HEMA311 LEC

BSMLS 3-YA-1
MACROCYTIC AND NORMOCYTIC ANEMIAS
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MACROCYTIC ANEMIA

❖ Characterized by an MCV of >100fL with large RBCs (>8um)

❖ Arise from conditions that result in megaloblastic or non-

megaloblastic red cell development in the bone marrow

❖ ASSOCIATED CONDITIONS:
➢ MEGALOBLASTIC ANEMIA → impaired DNA synthesis
leading to abnormal nuclear maturation
✓ Vitamin B12 (Cobalamin) deficiency
✓ Folate deficiency
✓ Myelodysplasia
✓ Erythroleukemia
➢ NON-MEGALOBLASTIC ANEMIA → due to disruption of the
cholesterol-to-phospholipid ratio
✓ Aplastic anemia
✓ Chronic liver disease
✓ Alcoholism
MEGALOBLASTIC ANEMIA
VITAMIN B12 DEFICIENCY
❖ Defective DNA synthesis causes abnormal nuclear
maturation; RNA synthesis is normal, so the cytoplasm is not ❖INADEQUATE INTAKE
affected (megaloblastic: RBC precursors ; macrocytic: mature ➢ Rare in strict vegetarians who do not eat meat, eggs, and
RBC) dairy products
❖ Associated with Vitamin B12 and folate deficiency (leads to ➢ Best sources are liver, dairy products, fish, shellfish, and
impaired thymidine production) eggs
➢ Folate deficiency → preventing methylation of dUMP ❖INCREASED DEMAND
(deoxyuridine monophosphate) ➢Pregnancy, lactation, and periods of growth
➢ Vitamin B12 deficiency → preventing production of THF from 5- ❖ IMPAIRED ABSORPTION
methyl THF (5-methyltetrahydrofolate) ➢Failure to separate vitamin B12 from food proteins ➢Failure
to separate vitamin B12 from haptocorrin
PATHOPHYSIOLOGY: ➢ Malabsorption
➢ Thymidine deficiency leads to the production of non- ➢Inherited disorders of vitamin B12 absorption and transport
functional and impaired DNA replication ➢Competition for vitamin B12
➢ Cell division is halted which leads to lysis or apoptosis of ➢Lack of intrinsic factor (IF)
many erythroid precursors (INEFFECTIVE E.)
➢ Red blood cells have an abnormal nuclear maturation and IMPAIRED ABSORPTION
imbalance between nuclear and cytoplasmic maturation; the
premitotic interval is prolonged; results in a large nucleus,
increased cytoplasmic RNA, and early synthesis of hemoglobin ❖ Failure to separate vitamin B12 from food proteins
(INEFFECTIVE ERYTHROPOIESIS) ➢ Known as food-cobalamin malabsorption is characterized by
➢ Also associated with impaired maturation of other myeloid hypochlorhydria and the resulting inability of the body to
cells ; ASSOCIATED WITH PANCYTOPENIA release vitamin B12 from food or intestinal transport proteins
✓ Leukopenia with hypersegmented neutrophils for subsequent binding to intrinsic factor
✓ Thrombocytopenia with giant platelets
❖ Failure to separate vitamin B12 from haptocorrin
➢ Due to chronic pancreatitic disease ; lack of gastric acidity or
FOLATE DEFICIENCY
lack of trypsin
❖ INADEQUATE INTAKE ➢ synthesized by microorganisms
❖ Malabsorption
and higher plants ; good sources are leafy green vegetables,
➢ Same conditions interfering with folate absorption, such as
dried beans, liver, beef, fortified breakfast cereals, and some
celiac disease, tropical sprue, and inflammatory bowel disease
fruits, especially oranges
❖ Inherited errors of vitamin B12 absorption and transport
❖ INCREASED DEMAND ➢ During pregnancy and lactation ;
➢ Imerslund-Gräsbeck syndrome and Transcobalamin
periods of growth
deficiency
❖ IMPAIRED ABSORPTION ➢ Deficiency in folate transporter
❖ Competition for vitamin B12
protein (PCFT) ➢ Intestinal diseases ✓ Sprue/Tropical sprue
➢ Fish tapeworm disease (Diphyllobothrium latum infections)
and Celiac disease (gluten-induced enteropathy) ❖ IMPAIRED
➢ Blind-loop syndrome - portions of the intestines that are
USE OF FOLATE ➢ Drugs
stenotic as a result of surgery or inflammation
❖ EXCESSIVE LOSS OF FOLATE ➢ occurs through the
kidneys ; dialysis patients
❖Lack of intrinsic factor (IF)
HEMA311 LEC
BSMLS 3-YA-1
MACROCYTIC AND NORMOCYTIC ANEMIAS
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
➢ Lack of intrinsic factor constitutes a significant cause of
impaired vitamin B12 absorption
➢ Associated with Pernicious anemia, Helicobacter pylori
infection, total or partial gastrectomy

❖PERNICIOUS ANEMIA
➢ is an autoimmune disorder characterized by impaired
absorption of vitamin B12 because of an intrinsic factor
deficiency
➢ Associated with Lymphocyte-mediated destruction of gastric
parietal cells and autoantibodies to parietal cells (anti-parietal
cell antibodies) ; also anti-IF antibodies
➢ PARIETAL CELLS → cells that secrete intrinsic factor

OTHER CAUSES OF MEGALOBLASTIC ANEMIA MEGALOBLASTIC ANEMIA

❖Myelodysplastic syndrome (MDS) ❖ CLINICAL FINDINGS (COMMON IN PERNICIOUS

❖Acute erythroid leukemia (AML) → FAB M6 ❖Congenital ANEMIA)
dyserythropoietic anemia (CDA) ➢Normoblasts in the bone ➢ Weakness, shortness of breath
marrow show multinuclearity, karyorrhexis causing ➢ Gastrointestinal symptoms
dyserythropoiesis (asynchrony of nuclear and cytoplasmic ➢ Paresthesias (numbness of the fingers of the extremities)
maturation: megaloblastic) ➢ Megaloblastic madness (Severe vitamin B12 def) → emotionally
➢Associated with refractory anemia and ineffective unstable
erythropoiesis ➢ ➢ Achlorhydia
3 types: Type I, Type II, Type III (based on cellular ❖ LABORATORY FINDINGS:
morphology) ➢ Complete blood count: Slight macrocytosis, giant platelets,
decreased hemoglobin (7-8 g/dL) and hematocrit,
❖TYPE I CDA PANCYTOPENIA, reticulocytopenia, increased MCV (>120 fL),
➢Associated with neonatal jaundice and splenomegaly increased MCH, normal MCHC, oval macrocytes,
➢LABORATORY FINDINGS: hypersegmented neutrophils, low reticulocyte count, teardrop
✓Basophilic stippling and cabot rings cells, fragmented RBC, microspherocytes, nucleated RBC,
✓ Mild macrocytosis, anisocytosis, poikilocytosis ✓Binucleated Howell-Jolly bodies, basophilic stippling, and cabot rings
cells, cells with incompletely separated or multi-lobulated nuclei ➢ Increased bilirubin and LDH: indication of hemolysis
✓Feulgen (+): presence of internuclear chromatin bridges ➢ Bone marrow examination → confirmatory test to identify the
joining two normoblasts megaloblastic appearance (characterized by a nuclear-cytoplasmic
asynchrony) of the developing erythroid precursors especially in the
❖ TYPE II CDA polychromatic normoblast
➢ Most common CDA; aka as HEMPAS (Hereditary
Erythroblast multi-nuclearity with positive acidified serum test) ❖ SPECIAL DIAGNOSTIC TESTS:
➢ presence of the HEMPAS antigen ; hepatosplenomegaly ➢ Schilling’s test → measures radioactive vitamin B12 in the urine
and jaundice ➢ Homocysteine and Methylmalonic acid (MMA) assays
➢ LABORATORY FINDINGS: ➢ Gastric analysis → to confirm achlorhydria in pernicious anemia
✓ Anisocytosis, poikilocytosis, basophilic stippling ➢ Serum gastrin → increased as a result to gastric achlorhydria
✓ Ham’s test positive (Ham’s acidified serum test) , sugar ➢ Antibody assays → antibodies to intrinsic factor and parietal cells
water/sucrose hemolysis test negative ➢ Holotranscobalamin assay → decreased in Vitamin B12 deficiency
✓ Paroxysmal Nocturnal Hemoglobinuria (PNH): Both positive ➢ Stool analysis for parasites → for D. latu

