Lab 5 - RBC Flashcards
Name the parameters of RBC’s that are the most important to check during pathological changes
RBC count and haemoglobin conc and/or function are the two most important parameters to check when looking for characteristics of pathological processes.
The rbc count and hb function/concentration parameters can check for which abnormalities?
evaluate polycythemias and anemias caused by different external and internal causative agent.
Which method is used for the Haemoglobin (Hgb) Measurement
Spectrophotometric Method (Drabkin-method)
What is the method and mechanism of the Drabkin method/spectophotometric method
Put 20microliter of whole blood sample to 5 ml reagent (K3Fe(SCN)6 hemolyses RBCs and forms Fe3+, from Fe2+ in the haemoglobin molecule, and this is further oxidised by KCN to cianidmethaemoglobin), mix it and measure the amount of orange coloured end product by spectrophotometer at 540 nm wave length.
How do we find the result from a spectophotometer reading
Using standard solutions or a standard curve.
(Esample/Estandard) * standard conc = result (in the unit of
measurement of the standard)
E= extinction
Hgb is bound and free and rbc form -
How are these measured during the spectophotometric method?
The measured Hgb concentration is a sum of Hgb molecules from the haemolysed RBCs (haemolysed by the reagent, this is almost 100% of the whole) and the very small amount of free Hgb content of the plasma, which is usually bound to a carrier protein (haptoglobin). Therefore there is no notable increase in Hgb concentration in case of intravascular haemolysis!!
Oxigen binding capacity of Hgb is ↑ increased by:
- decreased ↓ 2,3 DPG level in RBCs,
- decreased ↓ pCO2 level in the blood (for example in case of respiratory alkalosis), -increased ↑ pH of the blood (alkalosis, metabolic or respiratory),
- decreased ↓ temperature of blood (hypothermia!).
Oxygen binding capacity of Hgb is ↓ decreased by
- increased ↑ 2,3 DPG level in RBCs,
- increased ↑ pCO2 level in the blood (for example in case of respiratory acidosis), -decreased ↓ pH of the blood (acidosis, respiratory or metabolic),
- increased ↑ temperature of blood (hyperthermia!).
Oxygen saturation, definition and normal range arterial and venous
(SAT %) is the percentage (proportion) of oxygenated Hgb molecules compared to the whole amount of Hgb molecules in one unit of blood. Normal values in arterial blood: 95-99 %, in venous blood: 80-90 %
Toxins decr hgb oxygen affinity: CO
Color of blood: cherry red
Name/function: carboxyhemoglobin, hugher affinity so slow release - gradual suffocation
Toxins decr hgb oxygen affinity: Free radicals
Color of blood: dark brown
Name/function: methemoglobin. Due to oxidative damage fe2+ into fe3+, NADH can reverse it. Normal to have some in blood
Toxins decr hgb oxygen affinity: CN-
Color of blood: cherry red
Name/function: blocks respiration - does nothing to the hgb other than irreversibly binding to it
How can you estimate the RBC count?
If we suspect a normal average RBC volume (MCV, see later):
(Ht L/L / 5) x 100 = RBC count x 1012/L
Describe the mechanism of the automatic cell counters
Counting red blood cells is based upon the electric impendance of the particles. Impedance is in correlation with the size.
The impedance change(aka electrical resistance) when the rbc are sent through an aperture, this is measured and the impedance is measeured
Histogram: Axis ‘x’ shows the size (fl), axis ‘y’ shows the number
Aggregates are not counted
List the derivative parameters of RBC, and what are needed to calculate them
MCHC, MCH, MCV, RDW give info about size and color(influenced by hgb)
Indices: Ht, rbc count and hg are needed to calc these
Mch: definition, calculation and normal value - terms for abnormal high and low MCH
indicates average Hb content of RBCs.
