Automated Analyzers and Erythron

Erythrocytosis definition

too many red blood cells

Polycythemia definition

too many red blood cells

Polycythemia (Erythrocytosis) is classified into what 2 groups?

Relative and Absolute

Define relative polycythemia

change in RBC numbers with respect to plasma.

Not a true increase in RBC mass

Examples of relative polycythemia

Dehydration- RBCs increased because plasma volume decreased

Splenic contraction- excited animal

Define Absolute polycythemia

RBCs are increased

Examples of absolute polycythemia

RBC tumor in marrow

Increased erythropoietin from kidney tumor

Hypoxic disorders such as respiratory/ cardiac disease

What is anemia classified by?

Morphology

Marrow response

Pathophysiologic mechanism

RBC indices that classify morphology

MCV- average size of RBCs (microcytic, normocytic, macrocytic)

MCH- Average amount of hemoglobin (hypochromic, normochromic)

MCHC- Hb/ RBC= average amount of hemoglobin in each RBC (hypochromic, normochromic)

bigger RBC have more hemoglobin

Classification of marrow response

non regenerative

regenerative

Causes of non regen anemia

• chronic kidney disease

• anemia of inflammatory disease

• marrow tumor

• nutritional deficiency

Early anemia as all anemias are initially non regenerative in the first how many days?

3-5 days

Regenerative anemia suggests what?

extra marrow cause such as hemorrhage or hemolysis

What is macrocytosis due to

Polychromatophils (immature RBC larger than normal)

What are reticulocytes

polychromatophils with new methylene blue stain

When counting cat reticulocytes, what do we only count?

aggregate reticulocytes

What do metarubrocytes and basophillic stippling without anemia suggest?

lead poisoning

What is basophilic stippling

RNA remnants that stain blue, common in youngs ruminants with regen anemia

features of RBC regeneration

Howell jolly bodies

Basophilic stippling

Metarubricytes

Macrocytosis

Reticulocytes

Pathophysiological Mechanism causes

Blood loss (hemorrhage)

Erythrocyte destruction (hemolysis)

Reduced/ defective erythropoiesis

How does blood loss anemia occur externally?

Iron loss occurs with external bleeding

Iron access from storage is slow

storage iron gets depleted

RBCs are lost with external hemorrhage

How does blood loss anemia occur internally?

Reutilization of RBCs

recycling of iron from internal bleeding sites occur

Classifications of blood loss anemia

acute and chronic

Examples of acute blood loss

trauma, surgical bleed, hemangiosarcoma rupture

What happens with acute blood loss anemia

hypovolemia occurs

PCV and plasma protein appear normal until blood volumes restored over several hours

What happens in Acute blood loss anemia as blood volume is restored in non regen?

PCV ad plasma protein decrease

After 3-5 days after acute blood loss, what happens in regen?

a regenerative anemia becomes evident

Reticulocyte count high

plasma proteins restore to normal

by 1-2 weeks PCV and plasma proteins are back to normal after single bleed

What is associated with a common splenic tumor called hemangiosarcoma?

Acanthocytes

Examples of chronic blood loss

parasitism

GI ulcers

tumors

thrombocytopenia

What occurs during chronic blood loss

anemia develops slowly and hypovolemia does NOT occur

What happens if chronic blood loss is external?

depletion of iron stores and non regen anemia (microcytic, hypochromic) develops

What develops due to chronic blood loss, apart from anemia?

Hypoproteinemia develops as the liver cant keep up with the manufacture of plasma proteins

Common limitations of all automated analyzers

• partially clotted samples/ clumped cells arise due to a difficult blood draw

• overfilled EDTA tube not enough anticoagulant causes partial clots

• These present problems to analyzer as it tries to sort and count cells

issues with many types of analyzers

nucleated RBCs counted as lymphocytes

bands and other immature neutrophils not distinguished

large lymphocytes, including lymphoblasts may be counted as monocytes

Which idexx machine flags bands/ nucleated RBCs

Idexx procyte

What will clumped platelets look like on a dot plot?

