Hemoglobin Flashcards
Normal PO2 level in arterial blood
80-100 mmHg
Normal PO2 level in venous blood:
30-50 mmHg
Shift in the curve due to changes in pH; demonstrates relationship of blood pH and Hgb-O2 affinity
Facilitates the ability of hemoglobin to exchange oxygen and CO2
BOHR EFFECT
Hgb-O2 binding promotes release of CO
HALDANE EFFECT
Increased Hgb-O2 affinity, Decreased dissociation, decreased oxygen, increase release
SHIFT TO THE LEFT:
Decreased Hgb-O2 affinity, Increased dissociation, increase oxygen, decrease release
SHIFT TO THE RIGHT
True or false
Hgb-O2 affinity is inversely proportional with dissociation and oxygen release
True
High affinity for oxygen than HgbA1 due to weakened ability to bind 2,3-DPG.
High affinity allows more effective oxygen withdrawal from maternal circulation.
Hgb F
• REVERSIBLE oxidation of ferrous iron to the ferric state (Fe3+)
• Cannot bind and transport O2
Methemoglobin (MetHb or Hi)
can be acquired or hereditary
Aka toxic methemoglobinemia; occurs in normal individuals after exposure to an exogenous oxidant, such as nitrites, primaquine, dapsone, or benzocaine
METHEMOGLOBINEMIA
Toxic level of methemoglobin
<25%
Asymptomatic
Toxic levl of methemoglobin 30%
Cyanosis
bluish discoloration of skin due to decreased O2 in the tissues) and symptoms of hypoxia
Cyanosis
Toxic level of methemoglobin is >50%
Coma or death
amount of oxygen needed to saturate 50% of hemoglobin.
P50
Shift to the left mmHg?
27 mmHg
Normal mmHg of hemoglobin
26.52 to 27 mmHg
Autosomal recessive
<50% of total hemoglobin
Methemoglobin reductase/Cytochrome B5 reductase deficiency
Hydrogen sulfide derivative of hemoglobin; addition of a sulfur atom to the pyrrole ring of heme
Sulfhemoglobin (HgbS
Color of blood is Chocolate brown
Methemoglobin
Color of blood is Greenish pigment/ hemochrome * MAUVE-LAVANDER
Sulfhemoglobin (HgbS
IRREVERISBLE CHANGE in denatured/precipitated hemoglobin
Heinz bodies
colorless, tasteless gas, termed SILENT KILLER
Carbon monoxide
COLOR OF BLOOD: CHERRY RED
Carboxyhemoglobin (HgbCO)
increased affinity; prevent O2 release to the tissues
Shift to left of Carboxyhemoglobin
Absorption peak of hgbS
630 nm
Absorption peak of HgbCO
540 nm
TOXIC LEVELS of Carboxyhemoglobin (HgbCO)
20-30%
headache, dizziness, disorientation
Toxic levels of HgbCO >40%
coma, seizure, hypotension, cardiac arrhythmias, pulmonary edema, and death
controversial; it is primarily used to prevent neurologic and cognitive impairment after acute carbon monoxide exposure in patients whose level exceeds 25%
hyperbaric oxygen therapy
Reference method/Gold standard for hemoglobin determination because:
CYANMETHEMOGLOBIN METHOD/HEMIGLOBINCYANIDE (HiCN) METHOD
only standard used in hematology
HiCN standard are readily available
Absorption peak (wavelength): of CYANMETHEMOGLOBIN
540 nm
Principle of CYANMETHEMOGLOBIN
Colorimetric/Indirect/Spectrophotometric
Reagent used in CYANMETHEMOGLOBIN
Modified Drabkin’s reagent (contains anhydrous Dihy
light sensitive; contains cyanide which is highly toxic
DRABKIN’S REAGENT
DRABKIN’S REAGENT contains?
contains sodium bicarbonate
uses sodium lauryl sulfate (SLS) to convert Hgb to SLS-methemoglobin.
Automated instruments
HemoCue; converts hgb to azidehemoglobin (read at 570 nm and 880 nm)
POCT device
Converts methemoglobin to cyanmethemoglobin
Potassium cyanide
Converts hemoglobin (Fe2+) to methemoglobin (Fe3+)
Potassium ferricyanide
Improves RBC lysis and decreases turbidity from protein precipitation
Non-ionic surfactants/Detergent
Hastens conversion of Hgb to HiCN
Dihydrogen potassium phosphate (3 minutes) Sodium
bicarbonate (10 minutes
Hemoglobin molecules assume a negative charge and migrate toward the anode (positive pole
CELLULOSE ACETATE
CELLULOSE ACETATE alkaline pH?
