Hematopoiesis

Every day 200 billion RBCs, 100 billion WBCs and 100 billion platelets are made by an adult

Yolk sac (9-10d) Æ aorta-gonad mesonephros region Æ liver (5-6 wks) Æ bone marrow (5-6 months) x Primitive hematopoiesis occurs in yolk sac; little to no lymphoid or HSC generation x Definitive hematopoiesis occurs in the AGM region; capable of making all cell types and HSCs

Red marrow = primarily blood cells (from HSCs) in flat bones (pelvis, sternum, ribs, cranium) Yellow marrow = primarily fat; found in long bones

Progenitors are different from stem cells because they do not have self-renewal capacity x CFU-GM (colony-forming unit granulocyte macrophage) Æ most differentiated myeloid progenitor, make

WBCs x BFU-E (burst-forming unit erythroid) Æ make RBCs x CFU-MIXED Æ progenitor of CFU-GM and BFU-E

HSC Classes 1. Most primitive / high quality

a. Generate all lympho-hematopoietic lineages; life-long engraftment with extensive self-renewal capacity; rarely divide, resistant to disease

b. Phenotype = CD34+/-, Linneg, high ALDH, low HLA-DR c. ~10,000 in body, only ~1,000 active at a given time

2. Myeloid / low quality a. Lower self-renewal capacity (lasts 8-10years); target of most stem cell disorders/CAs b. Rapid but limited engraftment with committed progenitors c. Phenotype = more CD34, Linneg, lower ALDH, high HLA-DR

Stochastic (random) model Æ explains how 1000 high quality HSCs can produce so many cells x Asymmetric division whereby one daughter cell remains a HSC and the other differentiates to become more

committed x 2nd daughter  cell  undergoes  amplification  as  it  differentiates  and  makes  new  limited  “stem  cells”

Stem cell niche Æ critical for regulating self-renewal and differentiation of high quality stem cells

Growth factors promote survival; mutations in transcription factors or growth factor receptors lead to many diseases

Growth Factor Where  It’s  Produced Stimulates? Clinical Significance Erythropoietin Kidney

RBCs Anemia 2° renal failure

Thrombopoietin Liver, kidney, skeletal m. Platelets via survival of megakaryocytes

ITP Æ Ig attack/decrease platelets

Stem Cell Factor (SCF) Endothelial cells, fibroblasts

Mast cells; regulates stem cell niche

Allergic reactions

Flt-3 Dendritic cells and HSC Mutations lead to leukemia

G-CSF Endothelial cells, fibroblasts, macrophages

Neutrophils Tx chemo-induced neutropenia

Malignancy = unregulated clonal growth caused by 1 or more mutations in a stem (self-renewing) cell leading to increased proliferation or blocked apoptosis

x Cancer stem cell hypothesis: Tumor initiating cell acts similar to a stem cell Æ limited differentiation and limitless self-renewal

o Chemotherapy  that  does  not  kill  these  cancer  “stem  cells”  will  result  in  relapse x Most leukemia stem cells originate from low-quality HSCs x Pathways to bone marrow failure Æ oncogenesis/mutations, direct DNA damage, autoimmunity, viruses

Embryonic stem cells have the capacity to differentiate into any of the 3 germ lines (are pluripotent) Induced pluripotent stem cells (iPSC) can be generated from a differentiated cell that is reversed back to a pluripotent state with 4 transcription factors (though reverts to primitive hematopoiesis)

Anemia

Anemia = deficiency in RBCs and/or hemoglobin To increase oxygen delivery: Æ increase blood flow (Q), red cell mass (Hb) or oxygen unloading (%a-%v)

x Increase blood flow by: 1. Increasing cardiac output (↑HR, ↑pulse  pressure;  sx’s  =  murmur,  bruits, roaring in ears) 2. Change tissue perfusion: ↓to  skin/kidneys,  ↑to  vital  organs  (i.e.  brain  and  heart)

x Increase red cell mass (Hb) by increasing kidney synthesis of erythropoietin (EPO) o Stimulates RBC  production  in  marrow  (reticulocytosis);  sx’s  =  bone  pain o However, at some point, the ↑ in oxygen-carrying capability due to more Hb is off-set by the ↓ due

to increased blood viscosity (viscosity can also ↑  with  dehydration) x To increase oxygen unloading via increasing 2,3-DPG

o Synthesized during glycolysis pathway in RBC (only energy-production in RBC) Oxyhemoglobin Dissociation Curve

x Oxygen affinity Æ P50 = partial pressure of O2 at which half the Hgb is saturated

o Shift to the right = increased P50 = decreased O2 affinity = increased O2 unloading

o ↓ in pH, ↓in O2, ↑ in temperature, ↑  in  2,3-DPG,  ↑  H+    all shift to the right

x Cooperativity Æ partially saturated Hgb has a higher affinity for O2 o Deoxy or T = tense form (low affinity) o Oxy or R = relaxed form (high affinity)

Hemolysis releases hemoglobin from RBC Æ binds NO, resulting in vasoconstriction

x Can cause problems, especially with blood viscosity Æ can clot and cause stroke

Classification of Anemia Æ Diagnostic Approach 1. Determine the cause: ↓  RBC  production,  RBC  destruction,  or  RBC  loss  (i.e.  bleeding)? Reticulocyte count 2. Is the anemia microcytic (small RBC size), macrocytic (large RBC size) or normocytic (normal)? RBC indices 3. Is it hypochromic (decreased Hb per RBC) or normochromic (normal Hb)? RBC Indices 4. Is it associated with normal RBC morphology or abnormal RBC morphology? Peripheral blood smear

CBC Parameters and Analysis Test What it shows Calculation Indicates?

Hematocrit (Hct) Volume of packed RBCs per unit of blood (%)

# packed RBCs / ml blood

Hemoglobin (Hg) How much Hg in blood Grams Hgb / dL blood Reticulocyte How many young RBCs Hemolysis/bleeding Mean cell volume (MCV)* Size of the RBCs Hct / RBC count Micro/macro/normocytic Mean cell hemoglobin (MCH)

Weight of Hb in average RBC

Hb / RBC count

Mean cell hemoglobin concentration (MCHC)

Concentration of HV in average RBC

Hb / Volume packed RBCs Hypo/normochromic

*Normal RBCs are the size of a lymphocyte nucleus, are donut-shaped, and the center 1/3 is pale (without Hg)

Common causes for various types of anemia Hypochromic, microcytic Æ Iron/transferrin deficiency, thalassemia syndromes, sideroblastic anemia Macrocytic Æ Liver dz, newborns, drugs, marrow failure, reticulocytosis, megaloblastic anemias Normocytic, normal morphology Æ Hemorrhage/blood loss, unstable Hb, infections, chronic disease Normocytic, abn. morphology Æ Hemoglobinopathies, spherocytosis, autoimmune hemolysis, enzyme deficiency

VO2 = 1.39 x Q x HB x (SaO2 – SvO2) Uptake of O2 = 1.39 x blood flow x Hemoglobin x (% saturation arterial blood - % saturation venous blood)

Iron Deficiency Anemia

Heme: Fe-containing porphyrin ring; each Hgb molecule can bind 4 oxygen molecules at heme site Transferrin (Tf): plasma ion transporter protein Ferritin: multi-subunit protein (some in plasma, but mostly in cells) responsible for intracellular storage of iron Hemosiderin: insoluble form of ferritin (mostly in bone marrow and spleen), responsible for long-term iron storage

Iron Metabolism Distribution: 68% active use (Hb/mgb/enzymes); 40% storage (ferritin/hemosiderin); 0.1% transport (transferrin)

x 10-25mg ingested per day (mostly non-heme form) only 1-2mg absorbed via brush border in small intestine

Fe enters intestinal cell via DMT1; binds ApoTf to form Tf then exits into blood via FPN-1 and is picked up by soluble Tf x Tf binds transferrin receptors (TfR) on surface of cell that needs Fe Æ internalized into endosome, where

acid/H+ secretion releases Fe from Tf and into the cell (Tf is then recycled to serum) o Fe can then be used intracellularly or sent to mitochondria to be incorporated into heme for RBCs

The Iron Cycle (daily) Æ 1-2mg is circulating; 1-2 mg is lost in urine/skin; 1-2mg is absorbed from diet; 20mg are generated by destruction of RBCs

Pathophysiology of Iron Deficiency Depletion of Fe limits heme synthesis Æ limits Hb assembly Æ limits RBC production = microcytic/hypochromic RBCs

In response, the body increases absorption, transport and uptakes and decreases storage and utilization: x IRP-1 and IRP-2 (iron response proteins) stabilize 3' end of transferrin mRNA; ↑ transferrin receptor

production, thus improving iron transport x IRP-1/2 also bind to the 5' end of ferritin and ALA synthase mRNA, ↓ ferritin and ALA synthase production,

decreasing iron storage and utilization

A deficit in total body iron (requirement > supply) can be due to: 1. Blood loss 2. Failure to meet increased requirements (infant/adolescent growth, menstruation, pregnancy) 3. Inadequate absorption (2° diet or GI surgery)

Clinical Features of Iron Deficiency Anemia Symptoms common to all anemias Æ pallor, fatigue, weakness, dizziness, irritability Symptoms specific to IDA Æ Pagophagia (craving ice), pica (craving non-food), glossitis (smooth tongue), restless legs, angular stomatitis (cracked corners of mouth), koilonychia (spoon-shaped fingernails)

Diagnostic Tests x Peripheral blood smear Æ will show hypochromic, microcytic RBCs on smear x Serum ferritin & serum iron will both be low (low serum ferritin is very specific for IDA) x Bone marrow iron stain (gold standard in complicated cases) x RBC indices / CBC Æ will  show  nml  WBC,  ↓  H&H,  MCV,  MCH,  absolute  reticulocyte  count

