LabD 5 RBC Flashcards
(134 cards)
1
Q
Main function?
A
Oxygen transportation
2
Q
Problem with RBC causes?
A
Hypoxia in tissues
3
Q
What can cause disease of RBC?
A
Transitions in RBC metabolism, many diff substances is produced. Transitional products
4
Q
Most important RBC parameters?
A
RBC count, haemoglobin conc. and or function
5
Q
RBC count, haemoglobin conc. and or function, these parameters can help us evaluate?
A
Polycytaemias and anaemias caused by diff external and internal causative agent
6
Q
Haemoglobin measurement; spectrophotometric method (Drabkin method) how?
A
- Whole blood sample to reagent (potassium ferricyanide) which hemolyses RBCs and forms Fe3+ from Fe2+ in the haemoglobin molecule -> further oxidized by potassiumcyanide to cyanidmethaemoglobin
- Mix it and measure orange color enproduct by spectrophotometer
- Use standard solutions or standard curve to compare
7
Q
Spectrophotometer method (Drabkin method) formula
A
Esample/Estandard x standard concentration = result (in same unit as standard) (E = extinction)
8
Q
Normal value of haemoglobin in blood
A
18-20 mmol/L or 12-18 g/dl (g%)
9
Q
Drabkin method; hemolyzed RBCs contains almost the whole amount of haemoglobin in blood, but what and where about the rest?
A
- Very small amount of free Hb, bound to carrier protein Haptoglobin (acute phase protein from last lab)
- Therefore no notable increase in Hb conc in case of intravascular hemolysis
10
Q
Oxygen dissociation curve, left and right shift means?
A
- Left: decresed temp, decreased 2-3 DPG, decreased H+ conc, CO (increased affinity?)
- Right: reduced affinity, increased temp, increased 2-3 DPG, increased H+ conc
11
Q
Oxygen binding capacity of Hb is increased by?
A
- Decreased 2,3 DPG level in RBCs
- Decreased pCO2 level in blood (ex. resp alkalosis)
- Decreased temp of blood (hypothermia)
- Increased pH of the blood (alkalosis, metabolic or resp)
12
Q
Oxygen binding capacity of Hb is decreased by?
A
- Increased 2,3 DPG level in RBCs
- Increased pCO2 level in blood ( ex. resp acidosis)
- Increased temp of blood (hyperthermia)
- Decreased pH of blood (acidosis, respiratory or resp)
13
Q
What is oxygen saturation?
A
Percentage (proportion) of oxygenated Hb molecules compared to the whole amount of Hb molecules in one unit of blood
14
Q
Normal oxygen saturation values?
A
Arterial: 95-99%
Venous: 80-90%
15
Q
Causes of increased Hb conc?
A
- Usually associated with different types of relative (dehydration) or absolute polycytaemia
16
Q
Causes of decreased Hb conc?
A
- Usually associated with relative (hyperhydration) or absolute oligocytaemia
17
Q
Is Hb conc affected by age of animals?
A
- Usually not, except swine: young pigs have much lower -Hb conc than older ones
18
Q
Methaemoglobin?
A
- Hemoglobin containing Fe3+ –> unable to carry oxygen
- Small amount in blood, possible to reduce them to Hb by the methamoglobin-reductase enzyme
19
Q
Hemoglobin transports oxygen to the cells and then
A
it is used in the terminal oxidation phase of the metabolic process
20
Q
What factors can lead to increased methaemoglobin amount in blood?
A
- Severe oxidative damage tot he RBCs: ex. nitrites, free radicals, paracetamol, onion)
- Called Methaemoglobinaemia
21
Q
What species are especially sensitive to ox. damage?
A
- Hb molecules of cats, newborns and very young animals are sensitive to oxidative damage
22
Q
Methaemoglobinaemia, colour?
A
- Colour is dark brown and mucous membranes are deeply cyanotic
23
Q
Rough estimation formula for Hb conc?
A
PCV (1/) / 3 x 1000 = Hbg (g/L)
24
Q
RBC count methods?
A
- Burker chamber….