Decreased ↓ MCH - hypochromasia Increased ↑ MCH - hyperchromasia
Mean Corpuscular Volume (MCV) indicates what, calculation, normail value
indicates the average size of the RBCs. (macro-↑, normo-↔, microcytic ↓ RBCs)
Mean Corpuscular Volume diff spp
There is a great heterogenity in MCV among species, cats, horses have smaller RBCs, than other animals. Young RBCs are bigger. New born animals have large, adults have smaller RBCs. Japanese Aktia has small (55-65 fl) some poodles have very large (75-80 fl) RBC.
Some causes of microcytosis:
All linked to decr hgb!
chronic blood loss(iron def)
iron, copper, pyridoxine (vitamine B6) deficieny(decr cu cause iron def, B6 cofactor in porph ring synth needed in hgb synth)
portosystemic shunt (abnormal vein cause some of the blood to bypass liver during embryonic life - cause abnormal liver metabolism)
liver failure
akitas
Some causes of macrocytosis
(mostly regenerative anaemias)
erytheoblastic leukemia
vitamin B12, folic acid, cobalt deficiency - need B12 in nucleotide metabolism so it influence the maturation, get stuck in young form
erythroleukaemias - regen anemias, loads of young/big cells. Shift to young rbc eg. acute Blood loss
poodle macrocytosis
Mean Corpuscular Haemoglobin Concentration (MCHC) indicates what, calculation
MCHC indicates the average concentration of haemoglobin in erythrocytes (Hb concentration).
(hyper-↑, normo-↔, hypochromic ↓ RBCs)
(Hgb (g/l) / PVC) = MCH (pg) / MCV (fl) x 1000 = MCHC (g/l)
Decreased MCHC - hypochromasia reasons,
When MCV and MCH are low (eg.:chronic liver failure) MCHC can be normal, however there is anemic state.
newborn animals (decr hgb) regenerative anaemias iron deficiency anaemia chronic blood loss(iron def) iron, copper, pyridoxine (vitamine B6) deficieny(decr cu cause iron def, B6 cofactor in porph ring synth needed in hgb synth)
Increased MCHC - hyperchromasia
erythroleukemia (polycythaemia absoluta vera)
vitamin B12, folic acid, cobalt deficiency
immunhemolytic anaemia (inflamm disease, igs on rbc -> macroph eats bites off part -> spherocytosis) imp hemolysis example!
lead poisoning
splenectomy
regenerative anaemias: morphology, staining, general causes
Typical regen anemias: macrocytic, hypohromic: MCV⬆️, MCHC⬇️
(Reticulocytes⬆️)
Acute blood loss, hemolysis
Typical changes in non regenerative anaemias
normocytic, normochromic: MCV normal, MCHC normal, (bm hypoplasia, secondary bm supression)
normal or decreased MCH (iron deficiency)
Red Cell Distribution width (RDW), Platelet Distribution Width (PDW)
A ratio that is correlated with the range of the average size of the RBCs and platelets.
Rdw
width is the line between the two points (P1-P2) where horizontal line crosses the two side lines of the curve. We express RDW in the percentage of P1-P2 width compared to the distribution of all red blood cells (from 0 till approx. 100 fl) as 100%
Rdw normal values
RDW dog: 12-16% cat: 14-18%
PDW dog: 6-8 %, cat: 7-12%
Normally the histogram is symmetric Gauss-curve with slight right shift.
Short RDW means non regenerative processes. Large RDW means regenerative process.
What are reticulocytes
Young, but differenciated RBCs, with basophil punctates stained by Brylliant-cresil blue stain, are reticulocytes. Blue punctates in the reticulocytes are rRNA (ribosomal RNA) remnants. Reticulocytes containing big blue aggregates (aggregated forms), are younger, than those containing small punctates (punctated forms). In cats punctated forms are more common. No reticulocytes appear in horses and ruminants (they appear only in the bone marrow, not in the peripheral blood!).
Why do we count reticulocytes
- Appearance of reticulocytes is a sign of the regenerative function of bone marrow.
