Clinical pathology Flashcards
Define the term Haematopoiesis ?
The formation of blood cellular components.
- in mammals this occurs in the bone marrow and lymphoid organs.
- lympopoiesis occurs largely in extramedullary sites such as the spleen, thymus and lymph nodes.
What substance is considered to be the most important stimulus for erythroid production ? Where does it originate?
What determines its release?
Haematopoietic growth factors
- Erythropoietin
- Erythropoietin is produced by the peritubular interstitual cells of the kidney or less locally in bone marrow, liver.
- Low partial pressure of oxygen
Additional stimulatory factors = thrombopoietin, colony stimulating factors (CSF) and interleukins IL.
List 3 essential nutrients for erythroid production?
The three essential nutrients for erythroid production
- Iron (heme synthesis)
- Copper (release of iron from tissue into plasma transport developing erythroid cells)
- Vitamin B6 (cofactor in heme synthesis)
What does myelopoiesis encompass ?
Myelopoiesis
“myelo” refers to all aspects of bone marrow activity
Consist of
- Erythropoiesis
- Granulopoiesis
- Thrombopoiesis
- monocytes appear to be formed in the bone marrow as well as elsewhere
List the erythrocyte maturation sequence in order and be familiar with the basic morphological changes ?
Erthropoiesis (maturation of red blood cells)
Rubriblast
Rubricyte
Metarubricyte
Reticulocyte
Erythrocyte
The basic morphological changes
- at the rubricyte cell division ends when critical haemoglobin concentration ends
- cells decrease in size
- ribosomes, polyribosomes and chromatin condenses = reticulocyte
Which erythroid cells in peripheral blood indicate regeneration ?
Reticulocytes
Which species have reticulocytes in peripheral circulation in health ?
In cats and dogs it is normal to have 1-1.5% reticulocytes in the peripheral blood.
In health both cattle and horses do not have reticulocytes in peripheral blood. (in these species the reticulocytes mature within the bone marrow).
Explain how to interpret aggregate and punctate reticulocytes in cats and dogs ?
In health
Dogs
- immature aggregate type polychromatophils in peripheral blood circulation
Cats
- Mostly punctate (few to no aggregate reticulocytes) in peripheral blood circulation
- this is due to a relatively longer maturation time in cats and delayed degradation of RNA
List the granulocytic maturation sequences in order and know in which bone marrow compartment they belong ?
Order of granulocytic maturation
1. Granulocyte (associated with stromal cells away from vascular sinusses in the bone marrow).
2. Monoblast
3. Promonocyte
4. Monocyte
5. Macrophage in the circulation
Haematopoiesis and stems cells develop within different niches of the bone marrow.
Granulocytes develop associated with stromal cells away from vascular sinuses.
How long does it take the bone marrow to produce RBC’s and WBC’s after bone marrow stimulation ?
Why is this information clinically relevant ?
After bone marrow stimulation it takes
Erythrocyte
3-4 days to mature from rubiblast to metarubricyte production.
Leukocytes
Neutrophil 6-9 days transit
Clinical relevance
Which WBC type in addition to granulocytes also originates from the common myeloid precursor ?
Myeloid stem cell
In addition to the granulocytes (neutrophils, eosinophils and basophils)
The myeloid precursor cell may also develop into
- erythrocytes
- platelets
- macrophages
- mast cells
List the thrombocyte / platelet maturation sequence and know the basic morphological characteristics of each stage ?
Thrombopoiesis
(maturation of thrombocytes / platelets)
Promegakaryocyte
Megakaryocyte
Platelet
Maturation sequence
- the cells stop dividing but nuclear division still occurs (endomitosis)
- promegakaryocyte 2-4 nuclei (endomitosis)
- protrusions of cytoplasm (pro-platelets) extend from sinuses and are sheared off via force of flowing blood
Note; in non mammals nucleated thrombocytes are produced by mitosis of precursor cells.
What are the two main categories of haematopoietic neoplasia ?
Explain the difference between lymphoid leukaemia and lymphoma with a late leukaemic phase. In particular discuss their origin, peripheral blood characteristics and diagnosis ?
Name two non haematopoietic forms of neoplasia that may be identified in the bone marrow ?
Describe how haemoglobin deficiency affects Erythropoiesis ?
Erythropoiesis
Haemoglobin deficiency can result in an extra division of cells
- therefore iron deficiency can lead to decreased cell size
- microcytes
eg Iron deficiency.
Describe the changes in appearance of erythroid cells as the divide and mature ?
Why do these changes occur
Maturation of erythroid cells
leads to certain changes in the appearance of these cells.
- Early precursors have blue cytoplasm due to many basophilic ribosomes and polyribosomes synthesising globin chains.
- As cells divide and mature, their size decreases nuclear chromatin condenses, cytoplasmic basophilia decreases and HB progressively accumulates imparting a red colour to the cytoplasm.
List five types of bone marrow disorders and be familiar with a brief overview of the pathogenesis ?
Five types of bone marrow disorders
- Aplasia
- oestrogen toxicity, drugs, poisoning - Hyperplasia
- can be ineffective neutropenia bone marrow neutrophil hyperplasia FeLV - Dysplasia
- FeLV infection / myeloproliferative disorders - Myelopthisis
- replacement of normal haemopoietic cells with abnormal cells, myelofibrosis - Neoplasia
Describe the appearance of aggregate and punctate reticulocytes, and why they appear this way ?
Reticulocytes
Basic dye - new methylene blue
Aggregate reticulocytes
- maturation begins in bone marrow and is completed in the peripheral blood in dogs, cats and pigs
- contains a network of reticulum as an artefact, precipitation of ribosomal and ribonucleic acids and proteins secondary to staining
- ribosomes, polyribosomses and mitochondria are nessary for completion of HB synthesis.
Punctate reticulocytes
- the ribosomal material decreases seen as basophilic specs
Describe the process of maturation of white blood cells ?
Leukopoiesis
maturation of white blood cells
Mitosis and proliferation
- Myeloblast
- Promyelocyte
- Myelocyte
Maturation and storage within the bone marrow
Metamyelocyte
Band neutrophil
segmented neutrophil
Describe the appearance of myelocytes, neutrophils, basophils and eosinophils ?
Leukopoiesis
Myelocyte (precursor cell)
- contain specific granules identified by staining properties as neutrophils, eosinophils and basophils
- granules - peroxidase negative
- granules vary graetly in size, shape and concentration for domestic species
Neutrophils
- 7 times more neutrophils in the bone marrow than in circulation for the dog
- marrow transit time 6-9 days depending upon species (transit shortened with inflammation).
Eosinophils
- transit time one week
- significant storage pool of eosinophils
Basophils and mast cells
- same progenitor cell
- basophils mature in bone marrow
- mast cells mature in peripheral circulation
Describe what stimulate Thrombopoiesis ?
Thrombopoiesis
(maturation of thrombocytes)
TPO Thrombopoietin is the chief stimulator
- primarily produced by the liver
- kidney and bone marrow make a smaller contribution.
At which erythroid precursor stage in the bone marrow is the optimum haemoglobin concentration reached that stops dividing RBC’s from becomming smaller ?
A. Reticulocyte
B. Rubricyte
C. Metarubricyte
D. Rubriblast
Answer = B. Rubricyte
Know the blood collection system colour code and what is contained in each tube ?
Blood collection colour code - anticoagulant contents
Red = plain, no anticoagulant
Green = Lithium Heparin plasma or whole blood
Purple = EDTA whole blood
Grey = Sodium Fluoride (glycolytic enzyme inhibitor) whole blood or OXF plasma
Blue = Sodium citrate, whole blood or plasma
Demonstrate a familarity with the use of the following anticoagulants ?