Clumped platelets are variable in size and granularity and appear with the WBC population

what are automated analyzers good for?

identifying what is normal

What do you look for in 10x objective

platelet clumps, microfilaria, abnormal cell types (feathered edge)

RBC density, rouleaux, agglutination (monolayer)

What do you look for in 100X objective?

platelet count

RBC possibilities, leukocytes (toxic change, immature), or platelets with abnormal morphology

WBC differential

Causes of Hemolysis

immune mediated

neonatal hemolytic anemia

erythrocyte parasites

oxidants

microorganisms

Lab findings in hemolytic anemia

regenerative anemia after 3-5 days

-higher retics than hemorrhagic because iron is more available

+/- abnormal RBC morph

+/- hemoglobin/bilirubin in plasma/urine

depending on whether hemolysis is intra or extra

What is intravascular hemolysis

destruction of rbc within the blood stream

What happens internally in intravascular hemolysis

hemoglobin release into blood stream- present in plasma and small molecule that can pass through filtration and be present in urine (hemoglobinuria)

Destroyed rbc can release bilirubin- liver attempts to filter (presence in urine or blood)

What is extravascular hemolysis

macrophages (mainly in the spleen but also liver and bone marrow)

What happens internally in extravascular hemolysis

Hemoglobin trapped in macrophages- never released into bloodstream

usually rate of destruction is slower than intravascular and the liver can keep up with bilirubin conjugation and excreting. Usually do not see bilirubin in urine or plasma

Would you find hemoglobinemia/ urea in Intravascular or extravascular hemolysis

intravascular

Describe immune mediated hemolytic anemia

dogs

most common type of hemolysis

Cause often not identified

• primary idiopathic, secondary related to infections, other immune mediated diseases, live vaccines, neoplasia or drugs

• slightly more females than males

• prognosis is variable 25-50% mortality

Which breeds of dog are more susceptible to immune mediated hemolytic anemia

little white crusty dogs, poodles, collies, pinschers, sheepdog, English spaniel

describe Immune mediated hemolysis in cats

most commonly associated with FeLV

Lymphoid/ marrow neoplasia, primary IMHA (idiopathic)

Immune mediated hemolysis in horses

seen with penicillin/ other antibiotic use, clostridial infection and neoplasia

What disease causes Immune mediated hemolysis in cattle

reported with anaplasmosis

How to treat immune mediated hemolysis

The primary cause is identified and immune suppression (prednisone/ prednisolone, cyclosporine)

How does immune mediated hemolysis work internally

antibodies directed against the RBC membrane or immune complexes have attached to the RBC membrane

In Immune mediated hemolysis, what does the method of destruction depend on

type of antibody and its ability to activate complement

Describe what happens to the antibody IgG in immune mediated hemolysis

Macrophages have receptors for it, poor at activating complement

Describe what happens to the antibody IgM in immune mediated hemolysis

good activator of complement

What is the complement system

part of your bodies immune system

cascade system of a large number of plasma proteins

help fight off infection, induce inflammatory responses and can directly destroy microscopic organisms

IgG mediated Hemolysis

RBCs become coated with IgG

Macrophages (mainly in spleen) phagocytize these RBCs

Clinical signs include pale mm, weakness, potentially minimal jaundice

In spherocytes, the surface of the RBC is coated with what?

coated with antibodies. Macrophages phagocytize part of the cell membrane without loss of hemoglobin

What happens to spherocytes in the body

eventually trapped and removed from the bloodstream because they are not flexible enough to fit through the splenic filter

inflexible RBCs from any cause are removed this way ex. heinz and mycoplasma

What do spherocytes look like

small, dark, round RBCs, no central pallor

Describe IgM mediated hemolysis

RBC is coated with IgM and complement is activated

complement perforates the membrane causing rupture

clinical signs are acutely ill animal with pale mm, hemoglobinuria/emia, +/- jaundice, weakness and possible shock

Describe ghost cells in IgM mediated hemolysis

ghost rbcs are free hemoglobin are seen with intravascular hemolysis

+/- hemoglobinemia/ uria and/or bilirubinemia/ uria

Lab findings in immune hemolytic anemias

very regenerative anemia if more than 3-5 days

leukocytosis with high neutrophil/bands

agglutination in immune mediated hemolysis

occurs easily with IgM and sometimes IgG

antibodies are holding onto the rbcs and clumping them

can be macroscopic or microscopic

varying amounts occur

saline agglutination test

What does a Coombs test tell us

positive coombs test detects the presence of IgM, IgG or complements on RBC

How does a Coombs test work

RBCs are washed with saline several times to remove any unbound antibody/ complimet

reagent containing anti- IgG, anti-IgM and anti complement is added to the washed RBCs and the mixture is observed for agglutination

