Lab 5 - RBC Flashcards
1
Q
What are the red blood parameters?
A
- Red blood cell count
- Haemoglobin measurement
- Derivative parameters
- Reticulocyte count
- Osmotic resistance
- Morphology of the RBC
2
Q
What are the methods to determine the red blood cell count?
A
- Burker-chamber methods
- Estimated RBC-count
- Automated cell counters
3
Q
What is the normal RBC count
A
4,5-8 * 10^12 (T/l)
4
Q
What is the characteristics of the Burker-Chamber
A
- Counting chamber
- Need for dilution
- Uses the grid to manually count the nr of cells in a certain area of known size.
- Count RBC in 20 rectangles or 80 small squares *10^10
- Poor accuracy: 10-25% error can be estimated
- Not effective
5
Q
What is the characteristics of the Impedance method
A
- Based on Coulter Principle
- Blood cells are poor conductors of electricity, but are suspended in electrically conductive electrolyte solution
- Two Chambers filled with conductive fluid, connected by a small aperture
- Electric current is passed flows through the aperture
- As cells passing through the aperture impeded flow of current and a voltage pulse is generated
- Pulse amplitude (height) is proportional to cell size
- Number of the pulses indicates the cell number
6
Q
What is this
A
A Normal Histogram
7
Q
What characterize Flow Cytometric Haematology analysers
A
- Combination of impedance technology and flow cytometry
- Cells are directed through a laser beam
- Laser light is scattered and several detectors measure scattered light at different angles
- Low angle or forward scatter correlates with cell size, the amount of high angle or scatter correlates with cell granularity or density
- Not only size but also cell complexity/density is used to distinguish cell types
(PLT vs RBC)
8
Q
HEAMOGLOBIN
Normal value
A
120-180 g/l
9
Q
HAEMOGLOBIN
Hb functions
A
- Binding
- Transport
- Release
10
Q
HAEMOGLOBIN
Iron
A
Fe2+ = Functionally active
F3+ = Oxidised form - Methaemoglobin: Severe oxidative damages= Nitrites, paracetamol, onion
11
Q
HAEMOGLOBIN measurement
Characterize the method
A
Measurement
- Spectrophotometric method (DRABKIN-METHOD)
- Whole blood
- Drabkin-reagent + KCN
- ORANGE END PRODUCT
- 540 nm
12
Q
What does this curve repressent
A
Oxygen dissocication curve
13
Q
Oxygen dissociation curve
What increases the oxygen-binding capacity of Hb?
A
Decreased:
2,3 DPG level in RBC
pCO2 level in the blood (in case of respiratory ALKAlosis)
Temperature of blood ( Hypothermia)
Increased
PH of blood
14
Q
Oxygen dissociation curve
What decreases the oxygen-binding capacity of Hb?
A
Decreased
- PH of the blood ( Acidosis, respiratory or metabolic)
Increased
- 2,3 DPG level in RBC
- pCO2 level in the blood (respiratory acidosis)
- Temperature of blood (Hyperthermia)
15
Q
Red bloodcell indices
DERIVATIVE PARAMETERS
What is the DERIVATIVE PARAMETERS and what is it usefull for
A
Useful informations about the average SIZE and COLOR of RBC in small animals, humans
- MCV = Mean corpuscular vulume
MCH = Mean corpuscular Haemoglobin
MCHC = Mean corpuscular Haemoglobin Concentration
RDW = Red cell distribution WIDTH
16
Q
Characterize MCV
A
Mean corpusculat volume = MCV
= Indicates the average size of the RBCs
Increased = Macrolytic
Normal = Normolytic
Decreased = Microlytic
Normal: Depends on the species and the breed too
CAT= 40-60fl
DOG= 60-80fl (Akita= 55-60fl, Poodles 75-80fl)
17
Q
Characterize RDW
A
RDW = Red cell distribution WIDTH
= Describes the variability in RBC SIZE
More sensitive indicator of altered red cell size than MCV
Describes the entire population of RBCs instead of one average value
18
Q
Characterize MCH
A
Mean Corpuscular Haemoglobin = MCH
= Indicates the average Hb content of RBCs
