Clinical Haematology Flashcards
what are the erythrocyte (RBC) parameters shown on a haemogram?
manual reticulocyte count haemaglobin haematocrit number of RBC mean cell volume mean cell heamoglobin mean cell heamoglobin concentration
what value on a haemogram is most often wrong?
platelets
what are the main leukocyte (WBC) parameters on a haemogram?
total WBC neutrophils lymphocytes monocytes eosinophils basophils
what is the best tube to used for haematology?
EDTA blood tube
what does EDTA do to blood in the tube?
chelates (binds) calcium in the blood. This is required for clotting so EDTA prevents clotting.
when may heparin tubes be used?
in some exotic species as EDTA can cause lysis
what is essential when filling tubes for haematology?
respect the amount of blood required in the tube - only fill to the line
what must you do to haematology tubes to ensure good mixing of blood with anti-coagulate?
gently invert the tube 10-20 times and roll
how should blood smears be stored?
once dry - in slide containers
why should blood smears not be stored in the fridge?
condenses the cells and leads to water artefact
where should haematology samples be stored?
in the fridge until submission to the lab or running sample
why should haematology samples not be stored in the freezer?
causes cell rupture
how should haematology samples be packaged?
not right next to the ice pack as this may freeze them
what parameters evaluate RBC?
haematocrit
packed cell volume
RBC count
what parameters show average makeup of RBC/indexes?
mean corpuscular volume
mean corpuscular haemoglobin
mean corpuscular haemoglobin concentration
what test is used to assess RBC morphology?
peripheral blood smear exam
what parameters are directly measured by haematology analyser?
haemoglobin
red blood cell count
mean cell volume
what does the mean cell volume show?
average size of RBC
what can be calculated from parameters measured by haematology machine?
haematocrit
mean corpuscular haemoglobin
mean corpuscular haemoglobin concentration
how can haematocrit be calculated?
mean cell volume x red blood cell count
how can mean corpuscular haemoglobin be calculated?
Haemoglobin x 10/red blood cell count
how can mean corpuscular haemoglobin concentration be calculated?
Haemoglobin / haematocrit
what are the two types of haematology analysers?
flow cytometry
impedance
how do flow cytometry haematology analysers work?
individual cells pass through a laser beam absorbing and scattering light. Interruptions in light count cells and light scatter is used to determine size of cell and the internal complexity. Produces differential count
How is cell size measured by flow cytometry?
Interruptions in light count cells
how is cell size and complexity measured by haematology analyser?
light scatter
how do impedance haematology analysers work?
individual cells pass an isotonic solution between two electrodes. Cells are poor electrical conductors so when passed between electrodes they produce a change in electrical impedance that is proportional to the size of the cell
what form of haematology analyser is preferable?
flow cytometry can produce a differential count
what are the 9 common sample artefacts which interfere with automated CBCs?
clots of any size platelet clumps (may be read as a cell duce to lack of nucleus in mammalian RBC) macroplatelets RBC agglutination (RBC with nucleus) nRBC Heinz bodies Lipaemia leukocyte agglutination delay in sample handling (increased haemolysis)
what is packed cell volume?
PCV: percentage of RBC in a volume of blood
How is PCV read?
after centrifugation, the percentage of RBC in column of the capillary tube
what else can be assessed during PCV test?
buffy coat assessment - number of WBC
Plasma colour
total proteins
what does the buffy coat on a PCV test show?
WBC and platelets, should be small
what does plasma colour indicate?
straw/clear = normal
bright yellow = icteric
pink = haemolysed
milky and turbid = lipaemia
why is a blood smear so important?
morphology changes are not picked up by any analysers
will show discrepancies/errors in any analyser (QA)
help with quick clinical decisions
PCV+Blood smear are a low cost option when no machines available
what are the minimum levels of patients that should have their blood smear reviewed?
all critically ill patients
CBCs with unusual or suspicious results
what would indicate the need for performing a blood smear?
presence of:
nucleated RBC (indicating reduced RBC or high need for RBC)
neutrophil left shift (immature neutrophils)
unclassified or unidentified cells
automated WBC count that may not be accurate
what background would trigger a blood smear review?
unusual background matrix
unusual background colour
organisms or suspected organisms
what RBC parameters would trigger at blood smear review?
moderate to marked poikilocytosis of any kind (abnormally shaped RBC)
moderate to sever anaemias
any Heinz bodies in non-feline species (>10% in cats)
inclusions (organisms or suspected organisms)
Howell-Jolly bodies
abnormal MCV
what is poikilocyotsis?
strange shaped cells
what WBC parameters would trigger blood smear review?
left shift (marked or degenerative) leukopenia leukocytosis lymphocytosis unclassified cells organisms (or suspected) presence of granules in non-monocytes and abnormal granulation of any leukocyte
what is leukopenia?
decrease in number of WBC (<3,000 WBC)
what is leukocytosis?
