Hematology Flashcards
Most common antiseptic used for phlebotomy
70% alcohol
angle between skin and needle
15-30°
cuases of specimen hemolysis
- prolonged tourniquet application
- moisture/contamination
- too small bore needle
- excessive agitation
- frothing of blood
distance of tourniquet above the puncture site
3 to 4 inches OR 7.5 to 10 cm
usual type of EDTA used in the lab and the no. of inversions needed
action of anticoag
what’s the concentration per mL of blood
K2EDTA; 8
chelation of calcium
1.5 mg/mL
blood must be analyzed for CBC within how many hours?
if blood smear prep?
within 6 hrs of collection
within 3 hrs of collection
MPV is what?
how much does it swell when EDTA is used during the first hour?
mean platelet volume
20% increase
green top tubes contain what anticoag?
how many inversions?
what’s the anticoag concentration?
mode of action?
heparin
8
15-20 units per mL
binds antithrombin
3 types of heparin formulations
2 main tests heparin is used for
types: ammonium, sodium and lithium heparin
tests: OFT and blood gas studies
what anticoag causes cellular clumping which leads to pseudoleukocytosis and pseudothombocytopenia
heparin
light blue top anticoag?
no. of inversions?
action?
3.2% sodium citrate
3-4
chelation of Ca
factors that affect coag test (mnemonics)
in shortened? in prolonged?
Shortened: Hemolysis, Excessive agitation, Prolonged tourniquet, Excess needle manipulation, Plt contam
Prolonged: clots, inc anticoag conc.
black top ac and tests
3.8% sodium citrate for Westergren and ESR
pink top tube ac and uses
K2EDTA and blood bank tests, alternative whole blood hema determination
light blue top ac (2)
3.2% sodium citratie
CTAD citrate, theophylline, adenosine, dipyridamole
angle used for blood smear prep?
most commonly used technique for it?
30 to 45°
two glass slide method/manual wedge technique
distance of blood drop in smear from the frosted glass?
diameter of the drop of blood in blood smear?
length and shape of the smear?
scanning methods used? tail to head? back and forth serpentine?
1cm
2-3mm
2/3 or 3/4 and finger-shaped
Longitudinal and Battlement
Automated Methods: (SLIDE)
BLOOD FILM STAINING:
fixative:
stain:
buffer:
CellaVision HemaPrep®
Centrifugal (Spinner) Type
Coulter LH
Sysmex SP-10
methanol
Wright or Wright-Giemsa
0.05M Sodium Phosphate (pH 6.4)
defined as any stain which contains methylene blue (and/or its products of oxidation) and a
halogenated fluorescein dye (commonly eosin B or eosin Y)
- __ - a basic stain, it colors the nucleus and some cytoplasmic structures a blue or purple color (stained structures are described to be basophilic [e.g., DNA or RNA])
- __ – an acidic stain, it colors some cytoplasmic structures an orange-red color (stained structures are described to be acidophilic [e.g., proteins with amino groups]). most commonly used type of stain in the hematology laboratory
Romanowsky-type stain:
METHYLENE BLUE
EOSIN
STAINING TECHNIQUES FOR BLOOD FILMS:
- Flood the slide with Wright’s stain for 1 to 3 mins, buffer is then added
- Generally, 5 to 10 mins. to stain a batch of slides; ex. Midas III, Hema-Tek, Coulter LH, Sysmex SP-10
- 1 minute only, Uses modified Wright or
Wright-Giemsa stain filtered into a Coplin jar or a staining dish. aged distilled water is used as the buffer
COLORS:
macro: slide color
micro: rbc, wbc nuclei, neutrophil cytoplasm, eosinophil granules
MANUAL
AUTOMATED
QUICK
pink to purple
orange to salmon pink!!, purple to blue, pink to tan with violet to lilac granules, bright orange
- detect “snowplow” effect (presence of more than four times the number of WBCs per field at the lateral edges than the monolayer area) which is unacceptable
- Used also to estimate total WBC count:
Average no. of WBCs per hpf x 2,000 = estimated WBC ct. per μL - When the appropriate area of a blood smear with a normal RBC count is viewed, there are generally about 200 to 250 RBCs per 100x OIF
Used to examine the nuclear details of the white blood cells; Average number of platelets/OIF X 20,000 = estimated plt. ct. per μL
what is the formula for the platelet estimate?
storage of slides
10x Objective Examination
40x High-Dry or 50x Oil Immersion
Objective
100x Oil Immersion Objective
Average no. of platelets per field x total RBC count / 200 RBCs per field
7 days before disposal
___: process of blood cell formation
* Classical marker of hematopoietic stem cells: __
* considered to start around the nineteenth day of embryonic development after fertilization
Two Related Theories (Origin of Hematopoietic Progenitor Cells):
1.) ___
- suggests that each of the blood cell lineages is derived from its own unique stem cell
2.) ___
- suggests that all blood cells are derived from a single progenitor stem cell called a pluripotential stem cell. Most widely accepted theory among experimental hematologists
Hematopoiesis (aka: Hemopoiesis)
CD 34
Polyphyletic Theory
Monophyletic Theory
Three phases of Hematopoiesis:
site: yolk sac; 1st blood cells: ___
(formed during the first 2 to 8 weeks of life)
●Primitive Erythroblasts (PE); important in early embryo-genesis to produce hemoglobin (__ (3)) necessary for delivery of oxygen to the embryonic tissues. it occurs intravascularly (within developing blood vessels)
site: fetal liver; Hb F (Fetal hemoglobin) is the predominant hemoglobin; __ - first fully developed organ in the fetus -size of the thymus
increases during fetal development; major site of __production; __ - produce B cells
site: bone marrow; Hematopoiesis starts in the
bone marrow cavity before the 5th month of fetal development. The bone marrow becomes
the chief site by the end of 24 weeks’ gestation.
MESOBLASTIC/MEGALOBLASTIC; primitive erythroblast; Gower-1, Gower-2 and Portland
HEPATIC; Thymus; T cell; Kidneys and spleen
INTRAMEDULLARY/MEDULLARY/MYELOID
ADULT HEMATOPOIESIS
* Hematopoietic tissues of adults are located NOT ONLY in the __, but also in
the __(4).
* Bone marrow – contains developing erythroid, myeloid, lymphoid, and megakaryocytic cells
– considered to be a primary lymphoid organ with functions equivalent to that of __ (birds)
– is estimated to be capable of generating around:
__ billion RBCs
__ Granulocytes, and
__ Platelets
~ per kilogram of body weight DAILY
bone marrow; lymph nodes, spleen, thymus and liver; Bursa of Fabricius
2.5; 1 billion; 2.5 billion
process of replacing red marrow by __ marrow during development
⸙ between __ of age, adipocytes become more abundant and start to occupy the spaces in the long bones previously dominated by active marrow
⸙ eventually results in restriction of the red marrow in the adult to the:
- R - ____________
- S - ____________
- V - ____________
- P - ____________
⸙ Yellow marrow - has the ability of reverting back to active marrow in cases of increased demand on the bone marrow
what is the active and inactive?
RETROGRESSION; yellow; 5 and 7 years
ribs
sternum, skull, shoulder blades
vertebrae
pelvis and proximal ends of long bones
red: hematopoietically active
yellow: inactive
⸙ Primary lymphoid organs:
-consist of the __ where T and B lymphocytes are derived. Size of the thymus increases during fetal development (growth of this gland continues until puberty then undergoes atrophy with aging)
- lymphoid cells fated to become B cells undergo their early stages of differentiation within the __
⸙ Secondary (Peripheral) lymphoid organs
- MAJOR secondary lymphoid organs: __
- 2 major functions:
●Trapping and concentration of foreign substances
●Main sites of production of __ and the induction of antigen-specific T lymphocytes
bone marrow and thymus
bone marrow
spleen and lymph nodes; antibodies
LARGEST of the secondary lymphoid organs
Major organ in the body in which __ are synthesized and from which they are released into the circulation
Effects of splenectomy:
a. In children – often leads to an increased incidence of __ sepsis caused primarily by S. pneumoniae, N. meningitidis, and H. influenzae
b. In adults – less adverse effects, although it leads to some increase in blood-borne bacterial infections or bacteremia
Small ovoid, bean-shaped structures (normally < 1 cm in diameter) found in different areas throughout the body;
A related term: __ solid tumor neoplasm of lymphoid tissue
SPLEEN; antibodies; bacterial
LYMPH NODES; lymphoma
MINOR secondary lymphoid organs:
✓__ – to detect and respond to antigens in the respiratory and alimentary secretions
✓ __
✓__ – clusters of lymphocytes distributed in the lining of the small intestine; detect substances that diffuse across the intestinal epithelium
✓__ – general term for the unencapsulated lymphoid tissues present in regions underlying the mucosal areas
__ – can maintain hematopoietic stem cells and progenitor cells to generate various blood cells (a process called extramedullary hematopoiesis) as a response to infectious agents or in pathologic myelofibrosis of the bone marrow
Tonsils
Appendix
Peyer’s patches
MALT (mucosa-associated lymphoid tissue)
Liver
Bone marrow collection sites:
2 sites that are preferred for bone marrow aspiration in adults
- Sternum
preferred site for children under 2 yrs old
M:E ratio of patients with leukemia?
M:E (myeloid-to-erythroid) ratio is the proportion of myeloid cells to nucleated erythroid precursors in the bone marrow aspirate
-The monocytic and lymphoid __ and __ are EXCLUDED from the myeloid cell count. In healthy adult individuals, the M:E ratio varies from roughly __
Posterior superior iliac crest
Anterior superior iliac crest
Anterior medial surface of the tibia
10:1
precursors; plasma cells; 1.5:1 to 3.3:1.
- Recommended: at least 500 cells (preferably 1,000 cells) be counted. __ on each of 2 slides
❖ __: Disturbs bone marrow architecture; Spread as a smear on a slide, stained, and examined or hematologic or systemic disease - Used for analysis of __; Bone marrow smears – should be retained for _
❖__: Bone marrow removed intact without disturbing the bone architecture. A __ of gelatinous red marrow (fixed in formalin, sectioned, stained, and studied for hematologic or systemic disease)
* Advantage: gives a better picture of the real structure of bone marrow
* Used for analysis of bone marrow architecture
❖ __: Blood cell production outside the bone marrow. Occurs MAINLY in the __ (causes enlargement of the organs)
✓ Occurs under the following conditions; when bone marrow becomes dysfunctional such as in aplastic anemia, infiltration by malignant cells, or overproliferation of a cell line (for example, leukemia); When bone marrow is incapable of meeting the demands placed on it (as observed in hemolytic anemias)
Marrow differential; 500 cells
Bone marrow aspirate
individual cell morphology
10 years
Bone marrow biopsy; cylinder
Extramedullary hematopoiesis; liver and spleen
- process of RBC formation (takes place inside the bone marrow)
⸙ __: total mass of RBCs circulating in the peripheral blood AND the bone marrow RBC precursors
⸙ __: erythrocytes in the circulation
⸙Erythrokinetics: a term that describes the dynamics of RBC creation and
destruction
⸙ __: production of defective erythroid precursor cells. (they undergo apoptosis in the bone marrow before they have a chance to mature to the reticulocyte stage.)
✓ examples of conditions: (4)
⸙ __: decrease in the number of RBC precursors in the bone marrow (resulting in ↓ RBC production); examples of conditions: (3)
Erythropoiesis
Erythron
RBC mass
Ineffective erythropoiesis; vitamin B12 deficiency, folate deficiency, thalassemia, sideroblastic anemia
Insufficient erythropoiesis; iron deficiency, renal disease, acute leukemia
immature hematopoietic cell that is committed to a cell line but cannot be identified morphologically
immature hematopoietic cell that is morphologically identifiable as belonging to a given cell line
EARLIEST marker of erythroid differetiation; transferrin receptor (transport protein of iron in the blood)
progenitor cells (BFU, CFU)
precursor cells
CD 71
__: chief stimulatory cytokine for RBCs
- major hormone that stimulates the production of erythrocytes; thermostable, nondialyzable, glycoprotein hormone
- primary cell source: ______________________
- primary target cells: ______________________
-Normally, EPO is released from the kidney into the blood in response to __ (too little tissue oxygen).
- 3 major effects:
1.) allowing early release of reticulocytes from the bone marrow
2.) reducing the time needed for cells to mature in the bone marrow
3.) preventing __ (note: apoptosis rescue is the
MAJOR way in which EPO increases RBC mass)
- Some of the current/potential therapeutic applications:
⸙ anemia of chronic renal disease
⸙ autologous predonation blood collection
⸙ anemia in HIV infection to permit use of zidovudine (AZT)
“__”: some athletes illegally use EPO injections to increase the oxygen-carrying capacity of their blood (to enhance endurance and stamina) (can lead to deadly arterial and venous thrombosis)
Erythropoietin (EPO)
peritubular interstitial cell of kidneys
BFU-E and CFU-E
hypoxia
apoptotic cell death
blood doping
other hormones that stimulate rbc prod
Growth hormone
✓Produced by: ________________________
Testosterone
✓Produced by: ________________________
Prolactin
✓Produced by: ________________________
Estrogen
✓Produced by: ________________________
pituitary gland
testes
pituitary gland
ovaries
RBC STAGES OF MATURATION
*Note: __ is the EARLIEST committed progenitor.
* From BFU-E to CFU-E: __
* From CFU-E to Rubriblast: __
*It takes about __ for the BFU-E to mature to an erythrocyte, of which approximately 6 days are spent as recognizable precursors in the bone marrow.
RUBRIBLASTIC: stages? NORMOBLASTIC? ERYTHROBLASTIC?
Examples of supravital stains used for the demonstration of reticulocytes:
o _______ & ___________
*Polychromatophilic Erythrocytes and Diffusely Basophilic Erythrocytes are called when “reticulocytes” are stained with:
BFU-E
1 week
1 week
18 to 21 days
Rubriblast, Prorubricyte, Rubricyte, Metarubricyte, *Reticulocyte, *Reticulocyte
Pronormoblast, Basophilic/early Normoblast, Polychromatophilic/intermediate Normoblast Orthochromatic/late Normoblast
Proerythroblast, Basophilic/early Erythroblast, Polychromatophilic/intermediate Erythroblast, Orthochromatic/late Erythroblast
Brilliant Cresyl Blue (BCB) and New Methylene Blue (NMB)
Wright stains
ׁSIZE: 12 to 19 μm
ׁNUCLEUS: round or slightly oval, thin nuclear
membrane, central or slightly eccentric
ׁNUCLEOLI: 1 to 2
ׁCYTOPLASM: small in amount, dark blue
(because of the concentration of ribosomes),
homogenous
-N:C ratio = __
~ gives rise to ____ .
EARLIEST PRECURSORTO BE RECOGNIZED IN LM
RUBRIBLAST (PRONORMOBLAST); 4:1; 2 prorubricyte
ׁSIZE: 12 to 17 μm
ׁNUCLEUS: round, thin nuclear membrane,
smaller, slightly eccentric
ׁNUCLEOLUS: 0 to 1
ׁCYTOPLASM: deeper, richer blue and
appears more abundant than in normoblast
because of smaller nucleus
-N:C ratio = __
gives rise to ___
-Most helpful criteria in distinguishing the
prorubricyte from the rubriblast:
✓ ____ & ____
LAST STAGE WITH A NUCLEUS
1st STAGE OF HB SYNTHESIS
PRORUBRICYTE (BASOPHILIC NORMOBLAST); 4:1; 4 rubricyte; coarser chromatin and absence of nucleoli;
ׁSIZE: 11 to 15 μm
ׁNUCLEUS: round and smaller, thick nuclear
membrane, eccentric nucleus
ׁ NUCLEOLUS: none
ׁCYTOPLASM: gray
-N:C ratio = __
gives rise to _____.
LAST STAGE CAPABLE OF MITOSIS
1ST STAGE IN WHICH THE CYTOPLASM BECOMES __
RUBRICYTE (POLYCHROMATIC NORMOBLAST); 1:1; 2 metarubricytes; PINK
RUBRICYTE VS LYMPHOCYTE:
nuc? color of cytoplasm?
NUCLEUS:
RUB: checkerboard; LMYPH: crushed velvet
COLOR OF CYTOPLASM:
RUB: muddy or gray; LYMPH: sky blue or Robin egg blue
=enveloped extruded nucleus
=engulfed by bone marrow macrophages
=Frequently, small fragments of the nucleus are left behind if the projection is pinched off before the entire nucleus is enveloped (the fragments are called Howell-Jolly bodies when seen in the RBCs in the circulation).
**Howell-Jolly bodies are typically removed from the RBCs by the __ pitting process once they enter the circulation.
Pyrenocyte; splenic macrophage
ׁSIZE: 8 to 12 μm
ׁNUCLEUS: pyknotic (dense mass of degenerated chromatin)
ׁ NUCLEOLUS: none
ׁCYTOPLASM: __
-N:C ratio = __
-Nucleus is __ at this stage, and the
cell becomes a reticulocyte.
-Other names: Nucleated RBC (NRBC), Pyknotic erythroblast, Acidophilic normoblast
LAST STAGE WITH A NUCLEUS
METARUBRICYTE (ORTHOCHROMATIC
NORMOBLAST); salmon-pink; 1:2
extruded
ׁSIZE: 7 to 10 μm
ׁNUCLEUS: none
ׁNUCLEOLUS: none
ׁCYTOPLASM: predominant color is that of hemoglobin but with a __
because of some residual ribosomes and RNA
By the end of this stage, the cell is ___
LAST STAGE OF HEMOGLOBIN SYNTHESIS
* Young RBCs containing residual __ (last immature erythrocyte stage)
* Spends __ in the bone marrow and __ in the peripheral blood before developing into a mature RBC
* The shape of the reticulocyte is irregular
in electron micrographs.
RETICULOCYTE; bluish tinge
salmon pink
RNA; 2 to 3 days; 1 day
ׁSIZE: 6 to 8 μm
ׁNUCLEUS: none
ׁ NUCLEOLUS: none
ׁCYTOPLASM: salmon pink (with a central pallor occupying __ of the cell’s diameter)
*Shape: __
*Thickness: 1.5 to 2.5 μm
*Average life span: __
*Number of erythrocytes produced from each rubriblast: __
*Normal ratio of RBCs to WBCs is approximately __ and the normal ratio of RBCs to Platelets is approx. __
*Adult RBC contains no mitochondria (no protein or Hb synthesis)
●RBC membrane constituents: 8% carbohydrates, 40% lipids, and __(integral and peripheral)
MATURE ERYTHROCYTE; 1/3
biconcave disk; 120 days; 8 to 32;
600:1
15:1
52% proteins
__: Glucose transporter, supports ABH antigens
Anion
__: Water transporter
__: Anion transporter, supports ABH antigens
__: Transports negatively charged sialic acid, supports Ss determinants
__: Transports negatively charged sialic acid, supports Gerbich system determinants
Glut-1
Aquaporin 1
Band 3
Glycophorin B
Glycophorin C
- provide the horizontal or the lateral support for the membrane; The shape and flexibility of the erythrocyte depend on the cytoskeleton.
__: Primary cytoskeletal proteins
__: Caps actin filament
__: Regulates actin polymerization
♦SKELETAL PROTEINS
α-spectrin & β-spectrin
Tropomodulin
Tropomyosin
_: patients have an increase in the MCHC value
INH: Autosomal dominant (75%)!!!
Defect in protein(s) that disturbs vertical membrane interactions between transmembrane proteins and underlying cytoskeleton; loss of membrane and decreased surface area-to-volume ratio
procedure that makes RBC survival normal?
-a flow cytometry-based test; most sensitive and specific test to confirm the diagnosis**
Hereditary spherocytosis
splenectomy; EMA binding test
HER: Autosomal Dominant!!
Defect in proteins that disrupt the horizontal
linkages in the protein cytoskeleton; loss of mechanical stability of membrane
Hereditary elliptocytosis
HER: Autosomal recessive!!
Severe defect in spectrin that disrupts horizontal
linkages in protein cytoskeleton; severe RBC fragmentation
Hereditary pyropoikilocytosis
HER: Autosomal dominant!!
Defect in band 3 causing increased membrane rigidity; resistant to malaria; prevalent in some areas of Southeast Asia
Southeast Asian ovalocytosis (Hereditary
ovalocytosis)
HER: Autosomal dominant
Increased membrane permeability to sodium and potassium; increased intracellular
sodium causing influx of water, increase in cell volume, and decreased cytoplasmic viscosity; typical RBC morphology: __
(5% to 50%) and macrocytes
Overhydrated hereditary stomatocytosis; stomatocytes
This is the most common form of stomatocytosis
HER: Autosomal dominant
Increased membrane permeability to potassium;
decreased intracellular potassium, resulting in loss of water from cell, decrease in cell volume, and increasedcytoplasmic viscosity; typical RBC morphology: target cells, burr cells, stomatocytes (<10%), RBCs with “puddled” hemoglobin at periphery, desiccated cells with spicules
Dehydrated hereditary stomatocytosis
(Hereditary Xerocytosis)
✓ The __ values are ELEVATED in people living at a HIGHER ALTITUDE over what they would be at sea level. The difference is around 1 g Hb/dL at 2 km altitude and 2 g Hb/dL at 3 km.
AGE GROUP AND NORMAL RBC COUNT:
Children (8 to 13 y.o.):
Adult (male):
Adult (female):
RBC count, hemoglobin and hematocrit
4.00 to 5.40 X 10^12/L
4.60 to 6.00 X 10^12/L
4.00 to 5.40 X 10^12/L
RBC Metabolic Pathways:
● Glucose penetrates the red blood cell with no energy expenditure via _______ (a transmembrane protein).
●Handles 90% of glucose utilization in red blood cells.
●Non-oxidative, __ pathway
● Produces __ molecules of ATP
Embden-Meyerhof Pathway; GLUT-1
anaerobic; two
- An AUTOSOMAL RECESSIVE!! disorder
- Most common enzyme deficiency of the EMP and is the most common form of hereditary nonspherocytic hemolytic anemia (HNSHA).
- Possible PBS findings include: _______ (2)
- Tests:
o PK fluorescent spot test - __
o Autohemolysis test - screening test
o what is the confirmatory test?
PYRUVATE KINASE (PK) DEFICIENCY
Acanthocytes. Burr cells
recommended screening test
Quantitative PK assay
✓ These are the three alternate pathways that branch from the glycolytic pathway:
1. __
● aka: pentose phosphate pathway or phosphogluconate pathway
● aerobically converts glucose to __ and produces NADPH (nicotinamide adenine dinucleotide phosphate (reduced)
● NADPH reduces __ (it reduces peroxides and guards proteins, lipids, and heme iron from oxidation
- it prevents the denaturation of ___ by oxidation
- functionally dependent of G6PD
Glycolysis Diversion Pathways (Shunts)
Hexose Monophosphate Shunt (Aerobic Glycolysis); pentose; glutathione
GLOBIN!!!
