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Lab diagnostics > 5.Examination of RBC > Flashcards

5.Examination of RBC Flashcards

1
Q

What is essential for the body?

A

The ability of oxygen transport, binding, releasing and the metabolism of RBC.

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2
Q

Which parameters have to be measured in connection with RBC?

A

RBC count, haemoglobin concentration and function. We use these to evaluate polycythemias and anemias caused by different external and internal causative agents.

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3
Q

What is polycythemia?

A

Polycythemia (also known as polycythaemia or polyglobulia) = the hematocrit (the volume percentage of red blood cells in the blood) is elevated. It can be due to an increase in the number of red blood cells(“absolute polycythemia”) or to a decrease in the volume of plasma (“relative polycythemia”). Polycythemia is sometimes called erythrocytosis, but the terms are not synonymous, because polycythemia refers to any increase in red blood cells, whereas erythrocytosis only refers to a documented increase of red cell mass.

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4
Q

What is the goal of the spectrophotometric method?

A

The goal is to find the haemoglobin concentration from the RBCs.

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5
Q

How do we perform the spectrophotometric method?

A

We put 20 microliter of whole blood into 5ml reagent. The Potassium ferricianid will hemolyse the RBCs and form fe3+ from fe2+ in the hemoblobin molecule, then it will further be oxidized by KCN ti cianidmethaemoglobin. We measure the amount of orange coloured end product by sprectrophotometry.

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6
Q

what is the normal range of haemoglobin in the blood?

A

18-20 mmol/l or 12-18 g/dl

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7
Q

What are measured in the spectrophotometric method?

A

The sum of Hgb molecules from haemolyzed RBCs and the very small amount of free Hgb content in the plasma, which is usually bound to carrier protein. (haptoglobin).

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8
Q

How can the binding capacity of Hgb increase?

A

When the oxygen binding capacity of Hgb increases we say that the affinity is increased. Left shift. This can happen if the 2,3DPG level in RBCs decreases, the pCO2 level in the blood decreases (respiratory alkalosis), if the pH of the blood increases or the temperature decreases of the blood (hypothermia).

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9
Q

How can the O2 binding affinity of Hgb decrease?

A

When the oxygen binding capacity of Hgb decreases we say that the affinity is decreased. The oxygen dissociation curve shifts to the right. This can happen if the 2,3DPG level in RBCs increases, the pCO2 level in the blood increases (respiratory acidosis), if the pH of the blood decreases (acidosis, respiratory or metabolic) or the temperature increases of the blood (hyperthermia).

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10
Q

What is oxygen saturation and what is the normal values?

A

SAT % = the percentage or proportion of oxygenated Hgb molecules compared to the whole amount of Hgb molecules in one unit of blood. The normal values in arterial blood is 95-99%, in venous blood 80-90%.

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11
Q

What happens when the iron is in the Fe2+ form in the Hgb molecules?

A

This if functionally active Hgb. RBCs with these molecules are able to take up oxygen molecules in the lungs, carry them and deliver them to the cells where they are used in the terminal oxidation phase of the metabolic process.

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12
Q

What are methaemoglobin molecules and what do they do?

A

Hgb molecules which contain oxidized iron (fe3+). They are unable to carry oxygen. They can be redused by the methaemoglobin-reductase enzyme.

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13
Q

What can happen if there is a severe oxidative damage to RBCs? and what can cause this?

A

It can lead to increased methaemoglobin level in the blood which is clled methaemoglobinaemia. (chocolate blood og blue mucous). Can be caused by nitrites, free radicals, paracetamol, onion. Hgb molecules of cats, newborn or very young animals of any other species are very sensitive to oxidative damage.

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14
Q

How do we estimate roughly the Hgb conc?

A

PCV (l/l) 3 x 1000 = Hgb (g/l)

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15
Q

what can cause the Hgb conc to increase?

A

-Usually associated with different types of relative (dehydration, decrease in the volume of plasma) or absolute polycythemia ( increase in the number of red blood cells)

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16
Q

what can cause the Hgb conc to decrease?

A

-Usually associated with relative (hyperhydration, increase in the volume of plasma) or absolute oligocytaemia (anemia) (deficiency in the total number of red blood cells present in the body)

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17
Q

How do we do the burker-chamber method:

A

Put 50microliter (0.05ml) EDTA blood smple in 9.95ml physiological saline/0.9%NaCl, mix. Put one drop of this solution onto haemocytometer and count cells in 20 sqaures. Divid the number by 100 and thats gives the RBC count in tera. Poor accuracy (10-25%)

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18
Q

How do we estimate RBC-count?

A

If we suspect a normal average RBC volume: Htl/l / 5 x100 = rbc count in tera

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19
Q

How do we use an automatic cell counter?

A

Impedance method is based on the elctrical impedance change due to the transmission of particles through an aperture. The RBCs are impeding the electrical flow. The impedance is correlated with the size of the RBC.

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20
Q

What is the normal RBC count?

A

4.5-8 tera

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21
Q

what do we have to measure to get objective info about the average size and colour of RBCs?

