TV4101 - Regenerative Anaemia

Haemolytic anaemia – accelerated RBC destruction

DDX?

1. Immune mediated

2. Erythrocyte metabolic defect

a. Oxidative damage

b. Defect in ATP generation

3. Erythrocyte fragmentation

4. Infectious

5. Other (heparin, snake envenomation)

Immune Mediated Haemolytic anaemia

Causes inc?

1. Idiopathic (autoimmune)

• Immune response directed at normal self epitopes

2. Drug induced

• Develop abnormal antigens on erythrocyte cell membrane

3. Vaccine associated

4. Alloimmune

• Neonatal isoerythrolysis

• Blood transfusion reactions

IMHA - horses

Prevalence?

When it occurs it is usually secondary to?

Less common in horse c.f. dog

RBC parasites

Infection – Clostridial infection (20 -28%), Equine Infectious anaemia (EIA), Leptospirosis, Streptococcus equi.

Neoplasia – Lymphoma (13%), (melanoma)

Drugs – penicillin (13%), (trimethoprim- sulphamethoxazole)

IMHA - horses
Haemolysis location?
DX?

Extravascular more common
DX –multiple positives in a panel of tests (regenerative anaemia, persistent autoagglutination after washing, + Coomb’s test, spheroechinocytes, exclude other causes of regenerative anaemia)

IMHA - Neonatal Isoerythrolysis - Species?

Feline & Equine - Yes

Canine - 1 case

Bovine - Animals previously vaccinated (blood component)

Ovine - never

IMHA - Alloimmune mediated haemolytic anaemia

Caused by?

IMHA - Alloimmune mediated haemolytic anaemia (Neonatal Isoerythrolysis)

How does it work?

What is normally seen?

Important in who?

Immune mediated destruction (haemolysis or removal by

reticuloendothelial system) of foal/kitten RBCs caused by maternal antibodies ingested in colostrum

Born healthy → haemolytic anaemia → hallmarks pallor & icterus (0-8 days)

Horse and cats

IMHA - Alloimmune mediated haemolytic anaemia (Neonatal Isoerythrolysis) - Feline Neonatal Isoerythrolysis

How work?

Queen (type B) bred with Tom (type A) - kitten at risk if gets type A group
In colostrum type B blood with anti-A alloantibodies → kitten (type A or AB) ingests colostrum

Kitten has weak or no anti-B alloantibodies so can’t protect itself

IMHA - Alloimmune mediated haemolytic anaemia (Neonatal Isoerythrolysis) - Equine Neonatal Isoerythrolysis

How does it work? List the steps

1. Dam negative for RBC antigen (usually Aa or Qa)

2. Mare becomes sensitized & produce antibody to the offending antigen. (multiparous)

a. Previous blood transfusion

b. Previous pregnancy

c. Transplacental contamination with fetal RBCs earlier in current pregnancy (rare e.g. placentitis)

Certain RBC Ag factors are more antigenic: Aa & Qa

3. Foal inherits sire’s RBC antigen type

4. Foal ingests mare’s colostrum (concentrated with antibodies)

IMHA - Alloimmune mediated haemolytic anaemia (Neonatal Isoerythrolysis) - Equine Neonatal Isoerythrolysis

AB titer

Higher antibody titer at foaling – the higher the risk for NI

Highest titer – produced in a previously sensitized mare – that is reexposed to the same offending RBC antigen shortly before parturition

IMHA - Alloimmune mediated haemolytic anaemia (Neonatal Isoerythrolysis) - Equine Neonatal Isoerythrolysis

Clinical presentation?

Foal is normal at birth

Consumes colostrum concentrated with antibodies formed by mare including antibodies to sire’s RBCs (EAa or EQa)

If foal inherits sire’s RBC Ag type, within 24-48 hours foal is

- Anaemic (pallor)

- Icteric

- difficulty breathing

- reduced suckling

- requires intense medical treatment

- (can die acutely or present later)

MHA - Alloimmune mediated haemolytic anaemia (Neonatal Isoerythrolysis) - Equine Neonatal Isoerythrolysis

DX?