- An __ disorder
- Most common enzyme deficiency in the pentose phosphate pathway and is also the most common RBC enzyme defect (prevalence of 5% of the global population or approximately 400 million people worldwide)
- Possible PBS findings include: ______
- Tests:
*G6PD fluorescent spot test - reco
*Autohemolysis test - screening test
*Quantitative G6PD assay - confirm
GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD) DEFICIENCY
X-LINKED RECESSIVE
Heinz bodies and Bite cells
Classification of G6PD Deficiency Variants by the WHO:
Severely deficient!! <1% activity or not detectable
Chronic, hereditary nonspherocytic hemolytic anemia (HNSHA); severity is variable; rare
G6PD-Serres
G6PD-Madrid
Class I
Classification of G6PD Deficiency Variants by the WHO:
Mild to moderately deficient; __ activity
Episodic, acute hemolytic anemia associated with infections and certain drugs; self-limited
G6PD-A
G6PD-Canton
Class III
10% to 60%
Classification of G6PD Deficiency Variants by the WHO:
Severely deficient: __ activity
Severe, episodic acute hemolytic anemia associated with infections, certain drugs, and __; not self-limited and may require transfusions during hemolytic episodes
NOTE:
Only a small group of G6PD-deficient individuals demonstrate __, and most of these have the G6PD-Mediterranean variant!!
G6PD-Mediterranean
G6PD-Chatham
Class II
<10%
fava beans
favism
Classification of G6PD Deficiency Variants by the WHO:
Mildly deficient to normal: __ activity
None G6PD-B (wildtype); G6PD-A+ (may also manifest as Class III)
Increased: __ activity
None
FOOD THEY MUST AVOID: (4)
Class IV; 60% to 150%
Class V; >150%
fava beans, legumes, soya food, naphthalene (mothballs), soya food
✓ These are the three alternate pathways that branch from the glycolytic pathway:
____: __ - AKA cytochrome b5 reductase. maintains iron in __ in its reduced state (ferrous/Fe+2)
____: for the production of 2,3-BPG. 2,3-BPG binds to the hemoglobin and decreases the oxygen affinity of Hb
Two variables affecting the degree of association or dissociation between oxygen and hgb:
o partial pressure of oxygen
o affinity of hemoglobin for oxygen
- The affinity of hemoglobin for oxygen is dependent on 5 factors: ___
___:
o the curve produced when the 2 variables (partial pressure of oxygen and affinity of hemoglobin for oxygen) are plotted on a graph (oxygen saturation of hemoglobin versus the partial pressure of oxygen)
Methemoglobin Reductase Pathway; heme
Rapoport-Luebering Shunt
pH
Partial pressure of carbon dioxide
Concentration of 2,3-bisphosphoglycerate (2,3-BPG)
Temperature
Presence of other hemoglobin species that are nonfunctional
OXYGEN DISSOCIATION CURVE
SHIFT TO THE RIGHT
____ pH
____ PCO2
____ 2,3 – BPG
____ Temperature
SHIFT TO THE LEFT
____ pH
____ PCO2
____ 2,3 – BPG
____ Temperature
▪__:
- a shift in the curve due to an alteration in pH (or hydrogen ion concentration)
-effect of hydrogen ions and CO2 on the affinity of hemoglobin for oxygen
▪__:
-depicts the occurrence by which the binding of O2 to the hemoglobin promotes the release of CO2
inc, dec, dec, dec (increased affinity)
dec, inc, inc, inc (decreased affinity)
Bohr effect
Haldane effect
RED BLOOD CELL ANOMALIES:
- increased number of red cells with variation in size; normal RBCs (normocytes): 6-8 um in diameter (usually seen when MCV is 80-100 fL)
___:
-larger than normal RBCs (diameter > 8.0 um)
-usually seen when the MCV is > 100 fL
-associated with impaired DNA synthesis
___:
-smaller than normal RBCs (diameter < 6.0 um)
-usually seen when the MCV is < 80 fL
-associated with defective hemoglobin formation
MCV formula?
two parameters calculated from RBC histogram?
may cause a bimodal distribution curve include?
A __ curve on a histogram indicates more variation in the size of the cells (The cell population is NOT homogeneous.)
Using the RDW value
▪Red Cell Distribution Width – a calculated index (from the RBC histogram) given by hematology analyzers to help identify anisocytosis and provide information about its degree
ANISOCYTOSIS
Macrocytes
Microcytes
MCV = Hct/RBC ct x 10
MCV and RDW
✓ blood transfusion
✓ cold agglutinin disease
✓ hemolytic anemia with schistocytes present
wider or flattened
RED BLOOD CELL ANOMALIES:
General term for a variation in the normal coloration; Normally, RBCs have a central area of pallor approximately __ the diameter
- may also mean the occurrence of hypochromic cells and normochromic cells in the same blood smear; may be found in sideroblastic anemias, after treatment for IDA
__: central pallor >1/3 of diameter, usually microcytic
GRADING OF HYPOCHROMIA
1+, 2+, 3+, 4+
Anisochromia; 1/3; Hypochromic cells
1+, 1/2
2+, 2/3
3+, 3/4
4+ thin rim of Hb
RED BLOOD CELL ANOMALIES: (ANISOCHROMIA)
♦ Anulocyte: a.k.a.: ______
-RBC with a thin rim of hemoglobin and a large, clear center; may be observed in iron deficiency anemia
___: RBCs that lack central pallor even though they lie in a desirable area for evaluation
* These RBCs are actually caused by a shape change (such as that found in SPHEROCYTES)
* True hyperchromia occurs when MCHC is HIGH
* __
✓ basically the only disease in which the MCHC is high (above the reference range)
- Symptomatic HS has 3 key clinical manifestations:
⸙ _____________________
⸙ _____________________
⸙ _____________________ - DAT (direct antiglobulin test): negative
- MCV: normal to low
- MCH: normal
- MCHC: slightly increased
✓ some of the tests include:
* Autohemolysis test: greatly increased (but can be corrected with either glucose or ATP)
* OFT: increased osmotic fragility - NOT diagnostic of HS
* EMA binding test –↓ fluorescence (flow cytometry)
Pessary cell, Ghost cell
Hyperchromic cells
Hereditary Spherocytosis
splenomegaly, anemia, jaundice
RED BLOOD CELL ANOMALIES: (ANISOCHROMIA)
* larger than normal red cells with bluish tinge (Wright’s stain)
* bluish tinge – caused by the presence of __
* large numbers: associated with decreased RBC survival, hemorrhage or erythroid hyperplastic marrow
GRADING OF POLYCHROMASIA:
Slight: 1%
1+: 3%
2+: __
3+: 10%
4+:
Polychromatophilic erythrocytes;
residual RNA
5%; >11%;
RED BLOOD CELL ANOMALIES:
○ increased number of red cells with variation in SHAPE
○ Almost spherical in shape; Lacks the central pallor; Spherocytes may be wrongly reported if one examines the feathered edge of the blood film because the red blood cells in the said area lack central pallor.
POIKILOCYTES
SPHEROCYTE
RED BLOOD CELL ANOMALIES: POIKOLOCYTE
Elongated RBCs with a slit-like central pallor (may be considered as an artifact)
Dehydrated hereditary stomatocytosis (also
known as:________ )
*__: a dehydrated form of a stomatocyte; appears to have puddled at one end (half-light, half-dark)
- a.k.a.: thorn cell or ___; RBCs with irregularly spiculated surface; Abetalipoproteinemia also known as: ____ and ___
DSE:
-McLeod Syndrome - in BB
-Pyruvate kinase deficiency
STOMATOCYTE or mouth cell
hereditary xerocytosis; XEROCYTE
~
ACANTHOCYTE; spur cell
Kornzweig Syndrome and Hereditary Acanthocytosis
RED BLOOD CELL ANOMALIES: POIKOLOCYTE
___: RBCs with regularly spiculated surface
___: characterized by: ⸰ marked ↑ in plasma urea and other nitrogenous waste products, acidemia, electrolyte imbalance (K+ elevation); normocytic, normochromic anemia, uremic frost (dirty skin), generalized edema; foul breath; urine-like sweat; Pyruvate kinase deficiency
___: Elliptical (cigar-shaped) RBC
seen in: Hereditary Elliptocytosis, Thalassemia
___: Pear-shaped or teardrop shaped RBCs
seen in: Primary Myelofibrosis (PMF) and megaloblastic anemia, thalassemia, etc.
___: Fragmented RBCs; seen in patients with artificial heart valves, uremia, severe burns, MAHAs (microangiopathic hemolytic anemias):
-group of disorders characterized by RBC fragmentation and thrombocytopenia
-major MAHAs: TTP, HELLP, HUS, and DIC; characterized by narrowing or obstruction of small blood vessels by __ or __ resulting in the fragmentation of RBCs
- __ = helmet-shaped schistocyte that may appear in MAHA
___: it is also known as defibrination syndrome, consumption coagulopathy; generalized overactivation of the coagulation and fibrinolytic systems; some of the tests:_____, _______
BURR CELL a.k.a.: echinocytes; Uremia
OVALOCYTE
ELLIPTOCYTE
DACRYOCYTE a.k.a.: teardrop cell
SCHISTOCYTE a.k.a.: schizocyte
MAHAs:
○TTP (thrombotic thrombocytopenic purpura)
○HELLP syndrome (hemolysis, elevated liver enzymes, low platelet count)
○ HUS (hemolytic uremic syndrome)
○ DIC (disseminated intravascular coagulation)
MAHAs;
fibrin; platelet aggregates;
Helmet cell
DIC; D-dimer test; PF (prothrombin fragment) 1+2
RED BLOOD CELL ANOMALIES: POIKOLOCYTE
___: Sickle- or crescent-shaped RBCs; seen in Sickle cell anemia, Hemoglobin SC disease, etc.
Two forms:
___: -crescent-shaped
-with long projections
-when reoxygenated, they fragment
___: -have less pronounced projections
-when reoxygenated, they return to the original biconcave disk shape
____: RBCs which show a centrally stained area with a thin outer rim of hemoglobin; seen in Liver disease, certain hemoglobinopathies, thalassemia (a.k.a.: _________,) and LCAT
DREPANOCYTE a.k.a.: Sickle cell, Meniscocyte
ISC - irrversible sickle cell
Oat-shaped cells
TARGET CELL a.k.a. codocyte, platycyte, Greek helmet cell, Mexican hat cell, Bull’s eye cell, leptocyte
Hereditary leptocytosis, Mediterrenean anemia
RED BLOOD CELL ANOMALIES: POIKOLOCYTE
RELATED INFORMATION:
- ___ are RBCs with an increased cell membrane-to-volume ratio; In vivo and on electron microscopy, these cells appear bell-shaped.
___: Demonstrate a semicircular defect in their edge (resembles a bite mark); seen in: Glucose-6-phosphate dehydrogenase (G-6-PD)
___: Folded RBCs seen in Hemoglobin SC disease
___: Bipolar or central distribution of hemoglobin; seen in Sickle cell anemia
__ -As large as leukocytes; Pale-pink staining ghost of the red cell (the membrane remaining after the contents have been released); frequently seen in malaria and in other conditions causing __ hemolysis
Codocytes
BITE CELL a.k.a.: degmacyte
BISCUIT CELL
BRONZE ELLIPTOCYTE
SEMILUNAR BODY; overt
RBC INCLUSION BODIES:
* a.k.a.: punctate basophilia described as irregular, dark blue to purple granules evenly distributed within an RBC);
CONTENT: Aggregated RNA
STAIN: Wright stain (deep blue to purple) Supravital stain
-Lead poisoning (aka: _____)
-Arsenic poisoning
-Pyrimidine-5’-nucleotidase deficiency
-Anemias with impaired hemoglobin synthesis (ex: thalassemia)
-Refractory anemias
-Alcoholism
-Megaloblastic anemias!!!
Two forms of basophilic stippling:
a.) __ – usually observed when there is polychromatophilia
b.) __ – may be observed in plumbism or other disorders with impaired Hb synthesis, in megaloblastic anemia, and in other forms of severe anemia
BASOPHILIC STIPPLING
PLUMBISM OR SATURNISM
Fine stippling
Coarse stippling
RBC INCLUSION BODIES:
RBC INCLUSION BODIES:
__ (described as multiple dark blue irregular granules [in Prussian blue iron staining]) (described as pale blue clusters [in Wright staining])
CONTENTS: Intraerythrocytic collections of iron
STAIN: Iron stains (called as __) , New Methylene Blue, Wright stain (called as __)
-Sideroblastic Anemias =characterized by a dimorphic peripheral blood picture (presence of BOTH normochromic and hypochromic RBCs in the same blood smear)
-Thalassemia
-Hemochromatosis or
-Hemosiderosis
SIDEROTIC GRANULES a.k.a.: Pappenheimer bodies
siderotic granules; Pappenheimer bodies
RBC INCLUSION BODIES:
description: frequently appears singly in a cell (only one per cell), usually round, and <1 μm in diameter, blue to purple in color)
CONTENTS: round Remnants of nuclear chromatin (DNA)
STAIN: Wright stain, New Methylene Blue
____ (+) - biochem staining reaction for DNA
-Megaloblastic anemias
-After splenectomy
-Thalassemia
HOWELL-JOLLY BODIES
Feulgen reaction
RBC INCLUSION BODIES:
described as threadlike structures that appear as purple-blue loops or rings
CONTENTS: Mitotic spindle remnants
STAIN: Wright stain & Supravital stain
-Megaloblastic anemias
-Refractory anemia
-Lead poisoning
CABOT RINGS
RBC INCLUSION BODIES:
most of the time appear eccentrically along the inner RBC membrane, large, round, blue to purple materials
CONTENTS: Denatured and precipitated hemoglobin
STAIN: Supravital stains (examples:__)
~ what stain will dissolve the Howell-Jolly bodies?
-Glucose-6-Phosphate Dehydrogenase (G-6-PD) deficiency (**Favism) = occurs mainly in people of Mediterranean origin and in the Chinese who have glucose-6-phosphate dehydrogenase def
=may result in a severe hemolytic episode
-Drug-induced hemolytic Anemias (DIHA)
-Unstable hemoglobin disease (congenital Heinz body hemolytic anemia)
HEINZ BODIES;
BCB, NMB, methyl violet and crystal violet
Wright stain
RBC INCLUSION BODIES:
Inclusion bodies (described as small, multiple,
evenly distributed throughout the red cell, granular, greenish-blue bodies)
CONTENTS: Precipitated Hb H (an abnormal Hb composed of __ globinchains) (denatured β-globin chains)
Supravital stains examples: __ (2)
NOTE: Appearance of RBCs with Hb H Bodies: ____
Hb H Disease (a subtype of alpha thalassemia)
Hb H; 4 β
BCB and NMB
pitted golf ball appearance
RBC INCLUSION BODIES:
-Protozoa/Protozoans
Examples: Malaria’s Schuffner’s dots (aka: _____)
*Babesia spp. - “maltese cross”
STAIN: Wright stain and Giemsa stain
Parasitic infections
PARASITES; eosinophilic stippling
▪ Main component of the red blood cell
(approximately 95% of the cytoplasmic content of RBCs)
▪ also known as the: ______
▪ concentration of Hb within RBCs: approximately __
▪ molecular weight: approximately 64,000 Daltons
▪ Felix Seyler identified the respiratory protein
Hemoglobin (in 1862)
▪ About __ of cytoplasmic hemoglobin is produced before the nucleus is extruded, and the remaining __ is synthesized in the early
reticulocyte
▪ Single most common complex organic
molecule in vertebrates
▪ 1 gram of hemoglobin can carry ____ of
O2.
▪ 1 gram of hemoglobin can carry a constant
__________ IRON.
♦Complete adult hemoglobin molecule is composed of 4 different constituents:
1.) A protein component (globin) composed of
__ of two different polypeptide chains.
2.) __ of the nitrogenous
protoporphyrin IX.
♦Globin Chains in Hemoglobin:
Alpha and Zeta: __
what are the primary functions of hemoglobin?
HEMOGLOBIN; respiratory pigment
34 g/dL
65%; 35%
1.34 mL
3.47 mg
mnemonic: hemoglobin love ferrous: 3 letters, 4 letters, 7 letters (3.47 of iron, 1.34 of O2)
two sets; Four molecules
141 amino acids
delivery of O2; carry waste products; binding, inactivation and transport of NO (nitric oxide)
__: describes the amino acid sequence of the polypeptide chains
__: describes the chain arrangements in helices and nonhelices.
__: describes the arrangement of the helices into a pretzel-like configuration or formation
__: (TETRAMER) describes the complete hemoglobin molecule (complete hemoglobin molecule is spherical, has four heme groups attached to four polypeptide chains, and may carry up to four molecules of oxygen)
PRIMARY
SECONDARY
TERTIARY
QUATERNARY
HEMOGLOBINOMETRY (HEMOGLOBIN DETERMINATION):
Reference method: __
Uses Drabkin’s reagent - major components:
1. __: converts hemoglobin to methemoglobin
2. __: provides cyanide ions
✓ HiCN is measured at: ___________ (All types of Hb may be measured through this method,
EXCEPT: __________________.)
✓ Full conversion of hemoglobin to cyanmethemoglobin = ____________________
Cyanmethemoglobin (HiCN) Method
Hemoglobin (Fe2+) + K3Fe (CN)6 → methemoglobin (Fe3+) + KCN →cyanmethemoglobin
*Potassium ferricyanide (K3Fe(CN)6)
*Potassium cyanide (KCN)
540 nm; Sulfhemoglobin
10 mins at room temp
REMINDERS:
o Cyanmethemoglobin reagent is sensitive to __ (should be stored in a brown bottle or in a dark place)
o Another technique that has been used in some automated instruments involves the use of sodium lauryl sulfate (SLS) to transform hemoglobin to SLS-methemoglobin. This method does __.
o __ – an example of a commercially available handheld system to measure the hemoglobin concentration
- In here, hemoglobin is converted to __ and is read photometrically at two wavelengths (570 nm and 880 nm).
light
NOT produce toxic wastes
HemoCue
azidemethemoglobin
HEMOGLOBIN ELECTROPHORESIS:
♦ __ - movement of charged particles in an electric field
__:
* Considered as the primary screening procedure to detect variant (abnormal) hemoglobins
* In an alkaline buffer (8.4 to 8.6) hemoglobin is a __ charged molecule
* During electrophoresis, the Hb molecules travel toward the anode (+) because of their net negative charge
* The difference in the net charge of the Hb molecule defines its mobility and reveals itself by the speed with which it migrates to the positive pole.
* FASTEST (normal Hb) = _______
*NOTE: fast hemoglobins = ___________
* SLOWEST = __________
*NOTE: Hb S, Hb D and Hb G migrate to the same area at cellulos acetete electrophoresis
_______________________
* Used to confirm variant hemoglobins and further differentiates hemoglobin S from D and G, and hemoglobin C from hemoglobins E, OArab, CHarlem
Electrophoresis
* Cellulose Acetate (pH 8.4-8.6)
negatively
Hemoglobin A1
Bart Hb, Hb H, Hb I
HbC, HbA2, HbE, Hb C Harlem, Hb O Arab
●HEMOGLOBIN SYNTHESIS
A.) __:
o HEME a.k.a. ___
o Belongs to class of pigments known as porphyrins
o Site of heme synthesis: ________
o Heme biosynthesis:
-occurs in all metabolically active cells containing mitochondria
-most prominent in bone marrow and liver.
- Erythroid marrow is the MAJOR heme-forming tissue, generating __ of the daily heme requirement.
Ferrochelatase
- aka: Heme synthetase
- enzyme needed to insert the Fe+2 to the protophorphyrin IX ring
Heme Synthesis
Ferroprotoporphyrin IX
mitochondrion; 85%
B.) Globin Synthesis
o Site: __ in the normoblast cytoplasm
o Chromosome 16 = dictates the production of __
o Chromosome 11 = dictates the production of __
Theta: chromosome __
ribosomes
A and Z; B, E, D and G globin chain
16
FORMS OF HEMOGLOBIN (ACCORDING TO STAGE OF LIFE):
hemoglobins present in the embryonic life?
molecular structure of each?
what are the adult forms of hemoglobin? molecualr stucture and the proportions present in adult/baby?
other name for HbF
(zige, ez ean)
Portland - 2 zeta, 2 gamma
Gower I - 2 zeta, epsilon
Gower II - 2 alpha, 2 epsilon
(ah g, aba ang dali)
F - 2 alpha, 2 gamma (80% newborns; <1% adults)
A1 - 2 alpha, 2 beta (20% newborns; 97% adults)
A2 - 2 alpha, 2 delta (<0.5% newborns; 2.5% adults)
fetal hemoglobin
HEMOGLOBIN DERIVATIVES:
symbol: HbO2
Hb with Fe+2 + oxygen; found in arterial blood
conformation of Hb in the oxygenated state is: ___
Hb with Fe+2, NOT bound to oxygen
Found in venous blood (dark red)
conformation of Hb in the deoxygenated state is: ___
Oxyhemoglobin; R state/relaxed
Deoxygenated hemoglobin; T state “tense”
DYSHEMOGLOBINS:
-symbol: HbCO
Hb w/Fe+2, bound to CO (carbon monoxide)
CO gas - has __ greateraffinity to Hb than O2 (is tasteless, odorless, colorless
color of blood and skin in HbCO poisoning: ____
symbol: Hi
other names: ___
Hb with Fe+3 not bound to O2
color of blood (methemoglobinemia): ___
symbol: SHb
mixture of oxidized, partially denatured forms of Hb
some cases of SHb formation: prolonged, constipation, enterogenous cyanosis, bacteremia (caused by C. perfringens)
*In-vitro, sulfhemoglobin forms when hydrogen sulfide is added to Hb, thus the name sulfhemoglobin.
*In-vivo, sulfhemoglobin forms in the occasional patient as a result of Hb oxidation by certain drugs and chemicals.
color of blood (sulfhemoglobinemia): ____
Carboxyhemoglobin; 240x; CHERRY RED
Methemoglobin; ferrihemoglobin, hemiglobin; CHOCOLATE BROWN
Sulfhemoglobin; MAUVE-LAVENDER
LAYERS OF HEMATOCRIT:
1st to 4th
-“__”- actually pertains to the instrument used to determine packed cell volume [PCV]
-After centrifugation of an anticoagulated whole blood specimen, the red blood cells along with other formed elements (white blood cells and platelets) will settle at the bottom of the tube.
-The volume of the red blood cells that have settled is called the __ otherwise known as the hematocrit.