Increase in total WBC count (>30,000 WBC)
what is lymphocytosis?
elevation of lymphocytes (>10,000 cells)
what platelet parameters would trigger blood smear review?
> 900,000 platelet count
thrombocytopenia
abnormal MPV
suspected inclusions or abnormal granulation
what is thrombocytopenia?
low platelets (<100,000 cells)
what are the 3 main parts of a blood smear?
base/head
monolayer
feathered edge
what are the measured areas of a blood smear?
monolayer and feathered edge
how do RBC appear in the monolayer of the blood smear?
side by side, not overlapping
how should you start looking at a blood smear?
small - low magnification and at the feathered edge of the smear
what is the systemic approach to a blood smear exam?
start small at low magnification from the feathered end
go 2-3 fields back to the body of the smear in the monolayer
increase to oil and evaluate morphology
finish at the side, count 100 leukocytes into types looking for abnormal forms as you go
what do you look at regarding RBC in a blood smear?
numbers (does it loo anaemic)
do RBC look normal
is there evidence of regeneration
what do you look at regarding WBC in a blood smear?
number
type present
morphology
what do you look at regarding platelets in a blood smear?
number (estimate)
morphology (size)
what is the function of RBC?
oxygen carrying to tissues
how do RBC appear in dogs?
central pallor taking 1/3 of cell
how do RBC appear in cats?
small
no central pallor
all cells same size/colour
what is the role of neutrophils?
defence against invading microorganisms especially bacteria
when do neutrophils increase?
in inflammation and infection. Stress due to adrenaline and corticosteroids
what do neutrophils look like under a microscope?
ribbon shaped, segmented nucleus
pale cytoplasm
what do eosinophils do?
defence against parasites
allergic response
what do basophils do?
defence against parasites
allergic response as contian histemine
what do basophils do?
defence against parasites
allergic response as contain histamine
what are granulocytes?
collective name for neutrophils, basophils and eosinophils
how do eosinophils and basophils differ under a microscope?
basophils are blue toned. Eosinophils have pink granules with purple nucleus
what are lymphocytes involved in?
immunity both adaptive/cell mediated (T cell-) and humoral (b cells - antibody production)
how do lymphocytes appear under a microscope?
round cells with a large round nucleus
very little cytoplasm
what are monocytes?
precursor of macrophages
what are monocytes involved in?
phagocytosis
antigen presentation to T cells
immunomodulation
how do monocytes appear under a microscope?
have vacuoles
how do platelets appear under a microscope?
small
no nucleus
some granulation
what are platelets involved with?
haemostasis
formation of platelet plug
accelerating coagulation
inhibition of antithrombin III
in haematology what does the suffix philia or cytosis mean?
increase in number
in haematology what does the suffix penia mean?
decrease in number
what is thrombocytopenia?
reduction in number of platelets
what type of cells is the suffix philia used for?
granulocytes only
what is cytosis used for?
all other cells (apart from granulocytes)
in what species is the neutrophil the dominant cell type?
healthy cats, dogs, horses and camilids
in what species is lymphocyte the dominant cell type?
healthy cattle and rodents
what is WBC morphology like in rabbits and birds?
neutrophils are known as heterophils - have shiny pink granules like an eosinophil
in what species is the azurophil found?
reptiles and amphibions (monocyte with pinky/blue staining)
how do RBC appear in birds, reptiles, amphibians and fish?
nucleated
thrombocyte (platelet) is also nucleated - may look like a lymphocyte
counting is not performed by analysers due to presence of nucleus (cannot differentiate between RBC, thrombocyte or WBC)
what is used instead of a change in leukogram in horses, cattle and sheep?
haematology and measurement of serum acute phase proteins to detect inflammation
what can be diagnosed through blood smear?
thrombocytopenia regenerative vs non-regenerative anemia rouleux vs agglutination check machine WBC differentials/ manual differential WBC count normal and abnormal morphology
what can happen during automated counts of platelets in whole blood in EDTA?
done by analysers
commonly leads to artefactual (false) thrombocytopenia due to platelet clumping and macroplatelets
overlap in size between RBC and platelets often leads to false automated counts
under what circumstances should platelet numbers from blood in EDTA be checked by blood smear?
any animal with low automated counts
every animal with clinical signs of haemorrhage
what do platelet estimates on blood smears assume?
no platelet clumps on feather edge
how is platelet estimate carried out on blood smears?
