hematology - pathophysiology

8/14/2019 hematology - pathophysiology

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Pathophysiology: HematologyHematopoesis ......................................................................................................................................................................... 2

Consequences of & Approach to Anemia ............................................................................................................................... 4

Iron Deficiency Anemia ........................................................................................................................................................... 6

Folate & B12: Metabolism & Deficiency .................................................................................................................................. 9

Hemolytic Anemia ................................................................................................................................................................. 12

Hemoglobinopathies ............................................................................................................................................................. 16

Hemostasis ............................................................................................................................................................................ 20

Thrombosis............................................................................................................................................................................ 25


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Hematopoesis

Hematopoeisis (Gr. haimato, blood +poiesis, creation): The formation of blood cells in the living body

Stem cells: can self-renew (proliferate) or differentiate

Totipotent: can regenerate entireorganism (incl. extraembryonic tissues) Pluripotent (e.g. embryonic stem cells): can regenerate across germ layers (no extraembryonic tissues) Multipotent (e.g. adult stem cells): can regenerate cell types restricted by germ layer

Zen thought of the lecture: Every time a stem cell divides, its still a stem cell but a little less so (not stochastic)

Note on engraftment: both progenitors & primitive (high quality) stem cells can give rise to all elements; the difference is

in how long they can reproduce (lose graft after initial good result with low-quality stem cells)

Glossary Clone: cell population derived from single ancestral cell CFU: Colony-forming unit: represents the cell that gives rise to a colony (assayable growth in vivo / in vitro)

o E.g. CFU-S, spleen CFU (mouse low-quality hematopoietic stem cells, give rise to spleen colonies post-BMT-irradiation) Colony-forming assay: isolate mononuclear cells from marrow; let em grow (clones) CFU-GM: colonyforming unit granulocyte macrophage: most differentiated myeloid progenitor, no self-renewal

o White colonies on assay: makes white blood cells BFU-E: burst-forming unit erythroid: RBC progenitor

o Red colonies on assay; making RBCs CFU-Mixed / CFU-GEMM: progenitor of both CFU-GM and BFU-E (probably like CFU-S)

o Mixed coloration on assay; making WBC & RBC tooMalignancy

Malignancy: unregulated clonal growth; from 1+ mutations

Cancer stem cells:

tumor initiating cell with limitlessself-renewal, limiteddifferentiation

Treatments often dont target themLeukemic stem cells: mostly at low-quality HSC (myeloid precursor)

HEMATOPOETICSTEM CELLS(2 classes)

Most primitive

(high quality)

Myeloid

(low quality)

Precursor to: All lympho-hematopoetic

lineages

Granulocytes, RBCs,

platelets, B-cells(?)

Disease Rarely involved Moststem cell disorders

Engraftment Delayed but life-long Rapid but limited

PhenotypeCD34+/-, other markers

mostly -, smaller

CD34+, other markers +,

larger

Aldehyde DH +++ + (or low)

OTHER PLAYERS (IN STROMA)

Growth factor/cytokine Stimulates

Erythropoietin (kidneys) RBC

Thrombopoietin (liver) Platelets, HSC

Flt-3 Dendritic cells, HSC

Stem cell factor Mast cells, HSC

G-CSF PMNs

Pathways to malignancy

(KNOW THESE)

increased proliferation block in apoptosis

(more common)


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Acquired Aplastic Anemia Hypocellular bone marrow, severe pancytopenia, often in young patients Autoimmune disorder: CTLs target low-quality stem cells

o Reduced CD34 stem cell pool pancytopenia Treatment:cyclophosphamide,

o activated in liver, blows away lymphocyteso stops T-cell reaction against low-quality stem cellso High-quality stem cells are saved (have aldehyde DH,

inactivate cyclophosphamide)

Chronic myeloid leukemia BCR-ABL fusion protein (t9:22 Philadelphia chromosome) blocks

apoptosis

Called a myeloproliferative disorder is really a myelo-accumulative disorder

Diseases of Hematopoesis & where they come from

Notes about this crazy picture (down = more differentiation)

Top of the pyramid: high quality / primitive hematopoetic SC.

Few diseases at this levelNext: low quality / myeloid SC.

MOST DISEASES here.Third: lineage-committed progenitors (a bit more differentiated; like those colony-forming units).

Pediatric diseases are in this category; possibly why they do better clinically (more differentiated)Bottom: mature / differentiated blood cells.

Autoimmune diseases attack these more differentiated cells; lymphomas come from hereFinal random thoughts:

You can make human universal stem cells by transducing 4 genes into adult skin cells. Remember: epigenetics is important too

Pathways to bone marrow failure

(KNOW THIS)

1.Oncogenesis / mutations

(leukemia, MDS, dyskeratosis congenita)2.Direct DNA damage(radiation, benzene, chemo)

3.Autoimmunity(aplastic anemia, pure red cell aplasia)

4.Viruses(HIV, parvovirus)


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Consequences of & Approach to Anemia

Metabolic / Physiologic Responses to Anemia

O2 Delivery & Uptake: 2 = 1.39 (2 2)

Ways to increase oxygen delivery

1. Increase Blood Flow (Q)a. Cardiac output: HR, pulse pressure, murmurs, bruits,hyperdynamic precordium, tinnitus/roaring in earsb. Change tissue perfusion

i. shift from O2-insensitive (skin:pallor, kidney) O2 sensitive (heart, brain, muscles)

2. Increase Red Cell Mass (Hb)a. EPO (kidney)Reticulocytosis(see immature RBCs)

i. Usually lose 1% RBC/day so reticulocyte count is 1%;higher = reticulocytosis

ii. Clinical finding: bony pain, expansion of marrow (e.g. onimaging)

b. Increased hematocrit is good: to a point!

i. Increase too much: viscosity increases; overwhelms

increased ability to transport; oxygen transport actually

decreases!

ii. Hypervolemia helps a bit (increase volume, can fit more

RBC in) but still can be overwhelmed

3. Increase oxygen unloading (SaO2 -SvO2)

a. 2,3- DPG (decreased affinity: displaces oxygen from hemoglobin)

i. Generated from glycolytic pathways (anaerobic)

ii. RBC have mostly anaerobic metabolism (90%, 10% aerobic)

1. Allows RBC to generate ATP (maintain shape, flexibility, cation/H2O balance)

iii. More in women (better oxygen delivery)b. General characteristics ofoxyhemoglobin dissociation curve

(see below)

Oxyhemoglobin Dissociation CurveOxygen affinity (P50): P50 varies inversely with oxygen affinity

partial pressure of oxygen when Hb 50% saturated curve shifted to right: P50, oxygen affinity curve shifted to left: P50, oxygen affinity

INCREASED SHIFTS CURVE TO OXYGEN AFFINITY BECAUSE

2,3 DPG Right Decreases Youre bumping O2 off to deliver more to tissues (which iswhy your kidneys are cranking out 2,3 DPG in the first place)

pH Left IncreasesBohr effect: if carbon dioxide rises in a tissue, you need more

oxygen. Blood gets more acidic from CO2, pH drops, oxygen

affinity decreases, and more O2 gets dropped off

Oxygen Left Increases Youve got high O2 why not hang on to it?Temperature Right Decreases If its cold, your metabolism slows down, so you dont need as

much O2

VO2 = oxygen delivery

1.39 = constant(mL O2 that bind to 1 gm Hb)Q = blood flow (mL/min)