Sequential changes in IDA = First Ferritin Æ Iron Saturation Second Æ THEN MCV/Hb/Hct (Iron deficiency develops before anemia develops)

Treatment Æ Oral Fe (can cause constipation/gastric distress Æ non-compliance), IV Fe, RBC transfusion x Phytates (cereal/grains), tannins (tea) and antacids can inhibit oral iron absorption; ascorbic acid can increase

PO Fe absorption, but not usually enough for a significant difference x Response = ↑reticulocytes  in  7-10  days,  ↑  Hb/Hct  in  2-3 weeks, nml H/H at 2 mo., nml Fe stores at 4-5 mo. x Identification and treatment of the cause of Fe deficiency is more important than correcting the anemia

o GI blood loss (ulcers, tumors), excessive menstrual loss, rare conditions (footstrike hemolysis, PNH)

Differential diagnoses Æ thalassemia trait (low MCV, nml RDW), anemia of inflammation x In IDA Æ Ferritin, serum iron, transferrin saturation and marrow iron are LOW; sfTR/log Ferr is high x With inflammation Æ Low serum iron, transferrin sat. and sfTR/log Ferr, and HIGH ferritin

Iron Overload Syndromes Iron is highly conserved in humans (<0.05% lost daily); NO way to excrete excess iron Æ overload can occur from repeated blood transfusions (treated with iron chelation drugs) or hereditary causes

x Can cause long-term cirrhosis, many endocrinopathies, CHF, cardiac dysrhythmias

Folate and B12 Metabolism and Deficiency

Pernicious anemia is characterized by triad of gastric atrophy, megaloblastic anemia & neurologic degeneration x Enlarged RBCs (macrocytosis) and unusual appearing RBC precursors (megaloblasts) on blood smear

o Macroovalocytes of the RBCs and hypersementation of the granulocytes = most important findings x Tests  show  hemolysis  (↑bilirubin,  LDH),  thrombocytopenia/leukopenia,  elevated  gastric  pH

Vitamin B12 (Cobalamin/Coenzyme B12)

x Only synthesized by microorganisms (ingested in diet via liver, glandular tissue muscle, eggs, dairy, seafood) o Body stores 2-5 mg; daily need = 2-5ug (0.1% of store) Æ stores will last 1000 days

x B12 Absorption: B12 is released from ingested protein via pepsin and salivary enzymes o Binds to intrinsic factor in gastric secretions; B12-IF complex absorbed ONLY in terminal ileum o Transcobalmin II (TCII) binds B12 in the blood; taken up by cells with TCII receptors

x Mediates methyl transfer Æ Cofactor in methylation of homocysteine to generate methionine (crucial to producing thymine and purine in rapidly dividing cells)

x Mediates hydrogen transfer Æ Generation of succinyl CoA from methylmalonyl CoA (myelin maintenance)

Causes of B12 Deficiency 1. Decreased absorption is the most common cause, due to:

a. Loss of intrinsic factor (autoimmune, atrophic gastritis 2° H. pylori, gastrectomy, aging, H2 blockers) b. Pancreatic disease or terminal ileum disease (sprue, IBD, resection, CA/lesions)

2. Inadequate ingestion (vegans, breast-fed infants of vegans/B12 deficient mothers) 3. Congenital deficiency (Transcobalamin II deficiency, Imerslung-Grasbeck syndrome)

Clinical findings specific for B12 deficiency Æ low serum B12 levels, PNS/CNS disease, methylmalonic acidemia x Falsely high/normal B12 Æ intestinal bacterial overgrowth, liver disease, myeloid disorders x Falsely low B12 Æ Pregnancy, lymphoid disorders, aging, racial differences x Use measure of methylmalonic acid to confirm B12 deficiency (will be high; >500)

Therapy for B12 Deficiency Æ PO vitamin supplementation IM monthly for life (if problem with intestine absorption) Folic Acid / Dietary Folates

x Synthesized by plants and microorganisms; absorbed in proximal jejunum and throughout small intestine o Must be deconjugated in intestine before absorption o Reduced to FH4 (N5-methyl tetrahydrofolate) within intestinal cells before entering circulation

x Daily requirement is 50ug; body stores 5mg (deficiency occurs in 3-4 months)

Folate deficiency Æ slows/stops DNA synthesis 2° uracil mis-incorporation into DNA resulting in strand breaks x Bone marrow has heavy DNA synthesis requirement Æ megaloblastic anemia results (same as B12 def.) x Macrocytosis results from asynchronous maturation of nucleus and cytoplasm, slowed DNA synthesis and

slowed maturation

Causes of Folate Deficiency x Inadequate ingestion (poor diet, alcoholism, scurvy) or absorption (celiac disease, IBD, drugs) x Metabolic block in utilization or increased requirement (pregnancy, infancy, hemolytic anemia)

o 1998  FDA  mandated  cereal  grains  be  fortified  with  folic  acid  to  ↓  neural  tub  defects

Diagnosis of Folate Deficiency Æ serum folic acid (reflects recent status), red cell folate level (chronic status) x Folate and B12 are both required to generate

methionine from homocysteine (via methionine synthase)

x High homocysteine levels can be due to defect/lack of cystathionine B-synthase (CBS), B12/folate deficiency, chronic renal dz o High homocysteine = ↑ risk for vascular dz

x Giving folic acid bypasses initial step in cycle that regenerates methionine Æ cures anemia, but masks B12 deficiency

Folic Acid

Differentiating Between B12 and Folate Deficiencies B12 Folate Megaloblastic anemia Yes Yes Combined system degeneration Yes No Poor diet associated with deficiency Rare Common Other dietary deficiencies usually present No Yes Dietary source Muscle, liver, dairy, eggs Liver, leafy greens High homocysteine levels seen with deficiency Yes Yes Site of absorption Terminal ileum only Small bowel Intrinsic factor required Yes No Deficiency associated with high methylmalonic acid Yes No

Porphyrins and Heme Metabolism

Porphyrin = 4 cyclized pyrrole rings connected via methylene bridges, planar; (e.g. heme and chlorophyll) x Can be identified by characteristic wavelength absorption (Soret band) x Are modified with side chains Æ determines solubility and where in body they will be found x Synthesized in most nucleated cells using a common metabolic pathway:

o Glycine + succinyl CoA Æ ALA; 2 x ALA Æ PBG; 4 x PBG Æ tetrapyrrole Æ cyclize to porphyrin Hemoglobin = a tetramer of heme monomers

x Breakdown of heme produces bilirubin (a tetrapyrrole); central to liver metabolism x Heme is made in the bone marrow via ALAS2 (constitutively) and the liver via ALAS1 (inducible)

o 304 mg/day made in marrow for Hgb; 54 mg/day made in liver for p450, catalase, cytochromes Heme Synthesis in the liver is controlled at 3 steps:

1. ALA1 Synthase Æ critical enzyme that synthesized d-ALA; the first committed step x Highly regulated; controls porphyrin synthesis x ALAS1 in liver is induced when cytochrome p450 enzymes are requirement

o Increased activity can lead to accumulation of downstream intermediates x Abnormal functioning of the system affect many systems, is rare (porphyria)

2. PBG Deaminase Æ can be limiting in heterozygotes 3. Ferrochelatase

Chronic/Dermatological Forms of Porphyria: Variegate porphyria/photosensitivity & Porphyria cutanea tarda (PCT)

x Burns/ulcers/blistering with sun exposure, purple color (metabolites) in urine under UV light Acute Intermittent Porphyria (AIP) Æ an inducible metabolic disorder

x Autosomal dominant (heterozygous) defect in PBG deaminase resulting in high urine [PBG] and [ALA] o No skin changes; exquisite pain; central and peripheral neurologic damage also seen

x Due to heterozygosity; sx's don't develop until flux through pathway exceeds enzyme's capacity o Triggered by induction of ALAS1 due to drugs, chemicals, hormones, or diet

x Treatment Æ slow/stop ALAS1 activity (stop causative drug, negative feedback with glucose/heme) o No change to underlying genetic mutation

RBC Metabolism

Glycolysis Review Glucose Æ 2 pyruvate via glycolysis, but alternative products are made via shunts from the glycolytic pathway:

1. PPP generates NADPH (mediated by G6PDH) by branching at the 1st step in glycolysis (G6P) 2. 2,3-DPG is produced from 1,3-BPG (2nd step in pay off phase) Æ Rapoport-Leubering Shunt

o Use of 1,3-BPG prevents/sacrifices the production of an ATP at that step

Glycolysis characteristics specific to RBCs 1. Hexokinase I in RBCs converts glucose to the G6P (ATP-dependent) as the first step in RBC glycolysis

a. Has a lower Km than glucokinase or hexokinase IV (fully saturated at nml blood glucose levels) b. Is inhibited by G6P (negative feedback)

2. The 1st step of the payoff phase (G3P Æ 1,3-BPG) requires NAD+ Æ In RBCs, NAD+ must be replenished by lactate dehydrogenase (b/c RBCs have no mitochondria)

3. The 2nd/KEY regulatory point mediated by PFK-1 (F6PÆF16BP) is controlled by ATP/AMP/ADP ratio a. In RBC and all cells, small changes in AMP and ADP are more important than level of ATP

4. At the 3rd regulatory point, pyruvate kinase converts PEP Æ Pyruvate + ATP a. In RBCs, pyruvate kinase is positively regulated by AMP, F1,6BP, PEP and neg. regulated by ATP