Normal value: 12-30 pg
In young animals (+MCV) can be increased 28-32 pg
Decreased MCH= HYPOCHOMASIA
Increased MCH = HYPERCHROMASIA
19
Q
Characterize MCHC
A
Mean Corpuscular Haemoglobin Concentration = MCHC
= Indicates the mean concentration of hemoglobin per red cell
Increased = Hyperchromic
Normal = Normochromic
Decreased = Hypochromic
Normal = 300-350 g/l = Normochrom
20
Q
Anemia
Typical changes in derivated parameters
MACROLYTIC, HYPOCHROMIC
A
Increased
MCV
Decreased
MCHC
Regenerative anaemia
21
Q
Anemia
Typical changes in derivated parameters
MACROCYTIC, NORMOCHROMIC
A
Increased
MCV
Normal ()
MCHC
Impaired DNA synthesis - VITAMIN B12 = Normal in poodles
22
Q
Anemia
Typical changes in derivated parameters
MICROLYTIC, HYPOCHROMIC
A
Decreased MCV and MCHC
Decreased Hb synthesis
- Iron and Copper deficiency
23
Q
Anemia
Typical changes in derivated parameters
MICROLYTIC, NORMOCHROMIC
A
Decreased MCV
Normal ()
MCHC
Opposite from macrolytic normochromic (except MCHC = same)
JAPANESE AKITA = Normal
24
Q
Reticulocyte Counts
Where is it released from
A
Released from the bone marrow into the circulation
25
26
Reticulocyte Counts
When does it reach its full maturation
After a further 24-48 hours (after release?)
27
Characteristics about reticulocytes
**LARGER** than mature red cells (incr MCV, RDW)
Contains **LESS Haemoglobins** than mature red cells (MCHC decr)
Contain numerous clumps of **ribosomal RNA remnants**
- **Polychromatophillic** (**Blueish-pink**) color on **Giemsa staining**
**Dark clumps** with **vital staining**
Flourescent stains detected by flow cytometric haematology analysers
Reticulum network is lost as the red cells matures
28
Reticulocytes
- Polychromatophillic color with GIEMSA STAINING?
**(Blueish-pink)** color on Giemsa staining
29
Reticulocytes with **vital staining**
**Dark clumps**
30
Due to MANUAL reticulocyte Count staining -
What types of staining?
Which stain is used?
What does it stain?
Vital staining = Stains living cells
Stain= Bryllant-Cresil blue stain
Stains reticulum network of aggregated ribosomes, mitochondria, and organelles present in immature reticulocyte cells
31
What type of analyzer is this
**Flow cytometric** haematology analyser
32
What happens 3-5 days after acute blood loss due to reticulocytes
The reticulocytes must be interpreted in the light of the **degree of anemia**
**For manual methods:**
Absolute reticulocyte count (\*10^9/L)
=
**Observed % reticulocytes** \* **RBC count** (\*10^12/l)**\*10**
33
Determining the severity of the anemia due to hematocrit
MILD severity of anaemia
Ht (HEMATOCRIT) = 0,30-0,37
34
Determining the severity of the anemia due to hematocrit
MODERATE severity of anaemia
Ht = 0,29-0,20
35
Determining the severity of the anemia due to hematocrit
SEVERE severity of anaemia
0,19-0,13
36
Determining the severity of the anemia due to hematocrit
**VERY SEVERE** severity of anaemia
\<0,13
37
Degree of regeneration due to reticulocyte count
Inadequate or no regeneration
\<80
38
Degree of regeneration due to reticulocyte count
Mild degree of regeneration
80-150
39
Degree of regeneration due to reticulocyte count
Moderate degree of regeneration
150-200
40
Degree of regeneration due to reticulocyte count
Marked degree of regeneration
\>200
41
Characteristics of Osmotic resistance of RBC
= Evaluating RBC membrane function
Dilution line from NaCl solution (saline) from 0,3% to 2,5%
Incubation
Centrifuge (3000 rpm)
**Check the supernatant plasma for hemolysis**
42
Causes of membrane damage (Osmotic resistance of RBC)
- Uraemia
**- Immun-Haemolytic anaemia**
**- Hereditiary defects:**
Pyruvate-Kinase, Glucose-6-phosphate dehydrogenase deficiency
43
RBC 1, 2 3 on the picture are?