- Plain
- EDTA
- Heparin
The use of different types of anticoagulants
Plain / red
- general biochemistry
- bile acids
- serology
- endocrinology
EDTA
- Haematology
- Cytology (blood film evaluation)
- PCV
Heparin
- Biochemistry (plasma)
- reptilian avian
- haematology (whole blood)
Demonstrate a familarity with the use of the following anticoagulants ?
- Sodium Fluride
- Sodium Citrate
Demonstrate an understanding for the use of
Grey Sodium Fluride
- glucose
Sodium citrate
- whole blood or plasma
- clotting times PT and APTT
- VWB factor Ag
What collection tube is used to obtain serum ?
Plasma is the liquid which remains when clotting is prevented with an anticoagulant
Can use
- EDTA
- Heparin
- Citrate
What is the difference between serum and plasma ?
Serum vrs Plasma
Serum
Liquid component remaining after the blood has clotted
- 2.5x needed volume
- red top tube
- centrifuge
- aspirate the supernatent
- Reccomended serum gel seperator (uses the difference in specific gravity)
Plasma
Plasma is the liquid which remains when clotting is prevented with an anticoagulant
- EDTA, Heparin, Citrate tubes
- gentle inversion
- centrifuge
- aspirate the surpernatant
List the desireable qualities of an anticoagulant used for haematological examination ?
The desirable traits of an anticoagulant for haemotological examination
- preservation of cell morphology
- not clot
- no interference with stain
- reccomended EDTA tube (Potassium salt of ethylene-diamine-tetra acetic acid)
Why is the ratio of anticoagulant to blood important ?
Provide examples ?
The importance of blood to anticoagulant ratio
Excess anticoagulant
- shrinkage of RBSCs due to increased osmolality
- artefactural RBC shape and erroneous MCV and PCV
- automated HCT and PCV will not be comparable
- if anticoagulant is liquid will dilute causing artefactual low values
Excess blood
- could result in clotting (which may affect results and obstruct tubing)
Tubes should be filled exactly to the line and mixed well. Underfilling and overfilling of tubes will significantly affect results.
The blood collection tube in the photograph does not contain a sufficient amount of blood (underfilled), a blue line indicates the minimum volume of blood required.
A. What anticoagulant does this tube contain ?
B. List four errors that will occur if this sample is used by the laboratory ?
Answer
A. EDTA
B.
1. RBC distortion, shrinkage
2. Altered indices on MCV and MCHC
3. Altered HCT
4. Elevated protein on a refractometer
If you underfill a citrate tube will the PT and APTT results be shorter or prolonged, why ?
Underfilled citrate tube
Prolonged PT and APTT
- there is not enough coagulent
List sample handling principles (4) and explain the impact of incorrect procedures ?
Four sample handling protocols
- Samples collected in the field should be transported in an esky ?
- swelling of red cells affecting results
- pyknosis, karyorrhexis and karyolysis - Wrapped in tissue paper preventing direct contact with ice ?
- could result in freezing lysis - Blood smears should be made on site and transported seperately from the ice box ?
- freeze damage to cells
Why is vacutainer blood collection preferable to a syringe ?
Answer
Vacuatiners are manufactured with the appropriate negative pressure for venous draw.
Too much pressure will lead to haemolysis
What are the implications of sample haemolysis or lipaemia ?
The implications of
Haemolysis
- interfere with biochemistry test measured by spectrophotometer
- poor haematology (Erythrocyte) results
Lipaemia
- lipaemia may lead to sample haemolysis
- this is due to lipaemia indicing turbiditycausing falsely high concentrations
Demonstrate knowledge regarding the appropriate sampling site, syringe and needle size with respect to animals routinely treated in Veterinary practice ?
List the correct order of drawwhat collecting blood in vacutainers and explain why this is important ?
Order of draw
Blue - red- cream - green - lavender - grey
But why
- minimise contamination of sterile specimens
- avoid test result error, caused via carryover of additives between tubes
- reduce the effect of micro clot formation in tubes
Never pour blood from one tube into another tube.
Describe the order of draw ?
Vet
Red - blue - green -purple - grey
Why is patient preparation prior to blood collection important ?
Importantance of patient preparation
Lipaemia
- avoided by fasting 12 hours prior to blood collection
- lipaemia may lead to sample haemolysis
- both haemolysis and lipaemia interfer with biochemistry test collected by spectrophotometer
- affects haemotology (erythrocyte and haemoglobin measurement)
What pattern of biochemistry results would alert you to the possibility of EDTA serum sample contamination so that you avoid clinical diagnoses ?
Explain three ways in which EDTA contamination of a serum sample can occur ?
EDTA sample contamination
- Decanting and mixing of tubes
- Backflow of blood if venous pressure drops (eg removal of digital pressure) causing blood to flow out of the tube into the vein - the contaminated blood is then collected in the next sample.
- Lab technician error EDTA plasma only sample is used instead of serum.
Explain the difference between a PCV and a HCT ?
PCV vrs HCT
Both measure the proportion of blood composed of erythrocytes.
Approximately how much of the capillary microhaematocrit tube should be filled ?
As fill as possible - it does not matter if the amount of blood in the capillary tube is not exact.
As the tube has a linear scale to accomodate for the variation in volume (calibration)
There may be a slight difference between a PCV and a HCT; which one is slightly lower and why ?
HCT vrs PCV
Both are used to measure proportion of blood composed of erythrocytes.
HCT = calculated; via automated haematology haemoglobin
PCV = measured
The HCT can be slightly lower as there is no trapped plasma between the red blood cells in the reading.
Can you see if the patient has eaten a meal prior to blood sampling when evaluating the micro haematocrit tube ?
Yes in the plasma
Yellow pigmentation
- suggest icterus an increase of bilirubin in blood
- in large animals may be due to carotene pigments in the diet
Whit/opaque pigmentation
- lipaemia chylomicrons
- may be due to postprandial collection (patient has eaten a meal).
- or due to disease associated with abnormalities in lipid metabolism
Red discolouration
- Due to the pressence of haemoglobin in the blood haemolysis
- may be due to technique
- in vivo haemolytic anaemia (intravascular haemolysis)
Label the regions of a microhaematocrit tube on the diagram. What clinically relevant information may be measured from the region labelled A ?
Label on the microhaematocrit tube PCV
erythrocytes
buffy coat
and plasma
Section A = plasma
The plasma may be used to measure
- total plasma protein
- icterus
- haemolysis
- lipaemia
Estimate total plasma protein by means of a refractometer ?
Refractometer
Estimate the concentration of solute in fluid, since the solute bends lights passing through the fluid proportionate to the solute concentration.
use blunt tube end
Explain why the reading obtained of plasma protein is an estimate, and provide four possible occasions where the result may be erroneous ?
Refractometer provides an estimate of the total plasma protein. TTP
The protein concentration is an estimate, assuming other solutes in the serum are present in normal concentrations.
- relative to distilled water.
Erroneous measurements could be due to
- lipaemia
- Urea
- Glucose
- Cholesterol
In high concentrations these solutes cause an artificially high reading.
List the types of haematology analyzers available for use in Veterinary practice ?
The various types of haematology analyzers
- Quantitive buffy coat systems
- density gradient centrifugation Acridine orange dye - Impedance - Coulter counter
- blood passes through an electrically charged aperture
- each cell results in a change in electrical resistance
- pulse detected and amplified by the instrument - Laser and special stains
- impedance and laser flow cytometry
List the three groups automated CBC data may be partitioned into as part of the interpretation procedure ?
Automated haematology
Can be partitioned into
- RBC
- WBC
- Platelets (thrombocytes).
Why is it important to categorise the degree of change in laboratory data to mild, moderate and marked ?
