Hematology (Week 7) Flashcards
Blood and blood forming tissues
Erythrocytes (RBCs)
Leukocytes (WBCs)
Platelets
Bone marrow
Spleen
Lymph nodes and antibodies
Coagulation
Definition of blood
Blood is differentiated cells (generally nondividing) suspended in plasma
Plasma is composed of coagulation proteins in a solution of serum
Serum contains other proteins and solutes (antibodies, albumin)
Blood = cells + plasma + serum
Definition of bone marrow
Source of multipotential stem cells and their differentiated progeny
Source of cellular material of the blood
Source of immunologically active cells of the body (reticuloendothelial system)
Source of adherent bed of cells essential to hematopoietic proliferation, immunomodulation and cell survival
Where do you have bone marrow?
Everywhere from skull to axial bones to pelvis..
General flow of differentiation of blood cells
Hematopoietic stem cell (can self-renew and is pluripotent) –> committed stem cell (younger ones called “blasts”) –> differentiated cells
Pluripotent stem cell
Differentiates into myeloid and prelymphoid component, then inductive stimuli from bone marrow stroma cause cells to eventually become committed (neutrophil, basophil, erythrocyte, platelet, T cell, B cell, NK cell, etc)
(NOT embryonic stem cell, but close!)
How do pluripotent stem cells change as they differentiate?
As they differentiate, they get smaller
As they differentiate, they move from adherent bone matrix of marrow into marrow more
Where is blood formed in the growing embryo?
19 days: blood is formed in yolk sac
6 weeks: blood is formed in the spleen and liver (main site at weeks 9-24)
10-12 weeks: blood is formed in bone marrow (main site at >24 weeks)
2 weeks post-partum: blood formed only in bone marrow
What kind of cells does cord blood have?
Hematopoietic stem cells
Percent cells in the bone marrow
100 - age is percent cells in the marrow
(25 year old should have 75% cells in marrow and not too much fat)
Normal RBC maturation
Pronormoblast (proerythroblast)
Basophilic normoblast
Polychromatophilic normoblast (cytoplasm contains residual RNA that still stains slightly blue)
Orthochromic normoblast
Reticulocyte (no nucleus)
Mature erythrocyte
Why is it important that RBCs don’t have a nucleus?
Because whatever proteins/enzymes they have now is all they’ll ever have because they can’t do any more protein synthesis
What happens when RBCs get old and become senescent?
Senescent RBCs become rigid, cannot get through small places and are removed by the spleen
What should happen to reticulocyte cound if you’re anemic?
It should increase to compensate for the fact that you don’t have enough RBCs!
Note: only if you have hemolytic anemia or acute blood loss (NOT chronic disease, sideroblastic, iron deficiency, B12/folic acid deficiency, aplastic anemia)
Normal RBC count, hemoglobin, hematocrit, reticulocytes
RBC count (x 106 mm3): 4.4-5.9 male; 3.8-5.2 female
Hemoglobin (GM%): 13-18 male; 12-16 female
Hematocrit (%): 40-52 male; 35-47 female
Reticulocytes (%): 0.5-1.5
Reticulocyte count (x 106 mm3): 0.025-0.105
Reticulocyte Index
Correction to figure out how many reticulocytes are actually in the blood
1) Correct for degree of anemia: multiply reticulocyte % by Hgbpatient/Hgbcontrol
2) IF nucleated RBCs present, correct for 2-day lifespan of reticulocyte: divide number by 2
Note: reticulocytes still have residual ribosomal RNA (even though nucleus is gone!)
If you have anemia, what would you want to reticulocyte percentage to be?
Remember it’s usually only 1% and you need to compensate for destruction/decrease in RBCs
Depends on degree of anemia…
2% is not enough to compensate…maybe 3% and higher would be good compensation??
Mean corpuscular volume (MCV)
Average volume of RBC
HCT (%) x 10 / RBC count
Normal: 81-100 mm3
If microcytosis, low
If macrocytosis, high
Mean corpuscular hemoglobin concentration (MCHC)
Average concentration of hemoglobin per volume of RBCs
Hg x 100 / HCT
Normal 31-36 g/dL
If hypochromia, will be low
If spherocytosis, will be high (cell volume decreased by Hg content the same)
Mean corpuscular hemoglobin (MCH)
Average weight of hemoglobin per RBC
Hg x 10 / RBCs
Normal 27-34 pg
Reflects both size and Hg concentration
Usually varies in similar fashion to MCV
RBC terminology
Microcytic = RBC small
Macrocytic = RBC large
Hypochromic = less Hg/cell (larger central pallor)
Anisocytosis = variation in size of RBC
Poikylocytosis = variation in shape of RBC
Polycythemia = too many RBCs
Anemia = too few RBCs
Erythropoietin
Hormone that controls RBC production
Made in kidney (some in liver)
Anemia
Decreased RBC levels (or decreased hemoglobin levels?)
SIgns: weakness, fatigue, shortness of breath, pallor
Due to one of 4 things: decreased production, ineffective production, increased destruction
Diagnosis: reticulocyte count, evaluate blood smear, RBC indices (MCV, MCHC, MCH)
3 general causes of anemia
Hypoproliferative: impaired erythropoiesis
Ineffective: intact erythropoiesis but intramedullary hemolysis (die in bone marrow?)
Compensatory (hemolytic): intact erythroid production, egress from marrow but early erythrocyte destruction (exit bone marrow but die in peripheral blood?)
