EXAM Flashcards

Question

4. Importance, methods of acid-base balance evaluation - evaluate the given acid base blood test results: Respiratory alkalosis

Answer 1

- Increased loss of CO2: hyperventilation - Excitation - Forced ventilation (anaesthesia) - Epileptiform seizures - Fever, hyperthermia - Interstitial lung disease - Effects: hyperoxia, increased pCO2 : pO2 ratio -> may lead to apnoea. Increased elimination of HCO3- by the kidneys. - Treatment: mild sedative drugs (hyperexcitation), increase CO2 by closing nostrils and nose, paper bag over nose until normal breathing.

Answer 2

-Indication: assess effectiveness of gas exchange (ventilation) (eg. during dyspnoea or anasth.) -Sample: arterial for respiratory (venous gives only gross changes + how much O2 consumed) o Need anticoag blood, closed Astrup sample o Ca-equilibrated Li-heparinised plasma o Stored with no air, cause CO2 evaporate and contaminate (false incr pO2) o Within 15 min or stored on ice -Measure pO2 and pCO2 with ion selective electrode, 37 degrees -pCO2 and pO2 most important respiratory parameters o Determine normo-/hypo-/hyperventilation o Dissociation of gases depend on temperature of patient

Answer 3

-partial arterial pressure of CO2 - 40mmHg (35-45) -Indicates the ability of alveolar gas exchange to remove the CO2. Norm pvCO2 is higher than paCO2. -Increased: respiratory acidosis -Decreased: respiratory alkalosis -When combined with other parameters, indicate compensation mechanism of lungs: o Decreased respiration = hypercapnia o Increased respiration = hypocapnia

Answer 4

-partial pressure of oxygen -paO2: ability of lungs to oxygenate blood – Normal: 88-118 mmHg (saturation 97-100%) o Under 40-50mmHg: cyanosis -pvO2: not used to assess adequacy of oxygenation -FiO2: fractional inspired oxygen conc (21% room)

Answer 5

Increased respiration -> Hyperventilation (resp alkalosis) -paCO2 < 35mmHg. Hyperoxaemia usually together w. incr SAT. Causes: -Iatrogen: forced ventilation during anaest -seizures, epilepsy -excitation (mild/extreme eg. shock) -compensate of severe acidosis (kussmaul breathing)

Answer 6

Decreased respiration -> Hypoventilation (resp acidosis) -paCO2 > 45mmHg. Hypoxaemia +/- (depends on degree of hypercapnia and FiO2) Causes: -Upper airway obstruction -pleural effusion -drugs or disorders affecting central control of resp (eg. gen. anasth) -neuromuscular disease affecting resp system -muscle weakness -overcompensation of metab. alkalosis -Effects: Dyspnoea, cyanosis -Treatment: assisted ventilation, diuretics, mild anxiolytic/sedating -Ventilation- perfusion mismatch (V/Q): o Normal ventilation, inadequate perfusion: insufficient blood to alveoli for O2 o Inadequate ventilation, normal perfusion: not enough O2 o Patient with 100% O2 can have hypercapnia

Answer 7

1. ABE - Actual base excess (or demand/residue): metabolic parameter - Indicates amount of acid/base required to equilibrate blood to pH 7.4 (pCO2 is stabilized at 40mmHg on 37 degrees). - Ranges between – 3 (metabolic acidosis), + 3 (alkalosis) -> +/- 3.5mmol/l 2. SBE – Standard base excess (in vivo, base demand): residue in whole EC space, metabolic parameter. +/- 3 mmol/l

Answer 8

Spectrophotometric Method (Drabkin-method) 1. Put blood sample into reagent with K3(Fe(SCN)6) and KCN 2. Hemolyses RBC and Fe2+ -> Fe3+ in Hb molecule 3. Cyanide of KCN will bind to methHb and form cyano-methHb (irreversible) orange compound 4. Mix and measure end product by spectrophotometer at 540nm 5. Use standard solutions or curve • Esample/Estandard x Standard conc = result • The measured Hg conc is the sum of hemolysed RBC and small amount of free Hb content of plasma – usually bound to haptoglobulin (carrier protein). Therefore no notable increase in Hb if intravascular hemolysis! • Normal: 18 – 20 mmol/l, or 12 – 18g/dl

Answer 9

1. Oxygen dissociation curve -Describes how strongly O2 is bound to Hg -Left shift: high affinity, not let go of oxygen. o Oxygen binding capacity of Hgb is incr by: decr 2.3 DPG, decr pCO2 (eg. resp alkalosis), decr temp (hypothermia) and incr pH (met/resp alkalosis) -Right shift: reduced affinity. o Oxygen binding capacity of Hgb is decr by: incr 2.3 DPG, incr pCO2 (eg. resp acidosis), incr temp (hyperthermia) and decr pH (met/resp acidosis) 2. Factors influencing oxygen dissociation curve: • 2,3-DPG: Anionic organophosphate, created in RBCs during glycolysis o Important in adaptive mechanism because it incr for several conditions in absence of O2 -> enhances ability of RBC to release O2 near tissues that needs it the most. • pCO2 level in blood • pH of blood • temperature: any change causes decr in saturation o If give oxygen mask – increase o2 o Never give oxygen mask to hypothermic animal, because oxygen binds to Hb, so adding oxygen would make situation worse.

