Lab 5 Flashcards by Helene Berg

Haemoglobin (Hgb) Measurment:

Spectrophotometric Method (Drabkin-method)

Put 20 um of whole blood sample to 5 ml reagent (K3Fe(SCN)6), it hemolyses RBCs and forms Fe3+ from Fe2+ in the haemoglobin molecule, and this is furhter oxidised by KCN to cianidmethaemoglobin.
mix it and measure the amount of orange coloured end product by spectrophotometer at 540 nm wave length.
use standard solution or a standard curve.
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!

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Caluclation method for Sepctrophotometrid Method (Drabkin-method):

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Oxygen dissociation curve

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Oxygen binding capacity of the Hgb is increased by:

decreased 2,3 DPG level in RBC´s
decreased pCO2 level in the blood (eg in case of respiratory alkalosis)
increased pH of the blood (alkalosis, metabolic or respiratory)
decreased temperature of blood (hypothermia)

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Oxygen binding capacity of Hgb is decreased by:

increased 2,3 DPG level in RBC´s
increased pC02 level in the blood (eg. recpiratory acidosis)
decreased pH of the blood (acidosis, respiratory or metabolic)
increased temperature of blood (hyperthermia)

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What is Oxygen saturation?

(SAT %) is the precentage (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 %
normal values in venous blood: 80-90 %

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Hgb molecules containing Iron 2+:

When iron is in its 2+ form in the Hgb molecules (functionally active Hgb), RBC´s (containing these Hgb molecules) are able to take up oxygen molecules in the lungs, carry then, and deliver them to the cells, where they are used in the terminal oxidation phase of the metabolic process.

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Hgb molecules containing oxidised iron (3+ form):

are called methaemoglobin
these are unable to carry oxygen.
there is always a constantly small amount of methaemoglobin in the bood, because these are reduced to normal haemoglobin by methaemoglobin-reductase enzyme.
severe oxidative damage to the RBC´s (nitrites, free radicals, paracetamol, onion) can lead to increased methaemoglobin level in the blood which is called methaemoglobinaemia.
in this case the colour of the blood is dark brown (like chocolate) and the mucous membranes are deeply cyanotic.
Hgb molecules of cats and newborn or very young animals of any other species are very sensitive to oxidative damage.

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Causes of increased Hgb concentration:

usually associated with different types of relative (dehydration) or absolute polycytaemia

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Causes of decreased Hgb concentration:

usually associated with relative (hyperhydration) or aboslute oligocytaemia (anemia)

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Red blood cell count:

estimated RBC-count

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RBC count:

measured by automatic cell counter

counting red blood cells is based upon the electric impendance of the particles. Impedance is in correlation with the size.
Impedance method is based on the electrical impedance change due to the transmission of particles through an aperture.
the red blood cells are impeding the electrical flow.
the impedance is correlated with the size of the red blood cells.
Particles are taken generally as RBCs by the counter if their size is 40-100 fl.
histogram (printed out by the counters) shows the distribution of different sized cells (between 0-150fl), RBCs and thrombocytes.
Axis “x” shoes the size (fl), axis “y” shows the number of the counted cell particles.
if RBCs are forming aggregates (immunohemolytic anemia) they are not counted to be RBcs.
when you suspect cold agglutination, you should warm the blood samples to 37*C before cell counting to separate aggregated RBcs frome each other.

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Normal RBC count:

4.5-8 x 10^12/IT/L

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Derivative paramters:

in order to calculate the indices we must measure Ht or PCV, red blood cell count, haemoglobin concentration.
these indices give objective information about the average size and colour (which is influenced by Hb content) or RBC´s.
this information can also be gained by routine blood smear analysis, but the results are severly affected by the competence of the cytologist, so it is too subjective..
the indices are used in case of human, dogs and maybe cats.
in case of horses, ruminants these are alomst useless because of the big variance among parameters of RBCs of different animal induviduals, and also within individuals!

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Derivative parameters:

Mean Corpuscular Haemoglobin (MCH)

MCH indicates average Hb content of RBCs
normal: 12-30 pg
in young animals it (and MCV) can be increased 28-32 pg
Decreased MCH = hypochromasia
Increased MCH = hyperchromasia

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Equation for calculation of MCH:

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MCH (pg) in Horse, Ruminants, Dog, Cat:

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Derivative parameters:

Mean Corpuscular Volume (MCV)

MCV indicates the average size of RBCs
(Macro-increase, Normo, microlytic decreased RBCs)
normal: 60-70 fl

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Equation for calculation of MCV:

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MCV (fl) in horse, ruminants, dog, cat

cat: 40-53

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Heterogenity in MCV among species:

cat, horses: smaller RBC´s than other animals.
Young RBCs are bigger. Newborn animals have large, adults hae smaller RBCs.
Japanese Aktia has small (55-65 fl) some poodles have very large (75-80 fl) RBC

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Some causes of microcytosis:

chronic blood loss
iron, copper, pyridoxine (vit B6) deficiency
portosystemic shunt

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Some causes of macrocytosis:

mostly regenerative anemias

polycythaemia absoluta vera (erythroleukemia)
Vitamin B12, folic acid, cobalt deficiency

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Derivative parameters:

Mean Corpuscular Haemoglobin Concentration (MCHC)

MCHC indicates the average concentration of haemoglobin in eryhtrocytes (Hb concentration)
Hyper - increased, Normo, Hypocromic - decreased RBC)
normal: 300-350 g/l (30-35%)

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Equation for MCHC:

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MCHC % in Horse, ruminants, dog, cat:

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Decreased MCHC - hypochromasia, can be seen in case of:

newborn animals
regenerative anaemias
iron deficiency anaemia

Increased MCHC - hyperchromasia:

erythroleukemia (polycythaemia abosluta vera)
vitamin B12, folic acid, cobalt deficiency
immunhemolytic anaemia (spherocytosis)
lead poisoning
splenectomy

MCV vs. MCH vs. MCHC

When MCV and MCH are low (eg chronic liver failure) MCHC can be normal, however there is anemic state.

Typical changes in derivative parameters:

Red Cell Distribution width (RDW), Platelet Distribution Width (PDW):

these indices give a number that is correlated with the range of the average size of the RBCs and platelets
the distribution widt of the erythrocyte or platelet population derived from the histogram at 20% of peak.
RDW-s 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 ells (from 0 till approx. 100) as 100%.
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.

Reticulocyte count:

what are reticulocytes and how do they look

Young, but differentiated RBCs, with basophil punctates stained by Brylliant-cresil blue stain, are reticulocytes.
blue punctates in the reticulocytes are rRNA remnants.
reticulocytes containing big blue aggregates (aggregated form) are younger, than those containing small punctuates (punctuated form).
in cats punctuated forms are more common.
no reticulocytes appear in horse and ruminants (they appear in the bone marrow, not in the peripheral blood!)

Reticulocyte count:

Appearance of reticulocytes

Appearance of reticulocytes is a sign of the regenerative function of bone marrow.

Reticulocyes have the 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 (eg: vitamin B12-, or folic acid deficiency, or Feline leukemia virus) nucleated RBCs will never become reticulocytes, or mature RBCs.
that is why we differentiate between regenerative and non-regenerative anaemias by counting reticulocytes, and not nucleated RBCs.

Reticulocyte count:

regenerative anaemias

regenerative anaemias are usually diseases with favourable prognosis, because enough new RBcs are produced in the bone marrow to regenerate the anaemia, to replace the lost RBCs, and to reach normal RBC count quickly.
if bone marrow function is normal, RBC production is in positive correlation with the severity of anaemia.
in case of severe anaemia more intenstive RBC production, (a more significant increase in reticulocyte count) is needed to consider this as regenerative anaemia, than in case of mild anaemia.

Reticulocyte count:

staining

Preparing Brylliant-cresil stain:

put 0,04g Brylliant-cresil in 8ml physilogical saline, 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/vitall/RBCs).
incubate it at room temp for 2-3 hours, this is enough for the reticulocytes to take up stain. prepare a smear from this mixture.

Reticulocyte count:

counting

Count 100-1000 RBCs and take the percent of reticulocytes.

normal: 2-3%

The expected values for a proper regeneration:

in order to say that the bone marrow is capable for replacing RBC in an organism which lost red blood cells, we expect that the more severe anaemia will cause increased reticulocytic %.
the possible PCV changes we can expect a specific reticulocyte %.
the expected values for a proper regeneration can be read in the table:

What does it mean that Reticulocytic count sometimes should be corrected?

it means that it is correlated to the RBC count or the PCV of the sick animal.
this is because mature RBC damage, usually more mature RBCs are dead than the young ones.
the other reason for correction is that we need to consider the severity of anaemia to differentiate between regenerative and non-regenerative anaemias.
a slightly increased reticulocyte count in case of really severe aneamia does not mean regenerative process!

Equation of Corrected Reticulocyte Count (CRC):

CRC = reticulocyte % x RBC count

normal: <0,06 x 10^12/l (without aneamia!)