Hypoproliferative anemia
Most common type of anemia
Reticulocytopenia
Low or normal MCV
Impaired production of intact hemoglobin or impaired regulation of hematopoiesis
Specific causes of hypoproliferative anemia
Disorders of:
Erythrocyte production: congenital, acquired (deficiency of erythropoietin, chronic renal insufficiency, pure erythrocyte aplasia)
Production of mature hemoglobin: disorder of iron (deficiency, sequestration (anemia of chronic disease/inflammation; sideroblastic anemia)), disorder of heme (thalassemia, lead intoxication, hemoglobin E, sideroblastic anemia)
Hematopoietic stem cell
Bone marrow microenvironment
How is iron lost from the body?
No active secretion of iron
Iron lost only when cells lost (urine, skin, gut, menstruation)
Regulation mainly by absorption
Iron turnover
20-30 mg per day is turned over between RBC destruction and production
However, remember that only small amounts (1mg per day) are lost in gut, sweat urine that must be renewed by diet
What happens if you have iron deficiency?
O2 transport messed up
Electron transport messed up
Anemia
Muscle weakness
What happens if you have iron overload?
Oxidant damage affects:
Heart
Liver
Endocrine
Joints
Infection
Note: more of a problem in men because women at least have menstruation to get rid of some iron every month
Iron deficiency anemia
Cannot produce mature hemoglobin
Hypoproliferative anemia
Most common cause of anemia worldwide
Get microcytic, hypochromic RBCs, targets, anisocytosis, poikylocytosis
Negative iron stain (with Prussian blue) of marrow
Can be due to chronic blood loss (infancy, lactation, pregnancy, GI ulcer)
Mechanisms of iron deficiency
GI blood loss
Menstruation
Blood loss in pregnancy and lactation
Urinary blood loss
Less common: dietary deficiency (in baby on formula), intestinal malabsorption, atransferrinemia
Clinical manifestations of iron deficiency
Anemia (hypoproliferative, reticulocytopenia, microcytic)
Epithelial changes (koilonychia, depapillated tongue, esophageal webs and strictures)
Skeletal changes (growth retardation, skull changes)
Anemia of chronic disease (inflammation)
Cannot produce mature hemoglobin
Iron is sequestered in macrophages and have erythropoietin dysfunction
Lab: low serum iron, low TIBC, high ferritin, normal serum transferrin receptor
Iron necessary for microorganism growth and division but host binds iron with ovoalbumin, transferrin, lactoferrin and ferritin –> inflammation from disease leads to cytokine release (IL-1, TNF, IL-6) –> macrophages increase lactoferrin receptors to internalize more lactoferrin-bound iron, increase ferritin synthesis and decrease iron output from the macrophage –> overall iron sequestered in macrophages and withheld from both microorganisms and RBCs
Ineffective disorders of hematopoiesis
Nuclear-cytoplasmic dissociation (nucleus doesn’t mature normally and keeps cell very big so cannot get out into blood and is destroyed in bone marrow!)
Intramedullary maturation arrest and hemolysis
Reticulocytopenia (bc reticulocytes never get out of bone marrow!) with macrocytosis
May not be restricted to hematopoiesis
Folate deficiency
Causes megaloblastic anemia
Get mucosal changes
Measure low folate in serum and RBCs
Get folate deficiency if: poor diet, cancer, hemolysis, alcoholism, during pregnancy and lactation (increased demand), drugs, malabsorption
Folic acid does not need cofactor to be absorbed, is depleted in 5 months (“nutritional” megaloblastic anemia)
Folic acid does 1 carbon transfers to make thymidilate to make pyramidines and purines (for DNA synthesis)
Vitamin B12 (cyanocobalamin) deficiency
Causes megaloblastic anemia
Get neurologic symptoms (paresthesias in hands and feet, decreased vibration/position sense, ataxia, psychoses), mucosal changes
Measure low B12 blood levels
Get B12 deficiency if: deficiency in intrinsic factor activity (pernicious anemia), gastric resection/neoplasm, ileal resection/enteritis, fish tapeworm competition, diverticulosis, strict vegans
Get vitamin B12 from meat, dairy
Need intrinsic factor (secreted by parietal cells in stomach) to absorb B12 in terminal ileum
Takes years to deplete B12, so don’t just get nutritional deficiency!
Marrow and blood smear of megaloblastic anemia
Marrow shows young nuclei that are large and have no clumping of chromatin
Blood smear shows big RBCs with low hemoglobin (macrocytic and hypochromic?)