❖ TYPE III CDA

➢ Pronounced multinuclearity: up to 12 nuclei per cell
➢ Affects 30% of RBC precursors, gigantoblasts: (50 to 60 um
in diameter
HEMA311 LEC
BSMLS 3-YA-1
MACROCYTIC AND NORMOCYTIC ANEMIAS
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NORMOCYTIC ANEMIAS
❖ MCV is 80-100 fL (RBCs are 6-8 um)
❖ Characterized by normocytic, normochromic RBCs
❖ ASSOCIATED CONDITIONS:
➢ NORMAL OR DECREASED RETICULOCYTE COUNT:
✓ Aplastic anemia
✓ Myelophthisic anemia
✓ Anemia of renal disease
✓ Parvovirus B19 infections
✓ Anemia of chronic inflammation
➢ INCREASED RETICULOCYTE COUNT:
✓ HEMOLYTIC ANEMIAS
▪ INTRINSIC o RBC membrane, enzyme defects o
Hemoglobinopathies
▪ EXTRINSIC o Immune or antibody mediated o Non-immune RBC
injury (mechanical fragmentations) → Micro and Macro angiopathic
hemolytic anemia, Infectious agents, drugs, etc
APLASTIC ANEMIA ACQUIRED APLASTIC ANEMIA
❖ Associated with bone marrow failure ; is one of a group of ❖ Quantitative and qualitative deficiency of hematopoietic stem
disorders, known as hypoproliferative disorders, that are cells
characterized by reduced growth or production of blood cells, ❖ Classified into two major categories: idiopathic and
ALSO ASSOCIATED WITH MACROCYTIC RBCs secondary
➢ Idiopathic: No known cause
❖ BONE MARROW FAILURE: ➢ Secondary: with identified cause
➢ Destruction of hematopoietic stem cells as a result of injury ❖ CLINICAL FINDINGS:
by drugs, chemicals, radiation, viruses, or autoimmune ➢ Pallor, fatigue, and weakness
mechanisms ➢ Severe and prolonged anemia can result in serious
➢ Premature senescence and apoptosis of hematopoietic cardiovascular complications, including tachycardia,
stem cells as a result of genetic mutations hypotension, cardiac failure, and death
➢ Disruption of the bone marrow microenvironment that ➢ Symptoms of thrombocytopenia are also varied and include
supports hematopoiesis petechiae, bruising, epistaxis, mucosal bleeding, menorrhagia,
➢ Decreased production of hematopoietic growth factors or retinal hemorrhages, intestinal bleeding, and intracranial
related hormones hemorrhage
➢ Loss of normal hematopoietic tissue as a result of infiltration ➢ Fever and bacterial or fungal infections are unusual at initial
of the marrow space with abnormal cells presentation but may occur after prolonged periods of
❖ TYPES: ➢ Inherited → 15-20% ➢ Acquired → 80-85 neutropenia

❖ LABORATORY FINDINGS:
➢ Pancytopenia
➢ Hemoglobin: <10 g/dL
MCV is decreased or normal
Decreased reticulocyte count

INHERITED APLASTIC ANEMIA

❖Compared with acquired aplastic anemia, patients with
inherited/ congenital bone marrow failure syndromes present at
an earlier age and may have characteristic physical stigmata
❖TYPES:
➢Fanconi’s anemia (FA)
HEMA311 LEC
BSMLS 3-YA-1
MACROCYTIC AND NORMOCYTIC ANEMIAS
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
➢Pure red cell aplasia (PRCA) ➢Dyskeratosis congenita (DC)
➢Shwachman-Bodian-Diamond syndrome (SBDS
FANCONI’S ANEMIA
❖ Aka Congenital aplastic anemia ; chromosome instability
disorder characterized by aplastic anemia, physical
abnormalities, and cancer susceptibility ❖ CLINICAL
FINDINGS:
➢ Physical malformations at birth: skeletal abnormalities,
thumb malformations, skin pigmentations, short stature,
abnormalities in the eyes (strabismus), kidneys, and genitalia,
low birth weight, developmental delay
➢ Increased risk for cancer INHERITED APLASTIC ANEMIA
❖ LABORATORY FINDINGS:
➢ Pancytopenia, reticulocytopenia, and a hypocellular bone
marrow
➢ Increased Fetal hemoglobin (Hb F) and alpha-fetoprotein ;
INCREASED EPO
➢ Chromosome breakage analysis
→ diagnostic test for FA ✓ Breakage with DEB (diepoxybutane)
and MMC (Mitomycin C

PURE RED CELL APLASIA (PRCA)

❖ is a rare disorder of erythropoiesis characterized by a
selective and severe decrease in erythroid precursors in an
otherwise normal bone marrow
❖ Patients have severe anemia (usually normocytic),
reticulocytopenia, and normal WBC and platelet counts ❖
PRCA may be acquired or congenital
➢ Acquired: MYELOPTHISIC ANEMIA
✓ Primary PRCA may be idiopathic or autoimmune related ; in
children called as transient erythroblastopenia of childhood ❖ Myelophthisic anemia is due to the infiltration of abnormal
(TEC) cells into the bone marrow and subsequent destruction and
✓ Secondary PRCA occur in association with an underlying replacement of normal hematopoietic cells
thymoma, hematologic malignancy, solid tumor, infection, ❖ Metastatic solid tumor cells (particularly from lung, breast,
chronic hemolytic anemia, collagen vascular disease, or and prostate), fibroblasts, and inflammatory cells (such as
exposure to drugs or chemicals those found in military tuberculosis and fungal infections) have
➢ Congenital: been implicated
✓ Diamond-Blackfan anemia ❖ Cytopenia results from the release of substances such as
DIAMOND BLACKFAN-ANEMIA cytokines and growth factors that suppress hematopoiesis and
❖ is a congenital erythroid hypoplastic disorder of early infancy destroy stem, progenitor, and stromal cells
with an estimated incidence of 7 to 10 cases per million live
births
❖ CLINICAL FINDINGS:
➢ Physical anomalies, including craniofacial dysmorphisms,
short stature, and neck and thumb malformations (bilateral
syndactyly)
❖ LABORATORY FINDINGS:
➢ Severe macrocytic anemia with reticulocytopenia ; WBC
count is normal or slightly decreased, and the platelet count is
normal or slightly increased. Bone marrow examination

