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Low Power Bone Marrow examination
- @20x examine marrow to fat ratio
- normal= 1:1
- Hyper= >4:1, hypo = <1:4
High power BM evaluation
- @100x examine myeloid to erythroid ratio
- normal= 4:1 will also see macrophages, osteoblasts and osteoclasts
Why so many more WBC precursors in BM?
WBC dont live as long, so you need to be making more to keep up
Top 3 cells seen in BM
Neutrophils, erythroblasts, lymphocytes
increase in tissue due to cell proliferation, gross enlargement
Types of hyperplasia
- Infection, M:E 5:1 unless a chronic hemolytic process
- Dysplasia, increased number of precursors getting stuck somewhere in development
- Leukemia, >20% blasts
- Lymphoma, increased lymphocytes
abnormality of cell development, increases number of blasts
underdevelopment of tissue
Reasons for hypoplasia
- old age, fewer precursor stem cells left
- improper BM collection
- aplastic anemia, stem cell autoimmunity
- reticular fibrosis, fiber cells replacing BM
What does it mean when you see a nucleolus
cell is more transcriptionally active, less mature cell
What does it mean when you see increased cytoplasm?
More proteins have been made, more mature cell
Whats a give away for myeloid precursors?
primary and secondary granules
What is a give away for promonocytes?
folding within the nucleus
How do monocytes differ from lymphocytes?
cytoplasm is more grey, nucleus is folded, cytoplasm doesnt usually bend around RBCs
What cells look like ginger snap cookies?
What does a T cell do
What does a B cell do
bind antigen for specific antibody response
How does a lymph morphology change from non-reactive to reactive?
Non reactive lymphs are very round with course clumped chromatin, reactive lymphs spread out to attack cells
What is the clearing you see in plasma cells?
Golgi apparatus, making proteins
Acid Phosphatase stain
for llymphocytes (T cells)
+ in hairy cell leukemia
Alkaline phosphatase stain
- differentiates btwn leukemoid reaction (high) and leukemia (low)
- stains certain cytoplasmic components that are not present in abnormal cells
neutrophils and mast cells
Non-specific esterase stain
Monocytes, megakaryocytes and plasma cells
varies based on maturity of cell, differentiates btwn myelocytes
Sudan Black B stain
+ in granulocytes and monocytes, - in lymphocytes
stains iron stores, ringed sideroblasts
- carbohydrate stain
- differentiates btwn Guahcer's and Nieman Pick
Why is flow cytommetry better than staining?
it can identify cells by their surface markers which are more specific than staining
What are megaloblsatic changes
- Changes in cell morphology that occur due to issues with DNA replication
- hypersegmentation, discontinuity of development, etc.
What causes toxic granulation?
infection or activation of neutrophils
What are Dohle Bodies?
- Rough ER remnants
- Associated with stress, infection, exposre to cytotoxic agents, etc.
- look for bilobed neutrophils
- Cells act normally, but can be pre-leukemic
- Lipids in the cytoplasm of cells, looks a lot like toxic granulation
- cell function is normal but can point to Hurler's or Hunter's disease
- Giant fused granules in neuts and lymphs
- Cell can engulf but dont kill bacteria causing pyrogenic
- Autosomal dominant, blue Dohle-like inclusions and giant platelets
- cells function normalls but can have thrombocytopenia due to size of platelets
- Normal wright's stain
- Cells cant kill bacteria, lacking oxidative burst.
- accumulation of glucocerebroside in macrophages (tissue paper cytoplasm)
- Casues enlarged spleen and liver to try to process out the bad macrophages
- Foamy macrophage cytoplasm due to accumulation of sphingomyelinase, complete ensyme knock out
- Fatal before childhood
- sibling disease to HIV, also a retrovirus
- infects T cells diagnosed with antibody assay
- long incubation period
- attacks CD4+ T cells, kills the cells that perform the everyday immune responses
- Diagnosed with nucleic acid based tests
- now treated like a chronic disease, not a death sentence
- infectious mononucleosis, infects B cells
- diagnosed with monospot (heterophile Ab), although not 100% accurate
The overproduction of one or more of the myeloid cells, the brakes are off!