Corrected reticulocyte percentage (CRP):

Increased reticulocyte count can be caused by different types of regenerative anaemias:

acute blood loss (approx. 3-5 days are needed for the bone marrow to increase the reticulocyte count in the blood)
haemolytic anaemia
chronic blood loss
some types of nutrient deficiency anaemias.

Osmotic resistance of RBC is dependent on?

the pH of the plasma (normal is 7,4) and the reagents
the temp.
the osmotic concentration of plasma and the reagents (NaCl concentration) and the RBC membrane status
the regenerative status (reticulocytes are more restistant)
the HbF (fetal hemoglobin) content of the RBCs (fetal RBCs containing HbF are more resistant.

When/or why do we perform the Osmotic Resistance analysis?

in order to examine RBC membrane function (membrane-integrity), whether the membranes are damaged due to i.e

nephrophaty (uremia)
specific membrane damage (immunoheolytic anaemia)
increased physical damage (long term severe physical activity, eg long distance running)

In general, Osmotic resistance becomes decreased in case of?

chronic haemolytic anaemia types:

where there is extravascular hemolysis and the plasma colour does not reflect to the haemolytic process.
chronim immunemediated haemolytic anaemia is typical of this kind of RBC damage

There are some rare heriditary genetical defects causing decreased osmotic resistance (therefore decreased life span) of RBC, eg:

pyruvate-kinase or glucose-6-phosphate-dehydrogenase deficiency in dogs.
methaemoglobin-reductase deficiency in dogs and horses.

Osmotic resistance of RBC´s:

method 1

make a dilution line from NaCl solution 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 min.
centrifuge the test tubes (300 rpm) check the upper layer for hemolysis.
Prefer using distilled water with a pH around 7,4.

Osmotic resistance of RBCs

Method 1, table of Species, Average RBC diameter, total hemolysis NaCl and beginning og hemolysis NaCl

Osmotic resistance of RBCs:

method 2

prepare a hypotonic solution from NaCl:

add 2 ml of distilled water to 3ml of physiological saline (0,9% NaCl) for dogs (this is a 0,54% 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: 5 mlhypotonic NaCl solution for blood sample of the sick animal
tube 3: 5ml of hypotonic NaCl solution for blood sample of control (healthy) animal
put 5 drops of blood into each tube (tube 1+2: from patients blood, tube 3: from control blood)
incubate them for 10 min in room temp.
centrifuge them on 3000rpm/min
check the upper layer for haemolysis

Result: if the upper layer of tube 3 shows hemolysis (reddish dyscolourisation, “+” in tha table) repeat the analysis, because the reagents are not accurate.

Osmotic resistance of RBCs

Method 1: table of the 3 tubes

Erythrocyte morphology:

blood smear analysis

correct blood smear analysis begins with using proper staining methods, i.e May-Grunwald, Giemsa, Diff Quick etc.
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 (often in horse)

–> thormboyte aggregates

Erythrocyte morphology:

intensity of staining of RBCs

Polychromasia, hyperchromasia: more intensice staining, RNA, or nuclear elements, more Hb - regenerative process.
Hypochromasia: weak staining. Decreased Hb-content - iron, or other nutrient deficiency.

Erythrocyte morphology:

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

Erythrocyte morphology:

RBC types

Young and nucleated RBCs (in order of maturation):

proerythroblast
basophil erythroblast (normocyte, normoblast)
polychromatophil erythroblast (normocyte, normoblast)
acidophil erythorblast (mormocyte, normoblast)

Young but mature RBC without nucleus:

Reticulocyte

Appearance of young RBCs:

increased production (regenerative anaemia)
spleen or bone marrow disease
leukamia
extramedullar erythrocyte production
Pb toxicosis (with basophil punctates)
hyperadrenocorticism

Erythrocyte morphology:

appearance of Reticulocyte, Spherocyte, Stomatocyte, Acanthocyte and Schysocyte

Reticulocyte:

appearance: increased production (regenerative anaemia), chronic Fe deficiency anaemia, haemolysis, acute blood loss, chronic blood loss.

Spherocyte (spherical small polychomatophil RBC):

appearance: sensitive RBC membrane, immunemediated hemolysis

Stomatocyte (mouth shaped RBC):

appearance: increased RBC production (regenerative anaemia)

Acanthocyte (Spur cell - RBC with few long spikes)

Appearance: RBC membrane failure (lipid layer), lipid metabolism disorder, hepatopathies.