Assists with a diagnosis (greater recognition of pattern)
The automated haemogram does not provide a clinician with a direct diagnosis only a pattern of change.
Including descriptions such as mild, severe indicates the degree of change and can provide alot of information in the pattern of change assisting with diagnoses.
Which components of the CBC are measured and which components are calculated ?
Answer
Measured
- RBC
- HGB
- MCV
- absolute reticulocyte count
Calculated
- HCT
- MCHC
- RDW
Which haemogram parameter is calculated and not measured by an automated analyser ?
Calculated haemogram parameters on an automated analyser
- HCT
- RDW
- MCHC
Describe what RBC, HGB and HCT woould indicate on an automated haemogram ?
RBC = number of red blood cells / measured
HGB = haemoglobin concentration / measured
HCT = volume of red blood cells per litre of whole blood / calculated
Define the terms MCV and MCHC ?
MCV = mean cell volume / measured
MCHC = mean cell haemoglobin concentration / calculated
Define the terms polycythemia, macrocytosis, microcytosis and hypochromasia on an automated haemogram ?
On an automated haemogram what indices should be used to define mean haemoglobin conc, and mean cell volume ?
Use descriptors for changes in these values ?
In a microcytic hypochromic regenerative anaemia which automated result, MCV or RDW would provide the most information regarding the distribution of erythrocyte size ?
Explain ?
The limitation of MCV
MCV = average and dose not provide any informationregarding the distribution of the data.
- RDW describes the distribution of the data (coeficcient of distribution).
To identify pre-analytical or analytical error in haemogram results which three haematology rules should be assessed ?
The three rules of haematology
- Group RBC, HB and HCT - they should travel together
- HCT should be roughly 3x HGB (/1000)
- Any increase in MCHC = analytical thermometer
- this is not possible
- not physiologically possible to place more than the optimum.
- MCHC physiologically conserved across mammalian species 320-360 g/L
If these rules are transgressed we can apply the tools to identify diseases or avoid mis diagnosis.
If laboratory results contain an elevated MCHCor HCT is not 3x haemoglobin which two tools are helpful to clarify the causes ?
If rule one or two are broken
Tool kit to resolve
- PCV microcapillary tube
- Blood smear evaluation
List five sample conditions or erythrocyte features that may produce an erroneous HgB concentration when measured by automated haematology analyser ?
5 Erythrocyte features that could produce an erroneous HgB on automated haematology.
- nucleated red blood cells in avian and reptile species may be miscounted on automated haemolyser as white blood cells
- Aging causing agglutination - reducing red blood cell count
- Artificially high RBC count - due to counting thrombocytes as RBC (platelet clumps)
- Swelling
- Cells which have shrunk due to a partially filled EDTA tube
Why is it important to check that the HCT calculated by the automated analyser is roughly three times haemoglobin concentration ? Include two pre-analytical or analytical examples in your answer ?
Why should HCT be 3x haemoglobin conc
- Every red blood cell contains roughly 1/3 haemoglobin.
- HCT is calculated = the volume of red blood cells per litre L/L.
- thus HCT should = 3X HgB
Analytical - problem with measurement with
- MCV
- RBC count
Pre analytical
- MCV is abnormally high (aging and agglutination)
- RBC count is incorrectly high - eg platelets counted as red blood cells.
What are the two most common causes of microcytosis ?
Two most common causes of microcytosis
- Iron deficiency
- PSS Post systemic shunt
What is the most common cause of hypochromasia (low MCHC) and which other two laboratory changes would you expect to see ?
Hypochromasia low MCHC
(reduced mean cell haemoglobin concentration)
Common = chronic blood loss (loss of haemoglobin)
Two other expected laboratory changes
- low HGB
- low HCT
Is hyperchromasia possible ?
Explain why ?
Hyperchromasia is not possible
Why - it is physiologically impossible to place more than is optimum (HgB) in a red blood cell.
Why is the MCHC a good analytical thermometer ?
Explain what you should do if an error occurs ?
MCHC is a good analytical thermometer
- it is physiologically conserved across mammals 320-360 g/L
- not physiologically possible to be higher than optimum
If an error occurs recorrect through
- Blood smear
- Microhaematocrit tube
Work through the clinical case of peanuts the foal and determine if he has any abnormalities of clinical concern based upon the laboratory data ?
Peanuts a three month old foal
HCT low 0.25
MCHC erroneously high
- Breaking rule one of RBC, Haem and HCT traveling together
- This indicates a error in the collection of results
- Correction by collecting a PCV = 38%
This indicates an error with MCV or RBC count.
List five types of leukocytes ?
Demonstrate competance in determining the absolute concentration of each type of WBC, if you are provided with the total WBC count and the percentage of each of the five types of white blood cell ?
To calculate the absolutele WBC count from percentage
absolute count = WBC x 10 ^9 x % of each type
eg 70% neutrophils and a total WBC count 12 x10^9
absolute neutrophil count =
12 x 0.70 = 8.4 x 10^9
Explain by means of an example why the absolute differential count and not the differential percentage should be used when interpreting a leukogram ?
The importance of absolute counts
All animals (in example) have 70% neutrophils but depending on the total WBC count this can result in health, neutropenia or neutrophilia depending on the total RBC.
Thus the 70% is not useful when comparing to the reference range.
What can cause an erroneous calculated HCT on an automated haematology analyser ?
HCT is caluculated
The answer is based upon MCV and the number of red blood cells
- HCT = (MCV X RBC)/ 1000
- an erroneous measurement would imply either the volume of the cell is abnormal or the cell count is abnormal.
- platelets may be counted as RBC; causing an extremely high RBC count
- where as MCV volume could be artifactually increased via agglutination due to aging.
Demonstrate the ability to interpret basic changes in an automated haemogram including; RBC, HgB, HCT, MCV, MCHC, RDW
and absolute reticulocyte count ?
Interpretation of an automated haemogram
RBC + HGB + HCT
- increase erythrocytosis or polycythemia
- decrease anaemia
MCV
- mean cell volume
- macrocytosis, normocytosis or microcytosis
MCHC
- Hypochromasia
RDW
- significant macro or microcytosis, regeneration
Disscuss the proportion of the different WBC’s in canine, feline, equine and ruminant health ?
Species variation in proportion of different WBC
In what respect do canine, feline, equine and ruminant bone marrow responses differe when faced with a focus of inflammation in the body ?
Species variation in degree of WBC response
Horses and cattle have a less pronounced WBC response to inflammation compared to carnivores;
paradoxically modest increases in horses and cattle are morse significant.
Demonstrate the application of appropriate scientific terminology when interpreting laboratory results ?
When is it important to calculate the corrected WBC count ?
You should reassess your automated haemogram via corrected WBC equation when;
On a blood smear you detect > 5 nucleated RBC / 100WBC
Demonstrate the ability to apply the appropriate calculation when required to determine corrected WBC from the initial total nucleated count (TNCC) ?
To correct WBC or nucleated count
- Count nRBC’s per 100 leukocytes on a blood smear
- Corrected WBC = machine TNCC x (100 / nRBC + 100)
Describe the clinical application / limitation of automated haemogram ?
The automated haemogram
( It is reccomended, where possible to interpret WBC values over the course of several blood counts.)
Clinical application
- detect existence of inflammatory disease
- determine prognosis and monitor treatment
- detect some haematopoietic neoplasia
Clinical limitations
- does not identify specific aetiological agents
- can not indicate the site of inflammation
- may not even identify the preseence of inflammation eg chronic non invasivem, generalised inflammation etc
What is the difference between MPV and PDW ?
P = platelets
MPV = mean platelet volume (average)
PDW = Platelet distribution width (%)
Why is the platelet (thrombocrit) more helpful than the platelet concentration when evaluating haematology results from Cavalier King Charles Spaniels ?