Hb = hemoglobin (g/dL)

SaO2 -SvO2 = A-V % sat difference(ability to unload oxygen)


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Cooperativity:

when Hb partially saturated, affinity of remaining hemes increases markedly 2 Hb conformations: Tense (deoxy) & relaxed (oxy) Pictures like the one to the right are popular when discussing cooperativity

Classifying Anemia1. Cause: is there decreased RBC production, increased RBC destruction, or RBC loss (bleeding)2. RBC Size: microcytic / normocytic / macrocytic

3. Hb: hypochromic(Hb/RBC),normochromic (normal Hb/RBC)

4. Morphology: normal / abnormal (anisocytosis = varied morphology)

Clinical Tests & Definitions

Hct =PCV

Vblood; PCV = packed cell volume (packed RBC volume) Hb=

g Hb

dL blood

Mean Corpuscular Volume: MCV =Hct

RBC Countreflects average size / volume of RBC (in fl, femoliters)

Mean Cell Hemoglobin: MCH =Hb

RBC Countreflects weight ofHb in average red cell

Mean Cell Hemoglobin Concentration: MCHC =Hb

Hctindicates concentration ofHb in average red cell (%)

Reticulocyte = young RBC

Normal morphology: donut shape, center pallor 1/3 of red cell

Common causes for various types of anemia

Hypochromic, microcytic

Iron deficiency Thalassemia syndromes Sideroblastic anemia, transferring deficiency

Normocytic, normal morphology

Hemorrhage / blood loss Unstable hemoglobins Infection / inflammation / chronic dz

Macrocytic

Megaloblastic anemias (folic acid / B12 deficiencies) Liver disease, reticulocytosis Bone marrow failure syndromes, drugs (AZT, etc)

Normocytic, abnormal morphology

Hemoglobinopathies (SS, SC, CC) Hereditary spherocytosis Autoimmune hemolytic anemia; enzymatic deficiencies

QUESTION TEST

Is the patient anemic? CBC, Hb, Hct

RBC production/destruction/loss? Reticulocyte count (usually ~1%)

Micro/macro/normocytic

Hypo/normochromic

RBC Indices

Morphology Peripheral blood smear


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Iron Deficiency Anemia

Background: Iron Metabolism Distribution: mostly active use (60% Hb, 13% Mb / enzymes)

o also stored (ferritin/hemosederin, 27%); in transport (transferrin 0.1%) Intake: 10-25mg from food per day

o Most dietary intake is nonhemeiron (spinach, etc) but less bioavailable than heme iron (veal, meat)From food to blood:(remember that Fe is very oxidative / dangerous & body needs protection from it)

1. Absorption:brush border of upper small intestine via transport proteins2. Transport: Binds to apotransferrin in mucosal cell forms transferrin

exported to blood (intracellular apotransferrin recycled) exported to

blood bound to soluble transferrin in blood

3. Uptake: cells that need iron have transferrin receptors,e.g. erythroid precursors

4. Storage: mostly in Mofreticuloendithelial system(liver/spleen/marrow)

o Ferritin: PRINCIPAL IRON STORAGE PROTEIN. Multi-subunit;form shell around Fe molecules. Serum ferritinis proportional to

intracellular ferritin (lab test). Good for quick mobilization.

o Hemosiderin: insoluble ferritin (packed together until itprecipitates; can see on microscopy. Longer-term storage

Iron cycle:

Erythroid precursors: uptake via Tf receptors incorporateFe into hemoglobin & package into RBC

Also used for myoglobin in muscle Old RBC destroyedM pick up iron

o store as ferritin/hemosiderin (recycling)o release as transferrin into plasma when needed

Pathophysiology of Iron Deficiency

1. Iron stores depleted

2. Fe becomes limiting factor in heme biosynthesis

3. hemehemoglobin assembly4. Hb RBC production

5. Small RBC (microcytic / low MCV) & Hb-deficient RBC (hypochromic / low MCH)Responses to iron deficiency:

1. Increase absorption, transport, uptake of iron (more absorption, more Tf/TfR made)2. Decreasestorage & utilization (less ferritin, microcytosis, hypochromia)

MolecularMechanisms:

When plenty of iron is around:o transferrin mRNA made but unstable(Tf protein, transport)o ferritin provides iron storage; heme synthesis (ALA synthase) uses irono Fe bound to IRPs, IRPs inactive (see below)

lumenbloodmucosal cell

Fe

Apo

Tf

sTf

Fe

sTf

Fe

TfFe

ferritin

FeFe

ApoTf

monoFeTf

di FeTf

TfR


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Iron deficiency:Iron response proteins (IRP-1 & 2)o IRP-1, IRP-2 lose their iron atoms (iron around); start RNA-

binding (bind iron response element: IRE)

o Bind IRE in transferrin mRNA & stabilizetransferrinproduction (iron transport)

o Bind IREs in ferritin / ALA synthase mRNAs & block translationstart site

o IRP-1 is aconitase (cytoplasmic TCA cycle enzyme); losesenzymatic activity ifno iron (increasing iron for its own use!)

Iron sufficient state Iron deficient state

Aconitase TCA enzyme activity IRP-1 functions

TransferritinmRNA unstable

transport

IRP stabilizesmRNA

transport

FerritinmRNA transcribed

storage

IRP blockstranscription

storage

ALA synthasemRNA transcribed

heme synthesis

IRP blocks transcription

heme synthesis

Iron losses

Iron closely conserved in humans: NO PHYSIOLOGIC MEANS TO EXCRETE EXCESS IRONS Very small amounts lost (urine/bile/sweat, cells shedding from GI/urinary tracts; 0.05% Fe lost/day) Higher loss states: menses in post-pubertal females, pregnancy (to fetus), lactation (to breast milk)

Pathogenesisof iron deficiency Iron deficiency = deficit in total body iron: requirement > supply (intake+storage)

Causes:

recurrent / chronic / occult blood lost (e.g. GI bleed) failure to meet physiological requirements(rapid growth, menses /

pregnancy / lactation)

Inadequate intake(diet low in heme iron, e.g. strict vegans; GI disease,surgery, excessive milk intake in infants)

ALL SIGNS AND SYMPTOMS DEPEND ON:

DEGREE OF ANEMIA RATE OF DEVELOPMENT OF ANEMIA

Lab findingsPeripheral smear:

Microcytic & hypochromic anisocytosis (variable sizes) & poikilocytosis (variable shapes), ovalocytes / eliptocytes

Serum ferritin: LOW(best general indicatorof IDA), remember that this is proportional to ferritin in cells

Serum iron (iron saturation transferrin): expect low iron, high transferrin; ratio is less reliableBone marrow iron stain is gold standard (but only used in difficult cases)

CBC / RBC indexes:

WBC normal with low Hb, low Hct Low MCV (microcytosis), low MCH Platelet ct: normal/high, retic % low/normal, occasionally high, abs. retic ct low (others are better indicators)

OCCASIONAL FEATURES OF IRON-DEFICIENCY ANEMIA

pagophagia craving ice

pica craving of nonfood substances

(dirt, clay, laundry starch)

glossitis smooth tongue

angular stomatitis cracking of corners of mouth

koilonychia thin, brittle, spoon-shaped fingernails

blue sclerae

Iron sufficient state:

ACONI-TASE-1

Fe+++

TRANSFERRIN mRNA (unstable)

FERRITIN mRNA

ALA SYNTHASE mRNA

transferrin

ferritin

heme

IRE

IRE

IRE

Iron deficient state:

ACON-ITASE-1

TRANSFERRIN mRNA (stable)

FERRITIN mRNA

ALA SYNTHASE mRNA

transferrin

ferritin

heme

enzyme inactive;becomes IRP

IRPIRE

IRPIRE

IRPIRE

General symptoms (all anemias)

Pallor Fatigability, weakness Dizziness Irritability


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Sequential changes in lab values

1. Ferritin decreases: stores being depleted2. Iron saturation (iron/transferrin) decreases: youre iron deficient!3. MCV, Hb, Hctdecrease: anemia! Hb production is limited now

Generally, microcytosis develops before significantanemia

Therapy: RBC transfusion if severe; mostly iron salts (ferrous sulfate) po (IV if required).