- Estimated RBC count; formula used if we suspect a normal average RBC volume
- RBC count measured by automatic cell counter
25
- Estimated RBC count; formula used if we suspect a normal average RBC volume
Ht L/L / 5 x 100 = RBC count x 10^12/L
26
- RBC count measured by automatic cell counter
- Based on electrical impendance change due to transmission of particles through an aperture -> electric flow -> size of RBC
- Shows RBC and thrombocytes
- X axis shows size, Y axis shows number counted
27
RBC size taken up by automatic cell counter
40-100fl (0-150)
28
Automatic cell counter, why should blood be warmed to 37 C before counting?
Because if the blood is cold-agglutinated the cells in the aggregrations will not be counted. Warm -> separates cells
29
Normal RBC count
4,5-8 x 10^12 /L
30
Tests to gain info about size, number, colour (the indices)
- Ht, PCV, RBC count, Hb concentration (objective info), blood smear if by good cytologist (can be very subjective)
- They are useful for humans, cats and dogs, but for horses and cattle the parameters of RBCs are varying too much, also within the individual
31
Mean corpusclar Haemoglobin (MHC) indicates?
MCH indicates average Hb content of RGCs
32
Mean corpusclar Haemoglobin (MHC) formula
Hb (g/L) / RBC count x 10^12/L = MHC (pg)
33
Mean corpusclar Haemoglobin (MHC), normal value?
12-30 pg
In young animals it can be increased 28-31 (MCV can also be increased)
Horse - 12-20
Ru - 8-17
Dog - 15-24
Cat - 13-17
34
Decreased MCH is called?
Hypochromasia
35
Increased MCH is called?
Hyperchromasia
36
Mean corpusclar volume (MCV) indicates?
MCV indicates the average size of the RBCs (macro, normo, microcytic)
37
Mean corpusclar volume (MCV) formula?
PCV / RBC count x 1000 = MCV (fl)
38
Mean corpusclar volume (MCV) normal values?
```
60-70 fl
Horse - 37-58
Ru - 42-52
Dog - 63-75
Cat - 40-53
```
39
MCV, size og RBC in species, age, and some breeds
- Great heterogenity in size
- Cats, horses -> smaller RBC
- Young RBC -> large
- Newborns -> large
- Adults -> smaller
- Japanese Akita -> smaller
- Poodles -> very large
40
Some causes of microcytosis:
- Chronic blood loss
- Iron, copper, pyridoxine (vi. B6) deficiency
- Portosystemic shunt
41
Some causes of macrocytosis:
- Mostly regenerative anemias
- Polycythaemia absoluta vera (erythroleukemia)
- Vit. B12, folic acid, cobalt deficiency
- Erythroleukemias
42
Mean corpusclar Haemoglobin concentration (MCHC) indicates?
MCHC indicates the average concentration of haemoglobin in erythrocytes (hB concentration)
(hyper, normo, hypochromic RBCs)
43
Mean corpusclar Haemoglobin concentration (MCHC) formula?
Hb (g/L) / PCV = MCH (pg) / MCV (fl) x1000 = MCHC (g/L)
44
Mean corpusclar Haemoglobin concentration (MCHC) normal values?
300-350 g/L (30-35%) - normochrom
Horse - 31-37 %
Ru - 30-36 %
Dog - 32-36 %
Cat - 30-36 %
45
When MCV and MHC are low (ex. chronic liver failure) MCHC can be normal, however there is?
Anemic state
46
Decreased MCHC- hypochromasia causes:
- Newborn animals
- Regenerative anemias
- Iron deficiency anemia
47
Increased MCHC- hypochromasia causes:
- Erythroleukemia (polycythaemia absoluta vera)
- Vitamin B12, folic acid, cobalt deficiency
- Immunohemolytic anemia (spherocytosis-presence of blood in spherocytes)
- Lead poisoning
- Splenectomy
48
Increased MCHC- hyperchromasia causes:
- Erythroleukemia (polycythaemia absoluta vera)
- Vitamin B12, folic acid, cobalt deficiency
- Immunohemolytic anemia (spherocytosis-presence of blood in spherocytes)
- Lead poisoning
- Splenectomy
49
Typical changes in derivated parameter:
| macrocytic, hypochromic; MCV-up, MCHC-down, (reticulocytes-up)
Regenerative anemias
50
Typical changes in derivated parameters: normocytic, normochromic; MCV-unchanged, MCHC-unchanged, MCH normal or decreased
Non regenerative anemias
51
Typical changes in derivated parameters: microcytic, hypochromic; MCV-down, MCHC-down, (decreased Hb synthesis)
Iron, copper, piridoxine deficiency anemias, liver failure, portosystemic shunt
52
Typical changes in derivated parameters: microcytic, normochromic; MCV-down, MCHC-unchanged
Normal for Japanese Akita
53
Typical changes in derivated parameters: macrocytic, normochromic; MCV-up, MCHC-unchanged, impaired DNA synthesis
FeLV infection, vitamin B12, CO or folic acid deficiency, erythroleukemia, poodle macrocytosis
54
Red Cell Distribution width, Platelet Distribution Width (PWD) are indices that gives us info about?