Anaemia: PCV 10-20% (5%)

Predominantly extravascular, but if intravascular component occurs then haemoglobinaemia & haemoglobinuria may be present

Hyperbilirubinaemia

(most unconjugated - due increased RBC destruction)

Total bilirubin (up to 20 mg/dl)

MHA - Alloimmune mediated haemolytic anaemia (Neonatal Isoerythrolysis) - Equine Neonatal Isoerythrolysis

Describe each photo

Icterus - Neonatal isoerythrolysis

Haemoglobinuria Pigmenturia

Haemoglobinuric nephropathy

MHA - Alloimmune mediated haemolytic anaemia (Neonatal Isoerythrolysis) - Equine Neonatal Isoerythrolysis - DX on a blood film what do we see

Anaemia (increased spaces/reduced density), agglutination

Anisocytosis – normal RBCs, macrocytes, Spherocytes

Metarubricytes & Howell Jolly bodies

Ghost cells

IMHA - Alloimmune mediated haemolytic anaemia (Neonatal Isoerythrolysis) - Equine Neonatal Isoerythrolysis

Diagnostic tests we use?

Definitive diagnosis requires demonstrating?

Alloantibodies in the dam's serum or colostrum that target foal erythrocytes

Haemolytic cross match test, Coombs test, Jaundiced foal test

IMHA - Alloimmune mediated haemolytic anaemia (Neonatal Isoerythrolysis) - Equine Neonatal Isoerythrolysis

Haemolytic cross match test?

washed foal RBC, mare serum & exogenous source of complement (usually rabbits) (positive at dilution>1:16)

IMHA - Alloimmune mediated haemolytic anaemia (Neonatal Isoerythrolysis) - Equine Neonatal Isoerythrolysis

Coombs test?

Often false negatives (because some equine alloantibodies act only as hemolysins, agglutination tests may be falsely negative)

IMHA - Alloimmune mediated haemolytic anaemia (Neonatal Isoerythrolysis) - Equine Neonatal Isoerythrolysis

– Jaundiced foal test.?

Serial dilutions of mare’s colostrum are centrifuged with foal’s RBCs. A positive reaction – identified by agglutinated (clump) RBCs bottom of a tube at dilution > 1:16

Neonatal Equine Alloimmune thrombocytopenia

How does it occur?

Maternal Ab pass through the colostrum and attach to foal platelets

Can be asymptomatic as condition may be self limiting due to rapid removal of alloantibodies

Neonatal Equine Alloimmune thrombocytopenia

CX if seen?

CBC?

Clinical- prolonged bleeding from venipuncture sites (petechial haemorrhage might not be seen)

Platelet count may be as low as 10 x 10 9/L

Neonatal Equine Alloimmune thrombocytopenia

Test?

Ab in colostrum or serum from mare against platelets

Neonatal Equine Alloimmune thrombocytopenia

DD’s for thrombocytopaenia

Sepsis

DIC

Drug induced

Angiopathies

Erythrocyte metabolic defect

Types and cause examples?

a. Oxidative damage

i. Heinz body haemolysis

ii. Eccentrocytic haemolysis

iii. Methaemoglobinaemia

b. Defects in ATP generation

i. Hypophosphataemic haemolysis

ii. PFK & PK deficiency

Erythrocyte metabolic defect - Oxidative damage

Occurs in?

Factors that determine the outcome of oxidative injury?

Dogs, cats, horses & ruminants

Not well understood (one substance can cause Heinz bodies in one species and methemoglobinaemia in another for example)

Erythrocyte metabolic defect - Oxidative damage

Anaemia occurs how?

e/v – RBC’s more rigid – less able to pass through splenic sinusoids – trapped & removed by macros

i/v – more fragile due to damaged membrane – may rupture spontaneously in blood vessels

antigen formation – when Heinz body binds RBC membrane, bound by autologous antibodies & removed the splenic or liver macrophages

Oxidative damage haemolysis - diagnosis

Anaemia – mild to severe

Polychromasia & reticulocytosis (regenerative)

Heinz bodies (NMB & Wrights or Diff quick) &/or Eccentrocytes (Wrights or Diff quick stain)

Haemoglobinaemia & haemoglobinuria (if intravascular haemolysis)

Hyperbilirubinaemia & Bilirubinuria

Severe cases – perhaps – Methaemoglobinaemia (dark or chocolate coloured blood)

What these?

how form?

Appearance in stains?

Heinz bodies

Aggregates of denatured Hgb caused by oxidative damage. may be free floating in blood stain sometimes

NMB stain as pale blue, protruding, rounded structures

associated with erythrocyte membranes

Wright-stained films, Heinz bodies have nearly the same staining features as normal Hgb but appear as slightly pale structures that create membrane defects or protrude

Heinz Body pathogenesis steps?