A

Ht or PCV (packed cell volume), RBC count and haemoglobin concentration.

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22
Q

How do we calculate MCH?

A

(mean corpuscular haemoglobin) MCH indicates average Hb content of RBCs. Hgb (g/L) / RBC tera = MCH (pg =picogram)

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23
Q

What is the normal MCH?

A

normal: 12-30 pg, in young animals it can be increased 28-32 pg

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24
Q

what happens if MCH are increased or decreased?

A

Increased= hyperchromasia, decrease = hypochromasia.

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25
Q

MCH values in horse, ruminants, dogs and cats:

A

horse 12-20, ru 8-17, dog 15-24, cat 13-17 pg

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26
Q

Explain mean corpuscular volume:

A

Indicates the average size of the RBCs. Macro, micro or normocytic. We calculate it with : PCV/RBC count tera = MCV (fl,femto litre)

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27
Q

Normal MCV calues of horse,ru,dog and cat?

A

horse 37-58 fl ,ru 42-52 fl,dog 63-75 fl and cat 40-53fl, Normal 60-70fl

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28
Q

Are there natural differences in RBCs?

A

yes, cats and horses have smaller RBCs, young RBCs are bigger, New born animals have largers, adults have smallers. Akita has smaller 55-65fl, poodles have very large (75-80fl)

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29
Q

Causes of microcytosis:

A

-Chronic blood loss -Iron, copper, B6 deficiency, -Portosystemic shunt,

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30
Q

Causes of macrocytosis: (mostly regenerative anaemias)

A

-Polycythaemia absoluta vera, -vit B12, folic acid or cobalt deficiency -Erythroleukemia

31
Q

Explain the mean corpuscluar Haemoglobin concentration:

A

MCHC indicates the average conc of haemoglobin in erythrocytes. (hyper,hypo or normochromic) Calculate: Hgb (g/l) / PCV = MCH(pg) / MCV (fl) x 1000 = MCHC (g/l)

32
Q

Normal MCHC:

A

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

33
Q

Normal MCHC for horse, ru, dog and cat: (%)

A

horse 31-37%, ru 30-36%, dog 32-36% and cat 30-36%:

34
Q

Decreased MCHC: hypochromasia:

A

newborn animals, regenerative anaemias, iron deficiency anaemia.

35
Q

Increased MCHC - hyperchromasia

A

-Erythroleukemia, vit B12 folic acid or cobald deficiency, immunohemolytic anaemia, lead poisoning, splenectomy.

36
Q

what are the typical changes in case of : Regenerative anemias

A

Macrocytic, hypochromic, MCV increased, MCHC decreased, (reticulocytes increase)

37
Q

what are the typical changes in case of : non regenerative anaemias

A

Normocytic, normochromic, MCV normo, MCHC normo, normal or decreased MCH

38
Q

what are the typical changes in case of : iron,copper, piridoxine deficiency anaemias, liver failure or portosystemic shunt?

A

Microcytic, hypochromic, MCV decrease, MCHC decreased, decreased Hb synthesis.

39
Q

what are the typical changes in case of : a Japanese Akita

A

Microcytic, normochromic, MCV decrease, MCHC normo

40
Q

what are the typical changes in case of : FeL V infection, vit B12, Co or folic acid deficiency, erythroleukemia, poodle, macrosytosis

A

Macrocytic, normochromic, MCV increase, MCHC normo, imparied DNA synthesis.

41
Q

What is RDW and PDW?

A

Red cell distribution width and platlet distribution width. These indices give a number that is correlated with the range of the average size of the RBCs and platlets.

42
Q

RDW dog and cat:

A

dog 12-16%, cat 14-18%

43
Q

PDW dog and cat:

A

dog 6-8&, cat 7-12%

44
Q

What does the change in RDW mean?

A

short RDW means non regenerative processes. Large RDW means regenerative process.

45
Q

What are reticulocytes?

A

Young, but differenciated RBCs with basophil punctates stained by brylliant-cresil blue stain are reticulocytes. The blue punctates are rRBA remnants. Reticulocytes containing big blue aggregates are younger. Punctated are more common in cats. Reticulocytes are not in the peripheral blood of horses and ruminants.

46
Q

If we see appearances of reticulocytes what is that a sign of?

A

sign of regenerative function of bone marrow.

47
Q

What are the function of reticulocytes?

A

Reticulocytes have the same functional properties as mature RBCs, so they are able to carry oxygen. Young RBCs cannot do this.

48
Q

What is maturation arrest?

A

Maturation arrest can stop the development of the young nucleated RBCs, they will never become reticulocytes or mature RBCs. Thats why we differentiate between regenerative and non-regenerative anaemias by counting reticulocytes and not nucleated RBCs.

49
Q

Staining of reticulocyte count:

A

Brylliant-cresit stain; 0.04g stain in 8ml phys.sal. mix, then put 2ml Na-citrate (3.8%)into it, mix. -Mix EDTA blood and brylliant-cresil stain in the same proportion. Room temp for 2-3 hours. Count 100-1000 RBCs and take the percent of the reticulocytes. Normal: 2-3%

50
Q

What do we expect when PCV changes?