8.4 to 8.6
Fastest to migrate to the anode in cellulose lactate
Hgb H
Slowest to migrate to the anode in cellulose lactate
HgbC
Migrates with C: in cellulose lactate
Hgb E and O
Migrates with S: in cellulose lactate
Hgb D and Hgb G
Hemoglobins assume a negative charge and migrate toward the anode, whereas others are positively charged and migrate toward the cathode
CITRATE AGAR
CITRATE AGAR pH
(Acid pH 6.0-6.2
Quantification of fetal hemoglobin; used to quantitate the number of fetal Rh-positive cells because of fetomaternal hemorrhage (iFMH
KLEIHAUER-BETKE TEST (ACID ELUTION TEST
Maternal blood smear is treated with _________ and then stained with counterstain what stain?
Citric acid phosphate buffer (pH 3.2)
Shepard’s stain
Acid hematoxylin and Erythrosine B as counterstain is called
Shepar’ds stain
Pink (has fetal Hgb which is resistant to acid elution)
Fetal cells (w/ Hgb F)
Ghost cells (appear as pale pink and is susceptible to acid elution)
Maternal cells (w/ Hgb A):
Adding sodium metabisulfite, a reducing substance, to blood enhances deoxygenation of Hgb and sickling of Hgb S
➢ SODIUM METABISULFTITE TEST
➢ SODIUM METABISULFTITE TEST • Positive result: formation of?
Sickle cells holly leaf appearance
Other positive result for sodium metabisulfate
Rare sickling hemoglobins (Hbs S Travis, C Harlem), Hgb I, Hgb Bart’s
False negative results is sodium metabisulfite test
Hgb S concentration is less than 10% (as in very young infants) or if deoxygenation is inadequate (e.g., deterioration of reagent)
Most common screening test for Hgb S
DITHIONITE TUBE TEST/SICKLE SOLUBILITY TEST/HGB SOLUBILITY TEST
Positive result to DITHIONITE TUBE TEST/SICKLE SOLUBILITY TEST/HGB SOLUBILITY TEST
Turbidity
Turbud Deoxygenated polymerized Hgb S)
Negative result to DITHIONITE TUBE TEST/SICKLE SOLUBILITY TEST/HGB SOLUBILITY
Negative: Clear (non-sickling hemoglobin)
converts ferrous to ferric iron; ferric iron is unable to bind oxygen, converting hemoglobin to the deoxygenated form
Dithionite
dissolves membrane lipids, causing release of hemoglobin from RBCs
Saponin
Central pallor/pallor area occupies 1/3 of the cell Normal MCHC: 32-36 g/dL
Normochromic RBC
Central pallor/pallor area is >1/3 of cell Decreased MCHC: <32 g/dL of hypochromic rbc
Target cells and elliptocytes
Example of Hypochromic RBC
Target cells, Elliptocytes
Central pallor/pallor area is <1/3 of the cell Increased MCHC: >36 g/dL
Hyperchromic RBC (De facto)
Example of Hyperchromic RBC (De facto)
Spherocytes and stomaticytes
RBC with a thin rim of hemoglobin and a large clear center Seen in Iron deficiency anemia
Anulocyte/Pessary cell/Ghost cell
variation in cell shape
Abnormal shape rbc
POIKILOCYTOSIS
RBC Membrane abnormalities -
Intrinsic defect
Trauma/Physical damage
Extrinsic defect
Developmental macrocytosis
➢ RBC Membrane abnormalities - intrinsic defect
➢ Abnormal hemoglobin content
➢ Trauma/Physical damage
POIKILOCYTOSIS
common cells in hemolytic anemias
SPHEROCYTES
Thinner variant of a Target cell
Leptocyte
oval macrocytes
Megablolastic anemia
Large RBC, mostly oval (MCV= >100 fL
Macrocytic RBCs
Small, round, RBC with no central pallor (increased MCHC)
Hyperchromic red cells
Decreased surface area to volume ratio (increased OFT
Spherocytes/Bronze cells
Hereditary spherocytosis Pre & post splenectomy HDN WAIHA, MAHA Severe burns Jaundice
Spherocytes/Bronze cells
Megaloblastic anemia (Vit B12 and B9 deficiency) Chronic liver disease Myelodysplastic syndromes
Bm failure
Macrocytic RBCs
Hemoglobinopathies Thalassemia
Hepatic disease with or without jaundice
Target cells/Codocytes/ Mexican Hat cell
Central area of hemoglobin surrounded by colorless ring and a peripheral ring of hemoglobin (Maldistribution of hemoglobin
Target cell
Codocytes
Mexican hat cell
Seen in IDA
Pencil shape rbc
Gerbich null (Ge: -2,-3,-4)
Leach phenotype
Have a cigar, elliptical, pencil, egg shape Hemoglobin are concentrated at the two ends of the cell with normal pallor area
Elliptocyte