1= Hypertonic
2= Isotonic
3= Hypotonic
44
Osmotic resistance
What is the result of this cause?
Control blood in hypotonic NaCl- solution
Laboratory analytical error
- do it again
45
Osmotic resistance
What is the result of this cause?
PATIENT blood in hypotonic NaCl- solution
1. Laboratory analytical error
2. Intravasal Hemolytic Crisis (BABESIOSIS)
3. Membrane defect of RBCs (Immun-haemolytic anemia)
46
Osmotic resistance
What is the result of this cause?
PATIENTs sample in PHYSIOLOGICAL SALINE
1. Laboratory analytical error
2. Intravasal Hemolytic Crisis (BABESIOSIS)
47
MORPHOLOGY of RBC
Preparation of blood smare - STEPS
PREPARATION of BLOOD SAMPLE
* Soon after taking blood sample
* Drop of blood placed at one end of slide
* Extend smare to 2/3 of the length of the slide (square edge if possible)
* AIR DRY fully before staining
* Blod film examination
* Feathered edge - First lower magnification (\*200) then \*1000)
* Examine smeare at high power in THIN area where the cells are evenly distributed in monolayers
* DO NOT EXAMINE cells at the feathered enge or in thick areas of the smare
48
Polychromatia
Arrow =
Arrow = Reticulocytes
- Polychromasia
- Anisocytosis
- Macrocytosis
49
Target cell/Codocyte
50
Spherocyte
51
Spherocyte
52
Echinocyte/Crenated red cell
53
Acanthocyte/Spur cell
54
Schistocytes
55
Heinz Bodies
56
Howell-Jolly body
57
Microfilaria
58
Babesia Canis
59
Due to Iron circulation
Name iron deficiencies
* **Chronic blood loss**
* **Innadequate intake (piglets, calves)**
* **Maldigestion, malabsorbtion**
Impaired gastric, duodenal, jejenual function (reduction, transport, absorption)
60
TIBC
Characteristics of the analysis
Total iron-binding capacity (TIBC)
**Indirect measurement of transferrin**
**TIBC= 50-68 mmol/l**
Method of measurement:
* **Measuring sample seFe**
* **Serum sample is flooded with excess iron, which binds to all the available binding sites on transferrin = full saturation**
* With **absorbent** unbound iron is **removed**
* SeFE in the remaining sample is evaluated again
* TIBC = Serum iron Level (saturated transferrin)+Free transferring(not saturated)
NORMAL value = 20-55% (33%)
61
TIBC
Distinguish microlytic hypochromic anaemia
Iron deficiency
Anaemia chronic inflamatory disease (transferrin negative acute phase protein)
62
Serum Iron
Chracteristics
* Sample= Serum
- fibrinogen disturb the measurement
* Spectrophotometric method
- Red coloured CHELATE (ironman)
* Normal SeFe (serum iron) = 18-20 mmol/l
* Serum iron measurement should always be performed together with TIBC analysi
63
Normal values in arterial blood:
95-99%
64
Normal values in venous blood:
80-90%
65
Hbg molecules containing oxidised iron (3+) are called:
Methaemoglobin
66
What does methaemoglobin-reductase enzyme do?
Reduces methaemoglobin to normal haemoglobin
67
What do we call increased methaemoglobin level in the blood? What color of the blood?
68
Methaemoglobinaemia. Dark brown
69
Hgb molecules of which animals are sensitive to oxidation damage?
Cats, newborn or very young animals
70
Causes of increased ↑ Hgb concentration:
Ususally associated with different types of relative (dehydration) or absolute polycytaemia
71
Causes of decreased ↓ Hgb concentration:
Usually associated with relative (hyperhydration) or absolute oligocytaemia (anaemia)
72
Hbg concentration is much lower in which species?
In young pigs
73
What does MCH indicate?
Average Hb content of the RBCs
74
What is hypochromasia?
Descreased Mean Corpuscular Haemoglobin (MCH)
75
What is hyperchromasia?