Answer 10

•Proportion of oxygenated Hb molecules compared to whole amount of Hb in one-unit blood o Normal: arterial = 95-99%, venous = 8-90% o When Fe2+ in Hb, it is able to take up O2 molecules, carry and deliver to cells. o MethHb: have Fe3+ form, unable to carry oxygen. * Constantly small conc of MethHb in blood, but reduced to normal Hg by methHb-reductase. o Methaemoglobinaemia: Severe oxidative damage to RBC (nitrites, free radicals, paracetamol, onion), can also be inherited -> enzyme dysfunction * Leads to increased methHb level • Dark brown colour (chocolate) • Mucous membranes deep cyanotic • Hb of cats/newborn very sensitive to oxidative damage o Rough estimation of Hb conc: if normal mean Hgb conc of RBCs = MCHC -> PCV (l/l)/3 x 1000 = Hgb (g/l)

Answer 11

-See topic 1 with PCV for poly and oligocythaemia • Increased Hgb concentration o Causes: types of relative (dehydration) or absolute polycythaemia • Decreased Hgb concentration o Causes: relative (hyperhydration) or absolute oligocythaemia (anaemia)

Answer 12

1. Burker-chamber method: a) Dilute anticoag (EDTA) hemogenised blood (200x dilution of whole blood w. saline) b) Put one drop of solution onto haemocytometer (Burker-chamber) and count cells in 20 rectangles or 80 small squares c) The nr of counted RBCs divided by 100 = RBC count in tera /10^12/ per litre (T/l) • Accuracy is poor: 10-25% error can be estimated • Note: volume of the drop spread on the whole surface of the network in the Burker chamber is 1 microlitre ! • Placement of drop on Burker chamber: on top under the glass slide 2. Estimated RBC-count: if suspect a normal avg RBC vol (MCV): (Ht/5) x 100 = RBC x 1012/l 3. RBC count measured by automatic cell counter • Counting RBC using electric impedance of particles which correlates with size • RBCs detected by the counter if size is 40-100 fl • Histogram: shows distribution of different sized cells (bw. 0-150fl), RBCs and thrombocytes • Axis ‘x’ shows the size (fl), axis ‘y’ shows nr of the counted cell particles • If forming aggregates (IHA): not counted as RBCs o When suspect cold agglutination: warm blood samples to 37 before counting to separate aggregated RBCs. 4. Flow cytometry can also be used for RBC count. (see topic WBC) Normal RBC count: 4.5-8 x 1012/lT/l

Answer 13

1. Normovolaemic Polycythaemia: Incr cell nr, norm vol -False: long sample storage with EDTA (corpuscular volume of RBCs increases) -Physio: species, breed, characteristics, hypercapnia, high altitudes, tumors, incr EPO -Patho = Absolute polyc. (incr RBC prod) o Primary: NON EPO effect -> without incr EPO * Polycythaemia absolute vera (eg. BM neoplasia-chronic leukaemia of RBCs) o Secondary: EPO effect -> due to incr EPO a. TRUE: caused by long term hypoxia - due to chronic resp or circ disorders (can be physio - low atmospheric O2, training) * More EPO which incr RBC prod. Eg. brachycephal syndrome in dogs, ROA – recurrent airway obstruction in horses, right-left shunt in the heart) b. NOT TRUE: without hypoxia: autonomous incr of EPO due to kidney disease or juxtaglomerular apparatus tumour. 2. Hypovolaemic Polycythaemia: Incr cell number with decr volume - = Relative polycythaemias -Dehydration: lack of drinking water, vomit, diarrhoea, PU, loss of plasma (eg. burns) -Splenic contractions (stress) -Since fluid loss, increased cell count -> more conc 3. Hypervolaemic Polycythaemia: Incr cell number and incr volume -Complex disorder -Transfusion overdose, splenic vessel contraction, life threatening acute stress or extreme physical exercise, fever or hyperthermia (concurrent constriction of blood vessels and the spleen)

Answer 14

-Erythrocyte morphology: 1. Begin with proper staining methods (May-Grunwald, Giema, Diff quick) 2. Smear prepared using fresh samples 3. Check blood film on low (200x) then high (1000x) 4. Check gross signs: o Rouleau formation: coin arrangement (Eq often, dog cat, su sometimes, cattle rare) o Aggregates from RBC and thrombocytes, large cells (eq) -Intensity staining RBCs: *Polychromasia, hyperchromasia: intensive (RNA, nuclear remnants, more Hb – reg. proc) *Hypochromasia: weak staining, decreased Hb content, Fe or other deficiency -Size of RBCs: *Macrocytosis: big cells *Microcytosis: small cells *Anisocytosis: variable cell size (Fe def, reg. proc) *Poikylocytosis: variable size + colour -Types RBCs: *Young, nucleated RBCs: in order of maturation: o Proerythryoblast -> basophil- -> polychromatophil- -> acidophil erythroblast *Young, mature RBC, no nucleus: reticulocyte *Young RBCs appear -> incr prod (reg. anaemia) o spleen or bone marrow disease o leukaemia o extramedullary erythrocyte production o Pb poisoning (basophil punctates) o Hyperadrenocorticism

Answer 15

1. Reticulocyte: Reg anaemia (incr RBC prod) - Chronic Fe deficiency, Haemolysis, Acute/chronic blood loss 2. Spherocyte (small polychromatophil RBC): Sensitive RBC membrane - IHA 3. Stomatocyte (mouth shaped): Reg anaemia 4. Acanthocyte (RBC with long spikes): RBC membrane failure - Lipid metabolism disorder, hepatopathies 5. Schysocyte (RBC fragment): Traumatic/toxic damage Uraema, blood parasites, LT physical activity, DIC 6. Anulocyte (shape like RBC): Iron def anaemia - Fe def 7. Codocyte (target): Reg process 8. Echynocyte (many small spikes): Lab error - Too quick dry, uraemia, DIC 9. Sickle cell: RBC damage - Hb globin chain malformation in Hu Inclusion bodies: 10. Heinz Body (new meth blue stain): Desaturated Hb O2 effect, oxidative damage to RBCs (cat!! MethHbaemia), GSH def 11. Howell-Jolly body: Nuclear membrane remnants - Vit B12 def, Increased RBC prod, Splenectomy 12. Basophilic punctates: Nuclear remnants - Reg process, Young RBC’s cat, Physiological in RU, Pb poisoning 13. Hb inclusions: Hb damage - Incr RBC prod, Reg anaemia