Cavalier King Charles Spaniels
Like the HCT it is the percentage of blood volume filled by platelets (typically <1%)
-
What level of thrombocytopaenia (platelet count) can be associated with spontaneous haemorrhage ?
Level of thrombocytopaenia
<30 X 10 ^9/L can lead to spontaneous haemorrhage
Which species is notorious for incorrect platelet counts on an automated haematology analyser and why ?
Grey hounds
Increasec red blood cell cells and lower platelet numbers on average.
- normal to have thrombocytopenia
Blood smears are a powerful diagnostic tool; what information can be gleaned from a good blood smear ?
What we can learn from a good blood smear
- platelet clumps
- verify automated count WBC, platelets and RBC density
- assess distribution of WBC ( abnormalities - left shift, toxic or neoplastic change).
- Evaluate RBC morphology ( rouleaux, agglutination and identify any abnormal cells
- identify parasites.
Explain why EDTA is the anticoagulant of choice for collecting samples to prepare blood films ?
EDTA the anticoagulant of choice for fresh blood smear
(Purple top)
- inhibits clotting by removing or chelating calcium from the blood
- dose not affect red blood cell morphology
- Heparin may cause clumping, citrate dilutes the cells
Describe what happens to each of the 3 blood cell lines when the sample is stored too long before preparing blood smears ?
When its stored to long
Cells change in EDTA if stored for a few hours (in transit)
Platelets
- start to clump
WBC
- become pyknotic (nucleus becomes dense and compact).
- eg neutophil start to appear as bands
RBC
- swell (>24hrs = macrocytic hypochromic RBCs) some RBC may even lyse
List 3 important steps for proper storage and packing of EDTA and blood smears transported to a laboratory ?
Three important steps for EDTA packing and transport
- Bloode smear should be made fresh as storage in EDTA tubes leades to clumping, RBC swelling and pyknotic WBC
- EDTA tube stored in fridge or with ice apck in transit (preventing contact with ice as this may cause lysis.
- Blood smears should not be placed in fridge or exposed to formulin as this may cause lysis of cells.
Why is it important to wipe glass slides and spreader slides prior to making blood smears ?
The importance of wiping
- slides become contaminated even while in packaging
- bacteria, fungi, foreign materials and oils could affect dying or slide prepartion
When preparing blood smears from samples with “normal” viscositiy, what is the correct angle of the spreader slide relative to the blood film slide ?
45 degree angle when pushing the slide in a forward motion
What would you do to correct a blood smear which is too long or too short ?
Correction of a blood smear
The smear is too short and thick
- make smear longer
- larger drop of blood
- reduce speed of spreader
- decrease angle of spreader
The smear is too long
- to shorten your blood smear
- use a smaller drop of blood
- increase the speed of the spreader
- increase the angle of the spreader
Describe how you would make a blood smear narrower or thicker through technique ?
Correcting a blood smear
To make it more thick
- increase time for capillary action by moving the slide slower
To make your slide thinner
- decrease the time for capillary action by moving the slide faster
Describe how you could correct a blood smear which is two thick at the feathered edge ?
Blood smear to thick at the feathered edge
This occurs when blood is infront of the spreader
- ensure firm contact while pulling slide backwards into the application point.
Identify the regions (black arrows) on a blood smear and list which features you would look for or evaluate in each region ?
Blood smear
- Application point
- Body
- Monolayer
- evaluaute cell morphology and concentration - Feathered edge
- assess for platelet clumps, large cells parasites and WBC clumps (poor technique)
Describe in detail how you would carry out a systematic examination of a blood smear ?
Blood smear systematic examination
**Step one 10X
Scan body, monolayer (find optimal area) and feathered edge **
- assess smear quality, distribution and staining
- feathered edge - platelet clumps, parasites large or neoplastic cells and WBC clumping
- locate optimal area for high power magnification: monolayer
Step 2. on 100X
Estimate platelet count and observe morphology
- note feline platelets are prone to clumping (cause erroneous automated counts)
- roughly 2 fields back from feathered edge
- count number in three fields / average X by 20 = platelet concentration 10^9
**Step 3. 10X then 100X
Estimate WBC count, then on 100X perform WBC differential count
Step 4. 10X then 100X
Assess RBC arrangement + density, then on 100x observe RBC morphology
On a blood smear how do we determine the differential and absolute WBC count ?
Systematic evaluation of a blood smear
Step three; Estimate WBC count 10X, then on 100X perform WBC differential count
10X WBC count, Estimate WBC count
- first assess for an even distribution
- count number of WBC in 3 fields on 10X objective
(answer/3) / 4 X 10^9/L = WBC count X 10^9/L
Differential count 100X objective
- monolayer
- count cells using a variation of battlement technique
- 100 squares
On a blood smear describe how you would estimate platelet concentration ?
100X objective (oil)
- count number in three fields (ideal ten fields)
- determine average platelet count / 3
- multiply by 20 = platelet concentration X 10^9/L
Estimation plateconcentration on a blood smear
What is the minimum number of platelets on a blood smear per high power oil objective you would expect in a healthy patient ?
Healthy patient
10 platelets minimum per field view
or 200 X 10^9/L automated analyser
Note - platelet numbers may be artefactually low in cats as they are prone to platelet clumping.
Describe how you would differentiate between a band and segmented neutrophil ?
Segmented neutrophil
- when an area of the nucleus has a diameter <2/3 of any other area
- classify mature neutrophil
Band neutrophil
- immature
Identify this cell type ?
Monocytes
If struggling to identify go to a lower power.
Monocytes are comparatively larger cells
Identify this cell type ?
Eosinophils
Identify this cell type ?
Basophils
Confidently identify a species (dog, cat, horse, ruminant and bird)
Guess the species
A = dog
B= cat
C= horse
D= cow
Which domestic species has the largest RBC’s with the greatest central pallor ?
Dogs
- largest in size
- greatest central pallor
Which two domestic species demonstrate 1+ anisocytosis of their RBC’s ?
1+ Aniscytosis
- cattle
- cats
- sheep
Which two domestic species demonstrate Rouleaux in health ?
Rouleaux
Horse > cats
Which domestic species may have basophilic stippling of erythrocytes in health ?
Basophilic stippling
Cow
sheep
goat
Which species have oval erythrocytes in health ?
Alpacca / Llama
elliptical in shape
lack central pallor
Confidently identify rouleaux and agglutination on a blood smear or image of a blood smear ?
Identify Rouleaux and Agglutination
Rouleaux
- rolls
- linear branching or non branching aggregate of RBC’s
- resembling a stack of coins
- formation involves interactions between RBC membranes and plasma macromolecules
Agglutination
- Aggregates of RBC held together via antibodies
Describe the procedure to distinguish between Rouleaux and agglutination ?
Differentiation anisocytosis and Rouleaux
- saline agglutination test
- agglutination will persist due to antibody bridges
- rouleaux dispereses into individual RBC when TPP is diluted
Identify the pictured cells and what species they belong to ?
Reptile bird
Erythrocyte = nucleated RBC
Heterophil = lobulated nucleaus and elongate red cytoplasmic granules
Which conditions would lead to an increased degree of Rouleaux ?
Rouleaux
Increased with hyperglobulinaemia and hyperfibrinogenaemia
What pathological process leads to agglutination ?
Agglutination
Inflammation
Erythrocytes stuck together via antibodies
Image A is a blood smear from a healthy patient and image B is from a blood smear from patient which presented with a clinical history of lethargy and pale mucous memebranes.
- Identify the pathophysiological process in B ?
- List which cell type you would see on a modifoiied Wrights stain or a Diff Quick stain to determine if this pathological process is regenerative or not ?