Increased Mean Corpuscular Haemoglobin (MCH)
76
Normal and young animals MCH:
12-30 and 28-32
77
What does Mean Corpuscular Volume (MCV) indicate?
78
They have smaller RBCs than other animals
Cats and horses
79
They have bigger RBCs
Newborns and young animals
80
Causes of microcytosis:
Chronic blood loss
Iron, copper, vit b6 deficiency
Portosystemic shunt
81
82
Causes of macrocytosis
Polycythaemia (erythrileukemia)
Vit b12, folic acid, cobalt deficiency
Erythroleukemia
83
What does Mean Corpuscular Haemoglobin Conentration (MCHC) indicate?
The avergae concentration of haemoglobin in erythrocytes (hb conc)
84
What is normal MCHC?
300-350 g/l (30-35%)
85
Decreased MCHC -
hypochromasia newborn animals
regenerative anaemias
iron deficiency anaemia
86
Increased MCHC -
hyperchromasia:
Erythroleukemia (polycythaemia absoluta vera) vitamin B12, folic acid, cobalt deficiency
immunhemolytic anaemia (spherocytosis)
lead poisoning
splenectomy
87
What does short RDW mean?
Non generative processes
88
What does large RDW mean?
Regenerative process
89
What are reticulocytes?
Young but differenciated RBCs with basophil punctates
90
What does it means if a reticulocyte have big blue aggregates?
They are younger than does containing small punctuates
91
Punctated forms are more common in which species?
Cat
92
Which species have reticulocytes only in the bone marrow and not in the peripheral blood?
Horses and ruminants
93
Apperance of reticulocytes is a sign of what?
The regenrative function of bone marrow
94
Why are nucleated RBCs not able to function as RBSs?
They are too young and therefore not able to carry oxygen
95
Why do we differentiate btw regenerative and non regenerative anemias?
Because nucleated RBCs will never become reticulocytes or mature RBCs, so nucleated RBCs are never counted
96
Why are regenerative anemia usually diseases with favourable prognosis?
Because enough new RBCs are produced in the bone marrow to regenerate the anaemia, to replace lost RBCs and reach normal RBC count quickly
97
Characteristic for vital staining:
```
Mix fresh (EDTA) blood and Brylliant-cresil stain in the same proportion Incubate at room temp for 2-3 h
Prepare a smear
```
98
Reticulocyte counting:
Count 100-1000 RBCs and take the percent of the reticulocytes 2-3% normal
99
Usually more mature RBCs are dead than young ones. Why?
Because mature RBCs are more sensitive to damage than young RBCs and reticulocytes
100
How many days are needed for the bone marrow to increase the reticulocyte count in the blood?
2-3 days
101
Increased reticulocyte count can be caused by different types of regenerative anaemias:
acute blood loss,
haemolytic anaemia,
chronic blood loss,
some types of nutrient deficiency anaemias
102
Osmotic resistance of RBCs is dependent on what?
pH of the plasma,
reagents,
temp,
osmotic concentration of plasma
103
Size of RBCs Macrocytosis:
many big cells
104
Size of RBCs Microcytosis:
many small cells
105
Size of RBCs
Anisocytosis:
variable cell size - iron deficiency and regenerative process
106
Size of RBCs Poikylocytosis:
variable size and colour
107
Reticulocyte appearance:
- increased production (regenerative anaemia)
- chronic Fe deficicency anaemia,
haemolysis,
acute blood loss,
chronic blood loss
108
Spherocyte (spherical small polychromatophil RBC) appearance:
Sensitive RBC membrane,
immune-mediated hemolysis
109
Stomatocyte (mouth-shaped RBC) Appearance:
increased RBC production (regenerative anaemia)
110
Acanthocyte (Spur cell - RBC with few long spikes) appearance:
- RBC membrane failure (lipid bilayer)
– lipid metabolism disorder,
- hepatopathies
111
Schysocyte (RBC fragment) appearance:
Traumatic or toxic damage (uremia, blood parasites, long term severe physical activity, DIC)
112
Anulocyte (0 - like RBC) appearance:
iron deficiency anaemia
113
Codocyte (Target cell, like a target) appearance:
regenerative process
114
Echynocyte (Burr cell, crenation, RBC with many small spikes) appearance:
laboratory error (too quick drying of blood film, uremia, DIC)
115
Sickle cell appearance:
RBC damage, Hb globin chain malformation in humans
116
Inclusion bodies in RBCs Heinz body (NMB - new methylene blue stain):
Denaturated Hgb appearance: O2 effect, oxidative damage to RBCs
(cat!, for example methemoglobinaemia!), GSH deficiency
117
Howell-Jolly body : nuclear membrane remnants appearance:
vitamin B12 deficiency,
increased production of red cells,
splenectomy
118
Basophilic punctuates :nuclear remnants appearance:
regenerative process,
young RBCs of cat,
physiological in ruminants,
lead poisoning
119
Hb inclusions appearance:
Hb damage,
increased RBC production,
regenerative anaemia
120
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 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
121
What test can we use if we suspect iron deficiency, especially due to chronic blood loss?