They give a number that is correlated with the range of the average size of the RBCs and platelets
55
Where is RDWs width on the graph?
It is the line between the two points (P1-P2) where the horizontal line crosses the two sidelines of the curve. We express it in the percentage of P1-P2 width compared to the distribution of all RBC (from 0-approx100 fl) as 100%
56
RDW dog and cat:
Dog 12-16%
Cat 14-18%
Normally the histogram is symmetric Gauss-curve with slight right shift
57
PDW dog and cat:
Dog 6-8%
Cat 7-12%
Normally the histogram is symmetric Gauss-curve with slight right shift
58
Short RDW means?
non regenerative processes
59
Large RDW means?
regenerative processes
60
What are reticulocytes
Young, but differentiated RBCs with basophil punctates stained by Brylliant-cresis blue stain. Blue punctates are rRNA remnants.
61
Reticulocytes containing big blue aggregates (aggregated forms) are?
Younger forms
62
Reticulocytes containing small punctates (punctated forms) are?
Older forms. In cats punctated forms are more normal.
63
No reitculocytes in what species?
Horses and ruminants. They exist only in bone marrow, not in the peripheral blood
64
Appearance of reticulocytes are a sign of?
regenerative function of the bone marrow
65
Does reticulocytes have the same functional properties as mature RBCs?
yes
66
Does reticulocytes have the same functional properties as mature RBCs?
yes, able to carry oxygen
67
Why do we count reticulocytes and not nucleated RBCs when differentiating between regenerative and non-regenerative anemias?
Because in case of a maturation arrest of RBCs, nucleated RBCs will never become reticulocytes or mature RBCs. Nucleated RBCs are too young to carry oxygen, reticulocytes are
68
What can cause maturation arrest of nucleated RBCs?
ex. vitamin B12 or folic acid deficiency, or FeLV
69
Regenerative anemias are ususally diseases with favourable prognosis, because?
Enough new RBCs are produced in the bone marrow to regenerate the anemia, to replace the lost RBCs and to reach normal RBC count quickly.
70
If the bone marrow function is normal, RBC production os ?
In positive correlation with the severity of anemia
71
In case of severe anemia more intensive RBC production (a more significant increase in reticulocyte count) is needed to?
consider this as a regenerative anemia, than in case of mild anemia
72
Reticulocyte staining
- Brylliant-cresil blue in physiological saline, then add Na-citrate in solution, mix.
- Vital staining: mix fresh EDTA blood and Brylliant-cresil in same proportion, (stain fresh/alive/vital/RBCs)
- Incubate in room temp, take up stain, prepare smear
73
Reticulocyte counting
- Count 100-1000 RBCs and take the percent of the reticulocytes
74
Normal reticulocyte count
2-3%
75
In order to say that the bone marrow is capable fro replacing the red blood cells in an organism which lost RBCs, we expect that the more severe anemia will cause?
increased reticulocytic %. The possible PCV changes we can expect a specific reticulocyte %. The expected values for a proper regenration can be read by table in lab
76
Corrected reticulocyte count (CRC) formula
CRC = reticulocyte % x RBC count
77
Normal reticulocyte count?