Phytates (cereal grains), tannins (tea), antacids can inhibitFe absorption; vitamin C (ascorbic acid) helps itCorrecting iron deficiency: need to ID & TREAT the UNDERLYING CAUSE

GI blood loss (ulcer/tumor parasite); excessive menstral loss (tumor / bleeding disorders), Rare conditions (pulmonary hemosiderosis, paroxysmal nocturnal hemoglobinuria)

Differential diagnosis of IDA Thalassemia trait: imbalance of globin chain reduction. Also microcytic / hypochromic but iron tests normal Anemia of chronic disease: decreasing Fe utilization with adequatestores

o Blood tests can look like IDA but usually ferritin / transferrinnormal)Anemia of Inflammation (Anemia of Chronic Disease)

Causes:

Chronic Infections: osteomyelitis, pneumonia, abscesses, bacterial endocarditis, meningitis,HIV, fungus, mycobacteria

Autoimmune disease - lupus, rheumatoid arthritisMalignancy - Hodgkin disease, lymphoma, metastatic carcinoma, sarcoma, multiple myelomaOther chronic diseases - congestive heart failure, liver disease, inflammatory bowel disease

Pathophysiology: want to hide iron from bacteria!

1. InflammationIL-6 release (endothelial/Kupffer cells)2. IL-6: makes hepatocytes release hepcidin3. Hepcidin: inhibits intestinal iron absorption & iron release from M that

have taken up old RBC

DDx vs IDA can be difficult:

ferritinincreases in ACD (want to store more iron) total iron binding capacity ( transferrin)increases in IDA (transport more) Gets even crazier if both present at once

Iron Overload Syndromes Remember: no route for iron excretion Heart (cardiomyopathy / CHF / arrhythmias) and exocrine glands (e.g. liver cirrhosis) are main organs affected

Causes:

Transfusional hemochromatosis: pts. getting frequent blood transfusions (e.g. -thalassemia major); get largecumulative load, Rx with iron chelation drugs

Hereditary hemochromatosis: genetic disorder of excessive iron absorption in gut (enterocyte transportermutation); Tx with periodic phlebotomy.

Response to therapy

Peak reticulocytecount 7 - 10 days

IncreasedHb and Hct 14 - 21 days

NormalHb and Hct 2 months

Normal ironstores 4 - 5 months

FerritinFerritin

IDAIDA InflammationInflammation

Serum IronSerum Iron

TIBCTIBC

sTfRsTfR

BothBoth

NoNo

??

??

Marrow IronMarrow Iron NoNo

Iron deficiency vs. Anemia of Inflammation

FerritinFerritin

IDAIDA InflammationInflammation

Serum IronSerum Iron

TIBCTIBC

sTfRsTfR

BothBoth

NoNo

??

??

Marrow IronMarrow Iron NoNo

Iron deficiency vs. Anemia of Inflammation


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Folate & B12: Metabolism & Deficiency

Megaloblastic anemias: from reduction in rate of DNA synthesis; RNA/protein synth normal.

Subset ofmacrocytic anemias (MCV > 100; abnormally large cells) Nuclear-cytoplasmic asynchrony(cytoplasm matures faster than nucleus)

can have 2 results:

o cell dies (intermedullary hemolysis / ineffective hematopoesis)o terminal division omitted (big macrocyte formed)

Unbalanced growth in all rapidly proliferating cells (bone marrow, tongueepithelium, small intestine, uterus): look for clinical manifestations here.

Pernicious Anemia (B12 defiency) Macrocytosis (big RBC) + megaloblastosis (lots of RBC precursors)

Clinical presentation:

Key triad:

1. Diminished gastric secretions / achylia gastric

2. Megaloblastic anemia

3. Neurologic degeneration (posterior/lateral columns)

Patient:older, especially Irish / Scandinavian / English

Signs / Sx: often develops slowly, Asx at first (sometimes neuro abnormalities early)

Most least frequent: anemia, paresthesias, GI complaints, glossitis (soretongue), difficulty walking

Lab:

Macroovalocytes of RBC & hypersegmentation of granulocytes; Hypercellular bone marrow with lots of erythroid precursors Hemolysis: LDH,hyperbilirubinemia, Fe

Vitamin B12Early studies: massive liver feedings helped PA; extrinsic factor (B12) in liver & intrinsic factor missing in ptsB12: Synthesized by microorganisms; dietary from flesh/milk of ruminant animals

liver, glandular tissue, muscle, eggs, dairy products, seafood Normal body stores 2-5mg, > 1mg stored in liver; daily requirement 2- 5 g (0.1%) Significance: B12 stores last at least 1,000 days after absorption stops

Structure: Dorothy Crowfoot Hodgkin

Porphyrin-like ring with cobalt in center (cobalamin) pharm forms are substituted at cyano group & converted by metabolism in vivo

Absorption, Transport, etc.

Bound to protein in food; released by pepsin (need acid pH) in stomach,binds to R-substance in gastric juice

Released from R-substance by trypsin injejunum Intrinsic factor (IF) secreted by gastric parietal cells & complexes with B12 IF-B12 complex is absorbed EXCLUSIVELY BY TERMINAL ILEUM

(only cells that have IF receptors)

B12 bloodstream bound to transcobalamins(TCII is physiologically relevant)

TCII receptors in tissues uptake for use in cell division

DDx: macrocytic anemia

Reticulocytosis (e.g.hemolytic anemia)

Liver disease, alcoholism Drugs Some myelodysplasias

DDx: megaloblastosis:

Interference with DNA synthesis

Chemotherapeutic drugs Acute leukemia Rare inherited disorders

(B12 or folate deficiency too!)

Wikipedia on DCH:Hodgkin's scientific mentor

Professor John Desmond Bernal

greatly influenced her life both

scientifically and politically. He

was a distinguished scientist of

great repute in the scientific world,

a member of the Communist

party She always referred to him

as "Sage" and loved and admired

him unreservedly; intermittently,

they were lovers.


