Diagnostic Approach to Small Animal Bleeding Disorders

งานประชุมวชิาการ คณะสัตวแพทยศาสตร์ มหาวทิยาลยัเชียงใหม่ 2563

Multi Systemic Disease

Diagnostic Approach to Small Animal Bleeding Disorders

Nawin Manachai (DVM., MSc., PhD.)

Small Animal Clinic

Department of Companion Animal and Wildlife Clinic

Faculty of Veterinary Medicine

Chiang Mai University

Outline

Clinical approach to patients with bleeding disorders

Normal hemostatic models

Primary hemostatic disorder

Secondary hemostatic disorder

Diagnostic approach for coagulation disorders

Common disorders associated with hemostasis in small animal

Clinical approach to patients with bleeding

1. Is the bleeding appropriate?

2. Localized or Systemic

3. Congenital or Acquired

4. Primary or Secondary hemostasis

5. Medication and other disorder : liver disease, renal failure, BM disease or malignancy

Abnormal?

-Spontaneous-Not related to injuryMultiple sits

Primary or Secondary ?

Primary hemostatic disorders Platelet and vessels Petechiae (thrombocytopenia) Small and multiple ecchymosis

Mucocutaneous bleeding

Immediate. Persistent bleedingFrom superficial cuts

Secondary coagulationand fibrinolysis

-Deep hematoma-Large ecchymosis solitary

-Deep tissue bleeding-Intramuscular bleeding-Delayed bleeding

Congenital or acquired- Onset- Family history

1 2 3


Not all diseases can be clearly characterized by these definitions, as is the case with disseminated intravascular coagulation (DIC) and some platelet and vascular anomalies


activation

aggregation

Fibrin degradation

Coagulation systems

Fibrinolysis(plasmin)

Natural(endogenous)anticoagulants

(PC, PS, AT)

Vessels injury

adhesion

Platelet plug+

Fibrin plug

• Primary hemostasis

• Secondary hemostasis

Normal Hemostasis

Primary or secondary hemostatic disorders?

1. Endothelial or vascular Injury

2. Platelet adhesion

3. Platelet activation

4. Platelet aggregation

GP Iib/IIIa

fibrinogen

Platelet aggreation

Platelet adhesiom

GP Ib-IXVWF

Serotonin ADP

Primary hemostasis

Primary hemostasis: platelet

Thrombopoiesis refer to platelets production from megakaryocytein BM undergoing TPO stimulation, majority

Platelets (thrombocytes) in mammals are anucleate cells

that contain small pink-red granules

Shed into the blood from megakaryocytes

in bone marrow

key players in the hemostatic process in primary hemostasis

Though most platelets are smaller than red cells

http://eclinpath.com/hematology/morphologic-features/platelets/normal-platelets/canine-platelets/


-moderately variable in size-well-stained of granules

-several large platelets and platelets

with dendritic processes

(possibly activated)

-degree of variation in platelet size is

normal for this species.

-the inset shows a large platelet.

• Feline platelets

Primary hemostasis: platelet

• Canine platelets



Primary hemostasis: platelet assessment

A part of CBC

Quantitative (platelet number /µL)

Platelet distribution width (PDW)

Mean platelet volume (MPV)

Semi-quantitative (adequate or decrease)

Distribution (clumping or aggregation)

Alteration of platelet morphology

(giant or macro-platelet, micro-platelets)

peripheral blood smear (PBS)

automate hematology analyzer

1. Clotting in blood sample

2. EDTA-induced platelet clumping/platelet satellitism

3. Hemodilution

4. Large/giant platelet

………………………………………………………………………

• Platelet aggregation (clumping) results in a falsely decreased countClumping: increase the mean platelet volume (MPV)

• Platelet satellitism form of platelets adhering to polymorphonuclear leucocytes imparting a rosette-like appearance

Need to examination peripheral blood smear

• What causes false thrombocytopenia?


platelet satellitism platelet clumping

Platelets estimation by counting the average number of platelets seen per 100x oil immersion field in the monolayer. in general, 10 oil immersion fields

• Estimated platelet count/µL = average count in 10 fields x 20,000

Report : the reference interval for the species

• Increased• Adequate• Decrease (Low)• Very low: below a medical decision limit associated with spontaneous hemorrhage

<30,000/µL

Reference: Semi-quantitative estimate of platelet numbers guideline by Cornell University


For example

if an average field contains 8 platelets

estimated platelet count/µL = 8 x 20,000

= an estimate of 160,000/µL

This value would be compared to the normal range for the species in canine spp.

