Exam 1

PTT

appropriate tube for CBC (complete blood count)

RTT

appropriate tube for serum diagnostic profile

BTT

appropriate tube for coagulation assays

gray top tube

appropriate tube for glucose when serum cannot be separated from the clot

pre-analytical

* Any part of the testing process that occurs BEFORE the actual measurement of the analyte(s)


* Includes pt ID, sample labeling, pt prep, clean venipuncture, proper tubes, proper filling, proper handling of tubes after filling, etc

Errors that occur when EDTA contaminated serum for diagnostic profiles

* Blood in RTT may not clot


* EDTA chelates bivalent cations such as calcium, magnesium, and iron, causing falsely low determinations of these analytes
* EDTA consists of a potassium salt, it adds potassium to the sample
* Severely low calcium, magnesium, and markedly high potassium

errors that result when blood is collected inappropriately from indwelling catheters

the blood may be overly contaminated w the other components. In other words, if the animal is receiving calcium gluconate, the analyzed serum may have calcium that is artifactually high. The same w dextrose or potassium supplementation

errors that occur when blood is not separated soon enough for diagnostic profiles in horses

RBC, which contain a high concentration of potassium (diff from other spp), can leak potassium into the serum and cause artifactually high potassium concentrations in the serum submitted for analysis (HYPERKALEMIA)

inadequately filled PTT

* excess EDTA
* RBCs shrink, decrease in PCV

inadequately filled BTT

* excess citrate
* prolonged clotting times in coag testing

heparin

Green top tube additive

sodium citrate

BTT additive

K2- or K3 EDTA

PTT additive

artifactual hypoglycemia and artifactual hyperkalemia

delayed removal of serum from RTT can cause

cellular elements (mostly RBC) consume glucose

causes artifactual hypoglycemia in delayed removal of serum from RTT

cellular elements leak cytoplasmic contents

causes artifactual hyperkalemia in delayed removal of serum from RTT

fibrinogen

RTT provides clotting factor and ____ free serum

10-15x

minimum amount of times to mix blood prior to testing

packed cell volume (PCV)

% of whole blood composed of RBCs

HCT (hematocrit)

ratio of volume of RBCS to volume of blood

HCT on a CBC

derived from a CALCULATED value using mean cell volume (MCV) and RBC count

* (MCV x RBC count)/10

buffy coat

layer btwn plasma and RBCs in PCV

* composed of leukocytes, platelets, and nRBCs

bottom of buffy coat

read PCV here

icterus

yellow plasma on PCV

increased \[bilirubin\] in blood

icterus w PCV indicates

plasma icterus in large animals

* not reliable
* high levels of diet-associated carotene pigment give plasma NORMAL yellow color

lipemia

hazy to opaque white plasma on PCV

increased lipids in circulation

lipemia on PCV indicates

Causes of lipemia on PCV

* Post-prandial (after meal) blood collection
* Diseases associated w abnx lipid metabolism

hemolysis

red colored plasma on PCV

presence of free hemoglobin from ruptures RBCs in plasma

hemolysis in PCV indicates

causes of hemolysis in PCV

* May be **in vitro** artifact
* Collection technique
* Erythrocyte fragility in lipemic samples
* Suspect artifact if PCV nx or high


* May result from **in vivo** (pathologic) hemolysis
* Intravascular hemolysis due to hemolytic anemia or other disorders
* Suspect pathologic hemolysis if PCV low

Plasma protein estimation by refractometer

* **ESTIMATED** protein concentration based on refractive index
* Assumes other solutes in plasma are present in nx concentrations

clumps, critters, cancer

things found on the feathered edge of a smear

Body of smear

* too dense and WBCs too rounded to ID
* May be useful for detecting microfilaria and platelet clumps
* Evaluate briefly as part of blood film review

Monolayer

* Cell details visible


* Perform nucleated cell differential, RBC morphology assessment, and platelet evaluation here

* Differential cell count - confirm automated analyzer data
* Evaluate morphologic abnormalities - leukocytes, erythrocytes, and platelets
* Examine for blood parasites - never detected by analyzer

Reasons to look at a blood film even if our instrument performs a differential count

Differential nucleated cell count

* Count 100 nucleated cells w/in the monolayer


* Classify nucleated cells as
* Segmented neutrophils ("segs")
* Band neutrophils ("bands")
* Lymphocytes ("lymphs")
* Monocytes ("monos")
* Eosinophils ("eos")
* Basophils ("basos")
* Nucleated RBCs (nRBCs)
* Other