distinguishes DBA from the hypocellular marrow in aplastic HEMOLYTIC ANEMIAS

anemia ❖Increase in erythrocyte destruction initiated primarily by
➢ Marked decreased in red cell precursors ; normal WBC and trapping of cells in sinuses of the spleen or liver and producing
platelet count a decrease in the normal average life span of the erythrocyte
➢ Increased EPO and Decreased CFU-E the platelet count is ❖Associated with increased bone marrow activity (increased
normal or slightly increased. Bon reticulocyte count)
FANCONI’S VS DIAMOND-BLACKFAN ANEMIA ❖ASSOCIATED CONDITIONS:
➢ INTRINSIC (INHERITED)
✓ RBC membrane, enzyme defects
✓ Hemoglobinopathies
➢ EXTRINSIC (ACQUIRED)
✓ Immune or antibody mediated
HEMA311 LEC
BSMLS 3-YA-1
MACROCYTIC AND NORMOCYTIC ANEMIAS
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
✓ Non-immune RBC injury (mechanical fragmentations) ❖It is common in the malaria belt of Southeast Asia, where its
→ Micro and Macro angiopathic hemolytic anemia, Infectious prevalence can reach 30%
agents, drugs, etc ❖The inheritance pattern is autosomal dominant, and all
patients identified are heterozygous
ERYTHROCYTE MEMBRANE DEFECTS ❖LABORATORY FINDINGS:
➢ Mild or absent hemolysis
HEREDITARY SPHEROCYTOSIS ➢30% ovalocytes
OVERHYDRATED HEREDITARY
❖ a heterogeneous group of hemolytic anemias caused by STOMATOCYTOSIS/HEREDITARY HYDROCYTOSIS
defects in proteins that disrupt the vertical interactions between ❖A very rare hemolytic anemia that results from a defect in
transmembrane proteins and the underlying protein membrane cation permeability that causes the RBCs to be
cytoskeleton overhydrated; it is inherited in an autosomal dominant pattern
❖ Deficiency in spectrin, ankyrin, band 3, protein 4.2 ❖RBC membrane is excessively permeable to sodium and
❖ Most common membrane defect; autosomal dominant; potassium at 37° C; there is an influx of sodium into the cell
characterized by splenomegaly, variable degree of anemia, that exceeds the loss of potassium, which results in a net
spherocytes on the peripheral blood smear increase in the intracellular cation concentration
❖ LABORATORY FINDINGS: ❖As a result, more water enters the cell, and the cell swells
➢ Decreased MCV (severely decreased if microspherocytes), and becomes stomatocytic
hemoglobin, retics count (aplastic crisis) ❖LABORATORY FINDINGS:
➢ Increased MCHC ➢ Moderate to severe hemolytic anemia
➢ Positive autohemolysis test ➢ 5-50% stomatocytes
➢ Normal RDW ➢ Microspherocytes ➢ Increased MCV and decreased MCHC
➢ INCREASED OFT ➢ Increased OFT → Decreased surface-area:volume ratio
➢ Negative DAT (Direct antiglobulin test) → DAT (+) if an antibody
is involved ➢ Decreased Eosin-5’-maleimide (EMA) binding test DEHYDRATED HEREDITARY
(low mean fluorescence intensity) – confirmatory test for HS ; STOMATOCYTOSIS/HEREDITARY XEROCYTOSIS
binds to band 3, Rh, RhAg, and CD147 ❖Autosomal dominant hemolytic anemia as a result of a defect
in membrane cation permeability that causes the RBCs to be
❖AUTOHEMOLYSIS TEST dehydrated; it is the most common form of stomatocytosis
✓ Uses defibrinated blood incubated at 370C for 48 hours ❖RBC membrane is excessively permeable to potassium, the
✓ Normal (without glucose): 0.2-2.0% potassium leaks out of the cell, but this is not balanced by an
✓ Normal (with glucose): 0-0.9% increase in sodium; because of the reduced intracellular cation
✓ Hereditary Spherocytosis: Increased concentration, water is lost from the cell
❖OTHER TESTS: ❖LABORATORY FINDINGS:
✓ SDS-PAGE ➢ mild to moderate anemia, reticulocytosis, jaundice, and mild
✓ Ektacytometer to moderate splenomegaly
✓ Acidified glycerol test ➢ Crenated RBC
✓ Hypertonic cryohemolysis test ➢ Decreased MCV
HEREDITARY ELLIPTOCYTOSIS ➢ Increased MCHC
❖ Heterogeneous group of hemolytic anemias caused by ➢ Decreased OFT → increased surface-area:volume ratio
defects in proteins that disrupt the horizontal or lateral ➢ Target cells, burr cells
interactions in the protein cytoskeleton
❖ Due to defective spectrin dimer-dimer interaction and protein OTHER CONDITIONS ASSOCIATED WITH
4.1 ❖ Membrane defect is caused by polarization of STOMATOCYTES
cholesterol at the ends of the cell rather than around pallor ❖ Familial pseudohyperkalemia
area ➢excessive potassium leaks out of the RBCs at room
temperature
HEREDITARY PYROPOIKILOCYTOSIS ➢ Mutations in the ABCB6 gene
❖ Cryohydrocytosis
❖ Due to defective spectrin dimer-dimer interaction, increased
➢cold-induced leakage of sodium and potassium from the
calcium
RBCs
❖ CLINICAL FINDINGS: Severe transfusion related hemolytic
➢RBCs have marked increase in cation permeability, cell
anemia, jaundice, splenomegaly, gallbladder disease
swelling, and hemolysis when stored at 4°C for 24 to 48 hours
❖ LABORATORY FINDINGS:
❖Rh null disease/Rh deficiency syndrome → defect in RhAg protein
➢ Extreme microcytosis (MCV 25-55 fL)
(synthesis of Rh antigens)
➢ Increased: retics count
➢ Presence of microspherocytes, fragmented cells,
HEREDITARY ACANTHOCYTOSIS
elliptocytes, bizarre forms
❖ Associated with neuroacanthocytosis and
abetalipoproteinemia ; mutations in the MTP (microsomal
HEREDITARY OVALOCYTOSIS
triglyceride transfer protein)
❖Hereditary ovalocytosis or Southeast Asian ovalocytosis
➢ Autosomal recessive; mild anemia associated with
(SAO) is a condition caused by a mutation in the gene for band
steatorrhea, neurological and retinal abnormalities; 50-100% of
3 that results in increased rigidity of the membrane and
erythrocytes are acanthocytes
resistance to invasion by malaria
HEMA311 LEC
BSMLS 3-YA-1
MACROCYTIC AND NORMOCYTIC ANEMIAS
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
➢ Increased lecithin:sphingomyelin ratio in the membrane due
to abnormal plasma lipid concentrations; absence of serum β-
lipoprotein needed for lipid transport
❖ OTHER CONDITIONS WITH NEUROACANTHOCYTOSIS:
➢ Mcleod syndrome → absence of the KX gene
➢ Chorea acanthocytosis (ChAc)
✓ mutations in the VPS13A gene (codes for the protein
chorein)
✓ Chorein deficiency - lead to abnormal membrane protein
structure and acanthocyte formation
PAROXSYMAL NOCTURNAL HEMOGLOBINURIA (PNH)
❖ Rare chronic intravascular hemolytic anemia caused by an
acquired clonal hematopoietic stem cell mutation that results in
circulating blood cells that lack glycosylphosphatidylinositol
(GPI)-anchored proteins on their surfaces, such as CD55 (DAF
or Decay accelerating factor) and CD59 (MIRL or membrane
inhibitor of reactive lysis)
❖ Mutations in the PIGA gene → codes for phosphatidylinositol N-
acetylglucosaminyltransferase subunit A ❖ Absence of CD55 and
CD59 on the surface of the RBCs renders them susceptible to
spontaneous lysis by complement ❖ Also found in platelets,
granulocytes, monocytes, and lymphocytes
❖ TYPES:
➢ Classic PNH
➢ Subclinical PNH
➢ Hypoplastic PNH
❖CLINICAL FINDINGS:
➢Hemolytic anemia
➢Thrombosis → leading cause of death
➢Smooth muscle dystonia
➢Hemoglobinuria and jaundice ❖LABORATORY FINDINGS:
➢Chronic renal failure ➢ Decreased: Hemoglobin (<6 g/dL) ; serum haptoglobin
➢ Increased: plasma hemoglobin, serum indirect bilirubin and
lactate dehydrogenase (LDH)
➢ Hemoglobinuria, and hemosiderinuria
➢ Reticulocyte count mildly to moderate increased
➢ Increased MCV
➢ DAT negative
➢ SCREENING TEST:
➢ Sucrose hemolysis test/Sugar water screening test, Ham’s
acidified serum test
➢ CONFIRMATORY TEST:
➢ Flow cytometry using anti-CD59 and anti-CD55
➢ Flow cytometry using FLAER (Fluorescein-labeled
proaerolysin variant) using antiCD24 (granulocytes) and anti-
CD14 (monocytes

ERYTHROCYTE ENZYME DEFECTS/ ENZYMOPATHIES

G-6-PD DEFICIENCY
HEMA311 LEC
BSMLS 3-YA-1
MACROCYTIC AND NORMOCYTIC ANEMIAS
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
❖ is the most common RBC enzyme defect (part of HMS), with
a prevalence of 5% of the global population, or approximately
400 million people worldwide
❖ Some variants CONFERS RESISTANCE TO MALARIA
❖ Defect in the hexose-monosphosphate shunt pathway ;
Reduced glutathione levels (decreased GSH)
are not maintained because of decreased NADPH generation
❖ Results in oxidation of hemoglobin to methemoglobin;
denatures to form Heinz bodies
❖ CLINICAL FINDINGS:
➢ are asymptomatic throughout their lives
➢ However, some patients have clinical manifestations. The
clinical syndromes are acute hemolytic anemia, neonatal
jaundice (hyperbilirubinemia), and chronic hereditary
nonspherocytic hemolytic anemia (HNSHA)