Hereditary elliptocytosis Iron deficiency anemia Thalassemia major Sickle cell anemia Pernicious anemia Myelofibrosis
Elliptocyte
Liver disease Renal insufficiency (uremia) Pyruvate kinase deficiency
Echinocytes/ Crenated RBC/ Sea urchin cell / Burr cells
not evenly distributed blunt serrated edges/short projections
Burr cell
Evenly distributed blunt serrated edges/short projections
Echinocytes
Due to plasma abnormalities, osmotic changes; decreased ATP
In vivo
❖ prolonged standing of blood film with AC, moist slide, and stored blood
May be an artifact
In vitro
Acute, severe hemolytic anemia
G6PD deficiency
Hereditary lipoprotein deficiency
Pyknocytes (Turgeon)
Are distorted, contracted erythrocytes that are similar to burr cells
Pyknocytes (Turgeon)
Contains uneven spaced, pointed projections without central pallor
Acanthocytes/Spur cells/Thorn cells
Abetalipoproteinemia/ Bassen-Kornzweig syndrome McLeod syndrome
Acanthocytes
Spur cells
Thorn cells
absence of Kx gene,
Due to the changes in the ratio of plasma lipids (lecithin and sphingomyelin) absence of Kell antigens
McLeod syndrome
Due to the changes in the ratio of plasma lipids ________
_________
lecithin and
sphingomyelin)
Characterized by elongated/slit/mouth like pallor area instead of circular pallor
Stomatocytes
Piezo type” ➢ Increased membrane permeability to
potassium leading to loss of water from cell water efflux
Dehydrated stomatocytosis
increased membrane permeability to sodium and potassium (water influx) __________
Rh null disease
➢ increased membrane permeability to
sodium and potassium (water influx)
Rh null disease
Overhydrated stomatocytosis
Rh Null disease/Rh deficiency syndrome
Stomatocytes
Can appear as an artifact
Can be seen with puddled hemoglobin at the periphery of cells with spicules
Stomatocytes
Fragmented RBC (about half the size of a normal RBC
Schistocytes/ Schizocyte
Helmet/Hornlike
Kerocyte
Triangular, resemble
“pinch-bottle”, RBC with 2 central pallors
Knizocyte
➢ Variety of small, irregular shapes
Blister cells
Causes of fragmentation:
❖ Altered vessel walls
❖ Presence of fibrin
❖ Prosthetic heart valves
❖ Renal transplant rejections
Schistocytes
Moschcowitz syndrome, UpshawSchülman syndrome
Microangiopathic hemolytic anemia (MAHA)
TTP
HUS
DIC
Schistocytes
Disk shaped cells with smaller volume Due to thermal damage to cell membrane protein spectrin, no pallor area
Pyropoikilocytes/ Microspherocytes
Severe burns Hereditary pyropoikilocytosis
Pyropoikilocytes/ Microspherocytes
Laboratory results:
❖ 2-3um in
diameter
❖ MCV = <60fL
Pyropoikilocytes/ Microspherocytes
Tear drop, pear drop shaped with blunt pointed projection
Due to squeezing of red cells through small openings or splenic sinuses and remains behind
Dacryocytes
Myeloid metaplasia Primary myelofibrosis
MMM
Myelopthisic anemia
Pernicious anemia
Beta thalassemia Tuberculosis
Heinz body formation
Dactocytes
Halfmoon cell, half crescent cell Large, pale pink staining ghost of red cell
Semilunar bodies
Malaria
Causes overt hemolysis
Semilunar bodies
Long, rod, crescent shaped, with thin and elongated with pointed ends
Polymerization of deoxygenated hemoglobin (Hgb S)
Sickle cells/ Drepanocytes/ Menisocytes
Decreased blood pH
Influx of sodium ions
Increased intracellular calcium
Polymerization of deoxygenated hemoglobin (Hgb S)
Can be due to amino acid substitution resulting to cell membrane alterations (glutamic acid to valine ; 6 th A.A. of the beta chain
Hemoglobin S
Homogeneous: hexagonal with blunt ends
Bar of gold/Clam shell appearance
Hemoglobin CC crystals
Hgb CC disease
Hemoglobin cc crystals
Confirmed with hemoglobin electrophoresis
Hemoglobin CC crystals
Dark-hued crystals of condensed hemoglobin
Hemoglobin SC crystals
Crystals appear straight with parallel sides and one blunt, point, protruding end, fingerlike (Washington’s monument appearance
Hemoglobin SC crystals
Hgb SC disease
Hemoglobin SC crystals
Small, 1-2 um in size, nuclear fragments of DNA Normally pitted by splenic macrophages and are not seen in normal RBCs
Howell-Jolly bodies