What antibody is run at 37C for the coombs test

IgG

What antibody is run at 4C for the coombs test

IgM

What is neonatal isoerythrolysis (NI)

Hemolytic anemia of the newborn

Describe NI

Animal blood type incompatibilities are rare

normal at birth, but within 1-2 days they become weak, pale, anemic and icteric

Blood groups in horses

A, C, D, K, P, Q, U

How many surface molecules (factors) are identified in horse blood

over 30 factors

How many horse blood types

400,000 possibilities

Which RBC antigens produce a strong immune response

Aa or Qa

What of the requirements for everything to go wrong in a NI case

Mare is not blood Aa or Qa

stallion has Aa or Qa and passes along to foal

mare has antibodies to this blood type from previous exposure

once foal is born, ingest colostrum containing mare antibodies

hemolysis occurs

How to prevent NI in foals

1) identify at risk mares

2) blood test the pregnant mare

3) perform a jaundiced foal agglutination test pre-suckle

Types of RBC parasites

Mycoplasma in cats most common

anaplasma marginale in cattle

Mycoplasma in pigs

Babesia- dogs, cattle, horses, rarely cats

What was Mycoplasma hemofelis called before

hemobartonella

How would you test for mycoplasma hemofelis

careful examination under oil, also PCR test

How is Mycoplasma hemofelis transmitted

arthropod transmitted (primarily flies)

Clinical signs of mycoplasma hemofelis

depressed, febrile, pale, anorexic, +/- jaundice

FeLV/ FIV 50% are positive

treatment of mycoplasma hemofelis

doxy, immune suppression with prednisone, blood transfusion if critical

What can hemoglobin oxidation result in

1) methemoglobin- iron is oxidized and cant carry oxygen, blood appears brown

2) Heinz bodies- globin is oxidized and precipitated. Visible as eccentrically located refractile bodies or blebs on RBC. Stain blue with NM blue. But normal in cats up to 10%

3) eccentrocytes- RBC membrane is oxidized. Visible as an eccentric

Plants that cause oxidant hemolytic anemia

onions, chives, garlic

cabbage kale, canola affect ruminants

wilted/ dry red maple leaves in horses

Drugs/ chemicals causing oxidant hemolytic anemia

acetaminophen in cats

zinc in dogs

copper in ruminants, esp sheep

propofol in cats

Diseases that cause oxidant hemolytic anemia

diabetes mellitus (ketones cause oxidative damage)

What is EIA

equine infectious anemia

Describe equine infectious anemia

viral disease

spread by biting infects (flies)

fever, weakness, low platelet, icterus, edema

Intravascular hemolysis due to virus binding RBC’s

Coggins test is used

No treatment

Microorganisms causing hemolytic anemia

leptospirosis in cattle, sheep and dogs

Clostridium hemolyticum in cattle and sheep

Describe what happens from leptospirosis and clostridium

intravascular hemolysis due to organisms producing hemolysin toxin

icteric and hemoglobinemia/uria

control with vaccines

What causes anemia from defective/ reduced erythropoiesis

1) marrow destruction

2)nutritional deficiencies

3) lack of erythropoietin

anemia is non regenrative

What causes defective erythropoiesis anemia from bone marrow destruction

radiation

chemicals

tumors

What might anemia look like in nutritional deficiencies from lack of iron

lack of iron due to dietary deficiency or blood loss

anemia can look:

-microcytic

-hypochromic

nutritional deficiency anemia from copper

low copper due to animals grazing on forage low in copper

copper is a component of ceruloplasmin

anemia is microcytic and hypochromic

nutritional deficiency anemia from protein

protein is required for many aspects of RBC production

anemia is normochromic and normocytic

anemia of chronic inflammation or disease

often develop mild non regenerative microcytic, hypochromic anemia

can be due to iron being diverted into hemosiderin stores, becoming unavailable for rbc production

decrease iron absorption

decrease rbc lifespan

anemia from lack of erythropoietin causes

kidney disease

decreased erythropoietin production

anemia is normochromic and normocytic