<0,06 x 10^12/L - without anemia
78
Corrected reticulocyte percentage (CRP) formula
CRP = Ht patient / Ht average (0,45 dog - 0,37 cat) x reticulocyte %
79
Normal corrected reticulocyte percentage (CRP)
<1-2% - without anemia
80
Increased reticulocyte count can be caused by diff types of regenerative anemias:
- acute blood loss (approx 3-5 days are needed for the bone marrow to increase the reticulocyte countin the blood)
- hamolytic anemia
- chronic blood loss
- some types of nutrient deficiency anemias
81
Osmotic resistance of RBCs is dependent on?
dependent on the pH of the plasma (normal is 7,4), the reagents , temperature, osmotic conc of plasma and the reagents (NaCl conc), RBC membrane status, regenerative status (reticulocytes are more resistant), HbF (fetal Hb) content of RBCs. (fetal RBCs containing HbF are more resistant)
82
What test can we perform to examine the RBC membrane function? (membrane integrity)
Osmotic resistance analysis
83
Normal causes of membrane damage
- Nephropathy (uremia)
- Specific membrane damage (immunohemolytic anemia)
- Increased physical damage (long term severe physical activity, ex. long distance running)
84
In general, osmotic resistance becomes decreased in case of?
- Chronic hemolytic anemia types, where there is extracellular hemolysis and the plasma color does not reflect to the hemolytic process
- > Chronic immunemediated hemolytic anemia is typical for this kind of RBC damage
- There are some rare hereditary genetical defects causing decreased osmotic resistance (therefore decreased lifespan) of RBCs: pyruvate kinase or glucose 6 phosphate dehydrogenase deficiency in dogs, methaemoglobin reductase deficiency in dogs and horses etc.
85
Method 1, osmotic resistance of RBCs
- Dilution line of NaCl from 0,3 % to 2,5%
- Drip blood samples into the saline test tubes, incubate in room temp for 10 min
- Centrifuge -> check upper layer for hemolysis
- Distilled water with pH around 7.4
86
Method 2, osmotic resistance of RBCs
- Hypotonic solution of NaCl
- Prepare three tubes; Tube 1 - physiological saline for blood sample of the sick animal, Tube 2 - hypotonic NaCl solution for blood sample of sick animal, Tube 3 - hypotonic NaCl for blood sample of control healthy animal
-> 5 drops of blood in each tube, incubate 10 min, centrifuge, check upper layer
Result: if the upper layer of tube 3 shows hemolysis, repeat analysis (analytical error)
Tube 1,2 - intravasal hemolytic crisis ex. babesiosis, immunehemolytic anemia
87
Erythrocyte morphology; correct blood smear analysis begins with?
- Using proper staining methods, ex. May-Grunwald, Giemsa, Diff quick ex.
- Smears must be prepared by using fresh samples
88
Erythrocyte morphology; correct blood smear analysis
- Staining method
- Fresh sample
- Check blood films on low then high magnification (200-1000x)
- Check gross signs: rouleau formation - coin arrangement, RBC aggregates, large cells (horse often), thrombocyte aggregates
89
Gross signs in smear: rouleau formation - coin arrangement, what species?
Horse often, dog, cat, swine sometimes, cattle rare
90
Intensity of staining RBCs; polychromasia, hyperchromasia
More intensive staining
| RNA, or nuclear remnants, more Hb - regenerative process
91
Intensity of staining RBCs; hypochromasia
Weak staining
| Decreased Hb content, iron or other nutrient deficiency
92
Size of RBCs; macrocytosis
many big cells
93
Size of RBCs; microcytosis
many small cells
94
Size of RBCs; anisocytosis
variable cell size, iron deficiency and regenerative process
95
Size of RBCs; poikylocytosis
variable size and colour
96
RBC types: young and nucleated RBCs (in order of maturation)
- Proerythroblast
- Basophil erythroblast (normocyte, normoblast)
- Polychromatophil erythroblast (normocyte, normoblast)
- Acidophil erythroblast (normocyte, normoblast)
97
RBC types: young but mature RBC without nucleus
Reticulocyte
98
RBC types: Appearance of young RBCs
Increased production (regenerative anemia), spleen or bone marrow disease, leukemia, extramedullar erythrocyte procuction, Pb toxicosis (with basophil punctates), hyperadrenocorticism
99
RBC types: reticulocyte appearance
Increased production (regenerative anemia) - chronic Fe deficiency anemia, hemolysis, acute blodd loss, chronic blood loss
100
RBC types: Spherocyte (spherical small polychromatophil RBC) appearance
sensitive RBC membrane, immunemediated hemolysis
101
RBC types: Stomatocyte (mouth shaped RBC) appearance
increased RBC production (regenerative anemia
102
RBC types: Acantocyte (spur cell - RBC with few long spikes) appearance
RBC membrane failure (lipid bilayer) - lipid metabolism disorder, hepatopathies
103
RBC types: Schysocyte (RBC fragment) appearance
traumatic or toxic damage (uremia, blood parasites, long term severe physical activity, DIC)
104
RBC types: Anulocyte (0-like RBC) appearance
iron deficiency anemia
105
RBC types: Codocyte (target cell, like a target) appearance
regenerative process
106
RBC types: Echynocyte (Burr cell, crenation, RBC with many small spikes) appearance
laboratory error, too quick drying of blood film, uremia, DIC
107
RBC types: Sickle cell appearance
RBC damage, Hb globin chain malformation in humans
108
Inclusion bodies in RBCs: Heinz body (NMB - new methylene blue stain); denatured Hb appearance
02 effect
Oxidative damage to RBCs (Cat: ex. methaemoglobinaemia)
GSH deficiency
109
Inclusion bodies in RBCs: Howell-Jolly body; nuclear remnants appearance
vitamin B12 deficiency
increased prod of red cells
splenectomy
110
Inclusion bodies in RBCs: Basophilic punctuates; nuclear remnants appearance
regenerative process
young RBCs of cat
physiological in Ru
lead poisoning
111
Inclusion bodies in RBCs: Hb inclusions appearance
Hb damage
increased RBC production
regenerative anemia
112
RBC parasites!