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Functions: co-factor for the following reactions

1. Methyl transfer: homocystine + methyl-THFmethionine + THFo B12 = obligate cofactor for certain folic acid functions

2. Hydrogen transfer: methylmalonyl coA succinyl coA

o Not involved in folic acid pathwayo High urinary/serum methylmalonyl coA helps distinguish

B12 deficiency from folate deficiency

Clinical findings specific to VitaminB12deficiency

1. Low serum B12 levels

2. Peripheral / central nervous system disease

a. Classic presentation: combined system degeneration

dorsal/lat column probs (position / vibratory sense), peripheral neuropathy, cortical abnormalities (megaloblastic madness)

3. Methylmalonic acidemia (see above)

4. Abnormal Schilling test

Causes

Acquired deficiency state:1. Decreased absorption

a. Loss ofintrinsic factor(gastric atrophy, autoimmune-associated gastric atrophyis #1, also

Ab against intrinsic factor / gastrectomy)

b. Terminal ileum disease(ileal resection, gluten-induced problems, non-tropical sprue, cancer,

granulomatous lesions, regional enteritis, bacterial overgrowth)

c. Food cobalamin malabsorption(chronic achlorhydria, proton pump inhibitors, loss of salivary gland function)

2. Inadequate ingestion(vegans / breast-fed infants of vegan moms)Congenital deficiency state: usuallyin infancy(failure to thrive, developmental delay, neuro abnormalities, anemia)

Autosomal-recessive conditions (cobalamin absorption or transport)

Treatment of pernicious anemia: parenteral cyanocobalamin; treat terminal ileum disease or microbes if present

Folic acidFolate metabolism: we dont synthesize it; half of body stores are in liver; body stores last about four months

Most absorbed in proximal jejunum but: DIFFUSE DISEASE ofINTESTINE is NEEDED for FOLATE MALABSORPTION to occur Dietary sources: green leafy veggies, liver, kidney, fruits, mushrooms

Structure / metabolism / function:

Dietary folate: conjugated with multiple glutamic acids;intestinal deconjugation is needed for absorption Needs to be reduced x4 (dihydrofolate reductase) to THF for activity (methotrexate, trimethoprim target) Most important function: methyl transfer (e.g. for thymidilate & subsequently DNA synthesis) MEGALOBLASTOSIS is from IMPAIRED THYMIDYLATE SYNTHESIS in folate deficiency.

Causes: dietary deficiency is most common (alcoholics, indigents, malnourished)

folate requirements with pregnancy / lactation / chronic hemolytic anemia

Schilling test: Pernicious anemia

1. Phase 1:

a. Give oral radioactive

cyanocobalamin + bolus dose

unlabeled B12 to block tissue

binding in B12-deficient individuals

b. 24h urine collection (need to take

up B12 to get radioactivity into

urine instead of feces).

c. Abnormal (


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Impaired deconjugation or diffuse intestinal disease can lead to malabsorption Blocked utilization (cancer chemotherapy) too

Treatment: 1 mg folic acid PO qd

If caused by methotrexate, coadminister leucovorin (FH4)Compare & contrast: B12 vs Folate

B12 FOLATE

Megaloblastic anemia Yes Yes

Combined system degeneration Yes No

Dietary Deficiency Rare Common

Dietary Source Muscle, liver, milk, eggs Liver, leafy greens

Deficiency induces hyperhomocysteinemia Yes Yes

Deficiency induces methylmalonic acid Yes No

Site ofabsorption Terminal ileum Small bowel

Intrinsic Factor required Yes No

B12, Folate, DNA replication, Neural Tubes, Vascuar Disease, and Cereal

Vitamin B12 deficiency: might trap folic acid as N5-methyl

FH4,

predominant dietary form (cant be converted to N-5,10-methylene FH4 for thymidilate & DNA synthesis)

Oral folic acid can mostly correct B12 ANEMIA(possible explanation for why)

Treatment ofB12 deficiency with folic acidDOES NOT

CORRECT NEUROLOGIC DEFECTS

Dietary folateenters @ folic acid on top diagram;via methionine synthase & with B12 participation can

generate methionine (needed for myelin synthesis) Pharmacologic folatedoesnt generate methionine,

formate, or SAM (enters at THF stage)

Cure anemia (can still bump up DNA production) buthide worsening B12 problem: MUST DDX B12 VS

FOLIC ACID PROBLEM

Folate deficiency & fetal malformations

Folate deficiency associated with neural tube defects 0.8 mg folic acid po qd prevents 1st occurrence, 4 mg

prevents neural tube defects in subsequent dose

USPHS: women in reproductive years should take 400g/day of folate supplements

Hyperhomocysteinemia & vascular disease: associated with increased incidence of atherosclerosis / heart disease; folic

acid supplementation reduces homocysteine levels but doesnt prevent venous/arterial thrombotic disease;

homocysteine may be marker of vascular disease instead of causative agent

Cereal: FDA mandated in 1998 that all cereals be fortified with folic acid to try to bump up folic acid intake; designed for

100g / day increase (falls well short of USPHS guidelines) efficacy of program not known.

Diagnosis offolate defiency

serum folic acid (


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Hemolytic Anemia

Hemolytic disorder: any condition where survival time of erythrocyte in circulation < 120 days (normal RBC)

Etiologies:

Primary (usually congenital): membranopathy, enzymopathy, hemoglobinopathy Secondary (usually acquired): immunologic, chemical, physical

Physiologic response(independent of etiology): rate of delivery of new RBC ( reticulocytes)

1. Compensatedhemolytic disorder: new increased rate can match destruction

2. Hemolytic anemia: when you cant compensate (more destruction than new delivery)

a. Max increase is usually 6-8x normalb. So if RBC life < 15-20 days (1/6-1/8 normal), then hemolytic anemia ensues

Lab techniques

Direct techniques: look at RBC survival time Ashby test (historical): transfused mismatched RBCs; follow % cells surviving; problem: not looking @ endogenous RBC) Radioactive chromium: most common; binds to hemoglobin (labeling pts own RBC)

o Problems: chromium elutes 1%/day from Hb, so have to correct; rate ofelutionvaries with differentHbs (e.g. faster from SC than normal); has higher affinity for retics, cant tellblood loss vs hemolysis

Indirect techniques: look at RBC production

Reticulocyte count (1% circulating RBC normally): increased in hemolyticdisorder (abs & %)o Reticulocyte = cell after most mature nucleated red cell precursor in BM loses nucleuso Hard to tell in peripheral blood smear: use supravital staining (methylene blue) to clump ribosomes / mitochondriao Flow cytometry mostly used these days

Peripheral blood smear:o young RBC are macrocyteso polychromatophilia (reticulocytes have diffuse basophilia, look blue when released early from BM) o nucleated red cells (early release from BM)

Bone marrow: see more erythroid precursors; usually not required. Other: Can see medullary expansion (hair-on-end appearance) on radiography; maxillary prominence & expansion of

bones (skull, ribs, hands); sometimes hepatosplenomegaly too (extramedullary hematopoesis & increased sequestration)

BilirubinElevation of indirect bilirubin in hemolytic anemia

Bilirubin review:

1. Breakdown ofHbheme, globin + Fe released2. Hemebiliverdin (via heme oxidase, CO2 released)3. Biliverdinbilirubin4. Bilirubin bound to albumin when it circulates.

(Unconjugated bilirubin a.k.a. indirect bilirubin =insoluble, so it doesnt appear in urine; goes to fat

instead e.g. sclerae)

5. Conjugated bilirubin formed in liver (direct bilirubin,

water-soluble & can be seen in urine)

6. Enterohepatic recirculation from GI to liver or

excretion as urobilinogen from kidneys / stercobilin in

feces


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Hemolysis: degradation of Hb bilirubin; exceed liver conjugation abilities; see INDIRECT BILIRUBIN in blood Conjugated/ direct bilirubin doesnt accumulate (liver can still excrete it)

Liver disease: liver cant excrete conjugated bilirubin, so DIRECT BILIRUBIN increases in blood

Hemolysis: extravascular & intravascularExtravascular hemolysis:

RBC trapped in reticuloendothelial system (liver/spleen/bonemarrow)

Heme breakdown proceeds through bilirubin pathway; seeincreased indirect bilirubinin blood, CO & Fe

Intravascular hemolysis:

RBCs destroyed within circulation, Hb released directly intocirculation

In addition to increased indirect bilirubin,CO & Fe, seehemoglobin in urine (hemoglobinuria).