Total nucleated cell count (TNCC or NCC)

* Includes nucleated RBCs


* Usually automated for mammals
* WBC and TNCC often used interchangeably
* But not the same if nRBCs present
* By itself is NOT v useful for interpretative purposes
* used primarily to CALCULATE the concentration of specific leukocyte types

components of blood

plasma, RBCs, WBCs, platelets

fxn of RBCs

transport O2 & CO2, buffer plasma pH

20%

how much total blood volume can be lost w/o complication

1%

how much of blood we can take per BW

10%

estimate of blood volume as a % of body wt

hemoglobin (Hb)

* (g/dL)
* Indicates the O2 transport capacity of the blood
* \~1/3 x Hct

hematocrit (Hct)

* (%)
* % of blood volume composed of RBCs
* (%) = RBCs (millions/uL) x MCV (fL) /10
* Reported by automated cell counters

 mean cell hemoglobin (MCH)

* (pg)
* Avg amount of Hb in RBCs
* (pg) = \[Hb(g/dL) x 10\] / RBC count

 mean cell hemoglobin concentration (MCHC)

* (g/dL)
* Corrects the Hb measurement for RBC volume
* (g/dL) = \[Hb(g/dL)x 100\]/Hct(%)
* Low = hypochromasia (pale)
* High = increased extracellular Hb
* Decrease
* Reticulocytosis
* Iron deficiency
* Lead toxicity
* Increase
* Hemolysis
* Oxyglobin administration

mean cell volume (MCV)

* (fL)
* Automated cell counter measure size directly (doesn't usually get messed up)
* (fL) = \[Hct(%) x 10\] / RBC count
* Decreased = microcytosis
* Causes
* Iron deficiency
* Portosystemic venous shunt
* Heinz body anemia
* Fragmentation anemia
* Hyponatremia
* Asian dog breeds
* Increased = macrocytosis
* Causes
* Reticulocytosis
* FeLV (abnx maturation of RBC bc it affects bone marrow)
* Leukemia
* Nx greyhound RBCs (why they are used as donors=> carry more O2)
* Inherited malabsorption of cobalamin (B12) of giant schnauzers
* Congenital macrocytosis of poodles
* Hereditary stomatocytosis
* Alaskan malamutes, drentse-partrijshonds & mini schnauzers)
* agglutination
* There is spp variation

red cell distribution width (RDW)

* (%)
* Indicates variation in RBC size, if they are diff sizes, RDW goes up
* RDW = (SDmcv/MCV) x 100
* Increased RDW
* Reticulocytosis
* Microcytosis
* macrocytosis

Metarubricyte (nRBC)

* Last erythroid stage w a nucleus


* Hemoglobin concentration continues to increase
* Nucleus continues to condense

Reticulocyte (polychromatophilic RBC)

* Hemoglobin content high


* Nucleus has been extruded
* Ribosomes stain basophilic w new methylene blue stain
* Aggregate reticulocytes have larger amounts of mRNA, ribosomes and mitochondria

Erythrocyte (mature RBCs)

* Mammalian
* No nucleus, mitochondria, ribosomes, de novo protein synthesis
* Anaerobic glycolysis
* Pretty inactive cells, only job is to move around O2
* Fxns: CO2 transport, H+ buffer, O2 transport

factors that decrease O2 affinity of Hb

Increased H+, Decreased pH, Increased CO2, Increased temp, Increased 2,3-diphosphoglycerate (2,3-DPG)

factors that increase O2 affinity of Hb

Decreased H+, increased pH, Decreased CO2, Decreased temp, Decreased 2,3-DPG

Rouleaux

* stacking of RBCs in lines
* Nx in cats & horses
* Disperses in saline

Agglutination

* aggregation of RBCs in clumps/clusters
* Does NOT disperse w saline
* Indicates ongoing immune-mediated diseases

Anisocytosis

all diff sizes

Polychromasia

* staining of retics (blue)
* Usually correlates to regenerative response

Basophilic Stippling

* Nx in anemic Rums, good sign bc responding to anemia
* Lead toxicity

Siderocytes

* contain blue/black aggregates
* Stain blue w Prussian blue stain
* More centralized
* Iron excess
* causes : Lead poisoning, Hemolytic anemia, Dyserythropoiesis, Myeloproliferative dz: effect bone marrow production of RCs, Some drugs (chloramphenicol, hydroxyzine in dogs)

nRBCs

* metarubricytes or earlier erythroid precursors
* Low #s are appropriate during regenerative anemia or hypoxia
* Seen in piglets

