Hematopoiesis and RBC Evaluation

VMED 5175

What is clinical pathology?

Subspecialty of pathology that utilizes laboratory assays and microscopy to evaluate bodily fluids (blood, urine, cavitary effusions) or tissue aspirates to screen for disease

What is the level of clinical pathology you will experience as a first year student?

Introduction to hematology and clinical chemistry

What is a CBC?

Complete Blood count

What is the etymology of hematopoiesis?

Blood + to make

What cells are produced in hematopoiesis and why are they needed?

Erythrocytes (RBC)- carry oxygen to tissues

Leukocytes (WBC)- defend against infectious organisms

Thrombocytes (PLT)- form clots

What type of cell does hematopoiesis start with?

Hematopoietic stem cells (HSC)

long term self renewal and differentiation

gives rise to additional HSCs

What are the two possible paths of HSC?

1) HSC --→ Common Myeloid Progenitor

2) HSC → Common Lymphoid Progenitor

What is a progenitor cell?

no long term self renewal

divides only a limited # of times

What is a Common Myeloid Progenitor cell

megakaryotic (platelet precursors) and erythrocytic precursors

granulocyte and monocyte precursors

What is a Common Lymphoid Progenitor cell?

B lymphocytes or NK cells

Leaves marrow to form T lymphocytes in the thymus

What is meant by lineage specific precursors?

a name for the way blood cell precursors are grouped based on what they will become (erythropoiesis, granulopoiesis, and thrombopoiesis)

What is the progression of hematopoiesis in embryonic and fetal development?

yolk sac → aorta-gonad-mesonephros → liver >>> spleen → bone marrow

HSC seed into respective sites

What are the two kinds of hematopoiesis in adult animals that determine where it takes place?

Intramedullary and extramedullary

Where does Intramedullary hematopoiesis occur?

axial skeleton and epiphyseal region of long bones

PRIMARY

Where does Extramedullary hematopoiesis occur?

spleen and liver

What organ may be an additional site of hematopoiesis in lower vertebrates such as fish?

kidneys

Where in the bone is bone marrow found?

within cavities of cortical bone with trabecular bone radiating out of the bone cortex

Where are the hematopoietic spaces?

Between venous sinusoids, extracellular except in birds!

How are bone marrow sinusoids arranged to maintain the blood marrow barrier?

Trilaminar arrangement

What is the trilaminar arrangement to maintain the blood-marrow barrier?

1) endothelial cells

2) basement membrane

3) adventitial reticular cells

How are Bone marrow sinusoids arranged to create an entry point for maturing cells?

early precursors are closer to the bone, while later precursors sit closer to the barrier for easy access

What is the function of bone marrow sinusoids?

delivers nutrients, removes waste

How is the up/downregulation of hematopoiesis controlled?

Stromal cells, matrix, and cytokines

How does the bone marrow microenvironment signaling cascade control stem cell production

need based

* if anemia is present, accelerated erthyropoiesis expected
* if erythrocytosis is present, should be halted via apoptosis of erythroid precursors

how are cells released from the marrow into circulation?

* precursors lose adhesion proteins that anchor them to the matrix
* pericytes (aka adventitial reticular cells) retract cytoplasmic processes
* cells exit through the temporary pores in the cytoplasm of the endothelium (transendothelial migration)

where are the non-motile platelets and late erythroid precursors in relation to the sinusoids?

close with easy access to the blood

Where are motile neutrophils and monocytes located in relation to the sinusoids?

deeper in the hematopoietic cords and will migrate when they are mature

On a CBC, how is increased hematopoiesis marked?

erythrocytosis, neutrophilia, thrombocytosis

On a CBC, how is decreased hematopoiesis marked?

anemia, neutropenia, thrombocytopenia

How is abnormal hematopoiesis marked on a CBC?

Leukemia, dysplasia

What is Erythropoietin?

how are they synthesized?

how are they stimulated?

What is their job?

the main regulator of erythropoiesis, synthesized by renal peritubular interstitial cells

stimulated by hypoxia

prevents apoptosis of erythroid precursors

What are the other stimulators for erythropoietin?

glucocorticoids, growth hormone, thyroid hormones

What is the marrow transit time for a rubriblast to develop into a mature erythrocyte?

