Benign Heme Flashcards
Ataxia Telangiectasia
- rare, neurodegenerative, inherited disease
- impairs cerebellum, causing difficulty with movement and coordination.
- weakens the immune system, causing a predisposition to infection.
- prevents repair of broken DNA, increasing the risk of cancer (especially lymphoma/leukemia)
- sx appear in early childhood when children begin to walk
- caused by a defect in the ATM gene (manages cell’s response to multiple forms of stress including double-strand breaks in DNA)
Bloom Syndrome
Ashkenazi Jewish population
diabetes mellitus
decreased fertility
MDS
Down Syndrome
- overall risk of cancer is not changed, risk of leukemia and testicular cancer is increased and risk of solid cancers is reduced
- leukemia is 10 to 15x more common in Downs:
*ALL is 20x more common
*megakaryoblastic AML leukemia is 500 times more common
- TAM affects 3–10% of infants, resolves without treatment
- After TAM, 20 to 30% risk of developing ALL
HIV marrow findings
ALIP commonly seen (NOT dysplasia) increased plasma cells
Measured CBC parameters
Hgb RBC MCV RDW
Hemolyzed sample- what is still valid
Hgb (after all RBC lysis is the first step in the cyanmethemoglobin method for measuring Hgb)
3 methods automated instruments use to perform cell counts
- Impedance (size) 2. Optical (forward vs light scatter) 3. Combo of these
RBC count
Measures using Coulter principle: cells travel one by one through an aperture across which a current is flowing causing a momentary increase in impedance/electrical resistance V=IR I is constant, R is the increases resistance caused by a red cell, V momentary increase in voltage is interpreted as a single cell which is simultaneously counted/sized Particles measuring 36-360 fL are counted as red cells (includes WBC but their contribution is negligible)
Calculated CBC parameters
Hct MCH MCHC
Hct calculation
Hct= (MCV x RBC)/10
MCH calculation
MCH= (Hb/RBC) x 10
MCHC
MCHC= (Hb/Hct) x 100
Neoplastic causes of increased RBCs
RCC HCC Hemangioblastoma
WBC count
Lyse RBCs then count all particles >36 fL using Coulter principle (plt usually less than 36 fL)
WBC interferences
platelet clumps micromegakaryocytes nRBCs
Platelet count
Particles between 2-20 fL are counted as platelets using Coulter principle
How to fix platelet clumping/satellitism
Recollect sample in sodium citrate New platelet count should be multiplied by 1.1 to correct for dilution factor associated with this anticoagulant
Acanthocytes
- Abetalipoproteinemia
- Severe burns
- Liver dz
- Post-splenectomy
- McLeod phenotype (Kell null)
Fine basophilic stippling
Seen in reticulocytes as artifact of slow air drying
Coarse basophilic stippling
Ribosomal RNA aggregates, indicative of abnormal hemoglobin synthesis (lead poisoning, thalassemia)
Bite cells
Due to removal of Heinz bodies by splenic macrophages (suggestive of G6PD deficiency or drug induced oxidant hemolysis)
Burr cells
Usually artifactual; seen in uremia and pyruvate kinase deficiency
Cabot rings
Remnant of nuclear DNA; remnants of spindle fibers that form during mitosis (seen post splenectomy or in megaloblastic anemia)
Elliptocytes
Seen in iron deficiency anemia and hereditary elliptocytosis
Heinz bodies
Ppt hemoglobin, requires supravital dye to visualize (seen in G6PD deficiency and with unstable forms of Hgb)
Howell-Jolly bodies
Remnant of nuclear DNA (seen in post splenectomy, in hemolytic anemia, or in megaloblastic anemia)
Hypochromia
Seen in iron deficiency anemia, thalassemia, and sideroblastic anemia
Macrocytes
Seen with increased erythropoiesis (due to increased reticulocytes), megaloblastic anemia (oval macrocytes), and liver disease (round macrocytes)
Microcytes
Seen in iron deficiency anemia, thalessemia, and sideroblastic anemia
Pappenheimer bodies
Iron containing mitochondria (seen in sideroblastic anemia and post splenectomy)
Pencil cells
Hypochromic variants of elliptocytes that have long axis at least three times the length of the short axis (classic for Fe deficiency but also seen in anemia of chronic disease and beta thalassemia minor
Polychromasia
Due to residual ribosomal material
Sickle cells
Seen in sickling hemoglobinopathies (NOT seen in sickle cell trait)
Spherocytes
Seen in hereditary spherocytosis and immunohemolytic anemia
Stomatocytes
Usually artifact; may be seen in alcoholism, hereditary stomatocytosis, or Rh null phenotype
Target cells
Liver disease, HbC, SC disease, HbE, post splenectomy, thalassemia
Teardrop cells (dacrocytes)
Myelophthisic dz, splenic dysfunction
Dohle bodies
Thought to be remnants of rough ER, possibly agglutinated ribosomes
Toxic granulation
Prominent primary granules; seen in sepsis and GM-CSF (Neupogen) therapy
Prolymphocytes
Larger than lymphocytes (2x) with a moderate amount of cytoplasm, round nuclei (occasionally irregular), immature chromatin, and prominent central nucleolus
LGL
May be T or NK phenotype; very common to be increased post transplant
Manual retic count
Use supravital stain (eg new methylene blue)
Automated retic count
RNA binding dyes that can be detected by fluorescence (but Coulter still uses detection of light scatter after staining with new methylene blue)
Immature retic fraction
Cells with the highest fluorescence intensity (highest RNA content)
Retic count
% retics (# per 100 RBCs)
Corrected retic count (CRC)
(retic%)(hct/45) -takes into account normal RBC production for a given hematocrit
Reticulocyte production index (RPI)
CRC/correction factor -1.0 for hct 40-45 -1.5 for hct 35-39 -2.0 for hct 25-34 -2.5 for hct 15-24 -3.0 for hct
For how long do reticulocytes circulate?