Howell-Jolly Bodies

* basophilic nuclear remnant
* causes: Splenectomy, Chemotherapeutics, Glucocorticoids, Regenerative responses

Heinz Bodies

* denatured & precipitated Hb
* Caused by oxidative damage
* Stain light red w Romanowsky stains
* Stain blue w NMB stain
* Up to 5% of the RBCs in nx cats
* causes: Onion, Kale (RUMs), Winter rye (cattle), Red maple leaves (horses) , Copper toxicity (sheep, goats), Zinc (dogs, eating pennies), Acetaminophen (cats), Methylene blue (cats, dogs), Methionine (cats), Phenazopyridine (cats), Vitamin K3 (dogs), Phenothiazine (horses), Crude oil (marine birds), DM, Lymphoma, Hyperthyroidism (cats)

Poikilocytosis

irregular shapes

Echinocyte

* burr cell, crenated cell
* Drying artifact, nx in pigs
* causes: Electrolyte imbalance, Uremia, Glomerulonephritis, Neoplasia, Doxorubicin toxicity, Coral/rattlesnake venom, Transfusion w stored blood

Dacrocytes

* tear-drop shaped
* Nx in goats
* causes: Iron deficient camelids, Glomerulonephritis, Myeloproliferative disorders

Elliptocytes (Ovalocytes)

* Nx in camelids
* causes: 4.1 band deficiency (dog), Iron deficiency, Myelodysplasia, Glomerulonephritis, Portosystemic shunts, Doxyrubicin toxicity, Hepatic lipidosis

Drepanocyte

Nx in deer

(Sickle Cell)

Acanthocytes

* irregular spicules
* Altered lipid:cholesterol
* causes: Hepatopathy, Hemangiosarcoma, Glomerulonephritis, Lymphoma, DIC, Young goats & cattle

Schistocytes

* Fragments formed by passing through fibrin strands or turbulent blood flow
* causes: DIC, Hemangiosarcoma, Vasculitis, Glomerulonephritis, Congestive heart failure, Myelofibrosis,

Doxorubicin toxicity, Severe hepatopathy, Iron deficiency, Dyserythropoiesis (dogs)

Eccentrocytes

Condensed Hb

Oxidative damage

Keratocytes

* Contained a vesicle
* (Helmet cells)
* causes: Oxidative damage, Iron deficiency, Hepatopathy, Doxyrubicin toxicity

Leptocytes

* Thin, too much central pallor
* causes: Portosystemic shunts, Hepatopathy, Iron deficiency

Target Cells

* Hb at the edges & center of the cell
* (Codocytes)
* causes: Hepatopathy, Iron deficiency

Stomatocytes

* Oval area of central pallor
* causes: Hereditary stomatocytosis, Hepatopathy, Iron deficiency

leukogram

numeric and morphologic info about WBCs

Segmented Neutrophil

* Most abundant circulating cell in most spp


* Basophilic cytoplasm
* Chromatin gets condensed

FXN: Inflammation

* Migrate to tissue site, phagocytize microorganisms if present


* Die in the tissues

Band Neutrophil

* Lack any nuclear constriction/ segmentation

FXN

* Immature neutrophil released from bone marrow
* Usually seen w marked inflammation

Heterophil

segmented nucleus

Basophilic cytoplasm

Exotic neutrophils

Monocytes

* Typically larger than neuts


* Less nuclear segmentation
* Nuclear chromatin less coarse/dense
* Bc still actively transcribing things
* Blue cytoplasm
* Often contain vacuoles

FXN

* Migrate into tissues to become macrophages
* Phagocytize foreign material, dead/dying cells
* Critical role in initiating, maintaining, and resolving inflammation
* Antigen presentation
* Cytokine production

Eosinophils

* Pink orange staining cytoplasmic granules
* Shape, density, staining can very among spp

FXN

Worms, wheezes, weird diseases

* Parasites
* Hypersensitivity, allergy
* Inflammation of skin, gut, respiratory system
* Addison's disease, etc

Basophils

* Variable density of purple granules


* Large animals typically contain abundant, dark granules
* Dogs have sparser granules
* Cats have paler granules

