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Lab D > Lab 5 - RBC > Flashcards

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
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2
Q

What are the methods to determine the red blood cell count?

A
  • Burker-chamber methods
  • Estimated RBC-count
  • Automated cell counters
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3
Q

What is the normal RBC count

A

4,5-8 * 10^12 (T/l)

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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
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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
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6
Q

What is this

A

A Normal Histogram

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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)

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8
Q

HEAMOGLOBIN

Normal value

A

120-180 g/l

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9
Q

HAEMOGLOBIN

Hb functions

A
  • Binding
  • Transport
  • Release
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10
Q

HAEMOGLOBIN

Iron

A

Fe2+ = Functionally active

F3+ = Oxidised form - Methaemoglobin: Severe oxidative damages= Nitrites, paracetamol, onion

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11
Q

HAEMOGLOBIN measurement

Characterize the method

A

Measurement

  • Spectrophotometric method (DRABKIN-METHOD)
  • Whole blood
  • Drabkin-reagent + KCN
  • ORANGE END PRODUCT
  • 540 nm
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12
Q

What does this curve repressent

A

Oxygen dissocication curve

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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

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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)
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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

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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)

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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

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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

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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

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20
Q

Anemia

Typical changes in derivated parameters

MACROLYTIC, HYPOCHROMIC

A

Increased

MCV

Decreased

MCHC

Regenerative anaemia

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21
Q

Anemia

Typical changes in derivated parameters

MACROCYTIC, NORMOCHROMIC

A

Increased

MCV

Normal ()

MCHC

Impaired DNA synthesis - VITAMIN B12 = Normal in poodles

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22
Q

Anemia

Typical changes in derivated parameters

MICROLYTIC, HYPOCHROMIC

A

Decreased MCV and MCHC

Decreased Hb synthesis

- Iron and Copper deficiency

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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

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24
Q

Reticulocyte Counts

Where is it released from

A

Released from the bone marrow into the circulation

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25
Q
A
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26
Q

Reticulocyte Counts

When does it reach its full maturation

A

After a further 24-48 hours (after release?)

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27
Q

Characteristics about reticulocytes

A

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

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28
Q

Reticulocytes

  • Polychromatophillic color with GIEMSA STAINING?
A

(Blueish-pink) color on Giemsa staining

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29
Q

Reticulocytes with vital staining

A

Dark clumps

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30
Q

Due to MANUAL reticulocyte Count staining -

What types of staining?

Which stain is used?

What does it stain?

A

Vital staining = Stains living cells

Stain= Bryllant-Cresil blue stain

Stains reticulum network of aggregated ribosomes, mitochondria, and organelles present in immature reticulocyte cells

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31
Q

What type of analyzer is this

A

Flow cytometric haematology analyser

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32
Q

What happens 3-5 days after acute blood loss due to reticulocytes

A

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

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33
Q

Determining the severity of the anemia due to hematocrit

MILD severity of anaemia

A

Ht (HEMATOCRIT) = 0,30-0,37

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34
Q

Determining the severity of the anemia due to hematocrit

MODERATE severity of anaemia

A

Ht = 0,29-0,20

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35
Q

Determining the severity of the anemia due to hematocrit

SEVERE severity of anaemia

A

0,19-0,13

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36
Q

Determining the severity of the anemia due to hematocrit

VERY SEVERE severity of anaemia

A

<0,13

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37
Q

Degree of regeneration due to reticulocyte count

Inadequate or no regeneration

A

<80

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38
Q

Degree of regeneration due to reticulocyte count

Mild degree of regeneration

A

80-150

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39
Q

Degree of regeneration due to reticulocyte count

Moderate degree of regeneration

A

150-200

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40
Q

Degree of regeneration due to reticulocyte count

Marked degree of regeneration

A

>200

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41
Q

Characteristics of Osmotic resistance of RBC

A

= 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

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42
Q

Causes of membrane damage (Osmotic resistance of RBC)

A
  • Uraemia

- Immun-Haemolytic anaemia

- Hereditiary defects:

Pyruvate-Kinase, Glucose-6-phosphate dehydrogenase deficiency

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43
Q

RBC 1, 2 3 on the picture are?

A

1= Hypertonic

2= Isotonic

3= Hypotonic

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44
Q

Osmotic resistance

What is the result of this cause?

Control blood in hypotonic NaCl- solution

A

Laboratory analytical error

  • do it again
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45
Q

Osmotic resistance

What is the result of this cause?

PATIENT blood in hypotonic NaCl- solution

A
  1. Laboratory analytical error
  2. Intravasal Hemolytic Crisis (BABESIOSIS)
  3. Membrane defect of RBCs (Immun-haemolytic anemia)
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46
Q

Osmotic resistance

What is the result of this cause?