Blood smear also shows hypersegmentation of neutrophils
Hemolysis
Premature destruction of erythrocytes:
Intravascular vs. extravascular
Intracorpuscular vs. extracorpuscular
Lab evaluation of hemolysis
Reticulocytosis (trying to make up for RBC loss/lysis) with any MCV
Polychromatophilia of RBCs
Erythroid hyperplasia of bone marrow –> increased indirect bilirubin, increased urinary and fecal urobilinogen, increased endogenous carbon monoxide production
Depleted unbound haptoglobin (because lots of free hemoblobin to bind haptoglobin)
Findings in a patient with hemolysis
Increased indirect bilirubin
Scleral icterus
Serum is yellow from indirect bilirubin
Peripheral blood smear used to determine cause of hemolysis
Erythrocyte features: fragmentation, spherocytosis, distinct erythrocyte morphology, erythrocyte inclusion
Autoimmune hemolytic anemia
IgG eats up membrane of RBC
On peripheral blood smear, see spherocytes
Different kinds of hemolytic anemia
Trauma to RBC: heart valve shears RBCs –> fragmented RBCs on smear
Chronic liver or kidney disease: RBC membrane becomes pickled due to abnormal distribution of membrane lipids
Infection: Plasmodium falciparum infects RBCs and causes RBC lysis
Different sites of erythrocyte injury
Splenic consumption
Vasculature
Plasma
Erythrocyte membrane
Cytoplasm
Hemoglobin
Erythrocyte enzymatic machinery
Infection
Spleen
Normal spleen 200-300 cc/minute (4-5% cardiac output)
Half cells capable of phagocytosis
White and red pulp, marginal zone and germinal centers
Differential diagnosis of splenomegaly
Portal HTN
Infiltrative disorders of spleen (lymphoma)
Cardiomyopathy
Autoimmune disease
Subcapsular hemorrhage
Hematologic disorders (hemolysis, hemoglobinopathy, neoplastic)
Vascular disorders causing hemolytic anemia
Macroangiopathic hemolytic anemias (heart valve shearing RBCs)
Microangiopathic hemolytic anemias (DIC, malignant hypertension, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome)
Plasma disorders causing hemolytic anemia
Membrane lysins
Toxins and envenomations (clostridial sepsis, spider bites, snake bites, chemical lysins)
Membraneopathies causing hemolytic anemia
Congenital: hereditary spherocytosis, elliptocytosis, stomatocytosis, acanthocytosis
Acquired: immunohemolytic anemias, immune hemolysis, Rh incompatibility, autoimmune hemolytic anemia, drug-induced hemolytic anemia
Hemoglobinopathies
Change in AA can give new characteristics to hemoglobin and lead to:
Sickle cell hemoglobin (HbS): increased hemoglobin precipitation
Unstable hemoglobin
Methemoglobins: inability to keep iron in reduced form within hemoglobin molecule
High/low affinity molecules: altered O2 affinity of hemoglobin molecule
Hemoglobin genes
Alpha on chromosome 16 (4 genes total)
Betas on chromosome 11 (2 genes total)
Also, gamma and delta on chromosome 11
Note: easier to develop beta thalassemia because only 2 beta genes!
Normal hemoglobins
HgA: alpha2beta2 = major adult Hg (>95%)
HgA2: alpha2delta2 = minor adult Hg (<3%)
HgF: alpha2gamma2 = major Hg in fetus (<2% in adults)
Sickle cell disease (HgSS)
Qualitative problem
Point mutation at 6th AA position of beta globulin gene from hydrophilic glutamic acid to hydrophobic valine –> when hemoglobin deoxygenated, beta globulins interact with each other so hemoglobins form polymers within RBC –> rigid, sickled RBC
10% of American Blacks have S gene
Age of onset is variable (6 months - 2 years)
Lab: low grade anemia, erythroid hyperplasia, extravascular hemolysis (in the spleen? indirect hyperbilirubinemia)
Symptoms: pain, bone infarcts, lungs, CNS, heart, renal, autosplenectomy, infections
Treatment: analgesia, fluid if dehydrated, alkalinization if acidotic, antibiotics if infected, transfusions, hydroxyurea (only FDA approved), bone marrow transplant (?)
85% survive to age 20; 60% survive to age 50
Cause of death in kids is infection (spleen infarcted –> encapsulated bacterial infection); cause of death in adolescents/adults is acute chest syndrome or infection
Carrier state (HgAS usually asymptomatic and resistant to Malaria)
Thalassemias
Quantitative problem
Decrease in synthesis of a globin chain (globin gene missing!) resulting in unbalanced synthesis of globin chains and decreased hemoglobin production
Microcytic, hypochromic RBCs
Beta Thalassemia Major: homozygous; severe anemia, hepatosplenomegaly, hypercellular marrow, bone changes, iron overload (due to transfusions AND hemolysis of bad RBCs), infections, HgA 0; Hg 2-6
Beta Thalassemia Minor: heterozygous; mild anemia or asymptomatic, may worsen with infections or pregnancy; Hg >9
Hydrops fetalis: missing all 4 alpha genes; fetus has “Barts Hg” (gamma 4 tetramers) and dies
Hemoglobin H disease: missing 3 alpha genes so get HgH which is tetramer of beta chains only; intra-erythrocytic inclusions because they precipitate; hemolytic anemia, microcytic, hypochromic target cells
Alpha Thalassemia Minor: missing 2 alpha genes; mild microcytic, hypochromic anemia or asymptomatic
Silent carrier: missing 1 alpha gene; asypmtomatic
Enzymopathies that can cause hemolytic anemia
G6PD deficiency
Pyruvate kinase deficiency
Hemolytic anemias caused by other derangement of Embden-Meyerhoff pathway (glycolysis)
Abnormalities of nucleotide metabolism
Erythrocyte infections that can cause hemolytic anemia
Malaria
Babesiolsis
Other protozoal infections
Bartonellosis
If absolute neutrophil count (ANC) is <500, what are patients at risk for?
Bacterial infection
Hyphal fungal infection
3 types of WBC disorders
1) Too many WBCs (leukocytosis): reactive (infection) vs. neoplastic (leukemias, lymphomas)
2) WBC dysfunction: congenital, toxic, neoplastic
3) Too few WBCs (leukopenia): decreased production, increased destruction, or splenic sequestration
Aplastic anemia
Decreased marrow production of (usually) all blood cells
Get pancytopenia (decreased erythroid, myeloid and megakaryocytic cell lines); only cells that remain are ones that live a long time (plasma cells and lymphocytes)
Bone marrow is hypocellular
Symptoms: weakness, fevers, infections (bc low WBC), bleeding (bc low platelets)
Signs: peticheae, hemorrhage, pallor, fever
Causes: idiopathic, drugs/toxins (benzene, chloramphenicol), infection, radiation, immune mediated, paroxysmal nocturnal hemoglobinuria
Treatment: transfusion, antibiotics, immune suppression (ATG = horse serum), hematopoietic stem cell transplantation
Lymphoproliferative disorders
Abnormal production or accumulation of lymphoid cells with clinical behavior reminiscent of ontogeny of the cells
Note: you can only get cancer in dividing cells so there is no such thing as neutrophilic leukemia because neutrophils can’t divide
Suffixes for decreased and increased numbers of cells
Decreased: cytopenias
Increased: cythemias, or cytoses
Two different reasons why you may have leukocytosis
1) Primary marrow abnormality (neoplastic or preneoplastic)
2) Secondary (appropriate marrow response to external signals, like infection!)