-160,000/µL

Report : adequate (for dog)

(within reference intervals)

Peripheral blood smear (PBS)from male dog 3 yrs.


Exclude false thrombocytopenia

Underproduction-Bi or pancytopenia-Drug/alcohol-PV, retrovirus- Chronic E canis- FeLV-Radiation-BM diseaseMalignancies-ITP

Immune • ITP (1 or 2)• Infection:

hemoparasite• Evan syndrome• Drug/Toxin/vac• Autoimmune• Viral causes• Malignancy

Non-Immune • DIC, TTP, HUS• Kasabach-merritt syndrome• HELLP syndrome• Major bleeding/operation• Trauma• Hemophagocytosis• Sepsis• Drug/Toxin

SequestrationThe excessive platelet pooling in an enlarged spleen (hypersplenism) liver disease

Mild to moderate

moderate to marked particularly in immune-mediated causes

mild.

Destruction/consumption

Primary hemostasis: approach to thrombocytopenia

True thrombocytopenia


Greater than 5 µm in diameter (as large as or larger than feline red blood cells)Low numbers of giant platelets : normal cats

• Increased numbers of macro-platelets in thrombocytopenic dogs suggests increased thrombopoiesis

• Seen in MPD and MDS • Hereditary congenital macrothrombocytopenia

platelet function defects in Cavalier KingCharles Spaniels

• Superficial and/or mucosal surfaces

-petechial or ecchymotic hemorrhages in the skin and

mucous membranes, retinal hemorrhage. epistaxis,

melena, hematochezia and/or hematuria; essentially,

bleeding from small vessels : thrombocytopenia

• Clinical bleeding in primary hemostatic disorder

- platelet count drops below 50×103/L (usually <30×103/L)

- approximately 3–4 platelets/ HPF

Primary hemostasis: clinical bleeding

Platelet count drops below 50×103/L (usually <30×103/µL)

Platelet count drops not more than 50×103/µL

+ Secondary hemostatic disorders(PT, aPTT, TT,…)

• Primary hemostatic disorder

Clinical bleeding tendency

• Primary hemostatic disorder

• Secondary hemostatic disorder

Normal in platelet numbers and coagulation times

Investigation into platelet function defects platelet function tests

• Diseases associated with platelet functions-Hereditary (Basset Hound thrombocytopathiaChédiak-Higashi syndrome,Cyclic hematopoiesis in Grey Collies,Glanzmann’s thrombasthenia of Great Pyrenees and Otterhounds)

-Acquired (drugs, toxins, infections, neoplasia, hyperglobulinemia)uremia from advance stage of CKD

Clinical bleeding tendency

• Increased platelet destruction by anti-platelet antibodies (mainly IgG )

• Premature destruction by macrophages in the spleen and liver

• Decreased platelet production (emerging pathogenesis)

• Epitopes GP IIb/IIIa target antigen in dog with ITP

B-cell

Plasma cell

Splenic macrophage

Fc-receptor

Cellular pathogenesis of ITP

Immune-mediated thrombocytopenia (ITP or IMT)

Immune-mediated thrombocytopenia (ITP or IMT)

Primary or Secondary as well as IMHA

Primary ITP or idiopathic thrombocytopenic purpura (pITP)

absence of other identifiable disease

Secondary ITP- underlying diseases

• Systemic immune-mediated disorders

• Systemic lupus erythematosus (SLE)