5. Many glycolytic enzyme deficiencies are related to hemolytic anemia

2,3-DPG Generation from the Glycolytic Pathway in RBCs x 1,3-BPG + Bisophosphoglycerate mutase Æ 2,3-BPG, which binds Hgb and reduces O2 affinity

o Delivers increased amount of O2 to tissues (elevated at high altitude and late pregnancy) x In RBCs, ~20% of glucose is metabolized to 2,3-DPG (lose 1 ATP that could be generated at that step) x Lower 2,3-DPG levels are seen with hexokinase and PFK-1 deficiencies x Elevated 2,3-DPG levels (and therefore ATP depletion) result from pyruvate kinase deficiency

Pentose Phosphate Pathway has 3 important functions: 1. Supplies cell with NADPH (provide reducing power, serve as a biochemical reductant, used in cytochrome

system, electron source for reduction in DNA synthesis ) 2. Convert hexoses into pentoses (essential components of ATP, CoA, NADP+, FAD, RNA, and DNA) 3. Enable complete oxidative degradation of pentoses by converting them to hexoses and trioses that can enter

the glycolytic pathway

The most important function of the PPP in RBCs is to maintain glutathione in a reduced state x To serve as a sulfhydryl buffer that maintains reduced cysteine residues in Hgb and other RBC proteins x To maintain iron in the ferrous/reduced state (Fe++) x To detoxify by reacting with hydrogen peroxides and organic peroxides (calayzed by glutathione peroxidase)

o Reduced glutathione is essentials for maintaining normal RBC structure (prevents hemolysis)

G6PDH Deficiency Can Lead to Hemolysis Æ Impairs the ability of erythrocytes to form NADPH x G6PDH catalyzes 1st step in PPP ( deficiency affects NADPH generation and hexose to pentose conversion)

o NADP+ is the rate-limiting substrate (NADPH inhibits via negative feedback)

x Can be induced by some drugs (primaquine for malaria), and cause hemolytic anemia

x Chemicals that increase oxidative stress induce symptoms in someone with G6DPH deficiency

o Include sulfonamides, ASA/NSAIDs, quinadine/quinine, mothballs/napthlane, fava beans

x Favism Æ Usually male children (1-5); peak in April/May around harvest time

o Sx's = 5-24 hrs after ingestion of fava beans, onset of hemolytic anemia, nausea, back pain, chills, fever, jaundice, hemoglobinuria, acute fall in Hgb requiring transfusion

Hemolysis

Hemolytic disorder = any disorder in which the survival time of an erythrocyte is < 120 days (normal) x Primary causes (usually congenital): membranopathy, enzymopathy, hemoglobinopathy x Secondary causes (usually acquired): immunologic, chemical, physical

Compensated hemolytic disorder = Increased rate of RBC production compensates for destruction Hemolytic anemia = when rate of RBC destruction cannot be compensated by increased production

x < 1/6 to 1/8 normal life span (<15-20 days)

Diagnosing a Hemolytic Disorder x Direct methods Æ RBC survival studies

o Ashby (mostly historical) Æ transfusion of mismatched RBCs; determine % surviving � Disadvantage = doesn't assess pt's endogenous cells

o Radioactive chromium, Cr51 (most common) Æ binds to pt's own hemoglobin � Disadvantages = Cr elutes from Hgb, higher affinity for reticulocytes, can't differentiate

between blood loss and hemolysis o DIFP labeled with P32 Æ binds irreversibly to red cell receptors

x Indirect methods Æ Measure  metabolic  products  of  RBC  destruction,  show  ↑d  RBC  production o Increased % or absolute reticulocytes seen w/ supravital staining (methylene blue) o Peripheral blood smear Æ large cells, diffuse basophilia, nucleated RBCs (prematurely released) o Bone marrow (increased erythroid precursors, medullary expansion)

� Classic bone sx's = maxillary prominent, expansion of ribs, hands, and skull (hair on end)

CO is liberated proportionally to RBC degradation Æ rate of CO production correlates linearly with RBC survival x Difficult technique, can't be used for smokers (only useful for pts with low CO levels normally)

Indirect bilirubin = unconjugated bilirubin = water insoluble (not in urine)

x Indirect bilirubin is elevated with hemolytic anemia because the liver cannot conjugate fast enough Direct bilirubin = conjugated bilirubin = water soluble (found in urine) Æ conjugated in liver

x Direct bilirubin is elevated in liver disease because the deficiency is in excretion

Haptoglobin =  α2  globin  which  binds  hemoglobin  on  a  1:1  ratio  basis  Æ an acute phase reactant seen when RBC survival drops below 90 days Hemopexin =  β  globulin  which  binds  heme  on  a  1:1  ratio  basis  Æ not an acute phase reactant, seen with both, but more specific for intravascular hemolysis Extravascular Hemolysis = RBCs are trapped in the reticuloendothelial system Intravascular Hemolysis = RBCs are destroyed within the systemic circulation

x NO depletion in intravascular hemolysis may contribute to pulmonary HTN, thrombosis, and ED x Specific markers: ↑  plasma  and  urine  Hgb,  ↑  urine  hemosiderin,  ↑  serum  methamalbumin

General  markers  for  hemolysis:  ↑Carboxyhemoglobin,  ↑serum  bilirubin,  ↑urinary  urobilinogen,↓haptoglobin,  ↓hemopexin  (hemopexin  most  common  in  intravascular  hemolysis) Intracorpuscular defects (within the RBC) Æ membranopathies (elliptocytes, spherocytes), hemoglobinopathies (sickle cell), enzymopathies (pyruvate kinase deficiency) Extracorpuscular defects (outside RBC) Æ thermal injury, mechanical injury, toxic injury, auto-immune antibodies Examples of pathophysiology leading to recognizable morphologic abnormalities:

1. Hereditary spherocytosis (intracorpuscular defect) = problem with protein interaction b/n membrane and cytoskeleton Æ decreased surface area Æ spherical shape

2. Autoimmune hemolytic anemia (extracorpuscular) = antibodies bind RBCs and remove parts of membrane when removed

a. Direct Coomb's test (detect Ig on RBC surface) Æ + in cases of autoimmune hemolytic anemia b. Indirect Coomb's test (detect anti-RBC Ig in serum) Æ + after FB exposure (transfusion, pregnancy)

3. G6PD Deficiency (extra and intracorpuscular) Æ Glucose cannot be shunted through PPP during oxidative stress Æ insufficient NADPH Æ Hgb precipitation Æ Heinz body formation Æ RBC destruction

Anemia in Systemic Disease Anemia = Hgb < 13 g/dL for men, <12 for women

x Low Hgb is an indicator of poor outcome Æ associated with  ↑mortality,  ↓strength,  and  ↓mobility x 10% people >65 yrs old are anemia; 20% of people >85

o 1/3 due to nutrient deficiency, 1/3 due to chronic disease, 1/3 no obvious cause/unexplained

Anemia of Inflammation and Chronic Disease (AICD) x Innate immune response in individuals with inflammatory disease Æ decreased absorption

o Low serum iron, increased macrophage iron, erythropoietin hyporesponsiveness x Characterized  by  ↓proliferation  of  erythroid  progenitors,  ↓erythrocyte  survival  &  functional  iron  deficiency

o Normocytic, normochromic on smear (sx's due to reduced number of RBCs) Steps of Erythropoiesis - Review

1. Proerythroblast (EpoR+, TfR+ ) Æ Requires EPO for survival 2. Erythroblast (TfR+) Æ Focuses on Hgb production; expresses

high level of transferrin receptors to get iron 3. Reticulocyte (TfR-, no nucleus) Æ Released into circulation;

continues to mature for several days 4. Erythrocyte circulates for 120 days, then endocytosed by tissue

macrophages, especially those in the spleen a. Some iron is stored in macrophage, some is released

to circulation to bind transferrin Molecular Mechanisms that contribute to AICD:

1. Cytokines Æ can inhibit various stages of erythropoiesis (IL-6 most closely associated with anemia) 2. Erythrocyte Turnover Æ results in anemia if production cannot compensate for rate of turnover 3. Iron availability

Hepcidin = acute phase response hormone made in the liver; binds to ferroportin (transporter responsible for iron egress in macrophages and enterocytes)

x Has both positive (IL-6, HFE, TFR-2, HJV) and negative regulators (sHJV, TMPRSS6) x Blocks iron recycling from macrophages and decreases iron uptake from diet x Assays for hepcidin Æ SELDI-TOF MS, C-ELISA, are complicated by hepcidin's role in iron homeostasis x Overexpression causes iron sequestration/AICD; deficiency leads to hereditary hemochromatosis

Hereditary Hemochromatosis Æ increased iron absorption, increased serum iron, low iron levels in macrophages

x Excessive absorption due to lack of expression of hepcidin Treatment for AICD Æ Treat the underlying disease (problem = many diseases are managed, but not treated)

x Use of erythroid stimulating agents (ESAs) with chronic renal disease x DRIVE trials Æ EPO doesn't always correct Hb, serum ferritin is a poor marker of available iron, pts may

benefit from IV iron and Hepcidin antagonists Diagnostic Differences Between IDA and AICD IDA AICD Blood Smear Microcytic, hypochromic Normocytic, normochromic Hemoglobin Low Low Serum Iron Low Low Serum Transferrin Low High Serum Transferrin Receptor High Normal Serum Ferritin* Low High *Serum ferritin is elevated in the context of inflammation and thus a poor marker of iron stores in AICD

Hemoglobinopathy and Thalassemia Hemoglobinopathies result from mutations at the globin gene locus that affect Hgb tetrameric structure

x Sickling disorders are due to change in charge or hydrophobicity of a surface AA x Thalassemias result from mutations anywhere that affects globin gene expression, which alter the

expression of the globin chains (imbalance  of  α/β  ratio) x Disorders of Hgb are the most common inherited diseases in the world Æ often have a genetic advantage

for heterozygotes (prevalence is higher in areas endemic for malaria )

