Week 4

Question 1

in adults where is the hematopeotic tissue located

Answer 1

BM and lymph nodes, spleen , liver and thymus

lymphoid cells develop in two places primary and secondary lymphoid tissue

the primary lymphoid tissue is the BM and thymus where B and T lymphs develop is the 2ndary

BM - has precursor cells

Question 2

What is the primary site of hematopoisis in adults

Answer 2

BM - medurally - central cavity with yellow and red marrow

Can take place outside the BM because of infection = Extra medullary non BM

Its not always pathological because it occurs during fetal stages

Question 3

What are the fetal stages of hematopoiesis

Answer 3

Mesoblastic -

Hepatic

Medullary

Question 4

What happens at the Mesoblastic -
stage

Answer 4

-in mesoderm of yolk sac
-production of primitive erythroblasts in forming blood vessels
-production of hgb

Question 5

What happens at the hepatic stages

Answer 5

-5-7 weeks
-occurs extravascular in the liver
-fetal liver is main site and continues 1/2 weeks after birth
-peak of hematopoises by 3rd month and decline at 6 months
-production of RBC, WBC, megakaryocytes mostly in liver and spleen
-spleen( make b cells) , kidney,( make b cells) thymus (make t cells) and lymph nodes help

Question 6

What happens at the medullary stage

Answer 6

-hematopoiesis starts in BM at 5 months
-at 24 weeks it is the primary site of hematopoiesis
-all cell lines at different stages of maturation seen

Question 7

What types of changes would we see in the BM

Answer 7

Infants have all red marrow and in 5-7 years it is replaced by adipose tissue
-red is hematopoietic reactive marrow
with developing RBC and progenitors
-active marrow

By adulthood , all long bones are yellow marrow
-sternum, vertebrae, scapulae, pelvis, ribs, skull and proximal parts of long bones are the only ones that are active
-yellow can reactivate when responding to hemolysis or bleeding
-yellow is hematopoietic but made up of up adipose tissue
-inactive marrow

Question 8

How is the marrow supplied with its nutriets and oxygen

Answer 8

nutrient artery which enters the bone and branches out into the sinuses

Question 9

how is the red marrow arranged

Answer 9

in extravascular chords made up of hematopoietic cells that are found in between vascular sinuses and supported by trabeculae , stroma cells, extracellular proteins, collegen

red marrow is active
yellow is adipose tissue

Question 10

What are stroma cells comprised of

Answer 10

Macrophages - phagocytosis, cytokine secretion

fibroblasts - extracellular matrix formation

osteoclasts - absorbs/ removes bone

osteoblasts - forms bone

endothelial cell- line the sinus and control passage of hematopoietic cells into circulation

Reticular cells - forms meshwork

stroma cells secrete fluid extracellular matrix

Question 11

What does the extracellular matrix secreted by stroma cells do

Answer 11

• anchors developing hematopoietic cells in the bone cavity
  -made up of reticular fiber, collagen , growth factors and cytokines that stimulate hematopoiesis

Question 12

How are hemotopoeitic cords arranged

Answer 12

• grouped in islands
  -islands and megakaryocytes develop close to sinuses
  -while myeloid cells develop deep in the cords

Question 13

What do erythropoietic islands contain

Answer 13

• maturing erythrocytes that surround macrophages containing iron
  -macrophages form the island base so they are able to be an anchor for erythroid precursors to control their release into circulation via stromal cells
  -macrophages release cytokines to help with red cell maturation and are the source of ferritin in hgb production
  -the rest of the ferritin is provided by transferrin -the most

Question 14

What controls erythropoiesis

Answer 14

Epo- hormone and stimulatory cytokine for erythropoiesis
-produced by peritubular cells in the kidney when you are in a state of hypoxia
-this hormone fluctuates to maintain homeostasis in O2

Question 15

What causes the hypoxia that induces EPO production

Answer 15

Low RBC count via hemolysis or bleeding
-abnormal hgb

not related to red cells - lung function or high altitude

epo binds to receptor on RBC precursors that leads to cell division, maturation with more cells entering circulation
-BM stores more RBC precursors than needed in case there is increased need because you cant store mature RBC
-when these RBC are not needed they will undergo apoptosis and macrophages will remove dead cells