Usually present in blood for 24 hours before they extrude residual RNA and become erythrocytes; if released early from marrow, immature retics may persist in PB for 2-3 days (RPI corrects for presence of immature retics)
RPI less than 2
failure of BM RBC production (hypo proliferative anemia)
RPI>3
marrow hyperproliferation or appropriate response to anemia
Constitutional aplastic anemia
Fanconi anemia Dyskeratosis congenita Diamond-Blackfan anemia Congenital amegakaryocytic thrombocytopenia Schwachman-Diamond syndrome Severe congenital neutropenia (Kostman syndrome)
Acquired aplastic anemia
-PNH -Idiopathic: (theory: destruction of CD34+ progenitor cells through apoptotic mechanisms stimulated by cytokine released from activated BM cytotoxic T cells) -Secondary to drugs (cytotoxic drugs, chloramphenicol, gold), radiation, toxins (benzen), infections (parvovirus), neoplasms (thymoma), pregnancy
Constitutional red cell aplasia
Diamond-Blackfan anemia
Acquired red cell aplasia
P-parvovirus infection I- idiopathic T- transient erythroblastopenia of childhood (most likely due to antecedent viral infection) A- antierythropoietin ab induced red cell aplasia (occurs in some patients receiving recombinant epo) S- sustained pure red cell aplasi (secondary to neoplasms such as T cell LGL leukemia, immune d/o, chronic viral infections, and drugs)
Fanconi anemia genetics
-AR (rarely X-liked recessive) -13 genes on many different chromosomes (one being BRCA2) -cells characteristically show abnormally high frequency of spontaneous chromosomal breakage and hypersensitivity to DNA cross-linking agents such as diepoxybutane (DEB) and mitomycin C
Fanconi anemia diagnostic test
increased chromosomal breakage seen in FA cells compared to normal controls after exposure to DEB or mitomycin C (AKA the DEB/MMC stress test)
Fanconi anemia clinical
-progressive BM failure (aplastic anemia with pancytopenia) and increased risk of malignancy (AML- often with monocytic features, solid tumors like cutaneous malignancies, HCC, gastric carcinoma) -cafe au lait spots, microcephaly, hypoplastic thumbs, absent radius, scoliosis, MR, horseshoe kidney, duodenal atresia, cardiac defects, neurologic abnormalities (1/3 no abnormalities) -present toward end of 1st decade -anabolic steroids (oxymetholone) produce useful trilineage hematopoietic response in 50-70%, but most become refractory -tx HSCT
Dyskeratosis congenita genetics
-X-linked recessive (80% cases), but AD and AR subtypes are recognized -DKC1 gene on Xq28 -genomic instability d/o similar to Fanconi anemia (however, no increased chromosomal breakage)
Dyskeratosis congenita clinical
Inherited BM failure syndrome characterized by mucocutaneous triad (mostly males): 1. abnormal skin pigmentation 2. nail dystrophy 3. oral leukoplakia -skin and nail changes appear first, usually by age 10 -BM failure by age 20 -like Fanconi anemia, main cause of death is BM failure -other causes of death include pulmonary complications and malignancy -again, like FA, anabolic steroids (oxymetholone) produce useful trilineage hematopoietic response in 50-70% -tx HSCT
Schwanchman-Diamond syndrome genetics
-AR, 90% due to mutation of SBDS gene (chr 7q11) -results in deficiency of SBDS protein (can see loss by IHC) -protein plays regulatory role in RNA metabolism in nucleolus by stabilizing mitotic spindle -can see an isochromosome 7q
Schwanchman-Diamond syndrome clinical
-2nd most common cause of pancreatic insufficiency in childhood after CF -exocrine pancreatic insufficiency (86%), BM failure-mostly as neutropenia (98%), anemia (42%), pancytopenia (19%), skeletal abnormalities (50% particularly metaphyseal dysostosis), growth retardation (56%), and increased risk of MDS/AML (30%) -tx with pancreatic enzymes and G-CSF -main cause of death infection/bleeding -at risk for complications from HSCT
Diamond-Blackfan anemia genetics
-DBA1 gene AKA RPS19 - encodes ribosomal protein S19 and is NOT a regulator of erythropoiesis rather is a ubiquitous ribosomal protein -AD with incomplete penetrance -heterozygosity for mutations of gene (genetic heterogeneity of DBA)
Diamond-Blackfan anemia clinical
-presents in early infancy (around 8 weeks) with sx of anemia -hallmark is selective decrease in erythroid precursors and normochromic macrocytic anemia -craniofacial, thumb, cardiac, and urogenital malformations -modest increase in MDS/AML/osteosarcoma -tx with corticosteroids and transfusions; HSCT
Diamond-Blackfan vs Shwachman-Diamond vs Fanconi Anemia
DB: pure RBC aplasia SD: neutropenia FA: pancytopenia
Diamond-Blackfan anemia lab tests
