Hepatic cases Kathleen Tennant BVetMed Cert SAM Cert VC FRCPath MRCVS Clincial Lead Diagnostic Laboratories Langford Veterinary Services

Case 1 2 year old Norfolk terrier Presented depressed, vomiting and lethargic after scavenging in dustbin including baby nappies, chocolate cake and sugar free gum Clinical exam – slightly icteric

Haematology Haemoconcentrated (PCV 59 L/L) Platelet count through the machine 100 (200 – 500 x 10^9/L) Leucocytes unremarkable (numbers and morphology)

Test Result Range Units Total protein 65 49 - 71 g/L Albumin 30 28 - 39 Globulins 35 21 - 41 Sodium 155 146 - 155 mmol/L Potassium 4.0 4.1 – 5.3 Chloride 107 107 - 115 Calcium 2.3 2.13 – 2.7 Inorganic phosphorous 3.2 0.8 - 2 Urea 9.3 3 – 9.1 Creatinine 162 98 - 163 Umol/L Cholesterol 9.0 3.3 – 8.9 Total bilirubin 16 0 – 2.4 AST 402 0 - 28 U/L ALT 4 13 - 88 ALP 2900 19 - 285

How do we best interpret the ALT value? Rules out hepatocellular damage 1. Low due to lack of remaining hepatic parenchyma 2. Likely to be laboratory or technical error 3. There was hepatocellular damage, but this is in the past, hence low ALT but AST still elevated 4.

Test Result Range Units Total protein 65 49 - 71 g/L Albumin 30 28 - 39 Globulins 35 21 - 41 Sodium 155 146 - 155 mmol/L Potassium 4.0 4.1 – 5.3 Chloride 107 107 - 115 Calcium 2.3 2.13 – 2.7 Inorganic phosphorous 3.2 0.8 - 2 Urea 9.3 3 – 9.1 Creatinine 162 98 - 163 Umol/L Cholesterol 9.0 3.3 – 8.9 Total bilirubin 8.2 0 – 2.4 AST 402 0 - 28 U/L ALT 4 13 - 88 ALP 2900 19 - 285

Internal consistency AST and ALT often both elevate with hepatocellular damage, with 2 x top of reference interval elevations mild, 4 x moderate and 10 x taken as marked It would be unusual for ALT to be below reference if AST is this elevated in hepatocellular damage as AST generally elevates with more, not less, damage. The half life of AST is actually shorter than that of ALT (< 1 day vs 2 -3 days), so severity and timing play a role in the final values Low ALT due to absence of remaining hepatocytes extremely unusual, and not expected if AST still high AST elevation without ALT may be due to muscle damage No history or suspicion of muscle damage – could use creatine kinase to confirm its presence or absence ? Falsely low ALT

Trouble - shooting single dubious results Check the gross appearance of the sample or machine flags for hemolysis, icterus or lipaemia if these interfere with the assay This sample is icteric – does not affect ALT but: Inorganic phosphorous 3.2 (ref 0.8 - 2mmol/L) – is affected by icterus – reject result

Trouble - shooting single dubious results (ALT) Check the quality assurance/ quality control for that analyte Usually by checking the machine’s log for low, medium and high control performance Should indicate if there is a problem with reagents, and can be run again if needed to check for a machine glitch (e.g. bubble in the system) Check the results of samples either side of this one In this case – all fine, so re – run to rule out a ‘pipetting’ error Also run in x 10 and x 100 dilution

ALT re-run results Neat sample – ALT 0 (13 – 88 U/L) X 10 dilution - ALT 3502 (13 – 88 U/L) X 100 dilution – ALT 3560 (13 – 88 U/L) Some wet chemistry dynamic assays, confronted with extremely high activity in their target enzyme will ‘miss’ the activity because it’s all over too quickly to measure – default to 0 or a very low level

Falsely low readings with high value rate of change assays Substrate depletion Substrate depletion Absorbence Absorbence delay delay measure measure

Test Result Range Units Total protein 65 49 - 71 g/L Albumin 30 28 - 39 Globulins 35 21 - 41 Sodium 155 146 - 155 mmol/L Potassium 4.0 4.1 – 5.3 Chloride 107 107 - 115 Calcium 2.3 2.13 – 2.7 Inorganic phosphorous N/A 0.8 - 2 Urea 9.3 3 – 9.1 Creatinine 162 98 - 163 Umol/L Cholesterol 9.0 3.3 – 8.9 Total bilirubin 16 0 – 2.4 AST 402 0 - 28 U/L ALT 3502 13 - 88 ALP 2900 19 - 285

What else SHOULD have been measured at the beginning, given the clinical history? Presented depressed, vomiting and lethargic after scavenging in dustbin including baby nappies, chocolate cake and sugar free gum Clinical exam – slightly icteric

What else SHOULD have been measured at the beginning, given the clinical history? Bile acids 1. Glucose 2. PT/APTT 3. D – dimers 4. Ammonia 5.