1. RBC has Oxyhaemoglobin (Fe2+) that carries oxygen normally → Oxidant overwhelms this mechanism → RBC has methaemoglobin (Fe3+) and can’t carry oxygen

2. Hgb-Fe3+ undergo spontaneous

   conformational changes to form

   hemichromes or heme-depleted Hgb

3. Hemichromes precipitate to

   form Heinz bodies

   1. During this Sulfhydral groups are

      oxidized & form disulfide

      bonds

Heinz body pathogenesis - cats

In health?

When are they pathogenic?

Cats – up to 5% Heinz bodies in health

“large” Heinz bodies or multiple Heinz bodies on RBC’s or > 5% RBC’s have Heinz bodies

Heinz body pathogenesis - cats

DX is?

regenerative anaemia, evidence haemolysis (bilirubinaemia, bilirubinuria), Heinz bodies, history of exposure

Heinz body pathogenesis – aetiological causes

Notable causes and which species involved?

Onion

• All

Garlic

• Dog

• Horse (possibly)

• Rums (ryegrass)

Benzocaine

• Cat/dog

• Horse (Maple leaf - dry) & alpaca

• Rum (brassica spp - kale and rape)

Acetominophen (paracetamol)

• Dog and cat

Napthalene (moth ball) - dog

Propylene glycol (wet cat food) - cat

Zn? - Dogs

Vitamin K1&3 - Dog

Propfol - Cat

Phenothiazine - Horse

Copper - Rums

Heinz body pathogenesis - cats

Cats and drug of note?

How does this work?

Acetaminophen (paracetamol) in cats bad, cat lack glucuronyl transferase which conjugates drug

• No conjugation → acetaminophen becomes reactive metabolites → Glutathione conc depleted → Dec protection from oxidative injury

Heinz body pathogenesis - cats

Dz of note?

 Diabetes mellitus

 (> ketoacidotic)

 Hyperthyroidism

 Lymphoma

Zinc and IMHA?

Zinc can cause Heinz bodies & mimic IMHA

- Causes oxidative damage - forms Heinz bodies

- Band 3 clustering, opsonisation of antibody & spherocytes formation –

can lead to a miss diagnosis of IMHA

What this?

DX how?

Features?

Sheep with heinz bodies = chronic copper toxicity

Diagnosis: elevated serum, liver & kidney copper

•Preceded by chronic copper accumulation in hepatocytes

•Sudden release of Cu, often after stress, acute intravascular haemolysis, haemoglobinaemia, haemoglobinuria – death in 2-3 days

What is this?

Describe it

Eccentrocyte aka Bite cell or hemighost

the haemoglobin is pushed to one side of the cell leaving an opposing pale stain

Eccentrocyte pathogenesis

Oxidation → erythrocyte membranes bond → collapsed, peripheral, crescent - shaped region of the cell (sometimes called a blister cell) and the cell’s Hgb is displaced eccentrically

Eccentrocyte pathogenesis - Causes inc?

Causes are similar to some things that cause Heinz bodies

(dogs, horses & cattle)

Exogenous oxidants: onions, garlic, acetaminophen, propylene glycol, zinc.

Endogenous oxidants: in very sick patients e.g. diabetes mellitus

Methaemoglobinaemia

Causes inc?

Drugs/toxins

-Paracetamol (cats, dogs)

Plants

-Onions

-Red Maple Leaf (horses)

-Nitrite (nitrate in plant/fertilizer)

Methaemoglobinaemia pathogenesis - steps?

1. Oxidant overwhelms RBC mechanisms to keep Hgb in reduced state

2. RBC with oxyhaemoglobin (Fe2+) that carries oxygen becomes Methaemogobin (Fe3+) which can’t carry oxygen

Methaemoglobinaemia pathogenesis

Effects of Methaemoglobinaemia?

How much is normal amounts of methaemoglobin in the blood?

Initially cyanotic (cannot deliver O2)

Later as Fe3+ > 10% mucous membranes appear brown

Normally about 1 to 3% of blood is in methaemoglobin state

Methaemoglobinaemia pathogenesis

Reductive pathways to protect RBC from oxidative damage include?

1. Reduced by NADH dependent methaemoglobin reduction pathway

2. Glutathione reductase pathway

Erythrocyte metabolic defect

b. Defects in ATP generation inc?

i. Hypophosphataemic haemolysis

ii. PFK & PK deficiency

Defects in ATP generation - Hypophosphataemic haemolysis - pathogenesis

Severe hypophosphataemia can be life

threatening, y?