• Neoplasia : Lymphoma, myeloproliferative disorders

Various solid tumors

• Drugs: Sulfonamides, cephalosporin

Infections

Ehrlichiosis

Mycoplasma

Anaplasma

H. pyroli

Leptospirosis

CDV, FIV, FeLV

Leishmaniasis

Babesiosis

Histoplasmosis

Heart worm disease

Vaccine with modified live virus

Clinical findings

• Often no fever

• No clinical signs

• Surface bleedings petechiae in skin and mucosa, ecchymosis, gingival bleeding, melena, epistaxis, scleral and retinal bleeding, hematochezia, hematemesis, hemorrhagic vaginal discharge, hyphema, hematuria

Clinical manifestation: ITP (IMT)

• Lethargy and pale mucous membranes

• Spontaneous bleeding: platelet counts < 30,000-40,000/µl

• Degree of hemorrhage and platelet count often unpredictable

• Dogs with ITP may have platelet counts < 20,000/µl without evidence of bleeding or other clinical signs

Primary ITP: diagnosed by rule out non-immune causes of thrombocytopenia and exclusion of secondary causes (underlying diseases)

1. Isolated thrombocytopenia

2. Occurrence with anemia (IMHA) : Evans’s syndrome 20-30% of cases

3. Normal coagulogram: no prolonged PT and a PTT

4. Blood smear: True low platelet, no MAHA blood picture

No abnormal cells, no leukoerythroblastosis,

often giant platelet or micro-platelet (active thrombopoiesis)

5. Coomb’ s test may be positive (Evans’s syndrome often)

Diagnosis: ITP

Clinical epidemiology

ITP is reported in dogs of all ages.

80% of the pITP patients were younger than 6 years (middle-age)

In approximately 30% of the cases ITP and IMHA occur together (Evan ‘s syndrome)

Predisposing breed : Cocker Spaniels, Scottish Terriers, Poodles, and

Old English Sheepdogs and a genetic background is strongly suspected. Familial ITP is reported in humans

Gender predilection to many autoimmune disease is recognized. ITP

Occurs in female dogs approximately twice as frequently as in male dogs.

Extremely rare in cat

Diagnosis: ITP

Alternative diagnostic tools

Bone marrow biopsy : not recommended- Decreased megakaryocytes in BM

• Directed immune response against platelets• Directed immune response against megakaryocytes

- Anti-platelet immunofluorescence test (PIFT)

• Therapeutic trial by response to immunosuppressive drug

Diagnosis: ITP

Case-based evidence of canine ITP

C poodle F 9 yo

Previous medical record : Previous treatment E. canis with doxycycline 1 m.

2 m. later LN within normal limit 101.1 F

Cc: Haematuria and petechiae

Complete blood count

PCV = 45%

MCV = 67.6

MCH = 22.7

WBCs = 12670

Neu = 9270

Lymp = 1150

Mono= 1560 H

Eo = 680

Baso = 10

Platelet = 19,000 (giant)

Plt smear decrease

Problem listsIsolated (marked ) thrombocytopeniaMild monocytosis

Clinical breeding ?

Initial assessmentDestruction of platelet due to chronic blood parasite

Diagnosis plansPT = 5.4 sec (5.0-7,7)aPTT = 10.7 sec(8.7-14.6)

PCV = 45%MCV = 66.7MCH = 22.8WBCs = 14800Neu = 10,340Lymp = 1,050Mono= 2,690 HEo = 790Baso = 10Platelet = 270KPlt smear adequate

Secondary ITP ?

After prednisolone 1 mg/kg PO one week

• Progress note• Resolve thrombocytopenia


Platelet 19,000 cell/uLMacro plateletsNormal leukogram• PT = 5.4 sec N• aPTT = 10.7 sec N

Platelet 270,000 Monocytosis

Platelet 118,000Left shift

Taper-downPrednisolone 0.5 mg/kgFor 14 days

Platelet 30,7000Normal leukogram

Platelet 21,4000complete response by prednisolone Tx for 2 m.-no clinical bleeding and others-Plt return to normal

Prednisolone 1 mg/kgFor 1 m.