-elevated erythrocyte deaminase activity and elevated fetal hemoglobin (HbF) -i ag overexpressed on RBCs
Diamond-Blackfan: hematologic diagnostic criteria
- normochromic, macrocytic anemia developing in early childhood 2. reticulocytopenia 3. normocellular BM with marked selective deficiency of eyrthroid precursors (erythroblasts
Reticulocytes on supravital stain
clumped ribosomal RNA (not dots-those are Heinz bodies)
Babesiosis vs Malaria
- On right, RBC contains 4 small, red-purple dots (tetrad)
- On left, 2 RBCs contain trophozoites (ring forms), which closely resemble P. falciparum
- RBCs may show single chromatic dot or two or more chromatic masses; occasionally 4 ring forms in tetrad => maltese cross
- 4 visible dots is diagnostic and not seen in malaria; can also see extraeythrocytic forms which are not seen in malaria
- in malaria, RBCs are enlarged/ovoid vs babesia they are normal
- noncyclic fever and hemolysis
- coinfection with Lyme dz and flavivirus
Causes of basophilia
- allergic/hypersensitivity reactions
- inflammatory d/o (collagen vascular dz, RA, UC)
- endocrinopathy (DM)
- chronic renal failure
- infections (influenza, chicken pox)
- neoplasms (CML)
Chediak-Higashi Syndrome pathogenesis
AR
- PMNs impaired mobility (defective chemotaxis) and defective granulation
- primary defect in intracellular granules:
*leads to WBC death in marrow resulting in neutropenia and thrombocytopenia
*defective granules in grans, lymphs, and NK cells alter immune fx with resultant severe pyogenic infections
*abnormal melanin containing granules result in eye/skin hypopigmentation
- all WBCs contain large cytoplasmic granules (due to fusion of lysosomes)
- cytoplasmic inclusions contain both primary (MPO+) and secondary granules
Chediak-Higashi Syndrome clinical
- severe recurrent bacteria infections
- partial oculocutaneous albinism
- many develop EBV associated hemophagocytic syndrome (“accelerated phase”) or EBV induced lymphoproliferative d/o
- pts die in childhood
- tx HSCT
Congenital Dyserythropoietic Anemia general
-rare disorders characterized by profound morphologic abnormalities of erythroid elements and ineffective erythropoiesis
- some cases are AR
- all pts have anemia with marked anisopoikilocytosis, reticulocytopenia, marrow erythroid hyperplasia
CDA Type I
- erythroid precursors show megaloblastic changes with internuclear chromatin bridges
- occasional binucleate forms seen
CDA Type II
**The most common type**
- characterized by bi- and multinucleated erythroid precursors (usually 2-7 nuclei)
- positive acidified serum test (Ham’s test) due to an abnormal RBC antigen; lysis occurs in heterologoys serum only, in contrast to PNH in which lysis occurs in both autologous and heterologous serum
- also referred to as “hereditary erythroblastic multinuclearity with positive acidified serum” HEMPAS
- RBCs have very high density of i (recall Diamond-Blackfan too)
Causes of reactive eosinophilia
- allerigic/atopic disorders
- medications (L-tryptophan linked to eosinophilia-myalgia syndrome)
- parasites (must invade tissue to produce eosinophilia)
- cutaneous disorders (eczema, atopic dermatits, bullous pemphigoid)
- inflammatory disorders (IBD, collagen vascular dz, sarcoid, celiac)
- pulmonary disorders (Loeffler syndrome, CF, Churg-Strauss)
- neoplasms (T cell lymphoproliferative d/o, HL, LCH)
CDA Type III
-pronounced multinucleateion with up to 12 nuclei present in some erythroid precursors (“gigantoblasts”)
Eosinophil function
Release of secondary granules:
*major basic protein
*peroxidase
*arylsulfatase
*histamine oxidase
*eosinophil cationic protein
- modulate immediate hypersensitivity rxn
- destroy parasites
Erythrocyte Cytoskeleton
- Two isoforms of spectrin, and , form a loosely wound helix. Two helices are linked end to end to form a tetramer, which has binding sites for several other proteins, including other spectrin molecules.
- The spectrin tetramers are organized into a meshwork that is fixed to the membrane by the protein ankyrin.
- Ankyrin is itself connected to a transmembrane protein called Band 3 (AE1, anion exchanger).
- Band 4.2 (palladin) is thought to function to stabilize the link between ankyrin and Band 3.
- Spectrin is also linked to the transmembrane protein glycophorin C by the protein known as Band 4.1.
- Thus the meshwork is anchored to the membrane at multiple sites.
- Band 4.1 stabilizes the association of spectrin with actin, as does the protein adducin.
- Actin subunits form short microfilaments consisting of actin and tropomyosin.
- The protein tropomodulin is also associated with filamentous actin.
