bioanalytical pathology II
Terms
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- platelet production is stimualted by ________
- thrombopoietin
- __________ acts as a bridge bt platelets and subendothelial collagen resultsing in ______ of platelets to collagen
-
von Willebrand factor
adhesion - 3 steps of the platelet plug
-
1.adhesion
2.granule release reaction
3.aggregation - notable granular contnets of platelets
-
1.calcium:which helps stimulate platelet actions and plays a role in the coagulation system
2.von Willebrand factor: which promotes platelet adhesion to subendothelial collagen
3.fibrinogen: which causes platelets to adhere to each other
4.ADP:which induces expression of platelet surface receptors that bind to fibrinogen - When due platelets release there granules?
- when they adhere to subendothelial collagen
- platelet aggregation
-
under the influence of released platelet granule contents, platelets begin to adhere to each other.
Ultimately this results in the formation of an aggregate of platelets that plugs the defect in the vessel wall, and, therefore hemorrhage stops - When can primary hemostasis alone provide permaent hemostasis in an injured vessel wall
- in the defect is small and the blood flow in the vessel is slow
- intrinsic factors
-
XII
XI
IX
VIII - extrinsic factors
- VII
- common factors
-
X
V
II
I - factor III
- Factor III is tissue thromboplastin. It does not circulate, but ratheris attached to fibroblasts and smooth muscle cells where it helps promote the activity of the extrinsic system by activating factor VII
- factor IV
- Calcium
- factor VI
- there is no factor VI
- where are coagulation factors synthesized
-
most are made in the liver.
some are made in macrophages.
factor III is made in fibroblasts and smooth muscle cells - coagulation factors that are dependent on vitamin K
-
II
VII
IX
X - factor V
- accelerator that promotes more rapid activation of factor II
- factor VIII
- accelerator that promotes more rapid activation of factor X
- The _________ system is thought to be the primary system responsible for activation of coagulation
- extrinsic
- factor XIII
- fibrin stabilizing factor
- severe __________ can result in loss of large amounts of _____ in the urine with susequent thrombosis
-
glomerulopathies
AT3 - 2 most important anticoagulant proteins
-
1.Antithrombin III(AT-3)70%
2.alpha 2 macroglobulin 20% - anti thrombin III
- heparin at the endothelial cell surface complexes with AT-3, and this complex inactivates thrombin. AT-3 also inactivates factors IX,X,XI,XII
- Factor II
- prothrombin (thrombin when activated)
- alpha 2 macroglobulin
- inhibits thrombin, plasmin, and kallikrein
- fibrinolysis is performed by ________ and produces ____________
-
plasmin
small peptides and d-dimers (which are refered to as fibrin/fibrinogen degradation products-FDPs - increased bleeding time can occur with the what abnormalities
-
1.blood vessel abnormalities (esp. abnormal subendothelial collagen)
2.thrombocytopenia
3.abnormal platelet function - ACT is a test of the function of the ________ and _________ systems. May also be affected by severe _______
-
intrinsic
common
thrombocytopenia - Acticated partial thromboplastic time (APTT) is a test of the ________ and _________systems
-
intrinsic
common - prothrombin time (PT) is a test of the function of the ________ and _______ systems
-
extrinsic
common - petechia and ecchymosis are seen with what type of abnormalites
- vascular and platelet
- acquired vascular disorders which can lead to a disorder of hemostasis
-
1.vasculitis
2.scurvy: vitamin c is needed for normal callagen formation
3.hyperadrenocorticism- leads to decreased collagen production - typical laboratory findings in animals with hemorrhage resulting form vascular disorders
-