Answer 16

1. Haemobartonella Ca, Fe, Bo 2. Babesia Spp (Ca (common Hungary), Gibson) 3. Ehlichia ca, eq 4. Dirofilaria immitis, repens 5. Anaplasma marginale, centrale, ovis 6. Eperythrozoon wenyoni, ovis, suis, parvum 7. Citauxzoon felis 8. Theileria parva, mutans, annulata, hirci, ovis 9. Trypanosoma cruzi, congolense, vivax, brucei, evans, suis, equiperdum 10. Leishmania donovani

Answer 17

-To calculate the indices, must measure: o Ht or PCV o RBC count (Burker chamber, cell counter, qualitative blood count) o Hb conc. (spectrophotometry) • Gives objective info about avg size and colour (influenced by Hb content) of RBCs • Info also by routine blood smear analysis, but results affected by competence of cytologist = subjective • Use in case of humans, dogs and maybe cats • Not in: Eq, Ru -> big variance among parameters of RBCs of different animal individuals, and within individuals!

Answer 18

Mean corpuscular haemoglobin (MCH): average Hb content of whole blood MCH (pg) = Hgb/RBC -normal: 12 – 30 pg (young animals: MCH and MCV can incr: 28 – 32) Hb/RBC=MCH (pg) -if MCH increase = hyperchromasia -if MCH decrease = hypochromasia

Answer 19

Mean corpuscular volume (MCV): indicates average volume of RBCs MCV (fl) = PCV/RBCx1000 -normal: 60 – 70fl -Great heterogeneity in MCV among species: o Cats, horses: smaller RBCs o Young: bigger RBCs - Adult: smaller RBCs o Diff bw species: Akita (small 55-65), Poodle (large 75-80) -Microcytosis: chronic blood loss, Fe, Cu, vit. B6 deficiency, portosystemic shunt -Macrocytosis: polycythaemia absolute vera, vit B12, cobalt, folic acid deficiency, erythroleukaemias, can also be different regenerative anaemia

Answer 20

Mean corpuscular haemoglobin concentration (MCHC): avg Hb conc in erythrocytes MCHC (g/l) = MCH/MCVx1000 = Hb/PCV -Normal: 300–350 g/l (30 – 35%) = normochromasia -When MCV and MCH low (liver fail), MCHC can be normal, but anaemic state -Hypochromasia: newborn, regen. and Fe def. anaemia -Hyperchromasia: vit. B12, cobalt, folic acid def, IHA, Pb poison, splenectomy, erythroleukemia

Answer 21

a) Horse b) Ruminants c) Dog d) Cat MCH (pg): a) 12 – 20 b) 8 – 17 c) 15 – 24 d) 13 – 17 MCV (fl): a) 37 – 58 b) 42 – 52 c) 63 – 75 d) 40 – 53 MCHC (%): a) 31 – 37 b) 30 – 36 c) 32 – 36 d) 30 - 36

Answer 22

Red cell distribution width (RDW), Platelet distribution width (PDW): - These indices gives a number correlated with the range of the avg size of RBCs and platelets - RDW: draw vertical line from peak of RBC to axis x, draw horizontal line at half of vertical line. RDW is line between 2 points where horizontal line cross vertical (P1 and P2). * Express in % of whole interval of red cell size * RDW in dog = 12-16%, cat = 14-18% * PDW: dog = 6 – 8%, cat = 7-12 % * Normally the histogram is a symmetric Gauss-curve with slight right shift. * Short RDW = non-regenerative anaemia, equal size cells * Large RDW = regenerative anaemia, many unequally sized cells

Answer 23

1. macrocytic, hypohromic: MCV incr, MCHC decr (Reticulocytes incr): regenerative anaemias 2. normocytic, normochromic: MCV same , MCHC same normal or decr MCH: non regenerative anaemias 3. microcytic, hypochromic: MCV decr , MCHC decr (decr Hb synthesis): iron, copper, piridoxine, deficiency anaemias, liver, failure, portosystemic shunt 4. microcytic, normochromic: MCV decr , MCHC same: Japanese Akita (normal) 5. macrocytic, normochromic: MCV incr , MCHC same, impaired DNA synthesis: FeLV infection, vitamin B12, Co, or folic acid deficiency, erythroleukemia, poodle macrocytosis

Answer 24

1. Regenerative anaemia: -Usually dis. w. favourable prognosis: enough new RBCs prod in BM to reg the anaemia, to replace lost RBCs, and reach normal RBC count quickly. -If BM function is normal, RBC prod is in positive correlation with the severity of anaemia. -In case of severe anaemia more intensive RBC prod is needed to consider this as regenerative anaemia, than in case of mild anaemia. -Characterized by: oligocythaemia, macrocyotsis, decr MCHC and INCR reticulocyte. *Sometimes all three first criteria are there, but you don’t know if it is B12 def reg or non reg anaemia before checking reticulocyte count. -Eg: Vit B12, B9, cobalt deficiency, iron deficiency anaemia. 2. Non regenerative: -Characterised by normal vol of MCV and MCHC, but there is decr Hb synt and reticulocyte count is normal or decr. -BM can also work, but there is a reason the RBC produced is not maturing. Either they are nucleated and can’t mature to do the functional work of reticulocytes, or they are being killed right away by the immune system (IHA). Reticulocyte -> first mature RBC. -Eg: IHA, lead poisoning, B12 deficiency. Biggest help to differentiate anaemias: reticulocyte count: -Reticulocyte is only incr in regenerative anaemia’s o Reticulocytes are a sign of regenerative function of BM -> same functional properties as mature RBC -> able to carry oxygen. o Nucleated are too young – not functional RBC. o The nucleated can be able to measure if underlying reason is treated. But if there is not an incr in reticulocyte, the anaemia is non-reg.