-Micro-Hematocrit Tube:
o Approx. __
o Inner bore: __
o Can hold __ of bld.
o Plug: __ long (seal the capillary tubes at the end of the tube with the colored ring)
2 TYPES:
with RED band: has ___
with BLUE band: has ___
how many tubes are needed for collection? how much blood needed? which end is it filled? which end is sealed? RPM used? results for both tubes should agree __; which layer should not be included?
-REMINDERS:
o __ causes microhematocrit to be 1% to 3% (0.01 to 0.03 L/L) higher than the value derived from automated instruments that calculate or directly measure the hematocrit and are unaffected by the trapped plasma
o __ – a calculated value from RBC and MCV
o The difference in the hematocrit results is typically insignificant, UNLESS there is a more severe case of poikilocytosis and anisocytosis.
fatty layer>plasma layer>buffy coat>packed rbc
Hematocrit
packed cell volume
75 mm long; 1.2 mm
0.05 mL
4 to 6 mm
heparin anticoagulant
NO anticoagulant
2 heparinized capillary tubes, filled 2/3 of the tube on the end without a marked band and then sealed with wax on the marked band. centrifuged 10,000 RPM (to 15,000) for 5 mins.
within 1%; buffy coat
Trapped plasma
Automated hematocrit
- used for checking validity of test results
-works only on normocytic, normochromic specimens; formulas:
B.) ERYTHROCYTE INDICES
▪ __:
o Average volume of an individual RBC
o Formula: __; R.R.: 80 to 100 femtoliters
__:
o Average weight or amount of hemoglobin in an individual RBC
o MCH follows the MCV (smaller RBCs necessarily hold less hemoglobin; larger RBCs can hold more hemoglobin)
o Formula: __; R.R.: 26 to 32 picograms
__:
o Mean concentration of hemoglobin in the average RBC
o Formula:__; R.R.: 31 to 37 g/dL or %
RULE OF THREE
RBC ct x 3 = Hb +/- 3
Hb x 3 = Hct +/- 3
Mean Cell Volume (MCV);
MCV = Hct/RBC ct. x 10
Mean Cell Hemoglobn (MCH)
MCH= Hb/RBC ct. x 10
Mean Cell Hemoglobin Concentration (MCHC)
MCHC = Hb/Hct x 100
C.) POLYCYTHEMIA
* CLASSICALLY defined as __ above normal
* In the clinical setting: polycythemia occurs when __ and __ count are elevated (reflecting an elevation of the total erythrocyte volume.
* Two general kinds:
o Absolute polycythemia: elevated total __
o Relative polycythemia: normal total red cell mass, but __ because plasma volume is decreased
hematocrit level
hemoglobin and RBC
red cell mass; hematocrit is increased
Secondary polycythemia with appropriately EPO production
*Decreased oxygen __: hypoxia, high altitude; pulmonary disease; cyanotic heart disease; carboxyhemoglobinemia; methemoglobinemia; Hb M
*Decreased oxygen __: high oxygen affinity hemoglobinopathy, biphosphoglycerate deficiency
b.) Secondary polycythemia with __ EPO production
*Neoplasms: Wilms’ tumor, renal carcinoma
;cerebellar hemangioma; hepatoma
*Localized tissue hypoxia: polycystic kidney; renal artery stenosis
*Post-renal transplant
*Acute hepatitis
a.) Diminished plasma volume: dehydration; shock
b.) Spurious polycythemia (stress polycythemia; __)
c.) __ - *Primary familial congenital polycythemia (mutated Epo receptor); *__ (mutated VHL gene)
d.) Primary marrow disorders - *POLYCYTHEMIA VERA
PV – Polycythemia vera (aka: __)
- It is a panmyelosis (a case in which excessive proliferation occurs in RBCs, megakaryocytes and granulocytes).
- Manifested by erythrocytosis, leukocytosis, and thrombocytosis of varying degree
-Most patients (90%-97%) with PV has ________ mutation
- Hallmark: ___; Treatment of choice: therapeutic phlebotomy (at a frequency necessary to maintain the hematocrit at < 45%)
ABSOLUTE POLYCYTHEMIA
LOADING
UNLOADING
inappropriately
RELATIVE POLYCYTHEMIA; Gaisböck’s syndrome
Genetic polycythemia; Chuvash polycythemia
Polycythemia Rubra Vera
JAK2 V617F
PLETHORA
Rate of fall of RBCs settling to the bottom of the tube (distance in mm. that the RBCs fall in 1 hr.). It is ordered with other tests to detect and monitor the course of inflammatory conditions (such as rheumatoid arthritis, an example of a chronic inflammatory condition).
METHODS OF ESR DETERMINATION:
▪ __– results are determined after 1 hr. & after 2 hrs.; Anticoagulant used: _____
__ – results are determined after 1 hr; Most commonly used method; Recommended by the ICSH and CLSI; Anticoagulant used: ________; Diluents: ____; Diluted sample is placed in a 200-mm column with an internal diameter of 2.55 mm or more.
▪ __ – results are determined after 1 hr; Anticoagulant used: __; Left side (red) → 0 (top) and 10 cm (bottom) (for ESR); Right side (white) → 10 cm (top) and 0 (bottom) (for Macro-hematocrit)
___: a bench top analyzer designed to measure ESR in 20 samples; blood is obtained in special cuvets, and mixed by the instrument; samples are then left to sediment for a specific period
✓ __ slant of the tubes with respect to the vertical axis: causes acceleration in sedimentation, allowing results comparable to those of Westergren at the 1st hour to be obtained in only __, while those comparable to Westergren at the 2nd hour require only __
✓ The optoelectric sensors automatically read the RBC sedimentation level.
NORMAL VALUES IN MOD. WESTERGREN
~ Tilted ESR tube causes 30% error even with a 3deg tilt
ERYTHROCYTE SEDIMENTATION RATE
Westergren; 3.8% sodium citrate (black top)
Modified Westergren; EDTA; 0.85% NaCl or 3.8% sodium citrate
Wintrobe; double oxalate or EDTA
VES-MATIC 20 Instrument; 18-degree; 25 minutes; 45 minutes
Adult Males: 0 to 10 mm/hour
Adult Females: 0 to 15 mm/hour
MORPHOLOGIC CLASSIFICATION OF ANEMIA:
(MCV and MCHC =both normal); Normal or Decreased Reticulocyte Count (mnemonic)
Increased Reticulocyte Count:
- PNH, PCH (Donath-Landsteiner Hemolytic Anemia), Sickle cell disease, Enzyme def (G6,PK), other hemolytic anemias
___:
o described as a rare but potentially deadly bone marrow failure syndrome; characteristic features:
* pancytopenia (marked ↓ in the number of RBCs, WBCs, and platelets in the blood)
* reticulocytopenia
* bone marrow hypocellularity
* depletion of hematopoietic stem cells
May be classified as:
__; approximately 80 to 85% of aplastic anemia cases; may be idiopathic or secondary acquired. (from chems, drug, viruses)
__: approximately 15% to 20% of aplastic anemia cases; associated diseases are:
(1) Dyskeratosis congenita
(2) Shwachman-Bodian-Diamond syndrome
(3) __ - most common of the inherited aplastic anemias; a chromosome instability disorder; characterized by: aplastic anemia, cancer susceptibility, and physical abnormalities
___:
o A.k.a.: _______; caused by deficiency of:
▪ DAF (decay-accelerating factor, or CD55)
▪ MIRL (membrane inhibitor of reactive lysis, or CD59)
○ Tests for PNH:
⸙ Ham’s Acidified Serum Test
⸙ Sugar Water Test or __
⸙ Flow cytometry (using FLAER) - ___
NORMOCYTIC, NORMOCHROMIC ANEMIAS
AKA (aplastic anemia, kidney disease and acute blood loss)
APLASTIC ANEMIA
I.) Acquired Aplastic Anemia
II.) Inherited Aplastic Anemia; Fanconi Anemia (FA)
PAROXYSMAL NOCTURNAL HEMOGLOBINURIA; Marchiafava-Micheli Syndrome
sucrose hemolysis test
confirmatory test for PNH
__ – defined as the decrease below normal of one or more of the following:
o Number of red blood cells
o Hemoglobin
o Volume of packed red cells (hematocrit)
ANEMIA
ANEMIAS:
With small cells that have increased central pallor on the smear (MCV and MCHC = both LOW); Microcytic anemia results from an iron level insufficient for maintaining normal erythropoiesis and is characterized by abnormal results of iron studies. Early development of a microcytic anemia may reveal reduced iron stores, but an obvious anemia has not developed.
Associated conditions [ mnemonic ] :
___
* was originally called __
* most common anemia among hospitalized patients
* associated with chronic infections such as tuberculosis, chronic inflammatory conditions such as rheumatoid arthritis, and tumors
* body stores have abundant iron; red cells are deficient in iron
* Central feature of ACI: sideropenia (↓ serum iron) despite abundant iron stores
* Usually PBS shows __; advanced states: __
* Acute phase reactants (APRs) that contribute to ACI: (3)
~ master regulatory hormone for systemic iron metabolism
-inactivates FERROPORTIN!!!
MICROCYTIC, HYPOCHROMIC ANEMIAS
CTAILS:
chronic blood loss
thalassemia
anemia of chronic inflammation
iron def anemia
lead poisoning
siderobalstic anemia
Anemia of chronic inflammation (ACI); ACD (anemia of chronic disease); normocytic, normochromic; microcytic, hypochromic
o Hepcidin; Ferritin; Lactoferrin
hepcidin
ANEMIAS: (microcytic hypochromic)
__: develop when the production of protoporphyrin or the incorporation of iron into protoporphyrin is prevented
* __ – nucleated RBC precursor with cytoplasmic iron granules
* __ – anucleate RBC w/ iron granules
* Iron is trapped in the __ (cannot be fully utilized in Hb synthesis)
* __ – hallmark of the sideroblastic anemias
* The “rings” in ring sideroblasts = __
* Dimorphic peripheral blood picture (normochromic and hypochromic RBCs)
___:
▪ __ – the most common anemia
▪ possible causes:
o Blood loss (especially menstruating women)
o Nutritional deficiency (infants)
o ↑iron demand (pregnancy, lactation, adolescence)
o Malignancies of gastrointestinal tract
o Hookworm infections
___: indirectly measures the concentration of transferrin by measuring its ability to bind iron
__: reveals the body’s tissue iron stores, good indicator of iron
storage status; useful in diagnosing iron deficiency (generally
the first laboratory test to become abnormal when iron stores begin
to decline); measured using radioimmunoassay
Sideroblastic anemias
SIDEROBLAST
SIDEROCYTE
mitochondria
Ring sideroblasts
mitochondria loaded with iron
Iron deficiency anemia (IDA)
Iron deficiency
TIBC
Serum Ferritin Test
ANEMIAS:
(MCV= HIGH, MCHC=NORMAL)
MEGALOBLASTIC ANEMIA
__ – may be caused by:
✓ inadequate intake
✓ increased need (occurs during pregnancy, lactation and growth)
✓ competition (ex.: D. latum infection)
✓ lack of intrinsic factor (IF) (ex.: gastrectomy, H. pylori infection, Pernicious Anemia)
___ – may be caused by:
✓ inadequate intake
✓ increased need
✓ renal dialysis
__ – generally characterized by the
presence of:
➢ Antibodies directed against PARIETAL CELLS
➢ Antibodies directed against INTRINSIC FACTOR (IF)
__ - found in the stomach; produce HCl and Intrinsic Factor (IF)
___:
* Liver disease
* Alcoholism (chronic)
* Bone marrow failure
MEGALO VS NONMEGALOBLASTIC ANEMIA
- Presence of hypersegmented neutrophils (with > 6 lobes)
- Shape of the macrocytes
- Presence of megaloblasts in the bone marrow
~ Normal neutrophils have __ nuclear lobes.
*Hypersegmented neutrophils - essentially pathognomonic for megaloblastic anemia
MACROCYTIC, NORMOCHROMIC ANEMIAS
Vitamin B12 deficiency
Folate deficiency
Pernicious Anemia
Parietal cells
NONMEGALOBLASTIC ANEMIA
MA: present. oval, present
NMA: absent, round, absent
3 to 5
__ - considered as root cause of megaloblastic anemia
➢ DNA synthesis is dependent on an important structure: __. This structure cannot be made unless it receives a methyl group from methyl tetrahydrofolate or folic acid. __ is the cofactor responsible for transferring the methyl group to methyl tetrahydrofolate. Adequate amounts of vitamin B12 and folic acid are important to the formation of TTP. If TTP cannot be made, then it is replaced by __. The synthesis of this structure results in nuclear fragmentation and
destruction of cells and impaired cell division.
➢ Vitamin B12 is also known as __. Folate is the general term used for any form of the folic acid.
➢ ___ - synthetic form of vitamin B12 (found only in supplements)
➢ ___ - naturally occurring form that may be obtained from either food sources or supplements
➢ In either folate or vitamin B12 deficiency, patients may experience: fatigue, weakness, and shortness of breath; glossitis (loss of epithelium on the tongue results in a smooth surface and
soreness); gastritis, nausea, or constipation
- In __ deficiency, neurologic symptoms may be noticeable and neuropsychiatric symptoms may also be present.
- Dietary deficiency of vitamin B12 is infrequent. However, this condition is possible for __ who do NOT eat meat, eggs, or dairy products.
Sources of Vitamin B12: meat, animal products, dairy (is not destroyed by cooking)
Sources of Folate: grains, fruits, vegetables (heat labile (overcooking of foods can ↓ their nutritional value)
what is the metabolically active form of Vitamin B12?
Impaired DNA synthesis
thymidine triphosphate (TTP)
Vitamin B12
deoxyuridine triphosphate (dUTP)
Cobalamin
Cyanocobalamin
Methylcobalamin
vitamin B12
vegans
holotranscobalamin (holoTC),
___:
- disorders characterized by impaired synthesis of HEME
- example: LEAD POISONING.
*Lead can inhibit many enzymes in the body, including __ (2)
* __: enzyme needed to completely remove the RNA remnants from reticulocytes.
___: due to differences in the arrangement of amino acids in the polypeptide chain; Major Groups: (4)
♦β-hemoglobinopathies
- both B genes: MUTATED
- HbA1 is absent
- abnormal hemoglobin becones the predominant Hb type
EX: sickle cell dse - Hb SS; Hb C disease Hb CC
- one of the B genes: NORMAL other is MUTATED
- Hb A1 ≥ Abnormal Hb
EX: sickle cell trait: Hb AS; Hb C trait: Hb AC
Examples of abnormal hemoglobins present in certain β-hemoglobinopathies:
1.___; most severe and most common; structural form?
2. __; 2nd most common; structural form?
3. __; 3rd most commoon; structural form?
*Porphyrias
ferrochelatase and pyrimidine-5’-nucleotidase
Pyrimidine-5’-nucleotidase
Hemoglobinopathies
Alpha-hemoglobinopathies
Beta-hemoglobinopathies
Delta-hemoglobinopathies
Gamma-hemoglobinopathies
Homozygous β-hemoglobinopathies
Heterozygous β-hemoglobinopathies
Hb S: A2B2 - 6GLU->VAL
Hb C: A2B2 - 6GLU->LYS
Hb E; A2b2 - 26glu->LYS
___:
✓ Glutamic acid on the 6th position of the β chain is replaced by valine
✓ When oxygenated, Hb S is fully __ (sickling happens when oxygen drops at the tissue level). When oxygen is released from the molecule, a conformational change happens which results in polymerization of hemoglobin molecules leading to the formation of ___ (in turn, this causes the cells to become rigid).
✓ Degree of sickling depends on the concentration of hemoglobin S in the RBC: If the concentration of Hemoglobin S is __ (as in sickle cell anemia), sickling of the red blood cells occurs readily at only slightly decreased oxygen concentrations
✓ If the concentration of Hemoglobin S is only ___ (as in the sickle cell trait), oxygen concentrations must be much lower before sickling occurs.
✓ Sickle cells obstruct blood flow to tissues and organs (resulting in tissue death, organ infarction, and pain).
TESTS FOR HEMOGLOBIN S
* Sodium metabisulfite method - Whole blood is mixed with a reducing agent which deoxygenates Hb; In such conditions, Hb S existing in the RBC causes the formation of sickle shaped RBCs; (+) RESULT IS?
* Sodium dithionite tube test (Solubility test); When RBCs are added to the working solution containing the reducing agent and saponin (hemolytic agent), the red cells immediately lyse; Hb S in the reduced state (in a concentrated buffer solution), forms liquid crystals and produces a __; (+) result is?
* Hemoglobin Electrophoresis (__); primary screening procedure to detect variant (abnormal) hemoglobins]; In an alkaline buffer (8.4 to 8.6) hemoglobin is a negatively charged molecule; During electrophoresis, the Hb molecules travel toward the anode (+) because of their net negative charge;
* Hemoglobin Electrophoresis (__): CONFIRMATORY TEST; Used to confirm variant hemoglobins and further differentiates hemoglobin variants; Migration distances of the different hemoglobins are based on the electrophoretic charge of the molecules and their adsorption to the agar compound
HEMOGLOBIN S
soluble
tactoids or crystals
80 to 100%
20 to 40%
POSITIVE (+) – presence of either sickle cells or “holly-leaf” form of the RBCs
turbid appearance; POSITIVE (+) – Turbid solution
(Cellulose Acetate)
(Citrate Agar)
__: Glutamic acid on the 6th position of the beta chain is replaced by lysine; ✓ Two crystals related to it:
(1) Hb SC crystals:
- characteristic appearance: ________________________
-found protruding the RBC membrane
(2) Hb CC crystals:
- characteristic appearance: ________________________
-found within the RBC membrane
HEMOGLOBIN C
Washington Monument
Bar of gold
Initially called “Thalassic (Greek for “great sea”) Anemia”
♦Other names: ________ , __________
♦ Reduction or total absence of synthesis of one or more of the globin chains; named according to the chain with reduced or absent synthesis
♦ Mutations affecting the ___ gene – most clinically significant (the reason: Hb A (α2β2) is the major adult hemoglobin)
♦ Thalassemia occurs in all parts of the world. However, its distribution is concentrated in the “thalassemia belt”.
o Thalassemia minor (heterozygous thalassemia) seems to impart resistance to __.
♦ Individual and family histories are important in thalassemia diagnosis. The ethnic background of the person should be investigated because of the increased prevalence of particular gene mutations in specific populations.
♦ Clinical findings that suggest thalassemia include:
✓ pallor (due to the anemia)
✓ jaundice (due to hemolysis)
✓ splenomegaly (due to sequestration of abnormal red cells, too much
extravascular hemolysis, and some extramedullary erythropoiesis)
✓ skeletal deformities (due to the massive expansion of the bone marrow cavities
SCREENING TESTS: (3)
CONFIRMATORY TEST:
Thalassemia; Hereditary leptocytosis, Mediterrenean anemia
α- or β-globin
malaria
Complete Blood Count (CBC), PBS and iron studies
Molecualr genetic test
THALASSEMIAS: In NORMAL individuals, genotype is: ____
___: typically caused by a deletion of one, two, three or all four of the α-globin genes (results in reduced or absent production of α chains)
___: One of four genes is deleted; genotype:__; is asymptomatic
___: three of four genes are deleted; genotype: __; mild micro, hypo anemia; __ found in adults; __ found in neonates; Hb H may coexist with __ (a more severe disease than Hb H)
___: Two of four genes are deleted; genotype: __; mild micro, hypo anemia
__: Four of four genes are deleted; genotype: __; MOST SEVERE FORM OF ALPHA THALASSEMIA
ALPHA THALASSEMIA
(aa/aa).
silent carrier state (α-thalassemia); (-a,aa)
Hb H disease; (–,-a); Hb H; Hb Bart; Hb Constant Spring
α-thalassemia trait/a-thalassemia minor; (–,aa) or (-a,-a)
Hb Bart hydrops fetalis syndrome; (–,–)
THALASSEMIAS: In NORMAL individuals, genotype is: __
caused by mutations that affect the β-globin gene complex
β silent / β; asymptomatic; normal hematologic parameters
β+/ β or β0/ β; asymptomatic; mild hemolytic anemia; micro, hypo RBCs
β+/β+ or β+/ β0 or β0/β0; MOST SEVERE FORM OF BETA THALASSEMIA; severe hemolytic anemia; microcytic, hypochromic
RBCs; transfusion-dependent
βsilent/βsilent or β+/βsilent or β0/βsilent; moderate clinical
symptoms, transfusion-independent, mild to moderate
hemolytic anemia, microcytic, hypochromic RBCs3
β-thalassemic genes:
β0 = β-globin gene mutation in which __ are produced
β+ = β-globin gene mutation that results in __ reduction in
β chain production
βsilent = β-globin gene mutation that results in __ β chain production
BETA THALASSEMIA; B/B
β-thalassemia silent carrier state
β-thalassemia trait/beta thalassemia minor
β-thalassemia major/Cooley’s anemia
β-thalassemia intermedia
no β chains
5% to 30%
mildly decreased
WHITE BLOOD CELLS:
☼Nucleated cells that function in body defense.
Reference ranges
◘ Adult: ___(SI) [or, 4,500 to 11,000 /mm3 (conventional)]
◘ Newborn: 13.5 to 38.0 X 10^9/L (SI) [or, 13,500 to 38,000/mm3 (conventional)]
Predominant WBC in an adult: ___________
☼ Predominant WBC in children <4 years of age: ___________
☼ On determining WBC maturity, MOST valuable and reliable criterion is: __________________
☼ WBC Classifications:
GRANULOCYTES? AGRANULOCYTES? (same as polymorphonuclear and mononuclear
what is the only wbc that is an immunocyte?