Hb also concentrated by renal tubular cells as hemosiderin,shed into urine (hemosiderinuria)

Lab stuff

Haptoglobin: 2 globin , binds hemoglobin 1:1, made in liver

Serum levels vary with age (0 in newborn, higher in olderchildren & adults); also an acute phase reactive protein

(increased in stress)

Reduced levels when RBC survival < 90 days REDUCED SERUM LEVEL in BOTH intra- & extra-vascular

hemolysis

Hemopexin: globulin, made in liver, binds heme 1:1

Serum levels vary with age (higher in older children & adults),not an acute-phase reactive protein

REDUCED LEVELS in INTRAVASCULAR hemolysis (mostly)Methemalbumin: when haptoglobins binding capacity exhausted, free Hb combines with albumin methemalbumin

(means that indirect hemolysis has been liberating Hb into bloodstream)

Carboxyhemoglobin: CO liberated in heme degradation

rate ofCO production directly related to rate ofheme degradation (directly related to red cell survival) BOTH intra & extra-vascular hemolysis can cause increased levels Technically difficult, not used routinely, results messed up in smokers or people with other CO exposure

Summary: distinguishing intravascular & extravascular hemolysis

Bothintravascular & extravascular hemolysis Intravascularhemolysis only

Indirect hyperbilirubinemia urinary / fecal urobilinogen haptoglobin / hemoplexin carboxyhemoglobin

Hemoglobinemia Methemalbuminemia Hemoglobinuria Hemosiderinuria


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Classification of Hemolytic Disorders: intracorpuscular vs extracorpuscular defects

Intracorpuscular defect: etiologic factor intrinsic to red cell; most often genetic

Membranopathy: see elliptocytes & spherocytes Hemoglobinopathy: e.g. sickle cell trait / dz Enzymopathy: e.g. pyruvate kinase deficiency

Extracorpuscular defect: etiologic factor extrinsic to red cell; most often acquired

Thermal injury, mechanical injury (e.g. Waring blender syndrome from heart valve, etc.), toxic injury, Antibodies (autoimmune hemolytic anemia)

Intra & extra-corpuscular defects combined too: drugs, for example

Mophologic abnormalities

Hereditary Spherocytosis: intracorpuscularmembranopathy

Membranopathy: disturbance in protein-protein interaction (within RBC cytoskeleton or links to cell membrane Spectrin abnormality (or spectrin binding proteins): cytoskeletal proteins involved in vertical (cytoskeleton

PM) and horizontal (cytoskeleton cytoskeleton) interactions

Unstable red cell membrane loss of membrane SA reducedsphere forms (smallest SA)o Cells rupture more easily in hypotonic solutions

(osmotic fragility test)

o Direct relationship between degree ofspectrindeficiency and: osmotic fragility, reticulocytosis,

depression of haptoglobin, severity of anemia

Autoimmune Hemolytic Anemia: extracorpuscular defect

Ab coat RBC; as RES removes Ab, bits ofmembrane get removed: reduction of surface

areasphering

Detection:Coombs testDIRECT COOMBS TEST INDIRECT COOMBS TEST

Detect antibody attached

to patientsRBC surface

Detect anti-RBC antibody in

patients serum

Use agglutinating

antibody or labeled anti-

human IgG

Expose control RBCs to patient

serum; look for Ab binding to

control RBCs surface

Positive for autoimmune

hemolytic anemia

Can be positive in autoimmunehemolyticanemia - if enough

Ab produced to exceed capacity

of RBC to bind it

Positive if antibodies are madeagainst foreign RBC antigens

(post-transfusion, feto-maternal

incompatibility)


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G-6-PD deficiency: combined extra- and intracorpuscular defect

Cells sensitive to oxidants: either an absence or dysfunction ofglucose-6-phosphate dehydrogenase Most common form: A- variant; enzyme functions but has a shorter half-life

o youngerRBC have a higher level of G6PDo If you induce oxidative stress (primaquine), anemia, reticulocytosis,

hemoglobinuria all go away

o Reticulocytosis pumps out more young RBC, so G6PD levels on average arebetter. See picture (Hb on top, retics on bottom, primaquine given at t=0)

When hemoglobin falls & red cells start to lyse (after an oxidative stress trigger):

Heinz bodies (intracellular precipitates of hemoglobin) Dark urine (hemoglobinuria/methemalbuminuria) Jaundice

Pathophysiology ofG-6-PD Deficiency

G-6-PD: catalyzes conversion ofG6P to 6PG, reducing NADP to NADPH

G-6-PD deficiency: Cells cant provide enough reduced glutathione following oxidative stress

(cant protect cellular elements against oxidation symptoms)Details of pathophys / metabolism:

G6P usually metabolized mainly through glycolysis, small proportionthrough pentose phosphate pathway

Normal patient:o Oxidative stress situation: increase in conversion ofNADP to

NADPH (more G6PDactivity, more 6PG produced, more

glucose utilization, and more pentose shunt activity)

o H2O2 & free radicals oxidize GSH, forming mixed disulfides ofHb and glutathione

o NADPH is used to return glutathione to reduced state& remove peroxides G6PD Deficiency: cant increase flow through PPP when oxidative stress occurs

1. NADPHcant be generated as in the normal patient

2. Cant regenerate GSH (glutathione GSH conversion needs NADPH)

3. Hb starts to precipitate (Heinz bodies); membrane lipids get peroxidatedred cells destroyed


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HemoglobinopathiesHemoglobin chains & forms

Genes

To make a hemoglobin: pick one from each cluster

Changes through the lifespan:

Erythropoesis happens in different organs throughout embryonic development (sequential):

yolk sac liver/spleen bone marrowHb expression

Just after conception: and chains expressed(22predominates)

Major switch #1: embryonic chainsreplaced byadult chains

(shortly after conception, 22predominates)o From this point on, its all chainsand no more o Embryonic replaced by fetal shortly thereafter

(22 predominates)

Major switch #2: fetal chainsreplacedby adult chains (22predominates)

o This change is INCOMPLETE and REVERSIBLE(basis for some therapy)

Adult hemoglobins: 95% Hb A (22), also 2% Hb A2 (22) and 2% Hb F(22)The locus control region(chromosome 11) along with part of the 5 -globin complex help modulate this switching

Transacting factors bind to LCR; LCR loops over & silences globin genes promoters (physical DNA rearrangement)

Mutations can mess up expression patterns; gene therapy (FDA-approved!) and other therapies can take advantage

Hemoglobinopathies: overview

Hemoglobinopathy: mutation ofeither the alpha or beta globin genes which leads to:

Varientglobin molecule, decreasedglobin production, or absence ofglobin production Every mutation type you can think of has been described in globin genes, including regulatory / noncoding areas

Five clinical syndromes:

Sickle syndromes (sickle cell trait, SC disease, etc) hemolytic anemia, vaso-occlusive events (pain / infarction) Unstable hemoglobins drug induced hemolytic anemia Oxygen affinity variations / M hemoglobin cyanosis / polycythemia Thalassemia( / types, microcytic anemia + iron load from transfusion dependence, from / imbalance)