Answer 25

-WBW counting use anticoagulated blood -Na2EDTA, K2EDTA, or Na-citrate - anticoagulant -Bird, reptile, fish samples: Li- or Ca heparin A) WBC counting by Bürker-chamber 1. Use Hagedorn pipette, aspirate 0.1 ml homogenised whole blood sample 2. Wash sample into 0.9 ml Türk-solution (acetic acid - hemolyser, gentiana-violet blue - stains nuclei) (10x dilution) 3. Incubate for 1-2 min. at room temp 4. Place one drop onto the Bürker-chamber and count the nr of WBCs above 25 large squares 5. Number divided by 10 -> WBCs in No x 109/l 2. WBC counting by haematology analyser -Analyse diff cell types according to light absorbency – more accurate -Automatic cell counter: use impedance technique o Dilute for WBC count, then haemolyse RBCs o Distribute cells according to greatness of electrical impedance o Reagents decr lymphocyte and incr size of neutrophils and macrophages -Some use laser technique, and some use reagents – Eg. to measure peroxidase activity -Laser cell counters: o Inner structure and size differentiation o Detect inner structure by laser beam technique o Scattered light detected by 2 light detectors o Low angular/forward -> detects size o High angular/side scatter -> detects inner structure o Put values together -> number on a graph -> cloud formed by the closer points -> cells with similar size and structure on same cloud, one type. Diff clouds. -Flow cytometry method