ACQUIRED/EXTRINSIC HEMOLYTIC ANEMIAS

❖ IMMUNE HEMOLYTIC ANEMIAS

➢ AUTOIMMUNE → associated with autoantibodies
✓ Warm autoimmune hemolytic anemia (WAIHA)
✓ Cold autimmune hemolytic anemia (CAIHA)
✓ Paroxysmal cold hemoglobinuria (PCH)
✓ Mixed type autoimmune hemolytic anemia
✓ Drug induced hemolytic anemia
➢ ISOIMMUNE/ALLOIMMUNE → associated with
isoantibodies/alloantibodies
✓ Hemolytic disease of the newborn (HDN)
✓ Hemolytic transfusion reaction (HTR)
❖ NON-IMMUNE HEMOLYTIC ANEMIAS
➢ Microangiopathic hemolytic anemia
➢ Macroangiopathic hemolytic anemia
➢ Infectious agents ➢ Caused by other red blood cell injury

AUTOIMMUNE HEMOLYTIC ANEMIA

WARM AUTOIMMUNE HEMOLYTIC ANEMIA

❖ RBCs are coated with IgG and/or complement
❖ 60% of cases are idiopathic; other cases are secondary to
diseases that alter the immune response; can also be drug
induced
❖ LABORATORY FINDINGS:
➢ Progressive weakness, Occasional acute fever, Pain,
Hemoglobinuria, Mild jaundice, Splenomegaly, Hepatomegaly,
Lymphadenopathy
❖ CLINICAL FINDINGS:
➢ Spherocytes
➢ MCHC may be >37 g/dL
➢ Increased OFT, bilirubin, reticulocyte count
PYRUVATE KINASE DEFICIENCY ➢ occasional nRBCs present; positive DAT helpful in
❖Autosomal recessive; most common enzyme deficiency in differentiating from hereditary spherocytosis
Embden-Meyerhof pathway WARM AU
❖Lack of ATP causes impairment of the cation pump that TOIMM
controls intracellular sodium and potassium levels ❖Decreased COLD AUTOIMMUNE HEMOLYTIC ANEMIA
erythrocyte deformability reduces their life span ❖RBCs are coated with IgM and complement at temperatures
❖Severe hemolytic anemia with reticulocytosis and below 37°C. Antibody is usually anti-I but can be anti-i
echinocytes ❖Can be idiopathic, or secondary to Mycoplasma pneumoniae
(anti-I), lymphoma, or infectious mononucleosis (anti-i)
LABORATORY TESTS FOR G6PD AND PK DEFICIENCY ❖CLINICAL FINDINGS:
HEMA311 LEC
BSMLS 3-YA-1
MACROCYTIC AND NORMOCYTIC ANEMIAS
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
➢Seasonal symptoms; RBC clumping can be seen both ➢ Rh negative woman is exposed to Rh antigen from fetus and
macroscopically and microscopically forms IgG antibody (anti-D); this antibody will cross the
➢ MCHC >37 g/dL; increased bilirubin, reticulocyte count placenta and destroy RBCs of the next fetus that is Rh positive
➢Positive DAT detects complement-coated RBCs UNE (D positive)
HEMOLYTIC ANEMIA ➢ Exchange transfusions in utero or shortly after birth ➢ No
PAROXYSMAL COLD HEMOGLOBINURIA longer a common problem with use of Rh immunoglobulin
❖ Caused by binding of Donath Landsteiner antibodies (IgG) (RhoGam)
to red cells ➢ LABORATORY FINDINGS: Severe anemia, nRBCs, positive
❖ Donath-Landsteiner antibody/autoanti-P → biphasic hemolysin ; DAT; very high bilirubin levels cause kernicterus leading to
binds at cold temperature and lyses at warm temperatures brain damage
❖ Antibodies binds to red cell antigen in the presence of
complement at 150C and shows specificity for the Pp blood
group system (autoanti-P)
❖ CLINICAL FINDINGS:
➢ headache, vomiting, pain in the abdomen, hemoglobinuria
❖ LABORATORY FINDINGS:
➢ Spherocytes, fragmented red cells, polychromasia
➢ DAT (+), Donath Landsteiner test (+)
AUTO
AUTOIMMUNE HEMOLYTIC ANEMIA

NON-IMMUNE HEMOLYTIC ANEMIA

❖ A common feature in the nonimmune extrinsic hemolytic
anemias is the presence of a condition that causes physical or
mechanical injury to the RBCs
❖ Mechanical fragmentation ; associated with schistocytes
(fragmented RBC)
ALLOIMMUNE HEMOLYTIC ANEMIA ❖ This injury can be caused by:
HEMOLYTIC ANEMIAAUTOIMMUNE HEMOLYTIC AN ➢ Abnormalities in the microvasculature (microangiopathic)
HEMOLYTIC TRANSFUSION REACTIONS ➢ The heart and large blood vessels (macroangiopathic)
❖ One of the most severe and potentially life-threatening ➢ Infectious agents
complications of blood transfusion is a hemolytic transfusion ➢ Chemicals
reaction (HTR) caused by immune-mediated destruction of ➢ Drugs
donor cells (Ag) by an antibody in the recipient ➢ Venoms
❖ The offending antibody in the recipient may be IgM or IgG, ➢ Extensive burns YTIC ANEMIAAUTOIMMUNE
complement may be partially or fully activated or not activated HEMOLYTIC
at all, and hemolysis may be intravascular or extravascular
❖ Can be acute HTR or delayed HTR
❖ ACUTE HTR:
➢ associated with ABO antibodies (IgM) with complement
activation, intravascular hemolysis
❖ Delayed HTR ➢ associated with Rh, Kidd, Duffy antibodies
(IgG) ; with or without complement activation ; extravascular
hemolysis HE