10 fields - oil immersion in monolayer count number of platelets do average multiply by 15 or 20 (depending on practice) estimated number x10^9/L
what can macroplatelets lead to?
false thrombocytopenia
regeneration / increased platelet production
in what breed are macroplatelets normally seen?
Caviler King Charles
how will non-regenerative and regenerative anemia present under a microscope?
non - regenerative: bone marrow producing no new RBC so they appear normal
regenerative - many different RBC shapes
what do agglutination vs rouleux show?
cell interaction with each other
how does agglutination appear under a microscope?
bunch of grapes
large 3D clusters
interference with machine
how does rouleux appear under a microscope?
stack of coins
no interference with machine
where is the leukogram taken from?
edge of smear within monolayer
what does a leukogram count?
number of WBC and the types present (in 100 cells)
what does anaemia reflect?
reduced oxygen carrying capacity
what is reduced red blood cell mass noted by?
reduced RBC
reduced haemoglobin concentration
reduced PCV and haematocrit
what intra-individual variables can affect RBC mass?
breeds bred for athleticism (greyhounds/thoroughbreds)
age - young animals have reduced RBC mass until 4-6 months of age
what are the clinical signs of anaemia?
mucous membrane pallor (pale) lethargy exercise intollerence tachycardia tachypnoea heart murmur collapse icterus melaena/haematuria/haemoglobinuria pica
what are the 3 areas anaemia is classified by?
RBC indexes
regenerative vs non-regenerative
severity of anaemia
what are the 3 main RBC indexes for anaemia?
macrocytic
normocytic
microcytic
what do macrocytic and microcytic RBC indexes mean?
macrocytic - large RBC
microcytic - small RBC
what is the difference between regenerative and non-regenerative anaemia measured by?
reticulocyte counts
what is severity of anaemia based on?
how low haematocrit is
do cats or dogs compensate for anaemia better?
cats
what are the tests used for investigation of anaemia?
PCV/HCT and haemoglobin concentration RBC indexes: MCV, MCHC/MCH reticulocyte count blood smear for morphology auxiliary tests are available
what are normocytic erythrocytes?
erythrocytes of normal size
what are microcytic (low MCV) erythrocytes?
smaller RBC
what can microcytic (low MCV) erythrocytes be caused by?
iron deficiency allows an extra cell division which is usually prevented by normal haemoglobin concentration.
what are macrocytic (high MCV)artic RBC?
larger than normal RBC
what causes macrocytic RBC?
immature RBC
what dog breeds show naturally higher RBC?
poodles
what can cause artificial macrocytic RBC?
artefact in stored/old blood samples
what is hypochromic/ low MCHC/MCH?
presence of immature RBC (not fully haemaglobinised)
what is hyperchromic/high MCHC/MCH?
not physically possible (cannot fill RBC with extra haemaglobin) down to artefact
what would macrocytic and hypochromic results suggest?
regenerative anaemia: haemorrhage or haemolysis
what would normocytic and normochromic results suggest?
normal. Non- regenerative anaemia or following acute blood loss before regeneration (pre-regenerative)
what would microcytic and hypochromic results suggest?
iron deficiency anaemia due to chronic external blood, or secondary to liver disease, portosystemic shunt
what are the 2 key types of anaemia?
regenerative and non regenerative
what can non-regenerative anaemia be divided into?
bone marrow related
systemic disease
what are the two divisions of regenerative anaemia?
haemorrhage
haemolysis
what are the 2 types of haemorrhage that may cause anaemia?
internal and external
what are the two types of haemolysis?
intravascular
extravascular
what is regeneration the bodies response to?
fall in oxygenation - kidneys release EPO
what is regeneration determined by in dogs and cats?
reticulocyte concentration
what length of time is required before there is significant blood reticulocytosis?
about 3-5 days
how may the regenerative response be seen in anaemia?
presence of polychromatophils (immature RBC - has lost nucleus)
what are reticulocytes?
precursors of RBC
how can reticulocytes be counted?
manually or by some of the newer haematology analysers
what are the 2 types of reticulocytes in cats?
aggregates
punctate
what are aggregate reticulocytes?
immature reticulocytes, appear like canine reticulocytes
where are aggregate reticulocytes formed?
in the bone marrow in response to anaemia
how do aggregate reticulocytes become punctate?
mature after 12-24 hours in circulation
what are punctate reticulocytes?
have only small amount of reticulum (mRNA) and have undergone a degree of maturation
how long can punctate reticulocytes remain in the blood stream?
up to 4 weeks after the anaemia has resolved
describe the stages of a manual reticulocyte count
mix an equal amount of blood and vital stain
incubate at room temperature
do a normal blood smear with the mixture
evaluate 500-1000 stained RBC to determine % of reticulocytes
use % reticulocytes and RBC to determine absolute value
what are the two types of stain used for manual reticulocyte counts?
new methylene blue
brilliant cresyl violet
how long should blood and stain be incubated for during a manual reticulocyte count?
new methylene blue - 10 mins
brilliant cresyl violet - 15 mins
what are RBC evaluated for on a blood smear for anaemia?