- What type of stain and cell type would provide a more quantitive assessment of regeneration ?
- Pathology anaemia
- Modified Wrights Stain = Polychromatophils
New Methylene blue stain = Reticulocytes
- New Methylene blue stain = reticulocytes
Identify this pathology on a blood smear and discuss its significance ?
Anissocytosis
(variation in cell size)
- often observed as one blood cell can be seen to fit inside of another
- in cattle this is observerd on a blood smear in health
Anissoctosis can be used as an indirect identifyer of regeneration on a blood smear.
Distinguish between specific and non specific indicators of regeneration on a blood smear by explaining the difference and listing them under appropriate headings ?
Indicators of regenerative anaemia
Specific indicators of regenerative anaemia
- absolute reticulocyte count ove ref range (new methyl blue stain)
- polychromatophils (modified wrights stain)
Non specific indicators of anaemia
(may occur for other reasons eg aniscytosis occurs in healthy cattle).
- basophilic stippling
- metarubicytes
- anissocytosis
Identify aggregate and punctate reticulocytes on a new methylene blue stained blood smear - what would each of these cell types look like on a modified wrights stain ?
Aggregate reticulocytes appear as slightly larger and darker red blood cells.
Punctate reticulocytes appear the same as mature erythrocytes on a Wright stained blood film ?
Why are aggregate and punctate reticulocytes interpreted differently ?
The different interpretation of aggregate and punctate reticulocytes
All species - count aggregate reticulocytes to determine regeneration
Aggregate
- in canines mature rapidly <24hrs to mature RBC
- feline aggregate mature within 12-24 hrs
- acute moderate or severe anaemia reflecting recent bone marrow activity
Punctate
- seen in cats take longer to mature and can survive in the circulation for upto ten days
- if you count punctate cells depends upon severity and duration of anemia
- punctate may be useful for mild or chronic anemia
Define hyperchromasia, normocyte, metarubicytes and poiklocytes ?
Definitions
Hyperchromasia = pressence of pale red blood cells that lack haemoglobin
Normocyte = red blood cell with normal colour
Metarubicytes = immature red blood cells / large amounts of haemoglobin in their cytoplasm which imparts a red colour to the cell and they are nucleated.
Poiklocytes = any abnormal cell
What is the reference interval of absolute reticulocyte counts in dogs and cats ?
Absolute reticulocyte counts
Dogs > 60-80 X10^9/L
Cats > 50 X10^9/L
Define the terms macrocytosis, microcytosis and hypochromasia, how would you identify this on an automated haemogram ?
How would you identify eryhtrocytosis (polycythaemia) on automated haematology results ?
Eryhtrocyttosis (polycythaemia)
- Is an increase in RBC in peripheral blood
- identification is through increased RBC, HCT and HgB on an automated haemogram
What is the difference between eryhtrocytosis and haemoconcentration ?
Eryhtrocyttosis
- Is an increase in RBC in peripheral blood
- identification is through increased RBC, HCT and HgB on an automated haemogram
- tube B
Haemoconcentration
Is increased concentration of blood components (RBC) due to decreased blood volume.
- decreased plasma volume
- tube C
Tube is normal A
Explain the different categories of erythrocytosis, indicate expected protein concentration where appropriate as part of you explanation ?
Erythrocytosis pathogenesis (6)
Physiological splenic contraction
- total plasma remains normal
Primary erthrocytosis (erythroid neoplasia, polycythemia vera)
Secondary appropriate eryhtrocytosis
Hypoxia erythrocytosis
- cardiac failure
- respiratory disease
- Hyperthyroidism cats
Secondary inappropriate eryhtrocytosis
- renal neoplasm + non renal neoplasm
What is the potential clinical impact of extreme erythrocytosis ?
In extreme cases of erythrocytosis
- sludging of blood which impairs blood flow
- poor tissue perfusion and oxygenation
- purplish mucous memebranes
- congested blood vessels
- seizures
Explain the reason for potentially secondary eryhtrocytosis in hyperthyroid cats. Is the erythrocytosis appropriate or inappropriate ?
Eryhtrocytosis hyperthyroid cats
MOA
- increased eryhtropoiten (EPO)
- increased tissue demand for oxygen due to increased metabolism
It is an appropriate response due to increased tissue demand
Describe what causes physiological eryhtrocytosis ?
Physiological erythrocytosis - splenic contraction
Adrenal mediated epinephrine (excitement, fright or exercise)
- common in the horse
- total plasma proetin remains normal
- mature neutophilia and lymphocytosis
Answer to Q1
Answer
2. Secondary haemolysis
Answer
2. Prevent inappropriate coagulation and thromboembolic disease
Answer = 1.
vWBP Ag < 35%
- Sample clot or haemolysis
Answer = 2. false
Describe two conditions which could lead to Haemoconcentration ?
Haemoconcentration
Increased concentration (include RBC) due to decreased plasma volume
Dehydration
- reduced fluid but the RBC actually stays the same
Endotoxic shock
- shift of fluid from intra to extravascular
- vessel damage - leak proteins - osmotic fluid gradient
- usually accompanied by inflammatory leukogram
What could result in primary erythrocytosis ?
Primary erythrocytosis
Eryhtroid cell proliferation is independant of EPO
- thus EPO should be normal
Erythroid neoplasia
Polycythemia
Discuss causes of secondary eryhtrocytosis ?
Secondary erythrocytosis
( red blood cell proliferation due to EPO)
Renal neoplasia (inappropriate) tumour secreting factors
Appropriate (sustained hypoxia)
Hyperthyroidism in cats
Hypoxia - cardiac failure and respiratory disease
List 3 instances where interpretation of a leukogram may be clinically helpful ?
Leukogram
1. existence of inflammation
2. determine a prognosis or monitor treatment
3. detect some haematopoietic neoplasia
It is recommended where possible, to interpret WBC values over the course of serial blood evaluation.
Describe the potential consequence of interventricular septal defect in a calf ?
interventricular septal defect
What are the clinical limitations of a leukogram ?
Which species can have the most pronounced inflammatory and stress leukogram ?
Dogs
Why is neutropenia common in cows with inflammatory disease ?
Cattle
Common inflammatory states (mastitis and pneumonia)
Why is neutropenia common in cows with inflammatory disease
- neutropenia
- due to small bone marrow storage pool
Use the correct terminology to describe a increase and decrease in neutrophils, lymphocytes and eosinophils ?
Which species is most prone to a physiological leukogram and explain why ?
Cats are most prone to having a physiological leukogram
Phys leukogram = neutrophilia and lymphocytosis (fight/flight) adrenaline
increased blood flow rate and decreased adherence (N + L)
Cats have the most pronounced physiological elukogram
- they have the greatest marginal pool : central pool ratio
- more erythrocytes to move from the marginal pool to central pool to be included in sample
- 2 URL dogs compared to 3URL cats
Which leukocyte in cattle may display morphological criteria suggestive of neoplasia but merely occurs with marked inflammation ?
Inflammatory states in cattle
Cause a marked lymphocyte atypia
- this is difficult to differentiate from neoplastic states
- atypical lymphocytes are activated cells often in response to infection
high levels of atypical lymphocytes may also be present in lymphocytosis
Discuss seven classic leukocyte patterns and indicate the expected changes in each type of leukocyte with each pattern ?
Leukogram
Physiological leukocytosis - increase N and L
Steroidal leukocytosis = up up (monocyte, neutrophil) down down (eosinophil, lymphocyte)
Acute inflammation = increase neutrophils (seg + band) decrease lymphocytes
Chronic inflammation = increase neutrophils, decrease eosinophils and lymphocytes
Acute overwhelming inflammation = decrease neutrohils and lymphocytes
acute inflammation and endotoxaemia = decrease neutrophils and decrease lymphocytes
Addison’s disease = decrease neutrophils and possible increase lymphocytes
How do you determine the magnitude of an increase in a leukocyte (or any laboratory parameter ?