Severe hypophosphatemia may lead to reduced RBC ATP production (energy) (& decreased glutathione) → unstable RBC membrane → haemolysis

Depressed myocardial function, rhabdomyopathy, seizure, coma, acute respiratory failure

Defects in ATP generation - Hypophosphataemic haemolysis

Causes inc?

1. Post Parturient Haemoglobinuria – cattle (most common)

2. Hyperinsulinism associated with hyperglycaemia (domestic animals)

3. Sporadic hypophosphatemia (horses, dogs & cats)

4. Artefact – bilirubin interference

Defects in ATP generation - Hypophosphataemic haemolysis - Post Parturient Haemoglobinuria – cattle

Pathophysiology

DX?

What can complicate it?

3 - 8 weeks post calving (sporadic multiparous cow)

Defective phosphorus mobilization from bone

Increased phosphorus loss via milk

Laboratory diagnosis

Anaemia – moderate to marked intravascular haemolysis (haemoglobinaemia & haemoglobinuria)

Hypophosphataemia

There is decreased ATP and Glutathione

Complicated with concurrent ketosis. Ketones potentially generate in vivo oxygen radicals – that can lead to formation of Heinz bodies

Defects in ATP generation - Hyperinsulinism hypophosphataemia haemolysis

Pathogenesis?

Hyperinsulinism – promotes movement of phosphate and glucose into cells other than RBC’s

(Insulin not needed for glucose to enter RBC’s)

Defects in ATP generation - Hyperinsulinism hypophosphataemia haemolysis

Cat signalment?

Pathogenesis?

Diabetic cats polyuria may lead to hypophosphataemia - hence haemolysis

Complicated like post parturient cattle they may have concurrent ketosis

Therefore combination of Hypophosphataemic & ketotic (Heinz body) haemolytic anaemia

Hypophosphataemia – artefact Bilirubin interference

Analyser & method dependent

Therefore be careful of interpreting the concurrent presence of hypophosphataemia and haemolytic anaemia in the presence of hyperbilirubinaemia

Regenerative anaemia – RBC fragmentation

Erythrocyte Fragmentation causes inc

1. Intravascular coagulation (localised or disseminated)

2. Vasculitis

3. Haemangiosarcoma

4. Rheological process - Cardiac valvular disease

Erythrocyte Fragmentation

What are these

Caused by?

Left - Schizocytes

Middle - keratocytes

Right - Acanthocytes

Form when blood forced to flow through an altered vascular channel or turbulent blood flow.

Erythrocyte Fragmentation

• Erythrocyte fragmentation in blood due to physical injury

Features and the ass. dz?

Fibrin – traumatic injury to RBC membrane

• Intravascular coagulation (localised or DIC)

• Vasculitis

Rheological forces (blood flow)

• Cardiac valvular Dz

• Caval syndrome or dirofiliariasis

“Membrane lipid changes” but cells are more fragile due to the forces - Haemangiosarcoma

Schistocyte or schizocyte

Which is correct for fragments, why?

Pathogenesis?

Schizo = cut

“any shaped” irregular small piece of RBC

Same conditions as keratocytes

DIC

Haemangiosarcoma

Endocarditis

Caval syndrome

What this?

Describe appearance

Keratocyte – Helmet or blister cell

RBCs with one or more ruptured/intact clear vesicles

• Ruptured ones make cell looks like it has 2 projections

• They are areas of apposed & sealed membrane rather than a true vesicle

Keratocytes - pathogenesis

Low numbers of keratocytes?

Chemicals?

When is it significant?

Low numbers seen in many situations but may not have significance e.g. healthy cats

Collection in EDTA may increase their presence. Make a blood smear immediately

When present in larger numbers or with other poikilocytes

Keratocytes - pathogenesis

When present in larger numbers or with other poikilocytes, keratocytes can indicate the following…

Fragmentation injury

Oxidant injury

Liver disease

Keratocytes - pathogenesis

When present in larger numbers or with other poikilocytes, keratocytes can indicate Fragmentation injury

Describe

Microangiopathic hemolysis (DIC, vasculitis, hemangiosarcoma) (fibrin) mechanical fragility, e.g. iron deficiency anemia. (schistocytes & acanthocytes)

Keratocytes - pathogenesis

When present in larger numbers or with other poikilocytes, keratocytes can indicate oxidant injury

Describe

Keratocytes may accompany eccentrocytes, & possible Heinz bodies, depending on the oxidant

Keratocytes - pathogenesis

When present in larger numbers or with other poikilocytes, keratocytes can indicate Liver disease

Describe

In cats, inc keratocytes in liver dz e.g. hepatic lipidosis

• Not sure why, may be due to mechanical fragility from altered phospholipid or cholesterol composition of the RBC membrane (membrane rigidity) or DIC

What this?