Schysocyte (RBC fragment):

appearance: tramatic or toxic damage (uremia, blood prarasites, long term severe physical activity, DIC)

Erythrocyte morphology:

appearance of Anulocyte, Codocyte, Echynocyte, Sickle cell

Anulocyte (0 - like RBC):

appearance: iron deficiency anaemia

Codocyte (target cell, like a target):

appearance: regnerative process

Echynocyte (Burr cell, crenation, RBC with many small spikes):

appearance: RBC damage, Hb globin chain malformation in humans

Erythrocyte morphology:

Inclusion bodies in RBCs

Heinz body (NMB - new methylene blue stain: denaturated Hgb

apperance: O2 effect, oxidative damage to RBCs (eg. methaemoglobinaemia in cat), GSH deficiency.

Howell-Jolly body: nuclear membrane remnants

appearance: Vitamin B12 deficiency, increased production of red blood cells, splenectomy

Basophilic puntuates: nuclear remnants

Appearance: regenerative process, young RBCs of cat, physiological in ruminants, lead poisoning.

Hb inclusions:

Appearance: Hb damage, increasd RBC production, regenerative anaemia.

Erythrocyte morphology:

RBC parasites

haemobartonella canis, felis, bovis
babesia spp. (canis, gibsoni), B. canis is very common in Hungary!
Ehrlichia canis, equi etc.
Dirofilaria immitis, repens
Anaplasma marginate, centrale, ovis.
Eperythrozoon wenyoni, ovis, suis, parvum
Citauxzoon felis
Theileria parva, mutans, annulata, hirci, ovis
Trypanosoma cruzi, congolense, vivax, brucei, evans, susi, epuiperdum
Leishmania donovani

Other laboratry measurments in connection with RBCs:

Serum iron measurments and Total iron binding capacity (TIBC)

Why use Serum iron measurement:

If we suspect iron deficiency, especially due to chronic blood loss, we can prove it by performing this test.

Normal SeFe: 18-20 umol/l

Serum iron measrument:

iron metabolism

Iron metabolism is closed, which means that normally very low amount of iron is lost from the body, most of it is reutilized.
daily iron need of adult animal is around 1mg.
Iron is in its Fe3+ form (oxalate, phosphate comples molecules) in the feed of animals, but in this form it can not be absorbed.
Gastric acid makes iron Fe3+ free from these complex molecules, then it is reduced to Fe2+ by ascorbic acid, cystein or glutathione in the duodenum, and now it can be absorbed.
in the mucosa of the small intestines iron is bound to apoferritin (or mucosa ferritin), and stored like this as ferritin.
from this place iron can be absorbed to the plasma or excreted to the faeces.
iron in the plasma is oxidised by coeruloplasmin enzymes to Fe3+ and it is transported by transferrin.
this acute phase protein is produced in the liver, and usually only 30% of transferrin molecules are carrying iron (saturated).
Iron is stored in easily available ferritin form in the spleen, bone marrow and liver; and is less utilizable haemosiderin form in macrophage cells.
approx. 80-90% of iron is reutilized for haemoglobin synthesis in the bone marrow.
when this metabolic circle is “opened” by chronic blood (and thus iron) loss, iron storages become depleted and at the end iron deficiency anaemia will appear.

Serum iron measrument:

method

serum samples are needed, because fibrinogen content of the plasma may disturb the measurment.
Fe3+ is reduced to Fe2+ by ascorbic acid.
Fe2+ reacts with ferrosin and forms a red coloured chelate (complex mlecule) which can be measured photometrically.
absorption maximum at 560nm

Causes of low and high serum iron concentration:

Low:

chronic blood oss
decreased intake (piglets, calves)
impaired gastric, duodenal, jejunal function (reduction, transport, absorption)

High:

iron toxicosis (overload)

What is Total Iron binding capacity (TIBC)?

test that gives information about the transferrin content
TIBC: 50-68 umol/l
serum iron measurment should always be performed together with TIBC analysis

Total iron binding capacity (TIBC):

determination

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)

Total iron binding capacity (TIBC):

causes of low and high TIBC

Low:

chronic inflammation (negative acute phase proteins)
chronic liver failure (decreased transferrin synthesis in the liver)
neoplastic disease

high:

iron deficiency anaemia (not severe: normal iron level + high TIBC. Severe: low iron level + high TIBC)

Total iron binding capacity (TIBC):

Iron saturation

Total iron binding capacity (TIBC):

Analysing methods of Ferritin, Transferin, RBC lifespan, Hgb, Vitamin B12, erythropoetin

Ferritin: RIA method. 12-300 ug/l
Transferrin: RIA, ELISA method
RBC lifespan: Cr51 method
Hgb electrophoresis: globin chain sequence analysis
Vitamin B12: RIA method
Erythropoetin: ELISA, higly species specific.

Laboratory findings in Hemolysis:

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Lab 5 Flashcards

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