(explanation must include a calculation).
How to determine the magnitude of increase
Interpretation = patient results / URL (upper reference limit
eg magnitude of increase 14.4 / 12.5 = 1.2x
3 magnitude increase = severe
<2 = mild
What is the most consistent change in a stress leukogram ?
Stress leukogram
(I monocytes, I neutrophils, D Eosinophils and D Lymphocytes)
The most consistent change observed is
- neutrophilia and lymphopenia
Why
- eosinophils are very low in health thus difficult to detect changes
- Monocytosis is a fairly consistent finding in dogs, but only occassionally seen in cats thus species dependent.
Describe what you would observe in physiological leukogram and its MOA ?
Physiological leukogram
(Neutrophilia and lymphocytosis)
MOA - Mechanism of action Adrenaline
Neutrophilia and lymphocytosis
- decreased adherence to blood cell wall
- increased blood flow rate
Note - should return to normal levels one hour after stimulus is removed.
What would you observe in a stress leukogram, and what is the mechanism of action ?
Stress Leukogram - cortisol
(up up down down)
MOA - Mechanism of action
Lymphopenia (most consistently observed)
- redistribution from CLP to BM
- reduced egress from lymph node
Neutophilia
- adhesion molecules are downregulated (double neutrophils)
- increased release of neutophils (mostly mature) storage pool
Monocytosis
- shift from MMP to CMP
- common response in cats and dogs due to glucocorticoids
- rarely observed response in cattle and horses to glucocorticoids
Describe the appearance of a classic steroidal/stress leukogram in a dog ?
Stress leukogram in a dog
(up up down down)
Monocytosis
Neutrophilia
Eosinopenia
lymphopenia
Why is accurate Eosinopenia rare ?
Describe what you would observe on a leukogram for a dog that has Addison’s disease ?
Addison’s disease = absence or reduced secretion of glucocorticoids
- lymphocytosis or normal
- neutropenia
Why are serial blood results often required when evaluating patient leukograms ?
Why are serial leukograms required in ill patients.
Health
- In health neutrophil production is orderly
- bone marrow production 7 days, and life span in circulation 6 to 10 hours
Ill health
- In ill health production occurs rapidly 2-3 days and lifespan is dramatically reduced
Bottom line; sequential blood smaples are needed to define pathology change and response to treatment
How would you identify a left shift and what does its presence mean in a dog versus a cow ?
Left shift
Definition
- cell maturation proceeds left to right
- so a shift to the left means more immature cells
- metamyelocytes, band neutrophils immature white blood cells indicate a left shift
Species variation
Dogs - this usually indicates inflammation due to accelerated bone marrow production releasing increased numbers of immature cells into the circulation
Cattle - poor reserve pool of neutrophils, so as neutrophils leave the blood to the site of inflammation / reduced replenishment and dose not nessarily carry a poor prognosis
Confidently identify the three different cell types pictured below ?
Identify cell types
M = Metamyelocytes
S = segmented neutrophil
B = Band neutrophil
Explain the difference between an appropriate left shift and a degenerative left shift and how this would influence your approach to a clinical case ?
Left shift
= an increase in immature neutrophils (usually bands but less mature precursors may be present) and is usually a sign of inflammation
Degenerative left shift
= A band neutrophil count higher than segmented neutrophils
- particularly in the pressence of low mature neutrophil count
- a degenerative left shift carries a guarded or poor prognosis
Identify the categories of left shift in patient one and two ?
Patient one = Leukocytosis, neutrophilia and left shift
Patient two = Neutropenia and left shift (degenerative left shift poor prognosis).
What is a toxic change ?
Toxic change
Morphological alterations
- accelerated bone marrow production
- defective maturation
- features of immature stages retained (organelles)
The morphological changes
- cytoplasmic basophilia
- Dohle body inclusions
- Cytoplasmic vacuolation
- toxic granulation
Toxic change - occurs when there is a very strong stimulus to the bone marrow due to the pressence of severe inflammation in the body.
Identify the hallmarks of toxic change, their origin and significance ?
The hall marks of toxic change (features of immature cells retained).
Cytoplasmic basphilia - retention of cytoplasmic RNA and ribosomes
Dohle body - retained aggregates of rough endoplasmic reticulum
Cytoplasmic vacuolation - loss of granule
Toxic granulation - retention of acid mucopolysaccharides
Significance - indicates a very strong stimulus to the bone marrow due to the pressence of inflammation in the body.
Identify the toxic change
Banded neutrophil
- toxic granulation
-
Explain why one can see a variety of leukogram changes from neutrophilia to neutropenia with inflammation ?
The balance of dynamics
The pattern observed in blood depends upon the balance between bone marrow production and tissue consumption
- BM exceeds > tissue consumption = neutrophilia
- BM < tissue consumption = blood neutropenia
Define acute inflammation and what you expect to see on a leukogram ?
Acute inflammation
occurs within hours of stimulus
- inflammatory mediators must enter blood stream and reach bone marrow within hours of stimulus
- increased neutrophils entering blood stream
- then the number of neutrophils entering tissue increased
Acute inflammation
increase total WBC
increase segmented and band neutrophils
decrease lymphocytes (lymphopenia)
monocytosis
Provide a differential list for acute inflammation ?
Acute inflammation
neutrophilia, lymphopenia
Differentials
- focal suppurative lesions pyometra
- pertonitis
- pneumonia
- abscess
Why can there be a severe inflammatory process in the brain in the absence of any evidence of inflammation in the peripheral systemic leukogram ?
Blood brain barrier
- mediators are unable to leave the protected environmnet
- blood brain barrier
Describe the MOA of acute inflammation ?
Neutrophilia
- band and segmented
- increase number entering blood stream
- increase number entering tissue
Lymphopenia
- increased margination and emigration into inflammed tissue
- increased homing to lymph node
Monocytosis
- herald recovery from inflammation (monocytes released first from bone marrow)
- increased release from bone marrow
How would you interpret the clinical significance of neutropenia in species commonly treated by veternarians dogs, cats, horses and cattle (reference bone marrow capacity) ?
Species differences in bone marrow response
dogs large reserve - neutropenia = severe lesion
cats + horses - intermediate reserve = generally a severe lesion
cattle small reserve but slow response = unusual finding regardless of severeity
If you identify reactive lymphocytes on a peripheral blood smear what does their pressence imply ?
Reactive lymphocyte
Defined lymphocytes that are immune stimulated
- with upregulated synthesis of inflammatory mediators and or immunoglobulins
Reactive lymphocytes in peripheral blood suggest active systemic antigenic stimulation secondary to either infectious or non infectious disorders.
Pictured below reactive lymphocytes;
Define chronic inflammation and describe what you would likely observe on a leukogram ?
Chronic inflammation
defined by nature not duration
- increase TWBC
- neutrophilia
- eosinophilia
- lymphocytosis
a dynamic process expands and contracts according to requirements
proliferation and production from bone marrow has increased.
Describe the MOA of chronic inflammation ?
Chronic inflammation
I WBC, I N, IE, and I L
Neutrophilia
- stimulus lasts atleast one week (hyperplasia bone marrow)
- Increased supply emigration to tissue = mature neutrophilia
- if supply < demand a left shift may persist (chronic inflammatory pattern not reached).
Lymphocytosis
- chronic antigenic, cytogenic stimulation = increased lymphopoiesis / production
- reactive lymphocytes may be seen
Describe what you would observe with overwhelming inflammation or endotoxaemia ?