HEMOGLOBIN TYPES:QUICK GUIDE

Hb A(adult hemoglobin) 22

Hb A2 (minor adult) 22

Hb F(fetal hemoglobin) 22

HbE(embryonic hemoglobin) 22

Produced only just after conception 22

Chromosome Genes

-globin cluster 11 Beta(), Delta (), Gamma (), Epsilon ()

-globin cluster 16 Alpha (), Zeta ()


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Sickle Cell Anemia

Hb S: a different type of Hb (like Hb A, F, etc.) where theres a 6

GLU to VAL mutation

HB C: a different type of Hb too (B6LYS to VAL);doesnt form polymers as readily as S (but more readily than A)

Remember that Hb A is normal adult Hb

Genotype Phenotype

AA Normal

SS Sickle celldiseaseAS Sickle celltrait: no sickling in vivo; same survival as AA

SC SC disease (compound heterozygote); less sx than SS

Pathophysiology

Polymer formation: 6 Glu Val mutation fits into ahydrophobic pocket in adjacent chain of another tetramer

Other interactions are favorable & stabilize too Hb in RBC is really concentrated (97-8% protein is

Hb!) these polymers are big deal!

Polymers & crisis:1. Polymer formation is concentration

dependent: in normal situations,

polymers are in reversible equilibrium

with monomers

2. After you hit a certain point (critical

polymer), growth of the polymer is thermodynamically favorable

3. Polymersrigidity, changes in cell shape, membrane distortion

(SICKLING!)

4. Classic theory:Rigid RBC obstruction ofblood flow tissuehypoxiatissue damage (localized pain + swelling); repeated lowgrade obstructionorgan damage (spleen, lungs infarct)

5. More current ideas: COMPLEX, MULTIPLE EVENTS (RBC, WBC,coagulation)

o RBCs block bifurcations in blood vessels, pre-capillary arterioles (why crises manifest in multiple places!o Membrane distortion of RBC expose new proteinsstick to endothelium (along with RBC)o NITRIC OXIDE: Intravascular hemolysis, arginase leakage,NO inflammation & vasoconstriction

(NO levels would explain some of the systemic nature of the crisis)

o Coagulation cascade activated too6. Genetic modifiers are key too: not simply a single gene

o WBC count, Hb F levels, cytokine production, endothelial surface proteins, even in opiate receptors(some patients dont respond as strongly; docs think theyre malingering!)Hb F and Sickle Cell Disease

Increased Hb F corresponds to less pain (Hb F doesnt form polymers) Newborns therefore have no symptomseven if SS (enough Hb F around to inhibit Hb S polymerization) Hydroxyurea (and two other drugs): can increase Hb F in SS patients (4%15%, 50% reduction in pain crises,

hospitalizations, mortality).

Other treatment: transfusions, bone marrow transplant if needed, others

FEATURES of SICKLE CELL ANEMIA

Hemolytic anemia (shortened red cell survival) Complex pathophysiologyvariable severity Gene-switching therapy

SC crisis:

Sudden in onset Unpredictable (and 80% SS have < 1 crisis/yr) Variable: even among individuals with

identical sickle mutations! MultifocalSuggests that classical theory is incomplete


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Lab tests for sickle cell(know how these compliment one another for the exam)

Sickle Prep (Sickledex, sickle solubility test)

1. Put drop of blood in sodium hydrosulfite solution2. RBC lyses, releases Hb

o Hb A goes into solution (clear)o Hb S precipitates (insoluble cloudy)

3. ALL THREE TYPES (SS, AS, SC) ARE POSITIVE (just screening for presence of Hb S)CANT DISTINGUISHHb Electrophoresis

1. Run sample on electrophoresis; measure migration2. CAN DISTINGUISH SS vs SC vs AS vs AA

Thalassemia

Mediterranean populations (Italians, Greeks, Arabs, Africans) & Southeast Asia

Normal ratio :alpha = 1.0 (beta thal 1)

Beta thalassemia

100+ mutations, decrease in globin production, dont alter -globin protein (normal Hb A, just less) Excess alpha chainsunstable 4 tetramersprecipitate RBC damagehemolysis

o chains are still around: make 22 (Hb F) in increased levelso chains are around in adults: make 22(Hb A2)in increased levels tooo Hb electrophoresis: see increase in Hb F and Hb A2relative to Hb A

Presentations:

Genotype Name Features

Heterozygote Thalassemiaminor, thalassemiatrait Small RBCs, minimal anemiaHomozygote Thalassemiamajor

(Cooleys anemia)

Transfusion-dependent (severe anemia)

Microcytic / hemolytic anemia

Compound heterozygote Thalassemia intermedia Thal major with 5+ alpha genes = more mild

Thal minor with 4- alpha genes = more severe

Diagnosis:

microcytic anemia (decreased Hb, low MCV, hypochromic, etc.) increased RBC number (marrow trying to compensate nucleated RBC in periphery) Elevated Hb F and Hb A2relative to Hb A Features of chronic anemia (thal major): Hepatosplenomegaly (extramedullary hematopoesis), brittle bones /

enlarged marrow space (hypertrophy of skull/facial bones, hair on end appearance)

Therapy:

prenatal dx (incidence has declined in countries where risk was high) hypertransfusion + iron chelation (would overload otherwise; no excretion mechanism) bone marrow transplant if severe; Hb F switching agents (e.g. hydroxyurea) might not make enough Hb F to help

Sickle Prep Hb electrophoresis

Speed Fast Slow

Can distinguish

AA/AS/SS/SCNope Yep

DISEASE OF GLOBIN CHAIN IMBALANCE

> Beta thalassemia

> Alpha thalassemia


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Alpha thalassemia

Excess beta chains unstable 4 tetramers precipitate RBC damage hemolysis In newborn: chains make 4tetramers (no alphas around)Hb Barts; can detect it but useless

o chains dont do you any good either, since youre low on chains! Nothing to pair it with.o Hb electrophoresis: Hb F, Hb A2dontchange relative to Hb A(all need chains, so all decrease!)

Common in Black, Italian, Greek, Arab, Asian, Indonesian populations

Tremendous selection factor (See map)Genotype Phenotype Features

4 alpha genes Normal Normal

3 alpha genes Silent carrier No Sx but can pass on gene

2 alpha genes Trait Microcytic anemia(like thalassemia trait &

mild iron deficiency anemia in severity)

1 alpha gene Hb H Severe hemolytic anemia

0 alpha genes Hydrops fetalis Inconsistent with life (die in utero)

Diagnosis:

Microcytic RBC anemia Often confused with iron deficiency Hb A2 / Hb F levels normal relative to Hb A so Hb electrophoresis not helpful! RDW / MCHC not reliable; family studies may not be helpful Rely on clinical history (race of patient); must rule out iron deficiency anemia as microcytic anemia cause

o E.g. put em on Fe and they dont get better! Think -thal!Methemoglobin (Fe

3+): a Hb variant

Doesnt lead to anemia; can cause decreased oxygen transport(pseudo)cyanosis Blood color (Hb color) altered: RED (Fe+2) BROWN (Fe+3) Can confuse with: pulmonary or cardiovascular disease

Causes: genetic or acquired

Aut-dom: congenital variants in alpha or beta chain that stabilize heme molecule in Fe+3 (ferric) state Aut-rec: deficiency ofenzymes in RBC that help keep heme molecules in Fe+2 state (Reduced) Acquried: oxidant damage of hemoglobin (drugs / toxins especially nitrates)

o Too much nitric oxide (used to close PDA in infant)o Diarrhea (nitrates overproduced by bacteria)o Poisoning (nitrates from fertilizer in well water)o Other drugs: nitroglycerin, sulfonamides, napthaline, others with nitrates.