- extreme elevation of myeloid cells in PB and BM
- Chronic phase: way too many cells, normal function
- accelerated phase: non-funtional mature cells
- Blast crisis: so many blasts, starting to make their way into circulation
In what disorder will you find the Philadelphia (Ph) chromosome? What does it do?
- Translocation is stuck in on, errors in cells accumulation due to such high production
How is CML most often found?
Usually incidentally, lab sees high WBCs
Signs of CML
- Can present with petechiae
- bone marrow ME ratio 10:1-50:1
- <20% blasts
What is Gleevec?
- Medication for CML based on understanding of molecular basis of the disease
- doesn't work in juvenile CML
How to distinguish Leukemoid reaction from CML
- CML: low LAP, blasts present, Ph chromosome,
- Leukemoid: dohle bodies, toxic granulation, vacuoles, high LAP
Chronic Idiopathic Myelofibrosis
fibroblast proliferation in BM replaces normal hematopoetic tissue
What to look for in Chronic Idiopathic myelofibrosis?
- Extramedullary hematopoesis due to fibrosis, BM will go wherever it an.
- Tear drop cells (dacrocytes), elevated LAP
Increased cells, especially RBCs (due to long lifespan). Stem cells extra sensitive to EPO
What are you looking for in Polycythemia vera
- a combination of things that say I have a lot of everything
- Increased RBC, splenomegaly, Thrombocytosis, etc.
Increased megakaryoctes, normal RBCs but decreased HgB
What is a myelodysplastic syndrome
- AKA BM failure, not enough healthy RBCs, brakes are on!
- decreased or ineffective cell cycling
What to look for in myelodysplastic disorders
- signs the DNA replication isnt working right
- oval macrocytes, siderocytes, howell-jolly bodies, etc.
- abnormal precursors
What is refractory anemia?
- anemia unresponsive to iron therapy
- reticulocytopenia, oval macrocytes
What is RARS
Refractory anemia with ringed sideroblasts, >15% of BM cells are ringed sideroblasts
What is RAEB?
- Refractory anemia with excess blasts
- up to 19% blasts, hypogranular neutrophils, pseudo pelger huet cells
Deletion of the q arm of chromosome s
What to look for in 5q syndrome
- mostly older women, refractory macrocytic anemia, thrombocytosis, erythroid hypoplasia
- Responds to lenalidomide
What is chronic myelomonocytic leukemia?
- Technically MDS/MPD
- peripheral blood looks like RA with high WBC count
- BM 5-20% blasts
What is the key thing to look for in AML?
>20% blasts in PB or BM
What is AML M3
Acute promyelocytic leukemia
What to look for in acute promyelocytic leukemia
- promyelocytes predominate in PB, folded nucleus, often auer rods
- Can by hyper or hypogranular
Stain results in acute promyelocytic leukemia
- Myeloperoxidase +
- chloroacetate +
- non specific esterase -
How do you treat acute promyelocytic leukemia? Why?
- High dose of vitamin A to push along maturation process
- Disease has a vitamin A receptor defect that prevents maturation
What is AML M0? What are its stain results?
- AML, minimally differentiatied, myeloblasts without granules
- Myeloperoxidase -
- chloroacetate -
- Non specific -
- all stains are negative because the cells are very immature, really can't be identified yet
What is AML M1? What are the stain results?
- AML without maturation, <10% myeloid cells show maturation, auer rods often seen
- Myeloperoxidase + (light)
- chloroacetate + (strongt)
- Non specific -
What is AML M2? What are the stain results?
- AML with maturation, 50% of the time leukocytosis, thrombocytopenia, myeloblasts in PB and BM
- Myeloperoxidase +
- chloroacetate +
- Non specific -
What is AML M4? What are the stain results?