HEMOLYTIC DISEASE OF A NEWBORN

❖ Also called as Hemolytic disease of the fetus and newborn
(HDFN)
❖ May be due to ABO or Rh incompatibility
❖ ABO incompatibility
➢ Group O woman develops IgG antibody that crosses the
placenta and coats fetal RBCs when fetus is group A or B; the
coated RBCs are phagocytized
➢ LABORATORY FINDINGS: Mild or no anemia, few
spherocytes, weakly positive DAT, slightly increased bilirubin
❖ Rh incompatibility
HEMA311 LEC
BSMLS 3-YA-1
MACROCYTIC AND NORMOCYTIC ANEMIAS
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NON-IMMUNE HEMOLYTIC ANEMIA
NO
MICROANGIOPATHIC HEMOLYTIC ANEMIA
❖ are a group of potentially life-threatening disorders
characterized by RBC fragmentation and thrombocytopenia
❖ RBC fragmentation occurs intravascularly by the mechanical
shearing of RBC membranes as the cells rapidly pass through
turbulent areas of small blood vessels that are partially blocked
by microthrombi or damaged endothelium
❖ Upon shearing, RBC membranes quickly reseal with minimal
escape of hemoglobin, but the resulting fragments (called
schistocytes) are distorted and become rigid
❖ The spleen clears the rigid RBC fragments from the
circulation through the extravascular hemolytic process
❖ ASSOCIATED CONDITIONS:
➢ Thrombotic Thrombocytopenic Purpura (TTP)
➢ Hemolytic Uremic Syndrome (HUS)
➢ HELLP Syndrome
➢ Disseminated Intravascular Coagulation (DIC)
N-IMMUNE
THROMBOTIC THROMBOCYTOPENIC PURPURA (TTP)
❖ rare, life-threatening disorder characterized by the abrupt
appearance of microangiopathic hemolytic anemia, severe
thrombocytopenia, and markedly elevated serum LD activity
❖ caused by a deficiency of the von Willebrand factor-cleaving
protease known as a disintegrin and metalloprotease with a
thrombospondin type 1 motif, member 13 (ADAMTS13)
❖ ADAMTS13 → regulates the size of circulating von Willebrand
factor (VWF) by cleaving ultralong VWF multimers (ULVWF) into
shorter segments ; preventing VWF from excessively binding and
activating platelets
❖ TYPES:
➢ Idiopathic → autoantibodies (IgG, IgM, IgA) to ADAMTS13 ➢
Secondary → infections, pregnancy, surgery, trauma, inflammation,
and disseminated malignancy
➢ Inherited → also called Upshaw-Schülman syndrome, is a severe
ADAMTS13 deficiency caused by mutations in the ADAMTS13
genE
HEMOLYTIC UREMIC SYNDROME (HUS)
❖ Characterized by microangiopathic hemolytic anemia,
thrombocytopenia, and acute renal failure from damage to
endothelial cells in the glomerular microvasculature
❖ TYPES:
➢ Typical/Shiga toxin-associated HUS, or Stx-HUS
✓ 90% HUS
✓ caused by E.coli (serotype O157:H7) and Shigella strains
that produce Shiga toxin and is preceded by an episode of
acute gastroenteritis, often with bloody diarrhea
➢ Atypical HUS
✓ 10% HUS
✓ uncontrolled activation of the alternative complement
system, which causes endothelial cell injury, activation of
platelets and coagulation factors, and formation of platelet-
fibrin thrombi that obstruct the microvasculature in the
glomerulus and other organs HEMOLYTIC ANEMI
HELLP SYNDROME
❖ serious complication in pregnancy, is named for its
characteristic presentation of hemolysis, elevated liver
enzymes, and low platelet count (HELLP)
❖ It occurs in approximately 0.5% of all pregnancies but
develops in approximately 4% to 12% of pregnancies with
preeclampsia and 30% to 50% of pregnancies with eclampsia,
most often in the third trimester
HEMOLYTIC ANEMI
DISSEMINATED INTRAVASCULAR COAGULATION
❖ Characterized by the widespread activation of the
hemostatic system, resulting in fibrin thrombi formation
throughout the microvasculature
❖ Major clinical manifestations are organ damage due to
obstruction of the microvasculature and bleeding due to the
consumption of platelets and coagulation factors and
secondary activation of fibrinolysis
❖ DIC is a complication of many disorders: metastatic cancers,
acute leukemias, infections, obstetric complications, crush or
brain injuries, acute hemolytic transfusion reactions, extensive
burns, snake or spider envenomation, and chronic
inflammation
❖ Thrombocytopenia with prolonged PT and APTT, decreased
fibrinogen levels HEMOLYTIC ANEM
.
MICROANGIOPATHIC HEMOLYTIC ANEMIA
❖ TRAUMATIC CARDIAC HEMOLYTIC ANEMIA
➢ Can occur in patients with prosthetic cardiac valves due to
turbulent blood flow through and around the implanted devices
❖ EXERCISED-INDUCED HEMOGLOBINURIA
➢ Also known as MARCH HEMOGLOBINURIA
Transient hemolytic anemia that occurs after forceful repeated
impact of the feet or hands on hard surfaces
❖ INFECTIOUS AGENTS
➢ Malaria, Babesiosis, Clostridial sepsis, Bartenellosis
❖ DRUGS AND CHEMICALS
➢ Causes oxidative denaturation of hemoglobin leading to
formation of methemoglobin and Heinz bodies
➢ Dapsone, Naphthalene, Primaquine, Arsine hydride, Copper
and Lead exposure
❖ VENOM
➢ Snakes, spiders, bees, or wasps
➢ Causes hemolysis by direct disruption of the RBC
membrane, alteration of the RBC membrane that results in
complement-mediated lysis, and initiation of DIC
❖ EXTENSIVE BURNS/THERMAL INJURY
➢ Can cause direct damage to the RBC membrane, producing
acute hemolysis, which is characterized by severe anemia with
many schistocytes and microspherocytes.
HEMOGLOBINOPATHIES
❖ Refers to a disease state (opathy) involving the hemoglobin
(Hb) molecule; are the most common genetic diseases,
affecting approximately 7% of the world’s population
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❖ More than 300,000 children are born each year with some ➢ Example is Hgb Constant Spring (alpha-Thalassemia) –
form of inherited hemoglobin disorder and approximately 80% addition of 31 amino acids
occur in mid- to low-income countries ❖ GENE FUSION
❖ Results from a genetic mutation in one or more genes that ➢ occur when two normal genes break between nucleotides,
affect hemoglobin synthesis switch positions, and anneal to the opposite gene
❖ Change in amino acid sequence can alter the structure of ➢ Example is Hgb Lepore → beta-delta (βδ or δβ) gene fusion
the hemoglobin molecule (structural defect) and its function NON-IMMUNE HEMOLYTIC ANEMIA
(qualitative defect)
ZYGOSITY
❖ refers to the association between the number of gene
mutations and the severity of the resultant genetic defect
❖ Beta gene mutations affect overall hemoglobin function to a
greater extent than the same number of alpha gene mutations
(2 copies of beta genes; 4 copies alpha genes)
❖ PATTERN OF INHERITANCE FOR BETA CHAIN
VARIANTS:
➢ HOMOZYGOUS (DISEASE):
✓ Both beta genes are mutated
O ✓ Ex: Hgb SS disease/Hgb S disease ➢ HETEROZYGOUS
LYTIC ANEMI (TRAIT)
NON-IMMUNE HEMOLYTIC ANEMIA ✓ Only one beta gene is mutated; other is normal
GENETIC MUTATIONS IN HEMOGLOBINOPATHIES ✓ Ex: Hgb AS trait
involving one or more of the adult globin genes- alpha (α), beta
(β), gamma (γ), delta (δ) NOMENCLATURE
❖ More than 1200 structural hemoglobin variants are known to ❖ Designated by letters of the alphabet; common and scientific
exist throughout the world, and more are being discovered designation
regularly ❖ Common name - discoverer and geographic area
❖ The mutations include point mutations, deletions, insertions, ❖ Scientific designation - indicate the variant chain, the
and fusions sequential and the helical number of the abnormal amino acid,
and the nature of the substitution designation

HGB S/HGB SS/SICKLE CELL ANEMIA/SICKLE CELL

DISEASE
❖ Most common form of hemoglobinopathy
❖ The term sickle cell disease is used to describe a group of
symptomatic hemoglobinopathies that have in common sickle
cell formation and the associated crises
❖ Patients with SCD are either:
➢ Homozygous for Hgb S (Hgb SS)
➢ Compound heterozygotes - Hgb S in combination with
another hemoglobin beta chain mutation (Hgb C or beta-
thalassemia)
❖ ETIOLOGY:
➢ Beta chain substitution; glutamic acid is replaced by valine (α2β2
6Glu→Val) at position 6
GENETIC MUTATIONS ➢ The change in the amino acid results in abnormalities in
polymerization with deoxygenation that leads to sickling which
❖ POINT MUTATION WITH AMINO ACID SUBSTITUTION
results in a permanently altered membrane protein (RBCs
➢ The replacement of one original nucleotide in the normal
undergo extravascular hemolysis)
gene with a different nucleotide
✓ Homozygotes – Sickling begins when oxygen saturation
➢ It also is possible to have two-point mutations occurring in
decreases to less than 85%
the same globin gene, which results in two amino acid
✓ Heterozygotes - Sickling does not occur unless the oxygen
substitutions within the same globin chain; more than 35
saturation of hemoglobin is reduced
mutations occur by this mechanism
❖ DELETION AND INSERTION
➢ involve the removal of one or more nucleotides, whereas
insertions result in the addition of one or more nucleotides; are
not divisible by three and disrupt the reading frame, which
leads to the nullification of synthesis of the corresponding
globin chain
➢ Example is Hgb Hill → deletion of 5 amino acids
❖ CHAIN EXTENSION
➢ occur when the stop codon is mutated so that translation
continues beyond the typical last codon
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✓ This acute, painful aspect of SCD occurs with great
predictability and severity in many individuals and can be
triggered by acidosis, hypoxia, dehydration, infection and fever,
and exposure to extreme cold
✓ Painful episodes manifest most often in bones, lungs, liver,
spleen, penis, eyes, central nervous system, and urinary tract.