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Hemostasis

Background: blood under pressure; breaks in vascular continuity exsanguination (need to seal it off)

Procoagulants: Platelets & coagulation proteins work together to stop bleeding1. Vessel walls contract (reduce vascular flowbleeding)2. Platelets adhere (vWF, etc.), provide phospholipid-rich environment for coagulation factors3. Cross-linked fibrin network (via coagulation cascade)

Anticoagulants: Balanced against procoagulants (1. Endogenousanti-thrombotic proteins (dampen procoagulant coag cascade)2. Fibrinolytic system (remodel / dissolve clots)

Hemostasis

General principles: basic goal is to get to formation & cross-linking offibrin

Extrinsic pathway more important in initiationo Requires extrinsic tissue factoro Produces some thrombin (IIa)o Tissue factor inhibitor shuts it down pretty quickly

(released by intact epitheliums endothelial cells)

Intrinsic pathway big in amplification / propogationo (heats up after thrombin production by extrinsic pathway)


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The Coagulation Cascade

THE PLAYERS

Serine proteases XII, XI, X, IX, VII, II (prothrombin), prekallikrein Circulate in blood as zymogens, cleave & activate others

Cofactors VIII (cofactor for IX) V (cofactor for X) HMWK* ( cofactor for XI & prekallikrein) Tissue factor (cofactor for VII)

Increase reaction kinetics (1000 fold) by localizing /

concentrating partners to membrane surfaces (optimize

reaction)

Glycoprotein Fibrinogen Becomes insoluble (fibrin) after thrombin cleaves it

Transglutaminase XIII

Cross-links fibrin strands covalently Links 2-antiplasmin to fibrin |

(inhibits plasmin, part of fibrinolytic system)

If no XII: clots fall apartVitamin-K-

dependent

II, VII, IX, X Proteins C, S Need vitamin K for synthesis (warfarin inhibits) Vit K needed for addition of -carboxy glutamic acid side

chains (allow proteins to bind phospholipid-rich

membranes in presence ofcalcium

No Vit K no side chains no binding, less activity*HMWK = high-molecular-weight kininogen

XII

XIIaIX

VIIIa

TF + VII

Surface kallikrein

HMW kininogen

Ca++

IIaVIII

Xa

XIa

II

fibrinogen

fibrin monomer

fibrin clot

XIIIa

Ca++

Phospholipid

XIIIIIa

Va

IXa

Ca++

Phospholipid

Ca++

Phospholipid

Extrinsic

Pathway

Intrinsic

Pathway

Ca++

IIa

XI

X

TF + VIIa

V


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Steps of Coagulation Cascade (in more detail)

EXTRINSIC PATHWAY

Step DescriptionRequirements Anticoagulant

Target?

Associated Diseas

State?Ca PLs Vit K

0 Factor VII normally circulates; small amounts ofVIIa also in blood (VII needs vitamin K)

1 Vascular damageTissue factor exposed in subendothelium (normally hidden)

2 Factor VIIa forms complex with tissue factor

3 VIIa + TF cleaves X Xa (X requires vitamin K)INTRINSIC PATHWAY

Step DescriptionRequirements Anticoagulant

Target?

Associated Dise

State?Ca+2

PLs Vit K

0Factor XII activated by exposure to highly negatively charged surfaces (e.g. endothelium)

Note: Not importantin vivo (just for assays)

1XIIa activates prekallikrein kallikrien; high molecular weight kininogen is a cofactor(concentrates prekallikrein on the membrane where XIIa generated)

1.5 Kallikrein activates more XII XIIa (positive feedback)2 XIIa also activates Factor XI (HMWK is cofactor, concentrates XI on membrane)

3XIa activates Factor IX IXa(needs Ca

++and phospholipids surface of activated platelets; IXa needs Vit K for synth)

ATIII (XIa XIi)Hemophilia B:

Factor IX deficienc

3.5 Meanwhile, VIII activated by thrombin (Factor IIa) VIIIa Proteins C/S(VIIIaVIIIi)

Hemophilia A:

Factor VIII deficien

Von Willebrand Dis

4 VIIIa + IXa form tenase complex, assembles on PL-rich platelets using Ca+2

5VIIIa+IXa (tenase) cleaves X Xa(X also requires vit K, binds to PL using Ca

+2using -carboxy-glutamic acid side chains)

ATIII (IXa IXi)

COMMON PATHWAY

Step Description Requirements AnticoagulantTarget?

Associated DiseState?Ca

+2PLs Vit K

0XXa either by VIIa/tissue-factor (extrinsic) or IXa/VIIIa (intrinsic)(X needs vitamin K, happens on platelet surfaces with Ca + PLs)

1 V Va via thrombin (IIa) activation Prot. C/S (VaVi) Factor V Leiden(hypercoaguable)

2Va + Xa (prothrombinase complex) cleaves prothrombin (II) thrombin (IIa)(thrombin needs Vit K for synthesis)

ATIII (Xa Xi)Thrombin deficien

embryonic lethal

3 Thrombin (IIa) cleaves fibrinogen fibrin monomers ATIII (IIa IIi)4 Fibrin monomers polymerize (loose fibrin clot, hydrostatic bonds

5 Thrombin activates XIII XIIIa4 XIII is a transglutaminase; catalyzes fibrin cross-linking & affixes 2-antiplasmin


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Observations:

When thrombin gets cleaved, things really start rolling: more cofactors! VIIIVIIIa (intrinsic), VVa (common)o Thrombin also activates platelets and protein C (anti-thrombotic protein!)

When vitamin-K-activated stuff is involved (II, VII, IX, X) the action needs calcium and takes place onphospholipids of platelet surfaces (thats where -carboxy glutamic acid side chains like to do their thing)

Lab TestsBasic idea for both PT & APTT:

1. draw blood into sodium citrate soln (chelate calcium)2. centrifuge (keep plasma only)3. add phospholipids (platelets were removed) and calcium4. add XII for APTT (intrinsic pathway) or tissue factor for PT (extrinsic pathway)

5. Measure time to clot formation based on light absorption

Prothrombin Time (PT) Activated Partial Thromboplastin Time (APTT, PTT)

Extrinsic Pathway YES (VII) no

Intrinsic Pathway no YES (XII, HMWK, prekallikrein, XI, VII)

Common Pathway YES (X, V, II, fibrinogen)

Factor XIII NEITHER (need test ofclot strength; these are just for clot formation)

Mixing test:

Prolongation of PT or APTT can be from either deficit in coagulation factor or Ab against coagulation factor Mix 1:1patient:normal plasma, re-run prolonged test.

o If it corrects, it was deficiency (50% of factor sufficient to normalize test)o If its still prolonged, abnormality is from antibodies (neutralizing factors in normal plasma too)

Factor levels

Isolate a specific factorthats the issue: use factor deficient plasma (all but one factor) and add patient plasmao Run test (APTT or PT depending on pathway you suspect)will be dependent on patients factor levelo set up standard curve with length of test vs % of factor (controls), determine patients level