Bleeding time: prolonged
ACT: Normal
APTT: Normal
PT: Normal
TT: Normal - typical laboratory findings in animals with hemorrhage resulting form thrombocytopenia
-
thrombocytopenia
bleeding time :prolonged
ACT: prolonged if less than 10,000 platelets
APTT: Normal
PT: Normal
TT: Normal - things that cause acquired platelet dysfunction
- NSAIDs, abnormal proteins produced in multiple myeloma, phenothiazines, waste products that accumulate during uremia, FDPs
- most common inherited platelet dysfunction problem in animals
-
von Willebrand disease
results in deficiency of von willenbrands factor - typical laboratory findings in animals with hemostatic defects cuased by acquired platelet function defects or inherited platelet function defects other than von willebrand disease
-
bleeding time: prolonged
ACT: Normal
APTT: Normal
PT: Normal
TT: Normal
von willebrand factor: Normal - typical labortory findings with von willebrand disease
-
bleeding time: prolonged
ACT: Normal to prolonged*
APTT: Normal to prolonged*
PT: Normal
TT:Normal
von willebrand factor: decreased or absent
*ACT and APTT may be prolonged due to the close association of von willebrand factor and factor VIII. decreased vWF may reduce the activity of factor VIII:C even though it is present in adequate concentration - acquired coagulation disorders
-
1.vitamin K
2.hepatic failure
3.DIC - decreased active vitamin K can result from
-
1.low dietary intake
2.malabsorption
3.prolonged bile duct obstruction
4.warfarin
5.coumarin (moldy sweet clover - laboratory findings in acuired coagulation disorders resulting from decreased active vitamin K
-
bleeding time: normal
ACT: prolonged
APTT: prolonged
PT: Prolonged
TT:Normal for vit K alone, prolonged in hepatic failure
von willebrand factor: normal - DIC laboratory findings
-
palelet count:decreased
bleeding time: prolonged
ACT:prolonged*
APTT:prolonged*
PT:prolonged*
FDP:increased
d-d9mer:increased
AT-3: decreased
*ACT,APTT, and PT may be shortened early in DIC due to the marked activation of the coagaulation cascade, but in most cases these are prolonged by the time the affected animal is presented to the veterinarian. TT can be expected to be prolonged if plasma fibrinogen concentration is decreased - reasons for doing a bone marrow
-
1.unexplained cytopenia
2.searching for the presence of neoplastic cells
3.staging of lymphomas
4.fever of unknown origin
5.principal means of evaluating the response to anemia in the horse-rarely done -
bone marrow aspiration preferred sites
1.cat and dog:
2.cow and hores:
3.bird: -
1.iliac crest, femur, humerus
2.sternum, rib
3.proximal tibiotarsal joint just below the femoral-tibiotarsal joint, sternum - bone marrow aspiration cytology versus core biopsy
-
aspiration:
1.quick turn around time
2.more cellular detail
3.No tissue archiecture
4.cellular density only estimated
5.the most useful and common evaluation
Core biopsy:
1.slower turn around time
2.no cellular detail
3.tissue architecture is intact
4.cellular density more accurately assessed
5.less commonly performed
-hypoplastic marrow
-myelofibrosis
-no cells on aspirate - _________ is the most consistent hematologic abnormality in both acute and chronic ehrlichiosis
- thrombocytopenia
- hematologic abnormalities in acute ehrlichiosis
-
thrombocytopenia-due to sequwstration, destructionor consumption
bone marrow:megakaryocytic hyperplasia, bone marrow hyperplasia
anemia: suppression of erythropoiesis, destruction of erythrocytes
leukocyte count is variable in the acute phase - laboratory abnormalities in chronic ehrlichosis
-
thrombocytopenia
pancytopenia <25% of dogs
severe bone marrow hypoplasia
bone marrow plasmacytosis
hyperglobulinemia 50-75%
protein eletrophoresis may be polyclonal or monoclonal - FeLV bone marrow:
- may produce hypoplastic bone marrow. hypoplasia may be restricted to one cell line or may be multiple
-
pure transudate:
modified transudate:
exudates: -
pure transudate:form due to hypoalbuminemia
modified transudate: form due to impaired blood or lymph flow
exudates:form due to increased capillary permeability - transudate or exudate
-
Transudate:
1.appearance-clear
2.total protein<3g/dl
3.NCC-<10,000/ul
4.clot form-no
exudate:
1.appearnce-cloudy
2.total protein>3g/dl
3.NCC->10,000
4.clot form-yes -
joint fluid analysis
inflammatory
A. suppurative:
B. monnuclear: -
A. usually immune-mediated disease. If septic difficult to see bacteria
B. degenerative disease or trauma - causes of lymphadenopathy
-
1.hyperplasia (antigenic stimulation)
2.reactive (antigenic stimualtion)
3.inflamed (lymphadenitis)
4.primary neoplasia (lymphoma)
5.metastaic neoplasia -
Indications for Transfusion
SEVERE ANEMIA- Numerical guidelines -
Acute anemia: Dog PCV < 20, cat < 12
Chronic anemia: Dog < 15, cat < 12
Must consider clinical signs!!! -
Indications for transfusion
Acute blood loss (surgery or trauma) -
Can use whole blood for RBC and volume
replacement
Packed cells and crystalloids preferable -
Indications for transfusion
Defect of hemostasis (eg Vit K antagonists) -
Replace coagulation factors
Must use fresh whole blood, fresh plasma, fresh
frozen plasma, or cryoprecipitates -
Indications for transfusion
Hypoproteinemia -
Albumin < 1.5 g/dL
Only for acute replacement
Difficult and expensive
Fresh plasma or frozen plasma -
Indications for transfusion
Severe thrombocytopenia -
Platelet-rich plasma
Used for acute replacement of platelets if
animal needs surgery
VERY difficult to increase platelet numbers
with fresh whole blood -
General principles of blood
typing -
Antibodies to KNOWN RBC antigens are
used to type
Antibodies are added to RBCs to be typed
Secondary antibodies (against antiRBC
antibodies), +/- complement
Agglutination or hemolysis of positive RBC -
Important blood groups of
domestic animals -
Dog erythrocyte antigens-
DEA 1.1, 1.2 (A), 7 most important
Cats- 3 blood groups
A, B, AB
Large animals
Zillions of blood groups -
Functional significance of
blood groups in animals -
Naturally occurring antibodies
antibodies against RBC antigens which occur
due to exposure to a different environmental
antigen (eg microorganism)
Can cause immediate transfusion reactions
Previous sensitization
Through transfusions, pregnancy or vaccines
with blood products - Crossmatching
-
Used to detect incompatibilities between
donor RBC and recipient serum
Detects the presence of antibodies in the
recipient serum which may react with donor
RBC to cause an immediate transfusion rxn
Naturally occurring antibodies
MAJOR crossmatch most important
Dogs may be transfused one time without a
crossmatch because they have few naturally
occuring antibodies - Minor crossmatch
-
Detects antibodies in donor plasma to recipient
RBC
Much less important
Used if large amounts of plasma are to be given
to a patient (eg failure of passive transfer) - Acute transfusion reactions
-
Acute hemolysis
Naturally occuring antibodies
Antibodies from previous sensitization
Intra- or extravascular hemolysis
Hemoglobinuria, hemoglobinemia
bilirubinemia, bilirubinuria, shock, DIC
Fever
Platelet, leukocyte or plasma sensitivity rxns
Allergic reactions
IgE mediated
anaphyllaxis
1-45 min post transfusion
urticaria, pruritis, erythema
Stop the transfusion!!
Circulatory overload- heart failure patients
Citrate toxicity- hypocalcemia
Ammonia toxicity- patients with liver
disease
Transmission of infectious agents - Delayed transfusion reactions
-
Hemolysis-
3-21 days after transfusion
due to transfusion of RBC with an
imcompatible blood group
Results in shortened life of RBC
This is not prevented by crossmatch!