❖ PATHOPHYSIOLOGY:
➢ SICKLING:
✓ Hgb S is freely soluble when fully oxygenated; when oxygen
is removed, Hgb S polymerizes
✓ Formation of tactoids (fluid crystals) that are rigid and
deform the cell
✓
OTHER CAUSES OF SICKLING: Decreased pH, increased
2,3-DPG, high cellular concentration of hemoglobin
➢ A strong interaction between the side chain of β6 valine and
the hydrophobic pocket of β85 phenylalanine and 88 leucine of
another Hgb S molecule is probably the basis of polymer
formation
➢ Sickle cells contain high calcium levels, which stimulate
potassium and water loss and exaggerate cell dehydration
➢ SICKLE CELLS OCCURS IN TWO FORMS
: ✓ Reversible sickle cells → Hgb S-containing RBCs that change
shape in response to oxygen tension; circulate as normal biconcave
discs when fully oxygenated but undergo hemoglobin
polymerization, show increased viscosity, and change shape on
deoxygenation
✓ Irreversible sickle cells → do not change their shape regard
less of the change in oxygen tension or degree of hemoglobin
polymerization; seen in peripheral blood films as elongated ❖ CLINICAL FEATURES/SIGNS AND SYMPTOMS:
sickle cells; REMOVED BY THE SPLEEN AND CAUSES ➢ SPLENIC SEQUESTRATION AND INFARCTS
VASOOCCLUSION ✓ Sudden trapping of blood in the spleen, which leads to a
✓ When sickled cells receive oxygen they return to their rapid decline in hemoglobin (<6 g/dL); occurs most
normal shape; repeated cycles of sickling and unsickling leads often in infants and young children whose spleens are
to permanent damage of RBCs leading to hemolysis. chronically enlarged
✓ Children experiencing splenic sequestration episodes may
❖ CLINICAL FEATURES: have earlier onset of splenomegaly and a lower
➢ Vary from no symptoms to potentially lethal state.; level of Hb F at 6 months of age; Crises are often associated
symptoms also vary between ethnic groups with Indian patients with respiratory tract infections
expressing a much milder disease than their African ✓ Autosplenectomy → gradual loss of splenic function; leads to
counterparts increased bacterial infections; presence of
➢ Many individuals with SCD undergo episodes of recurring Howell-Jolly bodies and Pappenheimer bodies
pain termed crises
➢ Various crises may occur: vasoocclusive or “painful,” splenic ➢ ACUTE CHEST SYNDROME
sequestration, chronic hemolytic, megaloblastic, and aplastic ✓ Acute illness with fever and/or other respiratory symptoms
(incuding cough, chest pain, and dyspnea) that
CLINICAL FEATURES/SIGNS AND SYMPTOMS: displays pulmonary infiltrates on chest radiograph;
➢ VASOOCCLUSIVE CRISIS (VOC) Approximately 40% of patients experience at least one
✓ The hallmark of SCD is vasoocclusive crisis (VOC), which episode of acute chest syndrome
accounts for most hospital and emergency department visits ✓ Second most common cause of hospitalization (third most
common cause of death among adults)
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✓ More than 10% of adults dying from complications linked to RBCs, SICKLE CELLS, TARGET CELLS, nucleated RBCs,
chronic lung disease and pulmonary along
hypertension with a few spherocytes, basophilic stippling, Pappenheimer
✓ Pulmonary infection, fat embolism, and pulmonary infarction bodies, and Howell-Jolly bodies
are the most common inciting factors, ✓ IMPORANT NOTE: Presence of sickle cells and target
resulting in a decreased alveolar oxygen tension that induces cells is the hallmark of SCD
Hgb S polymerization and sickle cell formation.

❖ CLINICAL FEATURES/SIGNS AND SYMPMTOMS:

➢ FAT EMBOLISM SYNDROME (FES) AND BONE MARROW
NECROSIS
✓ Rare but often fatal sequalae of SCD; Necrotic bone marrow
material and fat particles are released into
circulation and deposit in the lung, where they fragment
✓ Small fat droplets reenter circulation via pulmonary
capillaries and terminate in various organs, causing
Damage.

➢ BACTERIAL INFECTIONS
✓ Acute infections are common causes of hospitalization and
have been the most common causes of death,
especially in the first 3 years of life (Staphylococcus aureus, ❖ SPECIAL LABORATORY TESTS:
Streptococcus pneumoniae, and Haemophilus ➢ SCREENING TESTS
influenzae) ✓ SODIUM METABISULFITE TEST
✓ Bacterial infections of the blood (septicemia) are ✓ DITHIONITE TUBE TEST/SICKLE SOLUBILITY TEST/HGB
exacerbated by the autosplenectomy effect as the spleen SOLUBILITY TEST
gradually loses its ability to function as a secondary lymphoid ❖ CONFIRMATORY TESTS
tissue to effectively clear organisms from the ➢ HEMOGLOBIN ELECTROPHORESIS
blood ➢ Cation-Exchange High performance liquid chromatography
(HPLC)
➢ OTHERS: ➢ Capillary electrophoresis
✓ CHRONIC HEMOLYSIS ➢ Isoelectric focusing (IEF)
✓ PULMONARY HYPERTENSION ➢ DNA analysis – Gap PCR and DNA sequencing
✓ MEGALOBLASTIC AND APLASTIC EPISODES
✓ CARDIAC ABNORMALITIES ❖ SODIUM METABISULFITE TEST
o Severe anemia, cardiomegaly can develop as the heart ➢ Adding sodium metabisulfite, a reducing substance, to
works harder to maintain adequate blood flow blood enhances deoxygenation of Hb and sickling of Hgb S
and tissue oxygenation; risk of pregnancy ➢ Does not distinguish sickle cell anemia from sickle trait or
✓ BONE AND SKIN ABNORMALITIES other HgbS syndromes because all red cells sickle,
o Impaired blood supply to the head of the femur and humerus although sickling occurs more rapidly with greater amounts
results in a condition called avascular of HbS in the cells
necrosis (AVN); 50% of patients with SCD develop AVN by age ➢ Positive tests - may occur with other rare sickling
35 years hemoglobins (Hbs S Travis, C Harlem), HbI, and high
o Hand-Foot Dactylitis/Hand-Foot syndrome concentrations of Hb Bart’s. Hb I, an α-chain variant, has
✓ STROKE normal functional properties
o Hemorrhagic or ischemic stroke occurs in approximately 11% ➢ False-negative tests - may occur if Hgb S concentration is
of children with SCD before age 20 years less than 10% (as in very young infants) or if
o 24% of SCD patients have a stroke by the age of 45 years deoxygenation is inadequate (e.g., deterioration of
✓ RETINOPATHY reagent.
o occurs at a rate of 45% in patients with Hb SC, 11% with Hb
SS, and 17% with Hb S-b-thal by early
adulthood

❖ LABORATORY FINDINGS:
➢ Decreased hemoglobin (5 to 9.5 g/dL), hematocrit, RBC
count
➢ Increased WBC count (12,000 to 15,000 x 109
/L) ; up to
40,000
➢ Increased platelet count
➢ Increased RDW – moderate anisocytosis
➢ Increased reticulocyte count (10-25%); aplastic crisis –
decreased retics count
➢ PBS: marked poikilocytosis and anisocytosis with normal
HEMA311 LEC
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✓ Other hemoglobins (Hb C-Harlem (Georgetown), Hb C-
Ziguinchor, Hb S-Memphis, Hb S-Travis, Hb SAntilles, Hb S-
Providence, Hb S-Oman, Hb Alexander, and Hb Porte-Alegre)
➢ FALSE NEGATIVE:
✓ Results can occur in infants younger than 6 months
✓ Low hematocrits
✓ Severe anemia (decreased amount of Hgb S)

❖ HEMOGLOBIN ELECTROPHORESIS ➢ based on the

separation of hemoglobin molecules in an electric field
primarily as a result of differences in total molecular charge ➢
TYPES: ✓ CELLULOSE ACETATE ✓ CITRATE AGAR ➢
CELLULOSE ACETATE (Alkaline pH – pH 8.4-8.6) ✓
hemoglobin molecules assume a negative charge and migrate
toward the anode (positive pole) ✓ Replaced by agarose gel
medium.