Xylitol toxicity Marked elevation in insulin following ingestion Initial marked hypoglycaemia may lead to some of the central clinical signs Hepatocellular necrosis, development of D.I.C./ bleeding disorders Marked prolongations in PT and APTT and evidence of bleeding disorder, including petechiation, G.I. bleeding. D – dimers likely to elevate. Although they are likely to be abnormal, bile acids and ammonia should not take precedence over glucose and assessing clotting ability.

Case 2 8 y.o. FN Irish Wolfhound 3 week history of vague lethargy, inappetance and intermittent diarrhoea Owners noted a bloated abdomen that morning, followed by collapse – rushed in

Clinical findings Poor body condition (3/9) Fluid thrill in abdomen – structures could not be palpated Tachycardic, bounding pulses

Haematology – what describes the red cell picture? Hb 10.00 (12 – 18 g/dl) HCT 27 (35 – 55 %) RBC 5.0 (5.4 – 8.0 x 10^12/L) MCV 64 (65 – 75 fl) MCH 21 (22 – 25 pg) MCHC 32.8 (34 – 37 g/dl) This slide takes a while to look at.

What best describes the red cell picture? Mild normochromic anaemia 1. Moderate normochromic normocytic anaemia 2. Mild hypochromic normocytic anaemia 3. Mild hypochromic microcytic anaemia 4. Mild regenerative anaemia 5.

Haematology Hb 10.00 (12 – 18 g/dl) Mild anaemia HCT 27 (35 – 55 %) RBC 5.0 (5.4 – 8.0 x 10^12/L) MCV 64 (65 – 75 fl) Microcytic MCH 21 (22 – 25 pg) MCHC 32.8 (34 – 37 g/dl) Hypochromic

Smear examination – red cell morphology

What best describes the red cell morphology on the smear? Unremarkable 1. Microcytic, hypochromic 2. Echinocytes 3. Acanthocytes 4. Schistocytes 5.

Erythrocytes in liver dysfunction Poor iron handling compromises erythroid precursor haemoglobin manufacture – less haemoglobin per cell Slowness in achieving a haemoglobin content threshold allows an extra precursor division, resulting in smaller cells These will generally be picked up by the machine BEFORE they are obvious on the smear No evidence of regeneration in this population – no obvious polychromatophils

Acanthocytes and schistocytes Acanthocytes (black arrows) are red cells with large cytoplasmic projections. These are associated with liver diseases, lipid disorders and shear injury to the red cells Schistocytes (red arrow) associated with shear injury to red cells

Test Result Range Units Total protein 65 49 - 71 g/L Albumin 26 28 - 39 Globulins 20 21 - 41 Sodium 146 146 - 155 mmol/L Potassium 4.5 4.1 – 5.3 Chloride 107 107 - 115 Calcium 2.2 2.13 – 2.7 Inorganic phosphorous 1.2 0.8 - 2 Urea 7.8 3 – 9.1 Creatinine 158 98 - 163 Umol/L Cholesterol 7.2 3.3 – 8.9 Total bilirubin 3.3 0 – 2.4 AST 210 0 - 28 U/L ALT 880 13 - 88 ALP 810 19 - 285

Is this more a cholestatic or a hepatocellular damage picture? Both exactly the same 3.

Test Result Range Units Total protein 65 49 - 71 g/L Albumin 26 28 - 39 Globulins 20 21 - 41 Sodium 146 146 - 155 mmol/L Potassium 4.5 4.1 – 5.3 Chloride 107 107 - 115 Calcium 2.2 2.13 – 2.7 Inorganic phosphorous 1.2 0.8 - 2 Urea 7.8 3 – 9.1 Creatinine 158 98 - 163 Umol/L Cholesterol 7.2 3.3 – 8.9 Total bilirubin 3.3 0 – 2.4 AST 210 10 x ref 0 - 28 U/L ALT 880 10 x ref 13 - 88 ALP 810 3 x ref 19 - 285

Abdominal fluid – sampled through midline PCV 18 % Plt 5 x 10^9/L Nucleated cell count 3 x 10^9/L Total protein 30 g/L

Abdominal effusion – elsewhere on the slide

What best describes the nature of the fluid? Transudate 1. Modified transudate 2. Exudate 3. Iatrogenic haemorrhage superimposed on a transudate 4. Haemorrhagic 5.