4.5 to 11.0 X 10^9/L
Neutrophils
Lymphocytes
nuclear chromatin pattern
GRAN: Basophils, Eosinophils, Neutrophils
AGRAN: Lymphocytes, Monocytes
lymphocyte
GRANULOCYTIC SERIES:
14 to 20 μm; the earliest recognizable granulocytic precursor using the LM; __ of the nucleated cells in the bone marrow
THREE TYPES:
___: nucleus occupies most of the cell, with very little cytoplasm, slightly basophilic cytoplasm, fine nuclear chromatin, and two to four visible nucleoli, no visible granules when observed under light microscopy with Romanowsky stains
___: shows the presence of dispersed primary (azurophilic) granules in the cytoplasm; the number of granules does NOT exceed 20 per cell
___: have a darker chromatin and a more purple cytoplasm, and they contain more than 20 granules that do NOT obscure the nucleus; rare in normal bone marrows, but they can be seen in certain types of AML
MYELOBLAST
♦Type I myeloblasts:
♦Type II myeloblasts:
♦ Type III myeloblasts
GRANULOCYTIC SERIES:
16 to 25 μm; ___ of the nucleated cells in the bone marrow; relatively larger than the myeloblast; nucleus round to oval, often eccentric; “__” is usually seen in normal promyelocytes but NOT in the malignant promyelocytes of acute promyelocytic leukemia
*cytoplasm is evenly basophilic and full of primary (azurophilic/nonspecific) granules; __ nucleoli can be seen but may be obscured by the granules
~ PRIMARY GRANULES begins in the? (2)
PROMYELOCYTE
1 to 5%
hof
one to three
BEST IS: type II myeloblast, or promyelocyte
GRANULOCYTIC SERIES:
15 to 18 μm; 6 to 17% of the nucleated cells in the bone marrow; last stage capable of ___; stage of synthesis of secondary granules;
Two types:
I) ___: may look very similar to the promyelocytes
(EXCEPT that patches of grainy pale pink cytoplasm
*Source of the picture representing secondary granules start to become obvious in the area of the Golgi apparatus (called: ____)
II.) ___: smaller than promyelocytes (15 to 18 um), and the nucleus has considerably more heterochromatin , nucleoli are difficult to see by light microscopy
MYELOCYTE
MITOSIS
Early myelocytes
Dawn of neutrophilia
Late myelocytes
GRANULOCYTIC SERIES:
14 to 16 μm; __ of the nucleated cells in the bone marrow; Nucleoli are absent; Synthesis of ___ (also known as gelatinase granules) may begin at this stage. other name is: __
first stage of nuclear indentation!! (not more than 1/2 indentation of the nucleus
9 to 15 μm; __ of the nucleated cells in the bone marrow; other name is: ___; youngest granulocytic precursor to normally appear in the peripheral blood; __ (also known as secretory vesicles) may begin to be formed during this stage
*nucleus: elongated, curved, or __ with rounded ends (filaments NOT present)
* CLSI recommends that bands should be included within the neutrophil counts and NOT reported as a separate category (due to the difficulty in reliably distinguishing bands from
segmented neutrophils)
METAMYELOCYTE; 3 to 20%
tertiary granules
Juvenile cell
BAND CELL; 9 to 32%; Stab cell or Staff cell
secretory granules
sausage-shaped
MATURE GRANULOCYTES:
9 to 15 μm; __ of the nucleated cells in the bone marrow; Increased (↑) level in the blood is called: ____
*Some associated conditions:
- Bacterial infections (generally)
- Physiologic neutrophilia (a.k.a.
”Pseudoneutrophilia”)
Decreased (↓) level in the blood is called: ____
*__: (aka: EXTREME NEUTROPENIA) is neutrophil count: _____; associated drugs: amidopyrine and cephalosporin
SEGMENTED NEUTROPHIL
7 to 30%
neutrophilia
neutropenia
Agranulocytosis; <0.5 x 10^9/L
MATURE GRANULOCYTES:
9 to 15 μm Increased (↑) level in the blood is called: _____
*Some associated conditions:
✓ Asthma
✓ Scarlet fever
✓ Eosinophilic leukemia
✓ Parasitic infections - caused by ___; it may produce the highest eosinophil count
Decreased (↓) level in the blood is called: __
*Associated condition:
✓ ACTH administration
✓ Autoimmune disorders
✓ Steroid therapy
✓ Stress
EOSINOPHIL; eosinophilia
T. spiralis
eosinopenia
MATURE GRANULOCYTES:
10 to 16 μm; Increased (↑) level in the blood is called: ______
✓ Immediate hypersensitivity reactions
✓ Hypothyroidism
✓ Ulcerative colitis
✓ Estrogen therapy
Decreased (↓) level in the blood is called: ____
*Associated condition:
✓ Stress
✓ Hyperthyroidism
BASOPHIL; basophilia
basopenia
AGRANULOCYTIC SERIES: LYMPHOCYTIC SERIES
___: 15 to 20 μm; 4:1 N:C
___: 15 to 18 μm; 4:1 to 3:1 N:C
*Small = 6 to 9 μm; *Small = 4:1 to 3:1
*Large = 17 to 20 μm; *Large = 2:1
__ - predominant type of lymphocyte (normal adult blood)
__ - composed mostly of nucleus and has a scanty cytoplasm
LYMPHOBLAST
PROLYMPHOCYTE
MATURE LYMPHOCYTE
SMALL LYMPHOCYTE
AGRANULOCYTIC SERIES: LYMPHOCYTIC SERIES
(10 to 28 μm)
- Final maturation stage of: ______
- Nucleus: small, oval and __
: “__”, “__” or “clock face” in appearance
-Cytoplasm: ___ in color
: may contain round, discrete globules (called:______) that contain immunoglobulins
-Most common malignant disease of plasma cells: _______
PLASMA CELLS
B lymphocytes; eccentric
tortoise shell; cartwheel
dark blue/sea blue/cornflower; Russell bodies
plasma cell myeloma
AGRANULOCYTIC SERIES: MONOCYTIC SERIES
12 to 20 μm; 4:1
12 to 20 μm; 3:1 to 2:1
12 to 18 μm; 2:1 to 1:1
__ -slightly immature cell (immature macrophage); passing through the blood (from its point of origin to a targeted tissue location)
- its ultimate goal: to enter the tissues and mature into a macrophage, osteoclast, or dendritic cell
__: (size: 40 to 50 μm)
“monocytes” found in tissues; most abundant cell type in the body; Some of the functions of macrophages:
◘ Phagocytosis
◘ Synthesize nitric oxide (cytotoxic against viruses, bacteria, fungi, protozoa, helminths, and tumor cells)
◘ Release interleukin 1 (stimulates T lymphocytes)
◘ Produce transcobalamin II (the primary transport factor for vitamin B12)
MONOBLAST
PROMONOCYTE
MATURE MONOCYTE
MONOCYTE
MACROPHAGES
SPECIFIC NAMES OF MACROPHAGES and LOC:
Liver:
Lungs: (aka: ____________________)
Kidneys:(aka: ____________________)
Brain:
Skin:
Spleen: (aka: _____________________)
Intestines:
Peritoneum:
Synovial tissue:
Reproductive organ:
Bone:
Placenta:
Lymph nodes:
Kupffer cells
Alveolar macrophages; dust cells
Renal macrophages ; mesangial cells
Microglial cells
Langerhans cells
Splenic macrophages; littoral cells
Intestinal macrophages
Peritoneal macrophages
Type A cells
Reproductive organ macrophages
Osteoclast
Hoffbauer cells
Dendritic cells
●NUCLEUS: contain 3 to 5 lobes (separated by filaments)
●CYTOPLASM: Pink to tan with violet or lilac granules
✓ MOST COMMON WBC in the normal peripheral blood
✓ commonly the first phagocytes to reach the infected areas and are then followed by the __.
✓ Neutrophils move in a __ manner, but their movement changes into a straight line path if a chemotactic attractant or factor (e.g., a bacterium coated with particular components of complement) is within a particular distance.
✓ Neutrophils share a common progenitor with monocytes: ____
✓ Major stimulatory cytokine for neutrophil production: ____
✓ Half-life of neutrophils in the blood is relatively short (approximately 7 hours)
✓ 3 MAJOR FUNCTIONS:
(1) phagocytosis and destruction of foreign material and microorganisms
(2) generation of NETs (neutrophil extracellular traps when they die); __: unique form of neutrophil cell death results in the release of NETs
(3) secretory function (neutrophils are a source of a variety of cytokines and also a source transcobalamin I or R binder protein
Neutrophils
monocyte
zigzag
GMP (granulocyte-monocyte progenitor)
G-CSF (granulocyte colony-stimulating factor )
NETosis
- Formed during the promyelocyte stage
- Last to be released (exocytosis)
Contain: (7) - Formed during the myelocyte and metamyelocyte stages; Third to be released
- Contain: (7)
- Formed during the metamyelocyte and band stages; Second to be released
- Contain:
o Gelatinase
o Collagenase
o Lysozyme
o Acetyltransferase
o β2-microglobulin - Formed during the band and segmented neutrophil stages
- First to be released (fuse to plasma membrane)
- Contain (attached to membrane):
o CD11b/CD18
o Vesicle-associated membrane-2
o CD10, CD13, CD14, CD16
o Cytochrome b558
o Complement 1q receptor
o Alkaline phosphatase
o Complement receptor-1
PRIMARY (AZUROPHILIC) GRANULES (CLAMPED)
o Myeloperoxidase (MPO)
o Acid β-glycerophosphatase
o Lysozyme/muramidase
o Cathepsins
o Defensins
o Elastase
o Proteinase-3
SECONDARY (SPECIFIC) GRANULES
o β2-microglobulin
o Collagenase
o Gelatinase
o Lactoferrin
o Neutrophil gelatinase-associated lipocalin
o Transcobalamin I
o Lysozyme
TERTIARY (GELATINASE GRANULES)
SECRETORY GRANULES (SECRETORY VESICLES)
NEUTROPHIL POOLS:
A.) In the __:
(1) Stem cell pool: ___
✓ Hematopoietic stem cells
(2) Mitotic (Proliferation) pool
includes:
✓ CMP (aka: CFU-GEMMs) [colony-forming units-granulocyte, erythrocyte, monocyte, and megakaryocyte]
✓ GMP
✓ Myeloblast
✓ Promyelocyte
✓ Myelocyte
B.) In the __: has a ___ ratio
(1) Circulating Neutrophil Pool (CNP); found in the plasma
(2) Marginal Neutrophil Pool (MNP); found adhering on the blood vessel wall
(3) Storage (Maturation) pool
✓ Metamyelocyte
✓ Band
✓ Segmented neutrophils
BONE MARROW; CD34
PERIPHERAL BLOOD; 50:50
●NUCLEUS: Dark purple, usually has __
●CYTOPLASM: Filled with large, spherical granules of uniform size that stain __
▪ __- most specific cytokine for eosinophil lineage
– promotes: terminal maturation, functional activation, and prevention of apoptosis
▪ Tissue destinations of eosinophils under normal conditions appear to be the underlying __ in the respiratory, gastrointestinal, and genitourinary tracts.
▪ Eosinophilia is a hallmark of __ disorders.
*Eosinophil production is increased in helminthic infections, and in vitro studies have shown that this leukocyte is capable of destroying tissue-invading helminths by secretion of MBP and eosinophil cationic protein as well as the production of reactive oxygen species.
▪Eosinophils regulate mast cell function through the release of __ (causes mast cell degranulation and cytokine production), and they also produce nerve growth factor that promotes mast cell survival and activation
▪ Circulating half-life of eosinophils: roughly ___
▪ Survival time of eosinophils in human tissues ranges from __
EOSINOPHILS; two lobes; bright orange
IL-5
columnar epithelial surfaces
allergic
MBP [major basic protein]
18 hr
2 to 5 days
▪EOSINOPHIL GRANULES
✓ PRIMARY GRANULES
=Formed during the __ stage
=Contain: ___ protein
✓ SECONDARY GRANULES
=Formed throughout remaining __
=Contain:
o ___
o ___
o Eosinophil-derived neurotoxin (matrix)
o Eosinophil peroxidase (matrix)
o __
o __
o β-Glucuronidase (core and matrix)
o Cathepsin D (core and matrix)
o Interleukins 2, 4, and 5 (core)
o Interleukin-6 (matrix)
o Granulocyte-macrophage colony-stimulating factor (core)
o Others
✓ SMALL LYSOSOMAL GRANULES
- Acid phosphatase
- Arylsulfatase B
- Catalase
- Cytochrome b558
- Elastase
- Eosinophil cationic protein
✓ LIPID BODIES
- Cyclooxygenase
- 5-Lipoxygenase
- 15-Lipoxygenase
- Leukotriene C4 synthase
- Eosinophil peroxidase
- Esterase
✓ STORAGE(?) (SECRETORY) VESICLES
- Carry proteins from secondary granules to be released into the extracellular medium
promyelocyte
Charcot-Leyden crystal
maturation
Major basic protein (core)
Eosinophil cationic protein (matrix)
Lysozyme (matrix)
Catalase (core and matrix)
Four methods of eosinophil degranulation:
1.) __: granules move to the plasma membrane, fuse with the plasma membrane, and empty their contents into the extracellular space
2.) ___: granules fuse together within the eosinophil prior to fusing with the plasma
membrane
3.) ___; secretory vesicles remove specific
proteins from the secondary granules. These vesicles then travel to the plasma membrane and fuse to empty the specific proteins into the extracellular space.
4.) ___: happens when extracellular intact granules are deposited during cell lysis
Classical exocytosis
Compound exocytosis
Piecemeal degranulation
Cytolysis
●NUCLEUS:
-Difficult to observe because of overlying granules; Generally unsegmented or bilobed
●CYTOPLASM:
-Densely stained, dark violet to __ granules
(variable in size and unevenly distributed)
___: a dye that can bind with acid mucopolysaccharides in blood cells to form
metachromatic complexes (“metachromasia” – histochemically defined as a reaction product color that is considerably different from the color of the dye itself)
* Before the application of the toluidine blue stain, the films are fixed first in __.
* Basophil and mast cell granules are strongly ___
* Toluidine blue stain is valuable in identifying basophils and mast cells, especially neoplastic forms in which the number of granules may be considerably reduced.
-Some of the functions of the basophils are:
*Have IgE receptors on their surface membrane that, when cross-linked by antigen, result in granule release
*Induce B cells to synthesize __
*Involved in the control of helminth infections (promote eosinophilia & contribute to efficient worm expulsion)
-Least common WBC in normal peripheral blood -LIFE SPAN of basophils: __
____:
-NOT leukocytes
-Have several phenotypic and functional similarities w/ both basophils and eosinophil
-Connective tissue cell that has large basophilic granules containing heparin, serotonin, bradykinin, and histamine (these substances are released from the mast cell in response to IgE stimulation)
BASOPHILS
purple-black
TOLUIDINE BLUE
Mota’s fixative
metachromatic
IgE
60 hrs
Mast cell
●NUCLEUS:
- Round, ___ or lobulated, usually with some degree of folding or convolutions; Chromatin: “__” or “stringy”; Nuclear vacuoles may be present
●CYTOPLASM:
-Abundant, _, containing fine, indistinct granules called azure dust (__ appearance)
- Small pseudopods or blebs may be observed
-Cytoplasmic vacuoles may be present
-Largest cell of normal blood
-are best identified by their strong positive reaction with __ stain (by histochemical means)
NOTES:
“The nonspecific esterase enzymes alpha-naphthyl acetate and butyrate esterase are used clinically to recognize cells of __. If the enzyme is of monocytic origin, it is inhibited by __;
-Monocytes are said to be rich in __.
-Monocytes in the peripheral blood can be found in a marginal pool and a circulating pool (marginal pool of monocytes is 3.5 times the circulating pool).
-Monocytes remain in the circulation roughly: _____________
MONOCYTES; horse-shoe shaped; lace-like
blue-gray; ground-glass
NONSPECIFIC ESTERASE
monocytic origin; sodium fluoride
muramidase
3 days
●NUCLEUS: - Generally, __, round, oval, or indented; Nucleoli may be visible
●CYTOPLASM:
Generally: Sky-blue or “__” blue
-Lymphocytes NOT ONLY come from the bone marrow but also from the thymus and the lymphatic system included in lymphocytes?
-Three physiologic characteristics of lymphocytes:
1) predestined to migrate
3.) NOT an obligate end cell
2.) a heterogeneous group of cells
___:
-Nucleus: May range from extremely dense to pale and immature-looking
-Cytoplasm: Deeply basophilic to pale blue and usually abundant
-Seen in nonmalignant reactive disorders
-Synonyms: reactive lymphocytes, atypical lymphocytes, virocytes, stress lymphocytes, Downey cells, transformed lymphocytes, transitional lymphocytes, and glandular fever cells.
TYPES:
TYPE I or __: seen in German measles
TYPE II or Infectious mononucleosis cell: seen in IM, characterized by ___
TYPE III: nucleus is finely reticulated nuclear chromatin pattern
LYMPHOCYTES
deep purple
Robin egg
T cells, B cells, Natural Killer cells
VARIANT LYMPHOCYTE
Turk’s irritation cell or Plasmocytoid lymphocyte
“flared skirt” or “fried egg”
Pipets:
markings of 0.5, 1, 101; RED; vol: 100
markings of 0.5, 1, 11; WHITE; 10
used mainly to disperse blood cells to facilitate counting of cells
RBC THOMA PIPET
WBC THOMA PIPET
diluting fluids
●White Blood Cell Count
I.) Background on Manual Hemocytometry:
➢ Counting Chambers: __
✓ According to TYPE:
1.) (Examples: Spencer, Burker, Levy, Levy-Hausser)
2.) (Ex.: Thoma-Zeiss)
3.) Addis
4.) Exton
5.) Petroff
✓ According to RULINGS:
1). Thoma
2). Tuerk
3). Neubauer
4.) Improved Neubauer!!!
5.) Fuchs-Rosenthal
6.) Bass-Jones
___: This hemocytometer has two identical sides and both sides are counted.
▪ Chamber is __ (divided into 9 square millimeters)
▪ The 4 corner (large) squares – subdivided into __ ➔used for manual WBC count
▪ The middle (central) square – subdivided into __ ➔ used for manual RBC count
Hemacytometer/Hemocytometer
open type
closed type
Improved Neubauer Counting Chamber
3 mm by 3 mm
16 squares
25 squares
DILUTION:
diluting fluids for WBCs (leukocytes)?
types of dilution, obj and area
diluting fluids for platelets?
type of dilution, obj and area
diluting fluids for rbcs?
type of dilution, obj and area
WBC Count Procedure:
spx: __; wbc ct typical dilution: __
hemocytometer is cleaned with?
wbc dilution is allowed to sit for? performed within?
the hemocytometer is charged by holding the tube __; it is placed in a moist chamber for __
difference between the total cells counted on each side should be ___; formula for percentage difference?
cell count formula?
1% ammonium oxalate, OR 3% acetic acid, OR 1% hydrochloric acid
1:20; 10x; 4 mm2 OR 1:100; 10x; 9 mm2
1% ammonium oxalate; 1:100; 40x (phase); 1mm2
Isotonic saline; 1:100; 40x; 0.2 mm2 (5 small squares of central square)
EDTA or whole blood; 1:20
alcohol with a lint-free tissue.
10 minutes; 3 hrs
45-degree angle
10 mins
<10%
(V1-V2)/(V1+V2/2)x100
cells counted x dilution factor/area x 0.1 (depth)
CORRECTION OF WBC COUNT
✓ Performed if there are ___ (nucleated red blood cells) seen in 100 WBCs
✓ Formula: __
✓ Remember: result of the computation should be rounded off to the nearest hundreds.
●WBC Differential Count:
Types:
A.) ___ - Routinely performed
B.) ___ - Performed in instances when the WBC count is >40 x 10^9/L to increase accuracy of results
✓ NOTE:
- 200-cell differential may also be performed in cases when:
- __ differential performed in instances when the WBC count is >100 x 10^9/L to increase accuracy of results
- __ differential performed in instances when the WBC count is <1.0 X 10^9/L; Performed using a __
✓ Count 50 WBCs, then multiply the results by 2 to get a percentage
___: gives the number of specific WBC type per
100 WBCs; NOT as informative as the absolute count
___: gives the number of specific WBC type per cubic millimeter of blood (mm3); more informative than the relative count
-FORMULA:
> 5 NRBCs
uncorrected wbc ct x (100/100+NRBCs)
100-cell differential
200-cell differential
> 10% eo, >2% baso, >11% mono, more lymp than neutro
300 or 400-cell
50-cell; buffy coat smear
RELATIVE COUNT
ABSOLUTE COUNT
Relative count (%) x WBC count = Absolute count
(per mm3 of blood)
☼Shift to the Left and Shift to the Right:
__: increase in the number of young forms
- 2 types:
___: N to dec WBC ct; tuberculosis
___: inc WBC ct; appendicitis
___: increase in the number of old forms
Shift to the Left
Degenerative Shift to the Left
Regenerative Shift to the Left
Shift to the Right
☼Leukoerythroblastic Reaction
*Synonyms: __
*Presence on a blood smear of:
- immature neutrophils, nucleated RBCs, teardrop RBCs
*Often, but NOT always, it is accompanied by neutrophilia
*A nonspecific reaction but provides important evidence of underlying disease or stress to
the hematopoietic compartment
*This is strongly associated with PMF (primary myelofibrosis).
Striking and sustained leukoerythroblastic reactions are observed in conditions involving the presence of a space-occupying lesion in the marrow (___)
*Mild and transitory leukoerythroblastic reactions may be observed in:
✓ hemolytic anemia
✓ severe infections
✓ cardiac failure
✓ uremia
✓ megaloblastic anemia
Leukoerythroblastosis & Leukoerythroblastic anemia
myelophthisis
Some cells that may be found during the WBC Differential Count:
- nuclear remnants of lymphocytes; appearance similar to a thumbprint; structureless chromatin
-may be found normally in few numbers; may be associated with chronic lymphocytic leukemia (CLL) - nuclear remnants of granulocytic cells; netlike chromatin pattern; may be found normally in few numbers; may be seen in some leukemias
- granulocytic cells with pyknotic nuclei and an agranular cytoplasm; seen in prolonged exposure to EDTA; chemotherapy (rarely)
- neutrophils that have engulfed foreign bodies like bacteria and fungi; overwhelming septicemia; bacterial and fungal infections; erythrophagocytosis (in which neutrophil engulfs RBCs)
- large cells (20 to 30 um) which line the veins; stretched and ovoid in appearance; have a single nucleus with dense chromatin and no nucleoli; abundant cytoplasm which appears translucent; usually found at feather edge
-considered a contaminant of venipuncture (found occasionally in blood obtained by venipuncture); must not be confused with clumps of malignant cells - nude nuclei that stain dark purple; found in newborns; may be found also in aberrant platelet production, myelofibrosis, essential thrombocythemia
- varies according to cell maturity; found in newborns; may be found also in cases of hemolysis, leukemia, myeloproliferative disorders, and others
Smudge cells
Basket cells
Necrotic cells
Phagocytic cells
Endothelial cells
Megakaryocyte fragments
Nucleated red blood cells
(NRBCs)
WBC ANOMALIES:
-Deficiency of sphingomyelinase (an enzyme needed to break down lipids)
-Rare ___ disease
-More commonly seen in __
-Spleen and liver are greatly enlarged
-Signs of the disease begin in infancy with poor physical development
-Disease is often fatal by __ years of age
-characteristic cell: Pick’s cell (aka: Foam cell)
A MAC whose cytoplasm is swollen by many small lipid droplets (eaten my macrophages which in turn makes them a “foam cell”
-Defect or deficiency in the catabolic enzyme: β-glucocerebrosidase
- Most common of the __; An ___ disorder
- __; found in the bone marrow; large macrophage with small, eccentric nucleus; cytoplasm is distended by __
__: cells can be encountered in:
*thalassemia
*chronic myeloid leukemia
*acute lymphoblastic leukemia
*Non-Hodgkin lymphoma
*Plasma cell neoplasms
Niemann-Pick Disease (NPD)
autosomal recessive
Ashkenazi Jews
three
Gaucher Disease; lipidoses
autosomal recessive
Gaucher Cell
glucocerebrosides
Pseudo-Gaucher
WBC ANOMALIES:
-A rare __ disease of immune dysregulation
-Characterized by the presence of large, abnormal cytoplasmic granules in phagocytes (granulocytes and monocytes), and occasionally
in lymphocytes; Abnormal granules in phagocytes are: ___
-Abnormal granules in lymphocytes are: __
-numerous types of cells in the body are affected and show abnormally large lysosomes, which contain fused dysfunctional granules
- __ is observed due to abnormal packaging of melanosomes
-Patients exhibit the characteristic
triad of:
T - _____________________
I - ______________________
E - _____________________
-A rare ___ disorder
-T cells are decreased; B cells, T cells and NK cells, neutrophils and monocytes are dysfunctional which leads to
bacterial, viral and fungal infections
-Wiskott-Aldrich thrombocytes are structurally abnormal. The number of dense granules is low, and the thrombocytes are __ (microthrombocytes).