Answer 26

``` -Increased: o Chronic stress o Acute stress o Glucocorticoid therapy o Cushing´s disease - HAC o Chronic inflammation o Acute inflammation - 2. phase o Addison´s disease - hAC o Leukemoid reaction o WBC leukaemia - typical form o Regenerative left shift -Decreased: o Immunodeficiency disease o Cyclic neutropenia o Degenerative left shift o Acute inflammation - 1.phase o Leukaemia WBC/RBC - not-typical, hid in BM o Bone marrow suppression (eg. heavy metal poisoning, mycotoxins, chemotherapy, viral infection) -Inflammation: *Chronic: right -> stress leukogram *Acute: left -> degenerative or regenerative! *Mild/moderate inflam can be fixed by the storage pool ```

Answer 27

Haemopoietic (leukemic) tumours of animals -A lot of haemopoietic tumours known in animals, but mention most common: 1. Acute leukemic diseases o Typical cell types are “blast” cells - coarse chromatin pattern and nucleoli o Origin: evaluated by (immune)cytochemical, and BM analysis o Some cases neoplastic cells don’t appear in peripheral blood, but anaemia, leukopenia, thrombocytopenia *In these cases, BM evaluation is important o Acute lymphoblastic leukaemia o Acute myeloblastic, promyelocytic leukaemia o Acute erythroblastic leukaemia o Lymphoma of Stage V (metastatisis to BM) 2. Chronic leukemic diseases o Typical cell types are mature differentiated, or differentiated cells o Appear in huge amount in peripheral blood * Does not cause diagnostic issues if lymphoid, thrombocytes, or erythroid forms *But can be if mixed with leukemoid reaction •To diagnose these, BM evaluation is required o Chronic small lymphocytic leukaemia o Chronic myeloid (neutrophil granulocytic, eosinophil granulocytic, basophil granulocytic, monocytic leukaemia) o Polycythaemia absolute vera (overprod of mature erythrocytes), o Essential thrombocytosis (overprod of mature thrombocytes). 3. Lymphomas (also hemopoietic tumours) o Poorly differentiated lymphoid cells overprod in lymphatic organs and sometimes in other tissues o Some cases: tumorous lymphoid cells are overprod in BM also o Origin: in case of cats and cattle, only. In these species viral infection is the cause: *Bovine Leukosis Virus/BLV *Feline Leukaemia Virus FeLV.

Answer 28

Qualitative blood count 1. Prepare smear: can use several staining methods, e.g May-Grünwald, Romanowsky, Giemsa, Diff-Quick – Bürker chamber 2. Smears should be analysed at the edge of the slide and in the middle 3. Low power first, then high 1000x magnification by using immersion lens (and oil) 4. Count at least 50-200 cells and differentiate according to morphological pattern of the cell types and make the ratio in % among the different cell types

Answer 29

1. Neutrophil granulocyte cell line: Function: phagocytosis, elimination of Ag´s by lysosomal enzymes. Neutrophilic sp: dog, cat, human, horse (60%/35%) •myeloblast •promyelocyte •myelocyte •metamyelocyte (jugend) -> pathologic •band forms (stab) - young form •segmented forms - oldest form -> hypersegmented due to GCs 2. Lymphoid cell line: B-cells: humoral immunity, T-cells: cellular immunity, lymphokine. Lymphocytic sp: swine, cattle, sheep, goat, hamster (60%/35%) •lymphoblast •small and middle sized lymphocyte •reactive T lymphocyte 3. Eosinophil cell line: inactivation of histamine by lysosomal enzymes, phagocytosis •young (band nucleated) eosinophil granulocyte •segmented form eosinophil granulocyte 4. Basophil cell line: stim. elim. of Ag´s by lysosomal enzymes, heparin, serotonin, histamine. •young (band nucleated) basophil granulocyte •segmented form (basophil granulocyte) 5. Monocyte cell line: phagocytosis with lysosomal enzymes, present antigens, enzyme secretion, mediated phagocytosis •monocytic (young) form •reactive macrophage form

Answer 30

-Physiological leucocytosis (incr WBC count) •due to acute or chronic stress •catecholamine mobilizes cells from marginal to circulatory pools, happens in seconds •effect of GCs visible after some hours – neutrophilia, lymphopenia, eosinopenia -Pathological leucocytosis/leucopenia: A) Acute inflammation B) Chronic inflammation -Other

Answer 31

-Neutropenia: first period of inflam proc o Migrating factors (interleukins, leukotriens) from tissue cells and macrophages o Migrate neutrophils from blood to site of inflam -> decr in total WBC -Neutrophilia: later phases of inflam o G-CSF - Granulocyte colony stimulating factor + GM-CSF (macrophage) stim WBC prod in BM -> incr total WBC -Left shift: *More young WBC appear in circulating blood *Young metamyeloctyes (jugend) and band (stab) forms more visible and larger number 1. Regenerative left shift *Incr WBC count, neutrophilia, left shift (younger forms) *BM regenerates used neutrophils in circulation from storage, maturation and or mitotic pool *Most common cause of young neutrophil appearance *Favourable prognosis 2. Degenerative left shift *Low or normal WBC and neutrophil count. More band forms and immature neutrophils than mature/segmented neutrophils. *If the rate of usage is larger than regenerative capacity, then outflow of neutrophils is not enough to incr WBC o Young band form > old form by 50% o Widespread or severe inflammation o Abscesses, peritonitis, pleuritic, pyometra *Poor prognosis, very serious disease and need immediate treatment -Leukemoid reaction *Great stimulus for prod neutrophil granulocytes -> huge nr of neutrophils present in peripheral blood due to incr effect of G-CSF + GM-CSF. *Typical of: big abscesses, endometritis, some neoplastic dis. *Blood smear: easy confused w. chronic myelogenous leukaemia. -Toxic neutrophils *In very severe inflam proc: granulocytopoiesis can be disturbed. *When the granule prod. is not physio: some azurophilic, orange-red granules can be seen in basophilic cytoplasm = “toxic” neutrophils -Döhle-bodies *Due to toxic effect: angular, basophilic inclusion bodies can be seen in some neutrophil granulocytes -> remnants of the ER = Döhle-bodies (more common in cats)

Answer 32

Right shift: •Many segmented, old neutrophils (3-4 on 1 nucleus) seen in smear •Hypersegmented -> glucocorticoids •Appearance of older forms w. incr WBC typical for chronic inflam process •Also typical for glucocorticoid effects (drugs, incr. prod. in adrenal gland, Cushing´s) o GCs inhibit cellular proliferation (young cells disappear) and have membrane stabilizing effect (let neutrophils become old and hyper segmented) •Chronic inflam / chronic stress: GCs present in both o Chronic inflammation -> glucocorticoids -> lympholytic, but they have work to do so they stay -> lymphocytosis o Chronic stress -> glucocorticoids -> lympholytic, they leave due to GCs -> lymphopenia -> stress leukogram (right shift, leucocytosis, neutrophilia, eosinopenia and lymphopenia together)