1 week, shorted in presence of EPO

What morphologic changes occur during erythroid differentiation?

cytoplasm becomes less basophilic and more eosinophilic (loss of ribosomal RNA, gain of hemoglobin)

Cell and nuclear diameter decreases

nuclear chromatin condenses to a pyknotic state and is eventually extruded from the cell

What functions of RBC depend on hemoglobin?

gas exchange, acid-base buffering

Up to what stage must hemoglobin be synthesized with precursors?

reticulocyte stage

What does each hemoglobin molecule contain?

4 heme groups with central iron

4 globin chains (2 alpha, 2 beta)

O2 binds iron in heme group

How is iron gained in the body for hemoglobin to use?

acquired through diet and transferred to bone marrow to be stored in macrophages

macrophages provide iron to erythroid precursors

What is the fat of reticulocytes in most domestic animals? What is the exception?

released from the marrow and continue maturation in the spleen or in circulation

horses

how long do erythrocytes circulate?

until they are senescent (old)

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How long do erythrocytes circulate in horses and cattle?

150 days

How long do erythrocytes circulate in dogs?

100 days

How long do erythrocytes circulate in cats?

70 days

What happens to erythrocytes when they are senescent?

express markers that signal macrophages to phagocytize them

physiologic hemolysis

Describe normal erythrocytes

Anucleated discocytes

* biconcave disk maximizes SA and deformability
* central pallor most prominent in dogs

What are the exceptions to the anucleated discocytes?

Camelidae family- elliptocytes

White tailed deer- artifactual sickle shape

some goats- artifactual poikilocytes

non-mammals-nuclear elliptocytes

What are the three main functions of erythrocytes?

transport O2 to tissue

transport CO2 to lungs

buffer H+

What is the function of the erythrocyte membrane and what is it made of?

outer lipid bilayer

maintain deformability/durability

semi permeable for gas exchange

maintains environment for Hb function

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What are the three oxidants animals are exposed to?

hydrogen peroxide, superoxide, hydroxyl radicals

Why are RBCs extra susceptible to oxidants?

carry oxygen

exposed to chemicals in the plasma

What is the danger of oxidants to RBC? How do they protect themselves?

can damage hemoglobin, enzymes, and lipid membranes

generate antioxidants

What is erythron?

RBCs and their precursors in the blood and marrow

includes circulating erythrocytes, erythroid precursors, and sequestered erythrocytes (splenic storage pool)

What is the evaluation of the erythron?

an erythrogram, a component of a CBC

What are the components of the data gained from erythrograms?

quantitative- cell counts

qualitative- morphology

combination of instrumentation and blood smear eval

What values are included in the Circulating RBC mass?

hematocrit (%)

PCV (%)

Hb (g/dL)

RBC concentration (x10^6/microliter)

What values are included in erythropoietic activity?

Reticulocytes (X10^3/microliter)

RDW (%)

What values are included under Indices?

MCV (fL)- size

MCHC (g/dL)- Hb content

CHCM (g/dL)- Hb content

What is PCV and how is it found?

Packed cell volume (manual HCT/spun HTC), % of packed RBCs that occupy a given volume of blood

blood is centrifuged in a capillary tube, and packed erythrocytes at bottom are measured with a chart

What are the benefits of PCV?

Evaluate plasma color (icteric, lipemic)

plasma available for total protein and fibrinogen measurements

buffy coat assessment

Potential errors with PCV

plasma trapping w/i RBC will increase

Excess EDTA dehydrates RBC, falsely decreasing

What is HCT and how is it calculated?

hematocrit = MCV x \[RBC\]/10

generated by hematology analyzer

what is the major issue with HCT?

only as accurate as the values used to calculate it!

ensure analyzer is validated for species being tested

any artifact in MCV or RBC will affect

Within in what % should HCT be of PCV?

3%. If >3%, evaluate for errors

What test is preferred in human medicine for RBC mass?

Hemoglobin

what is the preferred test for RBC mass in vmed?

PCV or HCT

what % of RBCs are Hb?

33%, thus Hb of whole blood is 1/3 of HCT

How is hemoglobin measured?