-an autosomal dominant disorder
-characterized by the presence of gray-blue spindle-shaped inclusions [ ___ ] in the cytoplasm of __ and ___
-also characterized by leukopenia, variable thrombocytopenia and ___
Chediak-Higashi Syndrome
autosomal recessive
Peroxidase (+)
Peroxidase (-)
Partial albinism
Wiskott-Aldrich Syndrome (WAS)
thrombocytopenia
immunodeficiency
eczema
X-linked recessive
small
May-Hegglin Anomaly
Dohle body-like inclusions
granulocytes and monocytes
giant platelets
WBC ANOMALIES:
(aka: True/Congenital PHA)
-Failure of the neutrophil nucleus to segment
-Cell present: =__ or __ form of neutrophil nucleus
-Most common genetic disorder of WBCs
- __ disorder
- __ nuclear segmentation (bilobed, unilobed), coarse chromatin clumping pattern potentially affecting all leukocytes, although morphologic changes are MOST obvious in mature neutrophils;
UNILOBED = round, ovoid, or peanut shaped
__ = spectacle-like (“pince nez”) morphology with nuclei attached by a thin filament
-a result of a mutation in the __ β-receptor gene
-lamin β-receptor = an inner nuclear membrane protein; plays a major role in leukocyte nuclear shape changes that occur during normal maturation
___: all neutrophils are affected and demonstrate round nuclei
___: 55% to 93% of the neutrophil population are affected (there is generally a mixture of all of the aforementioned nuclear shapes)
-Neutrophils show __ granulation in true PHA.
-Neutrophils in PHA appear to function normally.
Pelger-Huet Anomaly
“pince-nez” or “spectacle”
autosomal dominant
decreased
BILOBED
lamin
Homozygous PHA:
Heterozygous PHA
normal
WBC ANOMALIES:
-Nucleus has ___ megaloblastic anemias,
myelodysplastic syndromes, hereditary neutrophil hypersegmentation, myelokathexis
(NOTE: ___ pertains to a rare hereditary condition characterized by normal granulocyte production; nevertheless, there is impaired release into the blood (leads to neutropenia).
Here, neutrophils appear hypermature. There may be hypersegmentation, hypercondensed chromatin, and pyknotic changes in this condition.)
___:
Usually a ___ that has ingested the antibody-coated nucleus of another neutrophil or has engulfed the homogenous, globular nuclear mass of a destroyed cell.
-Usually an in vitro phenomenon
-Found in systemic lupus erythematosus (SLE) but may also be found in comparable connective tissue disorders
___:
A __ that has ingested a whole lymphocyte or a nucleus (with an identifiable nuclear chromatin). May be seen in drug sensitivity
Hypersegmented Neutrophils; > 6 lobes
Myelokathexis
Lupus Erythematosus (LE) Cell; neutrophil
Tart Cell; monocyte
WBC ANOMALIES:
-Similar to normal ___ BUT the nucleus is notched, lobulated, and ___
-Found in __ or can be artificially formed through blood film preparation
-An abnormal ___ with a cytoplasm that is completely filled with Russell Bodies;
-Found in Multiple Myeloma (Plasma Cell Myeloma)
~ Some of the characteristics of patients with PLASMA CELL MYELOMA:
*Presence of ___; C R A B?
-Small ___ with little cytoplasmic projections; T R A P (+)?
-Found in Hairy Cell Leukemia; Nearly all blood cells contain __ nonerythroid isoenzymes of acid
phosphatase (0, 1, 2, 3, 3b, 4 and 5)
- ___ (tartrate resistant & is produced in abundance by these cells)
-A large __ which may demonstrate two nuclei (with eosinophilic nucleoli) and an abundant cytoplasm
-Presence of these cells is the definitive histologic characteristic of Hodgkin’s Disease
- Seen in Adult T Cell Leukemia
- Seen in NLPHL (Nodular Lymphocyte Predominant Hodgkin’s Lymphoma)
Rieder Cell; lymphocytes; cloverleaf-like
Chronic lymphocytic leukemia (CLL)
Grape Cell; plasma cell
(aka: Morula cell or Mott cell)
Bence Jones Protein;
C- hypercalcemia
R - renal insufficiency
A - anemia
B - bone lesions
Hairy Cells; lymphocytes
T - tartrate
R - resistant
A - acid
P - phosphatase
7; Isoenzyme 5
Reed-Sternberg Cell; lymphoid cell
Flower cells
Popcorn cells (aka: L and H cells)
WBC ANOMALIES:
-Characterized by a cerebriform nucleus; Seen in Mycosis Fungoides (an example of a Non-Hodgkin’s Lymphoma)
-Purple-red particles (precipitated mucopolysaccharides); Seen primarily in neutrophils, eosinophils, and basophils
-Also seen in monocytes and lymphocytes (occasionally); Can resemble very coarse toxic
granulation
-Most frequently seen in patients with Hunter, Hurler, and Maroteaux-Lamy types of genetic mucopolysaccharidosis
-Altered __ (present because of rapid cell maturation); Described as __ granules found in the cytoplasm of neutrophils; Seen in severe infections and chemical poisoning
-Linear projections of primary granules; Seen in certain types of acute Myelogenous leukemia (AML)
___: “bundles of sticks”; an abnormal WBC with bundles f Auer rods in its cytoplasm
-Round or oval blue-staining cytoplasmic inclusions found in __; (arranged in parallel rows and consisting of ribosomal RNA)
-Found in: pregnancy, severe burns, aplastic anemia, scarlet fever and other infectious diseases, and following administration of toxic
agents
Sezary cells
Alder-Reilly Inclusions;
Toxic Granulations; primary granules; dark-blue to black
Auer Rods
Faggot cell
Döhle Bodies; neutrophils
DOHLE BODIES VS MHA INCLUSIONS:
size, shape, PAS rxn, content?
LEUKEMIAS VS LYMPHOMAS:
___: Overproduction of various types of immature or mature cells in the bone marrow and/or peripheral blood (in most types of leukemia); Frequently involves WBCs of the myelogenous or lymphocytic cell types; Malignant cells easily trespass the ___
___: Solid malignant tumors of the lymph nodes and related WBCs in the bone tissue. The distinctive cell type is the __. Malignant cells are initially confined to the organs containing mononuclear phagocytic cells such as lymph nodes, spleen, liver, and bone marrow. Lymphomas can spill over into the circulating blood and demonstrate a leukemic-appearing
picture on a peripheral blood film.
DOH: smaller, round, positive, ribosomal RNA
MHA: larger, spindle-shaped, negative, messenger RNA
LEUKEMIAS; blood-brain barrier
LYMPHOMAS; lymphocyte
▪ a neoplastic disease characterized by an overproduction of immature or mature cells of various WBC types in the bone marrow or
peripheral blood; generalizations:
o more blasts: shorter, more fatal course of disease
o ↑ WBC count with shift to the left
o M:E ratio of __
o Type of anemia usually present in cases of acute leukemia: _______________
____:
-Described by symptoms of short duration
-Numerous immature cell forms in the bone marrow and/or peripheral blood
-Increased total WBC count
____:
-Described by symptoms of long duration
-Mostly mature cell forms in the bone marrow and/or peripheral blood
-Total WBC counts range from extremely elevated to lower than normal
✓ FRENCH-AMERICAN-BRITISH (FAB) CLASSIFICATION of LEUKEMIAS:
o based on morphology of cells in Romanowsky-stained smear; o based on cytologic and histochemical characteristics of cells involved
LEUKEMIA
10:1
normocytic, normochromic
ACUTE LEUKEMIAS
CHRONIC LEUKEMIAS
FAB CYTOCHEMICAL STAINS:
*Enzyme found in primary granules of: neutrophils and eosinophils, monocytes (to a certain extent)
*Used in differentiating blasts of ___
* (+) Peroxidase stain rules out __
POSITIVE: Neutrophilic granulocytes (except normal blasts), Auer rods, Leukemic blasts in FAB M1, M2, and M3, Eosinophils
WEAKLY POSITIVE or NEGATIVE: ___
NEGATIVE: Myeloblasts, Basophils, Lymphocytic cell series and Erythrocytic cell series
~ Peroxidase enzyme is sensitive to __. Smears
should be stained immediately or kept in the dark. Smears that are older than 2 weeks or that have been exposed to excessive light should NOT be reported as peroxidase negative.
MYELOPEROXIDASE (MPO)
AML from those of ALL
ALL
Monocytes
light
FAB CYTOCHEMICAL STAINS:
*Reactions parallel those of the MPO’s [in most cases]; Stains sterols, neutral fats, phospholipids (found in the primary and secondary granules of neutrophils and lysosomal granules of monocytes); __ - most sensitive stain for granulocytic precursors.
POSITIVE: Promyelocyte, myelocyte, metamyelocytes, bands, and segmented neutrophils:
WEAKLY POSITIVE or NEGATIVE: __ and __
STRONGLY POSITIVE: Leukemic blasts, Auer rods
and Eosinophils
NEGATIVE:
-Lymphocytes and its precursors, megakaryocytes and platelets and Erythrocytes
SUDAN BLACK B (SBB)
Myeloblasts and Monocytic cells
Sudan Black B stain
LYMPHOCYTIC LEUKEMIAS:
●Generally:
✓ Myeloperoxidase: _________
✓ Sudan Black B: ___________
___: most common form of childhood leukemia
CLASSIFICATION BY FAB:
___: 70% of childhood ALL; CALLA (CD10), TdT, CD19, CD20
-Homogeneous population of small blasts; nuc: Uniformly round, small; Single nucleolus; chromatin is slightly reticulated with perinucleolar clumping; Scant, blue cytoplasm;
- PAS and ORO ___
___: 70% of adult ALL; TdT; Heterogeneous
population of large blasts; Irregular nucleus; Single to several nucleolus; Fine chromatin; Moderate, pale cytoplasm
- PAS and ORO __
___: Rare in children and adults; sIg, CD19, CD20, CD22, CD24; Homogeneous population of large
blasts (with nuclear and cytoplasmic vacuoles); Round to oval nucleus; Two to five nucleolus; Coarse with clear parachromatin; Moderate, blue, prominently vacuolated cytoplasm.
- methyl green pyronine and ORO __
negative; negative
●ACUTE LYMPHOCYTIC LEUKEMIA (ALL)
L1; positive
L2; positive
L3; positive
characterized by the presence of increased numbers of smudge cells and Reider cells in the PBC; most common type of leukemia in the elderly; characterized by persistent lymphocytosis
CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)
●Generally:
✓ Myeloperoxidase: _________
✓ Sudan Black B: ___________
NON-LYMPHOCYTIC LEUKEMIAS/MYELOGENOUS LEUKEMIAS
positive; positive
ACUTE MYELOGENOUS LEUKEMIA (AML)
origin: Myelocytic
AML, minimally differentiated; MPO negative and SBB negative
M0
ACUTE MYELOGENOUS LEUKEMIA (AML)
origin: Myelocytic
AML, without maturation; may demonstrate Auer rods
M1
ACUTE MYELOGENOUS LEUKEMIA (AML)
origin: Myelocytic
AML, with maturation; most common type of AML; may demonstrate Auer rods
M2
ACUTE MYELOGENOUS LEUKEMIA (AML)
origin: Myelocytic
Acute Promyelocytic Leukemia (APL); associated with DIC and Faggot cells
~___: microgranular variant : cells have the
characteristic “butterfly”, “bowtie”, “coin-on-coin” or “apple core” nuclei
M3
M3V: APL,
ACUTE MYELOGENOUS LEUKEMIA (AML)
origin: Myelocytic, Monocytic
Acute Myelomonocytic Leukemia (AMML); AKA: __; 2nd most common subtype of AML; may demonstrate Auer rods
~ __: “AMML, w/ increased marrow eosinophils”
M4; Naegeli Monocytic Leukemia;
M4E
ACUTE MYELOGENOUS LEUKEMIA (AML)
origin: Monocytic
Acute Monocytic Leukemia (AMoL); AKA __
**__: “AMoL, poorly differentiated”
**__: “AMoL, well differentiated”
M5; Schilling leukemia
M5a
M5b
ACUTE MYELOGENOUS LEUKEMIA (AML)
origin: Erythrocytic, Myelocytic
Acute Erythroleukemia; AKA __; may demonstrate Auer rods, Type of anemia: macrocytic, normochromic; erythroid cells in AML Mx: PAS = ___
M6
Acute DiGuglielmo’s Syndrome
strongly (+)
ACUTE MYELOGENOUS LEUKEMIA (AML)
origin: Megakaryocytic
Acute Megakaryocytic Leukemia; requires immunochemical staining for accurate diagnosis (Factor VIII stain: ___)
~__: acute basophilic leukemia
~ leukemias that are PAS (+)?
M7
positive
M8
ALL L1 and L2, AML M6
also known as: _________
- characterized by the presence of Philadelphia Chromosome (Ph1); three clinical phases: Chronic phase, Accelerated phase, Blast crisis
___: -First described in 1960 by Peter C.
Nowell, a faculty member of the University of Pennsylvania, Philadelphia, as an unfamiliar small chromosome present in the WBCs of patients with CML; due to reciprocal translocation involving the long arms of chromosome ___ (results in the formation of the
___ fusion gene)
must be differentiated from ____- Excessive leukocytic response in the peripheral blood
- Confused with CML; WBC ct.: greater than 50 X 10^9/L (with neutrophilia and a marked left shift [presence of immature neutrophilic forms])
- LR most frequently refers to neutrophils, but the increased count may be due to an increase in other types of WBCs
CHRONIC MYELOGENOUS LEUKEMIA (CML); chronic granulocytic leukemia
Philadelphia chromosome; 9 and 22; BCR-ABL1
LEUKEMOID REACTION (LR)
GENERALLY used to distinguish LR from CML
Principle: ↑ activity can be observed in neutrophils that have undergone normal growth.
___:
* Principle: Hydrolysis of sodium alpha naphthyl phosphate by alkaline phosphatase produces a colored precipitate with a diazotised amine.
Reagents: what are their purpose?
- Methanol and formalin
- Brentamine-fast garnet salt
- Sodium alpha naphthyl phosphate
- Aqueous Mayer’s hematoxylin
- Propanediole
~ result is reddish-brown to black precipitate of alkaline phosphate granules
SCORE AND DESCRIPTION:
0: no reddish brown to black precipitate
1+: slightly diffused reddish brown to black precipitate
2+: moderately diffused reddish brown to black precipitate
3+: heavily diffused reddish brown to black precipitate
4+: very heavily diffused reddish brown to black precipitate
After which, total the individual scores of the 100 neutrophils.
- Normal Kaplow’s (LAP) Score = 15 to 100
- GENERALLY: Within normal or LAP score - ____
* decreased LAP score - _________
LEUKOCYTE (NEUTROPHIL) ALKALINE PHOSPHATASE (LAP/NAP) TEST
KAPLOW’S METHOD
fixative
initial stain
substrate
counterstain
buffer
leukemoid reaction
CML
Most common clinical application of flow cytometry: diagnosis of leukemias and lymphomas
✓ Originally designed to evaluate physical properties of cells based on their ability to deflect light.
✓ Most significant discovery that led to the improvement of flow cytometry and its subsequent widespread application in clinical practice: development of monoclonal antibodies.
✓ Although the term flow cytometry suggests the measurement of a cell, this procedure is successfully applied to study other particles, including chromosomes, microorganisms, and proteins.
✓ Flow cytometry measures physical, antigenic and functional properties of particles suspended in a fluid.
✓ A flow cytometer is composed of fluidics, a light source (laser), multiple detectors, and a computer.
✓ Main advantage of flow cytometry over other techniques: its ability to quickly and simultaneously analyze multiple parameters in a large number of cells
✓ Specimens most commonly analyzed: bone marrow, peripheral blood, lymphoid tissues, body cavity fluids and solid tissues.
✓ For peripheral blood and bone marrow specimens (___ as anticoag)
FLOW CYTOMETRY; heparin
o Differentiate acute granulocytic leukemias from monocytic leukemias
o SUBSTRATES: α-naphthyl acetate and α-naphthyl butyrate (__) & Naphthol AS-D Chloroacetate (__)
α-naphthyl acetate esterase (NSE)
▪ POSITIVE:
* ___ (strong positive reaction)
* Positive for other certain cell types
α-naphthyl butyrate esterase (NSE)
▪ POSITIVE:
* ___
* Positive for other certain cell types
Naphthol AS-D Chloroacetate esterase
▪ POSITIVE:
* ___ (5)
* Leukemic myeloblasts
* Auer rods
___:
o Stain for glycogen
o Useful in identifying FAB M6 leukemia
o RBCs in erythroleukemia (FAB M6) are STRONGLY POSITIVE
o Normal erythroid precursors are PAS negative
___:
o Enzyme marker for primitive lymphoid cells
o Strong TdT activity – observed in approximately 90% of patients with ALL
o Helpful in the recognition of the “lymphoblastic transformation” of CML
ESTERASES
nonspecific
specific
Monocytes
Monocytes
Promyelocyte, myelocyte, metamyelocyte, bands, seg. neutro
PERIODIC ACID-SCHIFF (PAS) REACTION
TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE (TdT)
Study of chemical constituents of cells
* Useful for acute leukemias
* Specimens: bone marrow, lymph nodes, peripheral blood
* For enzymatic techniques: fresh smears
* For nonenzymatic techniques (like PAS/SBB): may remain stable for months if stored at room temp.
CYTOCHEMISTRY
✓ __:
o immature, non-nucleated RBC which contains > 2 blue-stained, granulofilamentous materials (RETICULUM) after staining (supravital stains)
o continues to generate hemoglobin (last stage)
o normal maturation time for reticulocytes in blood: ___
o production of reticulocytes: 50 x 10^9/L/day
✓ __- permits effective assessment of RBC production by the bone marrow; a measure of EFFECTIVE ERYTHROPOIESIS
✓ Reference ranges:
o Adults: ___
o Newborns: __ (by 1 to 2 weeks of age, reference values are the same as for adults)
o Increased (↑) reticulocyte counts (reticulocytosis/polychromasia/polychromatophilia)
▪ considered as the __ of accelerated erythropoiesis and observed in hemolytic anemias, individuals with iron deficiency anemia receiving iron therapy, thalassemia, sideroblastic anemia, and in acute and chronic blood loss
o Decreased (↓) reticulocyte counts (reticulocytopenia):
▪ observed in aplastic anemia and in conditions in which the bone marrow is __
♦ calibrated disk placed in the ocular of the microscope
♦ __ is used for: counting RETICULOCYTES
♦ __ is used for: counting RBCs
(*minimum no. of cells that should be counted: __)
Reticulocyte Count
RETICULOCYTE
1 DAY
RETICULOCYTE COUNT
0.5 to 1.5%; 1.8 to 5.8%
first sign
NOT producing RBCs
Miller Disk
Large Square (A)
Small Square (B)
112
METHODS OF COUNTING RETICULOCYTES:
A.) ____:
1. Combine equal amounts of blood and supravital stain (2 to 3 drops, or around 50 μL each), and allow to incubate at room temperature for ___
2. Remix the preparation.
3. Prepare two blood smears.
4. In the region in which cells are near each other but NOT touching, count 1000 RBCs under the oil immersion objective lens (1000x total magnification). Note: Reticulocytes are included in the total RBC count (i.e., reticulocytes are counted as both an RBC and a reticulocyte).
5. To increase accuracy, have another medtech count the other blood smear; counts should agree within __.
6. Calculation: (formula)
ROUTINE LIGHT MICROSCOPE METHOD
3 to 10 minutes.
20%
Retic (%) = (No. of Retics. observed/1,000 RBCs observed) x 100
METHODS OF COUNTING RETICULOCYTES:
B.) ____:
✓ Count a minimum of 112 RBCs in small square (B).
✓ A reticulocyte in square B is counted as both an erythrocyte and a reticulocyte.
✓ At this point, theoretically, the number of retics in 1,008 RBCs has been counted.
✓ Computation:
Retics(%) =___
C.) ___:
✓ Most rapid, accurate, and precise method for reticulocyte count
✓ In this method, the reticulocytes are counted on the basis of optical scatter or fluorescence after treatment with fluorescent dyes or nucleic acid stains
✓ The test values in this method are reported in absolute and in relative terms.
✓ Example: Sysmex R-3500
CALIBRATED MILLER DISK METHOD
Retics(%) = (Total retics in Square A)/(Total RBCs in Square B x 9) x 100
FLOW CYTOMETRY
3 METHODS OF REPORTING THE RETICULOCYTE COUNTS:
I.) ____:
o Actual number of reticulocytes in 1 liter of whole blood
o Computation:
o Reference range: 20 to 115 X 10^9/L
II.) ____:
o Sometimes referred to as: Reticulocyte Index, Hematocrit Correction, or Poor Man’s Bone Marrow Aspirate
o Percentage of reticulocytes may appear increased because of early reticulocyte release into the circulation or because of a decrease in the number of mature cells in circulation.
o The CRC corrects the observed reticulocyte count to a normal Hct of 0.45 L/L to allow correction for the degree of patient’s anemia.
oFormula: _____
III.) ____:
o RPI (also known as shift correction) provides a further refinement of the CRC.
o A general indicator of the rate of erythrocyte production increase above normal in anemias.
o Calculation: RPI= ___
ABSOLUTE RETICULOCYTE COUNT
ARC = (reticulocytes (%) x RBC count (x10^12/L)/(100) x 1,000
CORRECTED RETICULOCYTE COUNT
CRC= Retics (%) x (Hct in L/L÷0.45 L/L)
RETICULOCYTE PRODUCTION INDEX
RPI= Corrected Reticulocyte Count ÷ Maturation Time in the peripheral blood
HEMATOCRIT (%) MATURATION TIME (Days)
40 to 45 =
35 to 39 =
25 to 34 =
15 to 24 =
<15 =
__ - generally indicates adequate bone marrow response
__ - generally indicates inadequate bone marrow response
1.0
1.5
2.0
2.5
3
RPI >3
RPI <2
Normal WBC histograms have three (3) distribution peaks:
1. First peak ( 45-90 fL.) = ___ population of cells (i.e., lymphocytes)
2. Second peak ( 90-160 fL ) = minor population of ___ (i.e., monocytes). = an increase in the number of cells in this size range can also represent abnormal cell types (such as the immature precursor of cell types found in patients with leukemia).