Abdominal fluid – sampled through midline PCV 18 % Plt 5 x 10^9/L Nucleated cell count 3 x 10^9/L Total protein 30 g/L Transudates TP < 25 g/L, nucleated cell count < 1 x 10^9/L Not expected here as serum albumin 26 g/L Previous and current haemorrhage….

Imaging - appearance of liver

Coagulation testing PT patient 9 secs , control 9 secs (expected < 11 s) APTT patient 25 secs, control 18 secs (expected < 21 s) D – dimers 0.5 (0 – 2 mg/dl)

Which of these fits best with the coagulation test results? Coumarin toxicity 1. Liver dysfunction 2. Hemophilia A 3. Hemophilia B 4. Consumptive coagulopathy (prolongation due to factors being used up attempting to stop bleeding from another cause) 5.

Coagulopathies and liver disease May be compromise in absorbing Vitamin K and other fat soluble vitamins Many clotting factors made by liver In some animals with liver disease, APTT may prolong ahead of PT Hemophiliacs often have very prolonged APTT values Can not rule out a consumptive coagulopathy, although often PT and APTT might be expected to prolong together Has anyone noticed we haven’t looked at platelets yet?

Platelet count through machine = 60 x 10^9/L, smear estimate 40 – 60 x 10^9/L Likely consumption….

Hepatic cytology 100x magnification

Hepatic cytology Normal Patient

Hepatic cytology 500x

What is the cytology most compatible with? Chronic hepatitis 1. Large cell lymphoma 2. Haemangiosarcoma 3. Hepatoma 4. Hepatic carcinoma 5.

Final diagnosis: Hepatic carcinoma - ruptured

Cytology correlation with histopathology Wang et al (2004): 94 cases, dogs and cats – overall agreement between histopathology and cytology in 30% canine and 51% of feline samples. Best agreement in vacuolar hepatopathy (7/11 dogs, 15/18 cats), poor correlation for inflammation (5/20 dogs, 3/11 cats) Kristensen et al (1990): agreement in 66% between histo/ cyto in 44 cases

Case 3

Case 3 9 y.o. N.F. miniature Dachshund Intermittent lethargy, abdominal pain, vomiting Recent rapid deterioration – collapse, pyrexia, dehydration, icterus

Haematology WBC 35 (5.5 – 17 x 10^9/L) Neutrophils 32 (3.0 – 11.5 x 10^9/L) Other findings through machine unremarkable Smear shows left shift and toxic change in neutrophils

Neutrophils

Test Result Range Units Total protein 84 49 - 71 g/L Albumin 26 28 - 39 Globulins 58 21 - 41 Sodium 149 146 - 155 mmol/L Potassium 5.1 4.1 – 5.3 Chloride 111 107 - 115 Calcium 2.3 2.13 – 2.7 Urea 9.3 3 – 9.1 Creatinine 162 98 - 163 Umol/L Cholesterol 13.2 3.3 – 8.9 Total bilirubin 54 0 – 2.4 AST 45 0 - 28 U/L ALT 210 13 - 88 ALP 1283 19 - 285 GGT 56 0 - 15

Which of these is true? ALP is a more sensitive or equally sensitive marker compared to GGT for cholestasis in the dog 1. ALP is a more sensitive marker than GGT for cholestasis in the cat 2. Bilirubin is a specific marker for cholestasis in the dog and cat 3. Cholesterol decreases in cholestatic disease 4.

Markers for cholestasis ALP is a sensitive marker for cholestasis in the dog, but in the cat, GGT tends to increase more in hepatic/cholestatic conditions other than hepatic lipidosis Due to potential elevations derived from increased haemolysis, bilirubin is not specific for cholestasis in the dog or cat Cholesterol increases in cholestasis (hence the name!)

Further testing Canine Pancreatic Lipase within normal limits PT/ APTT normal

Extracellular bile pigment

Gall bladder contents

Gall bladder contents

Final diagnosis Pure growth of E coli cultured from gall bladder contents aspirate, and from biopsies of liver parenchyma Ascending cholecystitis/ infection