Serum iron measurment
122
What the normal SeFe?
18-20 micromol/l
123
What is the daily iron need of adult animals?
**1 mg**
124
Iron is in it’s Fe3+ form in the feed of animals which cannot be absorbed, so how will iron be absorbed?
Gastric juice makes Fe3+ free from complex molecules. Reduced to Fe2+ by ascorbic acid, cystein or glutathione in the duodenum. Ready to be absorbed
125
What is iron bound to in the small intestines?
Apoferritin and stored like this as ferritin
126
Iron in the plasma is oxidised to Fe3+ by what enzyme?
Coeruloplasmin enzymes
127
How is iron Fe3+ transported?
By transferrin
128
How many % of transferrin molecules carry iron?
30%
129
How many % of iron is reutilized for haemoglobin synthesis in the bone marrow?
**80-90%**
130
When iron metabolism is not closed but opened by chronic blood loss, what happens?
Iron storage become depleted and at the end iron deficiency anaemia will appear
131
What may disturb the measurment of serum iron?
Fibrinogen content of the plasma, so serum plasma is needed for the analysis
132
What happens when Fe2+ reacts with ferrosin?
It forms a red-colored chelate which can be measured photometrically
133
Causes of low serum iron concentration:
```
chronic blood loss
decreased intake (piglets, calves)
impaired gastric, duodenal, jejunal function (reduction, transport, absorption)
```
134
Causes of high serum iron concentration:
iron toxicosis (overload)
135
Serum iron measurement should always be performed together with:
TIBC analysis
136
What test gives information about the transferrin content?
Total iron binding capacity (TIBC)
137
Normal TIBC:
50-68 micromol/l
138
Causes low TIBC:
chronic inflammation (negative acute phase protein, see later)
chronic liver failure (decreased transferring synthesis in the liver) neoplastic disease
139
Causes of high TIBC:
iron deficiency anaemia:
not severe: normal iron level+high TIBC,
severe: low iron level+high TIBC
140
Determination of TIBC:
1. Measure seFe then,
2. add Fe-solution to the plasma (by this method all transferrin molecules will be fully saturated),
3. then put absorbent to the solution,
4. centrifuge the absorbent (this binds to free Fe and goes to the sediment),
5. Use the upper layer and check seFe again.
TIBC =serum iron level (saturated transferrin)+free transferrin (not saturated)
141
Laboratory Findings in Hemolysis
```
PCV,
reticulocytes increase (regenerative anemia) polychromasia,
```
poikilocytosis leukocytosis, (neutrophilia)
spherocytosis,
total bilirubin increase
indirect bilirubin decrease,
lactate dehydrogenase (LDH) I,II ,
haptoglobin decrease,
RBC osmotic resistance decrease,
jaundice
hyperchromic stool,
urobilinogen and Hgb in urine increase
142
What is this a example of
Normal Histogram
143
What is this a example of
Non regenerative anaemia
144
What is this a example of
Regenerative anaemia
145
What is this an example of
Microcytic anaemia
146
What is this a example of
Macrocytic anaemia
147
What is this a example of
**Thrombocytopenia in the recovery phase** (macrothrombocytosis), often found in **cats**