Taper-down qodPrednisolone 0.5 mg/kgFor 14 days

VET MED CMU


Secondary hemostatic disorders

Nawin Manachai

Department of Companion Animal and Wildlife Clinic

Faculty of Veterinary Medicine

Chiang Mai University

activation

aggregation

Fibrin degradation

Coagulation systems

Fibrinolysis(plasmin)

Natural(endogenous)anticoagulants

(PC, PS, AT)

Vessels injury

adhesion

Platelet plug+

Fibrin plug

Primary hemostasis

Secondary hemostasis

Normal Hemostasis

Abnormal?

-Spontaneous-Not related to injuryMultiple sits

Primary or Secondary ?

Primary hemostatic disorders Platelet and vessels Petechiae (thrombocytopenia) Small and multiple ecchymosis

Mucocutaneous bleeding

Immediate. Persistent bleedingFrom superficial cuts

Secondary coagulationand fibrinolysis

-Deep hematoma-Large ecchymosis solitary

-Deep tissue bleeding-Intramuscular bleeding-Delayed bleeding

Congenital or acquired- Onset- Family history

1 2 3


Water-fall cascade model of coagulationHMWK, Prekallikrein

Intrinsic pathway

XIIa XII

XIXIa

IXIXa

VIIIVIIIa

XXa+

X

Va

VII VIIa

Tissue factors

Extrinsic pathway

Common pathway

IIa (thrombin)II

Fibrinogen Fibrin

Fibrin crosslink Fibrinolysis

XIII

(Prothrombin)

PT

aPTT

PT and aPTT

TT

Cell-based model of coagulation

Plasminogen Plasmin

Crosslinked Fibrin Fibrin degradation products (FDP)

Fibrin

Fibrin polymer

Crosslinked fibrin

Fibrinolysis

FDPs

D-dimer

PlasminPlasmin

Fibrinolysis

FXIII

Anti-fibrinolysis

Plasminogen Plasmin

Fibrin degradation products (FDP)

Tranexamic acid

Oral, IV, topicalEffective in areas with increased fibrinolysisPerioperative mucosal bleeding

-Oral operation-Urogenital operation-Prostate surgery

Contraindication in upper urinary tractbleeding or history of thrombosis

Global Objectives for Hemostasis Testing a systematic approach to the workup of a bleeding disorder helps to…

• formulate a diagnosis• establish prognosis• set up appropriate patient monitoring • develop corrective therapeutic measures

Basic tests for Secondary Hemostasis>>>>>>>>>>>>>>>>CBC (platelet)Peripheral blood smearActivated partial thromboplastin time (aPTT)Prothrombin time (PT)For Fibrinogen systemThrombin TimeFibrinogen levelFDP, D-dimer

Diagnosis of secondary hemostatic disorders

• Sample collection

• Direct Venipuncture- intravenous catheters not recommended

• Standard anticoagulant for coagulation testing: 3.2 or 3.8 % sodium citrate - PT, aPTT, TT, fibrinogen, FDPs, D-dimersas well as specific coagulation factors- 1:9 ratio of sodium citrate to blood

• Sample submission: as soon as possible• Sample storage: plasma separation and storage 24-48 hrs. 4 C


• Poorly collected blood sample• Mistake anti-coagulant

• Blood obtained from traumatic venipunctureof from indwelling catheters

• High hematocrit (Hct > 55%)relatively high anticoagulant

Cause False prolongation of coagulogram?

Pre-analytic error

General considerations

aPTT is more sensitive than the ACT, with prolongation of the aPTT detected after loss of approximately 65% of coagulation factor activity.

aPTT and PT : not affected by low platelet numbers

PT and aPTT will not prolong until factor level decrease to <30-40%

PT and aPTT are not sensitive for abnormalities of fibrinogen unless

fibrinogen < 100 mg/dL

TT is very sensitive to heparin/ direct thrombin inhibitor



Altered PT, aPTT

no pre-analytic error

Isolated aPTT prolongation Isolated PT prolongation

Combined PT and aPTT prolongation

• Isolated PT prolongation

PT• Early vitamin K deficiency• Vitamin K antagonist• Liver disease• DIC (early)• Factor VII deficiency (rare)

• Extrinsic and common pathway• Specifically factors VII, X, V, prothrombin (II).