Jaundice, fever, + Coombs - Blood donor selection- Dogs
-
Weight > 25kg
DEA 1 and 7 negative
Easily accessible veins (Greyhound)
Screened for infectious diseases
Initial CBC, chemistry panel, fecal exam
and urinalysis - Blood donor selection- cats
-
Weight 5-7 kg ideal
No universal donors in cats (types A, B,
AB)
PCV > 30%
Initial CBC, chemistry panel, fecal exam,
urinalysis -
Blood donor selection- large
animals -
No equine or bovine “universal donorsâ€
High degree of blood type polymorphisms
Crossmatch necessary - 3 categories of disorders of the immune system
-
autoimmune
lymphoproliferative disorder
immunodeficiency - clincal course of animals with complement deficiency
-
1.recurrent extracelluar bacterial infections
2.not usually fatal
3.Particular infections shown in people- Neisseria menigititis
4.Membranoproliferative glomerulonephritis later in life
5.Neutrophil migration and function appear normal
6.normal antibody response
7.similar incidence of toxocara infections in dogs
8. no adverse concequnces to vaccine with live virus - assessment of complement function
-
1.amount of complement in canine serum can be assessed by immunodiffusion assay
2.Human hemolytic complement assay can be used on veterinary species, must be serum and requires control animal sample
3. if one or both pathways are deficient consult lab for further tests - neutrophil failure to migrate clinical presentation
- bacterial infection with very high blood neutorphil counts
- chronic granulatmous diease can be caused by what type of neutrophil problem
- failure of intracellur killing
- assessment of neutrophil function
-
1.CBC
2.measure expression of cell surface proteins known to be involved in function-flow cytometry
3.measure functional response to activation:NBT assay, assay to measure oxidative burst
4.measure the ability of neutrophils to phagocytize-rare - rate of adaptive immune system deficiency by cell type
-
1.b-cell most common
2.b and t cell next most common
3.t cell least common - general characteristics of b cell immunodeficiency
-
bacterial infections, after 3-6 months when maternal antibody wanes
may be low AB with normal number of b cells or low numbers of b cells - general characteristics of t cell immunodeficiency
- infection with intracellular pathogens, fungi, bacteria, protozoa, viral infections- those organisms requiring t cell mediated killing or activation of macrophages for immunity
- general characteristics of deficiency of b and t cells
-
1.severe lymphopenia, hypoglobulinemia
2.usually survive the first few months of life but thats it
3.susceptible to all types of infections - enumerate lymphocyte subsets: flow cytometry
-
1.lymphocyts can be distinguished by their surface proteins:
t cells:cd8 or cd4
b cells:cd21
2.measures size and scatter of cells
and anitbody binding by fluroescence
lymphocytes are small and not complex - lympocyte function assays
-
1.lymphocyte blastogenesis:ability of t cells to divide
2.DTH:ability of t cells to divide and migrate
3.vaccine titers:both b and t cell function - to methods of phenotyping lymphocytes
-
1.immunohistochemistry in formalin fixed tissue-ab react to b or t cells
2.lymphocytes in suspension can be idifed by surface markers -
Whats your diagnosis:
lymphocyte pleural effusion in a cat
1.low serum to effusion triglyercide and cholestrol ratios
2.flow cytometry showing mixture of cd4 and cd8 t cells - chylothorax
-
Whats your diagnosis:
lymphocyte pleural effusion in a cat
1.high serum to effusion triglyceride and cholesterol ratios
2.flow cytometry shows mixture of cd4 and cd8 t cells and b cells - reactive
-
Whats your diagnosis:
lymphocyte pleural effusion in a cat
1.flow cytometry shows single phenotype of lymphocytes (all cd4 t cells,all cd8 t cels, or all b cells) - lymphoma
-
Whats your diagnosis:
lymphocyte pleural effusion in a cat
1.small lymphocytes
2.flow cytometry show unique phenotype (express both cd4 and cd8 at same time) - thymoma
- PARR assay
-
PCR for antigen receptor rearrangments:
used to determine if lymphocytes are derived from a single clone.
the presence of a predominat single clone is very specific for the presence of lymphoid neoplasia