Question 16

What is the action of EPO

Answer 16

Inhibition of apoptosis
-bind receptors on erythroid progenitor cells and promotes anti -apoptotic molecule production that turns off the apoptic signal in cell

Early release of retics
-reduces adhesion receptors so immature cells can leave the erythroid islands

Reduced maturation time
- decreased cell cycle time (decreases tiem spent in mitosis) ,
-increased cell processes “short boot camp; less time maturing in marrow”

increase in EPO = larger bluer cells in circulation because EPO forces cells into circulation before theyre ready = increased poly

Question 17

What are BM studies used for

Answer 17

diagnose and stage hematologic and non hematologic neoplasms
- NOT for anemia when you can diagnose by looking at cells and indices
-multilineage abnormalities, blasts in adults and unexplained pancytopenia can prompt marrow exam
-prohibited in pt with coagulopathies like hemophilia or Vit K deficiency
-bridging with coumadin or heparin is needed to prevent uncontrolled bleeding

Question 18

What secondary conditions can warrant a BM study

Answer 18

-solid tumors
-infections with FUO
-hereditary and acquired histiocytosis (abnormal increase in histocytes)
-unexplained organomegaly

Question 19

aspirate vs core biopsy
types of BM specimen

Answer 19

Aspirate
apritation of semi fluid marrow
cells not in native state- can look at morph

core biopsy
-intact bone piece is removed
-cells are in native state
cannot be taken from all sites

common sites
-posterior superior iliac crest- common in adults and fat kids - asp and core
-sternum -adult , asp only
-anterior superior iliac crest- asp and core
-spinal processes - rarely used

children 0 upper end of tibia - asp only

Question 20

What types of needles used in BM collection

Answer 20

Trephine - for circular piece of bone
like disposable Jamshidi needle
-Gauge is 11-15 bore size
-must be sterilized after use
-EDTA
-FORMALIN

Question 21

What is the procedure for BM collection

Answer 21

Informed consent
-iodine is the antispetic
-local anesthetic - 2% lidocaine
-cut with scalpel after frozen

do not collect more than 1.5 ml

Question 22

What is done with the aspirate collected

Answer 22

• put in a tube with EDTA or watch glass
  -MLT looks for spicules or marrow tissue = grey bits floating in blood and fat droplets
  -seperate spicules , pick up with pipette and put on slide ASAP with NO ANTICOAG

-2 types of smears
-push = like blood film
-squash = when you squash spicule between 2 slides and pull apart

label slides properly
-stain slides 4-6 with Wrights Geimsa
-keep a few that are unstained and unfixed for cytochemical staining

Question 23

What is the role of the MLT during the BM collection process

Answer 23

making slides at bedside
-aspirate is collected in EDTA and brought back to the lab
-send the remaining for C/S, Molecular diagnostics, cytogenetics (heparin), flow cyto and histo if anything is left

Question 24

How is the BM biopsy processed

Answer 24

• placed in B5 or 10% buffered formalin Zenkers or Carnoy
  -processes by histo -core cut and stained by H&E and PPB

Question 25

Advantages and disadvantages to bone marrow sampling

Answer 25

- Ad represents marrow and bone structure in natural state -demonstrates iron, reticulum and collagen disadvantage -fine cellular details lost in fixation and processing -little value when trying to diagnose a leukemia -dont use for differential heme like asp and histo like biopsy

Question 26

What does a complete BM examination include

Answer 26

Peripheral blood smear – CBC & complete morphological review done 24 hours before collection Bone marrow aspirate Bone marrow core biopsy

Question 26

What are the problems that come with using an aspirate

Answer 26

DRY TAP -correct site but sample cannot be aspirated can happen if marrow is too dense -extreme hypercellular marrow -fibrosis or marrow need to take a BM core biopsy sample -try and make slides from material around bone core = touch preps

Question 27

What is a touch prep

Answer 27

-biopsy held by forceps and touched or rolled onto a slide BEFORE placing into formalin -fixed and stained like BM aspirate -youll be able to see enough cells to assess maturation stages and types or do a differential