Reddish blue; Dark blue-purple with Wright’s stain (same with supravital
Howell-Jolly bodies
Feulgen positive reaction
Howell-Jolly bodies
Congenital absence of spleen
Splenic atrophy
Sickle cell anemia
Howell jolly bodies
Multiple, tiny, fine or coarse rRNA aggregates/precipitates
rRNA inclusions aggregates in drying & staining
Basophilic stippling/Punctuate basophilia
Hemoglobin appears homogeneous;
“Blueberry gel appearance”
Basophilic stippling/Punctuate basophilia
Dark-blue to purple with Wright’s stain (same with supravital
Basophilic stipplings
Thalassemia Lead Toxicity (plumbism)
Arsenic toxicity
Pyrimidine-5’-nucleotidase deficiency
Basophilic stippling
Aggregates of mitochondria, ribosomes, and IRON PARTICLES (unused iron)
Appears in the periphery of the erythrocytes
Pappenheimer bodies/ Siderotic granules
Stains with Perl’s Prussian blue (Rous test)
Pappenheimer bodies/ Siderotic granules
Pappenheimer bodies
Wright’s stains
Siderotic granules
Prussian blue stain
Sideroblastic anemia
Hemoglobinopathies
Thalassemias
Pappenheimer bodies/ Siderotic granules
Wright’s stain: Bluish tinge; also called
polychromasia
Dark blue RNA remnants in the cytoplasm
reticulocytes
Young cells with no nucleus but contains RNA remnants
Diffuse basophilia/ Diffusely basophilic RBC
Hemolytic anemia After treatment of iron, Vitamin B12 or folate deficiency
Diffuse basophilia/ Diffusely basophilic RBC
Thin ring like, circular, figure of eight, incomplete rings
Cabot rings
Remnants of microtubules from the mitotic spindle
Associated with Howell jolly bodies in the same RBC
Cabot ring
Reddish-violet (Wright’s stain)
Cabot ring
Megaloblastic anemia
Myelodysplastic syndromes
Cabot ring
Precipitated/denatured globin attached to the RBC membrane
Round, 0.2 to 2.0 um, seen with a supratival stains
Heinz bodies
RBC with pitted Heinz bodies
Bite cell / degmacyte
NOT VISIBLE ON WRIGHT’S STAIN Dark-blue purple (Supravital stain
Heinz bodies
Heinz body preparation
Crystal violet
Fava beans (Favisim
G-6-PD deficiency
Presence of unstable hemoglobins
Exposure to oxidizing agents
Oxidant drugs (Anti-malarial drugs)
Heinz body
Small, precipitated beta-globin chains of hemoglobin (4β)
Pitted golf ball appearance of RBC
Results to unstable, easily oxidized and easily precipitated hemoglobi
Hgb H
NOT VISIBLE ON WRIGHT’S STAIN dark blue or greenish granules (Supratival stain – BCB)
Hgb H
Hgb H disease
Alpha thalassemia
Granulo filamentous pattern in hgb H
Reticulocyte
Hgh H inclusions
Single body
morphologic classification of anemia
RBC indices
assess RBC production on response to anemia
Reticulocyte count –
morphological abnormalities
Peripheral blood film examination
– for unexplained anemia
Bone marrow examination
for microcytic, hypochromic anemias
Iron studies
hemolytic anemia
Urinalysis
occult blood and blood parasites
Fecalysis
Hemolytic anemia
Chemistry test
differentiate autoimmune anemias for other hemolytic anemia
DAT
are characterized by an MCV greater than 100 fL
Macrocytic anemia
characterized by an MCV of less than 80 fL
Microcytic anemia
characterized by an MCV in the range of 80 to 100 fL
Normocytic anemias
Ineffective erythropoiesis -
RPI <2.0
Decreased or ineffective RBC production (decreased reticulocyte count) –
w/o BM compensation
Excessive RBC loss (increased reticulocyte count) – w
/ BM compensation
Effective erythropoiesis -
RPI - >3.0
reacts with freed iron forming a colored complex that can be detected spectrophotometrically
Ferrozine
Indicator of available iron for transport Measures iron bounded to transferrin
Specimen – fasting specimen and collected early in the morning (highest levels)
Serum Iron (SI)
Indirect indicator of iron stores Measures total number of available transferrin sites for iron binding
Total Iron Binding Capacity (TIBC)
Serum iron (SI) represents the number of transferrin sites bound with iron
TIBC represents the total number of transferrin sites for iron binding
% Transferrin Saturation
Indicator of iron storage status
Quantitative assessment of body iron stores