- Haemobartonella canis, felis, bovis
- Babesia spp., B. canis very common in Hu
- Ehrlichia canis, equi etc.
- Dirofilaria immitis, repens
- Anaplasma marginale, centrale, ovis
- Eperythrozoon wenyoni, ovis, suis, parvum
- Citauxzoon felis
- Theileria parva, mutans, annulata, hirci, ovis
- Trypanosoma cruzi, congolense, vivax, brucei, evans, suis, equiperdum
- Leishmania donovani
113
Other Laboratory measurements in connection with RBCs:
- Serum iron measurement
- Total iron binding capacity (TIBC)
-
114
Serum iron measurement, why and what
If we suspect iron deficiency, especially due to chronic blood loss, we can prove it by performing this test
115
Serum iron measurement; normal values
Normal SeFe (serum iron): 18-20 umol/L
116
Serum iron measurement, method
- Serum sample is used, fibrinogen of plasma can 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
117
Causes of high serum iron conc:
Iron toxicosis (overload)
118
Serum iron measurement should always be performed together with?
TIBC analysis (total iron binding capacity)
119
Total iron binding capacity (TIBC), what and why
This test gives information about the transferrin content.
120
TIBC normal values:
50-68 umol/L
121
Blood needed for serum iron measurement and TIBC?
Serum iron measurement: serum, fibrinogen of plasma may disturb measurement
TIBC:
122
TIBC normal values:
50-68 umol/L,
| normal values of formula: 20-55% (30%)
123
TIBC, method
Measure seFe, add Fe-solution to the plasma (all transferring molecules will be fully saturated), put absorbent to the solution, centrifuge -> absorbent binds to free Fe and goes to sediment. Use upper layer and check seFe again.
124
Causes of low TIBC:
- chronic inflammation (negatove acute phase proteins)
- chronic liver failure (decreased transferrin synthesis in liver
- neoplastic disease
125
Causes of high TIBC:
iron deficiency anemia (not severe: normal iron level + high TIBC, severe: low iron level + high TIBC)
126
TIBC: iron saturation formula
SeFe / TIBC x 100 = iron saturation
127
Laboratory findings in Hemolysis:
```
PCV - down
Reticulocytes - up (regenerative anemia)
Polychromasia, poikilocytosis
Leukocytosis, (neutrophilia)
Spherocytosis
Total bilirubin - up
Indirect bilirubin - up
Lactate dehydrogenase 1, 2 - up
Haptoglobin - down
RBC osmotic resistance - down
Jaundice
Hyperchromic stool
Urobilinogen and Hgb in urine - up
```
128
Ferritin, RIA method normal values:
12-300 ug/L
129
Transferring, method:
RIA, ELISA
130
RBC lifespan, method:
Cr51 method
131
Hgb electrophoresis, method
globin chain sequence analysis
132
Vitamin B12 measurements
RIA method
133
Erythropoietin, method
ELISA method, highly species specific , a few laboratories can measure it
134
TIBC =
serum iron level (saturated transfering) + free transferrin (not saturated)