- no reticulocytes could mean maturation arrest and not reg/nonregen anemia, so we count reticulocytes instead of nucleated rbc`s to diff btw reg/nonregen anemia in case there is maturation arrest
Reticulocyte function
same functional properties as mature RBCs, so they are able to carry oxygen. Nucleated RBCs are too young, therefore they are not able to function as RBCs, they are not able to carry oxygen! In case of a maturation arrest of the RBCs (for example: vitamin B12 , or folic acid deficiency or feline leukaemia virus infection can cause this) nucleated RBCs will never become reticulocytes, or mature RBCs!
differentiate between regenerative and non-regenerative anaemias by counting reticulocytes, and not nucleated RBCs.
Define regenerative anemia
favourable prognosis, because enough new RBCs are produced in the bone marrow to regenerate the anaemia, to replace the lost RBCs. The more severe prod, more significant increase in reticulocyte count, lower PCV - higher reticulocyte count
If bone marrow function is normal, RBC production is in positive correlation with the severity of anaemia.
Osmotic resistance of RBC is dependent on
- pH of the plasma (remember: normal is 7,4!) and the reagents,
- temperature
- the osmotic concentration of plasma and the reagents (NaCl concentration) and the RBC membrane status
- the regenerative status (reticulocytes are more resistant)
- the HbF (fetal haemoglobin) content of the RBCs (fetal RBCs, containing HbF, are more resistant).
Changed performance of the cell membrane - reasons for incr hemolysis!
- damaged due to i.e. nephropathy (uremia)
- specific membrane damage (immunhemolytic anaemia)
- increased physical damage (long term severe physical activity
osmotic resistance analysis, why can it change
- decreased in case of chronic haemolytic anaemia types, where there is extravascular hemolysis, and the plasma colour does not reflect to the haemolytic process. Chronic immunemediated haemolytic anaemia is typical of this kind of RBC damage.
- There are some rare hereditary genetical defects causing decreased osmotic resistance (therefore decreased life span) of RBCs for example: pyruvate-kinase or glucose-6-phosphate- dehydrogenase deficiency in dogs, methaemoglobin-reductase deficiency in dogs and horses, etc.
What are the methods for measuring the Osmotic resistance of RBCs
- Dilution line of NaCl solutions
2. Making hypertonic NaCl solution
How do you perform Dilution line of NaCl solutions
Make a diluton line from NaCl solution (saline) from 0,3% to 2,5%
Drip your blood samples into the different saline solutions in test tubes, incubate them on room temperature for 10 minutes. Centrifuge the test tubes (3000 rounds per minute, rpm), check the upper layer for hemolysis
How do you measure the osmotic resistance of the rbc using hypertonic NaCl solution
Prepare a hypotonic solution from NaCl : – add 2 ml of distilled water to 3 ml of phys. saline (0.9% NaCl)for dogs (this is a 0.54% NaCl solution) and - 1 ml of distilled water to 4 ml of phys. Saline for cats (this is a 0.72% NaCl solution).
Prepare three tubes.
Tube 1 – 5ml phys. saline for blood sample of the sick animal
Tube 2 – 5ml hypotonic NaCl solution for blood sample of the sick animal
Tube 3 – 5ml hypotonic NaCl solution for blood sample of control (healthy) animal
Put 5 drops of blood into each tube (Tube 1, 2: from patient’s blood, Tube 3: from control blood)
Incubate them for 10 min. on room temperature.
Centrifuge them on 3000/min.
Check the upper layer for haemolysis.
Result:
If the upper layer of Tube 3 shows hemolysis (reddish dyscolourisation, ‘+’ in the table) repeat the analysis, because the reagents are not accurate (laboratory analytical error).
How do you perform a Correct blood smear analysis
- using proper staining methods, i.e. May-Grünwald staining, Giemsa staining
- Smears must be prepared by using fresh samples.
- Check blood films on low (200x) then high (1000x) magnification.