DiseasesHemophilia A: congenital factor VIII deficiency

X-linked, recessive (1/10k males) Deep tissue bleeds, hemarthroses (joint bleeds) Severityvaries with factorVIII level (mild > 5%, moderate 1-5, severe >1%)

Hemophilia B: congenital Factor IX deficiency

X-linked inheritance (1/50k males) Similar manifestations to hemophilia A

Von Willebrand disease: congenital vWF deficiency Autosomal, most common inherited bleeding disorder (1 in 1000) less vWF, which is a carrier protein for VIII in the bloodstream (protects

from C/S inactivation); ); less severe than hemophilia A

More important:adhesion of platelets to subendothelial collagen Bleeding from mucosal surfaces rather than deep tissue bleeds (platelets!) Tests:

o ELISA (vWF antigen test) used to measure vWF levelso Ristocetin Cofactor Assay (antibiotic; causes vWF-dependent platelet aggregation, tests vWF function

Treatment of hemophilia A:

Recombinant/plasma-derivedfactor VIII

DDAVP (desmopressin) torelease VIII & vWF (mild only)

Treatment of vWD:

mild vWDDDAVP (makesendothelial cells release vWF)

severe factor VIIIconcentrates (have vWF)

Treatment of hemophilia B:

Factor IX concentrates


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Vitamin K deficiency: MOST COMMONACQUIRED BLEEDING DISORDER

Immature forms ofII (prothrombin), VII, IX, Xo Also C and S

Half-lives vary: VII has shortest half-lifeprolonged PT 1st(extrinsic)o Eventually PT & APTT both abnormal

Treatment: Vitamin K (oral, sub-q, IV)

Liver disease: all coagulation proteins made in the liver Severe liver disease only: need ~90% loss of function before PT/APTT slowed; means poor prognosis Factor VIII synthesized outside of hepatocyte; not affected Treatment:plasma

Etiologies of Vit K deficiency:

broad-spectrum abxsurgerypoor nutritionexcessive biliary drainagewarfarin


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Thrombosis

Balance between procoagulant & anticoagulant forces; imbalancehemorrhage or thrombosis

Circulation & Endothelial Cells: AnticoagulantsCirculation: prevent local accumulation of activated coagulation factors & incidental

thrombous formation

Endothelial cells:

express thrombomodulin (binds thrombin, activates protein C) dontexpress tissue factor produce prostacyclin (PGI2): inhibits platelet function promote fibrinolysis (tissue plasminogen activator: activates plasminogen)

Endogenous anticoagulant proteinsAntithrombin III: inactivates IIa, IXa, Xa, XIa

1st to be discovered; synthesized in liver Serine protease inhibitor: suicidesubstrate for IIa, IXa, Xa, and XIa Inhibitory action increased 1000-fold ifheparin is around (used to treat thrombosis)

Proteins C & S: inactivate Factor Va & VIIIa

Both vitamin K-dependent; bind PL-rich surfaces like activated platelets when calciums around

Protein C: serine protease; Protein Cactivated protein C (APC)

Activated by thrombin when bound to thrombomodulin (endothelial cells)Protein S: in plasma; free (60%) and bound to C4b binding protein (40%)

Need free form to participate as cofactor for protein C C4b binding protein increased in pregnancy & estrogens / OCP (bind more protein S, hypercoagulable)

Activity: APC + free protein S make complex; inactivate Factor Va (Xa cofactor) and factor VIIIa (IXa cofactor)

Fibrinolytic systemPurpose: lyse fibrin clots

Plasminogen: major fibinolytic enzyme

circulates in inactive form in plasma, activated by two different enzymes (plasminogenplasmin)o Tissue plasminogen activator: from endothelial cells; mostly activates plasminogen on clot surfaceo Urokinase can activate plasminogenplasmin tooo Plasminogen activator inhibitor Iopposes this process (inhibits TPA / UK)

TPA / UK can be given for thrombotic disease (MI, PE)pharmacologically

Plasmin: cleaves fibrin into fragments (fibrin degradation products, FDP)

Opposed by 2-antiplasmin (connected to clot surface by FactorXIIIa); prevents premature clot lysis

Procoagulant:

platelets coagulation factors

Antithrombotic: circulation endothelial cells fibrinolytic system endogenous anti-

coagulant proteins

Fibrin FDP

TPA UK

Plasminogen activator inhibitor I

2-antiplasmin

Plasminogen Plasmin

Fibrin FDP

TPA UK

Plasminogen activator inhibitor I

2-antiplasmin

Plasminogen Plasmin


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Defects in fibrinolytic system not identified so far as venous thrombosis risk factor

Antiphospholipid Antibody Syndrome: predisposes to BOTH ARTERIAL AND VENOUS thrombosis

Ab against phospholipid binding proteins (e.g. beta-2-glycoprotein I; binds to endothelial-bound phospholipids) Ab binding triggers tissue factor expression by endothelial cells; C activated, endothelial damage

o End result: coagulation via extrinsic pathwaythrombosisSigns / sx:

Fetal loss (placental thrombosis) Thrombocytopenia (activation/consumption of platelets; immunological destruction

More common in pts with other autoimmune disorders (SLE, RA) but also viral infections (HIV) or cancers

Diagnosis: very important

Important: These pts have higher risk for recurrent thrombotic events; require longer durations of anticoagulant Tx

1. ELISA (phospholipid antigen substrate, add patient sample, detect with anti-human IgG/M/A)

2. Coagulation assays: sensitive to antiphospholipid Ab, which cause prolongation of clotting in vitro

APTT: use reagents with low phospholipid content, look for slowingo Mixing studiesto follow up (wont correct; Ab will still neutralize PLs)

Dilute Russell viper venom time (dRVVT)o Directly activates factor X in common pathway (DIC after a snake bite!)o Reaction & subsequent ones are phospholipid dependent; sensitive to anti-PL Abso Anything reducing X, V, prothrombin, fibrinogen would also slow test

Mixing studies to follow up (wont correct if inhibitor like antiphospholipid Ab present but will ifdue to warfarin, vitamin K deficiency, etc. normal pt plasma will have enough X, V, etc around)

Add purified phospholipids and repeat dRVVT: will correct (tons of PLs, bind all the antibodies,still have PLs around for reaction to take place)

Hyperhomocysteinemia Skipped over mostly in lecture; importance diminishing in

recent years

Acquired/congenital cause for both arterial & venousthrombosis

Deficiencies of folate / B12 / B5 result inhyperhomocysteinemia; mutations too

Risk increased for venous thrombosis and myocardialinfarction

High homocysteine tissue factor expression +endothelial damage; renal failure can result

need folate / B12 supplementation

Disseminated Intravascular Coagulation (DIC) Acquired coagulation disorder

Pathophysiology

excessive activation ofclotting cascadewidespread microvascular thrombosisconsumption ofclotting factors, platelets, endogenous anticoagulant

proteins & activation offibrinolytic system bleeding Common start: release of activators of coagulation (tissue factor / thrombin /

other activated serine proteases)

DIC: Associated with

Trauma Sepsis Snakebites Tumors Amniotic fluid emboli


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Plasmin working like crazy (fibrinolytic system)

digests fibrin clots into D-dimers (fragments) Can use ELISA to detect D-dimers in plasma (good in diagnosis)

Tx by detecting & correcting cause

Hemostasis: the whole big, ugly picture

DIC: LevelsD-dimers (plasmin!)APTT/PT (used up all your clotting stuff!)plateletsfibrinogen

hematology - pathophysiology

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