❖ DITHIONITE TUBE TEST/SICKLE SOLUBILITY TEST/HGB

SOLUBILITY TEST
➢ Most common screening test for Hgb S; capitalizes on the
decreased solubility of deoxygenated Hb S in solution,
PRODUCING TURBIDITY ➢ PROCEDURE: ✓ Blood is added
to a buffered salt solution containing a reducing agent, such as
sodium hydrosulfite (dithionite), and a detergent-based lysing agent
(saponin) o Dithionite → converts ferrous to ferric iron; ferric iron is
unable to bind oxygen, converting hemoglobin to the deoxygenated
form o Saponin → dissolves membrane lipids, causing release of
hemoglobin from RBCs ✓ Positive: Turbid (Deoxygenated
polymerized Hgb S) ✓ Negative: Clear (non-sickling
hemoglobin)

➢ CITRATE AGAR (Acidic pH – pH 6.0 – 6.2)

✓ some hemoglobins assume a negative charge
and migrate toward the anode, whereas
others are positively charged and migrate
toward the cathode

❖ DITHIONITE TUBE TEST/SICKLE SOLUBILITY TEST/HGB

SOLUBILITY TEST
➢ FALSE POSITIVE:
✓ Hyperlipidemia
✓ When too much blood is added to the test solution
HEMA311 LEC
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changes as they migrate through the pH gradient until the
hemoglobin species reaches its isoelectric point (net
charge of zero)

SICKLE CELL TRAIT (HGB AS)

❖ Heterozygous Hgb S is the most common
hemoglobinopathy in the United States
❖ It is present in about 8% of African Americans and in as
many as 45% of the population in certain areas of West Africa
❖ Individuals with SCT are generally asymptomatic and
present with no significant clinical or hematologic
manifestations
❖ Under extremely hypoxic conditions, however, systemic
sickling and vascular occlusion with pooling of sickled cells in
the spleen, focal necrosis in the brain, rhabdomyolysis, and
even death can occur
❖ LABORATORY FINDINGS:
➢ Normocytic, normochromic RBC, few Target cells
➢ Hemoglobin solubility screening test positive results
➢ Presence of Hb S and Hb A on hemoglobin electrophoresis
or HPLC
SICKLE CELL TRAIT (HGB AS)
✓ 40% or less Hb S, approximately 60% or more Hb A, Hb A2
level is normal or slightly increased, and Hb F
level is within the reference interval
✓ Levels of Hb S less than 40% can be seen in patients who
also have a-thalassemia or iron or folate
deficiency
❖ TREATMENT:
➢ No treatment is required for this benign condition, and the
patient’s life span is not affected
SICKLE CELL TRAIT (HGB AS)
HEMOGLOBIN C/HGB C/HGB CC/HGB C DISEASE
❖ Next hemoglobinopathy after Hb S to be described and in
the United States is found almost exclusively in the African
American population
❖ Glutamic acid is replaced by lysine in position 6 of the beta
chain (α2β2
6Glu→lys)
❖ Milder disease compared with SCD; mild splenomegaly and
hemolysis may be present; vasoocclusive crises do not occur
❖ LABORATORY FINDINGS:
❖ HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ➢ Mild to moderate, normochromic, normocytic anemia occurs
(HPLC) ➢ Some microcytosis and mild hypochromia
➢ Separates hemoglobin types in a cation exchange column ➢ Target cells; Slight to moderate increase in the number of
and usually requires only one sample injection reticulocytes (2% to 3%), nucleated RBCs
➢ Can identify and quantify low levels of Hb A2 and Hb F, ➢ Decreased MCV, MCHC is normal or increased
but comigration of Hb A2 and Hb E occurs (dehydration)
➢ Best used in the diagnosis of thalassemias rather than ➢ HGB CC CRYSTALS
hemoglobinopathies because quantification of low levels ✓ Hexagonal or rod-shaped crystals may be seen in
of normal and abnormal hemoglobin levels is necessary erythrocytes in the stained smear, especially after
to distinguish Thalassemias splenectomy or after slow drying of the smear
✓ “Bar of gold” appearance of RBCs
❖ CAPILLARY ELECTROPHORESIS ➢ Negative sickle cell solubility test; 90% Hgb C, <7% Hgb F,
➢ Separates hemoglobin types based on charge in an alkaline 2% Hgb A2
buffer but does so using smaller volumes and ❖ TREATMENT:
produces better separation than traditional agarose ➢ No specific treatment is required; becomes problematic only
electrophoresis if infection occurs, if splenomegaly becomes painful,
➢ Allow for the testing of multiple samples in parallel with or if mild chronic hemolysis leads to gallbladder disease.
computerized analysis of results
➢ Economical - can accommodate at least 3000 runs HEMOGLOBIN C/HGB C/HGB CC/HGB C DISEASE
❖ ISOELEECTRIC FOCUSING
➢ uses an electrical current to push the hemoglobin molecules
across a pH gradient. The charge of the molecules
HEMA311 LEC
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hemolysis, or splenomegaly
➢ Main concern is differentiating from IDA, Beta-thalassemia
trait, Beta-thalassemia major
❖ Heterozygous (Hgb E trait; Hgb AE trait)
➢ Asymptomatic
❖ Hemoglobin E-B-Thalassemia
➢ Symptoms more severe than Hb EE and more closely
resembles the severity Beta-thalassemia major, requiring
regular blood transfusion