- Acute myelomonocytic leukemia, myeloid and monocytic cells in PB and BM. >30% blasts in BM, increased serum muramidase (monocytic component)
- Myeloperoxidase +
- chloroacetate +
- Non specific +
- Lysozyme 3x normal
What is AML M5? What are the stain results?
- Acute monoblastic leukemia, most common in young 2 subtypes
- a= less mature cells, b= more mature cells
- Myeloperoxidase -
- chloroacetate -
- Non specific +
What is AML M6?
- Acute Erythroid leukemia, only seen in adults, erythroblast is predominant BM cell, can evolve into M1 or M2
- 2 subtypes
- erythroleukemia= significant myeloblastic component
- pure erythroid leukemia= no significant myeloblastic component
What is AML M7?
Acute megakaryoblastic leukemia, dry tap due to fibrosis from increase in megakaryocytes, ID'd by immunophenotyping and cytochemistry
What is the cell specific to Hodgkin's lymphoma?
Reed Sternberg cells
What are non-hodgkin's lymphomas?
- B-cell neoplasms
- small cell lymphocytic lymphoma- nodes
- chronic lymphocytic leukemia- PB and BM
What does CD tell you about the lymphocytes effected?
- <10 (excluding 5)= T cell
- >10 (plus 5)= B cell
If you see a smudge cell in a case study you think...
What cells predominate in Waldenstrom Macroglobulinemia?
Plasma cells (B cells)
What is Multiple Myeloma
malignant proliferation of plasma cells, lytic bone lesions, elevated serum calcium, bence jones proteins
What is MGUS?
Monoclonal ab present in the absence of plasma cell dyscrasia, can progress to multiple myeloma
What is sezary cell syndrome?
mycosis fungoides, T cell derived tumors of the skin, only bad prognosis if it progresses beyond the skin
What is ALL?
Most common in young children, typical findings are neutropenia, thrombocytopenia, anemia
What are the different types of ALL?
- Immature B cell (most common)
- Pre B cell
- T cell
- B cell
When is the TdT stain positive?
early B cell ALL
balancing of clotting and bleeding
deliver O2 and nutrients, take away waste
transport lymph fluid, water and cells
inert to platelets and coag factors, (i.e. blood vessels)
- formation of the primary platelet plug
- adhere via vWF
- stabilize and strengthen via XIII
- activate the coag cascade via VIIa after exposure to TF
in the BM by megakaryocytes
- in-vitro sampling, anticoag clumps
- remedy by using sodium citrate (blue top) tubes
Platelets shape change
3 stages of clot formation
adhersion, aggregation, secretion
- platelets roll and cling to non platelet surfaces to seal endothelial gaps
- vWF required
- platelets adhere to each other
- requires fibrinogen
- Platelets secrete granular contents, essential for coagulation
Types of platelet granules
- Alpha- larger, factor V, factor XI, fibrinogen, vWF, etc.
- Delta- smaller, Ca, Mg, serotonin, etc.
AKA delta granules
Gray platelet deficiency
alpha granule deficiency
vWF receptor, facilitates adhesion to exposed collagen
Fibrinogen receptor, allows platelets to bind to each other.