Answer 33

- Addison´s disease - Hypoadrenocorticism (see t. 40): no inhibitory effect of GCs - Pelger-Huet anomaly: normocytaemia and left shift. Inheritable problem causes diagnostic problem. - Cyclic neutropenia: inheritable disease, cyclic bone marrow production -> neutropenia occurs in weekly, monthly intervals. During these periods: sensitive to infection. - Bone marrow damage: leukopenia and neutropenia due to BM damage or decreased function, often w. thrombocytopenia and aplastic anaemia.

Answer 34

-Haemostasis: group of processes in order to stop bleeding during tissue or vascular injury -1st stage of healing process. Occurs in 3 steps: (1) Vasoconstriction (2) Temporary blocking of damage by platelet ‘plug’ (3) Clot formation -Major group of haemostasis disorders: 1. Vasculopathy – decr vasoconstrictive ability o Rare in animals as it is generally caused by vit C def 2. Thrombocytopathy – decr ability of platelets to aggregate, adhere to site of injury & form primary thrombocyte thrombus o E.g. thrombocytopenia – decr nr of thrombocytes in blood 3. Coagulopathy – problems relating to the intrinsic, extrinsic or common pathway of the coagulation cascade & formation of a fibrin network

Answer 35

-not necessarily accurate but easy to perform 1. Signs of increased bleeding tendency:  skin and mucous membranes – anaemia, petechial, ecchymosis, suffusion  thoracic cavity – haemothorax  abdominal cavity – haemoperitoneum  GI tract – haemetemesis, melena 2. Capillary resistance 3. Bleeding time 4. Appearance of first fibrin strand (clot time) 5. Appearance of clot (clotting time on different surfaces) 6. Clot retraction test

Answer 36

-normally human medicine •Ligature on arm, check palm side, see small reddish dots •If poor haemostasis -> petechia o Capillaries more fragile eg. if vasculitis (viral, autoimmune) or other diseases

Answer 37

Bleeding time, Buccal mucosal bleeding time (BT, BMBT): •Tests thrombocytopenia/pathys, vasopathy, NOT COAGULOPATHY •Incision inner part of ext. ear/buccal mucosa surface (some must be sedated) o Wipe blood from under, 20 – 30 sec, don’t touch wound – can remove thrombus o measure from start until finish bleeding o Normal: 3–5 minutes •BT,BMBT is dependent on thrombocyte function, platelet count and capillary function •No danger of clinical bleeding until platelet count > 50x109

Answer 38

•Tests for coagulopathies! •Important to do test ASAP, from fresh, native samples, no anticoagulants added •Two syringe method - do not use the first drops of blood for coagulation measurements, we change syringe after taking these first drops, and use the content of the second syringe. •Samples should be taken by a proper way, preferably with only one precise venipuncture, so that we don’t cause too much damage to surrounding tissues o Can increase tissue factor (factor III.) release from damaged cells → activates the coagulation cascade during sampling! (a) Appearance of first fibrin strand: drop blood onto slide, sink tip of needle into blood, move back and forth till first strand -> 1–2 min (good vision and lighting) (b) CT watch glass: sample on paraffin/wax treated glass – till whole amount is solid, like gelatine -> 7–15 min (c) CT plastic syringe: Put the fresh sample into syringe and check the time of the complete coagulation -> 10-12 min (d) CT glass tube: put fresh blood into glass test tube and check time  4–5 min (e) CT in activated clotting time (ACT) tube: contains SiO2 (brown cap), put in 37oC thermostat and check coagulation by slowly moving tube every 15-20 sec. -> 3 min. SiO2 activates factor XII (Hageman) which activates factor IX and kallikrein and kininogen (fibrinolytic pathway)

Answer 39

-Thrombocytopenia: o Decr prod of thrombocytes in BM (suppression) o Increased utilisation of thrombocytes (DIC) o Incr destruction of thrombocytes (AITP) o Incr seques. of thrombocytes (chronic splenomegaly) o Incr loss of thrombocytes (subacute/chronic bleeding) -Thrombocytopathy: o Improper development of platelets, hereditary glucoprotein def. o Von Willebrand´s disease o Uraemia, liver failure, myelo-, and/or lymphoproliferative diseases o NSAIDs treatment -Coagulopathy: o Intrinsic pathway:  Haemophilia A-factor VIII. deficiency  Haemophilia B-factor IX. deficiency  Von Willebrand´s disease o Extrinsic pathway:  Factor VII deficiency  Dicumarol toxicosis – first stage o Common pathway:  Liver disease -> decr production of coagulation factors  DIC  Dicumarol toxicosis – second stage  Factor X./V./II. /I./XIII. Deficiency

Answer 40

- Especially important when BT, BMBT is incr or petechie is visible on skin or mucous membranes - Use anticoagulated blood (Na2-/K2-EDTA) 1. Bürker chamber 2. Blood smear 3. Automatic cell counters 4. Clot retraction test

Answer 41

* Anticoagulated sample into 0.9ml physiological saline solution, mix and sediment 2 hrs * 1 drop of upper layer onto Bürker chamber (haemocytometer) * Count number of platelets in 10 rectangles * Process is quicker if sample is in NaCl solution and centrifuged 1500/min * Not accurate

Answer 42

* Find 1 platelet in 1 view by 1000x magnification  20 x 109/l platelet count * Very uncertain, but can see arrangement from different views in middle and edges of slide * Finding big thrombocyte aggregates suggest proper platelet function and can explain low thrombocytic counts measured by cell counters

Answer 43

•If volume is between 30 – 50fl, will be counted as platelets •Big if: o Regenerative process of bone marrow (many circulating young big platelets) o Physiologically: cats, King Charles spaniel (such high volume, that big platelets can be counted as RBCs) •Thrombocytic aggregates can be mistaken as WBC, so platelet count is measured to be normal/low and WBC count to be increased. •Evaluation of blood smear important step of diagnosing disorders o Arrangement, morphology •Normal 200 – 800 x 109 /l

Answer 44

-determines thrombocyte function •Leave clot in tube, will get smaller and serum appear due to contractile protein thrombostenin •Normal volume of serum released is 25% of whole volume of initial clot •If slower, or absent = suspect thrombocytopathy