PATIENTs sample in PHYSIOLOGICAL SALINE

A
  1. Laboratory analytical error
  2. Intravasal Hemolytic Crisis (BABESIOSIS)
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47
Q

MORPHOLOGY of RBC

Preparation of blood smare - STEPS

A

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
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48
Q

Polychromatia

Arrow =

A

Arrow = Reticulocytes

  • Polychromasia
  • Anisocytosis
  • Macrocytosis
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49
Q
A

Target cell/Codocyte

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50
Q
A

Spherocyte

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51
Q
A

Spherocyte

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52
Q
A

Echinocyte/Crenated red cell

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53
Q
A

Acanthocyte/Spur cell

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54
Q
A

Schistocytes

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55
Q
A

Heinz Bodies

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56
Q
A

Howell-Jolly body

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57
Q
A

Microfilaria

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58
Q
A

Babesia Canis

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59
Q

Due to Iron circulation

Name iron deficiencies

A
  • Chronic blood loss
  • Innadequate intake (piglets, calves)
  • Maldigestion, malabsorbtion

Impaired gastric, duodenal, jejenual function (reduction, transport, absorption)

60
Q

TIBC

Characteristics of the analysis

A

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
Q

TIBC

Distinguish microlytic hypochromic anaemia

A

Iron deficiency

Anaemia chronic inflamatory disease (transferrin negative acute phase protein)

62
Q

Serum Iron

Chracteristics

A
  • 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
Q

Normal values in arterial blood:

A

95-99%

64
Q

Normal values in venous blood:

A

80-90%

65
Q

Hbg molecules containing oxidised iron (3+) are called:

A

Methaemoglobin

66
Q

What does methaemoglobin-reductase enzyme do?

A

Reduces methaemoglobin to normal haemoglobin

67
Q

What do we call increased methaemoglobin level in the blood? What color of the blood?

A
68
Q

Methaemoglobinaemia. Dark brown

A
69
Q

Hgb molecules of which animals are sensitive to oxidation damage?

A

Cats, newborn or very young animals

70
Q

Causes of increased ↑ Hgb concentration:

A

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

71
Q

Causes of decreased ↓ Hgb concentration:

A

Usually associated with relative (hyperhydration) or absolute oligocytaemia (anaemia)

72
Q

Hbg concentration is much lower in which species?

A

In young pigs

73
Q

What does MCH indicate?

A

Average Hb content of the RBCs

74
Q

What is hypochromasia?

A

Descreased Mean Corpuscular Haemoglobin (MCH)

75
Q

What is hyperchromasia?

A

Increased Mean Corpuscular Haemoglobin (MCH)

76
Q

Normal and young animals MCH:

A

12-30 and 28-32

77
Q

What does Mean Corpuscular Volume (MCV) indicate?

A
78
Q

They have smaller RBCs than other animals

A

Cats and horses

79
Q

They have bigger RBCs

A

Newborns and young animals

80
Q

Causes of microcytosis:

A

Chronic blood loss

Iron, copper, vit b6 deficiency

Portosystemic shunt

81
Q
A
82
Q

Causes of macrocytosis

A

Polycythaemia (erythrileukemia)
Vit b12, folic acid, cobalt deficiency

Erythroleukemia

83
Q

What does Mean Corpuscular Haemoglobin Conentration (MCHC) indicate?

A

The avergae concentration of haemoglobin in erythrocytes (hb conc)

84
Q

What is normal MCHC?

A

300-350 g/l (30-35%)

85
Q

Decreased MCHC -

A

hypochromasia newborn animals
regenerative anaemias
iron deficiency anaemia

86
Q

Increased MCHC -

A

hyperchromasia:

Erythroleukemia (polycythaemia absoluta vera) vitamin B12, folic acid, cobalt deficiency

immunhemolytic anaemia (spherocytosis)
lead poisoning
splenectomy

87
Q

What does short RDW mean?

A

Non generative processes

88
Q

What does large RDW mean?

A

Regenerative process

89
Q

What are reticulocytes?

A

Young but differenciated RBCs with basophil punctates

90
Q

What does it means if a reticulocyte have big blue aggregates?

A

They are younger than does containing small punctuates

91
Q

Punctated forms are more common in which species?

A

Cat

92
Q

Which species have reticulocytes only in the bone marrow and not in the peripheral blood?

A

Horses and ruminants

93
Q

Apperance of reticulocytes is a sign of what?

A

The regenrative function of bone marrow

94
Q

Why are nucleated RBCs not able to function as RBSs?

A

They are too young and therefore not able to carry oxygen

95
Q

Why do we differentiate btw regenerative and non regenerative anemias?

A

Because nucleated RBCs will never become reticulocytes or mature RBCs, so nucleated RBCs are never counted

96
Q

Why are regenerative anemia usually diseases with favourable prognosis?

A

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
Q

Characteristic for vital staining:

A 
Mix fresh (EDTA) blood and Brylliant-cresil stain in the same proportion Incubate at room temp for 2-3 h
Prepare a smear
98
Q

Reticulocyte counting:

A

Count 100-1000 RBCs and take the percent of the reticulocytes 2-3% normal

99
Q

Usually more mature RBCs are dead than young ones. Why?

A

Because mature RBCs are more sensitive to damage than young RBCs and reticulocytes

100
Q

How many days are needed for the bone marrow to increase the reticulocyte count in the blood?