RBC density
RBC regeneration
RBC morphology (clues as to causes of anaemia)
what are 4 of the most common morphological changes seen in RBC?
anisocytosis
polychromasia
hypochromasia
spherocytes/ghost cells
what is anisocytosis?
different cell sizes
what3 questions must be asked when presented with anisocytosis?
is there macrocytosis (large RBC)
is there microcytosis (small RBC)
is there poly chromasia?
the presence of what red blood cell morphology in anaemic patients suggests RBC regeneration?
anisocytosis and polychromasia
what does polychromasia indicate?
regeneration
what are polychromatophils?
younger RBC - reticulocytes
what do polychromatophils look like?
more purple in colour - have lost nucleus
what is hypochromasia?
not enough haemoglobin
what do hypochromatic RBC look like?
larger pallor in the middle of the RBC (>1/3 of cell)
when is hypochromasia seen?
alongside microcytosis (reduced cell size) in iron deficiency anaemia
in what disease are spherocytes and ghost cells seen?
IMHA
what do spherocytes look like?
round, smaller and darker than RBC and have no central pallor
what are ghost cells formed of?
only outer RBC membrane with no internal structures
when are spherocytes often seen in low numbers?
concurrently with evidence of shear damage injury
what is polycythaemia?
increased red cell mass
what indicates polycythaemia?
increased: haemoglobin packed cell volume (PCV) haematocrit (HCT) red blood cell count (RBC)
what are the 2 types of polycythaemia?
relative
absolute
what is relative polycythaemia due to?
loss of plasma volume/dehydration
what is relative polycythaemia known as?
erythrocytosis
what is absolute polycythaemia due to?
red cell mass is increased
what is absolute polycythaemia known as?
true polycythaemia
what are the clinical signs of polycythaemia?
elevated PCV (70-85%)
hyperaemic mucous membranes (dark red or blueish)
sneezing
nosebleeds
neurological signs (hyper viscosity) - seizure, blindness, ataxia, behavioral changes
what must be checked first in polycythaemia?
whether it is persistent or the patient is dehydrated
what are the 6 steps to rule out secondary causes of polycythaemia?
haematology, biochemistry and urinalysis
check reticulocyte count
assess for signs of cyanosis
check blood gas (hypoxia)
abdominal ultrasound to identify neoplasia or renal disease
thoracic rads to detect respiratory or cardiac abnormalities
what is increased EPO (erythropoetin) level diagnostic for?
secondary polycythaemia (can be 50x normal)
what is thrombocytopenia?
low platelet numbers
when does spontaneous haemorrhage occur?
very low platelet counts (<50x10 to the 9/L)
what are the clinical signs of thrombocytopenia?
petechiae - tiny haemorrhages ecchymosis - bruising melaena epistaxis haematuria
how can you check for true thrombocytopenia?
check the blood smear for estimate of platelets
check for clinical signs (could be due to poor collection/artefact)
repeat haematology to check for persistence if no clinical signs
what are additional tests for thrombocytopenia?
other haemostasis tests
testing for infectious diseases
bone marrow analysis
what are the common leukogram changes associated with normal or increased WBC?
stress leukogram inflammatory leukogram adrenaline (white coat) leukogram inverted "stress" leukogram neoplasia
what is a stress leukogram due to?
chronic disease
exogenous steroids
what do you need to know when looking at inflammatory leukogram changes?
which leukocytes are increased
what is an inverted “stress” leukogram caused by?