3. Third peak (160-450 fL) = normal ___
staining that is considered the gold standard for assessment of body iron?
small mononuclear
large mononuclear cells
mature types of granulocytes.
Prussian blue staining
___:
- characterized by increased iron accumulation in body tissues leading to tissue damage
-example: __ (aka: __)
✓ Most common form of iron overload disease
✓ Autosomal recessive disorder that causes body to absorb and store too much iron
✓ Caused by mutations in the HFE gene (regulates the amount of iron absorbed from food)
✓ Arthritis, liver cirrhosis, congestive heart failure, impotence, bronze skin, diabetes, thyroid deficiency
✓ Treatment:
▪ Avoiding foods that contain iron
▪ Phlebotomy
▪ Deferoxamine (Desferal)
HEMOCHROMATOSIS
Hereditary Hemochromatosis; BRONZE DIABETES
___: Maintenance of blood flow within the vascular system; Important considerations in blood collection for hemostasis testing:
✓ If the patient has many bruises or mentions a tendency to bleed (a reason to expect excessive bleeding), the phlebotomist should extend the time for observing the venipuncture site from ___ and should apply a pressure bandage before dismissing the patient.
✓ Factors V and VIII - _________________
✓ Cold temperature (1 to 6 C) storage
o causes precipitation of: ______________
o activation of: ________________
o destruction of: ____________
✓ 0.105 to 0.109 M (3.2%) buffered Sodium Citrate
o found in __tops
o may inc the stability of FV and VIII
HEMOSTASIS
1 to 5 minutes
labile factors
von Willebrand factor
Factor VII
platelets
light blue tops
HEMOSTASIS
✓ CTAD [___]
o found in light blue top tubes
o For coagulation tests, and PF4 (platelet factor-4) and β-TG (beta-thromboglobulin) assays
✓ Factors that may affect coagulation test results (PT and/or APTT):
o Shortened test results: (mnemonic)
o Prolonged test results:
▪ ______________________
▪ ______________________
* Caused by:
o Short draw
o Elevated hematocrit (>55%)
if hematocrit is high, the ac is adjusted by using this formula ___
citrate, theophylline, adenosine, dipyridamole
HEPEP
▪ Hemolysis
▪ Excessive agitation
▪ Prolonged tourniquet application
▪ Excessive needle manipulation
▪ Platelet contamination
presence of clots
increased AC conc
C = (1.85 X 10-3) (100-H) V
✓ Needles for Hemostasis Specimens:
Adult with good veins, specimen < 25 mL: ___
Adult with good veins, specimens > 25 mL: ___
Child or adult with small, friable, or hardened veins: ____
20 or 21 gauge, thin-walled, 1.0 or 1.25 inches long
19 gauge, 1.0 or 1.25 inches long
23 gauge, winged-needle set; apply minimal
negative pressure
✓ Needles for Hemostasis Specimens:
Transfer of blood from syringe to tube: ___
Syringe with winged-needle set: ___
19 gauge, slowly inject through tube closure
20, 21, or 23 gauge, thin-walled; use only for small, friable, or hardened veins or specialized
coagulation testing
○ Petechiae (______)
○ Purpura (_______)
○ ___ (_______) (aka: Bruise); most common complication of phlebotomy
o __ – nosebleed
o __ – hemorrhage into a joint
o __ - expectoration of blood
from some part of the respiratory tract
o __ – vomiting of blood
o __ - unusually heavy or prolonged menstrual periods
o __ - passage of fresh blood per anus, usually in or with stools
o __ - passage of black, tarry stools
o __ – pathological formation of blood clots in veins/arteries that obstruct blood flow
o __ - reduced levels of fibrinogen in the blood
o __ - fibrinogen does not function normally or optimally
1 mm
3 mm
Ecchymosis (≥1 cm)
Epistaxis
Hemarthrosis
Hemoptysis
Hematemesis
Menorrhagia
Hematochezia
Melena
Thrombosis
Hypofibrinogenemia
Dysfibrinogenemia
Hemostasis Specimen Storage Times and Temperatures: (constant @ RT)
PT with no unfractionated heparin (UFH) in specimen: ___
PTT with no unfractionated heparin (UFH) in specimen: ___
PTT for monitoring UFH therapy: ___
PT when UFH is present in specimen: ___
24 hrs
4 hrs
Separate within 1 hour, test within 4 hours
Separate within 1 hour, test within 4 hours
involves the constriction of blood vessels and formation of platelet plugs!!!
involves the formation of fibrin meshwork!!! through activated coag factors & inhibition of activated coag factors
PRIMARY HEMOSTASIS
SECONDARY HEMOSTASIS
PRIMARY HEMOSTASIS:
Blood Vessels
A.) 3 coats (tunics) composing the tissue in a blood vessel wall:
1.) _________________________
- also known as tunica interna
- endothelium – simple squamous epithelium lining the blood vessels
- forms the smooth glistening surface of endothelium that lines the inner tubular cavity (lumen)
2.) _________________________
-composed of smooth muscle and elastic fibers
-thickest coat
3.) __________________________
-also known as tunica externa
-composed of fibrous connective tissue that contains autonomic nerve endings and the vasa vasorum (small networks of blood vessels that supply nutrients to the tissues of the wall).
tunica intima
tunica media
tunica adventitia
PRIMARY HEMOSTASIS:
Blood Vessels
B.) Types of Blood Vessels
(1) __ (size: 4 mm)
- distributing blood vessels that leave the heart
-have the thickest walls of the vascular system
-__ (30 μm): microscopic continuation of arteries that give off branches
called metarterioles, which in turn join the capillaries
(2) ___ (5 mm)
-collecting blood vessels that return to the heart
-larger, have a more irregular lumen than arteries
-___ (size: 20 μm) – microscopically sized veins; connect the capillaries to the veins
(3) ___ (8 μm)
- blood passes from the arterial to the venous system via the capillaries
- capillaries are the thinnest walled and most numerous of the blood vessels.
-composed of only one cell layer of simple squamous epithelium (unlike the vessels of the
arterial and venous systems), which permits a
more rapid rate of transport of materials between blood and tissue.
-__ – specialized types of capillaries found in locations such as the bone marrow,
spleen and liver
ARTERIES
Arterioles
VEINS
Venules
CAPILLARIES
Sinusoids
C.) Some of the substances released from or found on the surface of intact endothelial cells:
___: inhibits platelet activation;
-produced by the eicosanoid pathway in endothelial cells
-penetrates the platelet and binds at its IP receptor
___: (metabolic product of ATP and ADP)
-stimulates vasodilation
___: weakly enhances Antithrombin (previously called Antithrombin III)
Prostacyclin/Prostaglandin I2 (PGI2)
Adenosine
Heparan sulfate
C.) Some of the substances released from or found on the surface of intact endothelial cells:
___: a thrombin cofactor; binds thrombin and reduces its ability to participate in the clotting process
- thrombomodulin + thrombin activate =
▪ ___ - inactivates factor Va and VIIIa
▪ ___ - an antifibrinolytic enzyme
___: major plasminogen activator
- PLASMINOGEN →TPA→PLASMIN (other name:__ & function)
Thrombomodulin (TM)
○ Protein C (activated protein C - APC)
○ TAFI -thrombin activatable fibrinolysis inhibitor
TPA (tissue plasminogen activator)
fibrinolysin - enzyme that dissolves clot
C.) Some of the substances released from or found on the surface of intact endothelial cells:
- MAJOR FUNCTIONS:
● aids in platelet adhesion; acts as a carrier protein for factor VIII
- SITES OF SYNTHESIS:
● ___ (2)
- SITES OF STORAGE:
● _______
~ ___
- “a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13”
- aka: ________
-a plasma enzyme secreted by the: __________
- regulates the size of circulating VWF by cleaving ultra-long VWF multimers (ULVWF) into shorter segments (have less hemostatic potential)
- TTP - caused by deficiency of ADAMTS13
VWF (von Willebrand Factor)
endothelial cells and megakaryocytes
Weibel-Palade bodies (in blood vessels) and Alpha granules (in plts)
ADAMTS 13/VWF-cleaving protease
liver
thrombotic thrombocytopenic purpura
✓ aka: ____________
✓ arise from a bone marrow cell called megakaryocytes
✓ important in both primary and secondary hemostasis; described as cells with granular cytoplasm but no nuclear material
✓ platelets cluster with the RBCs near the center of the blood vessel
✓ platelets move back and forth with the WBCs from venules into the white pulp of the spleen
✓ LIFE SPAN: __
✓ On a Wright-stained PBS:
o platelets are spread throughout the RBC monolayer (7 to 21 cells per 100x field)
o platelets have an average diameter of __ (or, 2 to 4 μm)
-It is believed that thrombocytes enter the spleen initially, where they
remain for two days. After this period, thrombocytes are in either
the circulating blood or in the active splenic pool.
-AT ALL TIMES:
▪approx. __ of the total number of platelets are in the systemic
circulation, while __ of the platelets are in the spleen
Platelets/thrombocytes
9.0 days ± 1 day (8-10)
2.5 μm
2/3; 1/3
PLATELETS:
✓ 3 major functions:
1.) To form an aggregate plug of platelets that can slow down or stop blood loss
2.) To participate in plasma coagulation
3.) To preserve the endothelial lining of the blood vessels
✓ ___
o aka: _______________________
o appear in compensation for thrombocytopenia
o newly released from megakaryocytes and still contain RNA
o markedly larger than the usual platelets
(diameter in PBS: exceeds 6 μm [MPV reaches 12 to 14 fL])
o clinical use:
▪ can help differentiate bone marrow failure from peripheral
destruction in thrombocytopenia
▪ early predictor of bone marrow recovery after chemotherapy and transplantation
o ___ - potentially prothrombotic (may be associated with increased risk of cardiovascular disease)
SIZE OF THE PLATELETS
* Normal: __
* Mean Platelet Volume (MPV)
o Reference Range: __________________________
o Average volume of individual platelets in a specimen
o EDTA causes swelling of platelets (causes approximately ___ in MPV during the first hour).
o Should be based on EDTA specimens that are between 1 to 4 hours old
RETICULATED PLATELETS/ stress platelets
Reticulated Platelets
2.5 μm (average)
6.8 to 10.2 fL
20% increase
SMALL PLATELETS:
diseases involved are ___
___: inheritance X-linked recessive XLR
Wiskott Aldrich Syndrome (WAS)
TORCH:
toxoplasma
other agents
rubella virus
cytomegalovirus
herpesvirus
LARGE/GIANT PLATELETS
❖ ___: inheritance:_____________
❖ ___: inheritance:_____________
❖ MYH9 gene mutations: all are AD
examples:
*Fechtner syndrome
*Sebastian syndrome
*Epstein syndrome
*MHA
~ MHA (May-Hegglin Anomaly) -characterized by:
*leukopenia
*variable thrombocytopenia
*giant platelets
*Dohle body-like inclusions in leukocytes
BSS [Bernard-Soulier Syndrome]; AR
GPS [Gray Platelet Syndrome]; AR
PLATELETS:
SHAPE OF THE PLATELETS
✓ Resting and circulating platelets =_______________
✓ Activated platelets =_____________
CYTOPLASM
➢ On a Wright-stained PBS, platelets appear ___.
➢ two general parts:
o __ - (aka. granulomere) centrally-located, granular
o __ - peripherally-located, non-granular
biconvex/disk-shaped
spherical with pseudopods
lavender and granular
chromomere
hyalomere
MEGAKARYOCYTOPOIESIS
➢ aka: ____________________________________
➢ process by which megakaryocytes develop from hematopoietic stem cell (HSC)
➢ ___
o aka: MGDF (megakaryocyte growth and development factor); major regulator of platelet production
o Produced primarily by the: ________; 70,000 Dalton molecule
o Possesses 23% homology with erythropoietin (EPO)
o ___ receptor site present at all maturation stages (from BFU-Meg to PLTs)
o Some of the functions of TPO:
▪ Stimulates megakaryocytopoiesis (Some of the cytokines that function with it to stimulate this process include IL-3, IL-6, and IL-11.)
▪ Induces the proliferation and maturation of megakaryocytes
▪ Induces thrombocytopoiesis
megakaryopoiesis
THROMBOPOIETIN (TPO)
liver
MPL– TPO
MEGAKARYOCYTES:
➢ Megakaryocyte (general characteristics):
o largest cells in the bone marrow (size: ______)
o has a multilobulated nucleus and abundant granular cytoplasm
o ___________ of all bone marrow cells
o ____________ megakaryocytes per 10x low-power field
◘ MEGAKARYOCYTE PROGENITORS
✓ 3 megakaryocyte lineage-committed progenitor stages:
o ____ – least mature; participate in normal mitosis
o ____ - participate in normal mitosis
o ____ - most mature; loses its capacity to divide; performs endomitosis
~ all of these resemble the: ___
30-160um
<0.5%
2 to 4
burst-forming unit (BFU-Meg)
colony-forming unit (CFU-Meg)
light-density CFU (LD-CFU-Meg)
lymphocytes
◘ MEGAKARYOCYTE PRECURSORS (Terminal Megakaryocyte Differentiation Stages):
✓ stages wherein the observers are able to recognize the unique Wright-stained morphology of the cells in bone marrow smears or H and E-stained bone marrow biopsy sections
✓ ___: least differentiated; cannot be reliably distinguished from myeloblasts or pronormoblasts (light microscopy)
o begins to develop MOST of its cytoplasmic ultrastructure (including α-granules, dense granules, and the demarcation system (DMS))
✓ ___: identified by the appearance of nuclear lobularity
✓ ___: most abundant; easily recognized at 10X magnification (basis is size: 30 to 160 μm)
o At the full maturation of the cell, ___ proceeds.
o One megakaryocyte may shed _______ platelets.
o Platelet shedding – aka: ____/_____
MK-I stage (megakaryoblast)
MK-II stage (promegakaryocyte)
MK-III stage (megakaryocyte)
platelet shedding
2,000 to 4,000
thrombopoiesis/thrombocytopoiesis
PLATELET ULTRASTRUCTURE:
➢ studied using scanning and transmission electron microscopy, flow cytometry, and molecular sequencing
I. Platelet Plasma Membrane
= selectively permeable
= provides ___ that support platelet activation internally and plasma coagulation externally
=anchored within the membrane are __ and __
●PHOSPHOLIPIDS
a.) Neutral phospholipids: __ and __ (found in the outer, plasma layer)
b.) Anionic or polar phospholipids: (found in inner cytoplasmic layer)
⸙ phosphatidylinositol (support platelet activation by supplying arachidonic acid)
⸙ phosphatidylethanolamine, and
⸙ phosphatidylserine (flips to the outer surface upon activation and is the charged phospholipid surface on which ___ complexes assemble); what are those complexes? ___
● ___ - the platelet membrane surface
- cellular ultrastructural component unique to the platelet
- absorbs albumin, fibrinogen, and other plasma proteins through endocytosis
phospholipids
glycoproteins and proteoglycans
phosphatidylcholine and sphingomyelin
2 coagulation pathway
TENASE complex and PROTHROMBINASE complex!
GLYCOCALYX
✓ Platelet STR (“Seven-Transmembrane Repeat”) Receptors
RECEPTOR; what is the ligand?
PAR 1
PAR 4
P2Y1
P2Y12
TPα and TPβ
α2-adrenergic
IP (inhibits platelet activation)
~ the rest activates the plts
▪Additional Platelet Membrane Receptors
✓ ___ – a low-affinity receptor for the immunoglobulin Fc portion (has a role in a perilous condition known as heparin-induced thrombocytopenia [HIT])
✓ ___ – an integrin that helps platelet binding to endothelial cells, WBCs, and one another
- found on the α-granule membranes of the resting platelet but travels via the SCCS to the surface of activated platelets
- P-selectin or CD62 quantification by flow cytometry is a means for measuring in vivo platelet activation
LIGAND
Thrombin
Thrombin
Adenosine diphosphate (ADP)
Adenosine diphosphate (ADP)
Thromboxane A2 (TXA2)
Epinephrine (Adrenaline)
Prostaglandin I2 (PGI2) [aka: Prostacyclin]
FcγIIA (CD32)
P-selectin (CD 62)
III. Platelet Cytoskeleton
- microtubules, actin microfilaments, and intermediate microfilaments control platelet shape change, extension of pseudopods, and secretion of granule contents
◘MICROTUBULES
✓ Formed by ___________________
✓ Thick, circumferential bundle of these maintains platelet’s discoid shape
✓ reside just within, although NOT touching, the plasma membrane
✓ Platelets become round when microtubules disassemble in the cold. However, upon ___, they recover their original discoid shape.
✓ Aside from maintaining the platelet’s discoid shape, microtubules also move inward on activation to enable expression of α-granule contents.
✓ During platelet shape change, microtubules also reassemble in long parallel bundles to provide rigidity to pseudopods.
- Location: between the microtubules and the membrane
- Formed by _________________
- Actin:
- anchors the plasma membrane glycoproteins and proteoglycans
- also present throughout the platelet cytoplasm
- globular and amorphous (in resting platelets)
- As cytoplasmic calcium concentration increases, actin becomes filamentous and contractile.
▪NOTE: The cytoplasm also contains intermediate filaments (Desmin and Vimentin) - connect with actin and the tubules, maintaining the platelet shape.
tubulins
warming to 37◦C
actins
IV. Platelet Granules
● α-granules contents and lysosomes’ contents flow through the __; dense granules travel to the ___
◘ __:
- 50 to 80 granules in each platelet
- Stain ___ (osmium-dye transmission electron microscopy preparations)
- Most of the α-granule contents are proteins that participate in secondary hemostasis (called coagulation).
- As the platelets becomes activated:
* α-granule membranes fuse with the ___
* contents of the α-granule flow to the nearby environment
* In the nearby environment, the contents of the α-granule:
○ participate in platelet adhesion
○ participate in platelet aggregation
○ support plasma coagulation
SCCS; plasma membrane
ALPHA GRANULES
medium-gray
SCCS
IV. Platelet Granules
Some of the platelet α-granule proteins include:
◘ ___:
✓ 2 to 7 dense granules per platelet
✓ aka: ____________
✓ stain __ (opaque) when treated with osmium in transmission electron microscopy
✓ They migrate to the plasma membrane and release their contents directly into the plasma upon platelet activation.
✓ The dense granule contents are __ and __ that intensify primary hemostasis.
✓ AGONIST - a substance which initiates a response when combined with a receptor
DENSE GRANULES; delta granules
black
vasoconstrictors and platelet agonists
✓ Some of the components of dense granules include:
COMPONENTS REMARKS:
___: Supports neighboring platelet aggregation by binding to P2Y1 and P2Y12
___: Function unknown, but ATP release is detectable upon platelet activation
Phosphate: –
___: Vasoconstrictor that binds endothelial cells and platelet membranes
Histamine: –
___: Divalent cations support platelet activation and coagulation
Epinephrine (Adrenaline): –
◘ ___:
✓ Few in number
✓ Stain positive for arylsulfatase, β-glucuronidase, acid phosphatase, and catalase
✓ __ contents flow through the SCCS.
✓ __ probably digest vessel wall matrix components during in vivo aggregation and may also digest autophagic debris
ADP
ATP
Serotonin (5-HT)
Ca2+ and Mg2+
LYSOSOMES
V. ___:
✓ The plasma membrane invades platelet interior, producing this.
✓ Glycocalyx is less developed in _ and lacks some of the glycoprotein receptors present on the platelet surface.
✓ this is the route for: (3)
SCCS (Surface-Connected Canalicular System)
o endocytosis
o secretion of α-granule contents
o secretion of lysosome contents
VI. ___:
✓ “control center” for platelet activation
✓ Parallel and closely aligned to the SCCS
✓ a condensed remnant of the rough endoplasmic reticulum
✓ sequesters __ and bears a series of enzymes that support platelet activation
✓ These enzymes include:
- phospholipase A2, cyclooxygenase, and thromboxane synthetase (support the eicosanoid synthesis pathway that produces thromboxane A2)
- ___: (supports production of inositol triphosphate (IP3) and diacylglycerol (DAG))
Aspirin (aka. ___)
- most frequently used antiplatelet therapy
- cyclooxygenase inhibitor
DTS (Dense Tubular System)
calcium
phospholipase C
acetyl salicylic acid
COMMON TESTS FOR PRIMARY HEMOSTASIS:
A.) ___
❖ Reasons why platelets hard to count:
* Platelets adhere to foreign surfaces (like skin and dried walls of pipets).
* Platelets easily disintegrate.
* They are hard to differentiate from debris.
* Platelets are unevenly distributed in the blood because they tend to clump.
❖ Normal Value (general): ___ *platelet count decreases after 65 years old to 122 to 350 X 10^9/L in men and 140 to 379 X 10^9/L in women.
❖ Platelet count of ____ is the most common cause of clinically important bleeding.
_____:
❖ Reference method (described by Brecher and Cronkite)
❖ EDTA whole blood is diluted __ with ___ to lyse the nonnucleated RBCs.
❖ Platelets are counted in the ___ of the central large square (1 mm2) of the hemocytometer.
❖ The accuracy of the manual thrombocyte count must be verified by performing a platelet count estimate on a Wright-stained PBS made from the same specimen.
❖ Alternatively, a light microscope can also be used. However, visualizing the platelets may be more difficult.
IMPORTANT:
~ the charged hemocytometer is placed in a moist chamber for __
and is counted using 40x objective lens
✓ difference between the total cells counted on each side should be __
✓ greater difference could indicate uneven distribution (requires that the procedure be repeated)
PLATELET COUNT
150 to 450 x 10^9/L
< 100,000/μL (100 x 10^9/L)
PHASE-CONTRAST MICROSCOPE METHOD
1:100; 1% ammonium oxalate
25 small squares
15 minutes
< 10%.