• Isolated aPTT prolongation

aPTT

No bleeding Bleeding

Mixing test

Correctable• Hemophilia A, B• Factor XI deficiency• vWD

Uncorrectable• Factor VII inhibitor• Heparin induce

LupusFXII deficiency


• Mixing test (classical 1:1 mixing test)

Patient’s plasma

Pooled normalplasma

+1:1 • Recheck coagulogram after 60 min incubation @ 37 C

>50% Correctable (normal coagulation time)Deficiency

Principle: Normally coagulation factors are > 50-100%Prolong aPTT or PT occurs when coagulation factors < 30-40%

<50% Uncorrectable (abnormal coagulation time)Inhibitor



Mixing test for aPTT

Distinguish between..

• Clotting factor deficiency : correctable

• Inhibitor : UncorrectableFVIII inhibitors, Lupus anticoagulant

• Deficiency of factor in common pathwayFactor X, V deficiencyDefects in fibrinogen

• Multiple factor deficiencyVitamin K deficiency/antagonist, heparinLiver diseaseDICMassive transfusionMalignancies

• Combined PT and aPTT prolongation

Factor X deficiency is associated with plasma cell dyscrasia (myeloma)



• Combined PT and aPTT prolongation

Prolonged TT Normal TT

Factor X, V II deficiency

Multiple factor deficiencyVitamin K defienciy/antagonist, Liver diseaseDICMalignancies

thrombinFibrinogen ---> fibrin

Hypo/afibrinigenemiaDysfibrinogenemia(liver disease)Thrombin inhibitor

PKHMWKXII

XIIX

VIII

Tissue factor

VII

XVI

XIII

Fibrin

aPTT PT

Common pathway

Intrinsic pathway Extrinsic pathway

• Classical diagnostic approach of secondary hemostasis


Common disorders associated with hemostasis

in small animal

• Vitamin K deficiency (antagonist)

• DIC (Disseminated intravascular coagulopathy)

• Snake or other venom associated hematologic toxin

• Causes

Ingestion (direct or indirect) of Vit K antagonism

- Hydroxycoumarins

- Indanediones

- Warfarin

Block and impaired hepatic carboxylation of vitamin K dependent coagulation factors (Factors II, VII, IX, X) and vit K-dependent anticoagulant (Ps and Pc)

PcPS

AT

Vitamin K deficiencies (antagonist)

Clinical signs

Vit K antagonist

• delay 3-7 d after exposure

• Most severity

• Internal hemorrhage

• pleural space and lung –the most common

• Signs of hypovolemia shock

Diagnosis

• Hx –rodenticide exposure (risk)

• Prolonged PT, aPTT

• Treatment of Vit K antagonism

• Ingestion < 5 hr

• induce emesis

• activated charcoal 2-5 g/kg PO

• Specific therapy

• Vit K1 1.23 -2.5 mg/kg bid

Vitamin K deficiencies (antagonist)

Disseminated intravascular coagulation (DIC)

Syndrome, not a disease >> underlying etiology

Excessive or unbalanced intravascular activation of coagulationMultiple Organ Dysfunctions (MODs)

• Consumptive coagulopathy and a thrombo-hemorrhagic state• Primary, secondary and/or fibrinolysis

Any disease process that increases prothrombotic factors, decreases endogenous anticoagulants, causes endothelial dysfunction,or leads to defects in fibrinolysis can trigger DIC in small animals.Typically these are conditions associated with a heightened (systemic) inflammatory response (SIRs) (e.g., sepsis)

• Underlying causes…

• Severe trauma, burns, tissue necrosis• Disseminated neoplasia• Shock, heat stroke• Endotoxemia, sepsis• Pancreatitis• Malignancy• Endothelial cell injury, vasculitis• Hepatic diseases