Genetics of hemoglobinopathies Æ Autosomal recessive inheritance pattern x α  lineage (for ζ  &  α  globin  genes)encode  α  chains  and  are  found on chromosome 16 x β  lineage (for γ,  β,  ε  &  δ  globin  genes) is found on chromosome 11 x Three distinct switches in the genes produce 3 different types of globin chains/Hg

o Fetal Hgb (Hb F)  =  2  α  and  2  γ chains (from 8wks until 6 mo after birth) o HbA2 (minor  Hgb)  =  2  α  and  2  δ chains (used diagnostically) o Adult Hgb (Hb A) =  2  α  and  2  β chains (most common in adults)

x Polymorphisms in these genes cause >1200 variants, most of which aren't clinically significant Sickle Cell Disease Æ mutation  in  β  chain  =  hemolytic anemia, vaso-occlusive events, possible fatal complications

x Altered charge/hydrophobicity of a surface AA allows Hgb to aggregate while in deoxy form, causing long rigid fibers that disrupt the RBC membrane

o Valine in the Beta-6  position  binds  with  hydrophobic  pocket  of  β  chain  in  adjacent  tetramer   x Sickledex is a test using a reagent that lyses RBC if Hgb S is present

o Will be positive for any Hgb S production, so will be positive for SS, SC, and AS x Hemoglobin electrophoresis Æ separates Hgb by charge (A and S migrate differently) x HPLC gives quantitative value of how much of each Hgb is present

Sickle Cell Trait Æ Asymptomatic; however can be affected at high altitudes or under extreme circulatory stress x Trait = Hgb A > Hgb S Æ usually 60/40 ; Sickle cell disease = Hgb S > Hgb A

o 60% S and 40% A = sickle cell trait with B-thalessemia which reduces production of Hgb A x SS disease = close to 100% Hgb S; SC disease = 50/50 Hgb S and Hgb C

Thalassemia disorders

x Mutation leading to decreased synthesis of one of the globin chains in the Hgb tetramer o Precipitation of unstable globin chains Æ ineffective erythropoiesis/hemolysis + produces

inclusion bodies Æ cause damage to RBC cytoskeleton and membrane � RBCs with inclusion bodies are removed by macrophages before 120 days

x Results in RBCs that are hypochromic and microcytic as well as target cells o RBCs have less Hgb in them, so the body makes more Æ diagnostically ↑  RBC  ↓  MCH ↓MCV

Differentiating between beta and alpha thalassemia x Normally  production  of  α  globin  from  4  α  genes  and  β  globin  from  2  β  globin  genes  is  balanced

o Extra  α  chains  combine  with  δ chains Æ HbA2 (measure  with  HPLC  to  diagnose  β  thalassemia) o α  Thalassemia  diagnosed  clinically  after  excluding  β;  no  specific  test

x With  α thalassemia,  you  produce  extra  β  chains; with β  thalassemia  you  produced  extra  α  chains o Precipitation  of  excess  α or  β  chains  Æ premature RBC destruction in peripheral circulation

Alpha thalassemia x Will want to do α  gene  DNA  analysis for  α  thalassemia  diagnosis

o African Americans often have (aa/a-) or (a-/a-) mutations, at least 1 α  chain  gene  is  functional o SE Asians often have (--/aa) mutation Æ concern for (--/--)  complete  inability  to  make  α  chains

x 2 clinically significant disorders o Hemoglobin of Bart's /  α0 (--/--) Æ fatal;  excess  γ  chains form tetramers, lead to hydrops fetalis o Hemoglobin H- (a-/--) Æ Excess  β-chains form tetramers that can precipitate out as the RBC ages

and lead to shortened RBC survival (compatible with birth)

**Whether a reticulocyte count is normal depends on the hemoglobin level x Example: with very low Hgb, a very high absolute retic is expected Æ normal range would be abnormal

Aplastic Anemia - Bone Marrow Failure Cytopenia = Low circulating blood counts due to compromised bone marrow production

x Can include low WBCS (leukopenia), neutrophils (neutropenia/granulocytopenia), RBCs (anemia), or platelets (thrombocytopenia) Æ Most not caused by a 1° bone marrow pathology

x Characterized as unilineage or multilineage (broad affects on multiple lines and organ systems)

Amegakaryocytic thrombocytopenia = Marrow failure due to recessive loss-of-function of MPL, the thrombopoietin receptor (no thrombopoietin signal = no megakaryocyte lineage production)

x Megakaryocytes are absent, platelets are very low Æ can progress to multilineage aplasia or leukemia Inherited Bone Marrow Failure Syndromes (most present in 1st decade of life w/ marrow failure sx's)

1. Fanconi Anemia = multilineage congenital bone marrow failure due to autosomal recessive mutation x Presentation Æ upper limb malformations, 'café au lait' spots on skin, short stature, thrombocytopenia,

facies, multiple cytopenias; reduction in bone marrow cellularity replaced by fat in childhood x Due to DNA repair defect resulting in damage in replication Æ predisposed to many types of malignancies

o Progression to MDS and acute leukemia is common (avg age of CA onset = 16) x Treatment Æ stem cell BMT (ideally from HLA-identical donor) if very severe, to restore hematopoiesis

o Hypersensitive  to  toxicities  (chemo/radiation)  used  in  BMT;  ↑  risk  for  solid  organ  tumors

2. Dyskeratosis Congenita (DKC) Æ Defect in telomere maintenance caused by mutations in telomerase complex x Sx's = premature graying, pulmonary fibrosis, hyperpigmentation of skin, nail dystrophy, oral leukoplakia x Diagnosed via Flow FISH assay to determine telomere length x Tx Æ androgens, BMT (only in life-threatening marrow failure)

3. Diamond-Blackfan anemia = Congenital red cell aplasia due to defect in erythroid colony forming unit (CFU-E) x Presentation Æ Macrocytic anemia, reticulocytopenia, normal WBC/platelet counts x Sx's include facial defects and radial abnormalities Æ less leukemia than other two x >50% due to haploinsufficiency  of  a  ribosomal  subunit  (stress  ↑p53  levels,  ↑  apoptosis  in  RBC  lineage) x Treatment Æ corticosteroids, transfusions, BMT if severe

*memorize for exam* Acquired bone marrow failure syndromes

1. Aplastic Anemia = Pancytopenia with hypocellular marrow (can be inherited or acquired) x Congenital causes discussed above; most acquired cases are idiopathic or autoimmune (T-cells target

CD34) x Severe aplastic anemia(SAA) = hypocellular bone marrow + at least 2 markedly low blood counts

o Neutrophils < 500, platelets < 20,000 or corrected retic < 1% abs retic <60,000 x Very severe aplastic anemia (vSAA) = Above but with neutrophils < 200 x Non-severe aplastic anemia(nSAA) = Hypocellular marrow/pancytopenia but qualify as SAA x Treatment Æ allogeneic BMT, immunosuppresive therapy, high-dose cyclophosphamide x Late complication = PNH, MDS/leukemia, relapse (40%), after BMT, GVHD & other CAs

2. Paroxysmal nocturnal hemoglobinuria (PNH) Æ Somatic X-chromosome mutation PIGA in HSCs x Defective anchor protein that binds CD55 and CD59 (complement regulators) to RBC surface,  ↑ing  risk  of  

complement attack, which results in intravascular hemolysis Æ releases Hgb into circulation o Scavenges NO; causes sx's like hemoglobinuria, ED, abd pain, dysphagia, w/ hemolytic anemia

3. Acquired Pure Red Cell Aplasia Æ Retic < 1% & a paucity of erythroblasts in marrow (<0.5%) x CausesÆ immunologic (T-cell or Ig-mediated), LGL leukemia, neoplasia, drugs, pregnancy, parvovirus x Parvovirus induces in immunosuppressed and chronic hemolytic anemia patients b/c they cannot clear

infection within the lifespan of their RBCs

Disease Defect Cancer/Other Risks Faconi anemia DNA Repair MDS/leukemia, solid tumors

Dyskeratosis congenita Short Telomeres MDS/leukemia, solid tumor, pulmonary fibrosis Schwachman-Diamond Ribosomopathy MDS/leukemia

Diamond-Blackfan anemia Ribosomopathy Lower risk of leukemia

2yr mortality >70%

Normal Platelet Structure and Function

Hemostasis = process of stopping bleeding; involves blood vessels, platelets and plasma clotting proteins

x Primary hemostasis is the response to injury to a blood vessel Æ vessel constricts, platelets aggregate

x Secondary hemostasis fortifies primary Æ clotting cascade induces the formation of thrombin and conversion of fibrinogen to fibrin, which is deposited around the platelets

o 12 enzymes are involved in the clotting cascade

Platelets (nml range = 150,000 - 350,000/ul) x Produced in the bone marrow by multinucleated

megakaryocytes, which are stimulated by thrombopoietin o Platelet release from megakaryocytes has been likened to budding o They are pre-packed with all proteins they will need (don't produce any more) for 7-9 day lifespan

x ~1/3 body's mass of platelets are stored in the spleen x Histology Æ No nucleus, abundant mitochondria, dense granules (contain ADP, ATP, and serotonin), alpha

granules (contain plasma proteins), extensive system of microtubules (can change morphology) x Platelet development stages = HSC Æ BFU-Mk/CFU-Mk Æ Immature Mk Æ Mature Mk Æ Platelet shedding

Thrombopoietin (TPO) Æ growth factor produced in liver (a little in kidney) that simulates megakaryocyte production x A potent megakaryocyte-stimulating factor; works synergistically with cytokines (esp. IL-6)

o Also primes platelets to be more sensitive to platelet agonists x Is constitutively expressed in the liver Æ platelets act as a sponge, taking up TPO from circulation

o The amount of TPO in circulation depends on binding of platelets x Knockout of Tpo or c-Mpl (TPO receptor) in mice Æ profound thrombocytopenia w/o affecting other RBCs