PLATELET COUNT:
✓ formula for percentage difference:
- Average the number of platelets counted on the two sides. Using the average, compute the PLT count using the following formula: ___
(REMINDER: This is the general formula used for manual cell counts and can be used to compute any type of cell count.)
(V1-V2)/(V1+V2/2) x 100
No. of cells/μL = (cells counted x dilution factor)/area (mm2) x 0.1
____
◘ To determine the approximate number of platelets per field, examine the thin area of the slide (where only a few red blood cells slightly overlap) using the oil immersion objective.
◘ A normal (wedge) blood smear should demonstrate approximately __ cells per 100x field
◘For the estimation of the platelet count:
♦ Scan ten (10) oil immersion fields for the number of platelets
♦ ___ = estimated plt. ct. per μL
♦ In occasions of significant anemia or erythrocytosis, use the following formula for the platelet estimate:___
PLATELET COUNT ESTIMATION
7 to 21
Average number of platelets/OIF x 20,000
Average number of platelets x total RBC count/200 RBCs per field
B.) PLATELET AGGREGOMETRY
___:
✓ Designed to test platelet-rich plasma (plasma with a platelet count of 200,000 to 300, 000/uL)
✓ To prepare PRP:
o Sodium citrate-anticoagulated blood is checked visually for clots
o Then it is centrifuged at ___ with the stopper in place to maintain the pH.
o Supernatant ___ is transferred by a plastic pipette to a clean plastic tube
o The tube is then sealed and stored at 18 to 24 C (ambient temperature) until the test is begun.
✓ PRP-based light-transmittance aggregometry is initiated NO LESS THAN 30 minutes after the specimen is centrifuged and completed within 4 hours of the time of collection.
✓ To produce adequate PRP, the original specimen must measure ___. Light-transmittance aggregometry is UNRELIABLE when the patient’s whole-blood platelet count is < 100, 000/uL.
LIGHT-TRANSMITTANCE (OPTICAL) PLATELET AGGREGOMETRY
50 x g for 30 minutes
PRP
9 to 12 mL of whole blood
PLATELET AGGREGATION STUDIES:
Examples of Aggregating Reagents; what are the N and abN response?
A, C, E
○ ADP
○ Collagen
○ Epinephrine
Ristocetin
___
* measures platelet aggregation and ATP release (from dense granules)
* performed on: whole blood diluted with saline
* As ATP (adenosine triphosphate) is liberated, it oxidizes a firefly derived luciferin-luciferase reagent to produce cold chemiluminescence proportional to the ATP concentration.
N: BSS (Bernard-Soulier Syndrome) and VWD
abN: Glanzzmann’s Thrombasthenia
N: Glanzzmann’s Thrombasthenia
abN: BSS and VWD
PLATELET LUMIAGGREGOMETRY
DISORDERS OF PRIMARY HEMOSTASIS:
VASCULAR DISORDERS
*GENERAL laboratory test results:
NORMAL RESULTS in these tests = ____________________
ABNORMAL RESULTS in these tests= _____________________
plt ct, plt function test, coagulation test
bleeding time, Rumple-Leede Test
VASCULAR DISORDERS:
-most common inherited vascular bleeding disorder
Hereditary Hemorrhagic Telangiectasia (aka: Rendu-Osler-Weber Syndrome)
VASCULAR DISORDERS:
-characteristics include: hyperextensible skin, hypermobile
joints, joint laxity, fragile tissues, subcutaneous hematoma
formation
Ehlers-Danlos Syndrome (aka: Cutis Hyperelastica)
VASCULAR DISORDERS:
-most commonly seen in children
-vascular abnormalities: most probably caused by immunologic damage to the endothelial cells
-characterized by gastrointestinal hemorrhage and joint swelling
Henoch-Schonlein Purpura
(aka: Allergic Purpura or Nonthrombocytopenic Purpura)
PLATELET DISORDERS
A. QUANTITATIVE
Examples of conditions:
✓ Bernard-Soulier syndrome
✓ Fanconi anemia
✓ TAR (Thromobocytopenia with Absent Radius) syndrome
✓ Viral infections
✓ Leukemia
✓ Megaloblastic anemias
✓ WAS (Wiskott-Aldrich syndrome)
✓ MYH9 gene mutations (ex.: May-Hegglin Anomaly)
Examples of conditions:
✓ HUS (hemolytic uremic syndrome)
✓ DIC (disseminated intravascular coagulation)
✓ ITP (immune thrombocytopenic purpura)
✓ TTP (thrombotic thrombocytopenic purpura)
✓ HIT (heparin-induced thrombocytopenia)
~ __: platelets are activated by heparin-dependent IgG antiodies against a complex of heparin and PF-4
Examples of conditions:
✓ Gaucher disease
✓ Hodgkin’s disease
✓ Sarcoidosis
✓ Lymphoma
✓ Cirrhosis of the liver
✓ Portal hypertension
THROMBOCYTOPENIA (decreased platelet count)
Impaired or Decreased Platelet Production
Increased Platelet Destruction
HIT (heparin-induced thrombocytopenia)
Increased Splenic Sequestration
VASCULAR DISORDERS:
-characterized by defects in the synthesis of collagen and
hyaluronic acid
Scurvy (aka: Ascorbic acid (Vitamin C) deficiency)
●THROMBOCYTOSIS (increased platelet count):
✓ aka: secondary thrombocytosis
✓ characterized by: moderately increased platelet count
Examples: Recovery from splenectomy, Acute blood loss & Major surgery
A.) Reactive Thrombocytosis
●THROMBOCYTOSIS (increased platelet count):
✓ aka: Primaru thrombocytosis
✓ characterized by: markedly increased platelet count
Examples: (MPNs) Essential thrombosthemia, Chronic Myelogenous Leukemia, Polycythemia Vera, Primary Myelofibrosis
B.) Autonomous Thrombocytosis
RELATED INFORMATION:
▪ ____
-group of malignant neoplasms
-described as clonal proliferations of bone marrow stem cells
-characterized by elevations in one or more myeloid cell types in the peripheral blood
▪ ____
-an example of an MPN
-some of the characteristics: ·uncontrolled proliferation of bone marrow megakaryocytes
-platelet count: exceeding 1 million/μL
Myeloproliferative Neoplasms (MPNs)
Essential Thrombocythemia
B. QUALITATIVE
Examples of conditions with: PLATELET ADHESION DISORDER
▪ ___: deficiency of the GP Ib/IX/V; inherited as AR (autosomal recessive); Associated with ↓plt ct, giant plt, normal aggreg in A, C, E, abnormal aggreg in ristocetin
~ *___: process wherein platelets adhere to a foreign surface
- important in this process are:
*___ - primary platelet surface receptor for vWF
▪ ___: MOST COMMON CONGENITAL BLEEDING DISORDER
- generally described as the deficiency of vWF, inherited as autosomal dominant (Type I VWD); decreased factor VIII; Normal PT, Abnormal APTT; normal aggreg A, C, E; Abnormal aggreg in Ristocetin
Bernard Soulier Syndrome (BSS)
PLATELET ADHESION
GP Ib/IX/V
von Willebrand’s Disease (vWD)
TYPES OF VON WILLEBRAND’S DISEASE:
Bleeding Time: normal or increased
Bleeding Tendency: mild
Petechiae: none
Bleeding Time: increased
Bleeding Tendency: moderate
Petechiae: usually none
Bleeding Time: increased
Bleeding Tendency: often severe
Petechiae: occasionally
Type I - most common variant, mildest
Type II
Type III - rarest type, most severe
Examples of conditions with: PLATELET AGGREGATION DISORDER!!
*___: deficiency of GP IIb/IIIa; inherited as AR
○ characterized by very prolonged BT, abnormal clot retraction, abnormal aggregation response in A, C, E
○ Normal aggreration in Ristocetin
- ___: absence of fibrinogen in the blood
*___
– process wherein platelets adhere to other platelets
- important in this process:
*GP IIb/IIIa - _________________________
*Fibrinogen
Glanzmann’s thrombasthenia
Hereditary afibrinogenemia
Platelet aggregation
platelet fibrinogen receptor
Examples of conditions with: PLATELET SECRETION DISORDER!!!
▪Thromboxane pathway disorders
▪Cyclooxygenase or thromboxane synthetase deficiency
▪STORAGE POOL DISEASES – Alpha and Dense Granule Deficiencies
ALPHA GRANULE DEF:
____: inheritance: AR; characteristics: specific absence of morphologically recognizable α-granules in thrombocytes; lifelong mild bleeding tendencies, moderate thrombocytopenia, fibrosis of the marrow, large, gray platelets, plasma levels of PF-4 and β-thromboglobulin: INCREASED
____: AD bleeding disorder -results from a deficiency of multimerin (a protein stored in α-granules)
Gray Platelet Syndrome
Quebec platelet disorder
ALPHA GRANULE DEF:
Albinism: Oculocutaneous albinism
platelet count: Normal
Albinism: Partial albinism
platelet count: Low
Albinism: No albinism
platelet count: Low
Albinism: No albinism
platelet count: Normal
Hermansky-Pudlak syndrome (AR)
Chediak-Higashi syndrome (AR)
Wiskott-Aldrich syndrome (X-linked)
TAR syndrome (AR)
SECONDARY HEMOSTASIS:
COAGULATION FACTORS
✓ Coagulation factors are produced primarily in the ___ (except for Factors III and IV)
* __ – produced in a number of tissues (however, the major production site is the liver)
* The vWF portion of VIII:vWF - made by ___
* Increased in liver disease are ___
* __ has the shortest half-life (6 hours). With acute liver dysfunction, levels of factor VII are reduced early. __ is a very good test to assess liver function!!! in the acute setting because Factor VII is needed for the extrinsic pathway.
* All deficiencies of coagulation factors are transmitted as ___ with the exceptions of Factor VIII deficiency and Factor IX deficiency, which are transmitted as __.
LIVER
Factor VIII
megakaryocytes and endothelial cells
Factors I and VIII
Factor VII
PT
autosomal recessive
X-linked recessive
CHARACTERISTICS OF CLOTTING FACTORS:
Factor I; other name: ___
● Mol. Weight (Daltons): 340,000
● Half-life (hours): 100 to 150
● Mean plasma concentration: 200 to 400 mg/dL
♦ Most concentrated of all the plasma procoagulants
♦ If fibrinogen level is _________, PT and aPTT will be prolonged.
♦ Essential for platelet aggregation (links activated platelets through their GP IIb/IIIa platelet fibrinogen receptor)
♦ Platelet alpha granules absorb, transport and release abundant ___.
♦ increases approximately 10 mg/dL er decade in the elderly
Fibrinogen
<100mg/dL
fibrinogen
CHARACTERISTICS OF CLOTTING FACTORS:
Factor II; ___
● Mol. Weight (Daltons): 71, 600
● Half-life (hours): 60
● Mean plasma concentration: 10 mg/dL
Prothrombin
CHARACTERISTICS OF CLOTTING FACTORS:
Factor III; ___
● Mol. Weight (Daltons): 44,000
● Half-life (hours): insoluble
● Mean plasma concentration: None
○ originate from peripheral tissues: F3 doesnt exist, always present in tissues
Tissue Factor/Tissue Thromboplastin/Thrombokinase
CHARACTERISTICS OF CLOTTING FACTORS:
Factor IV; ___
● Mol. Weight (Daltons): 40
● Half-life (hours): N/A
● Mean plasma concentration: 8 to 10 mg/dL
Calcium ions
CHARACTERISTICS OF CLOTTING FACTORS:
Factor V; ___
● Mol. Weight (Daltons): 330,000
● Half-life (hours): 24
● Mean plasma concentration: 1 mg/dL
♦ ___ – mutant factor V (Dutch investigators from the city of Leiden first described this mutation)
♦Remember that factors V and VIII are inactivated by ___. However,
Factor V Leiden (mutated factor V) is NOT inactivated and leads to excessive clot formation.
○ Deficiency of Factor V is “___” or “___”
Proaccelerin/Labile Factor/Thrombogen
Factor V Leiden
protein C-protein S complex
Owren’s Disease or Parahemophilia
CHARACTERISTICS OF CLOTTING FACTORS:
Factor VII; ___
● Mol. Weight (Daltons): 50,000
● Half-life (hours): 6
● Mean plasma concentration: 0.05 mg/dL
○ First coagulation factor to be affected by Warfarin therapy
○ has the shortest half-life
Proconvertin/Stable Factor
CHARACTERISTICS OF CLOTTING FACTORS:
Factor VIII; ___
● Mol. Weight (Daltons): 330,000
● Half-life (hours): 12
● Mean plasma concentration: 0.01 mg/dL
♦ Free factor VIII is ___ in plasma (it circulates bound to vWF). During coagulation, ___ cleaves factor VIII from vWF and activates it.
○ Factor VIII deficiency is “___” or “___”
Antihemophilic Factor A (AHF-A)/Antihemophilic globulin (AHG)
unstable
thrombin
Hemophilia A or Classic Hemophilia
CHARACTERISTICS OF CLOTTING FACTORS: VIII SIDE NOTES
*Factor VIII circulates in the blood bound to vWF (“Factor VIII complex”)
*Factor VIII complex may be symbolized as follows (based on its characteristics):
(1) ___
- refers to procoagulant portion
- measured by standard Factor VIII assays and APTT
- markedly decreased in Hemophilia A
(2) ___
- refers to the antigenic properties
- measured by immunoassays
(3) ___
- refers to the portion responsible for platelet aggregation in the presence of ristocetin
- termed as ristocetin cofactor
(4) ___
- also termed the von Willebrand factor
- required for normal platelet adhesion
(5) ___
- antigenic portion of the von Willebrand factor
- was previously termed Factor VIII related antigen (VIIIR:Ag)
- measured by immunoassays
Factor VIII, Factor VIIIC, Factor VIII:C
Factor VIII: Ag
Factor VIIIR:RCo
Factor VIII:vWF
vWF:Ag
CHARACTERISTICS OF CLOTTING FACTORS:
● Mol. Weight (Daltons): 600,000 to 20,000,000
● Half-life (hours): 24
● Mean plasma concentration: 1 mg/dL
♦ largest molecule in the human plasma
♦ VWF has receptor sites for BOTH platelets and collagen (helps to bind platelets to exposed subendothelial collagen during platelet adhesion).
♦ ___ - primary platelet surface receptor for VWF
vWF von Willebrand Factor
GP Ib/IX/V
CHARACTERISTICS OF CLOTTING FACTORS:
Factor IX; __
● Mol. Weight (Daltons): 57,000
● Half-life (hours): 24
● Mean plasma concentration: 0.3 mg/dL
○ Factor IX deficiency is: “___” or “___”
Christmas Factor/Antihemophilic Factor B (AHF-B)/ Plasma Thromboplastin Component (PTC)
Hemophilia B or Christmas Disease
CHARACTERISTICS OF CLOTTING FACTORS:
Factor X; ___
● Mol. Weight (Daltons): 58,800
● Half-life (hours): 48 to 52
● Mean plasma concentration: 1 mg/dL
Stuart-Prower Factor
CHARACTERISTICS OF CLOTTING FACTORS:
Factor XI; ___
● Mol. Weight (Daltons): 143,000
● Half-life (hours): 48 to 84
● Mean plasma concentration: 0.5 mg/dL
○ Factor XI deficiency is “__” or “__”; >50% of cases seen in ___
Antihemophilic Factor C (AHF-C)/Plasma Thromboplastin Antecedent (PTA)
Hemophilia C or Rosenthal Syndrome
Ashkenazi Jews
CHARACTERISTICS OF CLOTTING FACTORS:
Factor XII; ___
● Mol. Weight (Daltons): 84,000
● Half-life (hours): 48 to 70
● Mean plasma concentration: 3 mg/dL
○ Factor XII deficiency has NO bleeding tendency; has ___
Hageman Factor/Glass Factor/Contact Factor
thrombotic tendency
CHARACTERISTICS OF CLOTTING FACTORS:
Factor XIII; ___
● Mol. Weight (Daltons): 320,000
● Half-life (hours): 150
● Mean plasma concentration: 2 mg/dL
○ To detect Factor XIII, one may use the ___ (aka. ___ Test)
○ Not impotant clot formation but for clot ___!!
○ if clot is soluble in 5M urea = __
○ if clot is insoluble in 5M urea = __
Fibrin Stabilizing Factor/Fibrinase/Laki-Lorand Factor
5M Urea Clot Solubility Test (aka. Duckert’s Test)
stabilization
Factor XIII deficiency
Factor XIII is still present
CHARACTERISTICS OF CLOTTING FACTORS:
(aka: Fletcher Factor)
● Mol. Weight (Daltons): 85,000
● Half-life (hours): 35
● Mean plasma concentration: 35 to 50 μg/mL
PK Prekallikrein
CHARACTERISTICS OF CLOTTING FACTORS:
Flaujeac Factor
● Mol. Weight (Daltons): 120,000
● Half-life (hours): 156
● Mean plasma concentration: 5 mg/dL
HMWK High-molecular-weight Kininogen/Reid Factor/Williams Factor/
Fitzgerald Factor/
●REMEMBER THESE:
➢ Platelet phospholipids (NOT in the list), particularly __, are considered coagulation factors also. They were once called collectively as platelet factor 3 (PF-3).
➢ __ coagulation factors are enzymes that circulate in an inactive form called zymogens. These are: __
➢ The coagulation factors that function as cofactors are:
o Tissue factor (III)
o Factor V
o Factor VIII
o HMWK
phosphatidylserine
Eight
(all else becomes serine protease)
o Prothrombin (II)
o VII
o IX
o X
o XI
o XII
o Prekallikrein
o XIII - becomes transglutaminase
GROUPS OF CLOTTING FACTORS:
INTRINSIC COAGULATION PATHWAY FACTORS?
XII, XI, IX, VIII
(12, 11, 9, 8)
GROUPS OF CLOTTING FACTORS:
EXTRINSIC COAGULATION PATHWAY FACTORS?
III and VII
(3 and 7)
GROUPS OF CLOTTING FACTORS:
COMMON COAGULATION PATHWAY FACTORS
X, V, II, I
(10, 5, 2, 1)
GROUPS OF CLOTTING FACTORS: According to Properties:
I, V, VIII, XIII (1,5,8,13)
-aka: thrombin sensitive grp.
-Absent in serum
-Vitamin K-independent
-Calcium-dependent
-INCREASED in:
*P- __________________
*I - ___________________
*S - __________________
*O- __________________
FIBRINOGEN GROUP
pregnancy
inflammation
stress
oral contraceptive
GROUPS OF CLOTTING FACTORS: According to Properties:
II, VII, IX, X (2, 7, 9,10)
-Present in aged serum, EXCEPT: __
-VITAMIN K-DEPENDENT
-Calcium-dependent
-Adsorbed from the plasma using either: * ______, or * ________
~ ▪ ___________: soag factors and coag control proteins formed under conditions of vitamin K absence or antagonism. do NOT precipitate participate in the coag process!
PROTHROMBIN GROUP
factor II
barium sulfate
aluminum hydroxide
PIVKA (protein induced by Vitamin K absence)
GROUPS OF CLOTTING FACTORS: According to Properties:
XII, XI, PK, HMWK (12, 11, Prekallikrein, High-mol.-weight kininogen)
- Vitamin K-independent
- Calcium-independent
CONTACT GROUP
NOTES:
▪ This concept of coagulation process is used extensively to interpret in vitro laboratory tests and to identify factor deficiencies; however, this does NOT sufficiently describe the complex interdependent reactions that occur in vivo.
▪ Thrombin - activates factors ___
▪ ___ – includes 9a, platelet factor-3 (PF-3), Calcium,
and 8a
▪ ___ – includes 10a, PF-3, Calcium, and 5a
▪ ___– involved in all phases of coagulation, except in the contact phase
▪ Stabilized fibrin clot – __ to 5M Urea
1,5,8, and 13
Tenase Complex
Prothrombinase Complex
Calcium
NOT soluble
CELL-BASED (IN VIVO, PHYSIOLOGIC) COAGULATION
-Aside from the procoagulant and anticoagulant plasma proteins, normal physiologic coagulation needs the presence of two cell types for formation of coagulation complexes:
(2)
-coagulation in vivo can be described as occurring in two phases:
(1) ___ - occurs on tissue factor-bearing cells and generates 3% to 5% of total thrombin produced
(2) ___ - occurs on platelets; produces > 95% of the total thrombin
(1) tissue factor-bearing cells (usually extravascular)
(2) platelets (intravascular)
INITIATION
PROPAGATION
COMMON TESTS FOR SECONDARY HEMOSTASIS:
I.) ___ - “partial” – because the reagent consists only of the PL (phospholipid) portion of the tissue thromboplastin (prepared from brain or plant phospholipids); “activated” – because there are negatively charged activators to be added
✓ Reference range: ___
✓ An example of a clot-based coagulation screening test
✓ Specimen requirements:
* Citrated platelet-poor plasma
(centrifugation for 15 minutes at 2500 X g)
✓ Materials and reagents:
*APTT reagent = Two components: (1) ___ – supplied to substitute for PF3
(2) ___ – added to achieve maximum activation of the contact factors
USES: to monitor patients in ___; to detect coag factor deficiency in the ___ and ___
APTT/aPTT
35 to 45 seconds
Platelet substitute (phospholipid)
Activator
UFH therapy
COMMON & INTRNSIC
II.) ___
✓ Reference range: 10-13 seconds (generally)
✓ clot-based coagulation screening test
✓ Specimen requirements:
* Citrated platelet-poor plasma
✓ Materials and reagents:
* Thromboplastin-Calcium Chloride (CaCl2) reagent (PT reagent; Simplastin)
✓ USES:
* to monitor patients in ___ therapy; to detect coag factor deficiencies in the __ and __
___:
- Standardized way of reporting PT (NOT a new laboratory test)
- It is merely a mathematical calculation (corrects for the variability in PT results caused by variable sensitivities of the thromboplastin agents used by laboratories).
- A target INR range of ___ is recommended for most indications. The physician adjusts the Coumadin dosage to achieve the desired INR of 2.0 to 3.0 (or ___ if the patient has a
mechanical heart valve).
~ appropriate test to monitor rat poison that was ingested (content is similar to warfarin)?
~ formula for INR
PT – Protime or Prothrombin Time
Warfarin
COMMON & INTRINSIC
INR (International Normalized Ratio)
2.0 to 3.0
2.5 to 3.5
PT
INR = (patient PT/mean normal PT)^ISI
ISI stands for International Sensitivity Index
RELATED TOPICS TO PT AND APTT:
Therapeutic Anticoagulants:
I.) ___
✓ Vitamin K antagonists (VKAs)
✓ Oral anticoagulants
✓ Examples: dicumarol, phenprocoumon, acenocumarol, warfarin (WARF?)