Overwhelming inflammation / endotoxaemia
Leukocytopenia
Neutropenia
(band neutrophils may increase or remain within reference interval)
Eosinopenia
Lymphocytopenia
Describe the MOA of overwhelming inflammation ?
Overwhelming inflammation
D WBC, DN, DE, DL
Neutropenia
- increased margination and emigration into tissues > than release from bone marrow
- occurs within hours of infection
- can have a left shift
( 2 days until we see the effect of increased bone marrow stimulation)
This occurs commonly in cattle - due to having a small storage pool and bone marrow response
How does neutropenia due to overwhelming inflammation differ in mechanism from neutropenia due to endotoxin ?
Neutropenia MOA
Neutropenia due to overwhelming inflammation
- increased margination and emigration of neutrophils into tissue > bone marrow response
- often occurs within hours of infection
Neutropenia due to endotoxaemia
- increased MNP
- rapid shift CNP to MNP (lasts 1-3 hours) increased endothelial adhesion
List two common causes of Eosinophilia ?
Eosinophilia
Mechanism of action
Parasitism
- ectoparasites, heartworms, tissue nematodes
Hypersensitivity (allergic disorders)
- flea bite dermatitis
Hyperadrenocorticism
Idiopathic eosinophilic conditions
Eosinophilic leukaemia
Provide a laboratory definition of anaemia ?
common clinical problem or pathological state
Laboratory definition
based upon low
RBC
Hgb
HCT/PCV
List three diagnostic classification schemes for anaemia ?
Three classification schemes for anaemia ?
- Bone marrow response (regenerative, non regenerative)
- Erythrocyte indices (morphological classification)
- Pathophysiology (mechanism)
List two mechanisms that cause regenerative anaemia ?
Two causes of regenerative anaemia
Haemorrhage
Haemolysis
How do you distinguish pre-regenerative anaemia from non-regenerative anemia ?
Anaemia
It takes the bone marrow 3-4 days to respond from onset of haemorrhage dogs + cats
Assessment take serial blood samples / blood smears
if the anaemia is regenerative should be able to see regeneration after four days
- reticulocytes
What are the laboratory indicators of a regeneration anaemia and where would we find that information ?
Is it regenerative anaemia
Automated haematology
Specific
- reticulocytes
Non speific
- MCV and RDW
Blood smear
Specific
- Reticulocytes
- polychromatophils
Non specific
- basophilic stippling (cattle)
- metarubicytes
- anisocytosis
If your patient has regenerative anaemia list two additional tests you could perform in house to refine your differential diagnosis ?
Regeneration the two additional testsadditional tests
Test plasma protein concentration - refractometer
Haemorrhage = decreased (except per acute or chronic)
Haemolysis = normal or potentially increased
Assess colour of plasma in haematocrit PCV
Haemorrhage = clear
Haemolysis = icteric or haemolysed
Explain why a patient can cope with a more severe degree of anaemia if the disease process is chronic compared to an acute onset ?
Acute vrs chronic anaemia
Acute
- sudden loss RBC
- ECF not yet replenished
- proteins won’t become diluted until the body has time to replenish the fluid loss
Chronic
- proteins replaced before blood cells
- anaemia has taken place over a long time thus the animal is able to learn to compensate.
Explain how you could distinguish between external haemorrhage, intravascular and extravascular haemolysis - you may include a table to organise this information ?
Haemorrhage
- decreased plasma protein
- clear plasma
Intravascular haemorrhage
- lysis of the RBC within the vasculature
- haemoglobinaemia, haemoglobinuria
- bilirubinuria, bilirubinaemia
- increased plasma protein
Extravascular haemorrhage
- cells identified as abnormal broken down intracellularly via macrophages
- increased plasma protein
- haemoglobinuria, haemoglobinaemia
- bilirubinaemia, bilirubinuria
Why can hyperbilirubinaemia and bilirubinuria develop in both intravascular and extravascular haemolsis ?
The rate limiting step is excretion of conjugated bilirubin.
Why do we try to determine if a haemolytic process is intravascular or extravascular ?
Describe the haematology and protein concentration laborartory results you might see in a clinical case of iron deficiency ?
(RBC morphological features + automated indices)
Automated and morphological of iron deficiency
Automated
- non regenerative anaemia
- initially compensatory regenerative but becomes non regenerative
Morphology
Microcytic normochromic - hypochromic
- microcytosis - extra cell division of rubicyte
(smaller cell required to achieve optimum HgB concentration)
- hypochromic later
- schistocytes and keratocytes
Clinical
- tarry faeces
- lethargy
Describe the pathology of Fe deficiency ?
Pathology Fe deficiency
Fe deficiency anaemia develops due to
- decreased production, maturation and release of RBCs
- increased RBC fragility - less deformable - reduced life span (schistocytes and keratocytes)
- blood loss
Why are young animals more at risk of developing iron deficiency ?
Young animals are more prone to iron deficiency
- less intake
- less storage
- greater requirements for growth
- more prone to parasites
Provide a differential list for non regenerative anaemia ?
Differentials lis non regenerative anaemia.
Reduced rate or erythropoiesis - anaemia takes longer to develop
- potential shortened RBC life span
- characteristics of RBC
How could you use the degree of non regenerative anaemia to assist in your diagnosis ? (normocytic, normochromic)
The degree of anaemia
Mild to moderate (normochromic, normocytic)
- usually only RBC affected from bone marrow
- inflammatory disease
- renal / liver disease
- retroviral infection
- hypothyroidism4hypoadrenocortism
Severe (normochromic, normocytic)
- usually > one cell line affected from bone marrow
- leukaemia
Explain if you have a low degree of normochromic, normocytic anaemia non regenerative anaemia what further diagnostic test would you look into ?
Anaemia of chronic inflammation is often mild to moderate and non regenerative
The underlying disease is identified on;
- CBC
- hormone testing thyroid + adrenal
- chemistry panel
- imaging
- urinalysis
- retroviral testing
- clinical signs and history
Describe how you would identify anaemia of inflammatory disease ?
Anaemia of inflammatory disease
Mild normocytic normochromic non regenerative anaemia
- MNEL; monocytosis, neutrophilia, eosinophilia and lymphocytosis
- hyperglobulinaemia and hyperfibrinogenaemia
- acute phase proteins
Provide 3 pathogenic machanisms for non regenerative anaemia due to inflammation ?
Inflammatory anaemia pathogenesis
mild normocytic normochromic non regenerative anaemia
1. Fe functional deficiency
- limit microbial access to Fe
2. Shortened RBC life span
- inflammatory mediators
3. Impaired eryhtropoietin mediated RBC production in bone marrow
- reduced amount and response to EPO
Describe what you would observe and the pathogenesis of anaemia due to renal disease ?
Anaemia due to renal dsiease
Mild to moderate normocytic normochromic anaemia
- azotaemia (elevation of nitrogenous products)
- isosthenuria (excretion of urine with a specific garvity = to that of plasma
- electrolyte abnormalities
Pathology
- reduced eryhtropoientin production
- reduced red blood cell survival time (reduced toxin clearance)
Describe how you would diagnose and the pathology underlying anaemia due to hypothyroidism ?
Hypothyroidism
Mild normocytic normochromic anaemia
- low T4 and T3
Pathology
- reduced metabolic rate
- decreased O2 requirements
- decreased EPO production
- new homeostasis with metabolic needs meet by lower RBC
Describe the haematological pattern of changes you might see in a patient with a PSS ?