A

We expect a specific reticulocyte %, severe anaemia will cause an increase of reticulocytes. Mature RBCs are more sensitive to changes.

51
Q

Corrected reticulocyte count: CRC

A

CRC= reticulocyte% x RBC count , normal: under 0.06 tera (without anaemia)

52
Q

Corrected reticulocyte percentage (CRP)

A

CRP= ht patient / ht average (0.45 dog , cat 0.37) x reticulocyte %, the normal: under 1-2% (without anaemia)

53
Q

what can increased reticulocyte count be caused by?

A

Different types of regerenerative 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.

54
Q

Osmotic resistance of RBCs are depended on:

A

pH of plasma, reagents, temp, osmotic conc of plasma and reagents, RBCs membrane status, regenerative status, the HbF (haemoglobin fetal) content of the RBCs (more = more resistant).

55
Q

Why do we perform the osmotic resistance analysis?

A

Examine RBC membrane function, whether the memebranes are damaged due to nephropathy (uremia), specific memebrane damage, increased physical damage.

56
Q

When does the osmotic resistance decrease?

A

in case of chronic haemolytic anaemia types, where there is extravascular hemolysis, and the plasma color does not reflect to the haemolytic process. Chronic immune mediated haemolytic anaemia is typical for this kind of RBC damage. Also some hereditary genetical defects: pyruvate-kinase or glucose 6P dehydrogenase deficiency in dogs or methaemoglobin-reductase deficiency in dogs ans horses.

57
Q

Method 1 for testing osmotic resistance:

A

Make a dilution line form NaCl from 0.3% to 2,5%, drip blood into test tubes in room temp for 10 min. Centrifuge, check upper layer for hemolysis.

58
Q

Method 2 for testing osmotic resistance:

A

Prepare hypotonic solution from NaCl: add 2ml water to 3ml (0.9%) phys.sal. for dogs(0.54%NaCl solution), 1ml to 4ml if cat (0.72%NaCl solution) Prepare 3 tubes: phys,sal, hypertonic, hypotonic. Sick blood in 1 and 2, healthy blood in 3. Room temp 10min then centrifuge, check upper layer for hemolysis.

59
Q

Size of RBCs

A

Macrocytosis: many big cells, Microcytosis: many small cells, Anisocytosis: variable cell size - iron deficiency and regenerative process, Poikylocytosis: variable size and color.

60
Q

Intensity of staining of RBCs:

A

-Polychromasia, hyperchromasia: more intensive staining, RNA or nuclear remnants, more Hb - regenerative process. -Hypochromasia: weak staining, decreased Hb-content - iron, or other nutrient deficiency.

61
Q

RBC types:

A
  • Young and nucleated
  • Young but mature
  • reticulocyte
  • Spherocyte
  • stomatocyte
  • acanthocyte
  • schysocyte
  • Anulocyte
  • Codocyte
  • Echynocyte -

Sickle cell

62
Q

Inclusion bodies in RBCs:

A
  • Heinz body
  • Howell-Jolly body
  • Basophilic punctuates
  • Hb inclusions
63
Q

RBCs parasites:

A
  • Haemobartonella canis, felis,bovis
  • Babesia spp.
  • Ehrlich canis, equi
  • Dirofilaria immitis
  • Anaplasma marginale, centrale, ovis
  • Eperythrozoon wenyoni,ovis,suis,parvum
  • Citauxzoon felis
  • Theileria parva,mutans,annulate,hirci,ovis
  • Trypanasoma cruzi, congolense
  • Leishmania donovani
64
Q

Serum iron measurment:

  1. Goals:
  2. normal value
A
  1. Goal: if we suspect iron deficiency, especially due to chronic blood loss, we can prove it by performing this test.
  2. Normal SeFe (serum iron): 18-20 micromol/l
65
Q

Method of serum iron measurments:

A

-Serum sample are needed for this analysis, because fribrinogen 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 which can be measured photometrically.

66
Q

What are the causes of low serum iron concentration:

A
  • Chronic blood loss
  • Decreased intake (piglets,calves)
  • impaired gastric, duodenal, jejunal function (reduction,transport,absorption)
67
Q

Causes of high serum iron conc:

A

Iron toxicosis (overload)

68
Q

what is TIBC?

A

It is a test that gives the information about the transferrin content. Total iron binding capacity: 50-68 micromol/L

69
Q

Determination of TIBC:

A

Measure seFe then ass Fe-solution to the plasma. (transferrin 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 sat)

70
Q

what can sause low TIBC?

A
  • Chronic inflam.
  • chronic liver failure
  • neoplastic disease
71
Q

What can cause high TIBC?

A

-Iron deficiency anaemia (not severe: normal iron level + high TIBBC, severe loe iron level + high TIBC)

72
Q

Iron saturation:

A

SeFe / TIBC x 100 = iron saturation

73
Q

Normal value of TIBC:

A

20-55 % (30%)

Lab diagnostics (6 decks)

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