Chromogenic Factor Xa Assay
- useful in monitoring warfarin in presence of a LA or DTI
- utilizes an activator of factor X and peptide substrate for factor Xa that is coupled to a chromogenic marker
- cleavage of peptide by factor Xa releases the marker and yields a colored product, the intensity of which is directly proportional to factor X levels
Ferritin
- serum level has a direct correlation with total amount of iron stored in the body (less invasive than BM bx)
- is a storage complex of iron and apoferritin protein
- ferritin is an acute phase reactant and often increase with infection, inflammation, or malignancy
- normal is 10-500 ng/mL
Serum Iron
- measures amount of circulating iron that is bound to transferrin
- serum iron concentrates show wide diurnal variation; should collect specimen in AM when level is highest
- normal is 60-180 µg/dL
TIBC
- measures the extent to which iron-binding sites in serum can be saturated
- becuase iron-binding sites in serum are almost entirely on circulating transferrin, this is really an indirect measurement of the amount of transferrin in the blood
- normal is 250-410 µg/dL
Transferrin Saturation
(serum Fe/TIBC) x 100
normal is approximately 30% (20-50%)
Unsaturated iron-binding capacity
UIBC= TIBC-serum iron
Serum soluble transferrin recepetor
- transferrin receptor is a transmembrane protein that transfers iron from plasma transferrin into the RBC
- most transferrin receptors are found in the cell membrane, but a portion is found in soluble form in the serum
- transferrin receptor levels reflect iron status, with receptor synthesis being rapidly induced by decreased iron levels; unlike ferritin it is not altered in inflammatory states
- useful to distinguish iron deficiency (increased) from thalassemia minor (normal) and anemia of chronic disease (normal to slightly increased)
- elevated levels may also be seen in pts with hematolymphoid neoplasms or conditions with increased hematopoiesis (eg HA, hemoglobinopathies, folate/B12 def) irrespective of iron status
Free erythrocyte protoporphyrin
(zinc protoporphyrin is similar)
- FEP levels are increased in pts with anemias associated with failure of iron incorporation into heme
- when insufficient iron is available for developing erythroblasts, excess protoporphyrin that was destined to be converted to heme accumulates as FEP
- FEP is high in iron deficiency anemia and in anemias associated with block in iron utilization (anemia of chronic dz, lead poisoning, sideroblastic anemia); FEP is normal in thalassemia (in thalassemia, globin synthesis is abnormal but heme synthesis is normal)
- useful to distinguish iron deficiency (increased) from thalessemia minor
- normal less than 100 µg/dL
Stainable bone marrow iron
- iron is stored in reticuloendothelial cells
- erythroid precursors that cotnain one or more particles of stainable iron are known as sideroblasts
- iron is stored as ferritin (iron complexed to the apoferritin protein) and hemosiderin (iron-protein complexes with a high iron content and denatured ferritin aggregates); hemosiderin is visible with Prussian blue stain
- when storage iron is present in the BM, anemia cannot be a result of iron deficiency (unless the pt has recently received parenteral iron)
- normally 20-40% of erythroid precursors are sideroblasts
G6PD Deficiency Pathogenesis
- G6PD reduces NADP+ to NADPH while oxidizing glucose-6-phosphate in the hexose monophosphate shunt
- NADPH is then used to convert oxidized glutathione to reduced glutathione
- reduced glutathione protects RBCs from oxidizing injury by catalyzing breakdown of oxidants such as H2O2 (O free radicals)
- deficiency of G6PD leads to insufficient glutathione, which renders RBCs susceptible to oxidant injury
- oxidative agents include free radicals generated during an infection or following exposure to various drugs (sulfonamides, nitrofurantoin, antimalarials)
- >400 mutations of G6PD gene are known; the two most common are the A variant, which occurs in 12% of AA males and the Mediterranean variant, which is more severe and can occur after ingesting fava beans
G6PD Deficiency Inheritance
- X linked recessive (the enzyme deficiency is present in all RBCs of affected males)
- heterozygous females have 2 populatins of RBCs (normal and G6pD deficient); the proportion of each is dependant on random inactivation of the X chromosomes (some patients healthy vs others appear fully affected)
G6PD Deficiency Clinical
- hemolysis begins acutely in infection and 1-3 days after drug/fava bean ingestion
- hemolysis often severe, leading to hemoglobinemia, hemoglobinuria, and jaundice
- G6PD-A deficiency the hemolytic anemia is usually self limited b/c reticulocytes have nearly normal G6PD levels; more severe forms may require transfusions
G6PD Deficiency Labs
PB smear: Heinz bodies on supravital stain (ppt hemoglobin) or bite cells
Fluorescent spot test (screening test):
- NADPH fluoresces when exposed to UV light; NADP+ does not
- RBCs mixed with soln of G6P, oxidized glutathione, NADP+ which is then spotted on filter paper
- Normally, when the spot is exposed to UV light, fluorescence appears within 5-10 min
- G6PD deficient samples, no fluorescence seen
- screening test may not be useful in females or post-hemolytic period (reticus more G6PD activity)
- confirmation of abnormal screening requires quantitative assay of G6PD based on spectrophotometric quantification of NADPH formation from NADP+
Gaucher Disease General
- most common lysosomal storage dz
- Ashkenazi Jewish pop (1 in 450; 1 in 100K of general pop)
- AR inheritance, mutation in glucocerebrosidase gene (GBA) with >200 mutations described
- defective function of ß-glucocerebrosidase
Gaucher Disease Morphology
PB: moderate-marked anisopoikilocytosis, mild microcytosis, hypochromasia, moderate thrombocytopenia
BM: cellular marrow admixed with individual clusters of Gaucher cells (PAS+, CD68+, Fe+)
Accumulation of glucocerebroside (a lipid) in histiocytes (spleen, LNs, BM), Kupffer cells (liver), osteoclasts (bone), microglia (CNS), alveolar macrophages (lung)
Gaucher Disease Types