Serum ferritin
Uses acidic potassium ferrocyanide Prussian blue stain of the bone marrow is considered the
GOLD STANDARD for assessment of body iron stores
Prussian Blue staining of bone marrow
readily seen as dark blue granules or precipitate
Ferric iron
stains readily, distinct blue granules
Hemosiderin
Indicator of functional iron available in cells Major advantage – IDA and ACD differentiation
Soluble transferrin receptor (sTfR) level/ Serum transferrin receptor
- associated with increased serum levels of
IDA
Ida- associated with increased serum level of tfrs because of increased membrane TfR1
Stfr
Indicator of functional iron available in cells
Ferritin index/ stfr
Increased binding of zinc to protoporphyrin IX when iron is not incorporated to heme
Free Erythrocyte Protoporphyrin (FEP)/ Zinc protoporphyrin level (ZPP)
BM iron store : decreased
Hgb: normal
SI: Normal
Ferritin: Decreased
TIBC: normal
Storage Iron depletion (Pre-Latent iron deficiency
BM stores: Absent
Hgb: normal
SI: decreased
Ferritin: Decreased
TIBC: Increase
Transport iron depletion (Latent iron deficiency)
BM iron stores: absent
Hgb: decreased
Serum iron: decreased
Ferritin: decrease
Tibc: increased
Iron deficiency anemia/ Frank’s anemia
➢ Cracks at the corners of the mouth
angular cheilosis
Spooning or clubbing of the fingernails
Koilonychias
severe iron deficiency, neurologic problems
➢ Pica/Pica syndrome
• most common in pica syndrome
craving for ice
Craving for ice is known as
Pagophagia
Aka Patterson Kelly / Plummer
Vinson syndrome
Impaired ferrokinetics (most significant cause)
Anemia of a chronic disease or inflammation
Acute phase reactant of Anemia of a chronic disease
Hepcidin
Other acute phase reactants (APR):
Ferritin, and lactoferrin
inhibits iron release of ferroportin from enterocytes → decreased serum iron
Increased hepcidin
promotes iron release of ferroportin from enterocyte increased serum iron
Decreased hepcidin
Caused by blocks in the protoporphyrin pathway resulting in defective hemoglobin synthesis and iron over
Sideroblastic anemia
Excess iron accumulates in the mitochondrial region of the sideroblasts (metarubricyte) in the bone marrow and siderocytes in the blood and encircles the nucleus
Sideroblast anemia
aka: PLUMBISM
Lead poisoning
Gum lead line that forms from blue/black deposits of lead sulfate
Lead poisoning
Hallmark of the sideroblastic anemias
➢ Contains at least 5 iron granules per cell, and these iron containing mitochondria must circle at least 1/3 of the nucleus
➢ 10-40% of nucleated cells in the bone marrow
Ringed sideroblast
❖ HEREDITARY: ➢ Common in males ➢ X-linked and autosomal - d-ALA synthase deficiency
Sideroblast anemia
❖ Are named according to the chain (alpha or beta) with reduced or absent synthesis
Thalassemias
Mild anemia; sufficient alpha and beta chains produced to make normal hemoglobins A, A2 , and F, but may be in abnormal amounts
Thalassemia minor/trait
Severe anemia; either no alpha or no beta chains produced
Thalassemia major
This underproduction of beta chains contributes to a decrease in the total erythrocyte hemoglobin production, ineffective erythropoiesis, and a chronic hemolytic process
❖ Unpaired, excess alpha chains precipitate in developing erythroid precursors forming Heinz bodies
Beta thalassemia
(COOLEY’S ANEMIA)
➢ Characterized by a severe anemia that requires regular transfusion
➢ Markedly decreased rate of synthesis or absence of both beta chains results in an excess of alpha chains; no
Hgb A can be produced; compensate with up to 90% Hgb
Beta thalassemia major
Decreased rate of synthesis of one beta chains
➢ Hemoglobin level – 11-15 g/dL
➢ Hemoglobin electrophoresis: Hgb A 1 is slightly decreased, but Hgb A 2 is slightly increased to compensate
BETA-THALASSEMIA MINOR/TRAIT ➢
Hereditary recessive microcytic anemia, which does not respond to oral iron therapy ➢ Mutation in the TMPRSS6 gene – encodes for matriptase-2 (MT-2) ✓ MT-2 – involved in downregulation of hepcidin
IRON-REFRACTORY IDA (IRIDA)