Leukemia vs. lymphoma
Leukemia: abnormal cells in blood and marrow
Lymphoma: abnormal cells in lymph nodes, thymus, spleen, or other lymphoid tissues (Peyer’s patches)
Note: this is a relative difference, not aboslute–they overlap obviously
Leukemias where you have too many lymphocytes (lymphocytosis)
Chronic lymphocytic leukemia (CLL)
Acute lymphocytic leukemia (ALL)
How can you tell if lymphocytosis is neoplastic or infectious?
Neoplastic will be clonal: all lymphocytes have either kappa or lambda light chain but not both
Infectious will be polyclonal because lots of different cells fighting infection
Chronic lymphocytic leukemia (CLL)
Lymphoproliferative disorder
Lymphocytosis, lymphadenopathy, hepatosplenomegaly, infections, immunologic abnormalities (hypogammaglobulinemia, immune cytopenias, paraproteinemias), secondary malignancies
Usually monoclonal mature B-cells, but rarely T cells, NK, Prolymphocytic, or Hairy Cell)
30% of all leukemias in US
Cytogenetic abnormalities: deletion 13q14.3, trisomy 12
Lab: coexpression of CD5 (usually T cell marker) with CD19 and 20 (B cell markers), anemia, thrombocytopenia
Treatment: only treat if symptomatic (alkylators, fludarabine, chemo, steroids to induce apoptosis of lymphocytes, Mab therapy, blood or marrow transplant in younger pts)
Staging of CLL
Stage 0: lymphocytosis of blood and marrow
Stage I: lymphocytosis + lymphadenopathy
Stage II: lymphocytosis + splenomegaly and/or hepatomegaly
Stage III: lymphocytosis + anemia (Hg<11)
Stage IV: lymphocytosis + thrombocytopenia (plt<100,000)
Why do people with CLL get frequent infections if too many immune cells?
Body tries to control B cell clone but actually ends up controling normal clones and neoplastic cells still grow
(reason why patients develop hypogammaglobulinemia?)
CLL patients get encapsulated bacteria infections
Hairy cell leukemia
Lymphoproliferative disorder
Blood and marrow lymphocytes with fine filamentous “hairy” projections
Usually B cells
Stain for tartrate resistant acid phosphatase (trap), monoclonal surface immunoglobulin and Fc receptors
Pancytopenia, splenomegaly, infections, immune abnormalities
Responsive to deoxycoformycin, alpha-interferon, splenectomy
1 week of nucleoside analog can produce 10 year remission!
Chronic T cell leukemias/lymphomas
Mycosis fungoides/Sezary’s syndrome: CD4+ lymphoma which produces cutaneous infiltrates, lymphadenopathy and can transform to erythrodermatous phase with circulating Sezary cells
Large granular lymphocytosis syndrome: T cell/NK cell disorder (CD8+), severe neutropenia, pancytopenia, rheumatoid arthritis, splenomegaly
Adult T cell leukemia/lymphoma: associated with HTLV-1, have lymphocytosis, lymphadenopathy, hypercalcemia, lytic bone lesions
Three stages in thrombus formation
1) Vasoconstriction (if have vascular disease and hardened vessel, can’t constrict and pt will bleed!)
2) Primary hemostasis: platelets
3) Secondary hemostasis: fibrin
What initiates primary hemostasis (platelet plug formation)?
Endothelium is damaged and exposes subendothelium below
von Wildebrand Factor (vWF) binds subendothelium
Platelets then bind vWF via GpIb receptor
Platelet adhesion vs. platelet activation
Platelet adhesion: unactivated platelets bind ??
Platelet activation: activated platelets expose adhesion molecules and adhere to subendothelium?
Steps in primary hemostasis
1) Adhesion
2) Activation and secretion
3) Aggregation
4) Procoagulant activity (assembly of factors in secondary hemostasis)
What prevents us from clotting all the time?
1) Endotheluim: covers subendothelium because as soon as subendothelium exposed, we clot
2) Fast flow of blood: things zipping by so fast that they can’t find each other (need high enough concentration to make clotting happen)
Basics of intrinsic and extrinsic pathways
Intrinsic pathway: 9 needs 8 as cofactor to activate 10 –> 10 needs 5 as cofactor to activate 2 (thrombin) –> thrombin turns fibrinogen to fibrin
Extrinsic pathway: 7 activates 10 –> 10 needs 5 as cofactor to activate 2 (thrombin) –> thrombin turns fibrinogen to fibrin
Which 4 enzymes are Vitamin K (Ca2+) dependent?
7, 9, 10, 2 (thrombin)
These trigger the clotting cascade
Serine proteases, synthesized in liver
These zymogens (proenzymes) need to be carboxylated by carboxylase, but carboxylase needs Vitamin K as cofactor –> once carboxylated, can bind Ca2+ which they need in order to become active
Coumadin (warfarin)
Coumadin inhibits carboxylase reaction on 7, 9, 10, 2 so that these clotting enzymes cannot become active
How low can enzyme/co-factor level get before coagulation is impaired?