Addisons disease
what neoplasia can be indicated by a leukogram?
leukaemias
stage V lymphoma
mast cell disease
describe the leukogram pattern of physiological (white coat) leukogram
increased: total WBC count, segmented neutrophils, lymphocytes
variable: monocytes
describe the leukogram pattern of a steroid/stress leukogram
increased: total WBC count, non-segmented neutrophils (mild), segmented neutrophils, monocytes
decreased: lymphocytes, eosinophils
(roller coaster)
describe the leukogram pattern of an acute inflammatory leukogram
increased: total WBC count, non-segmented neutrophils (mild), segmented neutrophils, monocytes
reduced: lymphocytes
variable: eosinophils
describe the leukogram pattern of a chronic inflammatory leukogram
increased: total WBC count, segmented neutrophils,
variable: non-segmented neutrophils, lymphocytes, monocytes
describe the leukogram pattern of a leukaemia leukogram
hugely increased total WBC count - cell types vary
where should WBC differential counts be taken from?
edge of monolayer
what is leukopenia?
no leukocytes
what is calculated during WBC differential counts?
percentage of different leukocyte types
what is neutrophilia?
increased neutrophils
when does neutrophilia occur?
inflammation/infection
part of stress leukogram
physiological leukogram
independent of demand due to neoplasia
why does neutrophilia increase with inflammation/infection?
due to cytokine release (can be seen with or without left shift or toxic change)
why does neutrophilia increase with physiological leukogram?
due to adrenaline release
what will neutrophilia be seen alongside in a physiological leukogram?
mild lymphocytosis
why does neutrophilia increase with stress leukogram?
due to endogenous or exogenous steroids
what will neutrophilia be seen alongside in a stress leukogram?
lymphopenia
monocytosis
eosinopenia
what neoplasia causes neutrophillia?
paraneoplastic (outside of BM)
neoplasia of BM
what are signs of increased demand on neutrophils?
band neutrophil - U or S shaped nucleus with parallel sides (minimal indentation or segmentation)
signs of toxicity - Dohle bodies, foamy cytoplasm, blueish cytoplasm, toxic granules
what is neutrophil left shift?
release of earlier granulocyte precursors from BM
what does neutrophil left shift indicate?
indication of increased neutrophil demand/consumption
inflammation/infection
what are neutrophil toxic changes?
in response to overwhelming demand immature neutrophils are seen. Organelles that are normally removed from the neutrophil in the BM, persist when the neutrophil is in circulation
what are toxic changes often seen with?
alongside left shift
what is seen during toxic changes?
cytoplasmic basophillia Doehle bodies cytoplasmic vacuolation persistent primary granules (toxic granulation) ring form nuclei giant forms
when may neutropenia be seen in normal animals?
breed variations (greyhounds) cats sit on low end of reference interval
what may lead to neutropenia?
secondary to decreased production with marrow disease/suppression
increased utilisation through marked inflammation or immune mediated destruction
what does complete marrow destruction or suppression cause the destruction of first?
neutrophils
what can marked neutropenia indicate?
predisposition to infection and sepsis
what is lymphocytosis?
increased lymphocyte count
is lymphocytosis “true” in young animals?
no as they are constantly immune stimulated
what can lead to lymphocytosis?
adrenaline release
mobilisation of cells (hypoadrenocorticism)
increased numbers to increased demand (immune stimulaton)
increased numbers independent of demand (lymphoproliferative disease)
how does normal lymphocytes appear?
around 2 RBC in size, little cytoplasm
how does a reactive lymphocyte appear?
lots of cytoplasm
how does a lymphoblast appear?
larger than 3 RBC
what is lymphopenia?
loss of lymphocytes
what can lymphopenia be caused by?
loss of chylous fluid
decreased production - viral infections, lympholytic drugs (chemo), immunodeficiency
redistribution - chronic stress, trapped in lymph nodes, lymphocytolysis, move from circulation into bone marrow and tissues
what is monocytosis (increased monocytes) caused by?
increased demand for macrophages - chronic inflammation, transient monocytosis
redistribution (part of stress leukogram) - increased glucocorticoid levels
increased production independent of demand - leukaemia
what is eosinophilia caused by?
increased demand - usually parasitism, can be allergy or inflammation of mast cell rich tissues
paraneoplastic
hypoadrenocorticism
eosinophilic leukaemia
is eosinopenia of clinical significance?
no
what leukogram findings indicate poor prognosis?
degenerative left shift leukopenia leukemoid reation toxic neutrophils severe or persistent lymphopenia
what is the reason for poor prognosis in degenerative left shift?
overwhelming tissue demands (exceeds BM production)
what is the reason for poor prognosis in leukopenia?
overwhelming tissue demands (exceeds BM production) or BM disease
what is the reason for poor prognosis in leukemoid reation?
excessive neutrophils due to marked inflammatory stimulus
what is the reason for poor prognosis in toxic neutrophils?
due to accelerated production. Associated with longer hospitalization, higher costs of treatment and increased morbility
what is the reason for poor prognosis in severe or persisttant lymphopenia?
indicates severe and persistent stress