Type I (nonneuronopathic, adult): 90% of cases; first appears in adult liufe with hepatosplenomegaly, bone pain, pathologic fx, or pancytopenia; no CNS involvement; dx by Gaucher cells on bx and enzymatic assays indicating reduced but detectable levels of gluocerebrosidase activity; life not markedly shortened
Type II (acute neuronopathic,infantile): infancy and assd with CNS dysfunction, including convulsions and progressive mental deterioration; no detectable glucocerebrosidase activity; die by age 2
Type III (chronic/subacute neuronopathic, juvenile): intermediate b/w I and II; presents in adolescents; slowly progressive; small amnt of glucocerebrosidase activity may be detectable
Niemann-Pick Disease General
- AR lysosomal storage dz
- in types A and B, deficiency of sphingomyelinase casues accumulation of sphingomyelin, a fatty substance present in every cell of body
- characteristic cell is a lipid-laden macrophage referred to as “foam cell;” cytoplasmic inclusions are relatively uniformly-sized globules
- may see vacuolated lymphocytes and monocytes in PB
Niemann-Pick Disease Types
_Type A (most common)_: occurs in infants and is characterized by jaundice, hepatomegaly, profound brain damage; children with this type rarely live beyond 18 months _Type B_: usually occurs in pre-teen years; pts have hepatosplenomegaly but no brain involvement _Types C and D_: may appear in early life or develop in teen or adult years; moderate splenomegaly, but brain damage may also be extensive; these type characterized by defect that disrupts transport of ***cholesterol*** b/w brain cells
Unstable Hemoglobins
Amino acid substitutions that affect:
1) binding of globin chains to heme OR
2) points of contact b/w globin chains
- affect protein stability and result in intracellular ppt of globin chains
- ppt globin chains attach to RBC membrane, giving rise to Heinz bodies
- Hg Koln is the most common variant; others include Hb Christchurch and Hb Sydney
- HbA will behave like unstable Hb when subjected to proportionately greater heat stress
- Heinz body positive hemolytic anemia (due to unstable Hb, NOT G6PD def) the heat stability and isopropanol tests can be used to confirm presence of an unstable Hb variant
Unstable Hb Tests
Heat Stability Test
Isopropanol Stability Test
Heat Stability Test
- when Hb is heated, stability of molecule decreases; under controlled conditions, unstable Hb ppt, whereas stable Hb remain in solution
- method: hemolysate is added to a Tris-HCl buffer and heated in water bath at 50 deg Celsius; tube is examined at 60, 90, and 120 minutes for ppt
Isopropanol Stability Test
- when Hb is dissolved in isopropanol, which is more nonpolar than water, the hydrophobic bonds are weakend and stability of molecule decreases
- method: hemolysate added to a 17% isopropanol buffer solution and heated in water bath at 37 deg Celcius; tube is examined at 5, 20, 30 min for ppt
*samples with increased HbF (5-10% or more) will produce a positive test; methemoglobinemia (usually due to prolonged storage) will also give a positive result*
-false negatives are avoided by continuing the incubation until a control sample undergoes ppt
PNH Pathogenesis
- acquired clonal genetic disorder that results in chronic intravascular hemolysis
- acquired mutation in the phosphatidylinositol glycan A (PIG-A) gene on the X chromosome
- PIG-A gene responsible for synthesis of glycosylphosphaidylinositol (GPI), which anchors certain proteins to the cell membrane; b/c GPI linked proteins inactivate complement, their absence renders affected blood cells unusually sensitive to lysis by complement
- mutation occurs in a pluripotent stemm cell (thus RBCs, WBCs, and plts will be deficient in GPI proteins
- most pts have a large pop of unaffected cells b/c the mutation affects only the clonal progeny of a single pluripotent stem cell
GPI proteins
RBCs: CD55 (DAF), CD59 (MIRL, potent inhibitor of C3 convertase that normally prevents spontaneous activation of complement)
Monos: CD14
Grans: CD24,CD66b,CD55,CD59
CD59
- plays the major role in development of hemolysis
- CD59 neutralizes cleaved portion of C5
- when CD59 is absent, cleaved part of C5 causes cell lysis
PNH and AA
- 50% of aplastic anemia pts have expansion of PNH clones
- ~5% of aplastic anemia pts develop PNH
- ~25% of pts receiving antithymocyte serum for the treatment of AA recover with evidece of PNH
- the somatic PIG-A gene mutation is relatively common; a PNH clone emerges b/c the normal cells are suppressed (by antithymocyte serum or by a native imunological attack)
PNH Clinical
- begins as insidious onset of anemia
- chronic hemosiderinuria is a constant feature and may result in Fe deficiency anemia
- thromboses (due to abnormal plt function) are most common cause of morbidity/mortality
- increased risk for AML and MDS
- tx with eculizumab (monoclonal Ab to C5, prevents it from being cleaved into active form)
- cure with BMT
PMH Labs
CBC: anemia, leukopenia, thrombocytopenia (may also occur b/c overall deficiency in hematopoiesis)
Chem: increased LDH (from RBC hemolysis), hemosiderinuria
Flow: of PB is the gold standard for dx; defect is often more apparent in grans and monos vs RBCs (b/c RBCs are more sensitive to complement-mediated lysis than leukocytes, often diluted by transfused RBCs)
FLAER: (fluorescent aerolysin, derived from from Aeromonas). Aerolysin binds GPI part of GPI-linked molecules on the cell surface (ie bind to normal cells but not PNH cells)
Positive sucrose hemolysis test (screening test): RBCS are incubated in serum and isotonic sucrose, which promotes complement binding; after incubation for 60 min, >5-10% hemolysis is a positive result
Positive acified serum lysis test (Ham’s test): acidifaction of serum activates complement leading to lysis of PNH cells
Erythroblast–>Reticulocytes
- erythroblasts mature into marrrow reticulocytes in about 6 days
- each erythroblast can give rise to ~8 reticulocytes
For how long do RBCs circualte?
120 days
For how long to PMNS circulate?
Only a few hours before moving into tissues
WBC differential automated measurements
Impedence: cell vol/size
Conductivity: cell complexity
Light scatter: cytoplasmic granularity
light scattter (x-axis) vs impedence (y-axis) creates the WBC histogram (axes are opposite of flow)
Evans syndrome
ITP + hemolytic anemia
How many platelets can each megakaryocyte produce?