Since enzymes/co-factors are not consumed in reaction, levels can get very low (<30%) before coagulation is impaired (recessive or X-linked mutations)
Mild bleeding: 30-5% activity of factors
Moderate bleeding: 5-1% activity of factors
Severe bleeding: <1% activity
Note: fibrinogen and vWF ARE consumed in reaction, so lower levels of those show anti-coagulation phenotype easily (autosomal dominant mutations)
Clinical findings of platelet defects
Petechiae and purpura (usually symmetric; small bleeds)
History of easy or spontaneous bruising
Mild to moderate mucosal membrane bleeding (gingival, menorrhagia, epistaxis)
Platelet disorders
Thrombocythemia (primary or secondary)
Thrombocytopenia (decreased production or increased destruction)
Loss of platelet function (congenital or aquired)
Primary thrombocythemia
Myeloproliferative disease (CML, PV, ET)
Platelets can have normal or abnormal function
Secondary thrombocythemia
Increased release of platelets from bone marrow
Due to steroids or stress, or cute phase reactant, iron deficiency, acute blood loss, post splenectomy, epinephrine, chronic infections
Platelets have normal function
Thrombocytopenia due to decreased production
Marrow replacement (space taken up by fibrosis)
Aplastic anemia
Viral infection
Drugs (chemical wiped out progenitors)
Congenital disorders (rare)
Thrombocytopenia due to increased destruction
Prosthetic valves
Hypersplenism
Immune mediated disseminated intravascular coagulation (DIC)
Medications (heparin, antibiotics, H2 blockers)
Causes of immune thrombocytopenia
Autoimmune (ITP) acute or chronic
Alloantibodies (neonatal or transfusion)
Drug induced (ie heparin) by creating new epitope
Disease association (make antibodies you shouldn’t): other autoimmune, lymphoproliferative, myeloproliferative, solid tumors, infection
Acute vs. chronic ITP (immune thrombocytopenia)
Acute: children 2-9 years; abrupt onset, after infection, <20,000 platelets (very dangerous!); lasts 2-6 weeks but then 80% spontaneous remission (don’t need tx other than support); variable response to immunosuppression or splenectomy
Chronic: adults 20-40 years; more female; gradual onset; no clear antecedent; 20-100,000 platelets still; lasts years and spontaneous remission is rare; usually respond to immunosuppression or splenectomy
What causes loss of platelet function?
Uremia (not clear why)
Liver disease
Prosthetic valves
Aspirin or NSAIDs (or other drugs)
Essential thrombocythemia
Congenital (vWD, intrinsic platelet defects)
Lab tests to assess platelet-type bleeds
Platelet count
Bone marrow (look for megakaryocytes to determine production vs. destruction)
Bleeding time (only if platelets >100,000)
Platelet aggregation assays
Risk of bleeding with thrombocytopenia
Normal platelet count: 150-350,000
Risk of excess bleeding with surgery: <50,000
Risk of spontaneous bleeding: <20,000
Imminent risk of GI or cerebral hemorrhage: <5,000
When would you use bleeding time as a screening test?
Very archaic, only used if you think there is a vascular problem (can’t test that with other lab tests!)
Normal bleeding time 10 minutes
Only do this if patient has >100,000 platelets and no liver disease, uremia, collagen vascular disease, prosthetic valves, etc because of course bleeding time will be increased!
Coagulation disorders (hemorrhagic)
Decreased factor production: acquired (liver disease, Vitamin K deficiency), congenital (hemophilia)
Increased factor consumption: acquired (DIC), congenital (rare: alpha2-anti-plasmin)
Congenital bleeding disorders
Von Willebrand’s Disease (autosomal dominant)
Hemophilia A (factor 8; X-linked)
Hemophilia B (factor 9; X-linked)
Other factors (autosomal recessive)
Fibrinogen (dominant or recessive)
Von Willebrand’s Factor (vWF)
Made in endothelial cells
Glues platelets down to exposed collagen to start primary hemostasis
Consumed in reaction so mutation is autosomal dominant (unlike enzymes!)
Also stabilizes factor 8, so can affect secondary hemostasis –> larger bleeds
Clinical findings in coagulation factor deficiencies
Common: bleeding in major muscles and joints, large bruises
Rare: mucosal hemorrhage, intracranial bleeds, bleeding from minor cuts and abrasions
Regulators of coagulation
Plasmin: degrades fibrin (degrades clot)
Protein C: serine protease (like 7, 9, 10, 2) with co-factor Protein S that degrades other co-factors 8 and 5; has shortest half-life
Anti-thrombin III: serine protease inhibitor; in presence of heparin, inhibits 2, 9, 10 (not 7 because doesn’t fit)
Heparin
Potentiates anti-thrombin III to inhibit factors 2, 9, 10
Starts working immediately!
If someone is clotting too much, which drug do you give first?
Give heparin first because starts anti-coagulating immediately (works to inhibit factors 2, 9, 10 by potentiating anti-thrombin III)
Give coumadin a few days later because coumadin only works on NEWLY synthesized factors (prevents carboxylation/inhibits protein C first to get slight clotting which you don’t wait–then prevents carboxylation/inhibits factor 7, 9, 10, 2 to anti-coagulate); coumadin will KEEP factors 7, 9, 10, 2 from working long-term
Coagulation disorders (thrombotic)
Note: clotting disorders are all autosomal dominant with incomplete penetrance (most symptomatic patients have >1 mutation and other contributing factors); homozygous mutation is incompatible with life!
