laboratory diagnosis of hemolytic anemia-190509145931.pptx

Dr. Deepa Anantha Laxmi N.V
Consultant Pathologist

Introduction
 Hemolytic anemias share the following features:
 A shortened red cell life span below the normal 120
days
 Elevated erythropoietin levels and a compensatory
increase in erythropoiesis
 Accumulation of hemoglobin degradation products
that are created as part of the process of red cell
hemolysis

Extravascular hemolysis
 Destruction of senescent red cells takes place within
macrophages
 Extravascular hemolysis is generally caused by
alterations that render the red cell less deformable
 Principal clinical features of extravascular hemolysis
are anemia, splenomegaly, and jaundice.
 Variable decreases in plasma haptoglobin and α2-
globulin.

Intravascular hemolysis
 Mechanical injury, complement fixation, intracellular
parasites (e.g., falciparum malaria, Chapter 8), or
exogenous toxic factors
 Intravascular hemolysis is manifested by anemia,
hemoglobinemia, hemoglobinuria, hemosiderinuria, and
jaundice.
 As serum haptoglobin is depleted, free hemoglobin
oxidizes to methemoglobin, which is brown in color
 Renal hemosiderosis
 Splenomegaly is not seen
 In all types of uncomplicated hemolytic anemias, the
excess serum bilirubin is unconjugated

Laboratory Features of Hemolysis
 Signs of Increased Red Blood Cell Destruction
 Signs of Intravascular Hemolysis
 Signs of Accelerated Erythropoiesis

1.Signs of Increased Red Blood Cell Destruction
 Serum Bilirubin: 0.2 to 1.2 mg/dL
 Hyperbilirubinemia is a hallmark of hemolytic anemia
 Serum Lactic Dehydrogenase:
 Not to as great an extent as in megaloblastic anemia.
 Non specific
 Serum Haptoglobin: 30-200 mg/dL
 Decreased in both intra and extra vascular hemolysis
 Erythrocyte Survival
 Only done in diagnostic difficulty.[51Cr][Biotin]-Flow cytometry
 Rate of Carbon Monoxide Production.

2. Signs of Intravascular Hemolysis
 Hemoglobinemia: (<5mg/dl)
 Particularly high values, up to 1,000 mg/dl
 Hemoglobinuria
 Exceeds the haptoglobin binding capacity
 Spectroscopy
 Urine Hemosiderin and Urinary Iron Excretion

3. Signs of Accelerated Erythropoiesis
 Reticulocytosis
 Correlate fairly well with the severity of the process.
 Some patients with acquired immunohemolytic anemia may have
normal reticulocyte counts.
 Aplastic crisis.
 Morphologic Findings in the Blood:
 Polychromatophilia and fine basophilic stippling are apparent on
routinely stained smears(EPO stimulation)
 When hemolysis is brisk, nucleated erythrocytes may be found in
the blood (erythroblastosis), usually in numbers below 1% of all the
nucleated cells.
 More striking in hemolytic diseases of newborn.
 Neutrophilic leukocytosis and thrombocytosis may accompany
hemolytic anemia(Acute)
 Large platelets (Chronic).
 Bone Marrow:
 erythroid hyperplasia(<M:E ratio)

4.Laboratory Tests Useful in the Differential
Diagnosis of Hemolysis
 Specific Morphologic Abnormalities
 Direct Antiglobulin Test
 Osmotic Fragility Test
 Tests for Hemolytic Disorders Associated with Heinz
Body Formation

4. Laboratory Tests Useful in the Differential
 Specific Morphologic Abnormalities

Laboratory Tests Useful in the Differential
 Specific Morphologic Abnormalities:
 Spherocytes
 hallmark of hereditary spherocytosis.
 Acquired immunohemolytic anemiasthermal injury,
hypophosphatemia, or certain kinds of chemical poisoning.
 Oval cells or elliptocytes:
 hereditary elliptocytosis(Marked poikilocytosis and
significant hemolysis)
 Stomatocytes:
 Disturbance in red cell cation content
 Rare inherited hemolytic disease and also in liver disease

 Specific Morphologic Abnormalities-cont….
 Acanthocytes: Disturbed erythrocyte lipid composition
 Abetalipoproteinemia and the spur cell anemia
 Liver cirrhosis
 Echinocytes (sea urchin cells)
 Uremia
 Sickle cell anemia
 Target cells
 Characteristic of thalassemia, Hb E syndromes, and Hb C disorders.
 Schistocytes/helmet cells,other fragmented red cells –
physical trauma to the erythrocyte/diseases affecting small blood
vessels.

 Bite cells are erythrocytes that look as if a semicircular
bite has been taken from one edge.
 Hemighosts are red cells that look as if the Hb has
shifted to one side of the cell, leaving the other side
clear. These hemighosts also are referred to as blister
cells and may appear to contain a coagulum of Hb that
has separated from the membrane .
 These cells are seen in patients with oxidant-induced
injury, such as in G6PD deficiency.