Answer 45

- 1-2 microm diam, granular centre, hyalomer part at edge - Eq, Ru: smallest 3-5 fl Car, Su: 7-8 fl Fe: largest 10-15 fl - In case of thrombocytopathies/penias/vasopathies, cannot expect signs of severe bleeding disorders because: prevented by formation of a fibrin thrombus (polymerised fibrin strands). - But coagulopathies can lead to severe bleedings because thrombi not stable without fibrin network, and if big vessel injury, blood flow can sweep the thrombus from the wound.

Answer 46

1. Platelet aggregation test: •Goal: if we suspect thrombocytopathy (von Willebr dis) •Sample: citrated blood plasma (citrate prevents coagulation by binding Ca-ions) and use upper layer (platelet rich plasma) •Method: Into cuvette for aggregometer, add drugs to exaggerate aggregation -> spectrophotometry 2. Prothrombin time (PT) -Info about ext pathway because cascade is triggered by tissue factor and calcium ions -Factors involved: VII, X, V, II, I, XIII o Perform 1 hour after sampling, decalcinate sample using Na-citrate o Centrifuge and separate plasma from sediment, 37. o Reagent contains Factor III (thromboplastin) and CaCl2 o Evaluation using coagulometer or test-tube o Reagent should be 2x vol when mix w. citrated plasma -Normal: 10-15 sec 3. Advanced partial thromboplastin time (APTT) - Info about intrinsic pathway, use surface activation and add PF3 & Ca2+ to activate X - Factors involved; XI, IX, VIII, X, V, II, I, XIII - Use citrated plasma again, reagent contain PF3 and micronized silica as contact activator o Normal: 20–30 sec 4. Thrombin time (TT) •Mix decalcinated plasma with reagent containing thrombin only •Coagulation time depends on conc of fibrinogen and XIII in plasma •Test for the common coagulation pathway, but also used when you need to know fibrinogen content of sample (if normal XIII in blood)

Answer 47

(warfarin, functional vit.K depleter, potent anticoag. drug) •In early stage: only PT incr, later: APTT also incr •It’s a competitive antagonist of Vit K which is responsible for gamma carboxylation of: o Proconvertin (VII) - will be deficient first (shortest half life) o Christmas (IX) o Stuart-Prower (X) o Prothrombin (II) o These are Ca dependent factors, so Vit K def will cause inability to bind Calcium. • PT incr when factor VII is deficient, so this test shows problem first.

Answer 48

* The fibrinolytic pathway is responsible for keeping the clot formation within normal limits * Clot inhibitors: antithrombin III, alpha-2-macroglobin, alpha-1-antitrypsin, heparin (bind to & neutralize thrombin) * After complete coagulation, fibrinolytic enzymes degrade the clot. * Kallikrein activates kininogen system -> forms bradykinin (activated kininogen) & activates plasminogen. * Plasminogen -> plasmin (activated form, an endopeptidase that cleaves fibrin strands) * Before complete degradation of fibrin, FDP’s can be detected in the blood (polymerised fibrin strands, incr level of fibrinolysis-products, fibrin dimers and monomers) * Incr fibrinolysis is determined by D-dimer level of blood (these originate only from fibrin, not fibrinogen) * Reagent contains Ig’s which agglutinate FDPs & D-dimers * Helpful in determination of DIC.

Answer 49

•Common acute disorder that requires quick and accurate lab. diag. •Usually a secondary disorder caused by primary diseases such as septicaemia, pancreatitis, widespread burns, necrosis of large tumours, shock, polytraumatisation etc. •Microthrombus & fibrinolysis are present in many places of the body simultaneously & so coagulation factors & platelets are consumed very quickly  consumption coagulopathy •Diagnosis of DIC: o Positive FDP or D-dimer test o Coagulation time, bleeding time, PT, APTT, TT, FDP & D-dimer - increased o Platelet count - decreased o Appearance of schysocytes & burr cells in blood smear

Answer 50

•Known in humans and Doberman pinchers •Often with hypothyroidism •Lack complete factor VIII (von Willebrand factor, responsible for platelet adhesion and aggregation) o Factor VIIIc: antihemophilic factor o Factor VIII related antigen determinable part bound strongly to VIII •Patients have: o Increased BT and BMBT o Decreased clot retraction ability, sometimes coagulation disorder • Detect lack of Von Willebrand Ag using ELISA

Answer 51

Protein metabolism: -Plasma protein functions: Maintain oncotic pressure, carrier molecules, haemostasis, buffer system, immune response, inflam. process -Main serum protein fractions: Albumin, globulins, fibrinogen. o Prod by hepatocytes. Except: g-globulins (in B-lymp.) Total protein concentration in blood plasma (TP): •Serum protein content depend on: Intake, synthesis, transformation, catabolism, hydration •Measure: chromatographic, electrophoretic, refractometry (depend on SG, quality of serum, temp) o Several TP measurements o Best determining range = biuret test •Average TP conc = 60–80 g/l •Smaller TP conc: in urine, CSF, body cavity - not determined correctly o So use Lowry method: Folin-Phenol reagent or by ultrasensitive TP where protein are directly bound to stain molecules 1. Biuret test: *Measure: TP photometrically *Reagent: KNaSCN, CuSO4, KI, NaOH *Blank: biuret reagent and physiological saline *Sample: serum and biuret reagent *CO-NH + Cu2++ alkaline -> purple complex - at 540nm *Esample/Estandard x 60=total protein g/l 2. Ultrasensitive TP analysis: Na-molybdate and pirogallol-red reagent -> complex molecule by binding proteins *Sensitivity: 0.2 – 0.4 g/l. Standards: 0.25, 0.5, 1, 2 g/l (TP conc also depends on water balance!!! If dehydrated -> TP incr , If overhydrated -> TP decr)

Answer 52

-small, negative charge: first in electrophoresis 1. Spectrophotometry: bromocresol green reagent, binds to albumin. At pH 4.2 produce blue-green complex detectable at 580nm. 2. Serum electrophoresis: expensive! o Use if protein fraction analysis is the aim o Gives %, so need to know TP conc. -Interpretation: • Increase: dehydration • Decrease: o Decr. synthesis: decr intake, abs. (malabs./digestion) o Decr. synthesis: liver failure, acute inflam. (is a neg APP) o Incr. usage: chronic disease (chronic inflam., neoplasm), physiological; pregnancy, work, exercise, growth, milk/egg production o Incr. loss: kidney (PLN), GIT (PLE), skin (burn), whole blood loss, sequestration into body cavities (not decrease of colloid P) o Other (relative): hyperhydration (eg. iatrogenic)

Answer 53

