal is converted into digital values
- photo-optical clot detection systems can be used for the determination of a wide of variety of assays ( APTT, PT, fibrinogen levels and thrombin time)
What are quantitative factor assays based on aPTT of photo-optical methods?
- factors VIII, IX, XI, and XII
What are quantitative factor assays based of PT of photo-optical methods?
- factors V, VII, and X
What is quality control from photo-optical methods of photo-optical methods?
- automatic checking of the optical system
- storage of standard and assay curves
Describe viscosity-based detection system (VDS)
- the natural thickening (viscosity) is monitored by the motion (amplitude of an oscillating steel ball in a specially designed cuvette), as a change in form takes place
- the final result in accurate and is insensitive to colored plasma, lipemic plasma, bilirubin, or turbid reagents, and it is a reliable measurement for the hemostasis laboratory
What is viscosity?
- as the resistance that a material has to a change in its form
What are the steps of VDS?
1) movement of the steel ball is triggered by two activating coils, working alternately to induce and maintain a natural oscillation
2) when the start reagent is added, the detection starts immediately
3) when the ball starts to oscillate left and right, a chronometer (clock) begins to time the clotting of the sample
4) as the ball oscillates left and right, the amplitude or motion is also measured. A peak is formed when the ball is detected in the center of cuvette
5) Amplitude is monitored during the entire clotting process
6) as the clot appears, the viscosity increases and the amplitude decreases
7) based on different algorithms, the chronometer is stopped even in the clot is peak, and/or the ball is still in motion
Describe platelet agglutination
- the ristocetin cofactor assay measures the ability of a patients plasma to agglutinate formalin-fixed platelets in the presence of ristocetin
- the rate of ristocetin- induced agglutination is related to the concentration of von Willebrand factor, and the percent normal activity can be obtained from the standard curve
- patient values are determined by comparison to a standard curve, allowing the quantitation of percent ristocetin cofactor activity
Describe platelet aggregation
- the principle of the test is that platelet-rich plasma is treated with a known aggregating agent
- if aggregated, cloudiness or turbidity patterns are determined by photometrically comparing the light transmitted through a suspension of aggregated platelets with that of a suspension of non aggregated platelets using an aggregometer
- depending of the agonist, the response may be only primary or both primary and secondary
—> primary response: reversible aggregation of platelets
—> secondary response: irreversible aggregation of platelets
Describe Newer automation of flow cytometry
- The Siemens PFA-11 in an automated system that incorporates a high shear flow system to stimulate the in vivo hemodynamic conditions of platelet adhesion and aggregation as encountered at a vascular lesion
- this system evaluates the ability of platelets to occlude an aperture in a biochemically active membrane
- results are reported as closure (CT)
- assess multiple facets of primary hemostasis, namely adherence, activation, and aggregation