SIRs >>> DIC >>> MODs


• Syndrome, not a disease hematological manifestation of systemic diseases and or local disease

Endothelial activation Endothelial injury

Consumption of coagulation factors

Consumption of platelets

Fibrin fibrinolysis

fibrinogen

D-dimer/FDP

MAHA +1,+2PlateletPT, aPTTFibrinogenD-dimer

1.Thrombocytopenia

2. Prolonged activated partial thromboplastin time (aPTT) / prothrombin

time (PT), hypofibrinogenemia

3. Elevated markers of fibrinolysis

fibrinogen degradation products (FDPs) or D-dimers

4. Blood smear: erythrocyte fragmentation (schistocytes, keratocytes,

acanthocytes) 40% of DIC cases

2 or more laboratory abnormalities from the following:

cytokines


Scoring system of international Society of Thrombosis and Hemostasis (STH)

-Thrombocytopenic severity

-PT prolongation

-Fibrinogen level

-FDP (D-dimer level)

1. Platelet count-Greater than 100K /µL -50-100K /µL-Less than 50K /µL

• 0 pt• 1 pt• 2 pts

3. D-dimer-No change-Moderate rise-Strong rise

• 0 pt• 1 pt• 2 pts

• 0 pt• 1 pt• 2 pt

2. PT prolongation-3 sec or less-3-6 sec-> 6 sec

4. Fibrinogen level- < 1 g/L- > 1 g/L • 0 pt

• 1 pt

• Total score 0-4 : DIC suspected treatment and monitor

• Total score ≥5 : overt DIC

The use of these drugs is typically guided by knowledge of the underlying disease process

Serious, life-threatening and monitor closely

Primary anticoagulant drug used for therapy of the procoagulant phase of DIC : Heparin for inhibition of secondary hemostasis

Unfractionated or low-molecular-weight heparins

• Heparin low dose 50-100 IU/kg SQ q 8 (unfractionated)

State of overt DIC

Blood products : fresh frozen plasma (consumed coagulation factors)

Dose of at least 6 to 10 mL/kg (up to 20 mL/kg)

for correction of bleeding from factor deficiencies

Monitor aPTT and PT

DIC: treatment and management

Topical ReviewUpdate on Disseminated Intravascular Coagulation: When to Consider It, When to Expect It, When to Treat ItAlan G. Ralph, DVM,a and Benjamin M. Brainard

C Female 8 yo Mixed vomit depress and feverCBC PCV =26% Hb=9 g/dL Plt = 71,000 cell/uLWBC = 89,320 cell/uLBlast = 27,689Band = 3,573Seg = 27,689PT = 11.7 (5-7.7) secaPTT = 25.9 (8.7-14.6) secBlood morphologyTarget cell +1Schistocyte+2Hypochromic +1BUN = 12.8 mg/dlCrea. = 0.67 mg/dlALT = 59 U/LALP = 436 U/L

DIC associated by malignancy case

MAHA blood picturewith thrombocytopeniaProlongation of PT and aPTT

Tantative DiagnosisDIC caused by…..

VET MED CMUVET MED CMU

VET MED CMU

DIC associated by malignancy case

Acute myeloid leukemia : M2 (suspected) Myeloblast with maturation

1. Is the bleeding appropriate?

2. Localized or Systemic?

3. Congenital or Acquired

4. Primary or Secondary hemostasis

5. Medication and other disorder?

Summary

1. Clinical bleeding

2. Primary hemostasis

Platelet and vessels

3. Secondary hemostasis

PT, aPTT and fibrinolysis

4. Both

?

Serious, life-threatening and monitor closely

First line of therapy for DIC, ideally

Recognition and therapy to address the underlying trigger

In the case of sepsis

appropriate antimicrobials

surgery (if indicated to address the source of infection)

Supportive care

Intravenous fluids to maintain euvolemia

Oxygen delivery to tissues:

Prevent tissue hypoxia and the inflammation associated with reperfusion of ischemic tissues

DIC: treatment and management

Documents

Diagnostic Approach to Small Animal Bleeding Disorders