- Check gross signs: rouleau formation - coin arrangement (horse often, dog, cat, swine sometimes, cattle rare)
- RBC aggregates large cells (horse often) thrombocyte aggregates
Intensity of staining of RBCs
Polychromasia, hyperchromasia: more intensive staining,
RNA, or nuclear remnants, more Hb - regenerative process Hypochromasia: weak staining
decreased Hb-content - iron, or other nutrient deficiency
Size of RBCs:
Macrocytosis: many big cells
Microcytosis: many small cells
Anisocytosis - variable cell size - iron deficiency and regenerative process Poikylocytosis variable size and colour
RBC types: age
young and nucleated RBCs (in order of maturation): Proerythroblast
Basophil erythroblast (normocyte, normoblast) Polychromatophil erythroblast (normocyte, normoblast) Acidophil erythroblast (normocyte, normoblast)
Young but mature RBC without nucleus: reticulocyte
Appearance of young RBCs: increased production (regenerative anaemia), spleen or bone marrow disease, leukaemia, extramedullar erythrocyte production, Pb toxicosis (with basophil punctates), hyperadrenocorticism
Reticulocyte
appearance: increased production (regenerative anaemia) - chronic Fe deficicency anaemia, haemolysis, acute blood loss, chronic blood loss !
Spherocyte
spherical small, polychromatophil RBC
appearance
When do we see it? sensitive RBC membrane, immunemediated hemolysis
(Inflamm process - igs bind on surface, phagocyte fires of some membr. Leavind the cell with less membr but same content)
Stomatocyte
mouth-shaped RBC
Appearance: increased RBC production (regenerative anaemia)
Acanthocyte
Spur cell - RBC with few long spikes
When: RBC membrane failure (lipid bilayer) – lipid metabolism disorder, hepatopathies
Schysocyte
RBC fragment
When: traumatic or toxic damage (uremia, blood parasites, long term severe physical activity, DIC!!)
Anulocyte
0 - like RBC
appearance: iron deficiency anaemia
Codocyte
Target cell, like a target(faded membr and nucleus)
appearance: regenerative process
Echynocyte/burr cell
RBC with many small spikes
Appearance: laboratory error (too quick drying of blood film, uremia, DIC!!)
Sickle cell
appearance: RBC damage, Hb globin chain malformation in humans
The 4 Inclusion bodies in RBCs
Heinz body (NMB - new methylene blue stain to see big violet lump): denaturated Hgb appearance: O2 effect, oxidative damage to RBCs (cat!, for example methaemoglobinaemia!, paracetamol, onions)
Howell-Jolly body : nuclear membrane remnants - one small/med sized. appearance: vitamin B12 deficiency, increased production of red cells, splenectomy
Basophilic punctuates :nuclear remnantsappearance, many! After proliferation.
regenerative process, young RBCs of cat, physiological in ruminants, lead poisoning
Hb inclusions - many small/med sized at periåhary - looks lumpy.
appearance: Hb damage, increased RBC production, regenerative anaemia
Name some RBC parasites
Haemobartonella canis, felis, bovis
Babesia spp. (canis, gibsoni), B. canis is very common in Hungary! Ehrlichia canis, equi etc.
Dirofilaria immitis, repens
Why do we do serum ironmmeasurement, normal range
If we suspect iron deficiency, especially due to chronic blood loss, we can prove it by performing this test. Normal SeFe (serum iron): 18-20 mol/l
How mechanism of serum iron measurement
- Fe3+ from feed, Fe2+ By ascorbic acid, cysteine or glutathione in duodenum to be absorbed.
- Bound to (APP) ferritin(3+) in the mucosal cells.
- Fe2+ to tp to plasma, bound totransferrin(3+) —> liver, spleen or bone marrow - 80-90% for hgb synth in BM, when iron stores are used up during blood loss, iron deficiancy anemia developes
Method of serum iron measurement:
Serum samples are needed for this analysis, because fibrinogen content of the plasma may disturb the measurement. Fe3+ is reduced to Fe2+ by ascorbic acid. Fe2+ reacts with ferrosin and forms a red coloured chelate (complex molecule) which can be measured photometrically. (absorption maximum at 560nm).
should always be performed together with TIBC analysis!