~2000-4000
Fe Deficiency Anemia- General
- accounts for 98% of anemia in children less than 4yo; in adults due to blood loss
- in mild Fe def anemia, decreased serum iron levels and increased TIBC usually precede changes in RBC morphology or CBC indices
- pts may have cheilitis, koilonychia (spoon nails), pica
Fe Deficiency Anemia- Temporal Progression
decrease ferritin→decrease transferrin sat→decrease in serum Fe→increase in ZPP→decrease in Hb, normocytic, normochromic→microcytic, hypochromic anemia
Fe Deficiency Anemia- Labs
- microcytic, hypochromic anemia w/ high RDW
- low ferritin
- low serum iron
- high TIBC
- low transferrin sat (often less than 10%)
- high serum transferrin
- high serum soluble transferrin receptor
- high free erythrocyte protoporphyrin
- absent marrow iron stores
- low serum hepcidin
Anemia of Chronic Disease- General
- defective iron cycling b/w macrophages and erythroid precursors, iron becomes trapped within macrophages and is unavailable for heme synthesis
- chronic inflammatory d/o (SLE, RA, IBD, sarcoidosis), chronic infections (HIV, TB, pyelo, osteo, chronic fungal infx, SBE), neoplasms, and end organ failure (chronic liver dz, endocrinopathies)
- anemia develops 1-2 months after onset of dz
- immunologic response to protect host from invading organisms/developing neoplasms by depriving of iron
Anemia of Chronic Disease- Labs
- normocytic normochromic
- normal-sl increased RDW
- normal or high ferritin
- low serum iron
- low TIBC
- low transferrin sat
- normal-sl increased serum soluble transferrin receptor
- high free erythrocyte protoporphyrin
- marrow iron stores increased in macrophages/reticuloendothelial cells, but decreased in sideroblasts
Thalassemia Minor- Iron Studies
- Fe phsyiology and heme synthesis are normal; defect is globin synthesis
- microcytic, hypochromic anemia
- normal RDW
- normal to increased RBC number
- often see basophilic stippling
- normal-high serum Fe
- normal-low TIBC
- normal-high transferrin sat
- variable serum soluble transferrin receptor (may be high)
- marrow iron stores are increased (due to ineffective erythropoiesis)
Summary of Iron Studies
Mentzer Index
MCV/RBC
less than 13: thalassemia
greater than 13: Fe deficiency
Sideroblastic Anemia- General
- iron overload state characterized by abnormal iron metabolism and heme synthesis within RBC
- Fe is sequestered in RBC mitochondria, making it unavailable for heme synthesis (mitochondria typically perinuclear-hence ring sideroblasts)
- may be hereditary (either X-linked or autosomal) or acquired later in life (as part of MDS or 2/2 toxic insult like drugs, EtOH, lead)
- hereditary forms may respond to tx with pyridoxine
Sideroblastic Anemia- Labs
- basophilic stippling on PB
- classically dimorphic pop of macrocytic cells and normocytic/microcytic cells- particularly in acquired forms
- high ferritin
- high serum iron
- normal-low TIBC
- high transferrin sat
- variable serum soluble transferrin receptor (may be high)
- increased marrow iron stores
Cyclic Neutropenia
- hereditary neutropenia (along with Kostmann syndrome)
- AD, some sporadic
- mutation in neutrophil elastase gene (ELA2)
- neutrophil elastase is a granule serine protease
- 21 day oscillating PMN count with ANC 0-1.5 x109/L (severe infx at low level)
- PB monos trend opposite ANC (high when ANC low)
- respond to G-CSF (shortens cycle length and increase amplitude of waves)
- does NOT progress to MDS/AML
Severe Congenital Neutropenia (Kostmann Syndrome)
- marked absolute neutropenia, usually less than 200/µL
- increased susceptibility to bacterial infections
- true Kostmann Syndrome is AR (other forms of SCN can be AD/sporadic)
- 60-90% cases have point mutation in neutrophil elastase gene (ELA2)- same gene but different mutations as cyclic neutropenia
- distinguish from Schwachman-Diamond Syndrome by lack of exocrine pancreas deficiency and growth retardation
- promyelocytic/myelocytic maturation arrest in BM
- increased monos and eos in BM (alternate fates of progenitor cell)
- 90% of pts respond to G-CSF (no toxic type granulation-weird)
- MDS/AML in 13%, cumulative risk with 8yrs G-CSF
- cure with BMT
Primary Myeloid Granules
Non-specific, azurophilic
- appear “late” in myeloblasts and promyelocytes, often remain visible in myelocytes
- coated with phospholipid membrane
- contain hydrolytic enzymes (MPO, lysozyme, acid phosphatase)
- visible in mature neutrophils as “toxic granulation”
Secondary Myeloid Granules
Specific
- appear in myelocytes
- like primary granules, secondary lysosomal granules contain numerous cytolytic enzymes (leukocyte alkaline phosphatase)
Cause of reactive monocytosis
*In reactive monocytosis, immature forms (ie monoblasts and promonocytes) are not seen*
- Infection (TB, listeria, syphilis, some protozoal/rickettsial infections)
- Neoplasms (lymphoma, carcinoma, myeloma)
- Miscellaneous (post-splenectomy, HA, ITP)
- Inflammatory d/o (IBD, collagen vascular dz, sarcoid, celiac)
- Chronic neutropenia (during recovery phase from acquired or cyclic neutropenia)
Gelatinase Myeloid Granules
- third type of granule subset
- recently identified in band and segmented neutrophils
Myelokathexis
- congenital d/o characterized by severe neutropenia
- chacteristic findings include degenerative changes and hypersegmentation of mature neutrophils and hyperplasia of BM myeloid cells (PMNS can have bisegmented nuclei connected by long filaments leading to bizarre ‘eyeglasses’ forms)
- recurrent bacterial infections (2/2 neutropenia AND depressed PMN fucntional activity)