Where are most coag factors made
Von willebrand Disease
- most common bleeding disorder in the US
- quantitative or qualitative vWF defect
Types of vWFD
- 1: you just don't have as much (most common)
- 2A: chopped up faster
- 2B: cleared out quicker
- 2M: less GP Ib, no primary plug
- 2N: factor VIII deficiency
- 3: absence of all multimers
platelet type, mutation in GP Ib gene instead of vWF gene
Bernard Soulier Syndrome
- deficiency/dysfunction of GP Ib (vWF receptor)
- Qualitative PLT disorder--> giant platelets
- deficiency/dysfunction of GP IIB/IIIA
- aggregation cannot occur unless treated with ristocetin
assessment of PLTs for adequate function via exposure to collagen and ADP or epi
- induces full aggregation and secretion of granular components
- conversion of fibrinogen to fibrin
binds PLT membrane to stimulate dense granule release and shape change
- Immune Thrombocytopenia Purpura
- PLT destruction due to autoantibodies
- labeled PLTs are removed quickly from the system leaving the patient with thrombocytopenia
- Thrombic Thrombocytopenia Purpura
- single acute episode in which clots form in the small vessels of the body
- leaves body with fewer clotting resources, PLT and vWF thrombi w/o fibrin
- clotting cannot occur without ristocetin
- Hemolytic Uremic Syndrome
- resembles TTP but in children
T/F Coag specimens should be kept refridgerated
- coag factors precipitate in the cold
If you see a fibrinogen level of <25..
be suspicious of a clot!
type of coag analyzer, if iron ball stops moving signifies a clot
scattered light detection to look for clots
- prothrombotic agent
- causes clotting in vivo, prolongs clotting in vitro
- anti-phospholipid protein complexes
- Thrombin Time
- Time to clot with thrombin added
- qualitative measurement of fibrinogen
- heparin interference
Like thrombin time but with reptilase
- prothrombin time
- evaluate extrinsic pathway using thromboplastin
- can be used to measure warfarin
used to normalize PT results regardless of strength of reagent
- Partial thromboplastin time
- measures the intrinsic pathway
accelerates inactivation of thrombin, AKA antithrombin
- vitamin K antagonist
- effects II, VII, IX, X, C+S
- heparin induced thrombocytopenia
- IgG antibody binds to heparin/platelet compound activating the platelet and starting the clotting cascade. Patient must be pulled off of heparin to remedy
- Direct thrombin inhibitor
- Can be used to avoid HIT
- binds thrombin active site on platelets without activation
- Factor XIII defiiency
- x-linked recessive disorder, "classic" hemophilia
- Factor IX disorder
- x linked recessive
- Christmas disease
- Factor XI deficiency
- autosomal dominant
Factor V Leiden
greater protein C resistance
- cuts fibrin
- forms split products like D dimers
- fibrin degradation product
- can be used to disagnose thrombosis
- disseminated intravascular coagulation
- Over-active coag factors can cause random small clots throughout the body and uses up factors and platelets, leaving the body low on them when they may be needede to stop bleeding. Can cause diffuse bleeding throughout the body
formation of blood cellular components
The stages of hematopoesis
multipotent stem cell --> pronormoblast --> basophilic normoblast --> polychromatophilic --> nRBC --> reticulocyte --> erythrocyte
Last stage of hematopoesis with a nucleus
Last division in hematopoesis
- hormone to regulat RBC production in bone marrow
- produced by kidneys, acts in BM
- normal levels replace 1% of RBCs per day
- high RBC count, hematocrit and hemoglobin
- associated with renal disease
ratio of rbc volume to total blood volume
What does the spleen do
- storage- 1/3 of wbcs and plts
- filtration- inspects cells for abnormalities
- piting- removes abnormalities with macrophages
- culling- phagocytose cells
- immunologic- produces IgM
different sized RBCs
different shaped rbcs
build up of porphyrina, Fe2+ can't incorporate into heme
What is the Embden Meyerhoff pathway?
- RBC glucose metabolism, 90-95%
- anaerobic pathway, nets 2 ATP
What is happens in the presence of 2,3-BPG
It competes with O2 for binding to Hgb and promotes the release of any remaining O2.
What is the oxygen dissociation curve?
- Plots sO2 vs. pO2
- Hemoglobin affinity to oxygen
- Left: wont let go
- Right: wont hold tight
What moves the oxygen dissociatin curve to the left?
- increased pH
- decreased, 2,3-DPG, pCO2, or temp
What moves the oxygen dissociation curve to the right?
- decreased pH
- increased 2,3-DPG, pCO2, or temp
What is the Kleihaure-Betke stain used for?