Sig in dogs?

Goats?

Acanthocyte

Dogs - Diseases with increased fragmentation e.g.

• Haemangiosarcoma

• Liver disease

• DIC

• glomerulonephritis

Young goats: normal as have haemoglobin C when young

Acanthocyte - pathogenesis

Dogs?

Mechanism is currently unknown. They might be present due to fragmentation (DIC, Fe deficiency)

Acanthocytes – clue for haemangiosarcoma (dogs)

Acanthocytes could be considered as a “blood biomarker” for haemangiosarcoma

Their presence in significant numbers should prompt?

Lab Aspects?

Basic workup including abdo(splenic or hepatic tumor) & cardiac (atrial tumor) US

Strongly regenerative anaemia if the spleen ruptures

Sometimes NRBC’s

Frequently accompanied by schistocytes & thrombocytopaenia suggesting fragmentation injury. (DIC)

Dogs and liver disease features acanthocytes, what else?

Presence of anaemia is variable, non or regenerative

depending on primary cause (hepatitis, infiltrative neoplasia (lymphoma), liver failure.

Regenerative anaemia - Infectious

Infectious agents associated with erythrocytes inc?

Anaplasma marginale

Anaplasma centrale

Babesia spp.

Distemper

Mycoplasma spp.

Mycoplasma haemocanis

Mycoplasma haemofelis

Candidatus Mycoplasma haemominutum

Theileria spp.

Regenerative anaemia - Infectious

Presence/Absence of organism?

Degree of clinical dz?

Many of the infectious agents have?

Don’t confuse what with agents?

Presence of an organism is diagnostic but its absence does not rule out the infection.

The degree of parasitaemia does not necessarily correlate with the degree of clinical disease

A concurrent immune mediated component (check for

spherocytes & agglutination)

Refractile artefact or stain precipitate

Regenerative anaemia - Infectious

Blood smear aspects?

Send fresh blood smears made at the time of collection of the EDTA, as epicellular organisms may dislodge from the erythrocyte membrane.

Capillary blood smears (prick ear) may be more helpful than normal jugular or cephalic vein.

Buffy coat smears may help concentrate some infectious agents

Infectious agents associated with erythrocytes

Mycoplasma aspects?

Thus, if confirmation of the parasite is sought from an outside consultant, what do?

Next steps in this case?

Mycoplasma are on outside of RBCs → blood smears should be made soon after collection lest they dislodge from membrane

• If dislodged they are free in extracellular space and look a lot like stain precipitate

Premade blood smears should be submitted along with an EDTA tube of whole blood

NEXT STEPS

Look for underlying disease (FeLV, neoplasia, spleen, immunocompromised)

Follow up with PCR

Check blood smear for agglutination as often a concurrent immunemediated component to the anaemia.

Other causes of haemolytic anaemia?

1. Heparin induced haemolysis

2. Envenomation (snakes)

Dots – not to be confused

Look around – look for the company they keep to help you distinguish

Describe this?

Howell – jolly bodies & basophilic stippling may be increased with a regenerative anaemia (polychromatophils)

Distemper usually has leukopaenia & lymphopaenia & inclusions can be present in RBCs & WBCs.

Dots - Which is what?

Heparin induced haemolysis

In who? How?

Pathogenesis?

Some horses - heparin anticoagulant therapy → RBC agglutination

• 6-8 hrs after TX

• Resolves after 5 days

Anaemia pathogenesis unclear - potentially RBC destruction in agglutinated groups in vivo

Hyperbilirubinaemia (due to inc RBC destruction)

Envenomation Venom from some snakes, spiders & insects may cause haemolysis

Mechanisms/pathogenesis include?

Activate complement (cobra venom factor)

Haemolysins causing direct haemolysis (e.g. phospholipase A2 in rattlesnake venom)

Bee stings

– haemolysins (phospholipase A2)

– spherocytic haemolytic anaemia

(altered membrane structure or antibody mediated)