- patholphysiology due to prolonged retention of PMNs in BM compartment (kathexis=retention)
- partially corrected by G-CSF
WHIM sydrome
- warts, hypogammaglobulinemia, infections, myelokathexis syndrome
- rare congenital immunodeficiency characterized by susceptibility to HPV-induced warts and carcinoma, neutropenia, B-cell lymphopenia and hypogammglobulinemia related infections, and BM myelokathexis
- caused by mutations in gene coding for chemokine receptor CXCR4, leading to mutant protein that causes abnormal apoptosis and migratory fx of leukocytes
Absolute Neutropenia
- less than 2.5 x 109/L in infants
- less than 1.5 x 109/L in kids and adults
- “severe” is less than 0.5 x 109/L
Causes of Neutropenia: Neonates
- infection
- maternal hypertension or drugs
- maternal ab production
- constitutional d/o (cyclic neutropenia, Kostmann syndrome, Chediak-Higashi, Schwachman-Diamond, myelokathexis, Fanconi anemia, dyskeratosis congenita)
Causes of Neutropenia: Infants/Children
- infection
- autoimmune neutropenia (BM shows macrophages with ingested PMNs)
- myelopthisic disease
- Idiosyncratic drug reactions
- 2/2 autoimmune neutropenia in collagen vascular d/o
- immunodeficiency
- myeloablative tx
Causes of Neutropenia: Adults
- infection
- idiosyncratic drug rxn
- myelophthisic dz
- myeloablative tx
- secondary autoimmune neutropenia in collagen vascular d/o
- autoimmune d/o
- aplastic anemia
- immunodeficiency
- hypersplenism (due to sequestration)
- megloblastic anemia
- Felty syndrome (triad: RA, splenomegaly, neutropenia)
Felty Syndrome
- RA
- splenomegaly
- neutropenia (risk for infections)
- affects women, 50-70 yo
Pearson Syndrome: pathogenesis
- rare, often fatal d/o of infancty
- due to large deletion of mitochondrial DNA (maternally inherited)
- deletions of certain components of electron transport chain cause defect in cellular oxidative metabolism; also impaired traslation of mRNA
- diagnose by detection DNA deletion via Southern blot
Pearson Syndrome: clinical
- severe, transfusion dependent macrocytic anemia begins early in infancy
- ring sideroblasts and vacuolated marrow precursors are variably present
- variable degree of neutropenia/thrombocytopenia
- lactic acidosis
- exocrine pancreatic insufficiency and often renal dysfunction
- progressive dz
- pts who survive infancy→ Kearns-Sayre syndrome:
- progressive external ophthalmoplegia
- weakness of skeletal muscles
- multisystemic d/o
Myelocytes
cell with visible Golgi likely to be myelocyte than promyelocyte
Lymphoblasts
scanty agranular cytoplasm, medium sized nucleus wtih delicate, evenly distributed chromatin, and 1 or more inconspicuous nucleoli
Myeloid blasts
larger cells with relatively abundant cytoplasm and prominent large nucleoli
Platelet estimate
on 100x, number of plts x 13,000
Dyserythropoiesis
Cells usually larger tahn normoblastic counterparts
- Megaloblastic type:
- N:C dyssynchrony, can be seen with hydrea therapy - Megaloblastoid type:
- more subtle, most prominent feature is abnormal chromatin pattern that is more condensed and clumped, with more prominent parachromatin spaces, also mild N:C dyssynchrony
N:C dyssynchrony in megas
monolobated nucleus with pink cytoplasm
Hematogones
- represent pre-pre-B cells and pre-B cells and are not seen in blood
- seen post chemo (recovering BM) and in children
- cells have condensed nucledar chromatin and very scant cytoplasm
- similar to B lymphoblasts on flow (positive for CD10, CD19, CD34, TdT; negative for surface light chain)
- on BM biopsy, small clusters of 5-6 cells
Megas in ITP and TTP
-small with immature (blue) cytoplasm and no granules
Megas in AML, CML, and MDS
small with mature (pink) cytoplasm
Mast Cells
dense, dark purple granules cover the cell and obscure the cytoplasmic edge of cell
Osteoclasts
- large multinucleated cells with multiple oval nuclei
- in contrast to megas, the nuclei are completely separate from one another
Hemophagocytosis
- associated with ertain infections (EBV), autoimmune dz, some malignancies (T cell lymphomas)
- internalized cells are visibly degenerated, in contrast to emperipolesis in which internalized cells are intact
Monocytosis
- monos are the first cell type to recover following BM insults (chemotherapy, aplastic anemia)
- other causes of monocytosis include chronic infection (listeria), collagen vascular diseases, and CMML
Sea Blue Histiocytes
- seen in MPNs (especially CML)
- storage diseases (Niemann-Pick dz, Fabry dz)
- occasionally seen in normal marrows
Core Biopsy: Myeloid
- maturation occurs predominantly along bony trabeculae
- Day 14: marrow should be 0% cellular (may have some plasma cells); if greater than 5% by morphology (not flow), then clinician will try to reinduce remission
- order of BM recovery after transplant: erythroid, megas, myeloid
- avg of 1-3 megas/50x field is normal
Core Biopsy: B Cell Aggregates
interstitial: more likely B9 or CLL
paratrabecular: follicular lymphoma
diffuse B cells: hairy cell
Naked Megas
may be seen in HIV infection
PAS to differentiate myeloid from erythroid
- clear cytoplasm in erythroid
- pink cytoplasm in myeloid and plasma cells
Centroblasts
-larger, blast like B cells with vesicular nuclei with fine chromatin and 2-4 membrane bound nucleoli (dark zone within germinal centers)
Immunoblasts
-cells with large central nucleoli, appreciable amount of basophilic cytoplasm, CD30+, CD20+, BCL2+
Paraimmunoblasts
-same as prolymphocytes (pale pseudofollicles in CLL/SLL)
Centrocytes