❖ LABORATORY DIAGNOSIS:
➢ Homozygous (Hgb EE)
HEMOGLOBIN AC TRAIT (HGB AC) ✓ Mild anemia (Hgb 11 to 13 g/dL)
✓ Decreased MCV (55 to 65 fL)
✓ Few to many target cells; normal reticulocyte count
❖ Prevalent in West Africans and in about 2% to 3% of black ✓ 95% Hgb E, 2-4% Hgb A2
people ➢ Heterozygous (Hgb E trait)
❖ The heterozygous state (HbAC) is asymptomatic, without ✓ normal hemoglobin levels, a mean MCV of 65 fL, slight
anemia, with normal or minimally reduced MCV and erythrocytosis, target cells; approximately 25% to
normal red cell life span 30% Hb E
❖ LABORATORY FINDINGS: ➢ Hemoglobin E-B-Thalassemia
➢ Normal or decreased MCV ✓ Patients with Hb E-B
➢ Slightly elevated MCHC +
➢ Target cells are present on blood film -Thalassemia have an anemia that varies in severity
➢ 60% Hb A and 30% Hb C (hemoglobin between 6.5 to 9.5
➢ When significant microcytosis is present, it is usually caused g/dL), depending on the beta-thalassemia mutation
by coexistent α-thalassemia, and HbC is reduced to ✓ Hb E-B
38%, 32%, and 24% in patients with three, two, and one α - 0
gene, respectively -Thalassemia have an even more severe anemia depending on
the degree of Hb F compensation
HEMOGLOBIN C-HARLEM/HEMOGLOBIN C- ❖ TREATMENT:
➢ No therapy is required with Hb E disease and trait; some
GEORGETOWN
patients may experience splenomegaly and fatigue
❖Aka Hgb SC-Harlem; Double substitution on the beta chain
➢ Patients with Hb E-B
❖Beta chain substitution:
0
1. Glutamic acid to valine at position 6 (α2β2
-Thalassemia are treated like beta-thalassemia major with
6Glu→lys)
chronic transfusion therapy, iron
2. Aspartic acid to asparagine at position 73 (α2β2
chelation therapy, splenectomy with hypersplenism, and
73asp→asn)
hydroxyurea in severe cases
❖Termed because the abnormal hemoglobin migrates with Hb
C on alkaline hemoglobin
electrophoresis; patients heterozygous for this anomaly are
asymptomatic, but patients with HEMOGLOBIN O-ARAB
compound heterozygosity for Hb S and Hb C-Harlem have ❖ Beta chain variant caused by the substitution of glutamic acid by
crises similar to those with Hgb S lysine at amino acid position 121 (α2β2121glu→lys);
(Sickle cell anemia) found in black and Arabic people
❖Positive Sickle solubility test ❖ Asymptomatic, except for mild splenomegaly in
❖Alkaline electrophoresis – Hgb C-Harlem migrates with Hgb homozygotes
C ❖ When inherited with Hb S (Hgb SO-Arab) severe clinical
❖Citrate agar electrophoresis – migrates with Hgb S symptoms like Hgb S (Sickle cell anemia)
❖ LABORATORY FINDINGS:
HEMOGLOBIN E (HGB E) ➢ Homozygous (Hgb O)
✓ Mild hemolytic anemia, Target cells on the peripheral blood
❖ First described in 1954; found mainly in the Asian region
film
❖ Hgb E is a beta chain variant in which glutamic acid is
✓ Negative result on the hemoglobin solubility test
replaced by lysine in position 26 (α2β2
✓ Presence of this hemoglobin variant must be confirmed
6glu→lys)
using electrophoresis or HPLC
❖ BOTH A QUALITATIVE DEFECT AND A QUANTITATIVE
❖ TREATMENT:
DEFECT with a beta-thalassemia phenotype
➢ Usually none
➢ Qualitative - amino acid substitution in the globin chain
➢ Quantitative – decreased production of the mutated globin
chain
❖ Homozygous (Hgb E) HEMOGLOBIN D (HGB D) AND HEMOGLOBIN G
➢ manifests as a mild anemia with microcytes and target cells; (HGB G)
not associated with clinically observable icterus,
HEMA311 LEC
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❖ Group of at least 16 beta chain variants (Hb D) and 6 alpha ➢ PBS: few sickle cells, target cells, Hgb SC crystals →
chain variants (Hb G) that migrate in an alkaline pH at the Washington’s Monument appearance
same electrophoretic position as Hb S ➢ Positive sickle solubility test
❖ Hemoglobin D ➢ 45% Hgb S, 45% Hgb C, Normal Hgb F
➢ Hgb D-Punjab and Hgb-D-Los Angeles – glutamic acid is
replaced by glutamine at position 121 in the beta chain
(α2β2121Glu→Gln) HEMOGLOBIN S-β-Thalassemia (Hb S-β
❖ Hemoglobin G-Philadelphia 0
➢ Asparagine is replaced by lysine at position 68 (α2 -thal)
68Asn→Lysβ2)
➢ Most common G variant encountered in African Americans ❖ Mild to moderate sickle cell anemia; severity of this
and is seen with greater frequency than the Hb D compound heterozygous condition depends on the beta chain
variants production of the affected beta-thalassemia gene
❖ Hgb D disease is associated marked by mild hemolytic ❖ TYPES:
anemia; heterozygous is asymptomatic for both variants and ➢ Hb S-β
chronic nonprogressive splenomegaly 0
❖ LABORATORY FINDINGS: -Thal
➢ Homozygous ✓ Hgb A is absent; HgbS is 75% to 90%, HbgF is 5% to 20%,
✓ Negative sickle solubility test and HbgA2
✓ Alkaline electrophoresis – same mobility as Hgb S is 4% to 6%
✓ Citrate agar electrophoresis – Hgb D and G can be ✓ Resembles sickle cell disease, except for the spleen, which
separated remains enlarged after childhood and into adult
✓ >95% Hb D, with normal amounts of Hb A2 and Hb F life
✓ MCV and MCH are decreased, and the HbA2 might be
COMPOUND HETEROZYGOUS WITH HGB S significantly increased
❖ Compound heterozygosity is the inheritance of two different ➢ Hb S-β
mutant genes that share a common genetic locus, in +
this case the beta-globin gene locus: -Thal
➢ Hgb SC disease ✓ HbA is 15% to 30%; HbS is over 50%, HbF is 1% to 20%,
➢ Hgb S-Beta-Thalassemia; Hgb S-Alpha-Thalassemia and HbA2 is 4% to 6%
(involves alpha chain) ✓ Resembles sickle cell trait (Hgb AS):
➢ Hgb SD and Hgb SG-Philadelphia o Sickle cell trait: Decreased Hgb S, increased Hgb A
➢ Hgb SO-Arab and Hgb SD-Punjab o Hb S-β
➢ Hgb S-Korle Bu → substitution of aspartic acid to asparagine at +
position 73 of the beta chain (α2β273asp→asn) -Thal: Increased Hgb S, decreased Hgb A
CONCOMITANT CIS MUTATIIONS WITH HGB S
❖ Involves a second mutation on the same gene along with Hb
S
❖ Variants: Hgb C-Harlem, Hgb S-Antilles, Hgb S-Oman

HEMOGLOBIN SC DISEASE

❖ Most common compound heterozygous syndrome that HEMOGLOBIN M

results in a structural defect in the hemoglobin molecule in ❖ caused by a variety of mutations in the alpha, beta, and
which different amino acid substitutions are found on each of gamma globin genes, all of which result in production of
two beta-globin chains methemoglobin (auto-oxidation of heme leading to
❖ SIGNS AND SYMPTOMS: methemoglobinemia)
➢ Milder form of sickle cell disease, delayed growth and ❖ Has iron in the ferric state (Fe3+
development ) and is unable to carry oxygen, which produces cyanosis
➢ May cause all the vasoocclusive complications of sickle cell ❖ Have altered oxygen affinity and are inherited as autosomal
anemia, but episodes are less frequent and damage is dominant disorders
less disabling ❖ Affected individuals have 30-50% methemoglobin;
➢ Mild to moderate hemolytic anemia, moderate associated with Heinz bodies
splenomegaly, retinopathy is more common and more severe ❖ Alpha chain variants:
in ➢ Hb M-Boston, Hb M-Iwate, and Hb Auckland
SCD ❖ Beta chain variants:
❖ LABORATORY DIAGNOSIS: ➢ Hb Chile, Hb M-Saskatoon, Hb M-Milwaukee-1, and Hb M-
➢ mild normocytic, normochromic anemia with many features Milwaukee-2
associated with sickle cell anemia ❖ Gamma chain variants:
➢ Hgb: 11-13 g/dL ➢ Hb F-M-Osaka and Hb F-M-Fort Ripley
➢ Reticulocyte count: 3-5%
HEMA311 LEC
BSMLS 3-YA-1
MACROCYTIC AND NORMOCYTIC ANEMIAS
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
UNSTABLE HEMOGLOBINS
❖ More than 170 variants
❖ Hgb variants result from genetic mutations to globin genes ❖ Hgb quickly release oxygen to the tissues, which results in
creating hemoglobin products that precipitate in vivo, normal to decreased hemoglobin concentration, slight
producing Heinz bodies and causing a hemolytic anemia anemia, and cyanosis
❖ More than 185 variants; majority are beta chain variants ❖ Oxygen uptake is impaired in the lung and the level of
❖ Aka as congenital non-spherocytic hemolytic anemia or deoxyhemoglobin is more than 5 g/dL, causing cyanosis
congenital Heinz body anemia; more properly called unstable
hemoglobin disease
❖ Variants:
➢ Hb Köln, Hb Hammersmith, Hb Zurich, Hb Gun Hill
❖ LABORATORY DIAGNOSIS:
➢ normal or show slight hypochromia and prominent basophilic
stippling
➢ Jaundice, Splenomegaly
➢ Dark pigmented urine – contains dipyrrole
➢ Before splenectomy: Hgb - 7 to 12 g/dL, with a 4% to 20%
reticulocyte count, Heinz bodies, bite cells
➢ After splenectomy: anemia is corrected, but reticulocytosis
persists with numerous Heinz bodies
➢ Not detect in Hgb electrophoresis – Migrates with Hgb A

❖SPECIAL TESTS:
➢ HEAT DENATURATION/INSTABILITY TEST
✓ Unstable hemoglobins are heat sensitive and cause partial
denaturation upon addition of phosphate
buffer/Tris buffer
✓ Specimen: Whole blood (EDTA or heparin)
✓ Incubated at 500C for 3 hours
✓ Positive result: appearance of flocculent precipitate (10-40%
of Total hgb) within 1 hour of incubation
➢ ISOPROPANOL PRECIPITATION TEST
✓ Precipitation of unstable hemoglobin in the presence of 17%
isopropanol at 37C
✓ Specimen: whole blood (any anticoagulant)
✓ Positive result: rapid precipitation within 5-20 mins (normal
precipitation: 45 mins)

HEMOGLOBIN WITH INCREASED OXYGEN AFFINITY

❖ More than 200 variants

❖ Hgb fails to release oxygen on demand and hypoxia results
❖ Kidneys sense the hypoxia and respond by increasing the
release of erythropoietin, which leads to a compensatory
Erythrocytosis

HEMOGLOBIN WITH DECREASED OXYGEN AFFINITY