Causes of low serum iron concentration:
chronic blood loss
decreased intake (piglets, calves)
impaired gastric, duodenal, jejunal function (reduction, transport, absorption) Causes of high serum iron concentration:
iron toxicosis (overload)
Total iron binding capacity (TIBC), normal range, for what
This test gives information about the transferrin content. TIBC (total iron binding capacity): 50-68 mol/l
How doe we measure Total iron binding capacity (TIBC)
measure seFe then, add Fe-solution to the plasma (by this method all transferrin molecules will be fully saturated), then put absorbent to the solution, centrifuge the absorbent (this binds to free Fe and goes to the sediment), Use the upper layer and check seFe again. TIBC =serum iron level (saturated transferrin)+free transferrin (not saturated)
Causes low TIBC:
chronic inflammation (negative acute phase protein, see later)
chronic liver failure (decreased transferring synthesis in the liver)
neoplastic disease
Causes of high TIBC:
iron deficiency anaemia (not severe: normal iron level+high TIBC, severe: low iron level+high TIBC)
How do we measure iron saturation, normal range
(SeFe/TIBC)x 100 = Iron saturation
normal: 20-55% (30%)
How do we measure Ferritin, Transferrin RBC life span Hgb electrophoresis vitamin B12 measurements erythropoetin
Ferritin (RIA method): 12-300 microg/l
Transferrin (RIA, ELISA method))
RBC life span (Cr51 method)
Hgb electrophoresis (globin chain sequence analyis)
vitamin B12 measurements (RIA method)
erythropoetin (ELISA method, highly species specific, a few laboratory can measure it)
Laboratory Findings in Hemolysis
PCV ⬇️
reticulocytes ⬆️renerative anaemia) polychromasia, poikilocytosis leukocytosis, (neutrophilia) spherocytosis,
total bilirubin⬆️
indirect bilirubin⬆️
lactate dehydrogenase (LDH) I,II ⬆️haptoglobin⬇️,
RBC osmotic resistance⬇️,
jaundice
hyperchromic stool, urobilinogen and Hgb in urine⬆️
How can we determine if we have regenerative anemia
Must look at reticulocytes to determine completely!!
How do you prepare for a reticulocyte count
Staining:
Preparing Brylliant-cresil stain: put 0,04 g Bryllant-cresil in 8 ml phys. sal., mix it then put 2 ml Na-citrate (3.8%) into the solution, mix it again. (Or buy the commercially available, ready to use Brylliant-cresil stain, like Reticount.)
Vital staining: mix fresh (EDTA) blood and Brylliant-cresil stain in the same proportion (stain fresh, alive /vital/ RBCs). Incubate it at room temperature for 2-3 hours, this is enough for the reticulocytes to take up stain. Prepare a smear from this mixture.
Reasons for correction of the normal possible PCV changes we can expect a specific reticulocyte %
- when sick animal, bc mature RBCs are more sensitive to damage, so there will be fewer mature than young - ratio is wrong!
- to consider the severity of anaemia to differentiate between regenerative and non-regenerative anaemias.
How do you preform for a reticulocyte count, normal range
Count 100-1000 RBCs and take the percent of the reticulocytes.
normal: 2-3 %
Causes of decreased ↓ Hgb concentration: ↓ MCH
1️⃣ hyperhydration: liver or kidney disease
2️⃣Iron def. Anemia
3️⃣ chronic blood loss: eg iron deficiancy
4️⃣ swine: young pigs have lower hgb conc than older ones
5️⃣ iron, copper(needed for fe absorption)
6️⃣ pyridoxine (vitamine B6) deficieny - iron production!
7️⃣ portosystemic shunt: very microcytic!!
Causes of increased ↑ Hgb concentration/ ↑ MCH
1️⃣dehydration (relative polycthemias: red water intake, kidney disease, diabetes)
2️⃣ acute erythroblastic anemia
3️⃣vitamin B12, folic acid, cobalt deficiency - stuck in young form (maturation)
4️⃣ polycythemia absoluta vera
5️⃣ immunhemolytic anaemia (spherocytosis)
6️⃣lead poisoning
7️⃣splenectomy
8️⃣ acute blood loss