-small B cells with irregular/cleaved nuclear contours and scant cytoplasm (light zone within germinal centers)
FDCs
-large, round, vesicular nuceli with open chromatin, one central nucleolus, often occur in pairs (like R-S cells)
Follicular Pattern in LN
- follicular hyperplasia
- follicular lymphoma
- HIV
- RA and Sjogren’s syndrome lymphadenitis
- syphilis
- Castleman’s dz
- PTGC
RA and Sjogren’s syndrome lymphadenitis
-reactive follicular hyperplasia with interfollicular plasmacytosis
Syphilis in LN
-similar to RA lymphadenitis, but also showing capsular and trabecular thickening with infiltration by plasma cells
Castleman’s Dz
- usually affects mediastinal nodes
- plasma cell variant associated with increased IL-6
Interfollicular Pattern
- viral infection (EBV-infectious mono, CMV, postvaccinial lymphadenitis)
- hypersensitivity reaction (dilantin)
- Kimura’s dz:
- young asian men, cervical lymphadenopathy
- florid follicular hyperplasia with interfollicular immunoblasts, increased vascularity, increased eos in paracortex, sinusoids, perinodal soft tissue, and within follicles
Sinus Pattern
- sinus histiocytosis
- Rosai-Dorfman disease
- Whipple dz (M:F = 10:1)
- dermatopathic change
- hemophagocytic syndrome
- vascular transformation of LN sinuses (abdominal nodes)
Granulomatous Lymphadenitis
-suppurative granulomas:
- cat scratch (stellate abscess)
- lymphogranuloma venereum
- tularemia
-necrotizing granulomas:
- TB/atypical mycobacteria
- Brucellosis
- fungal infection
- Yersinia
- non-necrotizing granulomas:
- sarcoid
- granulomas impinging on germinal centers:
- toxoplasmosis (triad: reactive follicular hyperplasia, epithelioid histiocytes encroaching germinal centers, monocytoid B cell hyperplasia)
- ill defined sheets of histiocytes with karyorrhexis:
- Kikuchi-Fujimoto
Diffuse LN Pattern
- EBV (may be interfollicular; node effacted by proliferation of T cells, plasma cells, immunoblasts that may be very atypical and mimic HL or large cell lymphoma)
- CMV (see typical inclusions)
- HSV (focal necrosis is characteristic, typical inclusions may be seen)
- measles (Warthin-Finkeldy giant cells)
- post-vaccinia
Megakaryocytic markers
-CD41, CD42b, CD61
Internal Control For CD30
- plasma cells
- immunoblasts
CD43
- T cell marker
- surrogate marker for clonality in B cells
CD45RO
T cell marker
BCL2 internal control
immunoblasts
Cellulose Acetate Gel
- initial method used; pH 8.6
- all Hb variants at this pH are negatively charged and migrate to anode
- starting at anode: A Fat Santa Claus
- band in S region: Hb S (solubility test +), Hb G, Hb D, and Hb Lepore (solubility test -)
- band in A2 region: C, E, O-Arab
Citrate Agar Gel
- pH 6.2
- starting at cathode: Fat Ass Santa Claus
- useful to separate bands at S and A2 on cellulose agar
- variants that co-migrate with Hb A: A2, D, G, E, O-Arab
Hb AC
-very mild microcytosis (if less than 75, consider concomitant thal), scattered target cells, pts asymptomatic
Hb CC
-mild hemolytic anemia, mild splenomegaly (pts mostly asymptomatic), mild microcytosis, numerous target cells, hexogonal or rod shaped crystals in RBCs in splenectomized pts
Hb SC
- sickling dz milder than SS
- true sickle cells and Hb C crystalss are rare; there are usually irregular shaped cells that contain misshapen crystals
Hb C Harlem
-solubility test is positive
Hb AE
-mild microcytosis, scattered target cells, no anemia
Hb EE
-MCV around 67, frequent target cells, mild or no anemia
Hb E
- SE Asians
- target cells, thalassemic indices
- mutation causing beta globin mRNA to be underexpressed and is functionally more like beta thal
- migrates with C, A2, and O on alkaline (AKA cellulose acetate)
- elutes with A2 on HPLC
Hb D
- clinically insignificant
- AKA HbD-Los Angeles or HbD-Punjab
- combo of HbD and HbS produces severe sickling disorder
HbG
- most common alpha globin gene mutation seen in US (mainly AA)
- clinically normal
HbO-Arab
- heterozygotes: no hematologic abornmalities
- homozygotes: mild anemia
- with HbS: produces severe sickling d/o similar to SS
Hb Lepore
- fusion of delta and beta genes during meiotic crossover (3 different forms exist)
- stable hemoglobins that are underproduced relative to normal beta chains, thus Hb Lepore gene functions as a thalassemic allele
- Mediterranean pts
- a minor band in the HbS position (10-15%) on alkaline (celluose acetate), negative solubility test, coelution with HbA2 on HPLC is virtually diagnostic of Lepore
- mild microcytosis and occasional target cells
Hb Constant Spring
- produces thalassemic indices
- results from mutation of the alpha stop codon, producing an abnormally long transcript that is unstable (and functionally more like thalassemia than hemoglobinopathy)
HbM
- rare; unable to maintain heme iron in reduced state or to reduce 02
- results in hereditary methemoglobinemia
Hereditary Persistance of Fetal Hemoglobin
elavated HbF into adulthood (20-40%)
deletional mutations:
- involve large deletions spanning the gamma and beta genes on chromosome 11
- MCV is normal-sl decreased, no anemia
- pancellular pattern on K-B acid elution test (all RBCs contain similar amount of HbF) or by flow cytometry using an anti-HbF ab
non-deletional mutations:
- typically due to point mutations in the promoter region of one of the gamma globin genes, resulting in persistent epression of HbF (the gamma globin switch is not turned off in adulthood)
- pancellular and heterocellular pattern on K-B test or by flow
- measurement of individual gamma chains usually show abnormal gamma G to gamma A ratio