Diagnosis PSS post systemic shunt
Mild to moderate microcytic normochromic (rarely hypochromic anaemia
- elevated dynamic bile acids
- if PSS is acquired we will see elevated liver enzymes but not if PSS is congenital
Pathology
Defective protein synthesis may lead to defective Fe transport and thus potential functional Fe deficiency (not total body Fe deficiency)
Why would a patient with cryptorchidism develop pancytopenia
Cryptorchid increases risk of testicular neoplastic production of oestrogens.
oestrogen can cause bone marrow suppression
Severe normocytic normochromic non regenerative anaemia
- pancytopaenia of oestrogen toxicosis (lower than normal number of red and white blood cells).
- signs of feminisation
- sertoli cell tumor or granulosa cell tumor
Evaluation of a blood smear may provide important clues regarding the cause of haemolytic anaemia -
List seven morphological abnormalities you might identify with haemolytic anaemia ?
Red cell abnormalities
regenerative anaemia due to haemolysis
- agglutination
- heinz bodies
- acanthocytes
- keratocytes
- spherocytes
- eccentrocytes
- schistocytes
- parasites
List five mechanisms that could cause haemolytic anaemia due to accelerated RBC destruction ?
Five mechanisms leading to increased RBC destruction
- Immune mediated haemolytic anaemia
- Eryhtrocyte metabolic defect (oxidative damage, defect ATP generation)
- eryhtrocyte fragmentation
- infectious
- 5 other - heparin, snake evenomation
Explain the three pathogenic mechanisms of immune mediated haemolytic anaemia ?
Pathogenesis of IMHA
There are three mechanisms - RBC coated with ESAIg and or C3
- extravascular haemolysis
- converted to spherocytes
- via macrophage removing part of the RBC membrane making the call fragile rigid - Antibodies (IgM) bind complement leading to activation of the complement cascadeand the formation of the membrane attack complex csb-9
What type of leukogram usually accompanies an IMHA ? Why does this occur ?
IMHA usually accompanied inflammatory leukogram
includes left shift;
The degree of neutrophilia and left shift correlate with the amount of tissue damage secondary to hypoxia and thromboeembolic disease.
What morphological features of IMHA would you observe on a peripheral blood smear ?
Blood smear IMHA
Mild to severe regenerative anaemia
Agglutination
Spherocytes
Others
- inflammatory leukogram - hypoxia
- bilirubinaemia
- haemoglobinuria, haemoglobinaemia
- Coombs test
If you identify spherocytes and agglutination IMHA is a Coombs test nessary ?
Coombs test
(direct antiglobin test)
Is not nessary when morphology of cells is present.
In what way are horses different to dogs with respect to IMHA ?
IMHA
In horses it is a relatively rare disease
- when it occurs it is usually secondary to RBC parasites
- clostridial infection / leptospirosis
- neoplasia
- drugs penicillin
Provide two reason for blood typing a horse ?
Blood transfusion reactions
Neonatal erythrolysis
Explain how eryhrolysis occurs in horses, include the blood group types most at risk ?
Equine neonatal isoerythrolysis
Immune mediated destruction of RBC haemolysis via maternal antibodies ingested in clostrum.
- icterus pallor
Horses
- dam negative for RBC antigen (Aa, Qa)
- mare becomes sensitised through previous exposure blood transfusion, prior pregnancy, transplacental contamination to antigen
- stallion Aa or Qa positive foal inherits these antigens
Certain RBC factors are more antigenic; Aa Qa
Provide a detailed explanation for how you would diagnose neonatal isoerythrolysis in horses ?
Neonatal isoeryhtrolysis horses
Foal born health then becomes anaemic, develops icterus and pallor
Diagnosis
- jaundiced foal agglutination test (agglutinate in saline +ve).
- identify antibodies in colostrum of mare
Blood film
anaemia
anisocytosis - normal RBC, macrocytes and spherocytes
metarubicytes
Howell Jolly bodies
Ghost cells
List two major pathogenic categories of eryhtrocyte metabolic defects that can lead to haemolytic anaemia ?
Eryhtrocyte metabolic defect
Oxidative damage
Defects in ATP production
Explain the 3 potential pathogenic mechanisms for causing anaemia due to oxidative injury ?
Anaemia pathogenesis due to oxidative injury
(Heinz bodies, eccentrocytes and methaemoglobinaemia)
Three mechanisms
1. Extravascular haemolysis RBC more rigid less able to pass through sinosoids and removed via macrophages.
2. Intravascular more fragile due to damaged membrane and may rupture spontaneously in blood vessel
3. Antigen formation - Heinz bodies binds RBC membrane bound by antibodies and removed by splenic or hepatic macrophages.
Describe how you would diagnose anaemia due to oxidative injury ?
Explain the pathogenesis of Heinz bodies in detail ?
Pathogenesis of Heinz bodies
They are aggregates of denatured Hgb caused by oxidative damage.
- Fe2+ carrys oxygen - oxidant overwhlems the RBC mechanisms to keep HgB in reduced state
- methaemoglobin Fe3+ can not carry oxygen
- Fe3+ Methaemoglobin undergo spontaneous conformational changes to form hemichromes (heme- depleted HgB
- Hemichromes precipitate to form Heinz bodies
How would you identify Heinz bodies on a Wrights, New methylene blue or Diff quick stain ?
Heinz bodies
They are aggregates of denatured Hgb caused by oxidative damage
Wright stain = same staining features as haemoglobin, but appear as slightly paler structures
NMB = pale blue and protruding
In what form of iron do RBCs carry oxygen ?
Fe2+
What roll do sulfhydryl groups play in Heinz body formation ?
Sulfhydryl groups 8 - disulphide bridges - when Heinz bodies precipiate
- cats 8
- dogs 4
- other species 2
Reductive capacity of RBC in cats is less; (cats can have upto 5% Heinz bodies in health).
Why are cats particularly susceptable to acetaminophen / paracetamol toxicity ?
Cats lack glucuronyl transferase
Glucuronyl transferase is used by most animals to conjugate paracetamol.
- lacking in cats
- unconjugated paracetamol is converted to reactive metabolites
- depele glutathione concentrations and therefore decrease protection from oxidative injury
List three causes of potential Heinz body formation in dogs, cats, horses and ruminants ?
Oxidative damage
horses, cats, dogs, horses and ruminants
Oxidative damage and Heinz body formation
- onion, garlic
- copper poisoning usually ruminants
- paracetomol in cats only
How do Heinz bodies form in chronic copper toxicity ?
Copper toxicity in ruminants
Chronic copper accululation in hepatocytes
- sudden release of Cu, often caused via stress
- Cu causes acute intravascular haemolysis
- haemoglobinaemia, haemoglobinuria and potential death
Describe the appearance of an eccentrocyte and demonstrate the ability to identify them on a blood film ?
Eccentrocyte
Eccentrocytes are mature erythrocytes in which the haemoglobin is pushed to one side of the cell leaving an opposing staining region.
Explain the pathogenesis of an eccentrocyte ?
Eccentrocyte
Eccentrocytes form
- oxidation leads to bonding of eryhtrocyte membranes and results in collapsed peripheral and cresent shaped region of the cell.
exogenous = onions, garlic, zinc
endogenous = diabetes mellitus
Explain how methaemoglobinaemia occurs ?
Methaemoglobinaemia pathogenesis
Oxidant overwhelms the RBC mechanisms to keep HgB in reduced state.
- Fe3+ methaemoglobin can not carry oxygen
Explain the pathogenesis of hypophosphataemic haemolysis in cattle (include what stage the are most at risk) ?
Hypophosphataemia low P
Defect in the ATP pathway - PFK and PK deficiency
Low levels of phosphorous in the blood
- phosphate is needed to generate ATP in the RBC eneergy pathway
Most at risk = 8 weeks post calving
sporadic multiparous cows
- defective phosphorous mobilization from bone increased loss of phosphorous in milk.