 Autoagglutination -characteristic of immunohemolytic
disease caused by cold agglutinins.
 Autoagglutination
 D/D rouleau formation-eg.multiple myeloma

 Direct Antiglobulin Test:
 The test used for detection of immunohemolytic anemia is
the direct antiglobulin or Coombs test
 Positive test results indicate that the red cells are coated
with IgG or complement components, especially C3
 2% to 5% of patients with immunohemolytic disease have
negative test results because the amount of globulin on the
cell surface is below the detection limits
 Positive tests are found in as many as 34% of patients with
AIDS without other evidence of immunohemolytic disease.

Diagnosis of Hemolysis-Coombs test

Diagnosis of Hemolysis-Direct antiglobulin test
(DAT)

Diagnosis of Hemolysis- Spherocytosis
 Osmotic Fragility Test:
 The osmotic fragility test is a measure of the resistance of
erythrocytes to hemolysis by osmotic stress.
 exposing red cells to decreasing strengths of hypotonic saline
 A symmetric, sigmoidal curve is obtained in most subjects,
Increased fragility is indicated by a shift of the curve to the left.
 Prior incubation of sterile blood for 24 hours
 Increased: Spherocytosis; decreased : Fe def anemia, thalassema,
sickle cell disease, liver disease(False negative!!).
 Osmotic gradient ektacytometry is more sensitive and specific
than the osmotic fragility.(The test is performed using the
Osmoscan LoRRca MaxSis ).

Osmotic gradient ektacytometry

Diagnosis of Hemolysis- Spherocytosis
 Other tests:
 Acidified Glycerol lysis time:
 Tests the rate of lysis of RBC and result is expressed in
length of time for 50% lysis.
 Shortened in HS, AIHA,Chronic renal insufficiency and
leukemias, gestation
 Hypertonic cryohemolysis test:
 More specific than OF for HS
 %cryohemolysis is observed after transferring cells from 37o
to 0o for 10 minutes.
 Normal-3-15%; HS>20%

Diagnosis of Hemolysis-Spherocytosis
 Eosin-5-maleimide (EMA) binding test:
 Rapid flowcytometric testing-EMA binds to band-3.
 Reduced fluorescence intensity in HS.
 Identification of deficient cytoskeletal protein:
 Molecular studies- Expensive

Spherocytosis-Diagnostic Nutshell
 Presentation in childhood
 +FH
 Spherocytes in peripheral smear
 Reticulocytosis
 RCI: inc MCHC, dec MCV.
 NegativeDAT
 Screening: OF, AGLT, Cryohemolysis, EMA binding
 Confirmatory: Electrophoresis of membrane
proteins

Disorders of hemoglobin
 Hemoglobinopathies
 Inherited disorder of the hemoglobin due to structural
alteration of globinpolypeptide.
 HBS, HBDpunjab,HBE.
 Thalassaemias
 Heriditary presence of fetal hemoglobin (HPFH)

Tests for disorders of hemoglobin
Clinical details-Hb, Ethnicity,
Indices, smear
Hb electrophoresis in alkaline ph
Suspected Hb
disorder
Inc HbF
Normal/Inc HbA2 Abnormal Hb
Investigate for
Heretozygous
Thalassemia
Citrate agar
electrophoresis@acid ph,
Sickling, Family studies
Investigate for
Homozygous
thalassemia

Hemoglobin electrophoresis at alkaline pH
 Principles:
 Alkaline ph 8.4-8.5- Tris EDTA borate buffer.
 Supporting media for separation of Hb-starch,
cellulose-aceate membrane, filter paper.
 Hb migrates from Cathode(-) to anode(+).
 Hb more +than HbA are near Cathode while Hb with
more _ charge are close to anode.

Hemoglobin electrophoresis at alkaline pH
 Procedure:
 Hemolysate
 Applied to one end of cellulose acetate strip
 Placed in electrophoresis chamber with Tris EDTA
borate buffer with point of origin towards cathode
 Electirc current till separation is achieved
 Removed and stained with protein stain- Ponceau S
and dried.
 Test compared with control.

Citrate agar electrophoresis at acid Ph
 Useful for further classification after elec in alkaline
ph.

HPLC-High performance liquid chromatography
 Detection and identification of hb variants.
 Quantification of HbA2 and HbF.
 Hb A, F, S,C, E/A2,DPunjab, O-Arab, DPhiladelphia can be
separated and identified.
 Hemolysate is introduced into a column packed with
silica gel.
 Different Hb is adsorbed into resin.
 Elution is obtained by changing the ph and ionic
strength.
 Hemoglobin fractions are detected as they pass
through the detector and recorded in computer.

Other tests
 Immonoassay for Hb variants:
 Commercial kits available
 Use monoclonal antibodies against specific Hb.
 HbS, HbA, HbE, HbC can be detected.
 Globin chain electrophoresis:
 α andβ chains can be separated by 6M urea and 6M
ethanol addition to buffer.
 Useful when routine methods fail.

Sickle cell anemia
 Extravascular hemolysis may occur by two
mechanisms: monocyte and macrophage recognition
and phagocytosis of red cells that have undergone
sickling- or oxidation-induced membrane changes
and physical entrapment of rheologically
compromised red cells.
 Intravascular and extravascular patterns of hemolysis
are believed to account for 1/3 and 2/3 of sickle
hemolysis, respectively.