RBCs are lysed to release Hb, which is converted to cyanmethemoglobin to be measured on a spectrophotometer

What substances can cause a falsely elevated Hb?

any that interferes with sample turbidity, including lipemia, intravascular hemolysis (hemolyzed plasma), and heinz bodies

What is \[RBC\]?

concentration of RBCs (millions/microliter) in whole blood, differentiated from platelets and WBCs

How is \[RBC\] measured?

automated impedance counter

* cells are diluted in a solution and drawn through an aperture
* electrical resistance across the aperture changes with each cell, recording size and number

What is the “gold standard” of \[RBC\] measurement? How?

automated flow cytometer

cells pass individually through a laser beam, information (size, #, internal complexity) obtained by the scatter of the laser at different angles

What can cause false \[RBC\] readings? In what direction?

agglutination and clotted samples can decrease

if anemia is present, how should bone marrow respond?

increased erythropoietin → Increased erythropoiesis → increased release of reticulocytes

What does the concentration of reticulocytes tell us veterinarians about anemia? What animal is the exception?

regenerative vs nonregenerative

horses

how are reticulocytes enumerated?

new methylene blue stains precipitate RNA for ID

can be performed manually or automated

What is polychromasia?

RNA remaining dispersed in reticulocytes and observed on routine staining via Wright’s stain

degree correlates with reticulocyte concentration

How is the absolute reticulocyte count calculated?

reticulocyte % multiplied by \[RBC\] to generate in 10^3/microliter

What absolute reticulocyte count represents regenerative anemia in dogs? in cats?

dogs: >80,000-100,000

cats: >60,000

How is manual reticulocyte count taken? What group of animals is this best for?

NMB stained bloodsmear used for % up to 500-1,000 erythrocytes

best for ruminants

calculation of absolute reticulocyte concentratoion

ARC= % retic X \[RBC\]

What are normal reticulocyte ranges in dogs?

1\.5% of erythrocytes are reticulocytes

What might cause reticulocytosis in dogs without anemia?

corticosteroids, NSAIDS, or dietary glucosamines

What is unique about reticulocytes in cats?

maturation is slower (over 2 weeks) resulting in both aggregate (clumped ribosomes, analogous to polychromatophils) and punctate (single, individual granules not counted) reticulocytes

What is unique about reticulocytes in cattle?

a % >1 is considered regenerative because of v low numbers except in cases of severe anemia

What is unique about reticulocytes in horses?

undergo maturation in marrow, not in blood

what is RDW? How is it calculated?

red cell distribution width, degree of variation in RBC size

analogous to anisocytosis

MCV could be WRI if both smaller and larger RBCs are present

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RCW= SD/Mean

What is MCV?

Mean corpuscular volume = average RBC size measured in femtoliters

picks up species differences in size and amount!

automated measurement by impedance or flow cytometry measuring size and not shape

What does increased MCV indicate?

larger erythrocytes, macrocytosis

What does decreased MCV indicate?

smaller erythrocytes, termed micricytosis

What artifacts can affect MCV

agglutination, hyperosmolarity, hypoosmolarity

what is agglutination in the context of MCV?

RBC doublets or triplets counted as 1 large RBC, and larger will not be counted at all

results in false low \[RBC\] and false high MCV

What is hyperosmolality in the context of MCV?

•RBCs flowing through hyperosmolar plasma (e.g., patients with hypernatremia or hyperglycemia) will become hyperosmolar in vivo to maintain osmolality



•When this blood is collected for CBC analysis, these RBCs are then diluted by the hematology analyzer with physiologic saline, which is hypoosmolar relative to the RBCs.



•Saline will then cause RBCs to swell, leading to artifactual macrocytosis

What does hypoosmolality cause in MCV readings?

artifactual microcytosis

What is MCGC? How is it calculated?

•Mean corpuscular hemoglobin concentration = relative concentration of Hb in RBCs



•Calculated value from aforementioned measured Hb; MCHC = Hb/HCT x 100

What is the normal value for MCHC? Why?

33g/dL (RBC contain 33% Hb)

What is increased MCHC? Is this possible?

hyperchromasia

No, it is always caused by artifacts (usually lipemia or hemolyzed plasma)

What is decreased MCHC?

hypochromasia

What is CHCM?

cell hemoglobin concentration mean = direct measurement of Hb w/i RBC

optical measurement uses flow cytometry based on light scatter, RBCs NOT LYSED

How is CHCM related to MCHC?

analogous, but will not be affected by plasma appearance such as lipemia. In these cases, MCHC will be elevated and CHCM will be WRI

What can affect CHCM?

RBC inclusions such as Heinz bodies or membrane changes like pyknocytes

anything that changes light scatter