Question 28

When you look at BM what do you look for

Answer 28

Cellularity Maturation of myeloid & erythroid series M:E ratio Cell distribution (Differential) Eosinophils/basophils Megakaryocytes Presence of other cells Lymphocytes Plasma cells Histiocytes Osteoclasts Fibroblasts Iron stores Abnormal infiltrates Fibrosis

Question 28

What to look for when evaluating a BM ASPIRATE

Answer 28

Relative cellularity - core is more accurate for cellularity Differential and M:E Iron stores Megakaryocyte Evaluation Metastatic tumor cells / Lymphoid aggregates- best on aspirate start on low power 10x to detect spicules -hematopoietic and stromal cells stained dark blue / purple -look for an intact area behind a spicule Assess general cellularity-all you can say is HYPO, HYPER or NORMAL cellular Abnormal cell clusters- seen on edges Quality of aspirate using Megakaryocytes 2-10 if more or less then pt condition

Question 29

What is the next step once you have found an intact area behind a spicule

Answer 29

- aspirate differential -done under 100x -count 300-500 nucleated cells to determine M:E ratio All nucleated cells: Myeloid series – Blasts – Segs: Neut, Eos, Baso Erythroid series Lymphs, Monos, Plasma cells – early cells as well in a normal BM a normal BM consists of myeloid and erythroids , all stages should be present but within their associated distribution -count and differentiate all

Question 30

What is your myeloid to erythroid ration

Answer 30

-derived from diff -normal ration with 1.5:1 - 3.3:1 -granulopoietic tissue occupies 1.5 to 3.3 times more space than erythroid precursors due to shorter survival of granulocytes in circulation -so changes in granulocytes and erytrocytes are reflected in M:E ratio -only take myeloid and erythroid cell #s from diff - exclude lymphs, monos, plasma

Question 31

how to evaluate iron stores in the bone marrow

Answer 31

use perls prussion blue -iron in BM is stored as intracellular hemosiderin in macrophages and in small amounts of normally developing erythroblasts -hemoglobin iron does not stain -positive iron is seen as blue deposits -aspirate is favored over biopsy for iron stain because when you process biopsy it tends to leach iron which can give you a false decrease or negative iron -enough spicules should be present because you need to know if the low iron is due to contamination or the pt lacks iron stores report iron as Absent Decreased Adequate Moderately or Markedly Increased

Question 32

how is the megakaryocyte evaluation doe

Answer 32

- scan the number on low power 10x -2-10 per lower power field -too unevenly distributed to count on diff so they are reported as increased or decreased -clusters can indicate hyperplasia -< 2 megakaryocytes / LPF (low power field) may indicate hypoplasia or poor quality aspirate -no spicules and no mega = low quality aspirate

Question 33

how to assess BM biopsy sample

Answer 33

look at cellularity The ratio of all nucleated hematopoietic cells (ANC) to fat cells cellularity in adults is approximately 50% ( 1:1) General rule to estimate normal cellularity for age: 100-age +/-10. Reported as Increased, Normal or Decreased so if youre 75 years old your cellularity is 15-35% A normal adult bone marrow displays 50% hematopoietic tissue and 50% fat. NORMAL CELLULARITY

Question 34

What does a BM report contain

Answer 34

Patient history, age, sex, etc. Description of samples received Peripheral blood data: CBC, ESR, Reticulocyte count, Manual differential, PBS Bone marrow differential count Description of the cellularity, M:E ratio Status of the iron stores Description of histological sections of biopsy Diagnostic conclusion

Question 35

What can cause BM failure

Answer 35

-reduction of cessation of blood cell production that affects 1 or more cell lines -no typical morph- just decrease in all lines : pancytopenia -Aplastic Anemia is a general BM failure syndrome pancytopenia, reticulocytopenia, bone marrow hypocellularity, low hetamo stem cells Destruction of HSC Premature senescence and apoptosis of HSC Ineffective hematopoiesis Disruption of BM microenvironment Decreased production of growth factors or hormones Loss of normal hematopoietic tissue due to infiltration of marrow by abnormal cells