Similar response patterns as eosinophils

Lymphocytes

* Small cells w smooth, dense nuclear chromatin and a small rim of pale blue cytoplasm
* Larger forms can be seen in smaller #s normally
* More abundant, pale cytoplasm
* Can have scant, fine cytoplasmic granules
* RUMs tend to be larger w more abundant cytoplasm

Lymphs w azurophilic granules

* Can be seen normally
* Marked increase or majority of lymphs
* Ehrlichia
* Canine CLL (CD8 T cell)
* Vax

Toxic Change in neutrophils

* Seen in CYTOPLASM:


* Increased basophilia - (persistence of cytoplasmic RNA)


* Dohle bodies- irregular margin (aggregates of rough ER)
* Vacuolization/foamy-(dispersing of organelles)
* Can start to resemble monocytes
* Mono chromatin is patchy, looks more condensed
* Neuts have more segmentation
* In bone marrow, accelerated production, can still do their job
* Indicates accelerated production/increased demand
* Inflammation
* Secondary to accelerated production/release from BM


* Persistence of organelles
* Indicate increased peripheral demand or inflammation

Degeneration of neutrophils

* Outside of circulation
* Seen in NUCLEUS
* Nuclear swelling
* Loss of segmentation
* Loss of coarse nuclear chromatin pattern
* In peripheral tissues, starting to fall apart, doing their job
* Found as neutrophils break down/die
* Can become prominent w bacterial infections

Hypersegmentation (neutrophil)

* More nuclear segmentation


* Not v significant clinically
* Aging
* Old blood sample
* A result of increased circulation time
* Coritcosteroids
* others

Pelger-Huet anomaly (neutrophil)

* Inherited (nuclear)
* Lack segmentation (eosinophils too)
* Virtually q neutrophil will look like band or earlier
* No toxic changes
* Impacts eos too
* Nucleus matures normally, but doesn’t shape normally


* Nx mature chromatin
* Nx fxn
* Seen in Australian shepherds
* Less commonly reported in foxhounds, Samoyeds
* Rarely reported in Arabian horses

Lysosomal Storage disorder (neutrophil)

* inherited(cytoplasmic)
* Cytoplasmic granulation helps ID issue
* Rare lysosomal trafficking or storage disorders
* Generally systemic dysfunction, physical abnormalities
* Can affect lymphocytes too
* Lysosomal defects; mucopolysaccharidoses (MPS)
* Clinical manifestations are generally readily apparent

Cytoplasmic vacuoles (lymphocyte)

* Most commonly an aging artifact
* Avoid by sending PTT and blood smear
* Some of the lysosomal storage disorders (inherited)
* Ingestion of plants containing swainsonine (acquired)
* Locoweed toxicity

Reactive lymphocytes

A subset of cell contain **increased amounts** of **deeply basophilic** cytoplasm

Associated w an immune response/inflammation

penia

Decreased concentration of cells in whole blood (cells/uL

Pancytopenia

decrease in all 3 cell lines

"philia" or "cytosis"

Increased concentration of cells in whole blood (cells/mL >RI)

Left Shift

* Increase in circulating immature neutrophils
* Typically involves band forms
* Can extend back to metamyelocytes when severe

regenerative left shift

When mature forms outnumber immature forms

degenerative left shift

When immature forms outnumber mature forms

* Total WBC may be nx or deceased
* Generally indicates severe demand, inadequate response
* Can occur early in less severe inflammation in cattle (bc they dont have as much stores)

Leukemia (WBC cancer)

* Presence of neoplastic hematopoietic cells in the blood or bone marrow
* Any of the cell lines can become neoplastic
* Myeloid, lymphoid, erythroid, megakaryocytic
* Lymphoproliferative disorders
* Neoplasms of lymphocytes and plasma cells
* Myeloproliferative disorders
* Neoplasms arising from bone marrow stem cells
* Can involve neutrophils, monocytes, erythrocytes,
* rarely eosinophils, basophils, megakaryocytes

Acute Leukemia

* (NOT commenting on how long it's been going on but rather what stage the cells are at)


* Immature cells; often blast forms w prominent nucleoli
* Aggressive disease
* Often result in other cytopenias
* Animals typically clinically ill
* Can be lymphoid or myeloid

chronic leukemia

* Well differentiated cells


* Less aggressive
* May be incidental finding (pre-op/dental bloodwork, annual exam etc)
* Myeloid forms are v rare
* Lymphoid forms seen more commonly in dogs and cats