Antiphospholipid syndrome (often seen in lupus)
Factor V Leiden mutation (resistance to Protein C)
Protein C deficiency
Protein S deficiency
ATIII deficiency
Prothrombin mutation
Homocysteinemia
Disseminated intravascular coagulation
Result of something else bad going on: major tissue trauma, brain trauma, shock (to treat DIC, fix initial problem; short term treatment has no protocol, either can fix clotting or bleeding)
Generalized intravascular clotting AND fibrinolysis (dissolving clots)
Disseminated microvascular thrombi cause tissue injury
Consumption of coagulation factors and platelets causes hemorrhage
See low platelets, factors, fibrinogen, and high fibrin degradation products
Trigger mechanisms in DIC
Direct intravascular factor activation by proteases: snake venom, proteases released in acute pancreatitis, crude factor concentrates
Release of cellular procoagulants (tissue factor) causes intravascular cell lysis (hemolysis, leukemia, granulocyte lysis in sepsis), extravascular cell lysis (tumor, trauma, surgery), ascitic or amniotic fluid emboli
Vascular factors: endothelial cell damage by endotoxin, hypotension and stasis (shock), hemangiomas
Note: with shock, coagulation factors going really slowly and can aggregate easier
Damage caused by DIC
Glomerular capillaries frequently affected because plugged with microthrombi (fibrin-platelet thrombi)
Widespread focal ischemia AND hemorrhage damages kidney, skin, brain, lung, GI, mucous membranes
Lab tests to evaluate hemorrhagic and thrombotic disorders
Prothrombin time (PT): 9-12 sec
Partial thromboplastin time (PTT): 22-33 sec
Fibrinogen: 200-400 mg/dl
Specific factor/co-factor assays: >50% activity
APC resistance/Factor V Leiden
D-dimer assay: should be negative (measures plasma degraded fibrin)
How various diseases/drugs affect PT or PTT
Remember, PT measures extrinsic pathway (factor 7) and PTT measures intrinsic pathway (factor 9 and 8)
Mutation in 7 –> prolonged PT
Hemophilia A –> prolonged PTT
Hemophilia B –> prolonged PTT
Mutation in 10 –> both
Liver disease –> both
Vitamin K deficiency –> both
Give heparin –> prolonged PTT (doesn’t affect 7!)
Give coumadin –> prolonged PT (7 has shortest half life!)
Part of marrow in normal adult where hematopoiesis occurs
Ends of long bones
Iliac crest
Nutrients required for RBC production
Iron
B-12
Folate
Where do reticulocytes mature?
2/3 mature in the marrow and then are released into circulation
1/3 are put into circulation and THEN mature in the circulation
If a patient is anemic, how high should the reticulocytes be?
Depends, but can be up to 10x higher % (normal is 0.5 - 1.5%, so could be 5 - 15%)
Proteins in the RBC membrane
Spectrin
Ankyrin
Actin
Note: these hold bilayer together and keep RBC in its normal shape; if defect in proteins, form spherocytes
Anemias with different RBC morphologies
Microcytic: iron deficiency, thalassemia
Macrocytic: folate or B12 deficiency
Normocytic but with abnormal shapes: hereditary spherocytosis, sickle cell disease
Clinical presentation in iron deficiency
Fatigue, breathlessness
Pica (persistent compulsive desire to ingest certain food or non-edible items like ice, clay, plaster)
Sore mouth, angular stomatitis, palor
Megaloblastic anemia
B12 and folic acid deficiency
Hypercellular bone marrow with increased megaloblasts
All hematopoetic lineages show nuclear to cytoplasmic dyssynchrony
Hypersegmented neutrophils
Treat with B12 and see increase in reticulocyte count in first week and disappearance of hypersegmented neutrophils in 2-3 weeks
Granules of neutrophils
Primary granules: MPO, elastase, defensins, cathepsins
Secondary granules: lactoferrin
Tertiary granules: cathepsin, gelatinase
Note: these enzymes play important role in killing microorganisms
Absolute neutrophil count (ANC)
ANC = WBC x (% bands + % mature neutrophils) x 0.01
Clinical presentation and treatment of severe neutropenia
Severe neutropenia <500 per mm3
Get infections (chills, fever, weakness), ulcerating, necrotizing oral/pharyngeal lesions with massive growth of bacteria and no granulocyte response
Treatment includes recombinant hematopoietic growth factors (G-CSF)
Multiple myeloma
Neoplasm of malignant plasma cells
Normal hematopoietic elements replaced by malignant plasma cells
Neoplastic plasma cells secrete paraproteins which cause kidney problems and interfere with normal antibody secretion by plasma cells
Clinical features: CRAB = calcemia, renal failure, anemia, bone lesions (lytic)
Diagnosis: M-protein (paraproteins) in serum or urine, bone marrow with clonal plasma cells or plasmacytoma, CRAB
Thrombopoetin
Hormone responsible for platelet production
Produced by liver and kidney
Normal platelet count but loss of function
Uremia (renal failure)
Liver disease
Prosthetic valves
Aspirin or NSAIDs (or other drugs)
Congenital (intrinsic platelet defects): Bernard Soullier syndrome (Gp1b deficiency), Glanzmann’s thrombasthenia
What does it mean for a diagnostic test if the prevalence of a disease in a population is low?
If prevalence of disease in a population is low, even tests with high specificity or sensitivity will have low positive predictive values
Makes sense because if prevalence is low, more positives will be false positive and more negatives will be truly negative
HIV testing is >99% sensitive and specific, is screening the population a good idea?
If very low HIV prevalence, positive predictive value (PPV) is very low but NPV is very high –> just have to do confirmatory test on positive results
Note: ELISA used for screening and nucleic acid test used for confirmation (not western blot anymore because that misses patients with early HIV infection)
Detuned HIV test
Strategy used to diagnose “recent” infection
First test with threshold of 50, see positive result, then test with threshold of 100 and see negative result –> means antibody titer is low which means person was just recently infected with HIV
This strategy “tunes down sensitivity of the test” (increase threshold to call test positive)
ABO blood groups
ABH antigens are located on transport proteins of the RBC membrane
Antigens differ only with respect to one terminal sugar
O: no terminal sugars (H antigen)
A: N-acetyl-D-galactosamine
B: D-galactose
What different blood types can receive
Blood type A (has A antigen) can receive RBCs from A, O; plasma from A, AB
Blood type B (has B antigen) can receive RBCs from B, O; plasma from B, AB
Blood type O (no antigens) can receive RBCs from O; plasma from O, A, B, AB
Cross matching
Add recipient plasma to donor RBC and see if there is a reaction (should be NO reaction if ABO matched!)