COUMARIN DRUGS
WARF:
Wisconsin
Alumni
Research
Foundation
Therapeutic Anticoagulants
I.) COUMARIN DRUGS
✓ ___ – most commonly used
o interferes with the normal synthesis of Factors II, VII, IX, and X as well as Proteins C and S
o brand name: Coumadin; for reversal of Coumadin overdose, the following may be given (depending on the patient’s current condition):
✓ Vitamin K (oral/intravenous)
✓ Fresh-frozen plasma
✓ Prothrombin complex concentrate
✓ Recombinant activated factor VII (rVIIa)
Warfarin
Therapeutic Anticoagulants
II.) ___
✓ Heparin has no anticoagulant activity of its own (acts as an anticoagulant by accelerating the binding of antithrombin to target enzymes (e.g., thrombin and factor Xa)).
✓ UFH (Unfractionated heparin) – used routinely in cardiac surgery. UFH therapy requires monitoring with partial thromboplastin time (PTT) or activated clotting time (ACT) assay. It is administered intravenously!!!
✓ For neutralizing UFH : protamine sulfate (a protein extracted from: ________ )
✓ ___:
o An example of a clot-based coagulation screening test
o A point of care assay that is used in clinics, at the inpatient’s bedside, in the cardiac catheterization laboratory, or in the surgical suite, and it is specifically useful at the high UFH dosages!!!; In this test, a particulate activator is added to blood, the mixture is rocked, and the interval to clotting is recorded.
UNFRACTIONATED HEPARIN (UFH)
salmon sperm
ACT (activated coagulation time)
___:
o produced by the controlled fragmentation of heparin; react with the regulatory protein antithrombin III to inhibit activated factor X (factor Xa) but not thrombin (factor IIa).
o administered by subcutaneous injection
o Some of the advantages of using this include:
▪ Lowered incidence of heparin-induced thrombocytopenia compared with unfractionated heparin
▪ Lowered incidence of hemorrhage and osteoporosis
o Some of the disadvantages of using LMWH include:
▪ Increased cost compared with traditional (unfractionated) heparin
▪ More complicated monitoring of LMWH than traditional heparin
Low-Molecular-Weight Heparin (LMWH)
● Coagulation Instrumentation
-Instrument methodologies used for coagulation testing are classified into five groups based on the end-point detection principle:
1.) ___
✓ Examples:
o BBL Fibrometer (uses electromechanical clot detection system that measures a change in conductivity between two metal electrodes
in plasma)
o Diagnostica Stago analyzers (In these analyzers, an electromagnetic field detects the oscillation of a steel ball within the plasma-reagent solution.
2.) ___
✓ Coagulometers that use this principle detect a change in plasma optical density
during clotting.
3.) ___
✓ A modification of photo-optical end-point detection
✓ Here, 90-degree or forward-angle light scatter, rather than OD, is measured
4.) ___
✓ Uses a synthetic oligopeptide substrate conjugated to a chromophore, usually para-nitroaniline (pNA)
✓ A means for measuring specific coagulation factor activity because it uses the factor’s enzymatic (protease) properties
✓ Useful to evauate specimens from patients who have circulating inhibitors or who are on anticoagulant treatment because the inhibitors do not interfere in the chromogenic assay.
5.) ___
✓ Based on antigen-antibody reactions similar to those used in nephelometry
MECHANICAL/ ELECTROMECHANICAL
PHOTO-OPTICAL (TURBIDOMETRIC)
NEPHELOMETRIC
CHROMOGENIC (AMIDOLYTIC)
IMMUNOLOGIC
___: may be used to identify specific factor deficiency by mixing correction reagents with
patient’s plasma and then performing the PT and/or APTT; may also be used to detect lupus anticoagulant and specific factor inihibitors
-uses the following: (4)
Substitution Test
1.) Fresh Plasma (Normal Plasma)
2.) Aged Plasma
3.) Aged Serum
4.) Adsorbed Plasma
Coagulation Factors present in:
1.) Fresh Plasma (Normal Plasma)
-_______________________________________________
2.) Aged Plasma
-_______________________________________________
3.) Aged Serum
-_______________________________________________
4.) Adsorbed Plasma
-______________________________________________
all factors (except F3 and 4)
all factors except factor 5 and 8 (labile f)
all factor except factors 1, 5, 8, 13, 2
all factor except factors 2, 7, 9, 10
Therapeutic Anticoagulants
III.) ____
- used to detect deficiencies in fibrinogen, prothrombin, and factors V and X
- differs from the PT in that deficiencies in factor VII are ___
- uses the coagulant properties of Russel’s viper venom, obtained from the snake ___
- venom is capable of bypassing the action of Factor VII and directly activating Factor X to Xa.
- may help differentiating ___ deficiencies
- Normal Value: 20 to 25 seconds
*____– used to detect lupus anticoagulants
- Normal Value: 30 to 35 seconds
Stypven time (Russel’s Viper Venom Time)
NOT detected
Vipera russeli
Factor VII and Factor X
Dilute Russel’s Viper Venom Time (dRVVT)
Therapeutic Anticoagulants
IV.) ___
- Prolonged thrombin times are found when the fibrinogen level is low, when function of fibrinogen is impaired, and in the presence of heparin, fibrin(ogen) degradation products and streptokinase
- TT is a sensitive test in detecting heparin inhibition
- Normal Value: 15 to 18 seconds
V.) ___
- Reptilase is an enzyme found in the venom of the ___ snake and is capable of converting fibrinogen to fibrin.
- Reptilase is unaffected by __
- Normal Value: 10 to 15 seconds
Thrombin Time
ReptilaseTime
Bothrops atrox
heparin
Therapeutic Anticoagulants
VI.) ___
* Slide or Drop Method; N.V.= 2 to 4 mins.
* ___ (Whole Blood Clotting Time) Method:
○ Place three test tubes (labeled as tube 1, 2 and 3) in 37 C water bath; Place 1 mL blood into each of the three tubes, starting with tube 1. tubes are checked in intervals
o N.V. = 7 to 15 mins.
Clotting Time
Lee and White
CIRCULATING INHIBITORS (CIRC. ANTICOAGULANTS)
- Acquired inhibitors of coagulation
- Usually detected by the prolongation of the PT or APTT which is NOT corrected by the addition of normal (fresh) plasma
-Two groups of inhibitors include:
1. ___: Antibodies against specific coagulation factors; Associated with bleeding
-Examples: Antibodies to factors VIII and IX (MOST COMMON)
2. ___: Ex:
○ Lupus anticoagulant :
- IgG, IgM, IgA; found in __ of SLE patients
- Normal or slightly ↑ PT and ↑APTT
- directed against phospholipids and are generally not associated with bleeding
- Tests : -Substitution test, Platelet neutralization test & dRVVT
○ Paraproteins
○ Fibrinogen degradation products
SPECIFIC INHIBITORS
NON-SPECIFIC INHIBITORS
10%
NATURALLY OCCURING INHIBITORS OF COAGULATION
●TWO MAJOR ANTICOAGULANT SYSTEMS IN THE BODY:
I. ___
▪ __ – activated by thrombin bound to thrombomodulin (TM)
▪ __ – inactivates Factors Va and VIIIa
▪ __ – co-factor to Protein C
II. ___
- Antithrombin (formerly antithrombin III) is the main serine protease inhibitor
- AT inhibits: IIa [thrombin], IXa, Xa, XIa, kallikrein, XIIa
Protein C/Protein S system
Protein C
Activated Protein C (APC)
Protein S
Plasma serine protease inhibitor (SERPIN) system
✓ Final stage of coagulation and is the dissolution of the formed clot
✓ Starts a few hours after fibrin polymerization and cross-linking
✓ TPA and UPA – two activators of fibrinolysis that are released in response to inflammation and coagulation.
✓ Fibrinolytic proteins accumulate on fibrin during clotting. Plasminogen, plasmin, TPA, UPA, and PAI-1 become incorporated into the fibrin clot.
✓ TPA and UPA activate fibrin-bound plasminogen several hours after clot formation, degrading fibrin and reestablishing normal blood flow during vascular repair.
FIBRINOLYSIS
PROTEINS OF THE FIBRINOLYSIS PATHWAY:
-a plasma zymogen produced by the __
-stored and transported in __
-fibrin-bound plasminogen become converted into active plasmin
-plasmin is a __ (bound plasmin digests clot and restores blood vessel patency)
-free plasmin is capable of digesting plasma fibrinogen, factor V, factor VIII, and fibronectin, causing a potentially deadly primary fibrinolysis (however, plasma α2-antiplasmin rapidly binds and inactivates any free plasmin in the circulation)
Plasminogen
liver
eosinophils
serine protease
PROTEINS OF THE FIBRINOLYSIS PATHWAY:
-serine protease secreted by activated endothelium, activates plasminogen
-circulating __ is bound to inhibitors such as PAI-1 and is cleared from plasma
Tissue plasminogen activator (TPA)
PROTEINS OF THE FIBRINOLYSIS PATHWAY:
-another intrinsic plasminogen activator
-secreted by urinary tract epithelial cells, monocytes, and macrophages
-UPA does NOT bind firmly to fibrin, and has a relatively minor physiologic effect
Urokinase Plasminogen Activator (UPA)
PROTEINS OF THE FIBRINOLYSIS PATHWAY:
-PAI-1 is the principal inhibitor of plasminogen
activation
-inhibits both TPA and UPA and thus preventing them from converting plasminogen to plasmin
Plasminogen Activator Inhibitor 1 (PAI-1)
PROTEINS OF THE FIBRINOLYSIS PATHWAY:
-inhibits plasmin, synthesized in the liver
-primary inhibitor of free plasmin
- ε-aminocaproic acid and tranexamic acid = antifibrinolytic (both inhibit the proteolytic activity of plasmin)
α2-antiplasmin
PROTEINS OF THE FIBRINOLYSIS PATHWAY:
-synthesized in the __
-becomes activated by the thrombin-thrombomodulin complex (the same complex that activates the protein C pathway; however, the two functions are independent)
-activated TAFI functions as an antifibrinolytic
enzyme
Thrombin-Activatable Fibrinolysis Inhibitor (TAFI)
liver
●TESTS FOR FIBRINOLYSIS:
A.) ___
✓ Plasmin lyses the fibrin and produces FDP that contain the portion called D-dimer.
✓ Plasmin will also lyse fibrinogen, but will NOT produce the D-dimer.
✓ The presence of D-dimer indicates that a stable fibrin clot has been lysed.
✓ FSP/FDP (+) = DIC or pathological fibrinolysis
-FDP(+), D-dimer (-) = ___
-FDP(+), D-dimer (+) = ___
D-dimer Test
Pathological fibrinolysis
DIC
●TESTS FOR FIBRINOLYSIS:
B.) ___
✓ __ -proteins that precipitate when plasma is diluted with water and acidified (1% acetic acid) -include plasminogen, plasmin, fibrinogen, and plasminogen activators
✓ PROCEDURE: - patient’s citrated PPP is diluted w/ water, acidified and refrigerated
- euglobulins will precipitate, which is then redissolved and clotted with thrombin
- if plasminogen (in the euglobulin fraction) is converted to plasmin, it will lyse the clot
- time needed for complete lysis (at 37 C) is the euglobulin lysis time
✓ Euglobulin lysis time is typically longer than 2 hours.
✓ Lysis in less than 2 hours = __
Euglobulin Lysis Time
Euglobulins
increased fibrinolytic activity
PLATELET ACTIVATION
● Platelet Activation Pathways:
o ___
▪ aka: guanine nucleotide-binding proteins
▪ control cellular activation for all cells (not just platelets) at the inner membrane surface
▪ Stimulate the two other platelet pathways: EICOSANOID SYNTHESIS PATHWAY and IP3-DAG PATHWAY
G proteins
PLATELET ACTIVATION
● Platelet Activation Pathways:
o ___
▪ aka: prostaglandin, cyclooxygenase, or thromboxane pathway
▪ __ : a mediator derived from the 20-carbon atom arachidonic acid (20 in Greek is “eicosa”) or a similar fatty acid
▪ Collagen, ADP, Thrombin, and Epinephrine bind their respective membrane receptors
(The combination activates phospholipase A2.)
▪ Phospholipase A2 releases ___ (also called 5,8,11,14-eicosatetraenoic acid) from membrane phosphatidylinositol.
▪ Arachidonic acid is acted upon by cyclooxygenase, peroxidase, and thromboxane
synthase to produce TXA2, which activates the _.
▪ TXA2 suppresses adenylate cyclase and reduces cyclic AMP concentration. This allows
the release of ionic calcium from the DTS. Ionic calcium supports the contraction of actin microfilaments, activating the platelet.
▪ REMEMBER:
* The eicosanoid pathway in the endothelial
cells is almost the same EXCEPT that prostacyclin synthase replaces thromboxane synthase. (thromboxane synthetase in platelets; prostacyclin synthase in endothelial cells)
* In endothelial cells, the eicosanoid pathway result is ___
* __ penetrates the platelet and binds to its IP receptor.
* Prostacyclin accelerates adenylate cyclase, increasing cyclic AMP, and sequesters ionic calcium to the DTS.
* In intact blood vessels, the endothelial cell eicosanoid pathway suppresses platelet activation through the mechanisms stated above.
Eicosanoid synthesis pathway
Eicosanoid
arachidonic acid
platelet
prostaglandin I2 (aka: prostacyclin)
Prostacyclin
PLATELET ACTIVATION
● Platelet Activation Pathways:
o ___
▪ G-protein activates ___, which then cleaves membrane phosphatidylinositol 4,5-bisphosphate to form IP3 and DAG.
▪ ___ promotes release of ionic calcium from the DTS, which activates actin microfilament contraction.
▪ IP3 may also activate ___
▪ DAG activates a multistep process: activation of phosphokinase C (which triggers phosphorylation of the protein pleckstrin (which regulates actin microfilament contraction).
Inositol Triphosphate-Diacylglycerol (IP3-DAG) pathway
phospholipase C
IP3
phospholipase A2
● Processes Involved in Platelet Activation:
☼ Blood vessel injury: (3)
✓ Exposes collagen
✓ Releases VWF
✓ Releases tissue factor
● Processes Involved in Platelet Activation:
A.) ___
➢ platelets adhering to a foreign surface
➢ initiated by exposure of the __
➢ damaged endothelial cells release __ from cytoplasmic storage organelles
➢ VWF unrolls (similar to a carpet) and adheres to the injured site. (NOTE: VWF circulates as a globular protein. However, it becomes fibrillar as it unrolls.)
➢ VWF exposes sites that partially bind the GPIbα portion of the platelet __ (This is a reversible binding process that “tethers” or decelerates the platelet.)
➢ Platelet and VWF interactions remain localized by ADAMTS-13 that digests “unused” VWF.
➢ The VWF-GP Ibα tethering reaction is temporary, and the platelet rolls along the surface unless GP VI comes into contact with the exposed collagen.
➢ When collagen binds platelet GP VI, it activates internal platelet activation pathways,
releasing ___ and ____ (agonists)
○ ____: trigger a reaction that increases the affinity of α2β1 (GP Ia/IIa) for collagen
➢ The combined effect of GP Ib/IX/V, GP VI, and α2β1 (GP Ia/IIa) causes the platelet to become firmly attached to the damaged surface, where it subsequently loses its discoid shape and spreads.
➢ ___; also secreted from the platelet to the microenvironment, there they activate adjacent platelets through their respective receptors; provide inside-out activation of αIIbβ3 (GP IIb/IIIa), which assists in platelet aggregation.
ADHESION
subendothelial collagen
VWF
GP Ib/IX/V
TXA2 and ADP
➢ TXA2 binds to TPα and TPβ
➢ ADP binds to P2Y1 and P2Y12
TXA2 and ADP:
● Processes Involved in Platelet Activation:
B.) ___
✓ platelets adhering to other platelets
✓ Blood vessel injury releases tissue factor (TF) from ___
✓ TF triggers generation of __ which reacts with platelet PAR1 and PAR4. This further activation generates the ___ (“collagen and thrombin activated”) platelet (integral to the cell-based coagulation model).
✓ Meanwhile, αIIbβ3 (GP IIb/IIIa) assembles from its resting membrane units αIIb and β3.
✓ ___ binds fibrinogen and VWF and supports platelet-to-platelet aggregation.
✓ Thrombocytes lose their shape and extend pseudopods.
✓ Phosphatidylserine flips to the outer layer (establishing a surface for the assembly of
coagulation factor complexes).
✓ As platelet aggregation goes on, membrane integrity is lost, and a syncytium of platelet cytoplasm forms as they use internal energy sources.
AGGREGATION
endothelial cells
thrombin
COAT platelet
αIIbβ3 (GP IIb/IIIa)
● Processes Involved in Platelet Activation:
C.) ___
✓ The activation of the platelets triggers actin microfilament contraction.
✓ Intermediate filaments also contract (moves the circumferential microtubules inward and compressing the granules).
✓ Phosphatidylserine is the phospholipid on which two coagulation pathway complexes assemble: ___ (both supported by ionic calcium) binds and stabilizes Factor VIII) are secreted and increase the localized concentrations of these clotting factors. Their presence further supports the action of tenase and prothrombinase.
✓ ___ contents and lysosomes’ contents flow through the SCCS
✓ dense granules travel to the __
✓ Most of the α-granule contents are proteins that participate in secondary hemostasis (called coagulation).
✓ The dense granule contents are vasoconstrictors and platelet agonists that intensify __.
✓ Lysosomes probably digest vessel wall matrix components during in vivo aggregation and may also digest autophagic debris
SECRETION
tenase and prothrombinase
α-granules
plasma membrane
primary hemostasis
OTHER TESTS FOR THE PRIMARY HEMOSTASIS
I.) ___
❖ Length of time a standard wound bleeds
❖ A screening test for detecting disorders of platelet function and VWD
❖ NOT affected by the coagulation mechanism
❖ Affected by:
1.) Platelet count and platelet function
2.) Thickness and vascularity of the skin
3.) Quality of the blood vessels
4.) Medications, such as:
▪ Aspirin or aspirin-containing products = ___ before bleeding time test
▪Ibuprofen, tolmetin sodium, and naproxen (nonsteroidal anti-inflammatory) = should be
discontinued ___
BLEEDING TIME
avoid 7 days
24 hours before the test
OTHER TESTS FOR THE PRIMARY HEMOSTASIS
I.) BLEEDING TIME
○ ___: Finger is punctured using a sterile lancet; Start timer as soon as the first drop of blood appears. Blot drop of blood with filter paper every 30 secs. (Filter paper must not touch the wound).
o N.V. = 2 to 4 mins.
Duke method
OTHER TESTS FOR THE PRIMARY HEMOSTASIS
I.) BLEEDING TIME
○ ___: Use sphygmomanometer (inflated to __ at patient’s upper forearm (above the elbow)
o Cleanse an area; Hold skin tightly and make 2 skin incisions (2 mm deep and 2 mm long) avoiding any subcutaneous veins.
o Start the timer and blot the drop of blood from each puncture site on 2 separate filter paper every 30 secs.
o N.V. = 1 to 7 mins.
Ivy method
40mmHg)
OTHER TESTS FOR THE PRIMARY HEMOSTASIS
I.) BLEEDING TIME
○ ___: A modification by C.H. Mielke, Jr. and colleagues of the original Ivy bleeding time test
o Used a template containing a standardized slit in place of the disposable lancets; Template has been replaced by several commercially made devices (examples: Simplate and Surgicutt)
o General reference range: 2 to 9 mins.
Template Bleeding Time
OTHER TESTS FOR THE PRIMARY HEMOSTASIS
II.) ___: an example of a capillary fragility test (particularly a tourniquet test)
- procedure:
✓ Determine the patient’s blood pressure.
✓ Apply pressure cuff on the upper forearm and inflate to a pressure midway systolic and diastolic for 5 minutes.
✓ Remove the cuff and observe for the presence of petechiae on the arm (volar surface) and on the dorsal area of hands and fingers.
✓ 15 minutes after release of pressure cuff, choose an area of 1-inch diameter anywhere on the areas mentioned above.
✓ Count the number of petechiae formed within the 1-inch diameter area and grade.
✓ Normal value: Grade 1+ (a grade of 2+ and above is indicative of capillary weakness)
RUMPLE-LEEDE TEST
OTHER TESTS FOR THE PRIMARY HEMOSTASIS
III.) ___
✓ Thrombosthenin – a contractile protein important for CR
✓ Principle:
o Normal blood clot contracts (in so doing, it expresses serum)
o Degree of clot retraction: measured on the amount of expressed serum
✓ Normal clot retraction requires: (4)
CLOT RETRACTION
o Normal level of fibrinogen
o Normal level of functioning platelets
o Calcium
o ATP
OTHER TESTS FOR THE PRIMARY HEMOSTASIS
III.) CLOT RETRACTION
____:
▪Procedure:
a.) Place few drops of castor oil into a clean test tube.
b.) Place 1 drop of fresh blood (without anticoagulant) into the castor oil then start timer.
c.) Observe for extrusion of droplet-like serum (“dimpling”) on top portion of the drop of blood
d.) Stop the timer as soon as “dimpling” of serum is detected.
▪N.V.: 15 to 45 mins.
HIRSCHBOECK/CASTOR OIL
OTHER TESTS FOR THE PRIMARY HEMOSTASIS
III.) CLOT RETRACTION
____:
▪Procedure:
a.) Place 5 mL of fresh venous blood into a clean calibrated tube.
b.) Insert a glass rod into the tube with the expansion immersed in the column of blood.
c.) Fit a cork into the glass rod by boring a hole at the center of the cork and allowing the projecting tip of the glass rod
pass through the hole. This will cover the tube.
d.) Place tube in 37◦C water bath and observe every 5 to 10 minutes for coagulation.
e.) One hour after firm clotting, allow tube to stand at room temperature for retraction to occur. (Retraction has taken place
if the clot has shrunk and has attached itself onto the glass rod).
f.) After retraction has taken place, remove the cork.
g.) Remove the glass rod and attached clot.
h.) Measure the amount of expressed serum directly against the calibration of the tube.
i.) Computation: ___
▪Normal Value: 44 to 67%
MAC FARLANE METHOD
%CR = (AMT. OF SERUM LEFT IN TUBE/AMT. OF BLOOD USED) X 100
▪REMINDERS:
o Interaction of platelets with fibrinogen and fibrin must also be normal for clot retraction to occur
o Abnormal Clot Retraction observed in: (4)
▪ Thrombocytopenia
▪ Low or abnormal fibrinogens
▪ Paraproteinemias (multiple myeloma, macroglobulinemia) that interfere with fibrin formation
▪ Glanzmann’s thrombasthenia (platelet is incapable of interacting with fibrin)