1. Roughly by diff. of TP and albumin: Globulin conc = TP – Albumin conc. 2. Serum electrophoresis – protein fraction analysis o Gives %, need to know TP conc. 3. Alb/globulin ratio o ESR and glutaric-aldehyde test o APPs -> inflam or processes related to neoplasm o Can be decr due to decr of albumin conc -Decreased globulin: • Decreased intake: Neonates, absorption disorder of neonates • Decreased synthesis: o Acquired of inherited immunodeficiency o Liver function impairment • Increased loss: o PLE, PLN o Via skin -> burn, inflammation o Whole blood loss, bleeding

Answer 54

1. If both plasma (separate from anticoag. blood) and serum (separated from clotted blood) then TP conc. Is measured the difference of these two. •Plasma – Serum TP = Fibrinogen conc. 2. Use heat labile character of fibrinogen: • One part of plasma used for TP, other part heated to 56-58degrees • Plasma centrifuged then measure TP concentration • Diff. bw the two is fibrinogen conc. 3. Thrombin time test • Since values depend on fibrinogen conc. • Reagent contains Bovine thrombin and Ca2+ • Clot formation determined by using standards of fibrinogen conc. •Add active thrombin, bind fibrinogen mol – prod by XIII •Thrombin time incr if low fibrinogen conc -Interpretation: •Increase: o acute inflammation (esp. Ru) o dehydration •Decreased: o liver function impairment o advanced protein deficiency o DIC o sequestration after bleeding to body cavity o chronic bleeding o blood loss o inherited afibrinogenaemia (St. Bernard)

Answer 55

-based on protein having amphoteric character -Examine globulin in blood o On paper with agarose gel – current to separate serum protein components into 5 classifications by size and electrical charge: Serum albumin, Alpha-1-globulin, Alpha-2-globulin, Beta globulin (IgM, IgE), Gamma globulin (IgG) o Rate is relative to charge and inversely to size: smaller and more charge --> fastest and furthest •SDS-PAGE (gel electrophoresis) o Protein samples are denatured by heating SDS o Coating of the polypeptides with negative charged SDS molecules -> all proteins highly negatively charged o Denatured proteins attached at + pole -> size decide how fast they migrate to the – pole when electric current is added * Larger = faster, smaller =slower.

Answer 56

1. Polyclonal gammopathy: Broad based peak in beta and/or gamma region •There is cloning of more than one cell. E.g.: parasite, bacteria, virus -> activate several AB because it has several Ags. •Common causes: chronic inflam., liver disease, FIP, occult heartworm disease, ehrlichiosis •Beta-gamma bridging: Occurs in disorders where IgA & IgM increases (lymphoma, chronic active hepatitis) 2. Monoclonal gammopathy: Sharp spike in beta or gamma region cause by both neoplastic and non-neoplastic diseases •Only one type of cell goes crazy and produce clones; normally B-cell or plasma cell o Neoplastic disorders - true monoclonal gammopathy because tumors of B-cells or plasma cells, BM going crazy: Multiple myeloma, Lymphoma, Chronic lymphocytic leukaemia, Extramedullary plasmacytomas, Macroglobulinemia o Non-neoplastic disorders - this is not only one cell going crazy, but it mimics the monoclonal graph even though it is polyclonal - have to treat it like a monoclonal (non-neoplastic): Occult heartworm disease, FIPV (rare), Ehrlichia canis, Lymphoplasmacytic enteritis, Lymphoplasmacytic dermatitis, Amyloidosis

Answer 57

1. Glutaric aldehyde test 2. Erythrocyte sedimentation rate ESR 3. C-reactive protein - CRP 4. Gammopathy

Answer 58

•Goal: examine incr of plasma fibrinogen and globulin cc •Fibrinogen is an APP -> marked incr during acute inflam. •Used in clinical practice of adult cattle to indicate inflam proc. •Sample: heparinised blood •Method: quick test -> mix blood sample with glutaric aldehyde o Observe clotting time: fibrinogen (incr during inflam) o Fast clotting -> severe inflammation • Interpretation: Coagulation time: < 1 min: ++++ / severe, widespread acute inflam. 1-3 min: +++ / acute inflam. 3-8 min: ++ / subacute inflam. 8-15 min: +/- / not severe > 15 min: - / no inflam. • Diagnose: reticuloperitonitits, sever mastitis or endometritis of cattle (poor clinical signs, but severe diseases)

Answer 59

* Goal: detect inflam by incr sedimen. of RBC´s, as APPs and other globulins attach to RBCs surface. * Method: Normally RBCs have neg. charged alb on their surface that prevents aggregation. High glob level (severe inflam or neoplastic diseases) causes glob attachments instead, which does not have a neg. charge, and therefore will aggregate and sediment quickly. * Sample: Westergreen (glass) tubes - contain Na-citrate, with mm scale. Whole on bottom and top of tube - connect with another tube, blood goes into tube and flow up in the tube with the scale until the zero point. Read from scale after 1 hour. In horses after 20 min. * Physiologically ESR = 0.5-3 cm/hour. * ESR inversely proportional with Ht/PCV, and proportional with serum viscosity, TP and fibrinogen conc. * Horse: speed of sedimentation decr due to inflam processes contrary to other species. * Obs! When there is hypoalbuminaemia (and therefore relatively incr. glob conc), ESR incr too. Eg. in chronic renal failure or glomerulonephropathy.

Answer 60

•C-reactive protein: APP that can bind to a protein of Pneumococcus bacteria called protein-C and cause precipitation. Produced in the liver, and in beginning of inflam. process cytokins stimulate it´s production. •Goal: examine acute inflam. before the clinical signs by looking at incr. in pos. APPs. •Theory: During acute inflammation processes: o Conc. of neg APPs (lactoferrin, transferrin) decr o Conc. of Pos APPs (CRP, haptoglobin, serum amyloid A) incr •In the beginning of acute inflam. process CRP values are incr, before appearance of clinical signs - therefore useful diagnostic tool for early detection. o Extremely imp. in immunosuppressed animals, eg. during chemotherapy or high dose of GC therapy. •Sample: serum •Method: immunological ELISA or immunturbidimetric method •General value: 8mg/l

Answer 61

Gammopathy - incr globulin, only seen on electrophoresis. •Monoclonal: one clone (b-cell or plasma cell) has gone crazy and are multiplying - true monoclonal, neoplasia. Non true, non-neoplasma monoclonal - only mimic the monoclonal but are more a polyclonal, e.g: FIP, heartworm, viral infection. •Polyclonal: infection, more antigens, more clones effected (salmonella).