Use secondary antibody to Fc region of human antibodies to allow for clumping if the recipient’s antibody did bind to donor’s RBC
What does giving one unit of packed RBCs do?
1 unit of packed RBCs = 250-300ml
One unit will increase hemoglobin by 1 gm/dL (Hct, 2-3%)
How do you decide when to transfuse RBCs?
Not based upon numbers, but upon symptoms!
To restore O2 carrying capacity in symptomatic anemia (exertional dyspnea, dyspnea at rest, fatigue, hyperdynamic state, lethargy and confusion, CHF, angina, arrhythmia, MI) or acute bleeding
In general, transfuse high risk patients (acute MI, unstable angina) at Hgb < 10g/dL; low risk patients at Hgb < 7g/dL
Note: if someone anemic (Hg = 6) but doesn’t have symptoms, don’t transfuse them!
Acute hemolytic transfusion reaction
What we’re most scared of during transfusion!
Occurs within minutes to hours after transfusion
Signs and symptoms: chills, fever, hemoglobinuria, hypotension, renal failure with oliguria, DIC (oozing from IV sites), back pain, pain at infusion site, anxiety
Management: supportive (maintain hydration, analgesics, pressors, hemostasis, follow-up labs), prevention
Incidence = 1:38,000 - 1:70,000
Etiology: clerical error 70% of time
ABO incompatible transfusions are the worst (can also have incompatibilities in other proteins?)
Hemolytic disease of the newborn
Occurs secondary to anti-D (anti-Rh) antibodies
Mother lacks antigen (Rh-)
Fetus possesses antigen (Rh+)
First baby’s fetal red cells stimulate maternal IgG response during birth (?)
If second baby is Rh+, antibody from mother crosses the placenta and binds/destroys fetal RBCs –> fetal anemia (cardiac failure and edema, hydrops fetalis, jaundice, kernicterus)
Note: mother can be sensitized by transfusion or previous pregnancy (maybe not enough of antibody response to cause anemia in first baby? Maybe just IgM with first baby..?)
Rhogam
Rho(D) immune globulin (Rhogam) is given to all Rh- mothers at 28 weeks gestation
If baby is Rh+, the Rho(D) immune globulins will coat baby’s RBCs so that mother never “sees” the antigen and never makes Rh antibodies!
When baby is born, can type the baby to see if it’s actually Rh+ –> if baby was Rh+, keep giving mother Rhogam until all baby’s blood out of her system?
How do you monitor mother/baby for potential hemolytic disease of the newborn?
Take serial titers every 2-4 weeks
Measure Rh antibody in mother
If baseline increase by two dilutions, means baby is at risk for hemolytic disease of newborn (baby is Rh+ and mother is reacting to it!)
Note: you can also look at the paternal genotype to see if he is Rh+
Can also use serial doppler ultrasound to measure peak systolic velocity of fetal MCA (if anemic, lower blood viscosity and increased cardiac output); this is performed at 18 - 35 weeks
Intra-uterine transfusion
Can do this if baby is anemic
Cord blood obtained by cordocentesis to measure hemoglobin level
Transfuse fresh O and Rh negative blood using umbilical vein
Goal is to suppress fetal red cell production
Do transfusions until birth
Transfusion of platelet products
Do this if someone has platelet count <10-20,000 (myelosuppression from chemotherapy or primary aplasa (ALL))
Apheresis platelets: platelets in small vol of plasma with minimal RBC/WBC; 150-250 ml/unit; raises platelet count by 30,000
Platelet concentrate (PC): platelets in small vol of plasma with minimal RBC/WBC; 50-70 ml/unit
Allergic transfusion reactions
Second most common reaction
IgE to donor plasma proteins (FFP > platelets > RBC > cryo)
Signs and symptoms: urticaria, pruritis, flushing
Therapy: stop transfusion, give antihistamine, then can restart transfusion; or give antihistamine prophylactically if know will have this rxn
Febrile non-hemolytic transfusion reaction
Antibody to donor WBCs, or transfusion of pre-formed cytokines in blood products (platelets most common)
Signs and symptoms: fever, chills, rigors, headaches, possibly changes in BP, HR, dyspnea, nausea
Not life-threatening but uncomfortable
Therapy: antipyretic; use leukocyte-reduced blood products
Fresh frozen plasma transfusion
FFP contains all coagulation factors, so give to someone who is not clotting well (liver disease, DIC, factor deficiency, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome being treated by plasma replacement, coumadin reversal)
Therapy guided by coag studies (PT, aPTT)
1 unit plasma increases most factors 2.5%; 4 units plasma increases most factors 10%
Contraindications: available specific therapy (factor 8, 9, vitamin K), volume expansion
Transfurion-related acute lung injury (TRALI)
Causes death more often than any other transfusion reaction (mortality 5-10%)
Leakage of fluid into alveolar space due to diffuse alveolar damage (antibody-mediated or cytokines)
Signs and symptoms: acute respiratory distress, frothy fluid coming from endotracheal tube, tachycardia, fever, hypo/hypertension, cyanosis
Consequences: lung injury and prolonged ventilatory time, predispose to pulmonary infection, death
Therapy: supportive care until recovery; test for WBC antibody (HLA, granulocyte) in donor and recipient
Note: this is a clinical diagnosis (act fast and lab test takes a week!)