Overview of Platelet Function 1. Adhesion

x Within 1-3 seconds, von Willebrand Factor (vWF) binds subendothelium, allows platelets to adhere o vWF circulates in plasma; is produced and released from Weibel Palade Bodies in epithelial cells Æ

it's receptor is the platelet glycoprotein Ib-IX 2. Activation

x As initial platelets adhere, they release ADP and thromboxane (the main platelet agonists secreted from platelet granules)

o Thromboxane is made by platelets, derived from arachidonic acidÆ causes blood vessel constriction o Thromboxane A2 is active & unstable Æ 30 sec. half-life before forming inactive Thromboxane B2

� ASA impairs the production of Thromboxane A2 by irreversibly acetylating COX-1, thus reduces platelet function and activation

x At the time of vessel injury and primary hemostasis, tissue factor activated the clotting cascade o Thrombin, the final end product of the cascade, stimulates maximum platelet secretion, and acts as

a protease to convert soluble plasma fibrinogen to fibrin, which can polymerize 3. Aggregation

x Platelets adhere to one another, forming a "plug" that results from the binding of fibrinogen to glycoprotein IIB-IIIA (fibrin = large, water-insoluble polypeptide that is the supporting structure of a normal blood clot)

Platelet-Endothelial Interactions (control mechanisms to prevent excessive platelet adhesion to subendothelium) x Prostacyclin (PGI2) Æ Induces vasodilation, inhibition of platelet aggregation by raising cAMP levels x ADPase (CD39) Æ Enzymatic destruction of ADP released by platelets, decreasing platelet activation x Nitric oxide (aka EDRF)Æ inhibits platelet secretion/function by decreasing intracellular cGMP

Platelet dysfunction is more commonly acquired than inherited (renal failure from toxin build-up, drugs like ASA)

x A variety of deficiencies can lead to platelet dysfunction Æ absent receptors for agonists, adhesion, aggregation, and biochemical pathways important for producing thromboxane

Hemostasis – Coagulation Cascade

Classes of Coagulation Factors 1. Serine proteases Æ Factors XII, XI, X, IX, VII, II/prothrombin (all present in blood as zymogens) 2. Cofactors Æ Factors VIII, V, high MW kininogen & tissue factor (↑ activity of serine proteases)

a. Lack of cofactors Æ bleeding disorders 2° insufficient serine protease activation/clot formation 3. Fibrinogen, an adhesive glycoprotein (fibrin) 4. Tranglutaminase/Factor XIII (catalyzes covalent cross-links fibrin polymers into a stronger clot)

Vitamin K-dependent coagulation factors Æ Factors II, VII, IX, X and protein C & protein S

x Vit K adds γ-carboxy glutamic acid side chains to these factors, allowing them to complex with Ca2+ x Vit K deficiency = Functional Factor II deficiency Æ Warfarin interrupts the process of forming side chains x All reactions with Vitamin K factors take place on phospholipid-rich membranes (activated platelets)

The extrinsic pathway requires tissue factor (extrinsic to circulation) and ONLY involves factor VII

x Tissue factor + factor VII Æ factor VIIa (a = activated), which then activates factor X (common pathway)

The intrinsic pathway does not require any outside components for activation

x Factor XII is activated by high negative charges in exposed subendothelium

o Activates PK (pre-kallikrein) Æ K (kallikrein), aided by cofactor HMWK (high molecular weight kininogen), which then loops back and activates more factor XII

o A large concentration of factor XII activates factor XI (also uses cofactor HMWK)

x Factor XI activates factor IX Æ Factor IX with cofactor Factor VIII activate Factor X (common pathway)

The common pathway is where the intrinsic and extrinsic pathways converge, and results in a fibrin clot

x Factor X (activated intrinsically by VIII/IX and extrinsically by VII/TF) + its cofactor, Factor V activate Factor II o Factor X + Factor V together are called the prothrombinase complex; Factor II is called thrombin

x Factor II catalyzes formation of fibrin polymers from fibrinogen to form a clot and reinforce platelet plug o Also activates Factor XIII, which catalyzes the covalent cross-linking of the fibrin polymers

x Thrombin is the main clotting factor ÆIn addition to fibrin and XIII, thrombin activates upstream factors VIII and V (cofactors for IX and X), and activates platelets and protein C (endogenous anti-thrombotic protein)

Revised model of coagulation Æ Clotting starts with the extrinsic pathway Æ turns off rapidly after a burst of activity, poorly activates factor X Æ Activated thrombin triggers the intrinsic pathway at factor XI which more efficiently activates factor X (propagation)

x TFPI (tissue factor pathway inhibitor) inhibits extrinsic path by binding to factor VII to turn off rapidly PT and PTT are tests that measure how long it takes a clot to form after adding sodium citrate and lipids to plasma

1. Prothrombin Time (PT) Æ Test that activates Factor VII, and observes the reaction of the extrinsic pathway

2. Activated Partial Thromboplastin Time (APPT) Æ activates factor XII; assesses the intrinsic pathway

o Will be prolonged with deficient factors XII, XI, IX, or VIII (hemophelia) o Deficiency in Factors X, V, or II would prolong both PT and APTT

Mixing studies Æ Test in which abnormal plasma sample is mixed with normal plasma and clotting time is observed

x Deficiency state Æ adding normal plasma will restore normal time to clot formation (nml PT/PTT) x Inhibitor (e.g. an anti-factor Ig) Æ adding normal plasma will NOT restore normal clotting time

Factor Assays Æ Mix a small amount of patient plasma with larger amount of plasma deficient ONLY in the factor you are testing for (normalizes all other factors, so only)

x Then do APTT and compare to standard curve Æ the longer clot formation takes, the less factor is present Hemophilia Æ X-linked recessive disorder characterized by hemarthrosis (joint bleeding) and tissue bleeding

x Types: Hemophilia A = Factor VIII deficiency (more common); Hemophilia B = Factor IX deficiency x APTT used as a screening test (prolonged) Æ factor assays for VIII and IX x Severe = < 1%; moderate = 1-5%; mild > 5% of the deficient factor

Von Willbrand Disease Æ autosomal dominant/recessive disorder more common than hemophilia (1/1000)

x Deficient in von Willebrand factor (platelet adhesion to collagen and protects FVIIIa from Protein C) x Pts present with mucocutaneous bleeding (commonly nasal or GI bleeding, heavy menses) x Diagnosis Æ APTT (low sensitivity), Factor VIII activity, vWf assay (concentration), Ristocetin cofactor assay

(vWf function) Acquired Bleeding Disorders

1. Vitamin K Deficiency Æ Low levels of Factors II, VII, IX, and X (also Protein C and S) Æ prolonged PT/PTT a. Caused by abx, malabsorption, surgery, coumadin; Tx = Vitamin K

2. Liver Disease Æ Deficiency of ALL factors except VIII, the only one not made in hepatocytes (Tx = plasma) Coagulation Cascade Diagram

Anti-Platelet Agents and Where they Act (for next lecture; see next page)

Pharmacology 1: Anti-Platelet Agents and Erythropoietin

All anti-platelet drugs are prophylactic and have no effect on an already formed thrombus Æ also all of these drugs include bleeding as a toxicity PLATELET AGGREGATION INHIBITORS

Salicylic Acid / Aspirin - Inhibits platelet aggregation and causes vasodilation x MOA Æ Covalently inhibits/acteylates cyclooxygenase, preventing formation of thromboxane A2

o Inhibition is long-lived because platelets are unable to synthesis new proteins x Low dose ASA purely inhibits COX; higher doses can also block prostacyclin I2, which naturally inhibits platelet

aggregation (too much negates the effects of COX inhibition)

Dipyridamole - Inhibits platelet aggregation and causes vasodilation x MOA Æ 1.) Increases intracellular cAMP via inhibition of cyclic nucleotide phosphodiesterase

2.) Inhibits nucleoside transport/uptake, preventing adenylate cyclase stimulation in platelets x Only effective in combination with warfarin or ASA Æ no prevention if used as monotherapy x Toxicity Æ HA and hypotension (especially with higher doses) in addition to bleeding

ADP Antagonists (Thienopyridines and Clopidogrel/Prasugrel) x MOA Æ bind irreversibly to ADP receptor, block ADP-mediated alpha and dense granule release, inhibit

fibrinogen binding to platelets, indirectly block activation of glycoprotein II/III receptor x Clopidogrel is an older, prodrug that requires activation via P450 enzymes; long-lasting effects

o The active metabolite causes anti-platelet activity (10% pts lack enzymes needed to activate) x Used to prevent stroke after stroke, TIA and MI x Toxicity Æ Neutropenia, thrombocytopenia, bleeding, diarrhea

*ASA and Clopidogrel effects last the lifespan of the platelet (~10 days); must be stopped 10 days before surgery PLATELET  GLYCOPROTEIN  IIB/IIIA  ANTAGONISTS  (very  expensive,  but  may  save  $  by  ↓  need  for  angioplasty  or CABG)

Anti-Platelet Antibodies (Abciximab) - Chimeric antibody x MOA Æ Blocks aggregation via binding/blocking GPIIb/IIIa receptor x PK Æ Half-life of 10 minutes, so given as a rapid, large bolus followed by a slow infusion for 18-24 hours

o Platelet aggregation inhibition lasts ~24 hours after treatment o Always given with heparin or ASA (possibly increasing risk of bleeding)

x Toxicity Æ Ig-mediated platelet clumping (pseudothombocytopenia); possible to develop anti-murine Ig

Anti-IIb/IIIa Peptide (Eptifibatide) x MOA Æ Mimics 3-AA sequence on fibrinogen and vWf, blocking binding to IIb/IIIa ( blocking aggregation) x PK Æ Eliminated via proteolysis to AAs, half-life 1-3 hours; given as rapid bolus, then slow infusion