A

2-3 days

101
Q

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

A

acute blood loss,

haemolytic anaemia,

chronic blood loss,

some types of nutrient deficiency anaemias

102
Q

Osmotic resistance of RBCs is dependent on what?

A

pH of the plasma,

reagents,

temp,

osmotic concentration of plasma

103
Q

Size of RBCs Macrocytosis:

A

many big cells

104
Q

Size of RBCs Microcytosis:

A

many small cells

105
Q

Size of RBCs
Anisocytosis:

A

variable cell size - iron deficiency and regenerative process

106
Q

Size of RBCs Poikylocytosis:

A

variable size and colour

107
Q

Reticulocyte appearance:

A
  • increased production (regenerative anaemia)
  • chronic Fe deficicency anaemia,

haemolysis,

acute blood loss,

chronic blood loss

108
Q

Spherocyte (spherical small polychromatophil RBC) appearance:

A

Sensitive RBC membrane,

immune-mediated hemolysis

109
Q

Stomatocyte (mouth-shaped RBC) Appearance:

A

increased RBC production (regenerative anaemia)

110
Q

Acanthocyte (Spur cell - RBC with few long spikes) appearance:

A
  • RBC membrane failure (lipid bilayer)

– lipid metabolism disorder,

  • hepatopathies
111
Q

Schysocyte (RBC fragment) appearance:

A

Traumatic or toxic damage (uremia, blood parasites, long term severe physical activity, DIC)

112
Q

Anulocyte (0 - like RBC) appearance:

A

iron deficiency anaemia

113
Q

Codocyte (Target cell, like a target) appearance:

A

regenerative process

114
Q

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

A

laboratory error (too quick drying of blood film, uremia, DIC)

115
Q

Sickle cell appearance:

A

RBC damage, Hb globin chain malformation in humans

116
Q

Inclusion bodies in RBCs Heinz body (NMB - new methylene blue stain):

A

Denaturated Hgb appearance: O2 effect, oxidative damage to RBCs

(cat!, for example methemoglobinaemia!), GSH deficiency

117
Q

Howell-Jolly body : nuclear membrane remnants appearance:

A

vitamin B12 deficiency,

increased production of red cells,

splenectomy

118
Q

Basophilic punctuates :nuclear remnants appearance:

A

regenerative process,

young RBCs of cat,

physiological in ruminants,

lead poisoning

119
Q

Hb inclusions appearance:

A

Hb damage,

increased RBC production,

regenerative anaemia

120
Q

RBC parasites:

A
  • 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
Q

What test can we use if we suspect iron deficiency, especially due to chronic blood loss?

A

Serum iron measurment

122
Q

What the normal SeFe?

A

18-20 micromol/l

123
Q

What is the daily iron need of adult animals?

A

1 mg

124
Q

Iron is in it’s Fe3+ form in the feed of animals which cannot be absorbed, so how will iron be absorbed?

A

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
Q

What is iron bound to in the small intestines?

A

Apoferritin and stored like this as ferritin

126
Q

Iron in the plasma is oxidised to Fe3+ by what enzyme?

A

Coeruloplasmin enzymes

127
Q

How is iron Fe3+ transported?

A

By transferrin

128
Q

How many % of transferrin molecules carry iron?

A

30%

129
Q

How many % of iron is reutilized for haemoglobin synthesis in the bone marrow?

A

80-90%

130
Q

When iron metabolism is not closed but opened by chronic blood loss, what happens?

A

Iron storage become depleted and at the end iron deficiency anaemia will appear

131
Q

What may disturb the measurment of serum iron?

A

Fibrinogen content of the plasma, so serum plasma is needed for the analysis

132
Q

What happens when Fe2+ reacts with ferrosin?

A

It forms a red-colored chelate which can be measured photometrically

133
Q

Causes of low serum iron concentration:

A 
chronic blood loss
decreased intake (piglets, calves)
impaired gastric, duodenal, jejunal function (reduction, transport, absorption)
134
Q

Causes of high serum iron concentration:

A

iron toxicosis (overload)

135
Q

Serum iron measurement should always be performed together with:

A

TIBC analysis

136
Q

What test gives information about the transferrin content?

A

Total iron binding capacity (TIBC)

137
Q

Normal TIBC:

A

50-68 micromol/l

138
Q

Causes low TIBC:

A

chronic inflammation (negative acute phase protein, see later)

chronic liver failure (decreased transferring synthesis in the liver) neoplastic disease

139
Q

Causes of high TIBC:

A

iron deficiency anaemia:
not severe: normal iron level+high TIBC,

severe: low iron level+high TIBC

140
Q

Determination of TIBC:

A
  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
Q

Laboratory Findings in Hemolysis

A 
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
Q

What is this a example of

A

Normal Histogram

143
Q

What is this a example of

A

Non regenerative anaemia

144
Q

What is this a example of

A

Regenerative anaemia

145
Q

What is this an example of

A

Microcytic anaemia

146
Q

What is this a example of

A

Macrocytic anaemia

147
Q

What is this a example of

A

Thrombocytopenia in the recovery phase (macrothrombocytosis), often found in cats

Lab D (11 decks)

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