Transfusion-associated circulatory overload (TACO)
Volume overload temporally associated with transfusion
Signs and symptoms: SOB, increased RR, hypoxemia, cough, tachycardia, JVD, headache
Therapy: upright posture, O2, IV diuretic, transfuse split unit
When people donate blood, what do we screen it for?
HIV
HepB
HepC
HTLV-1, 2
WNV
Syphilis
CMV (sometimes?)
Chagas (not yet FDA mandated)
Hemoglobin
Oxygen carrying protein within RBCs
Normal adult HbA contains 4 subunits: 2 alpha chains and 2 beta chains
Each subunit has a globin (polypeptide chain) and a heme (iron-containing prosthetic group)
Why do we see alpha gene defects in embryonic development but not beta gene defects?
Because alpha is expressed during fetal life and beta is not expressed until after birth
Globin gene developmental expression and globin switching
Ordered regulation of developmental gene expression
Genes in each cluster arranged in same transcriptional orientation and same sequential order as developmental expression
Embryonic and fetal hemoglobin
Embryonic hemoglobin: zeta2epsilon2
Fetal hemoglobin (HbF): alpha2gamma2 (predominates 5 weeks gestation to birth; 70% of total Hb at birth; <1% of total Hb in adulthood)
Adult hemoglobins
HbA: alpha2beta2 (nearly all is HbA by 3 months old)
HbA2: alpha2delta2 (<2% of adult Hb)
Thalassemias
Relative imbalance (NOT instability!) in relative amounts of alpha and beta chains, due to mutations resulting in decreased synthesis of one or more globin chains
Excess normal chains precipitate in red cell to damage cell membrane and destroy RBCs prematurely
Results in hypochromic microcytic anemia and tissue iron overload
Seen in Mediterraneans
Hereditary persistence of fetal hemoglobin (HPFH)
Clinically “benign” and not associated with disease
Mutations impair perinatal switch from gamma to beta globin synthesis
At least one gamma gene remains intact
Increased gamma chain production so increased HbF in adult
HPFH heterozygotes have 17-35% HbF
Sickle cell anemia
Autosomal recessive disorder HbSS
HbS is mutation of 6th codon of beta globin gene turning hydrophilic glutamic acid to hydrophobic valine
When deoxygenated, hemoglobin S interacts with other hemoglobin S to polymerize and cause RBC to be rigid and sickled
Found in “Malaria Belt” but mutation emerged outside of Africa separately too (heterozygote confers resistance to malaria)
Clinical features of SS disease
Presentation in first 2 years of life
Infections, anemia, failure to thrive, splenomegaly, dactylitis
Vaso-occlusive infarctions: strokes, acute chest syndrome, renal papillary necrosis, autosplenectomy, leg ulcers, priapism, bone aseptic necrosis, visual loss
However as many as 70% of people have no symptoms
Causes of death: progressive renal/cardio-pulmonary failure (in 30s and 40s), parvovirus infections (high risk of life-threatening aplastic anemias, temporary cessation of RBC production)
Alpha thalassemias
Disorder of alpha globin production
Affects formation of both fetal and adult hemoglobins (can cause intrauterine and postnatal disease)
In absence of alpha globin chains, beta globin tetramers form (gamma4 is Hb Bart’s and beta4 is HbH) which cannot release O2 to tissues normally
Normal = 4 functional alpha genes
Silent carrier = 3 functional alpha genes
Alpha-thal mild = 2 functional alpha genes
Alpha-thal HbH = 1 functional alpha genes
Hydrops fetalis = 0 functional alpha genes
Hydrops fetalis due to severe alpha thalassemia
High level of Hb Bart’s (gamma4)
Marked intrauterine hypoxia
Seen most commonly in Southeast Asia (high gene frequency, predominant form of alpha thal trait there is –/aa, so have risk of –/– whereas elsewhere it’s -a/-a!)
Note: hydrops fetalis is massive generalized fluid accumulation in utero
Milder alpha thalassemia (HbH)
Anemia develops because of gradual precipitation of HbH in erythrocytes
Beta thalassemia
Excess alpha chains
Alpha chains are insoluble, precipitate in RBC precursors and cause RBCs to be destroyed in bone marrow (ineffective erythropoiesis)
Not apparent until a few months after birth
Beta thalassemia minor = heterozygote
Beta thalassemia major = homozygous
Beta thalassemia minor
Heterozygote (one normal beta globin gene, the other mutated)
Hypochromic, microcytic anemia
May be misdiagnosed as iron deficiency anemia
HbA2 elevation only in these heterozygotes (alpha2delta2)
HbF is also increased (not due to reactivation of gamma globin gene but increased selective survival and possibly increased production of minor population of HbF-containing adult RBCs)
What is the mutation in beta thalassemia?
There are many different mutations in the beta globin gene that can lead to beta thalassemia!
Beta thalassemia major
Usually genetic compounds that create homozygote (two genes with mutations in beta globin though)
Severe anemia with phenotype due to combined effects of two alleles
Beta0 thal: no HbA present
Beta+ thal: HbA present
Severe hypochromic anemia
Treatment: blood transfusion and iron chelation; bone marrow transplantation if appropriate match
What happens if you have one gene that is beta thal and one that is beta S?
If the beta thal gene is beta0: like sickle cell disease
If the beta thal gene is beta+: may be mild phenotype
What happens if you have mutations in both alpha and beta loci?
Beta thal homozygotes (beta thal major) who also inherit alpha thal allele may have LESS severe beta thalassemia because there is LESS imbalance of alpha vs. beta globins!
Some symptoms of iron deficiency anemia
Pica (eating ice)
Glossitis (sore tongue)
Dysphagia (esophageal webs)
Hereditary spherocytosis
Due to defect in membrane skeleton protein of RBC
Intracorpuscular hemolysis