Anti-IIb/IIIa Small Molecule (Tirofiban) x MOA Æ Binds reversibly to IIb/IIIa receptor; inhibiting fibrinogen binding x PK Æ 2 hour half-life; given as large bolus/slow infusion; rapidly reversible due to fast clearance x None of the IIb/IIIa antagonists increase risk of intracranial bleed

ERYTHROPOIETIN

x MOA Æ Blocks apoptosis of erythroid precursor cells via binding and signaling via JAK-STAT pathway x PK Æ Given IV or subcutaneously; half-life = 10 hours; has sustained, long-lasting effect x Used to treat anemia of chronic renal failure, cancer,  AIDS  or  perioperatively  to  ↓  transfusion x Toxicity Æ aggravation  of  HTN  (↑  viscosity),  potential  increase  in  thrombus,  theoretical  risk  of  neoplasm

Platelet Disorders Quantitative Platelet Disorders:

THROMBOCYTOPENIAÆ LOW platelets; risk of bleeding varies: x > 100,000/ul = no excessive bleeding x 50-100,000 = may bleed longer than normal w/ trauma x 20-50,000 = bleeding with minor trauma x < 20,000 = possible spontaneous hemorrhage

Petechiae = microvascular leakage of RBCs through the endothelium; seen when platelets fall below 20-50,000

x Occurs because platelets play an important role in the integrity of the endothelium Æ bleeding occurs through disassembly of adherins junctions between endothelial cells

CAUSES OF THROMBOCYTOPENIA

1. Decreased production Æ caused by bone marrow hypoplasia/failure, leukemia, toxins, chemo/radiation, congenital thrombocytopenia

2. Increased Destruction Æ autoimmune Ig to platelets (can be drug-induced) drugs that induce Ig that bind platelets, activation of coagulation cascade/increased platelet consumption x Idiopathic thrombocytopenia purpura (ITP)/Autoimmune thrombocytopenia

o Ig against platelet glycoproteins; Ig-coated platelets are destroyed in the spleen x Disseminated intravascular coagulation Æacquired syndrome characterized by systemic intravascular

coagulation where there is intravascular deposition of fibrin (like getting caught in a fibrin web) o Coagulation factors are consumed along with platelets o Can have multiple causes (sepsis, shock, snake bite, incompatible blood transfusion)

x Thrombotic thrombocytopenic purpura (TTP) Æ Platelet consumption by impaired processing of von Willebrand factor, leading to small thrombosis in different organs

o It is potentially lethal disorder Æ if thrombosis of cardiac tissue � Schistocytes seen due to shearing when RBCs pass microscopic clots

o Inherited or acquired deficiency of ADAMST 13 enzyme (tx'd by giving plasma w/ the enzyme) x Associated with hemolytic anemia, thrombocytopenia, neurologic sx's, renal abnormalities and fever

3. Platelet Sequestration Æ if > 1/3 of platelets are stored in the spleen

THROMBOCYTOSIS Æ platelet count HIGHER than normal, can go as high as 2- 3 million platelets/ml

CAUSES OF THROMBOCYTOSIS

1. Most patients have reactive or secondary thrombocytosis Æ response to cytokines during an acute or chronic inflammatory/infectious disorders, iron deficiency, or acute blood loss

x Also seen post Splenectomy (? exam question Æ if spleen is removed,  platelets  ↑)

2. Primary/Clonal Thrombocytosis Æ due to clonal marrow disorder x Often pt with splenomegaly; giant platelets on blood smear

3. Familial Thrombocytosis Æ germline mutations of TPO receptor Qualitative Platelet Disorders = platelet # is normal; platelet receptors or secretory mechanisms are abnormal

x Acquired Æ Drugs like ASA = most common cause; Ig to platelet receptors, renal failure, platelet trauma

x Congenital Æ Adhesion, aggregation or secretion abnormalities x Sx's = mucocutaneous bleeding, (no hemarthrosis or deep tissue

bleeding), petechiae, nose bleeds, excess menstrual bleeding, bruising

Bleeding time test Æ one of the first coagulation tests used to evaluate platelet function x Not used anymore; replaced with platelet aggregation

Platelet aggregation test Æ When an agonist is added to resting platelets Æ become activated Æ undergo a shape change Æ primary aggregation wave.

x As platelets secrete substances (e.g. ADP) more platelets aggregate Æ secondary wave of aggregation Examples of Qualitative Platelet Disorders: 1. Bernard Soulier Syndrome Æ abnormal adhesion due to absence of receptor for vWf

x Characterized by mild-moderate thrombocytopenia, large platelets 2. Glanzman Thrombastheni Æ abnormal aggregation – Absence of IIB/IIIA receptor for fibrinogen

x Presentation = purpura, epistaxis, bleeding varies and decreases with age x Platelet count and morphology are normal

Platelet Storage Pool Disorders Æ impaired secretion due to absence of granules that store ADP

x Only primary wave observed in aggregation (no ADP release Æ no stimulation of other platelets) Platelets Role in Thrombosis: If there is an atherosclerotic plaque or vessel damage Æ platelets can adhere

o Platelets play a central role in arterial thrombus, (venous thrombi mostly from clotting factors) x Once platelets become active activate Æ thrombosis and ischemia result

Thrombosis

Pro-thrombotic factors = Platelets, coagulation factors Anti-thrombotic factors = Circulation, endothelial cells, fibrinolytic system, endogenous anticoagulants

x Intact endothelium o Expresses thrombomodulin (binds thrombin and activates Protein C) o Does not have tissue factor o Produces substances that inhibit platelet formation (prostacyclin/PGI2) and promote fibrinolysis

(tissue plasminogen activator)

Endogenous Anticoagulants x Anti-thrombin Æ inactivates and disposes of serine proteases (including activated factors II, IX, X, and XI)

o Circulates as a zymogen; upregulated when bound by heparin x Protein C and Protein S Complex Æ downregulate cofactors VIII and V

o Thrombomodulin (on intact endothelium) binds factor IIa and activates Protein C o Activated Protein C (APC) binds Protein S Æ complex cleaves/inactivates cofactors V and VIII

� Leiden V slows the inactivation of cofactor V by this complex o 60% Protein S is free in circulation (available); 40% is bound to C4b binding protein and how

much is bound/unavailable to Protein C depends on how much C4b is available � C4b can be increased by inflammation, oral contraceptives/estrogens, pregnancy

Fibrinolytic System Æ Lyses fibrin clots; plasminogen is central (which circulates in plasma in inactive form) x Synthesized in liver; activated by TPA (tissue plasminogen activator) & UK (urokinase) to plasmin

o Plasmin cleaves fibrin into fragments, fibrin degradation products (FDP) x System inhibited by plasminogen activator inhibitor-1 (binds TPA), α2 antiplasmin (binds plasmin)

*Fibrinolytic system defects are rare; no defects have been identified as a major cause of venous thrombosis

Thrombus formation is multifactorial Æ acquired and genetic risk factors can influence system balance x Surgery/trauma (vascular damage), old age (↑ in coagulation factor levels), drugs, cancer, and mobility x Estrogens/Pregnancy Æ estrogens increases synthesis of clotting factors VIII, vWf and fibrinogen as well

as increase C4b binding protein S

Factor V Leidin Æ Autosomal dominant mutation of Factor V that prevents inactivation of Factor Va x Thrombosis risk depends on hetero (5x) versus homozygous (50x) for mutation & acquired risk factors

o Prevalence 0-15% (mostly European ancestry) x Activated Protein C (APC) Resistance Assay Æ patient’s  plasma  +    Factor  V-deficient plasma

o Add APC Æ should significantly prolong APTT if pt has NORMAL Factor V Factor II mutation Æ Autosomal dominant mutation that causes increased mRNA translational efficiency

x (↑ prothrombin levels); rarer than Factor V Leidin, 1-2% in Europeans Antithrombin III Deficiency Æ Antithrombin III inactivates factors II, IX, X, and XI

x Heterozygotes have 15-20x increased risk for thrombus; homozygous mutation is embryonic lethal x Can be acquired; seen with liver disease and nephrotic syndrome (loss of protein into urine)

Protein C and Protein S Deficiency Æ Increases risk of thrombus 5-10x

x Genetic cause is rare; acquired through Vitamin K deficiency and liver disease o Protein S deficiency can also be acquired with estrogens/pregnancy due to increased C4b binding

x Diagnosed with lab testing Æ Protein C and S levels Antiphospholipid antibodies Æ Causes thrombosis, but unknown why because it has so many actions

x They activate platelets, upregulate tissue factor in monocytes, activate complement, bind to anti-phospholipid binding proteins, which disrupts/ exposes phospholipids and triggers coagulation

x Clinically associated with thrombocytopenia and fetal loss x Diagnosis Æ Anticardiolipin antibody test (high titers), APTT mixing studies (shows inhibiting substance),

Dilute Russell viper venom test (assesses Factor X activity Æ very prolonged APTT) Hyperhomocysteinemia Æ Increase in tissue factor expression leading to thrombosis and endothelial damage

x Caused by defects in pathways to form cysteine (CBS) or methionine (B12/N5 methy THF) o Congenital (homocystinuria) & Acquired (vitamin deficiency, renal failure)

x Treated with Folate/B12 +/- B6 Disseminated Intravascular Coagulation (DIC) Æ Massive activation of tissue factor and extrinsic pathway

x Triggered by trauma, sepsis, snake venom, cancer (to treat, must treat underlying cause) x Lab values seen: ↓platelets,  ↓fibrinogen,  ↓clotting  factors  (b/c  are  being  consumed),  ↑D  Dimer

D-Dimer = produced when plasmin cuts up a fibrin clot that has been cross-linked by FXIII Æ indicates the presence of a clot in the blood

Pharmacology II: Coagulation Drugs

Endogenous anticoagulants: x Prostacyclin (PGI2) Æ inhibits platelet aggregation x Antithrombin Æ protease inhibitor, interferes with several intrinsic and common pathway factors

o Acts as a "suicide substrate" by binding to the active proteolytic site x Heparan sulfate ÆAn endothelial cell surface (similar to heparin), acts with antithrombin x Protein C Æ Works with protein S to degrade activated factors V (Va) and VIII (VIIIa)

Clinical indications for anticoagulant therapy:

Anticoagulants (heparin/coumarin) are used as PREVENTION Æ Does not dissolve thrombus already present

Thrombolytics are used to actively DISSOLVE thrombi Æ Do not prevent thrombus formation, so are often followed by at least short-term anticoagulation with heparin/warfarin.