- test for fetal maternal hemorrahge
- stains fetal Hgb to compare to expected <1%
What do increased numbers of neutrophils point to?
What do large numbers of lymphocytes point to?
What so eosinophils point to?
Allergies or parasites
What points to May-Hegglin disorder?
Dohle body like inclusions, giant platelets
What points to Pelger Huet disorder?
What points to Alder-Reilly disorder?
Large, irregular, even granules
What points to Chediak-Higashi disorder?
giant fused granules, peroxidase +
What do monocytes do?
Mostly housekeeping via phagocytosis
What do basophils do?
exact purpose is poorly understood, some allergic response
Rule of 3
Hgbx3=Hct +/- 3%
graph of frequency of distribution, line graph of cell volumes
paired observations, often size vs. complexity
Manual cell counting equation
(cells counted)(dilution factor)/(square counted)(depth factor)
How does light scatter help identify cell type
- Forward light scatter shows cell size
- Side ligth scatter shows compexity
- RBC distribution width
- closely associated with anisocytosis
- really only concerned with increases
Microcytic Hypochromic Anemia
- Its all about the iron, aka IDA
- Decreased HgB, Hct, RBC, MCV, MCHC, Ferritin, transferrin sat.
- Increased RDW, TIBC
iron acquisition exceeding rate of loss
- defect in iron utilization
- impaired heme insertion, iron accumulates in mitochondira
- Look for pappenheimer bodies and ringed sideroblasts
disturbance of heme synthesis
When in DELTA, POR UR cop PROnto a cup of HEME
What happens in lead poisoning?
- acquired porphyria and sideroblastic anemia
- blocks protoporphyrin incorporation into heme and block ALA for heme synthesis
Anemia of chronic infection
- hepcidin causes macrophages to hold onto iron
- increased ferritin, decreased serum iron
- quantitative defects in Hgb production
- decreased globin chains
Alpha Thalassemia (aa/aa= normal)
- --/-- = death, hydrop barts fetalis
- a-/--= MCV <60, Hgb H disease
- aa/-- or a-/a- = microcytin, hypochromic
- aa/a- = silent carrier
Beta thalassemia (B/B =normal)
- B+/B = B thal minor, targets and eliptocytes, increased HgBH2
- Gamma to beta switch= B thal major, hypochomia with extreme inclusions
- B12 or folate deficiency causes ineffective erythrpoesis due to inability to incorporate thymidine into DNA
- effects all cell types
- Increased LDH, indirect bili
- decreased haptoglobin
antibodies against parietal cells, no IF secretion so no B12 absorption
- intravascular hemolysis: RBCs lyse in circulation, Hgb released into plasma, shistocytes
- Extravascular hemolysis: RBCs phagocytized by macrophages
- causes heinz bodies, causes bite cells
- unable to detoxify oxidative compounds to protect Hgb
- avoid fava beans
Sickle cell anemia
- abnormal Hbg forms strands that misshape RBCs, cells lose flexibility and cause painful crises
- shortens RBC lifespan to 10-20 days
Autoimmune Hemolytic anemias
- Cold agglutinin syndrome= IgM active @ RT, need to heat up to avoid
- Warm autoimmune= 95% DAT +, extravascular hemolysis, associated with CLL
- paroxysmal cold hemoglobinuria= intravascular hemolysis
- Microangiopathic Hemolytic anemia
- TTP (decreased ADAMS 13)
- HUS (TTP in children)
- DIC (pathologic hyperactiation of coag)
- Plasmodium, mosquito vector
- P. falciparum= multiple per RBC, rings <1/3 of RBC
- P. vivax= 1 per RBC, ring >1/3 of RBC
- Look for tetrads, otherwise very similar to
- Deer tick vector
- Borrelia recurrentis
- deer tick vector
- Loa Loa vector
- very large worm
Tse Tse fly or reduviid (cruz=america) bug vectors