ANTICOAGULANTS

1. Heparin x Measured in Units

o 1 Unit = amount of heparin required to prevent 1ml of plasma from clotting 1hr after adding CaC12.

x Heparin MOA Æ Increases thrombin inactivation (Helps antithrombin bind) o Inhibits intrinsic pathway more than the extrinsic (prolongs APTT predominantly) o Interferes w/ platelet aggregation; also activates an antithrombin homologue at high concentrations

x Pharmacokinetics are complicated Æ different half lives are seen depending on loading dose o Poor PO availability Æ given as continuous infusion, intermittent infusion, or subcutaneous

injection (must be followed closely by APTT levels) x Low-molecular weight heparins Æ inhibit Factor Xa but not thrombin (Factor IIa); do not prolong the APTT

o Bind to antithrombin, but not thrombin Æ Not as good an inhibitor of thrombin as HMW o Antithrombin III reacts with FXa and gives same effect (preventing and treating DVTs)

x Toxicity Æ Bleeding,  thrombocytopenia,  thrombosis  (white  clot  syndrome  =  ↑thrombosis,  ?  due  to  antibody  response), alopecia (telogen effluvium), osteoporosis

o Protamine = antidote to Heparin Æ a positively charged fish protein; immediate reversal � Saved for life-threatning cases due to risk of prophylactic reaction � Pts with IDDM more likely to have reaction b/c of pre-formed Ig to proteins 2° insulin

2. Warfarin/Coumarin x MOA Æ Vitamin K Antagonist (induces vitamin K deficiency)

o Inhibition of Vitamin K reductase, which blocks vitamin K recycling o ↓  synthesis  of  Vitamin K-dependent factors by 30-50%;  those  that  are  produced  have  ↓  activity  

x Pharmacokinetcs Æ nearly complete absorption and % bound to plasma protein (esp. albumin) o Effects depend on the half-lives of the different coagulation factors o PO dosing (good availability) Æ 99% bound to plasma protein (esp. albumin) o Half-life = 40 hours; renal clearance (major genetic component to clearance)

x Monitoring is required via PT/INR (International Normalized Ratio) x Highly susceptible to adverse drug interactions (metabolism induction/inhibition, displacement from

protein binding sites, can be overcome by a large amount of vitamin K) x Toxicity Æ Bleeding, skin necrosis (Protein C inhibition in already deficient pts), alopecia, teratogenicity

o Bleeding can be treated with FFP (acute, works immediately) or Vitamin K (delayed effect) � Vitamin K takes up to 24 hrs to work, and effects persist for days or weeks

3. Other Coagulants - Thrombin and Factor Xa Inhibitors x Thrombin inhibitors Æ Dabigatran, Bivalirudin, Argatroven x Factor Xa inhibitor Æ Ribaroxaban

THROMBOLYTICS

1. Streptokinase Æ Activates fibrinolysis by binding plasminogen and inducing plasmin activation x Produced by Strep bacteria Æ nearly all adults have pre-existing antibodies against it

o Very large loading dose must be given to overcome antibodies x Toxicity = bleeding, allergic reactions, anaphylaxis, fever

2. Recombinant Tissue Plasminogen Activator (rTPA) x Binds fibrin-bound plasminogen; activates plasmin to break down fibrin clots x Used for acute thrombus formation (MI or CVA) Æ given within 6 hours x Given in very high doses (continuous IV because of short 3 min half-life x Toxicity Æ bleeding, damage endothelial cells, thrombocytopenia

PROCOAGULANTS

1. Aminocaproic acid (Amicar) Æ Competitive inhibition of plasminogen/plasmin binding to fibrin x Toxicity Æ can cause pathological thrombus; rarely myopathy and muscle necrosis

2. Desmopressin Æ Analogue of vasopressin, induces release of endogenous stores of clotting factors (FVIII, vWf) x Used to treat von Willebrands Disease, but severe hemophiliacs don't respond d/t low endogenous FVIII x Toxicities = vasopressin-like activity Æ electrolyte imbalance and fluid overload

*The most common procoagulants are clotting factor concentrates and fresh frozen plasma

Granulocytes and their Disorders

WBCS = lymphocytes, monocytes and granulocytes (59% neutrophils, 3% eosinophils, <1% basophils)

Neutrophils Production: Myeloblast Æ promyelocyte Æ myelocyte Æ metamyelocyte Æ band cell Æ mature PMN

x Life cycle: Develop in marrow for 2 weeks Æ In blood for 6-8 hours Æ In tissue for 1-2 days o Enters tissue via chemotaxis following cytokines Æ rolling, adhesion

x Granules contain: Adhesion molecules, receptors, proteases, and antibacterial proteins Killing Mechanisms

1. Phagocytosis of opsonized bacteria Æ Myeloperoxidase system kills bacteria within the phagosome 2. Degranulation of antimicrobial substances 3. Neutrophil "nets" (apoptosis to release DNA and granules in area of bacterial infections)

Neutropenia = absolute neutrophil count <500 (severe), 500-1000 (moderate), 1000-1500 (mild)

x A QUANTITIATVE neutrophil problem

1. Acquired (very common) x Infection Æ viral bone marrow suppression, sepsis with exhaustion of neutrophil pool x Drug/toxin induced Æ impaired marrow production or induction of Ig-mediated response x Immune-mediated Æ maternal Ig (neonates), benign neutropenia of childhood, herald more significant

autoimmune disease (e.g. lupus; mostly in adolescents) x Nutritional deficiency in B12 or Folate x Bone marrow problems (aplasia, dysplasia, replacement) or large spleen problem

2. Congenital (rare) x Severe congenital neutropenia/SCN (Kostmann syndrome)

o Production/maturation defect, multiple mutations, ANC < 200, severe infections early in life � High risk of leukemia development

o Elastase, G-CSF, HAX-1 and WASp genes all associated with SCN x Cyclic neutropenia ÆElastase gene mutation (ELANE); 21 day cycles; often fevers/mouth ulcers x Myelokathexis Æ neutrophils mature, but can't get out of marrow (cytokine receptor defect) x Familial benign neutropenia Æ WBC and neutrophil count are genetically determined

3. Syndromes (rare, but many different causes) Æ Primary immunodeficiencies, metabolic disorders, bone marrow failure syndromes, and vesicle/organelle transport issues QUALITATIVE neutrophil problems (pt has normal neutrophil # with recurrent fungal/bacterial infections) 1. Problems getting to the infection:

x Absent/defective adhesion molecules Æ can be caused by steroids and epinephrine o Leukocyte adhesion deficiency (LAD) characterized by delayed umbilical cord separation

x Chemotaxis Æ Can be caused by cytoskeletal problems, hyper IgE syndrome, and alcoholism 2. Problems killing Æ Issues with phagocytosis (Fc receptor deficiencies), granule/vesicle formation (Chediak

Higashi), or oxidative killing (CGD) Neutrophilia Æ Leukocytosis (WBC >11,000), neutrophils >7,700, a left shift/bandemia (bands >700)

o Marked leukocytosis (>50,000) and ↑  in  early  myeloid  precursors  (bands,  metamyelocytes) x 2° causes are very common (acute infection, inflammation, steroids, smoking, stress, exercise, obesity)

o Dohle bodies and large vacuoles within neutrophils are associated with infection x 1° causes are less common (chronic myeloid leukemia, various problems with marrow production)

Eosinophilia (>5000 is severe, > 600 is mild) can be caused by allergic disease, infections, neoplasm, connective tissue disease, Addison's disease (hypeadrenalism)

x Hypereosinophilic syndrome Æ idiopathic; persistent eosinophilia, causing organ infiltration and damage Basophils Æ no known deficiency states; basophilia can be seen with chronic leukemias, infections (classically varicella), nephrotic syndrome

Principles and Practices of Blood Transfusion

Whole blood rarely used Æ donated blood is filtered to reduce WBC and separated/ transfused as components:

1. RBC Transfusion x Restores blood volume in situation of acute, rapid blood loss x Improves oxygen-carrying  capacity  (1  unit  ↑s  Hgb  by  1  and  Hct  by  3%) x Patients who require transfusion = hemorrhagic shock, anemic with clinical signs/symptoms

o 1942 recommendation Æ The 10/30 rule is still used, but is wrong (transfuse if Hgb <10, Hct < 30) o Most hospitalized, stable pts, transfusions should be considered when Hgb < 7-8 (Hct < 21-14%)

� Decision should incorporate pt's symptoms as well as Hb thresholds

2. Platelet Transfusion Æ treatment threshold = <10,000 for uncomplicated prophylaxis; <20,000 for prophylaxis in acute illness or tx of minor bleeding; <50,000 for major bleeding

x Transfer during at-risk periods; don't try to achieve an unattainable level

3. Fresh frozen plasma (FFP) Æ Contains FVIII and prothrombin complex (FX, IX,VI, V & thrombin) with concentrations similar to fresh plasma

x Biggest problem with FFP treatment is volume/ fluid overload x Used with bleeding 2° to liver failure, DIC, or warfarin x For prophylaxis, only given if PT and PTT > 1.5x normal and clotting factor deficiency is suspected

4. Cryoprecipitate Æ Contains fibrinogen, FVIII, XIII, vWf and fibronectin x Used for fibrinogen deficiency (most common), massive transfusion, DIC, uremia-associated bleeding

o Historically used for FVIII deficiency Æ NOT anymore; now give recombinant factors Red Cell Antigens: ABO Phenotype Æ based on different glycosyltransferases; controlled by 3 groups of genes

x H gene (FUT1) adds fucose to proteins/lipids on RBC surface Æ H antigen (O phenotype) is the precursor onto which A and B sugars are added via A- or B-transferase (A & B phenotypes)

o ABO Phenotypes in the US: 40% A, 11% B, 4% AB, 45% O o h is a rare, recessive nonfunctional H gene (hh = Bombay phenotype; anti-A, B, &H antibodies)

x Over 300 other blood groups Æ many other antibodies (unexpected antibodies) can cause reactions Transfusion Compatibility

Rh Blood Group Æ Highly immunogenic D antigen; high frequency of incompatibility (85% Rh+; 15% Rh-)

x Causes hemolytic disease of the newborn (HDN) Æ hemolysis of fetal RBC, resulting in hydrops fetalis o Rhogam given to prevent the formation of antibodies against the D antigen

Pre-Transfusion Testing Æ Verify patient antigen, ABO/Rh, antibody screen, compatibility test

x Type and Screen = ABO/Rh AND screen for unexpected antibodies inpatient's serum x Type and Cross = ABO/Rh, screen AND cross-match of pt serum with donor RBCS

Adverse Effects of Transfusion Æ Hemolytic, Non-hemolytic, GVHD, Circulation overload, Infection

x Stop transfusion immediately, report/document the detailed reaction & return unit to blood bank

1. Acute Hemolytic Transfusion Reaction Æ Destruction of transfused RBCs by pre-formed Ig against donor RBC Ag x Most often caused by clinical error; causes intravascular hemolysis 2° to complement activation

o Sx's Æ Renal failure, DIC, oozing at IV site, many systemic sx's x Lab finding Æ + DAT, + eluate (w/ allo-Ig on tranfused RBCs), hemoglobinemia,  hemoglobinuria,  ↑  LDH

2. Febrile Non-hemolytic Reaction (FNHTR) Æ more common (initially mimics acute hemolytic/sepsis reactions) x WBC antibodies in patient serum reacts with donor WBC or platelets (+ cytokines released)

3. Allergic Transfusion Reactions (ATRs) x Spectrum of hypersensitivity rxnsÆ most common rxn, especially with plasma-containing products

4. Transfusion transmitted infections Æ HIV, HDV, HBV, B19, prion, West Nile virus, bacteria

RBC Æ AB is the universal recipient; O is the universal donor (Type O, Rh- blood can be given to everyone) Plasma Æ AB is the universal donor (no anti-A, anti-B Ig in plasma); O is the universal recipients

Reference Hematopoiesis Chart: Blood Smear Indications

Blood Smear Finding Indication Hypersegmented PMNs, ovalocytes Megaloblastic hematopoiesis Howell-Jolly bodies Megaloblastic hematopoiesis, hyposplenism Red cell stippling Hemoglobinopathy, sideroblastic anemia, lead poisoning Spiculated red cells Liver disease, hypothyroidism, starvation Red cell fragmentation Mechanism destruction (vasculitis, abnormal valve, tumor,

consumption coagulopathy) Blister cells G6PD Deficiency induced hemolysis Target Cells Hemoglobinopathy, obstructive jaundice

Differentiating between diseases on blood smear appearance and lab tests

PLATELET DISORDERS DIsease Specific Findings Defect? ITP (idiopathic thrombocytopenic purpura)

Ig against platelet glycoproteins (destoyed in spleen)

TTP (thrombotic thrombocytopenic purpura)

Normocytic, schistocytes, sx's = organ failure/stroke Lab = ↓↓ platelets

Impaired vWf processing; thrombosis in various organs

DIC (disseminated intravascular coagulation)

Coag factors & platelets consumed

Clonal thrombocytosis Giant platelets/MKs, splenomegaly Clonal marrow disorder

Familial thrombocytosis Germline mutation of TPO receptor Bernard Soulier Syndrome Big platelets, mild-moder.

thrombocytopenia No vWf Æ abnormal adhesion

Glanzman Thrombastheni Nml platelet count/morph Sx's = purpura, epistaxis

No IIb/IIIa receptor Æ abnormal aggregation

Platelet storage pool disorders

Only primary wave of aggregation observed

Platelet absence of ADP-containing granules

Disease Blood Smear Findings Lab Findings Patient History Aplastic Anemia Spherocytes, ↓WBC/plateles,  ↑retic

pancytopenia

Extravascular Hemolysis Spherocytes No urine hemosiderin Jaundice, dark urine

Intravascular Hemolysis Schistocytes ↑  LDH,  ↑Indirect  bili,  ↓haptoglobin

Autoimmune hemolytic anemia (AIHA)

Normocytic/chromic, hyperproliferative

+ direct Coomb's test

Iron Deficient Anemia (IDA) Microcytosis, Hypochromia, Hypoproliferative

↓RBC/Hb,  ↓Serum  Fe, ↑TICB,  ↑RDW

Pica, Diet, Heavy Menstruation

B12 Deficiency Macrocytic, Normochromic, Hypoproliferative, Megaloblastic

↓Hb/Hct,  ↑MMA,  ↑homocysteine

Breast-fed infant of vegan mother

Hemolysis 2° G6PDH Deficiency

"Blister"/parachute cells

Hereditary Hemochromatosis

↑serum  Fe,  ↑Ferritin,    ↑  Transferrin saturation

Bronzed skin, diabetes, presents in 40s-50s

Megaloblastic Anemia Hypersegmented PMNs, oval macrocytes

Sickle Cell Anemia Normochromic/cytic, Hyperproliferative, sickled

↑WBC,  ↓RBC/Hg/MCV,  ↑RDW  

African heritage

Thalassemia Microcytic, target cells, inclusion bodies

↑RBC,  Normal RDW, ↓Hg/MCV,  ↑Retic

Mediterranean heritage

PRO

COAG

ULA

NTS

T

HRO

MBO

LYTI

CS

AN

TICO

AGU

LAN

TS

COAGULATION DISORDERS Pharmacology Overview

COAGULATION DISORDER DRUGS Drug MOA Dosing/Monitoring Toxicities Notes Heparin ↑  Thrombin  

inactivation No PO dosing , PTT monitoring

Bleeding,  ↓platelets  thrombosis, osteo- porosis, alopecia

Protamine = antidote in life-threatening rxns

Warfarin/Coumarin Vitamin K antagonist PO dosing, 40-hr 1/2 life; PT/INR

Bleeding, skin necrosis, alopecia, teratogenicity

Bleeding tx'd w/ FFP (acute) or Vit K (delayed)

Dabigatran Thrombin inhibitor Ribaroxaban Factor XA inhibitor Streptokinase Activates plasmin &

fibrinolysis Very large loading dose (overcome Ig)

Bleeding, allergic rxn Anaphylaxis, fever

Produced by strep; adults have Ig to it

Tissue Plasminogen Activator

Binds fibrin-bound plasminogen; activates plasmin

3 min 1/2 life; given in high doses

Bleeding,  ↓platelets  endothelial damage

Given within 6 hrs of MI or CVA

Amicar (Aminocaproic Acid)

Inhibits plasmin binding to fibrin

Thrombus, muscle necrosis/myopathy

Desmopressin Induces release of coag. factor stores

Fluid overload, electrolyte imbal.

Used to tx Von Willebrands Disease

Important Points from the Lab Notes:

x Pallor of the palmar creases isn't observed until Hgb < 7g/dL x If Hct < 25%; correct retic count should be divided by 2 as an additional correction x Elevated EPO levels in an anemic pt is an appropriate response and suggests marrow defect

o A low EPO level can be the problem, but doesn't rule out a marrow problem o Patients with renal disease will have a blunted response to EPO

x Aplastic anemia can only be diagnosed with a bone marrow biopsy x A reticulocyte with less RNA indicates that it is older x Only B12 deficiency causes neurologic symptoms Æ folate deficiency DOES NOT

o Only if pernicious anemia is the cause of B12 deficiency will it reverse with intrinsic factor (Schilling's test)

x Autoimmune hemolytic anemia o Warm agglutinin disease Æ IgG-mediated Æ smear shows spherocytes o Colg agglutinin disease Æ IgM-mediated Æ smear shows RBC clumping, not spherocytes

x Petechiae indicates a platelet or vascular disorder; joint/muscle bleeding indicates

DIsease Specific Findings Defect? Blood loss/Sepsis ↓RBC/platelet,  ↑nRBCs Hemophilia Prolonged PTT Factor VIII or IX Factor V Leiden Multiple thrombi, European Factor V mutation, prevent APC

inactivation Factor II Mutation ↑prothrombin  levels Increased synthesis of factor II Antithrombin III Deficiency Associated w/ liver dz and nephrotic

syndrome Antithrombin III isn't present to inactivate factors II, IX, X, and XI

Protein C/S Deficiency Seen w/ Vit K deficiency & liver dz Protein S & C unable to inactivate FVa Antiphospholipid antibodies Fetal loss, Exposes phospholipids Æ coagulation;

also  activate  platelets  and  ↑  tissue  factor Hyperhomocysteinemia Congenital or via vitamin deficiency,

renal failure Increased tissue factor expression d/t defect in CBS or methionine pathways

DIC ↓platelets,  fibrinogen,  clotting  factors;  ↑D-Dimer

Massive activation of TF/extrinsic pathway & consumption of platelets/coag factors