Sickle cell anemia-Nutshell
 MCV, MCHC-Low
 Sicle cells and ovalocytes in smear
 Sickling test:
 2% sodium metabisulfite or sodium dithionite.
 FN: Outdated ragent, Low Hbs in sample, Improper sealing of cover slip.
 FP: High conc of SMBS, Carryover due to inadequate washing of pipette, Mistaking
crenated red cells.
 Limitations: HBS- Trait diff not possible, not useful for mass screening.
 Solubility test:
 Addition of high phosphate buffer.
 HbS-reduced HbS
 Reduced HbSInsoluble polymersturbid solution.
 Quantitation of Hb A2 is performed by HPLC. Quantitation of Hb F can be carried
out by alkali denaturation, HPLC, or radioimmunodiffusion.
 The distribution of Hb F in red cells may be analyzed by its resistance to acid elution
or, more precisely, by Hb F-specific antibodies that measure the number of “F
cells”.(βthalssemia, δβthalassemia,SCA)
 HPFH: Pancellular distribution

Diagnosis of Hemolysis-Enzyme disorders of
hemoglobin
 The specific diagnosis of G6PD deficiency is made by
adding a measured amount of hemolysate to an assay
mixture containing substrate (glucose-6-phosphate)
and cofactor (NADP) and then spectrophotometrically
measuring the rate of NADPH generation.
 Other screening methods detect NADPH generation
indirectly by measuring the transfer of hydrogen ions
from NADPH to an acceptor.
 Methemoglobin reduction test: methylene blue is the
acceptor used for the transfer of hydrogen from NADPH
to methemoglobin, thereby facilitating its reduction.

Paroxysmal cold hemoglobinuria
 The Donath-Landsteiner test -patient serum
i+melting ice +washed group O, P-positive RBCs, and
fresh normal serum as a source of complement.
 Later, the tube is transferred to 37°C for a second
incubation.
 Lysis visible to the naked eye after the warm
incubation is a positive.

Diagnosis of Hemolysis-AIHA
 The hallmark of immune-mediated hemolytic
anemia is the presence of immunoglobulin,
complement, or both on the surface of the RBCs.
 In >95% of warm AIHA -DAT is positive.
 20% -66% have only IgG on the surface
 24% to 63% have IgG and C3,
 7% to 14% have only C3
 1% to 4% are DAT-negative.

Diagnosis of Hemolysis-Hemolytic disease in newborn

Diagnosis of Hemolysis-PNH

Diagnosis of Hemolysis-Aquired non immune
hemolyic anemia

Diagnosis of Hemolysis-Aquired non immune hemolyic
anemia
 Babesiosis:
 Laboratory features include hemoglobinuria, hyperbilirubinemia, normocytic
anemia, thrombocytopenia, and sometimes leukopenia.
 Both B. microti and B. divergens can be seen in RBCs on the peripheral blood
smear and can be confused with malaria.
 Serologic antibody tests and polymerase chain reaction– based assays are
available to aid in diagnosis
 Trypanosomiasis
 Normocytic anemia with reticulocytosis is prominent.
 Red cell survival is shortened and autoagglutination of erythrocytes with
accelerated erythrocyte sedimentation characteristically is observed.
 The results DAT test may be positive.
 Erythrophagocytosis by macrophages is seen throughout the
reticuloendothelial system

Diagnosis of Hemolysis-Aquired non immune hemolyic
anemia
 Visceral Leishmaniasis (Kala-Azar):
 Anemia, neutropenia, and thrombocytopenia
 The bone marrow is hyperplastic with dyserythropoietic changes, and the
diagnosis can usually be made by finding macrophages containing intracellular
parasites (Leishman-Donovan bodies).
 Red cell survival studies indicate that hemolysis is the major cause of anemia in
leishmaniasis
 Bartonellosis (Carrion’s Disease):
 The findings in the blood are characteristic of acute extravascular blood
destruction.103,109 As viewed in Wright- or Giemsastained blood smears,
numerous Bartonella organisms are apparent in the erythrocyte
 Clostridial Sepsis:
 Hemolysis is thought to result from the elaboration of a clostridial toxin, a
phospholipase that attacks erythrocyte membrane lipids to form highly lytic
lysolecithins.

Diagnosis of Hemolysis-DIHA
 Morphologic findings characteristic of hemolytic anemia caused by
oxidant drugs and chemicals include the following: Heinz bodies (seen
with brilliant cresyl blue supravital stains of blood during hemolytic
episodes)
 “bite cells” (seen in routine Wright-stained blood smear) as
erythrocytes that look as if a Semicircular bite has been taken from one
edge
 Hemighosts or Eccentrocytes: Erythrocytes that look as if the
hemoglobin has shifted to one side of the cell, leaving the other side
clear (Fig. 32.4C).
 These hemighosts also are referred to as “blister cells” and may appear
to contain a large vacuole. These RBCs contain a coagulum of
hemoglobin that has separated from the membrane, often leaving an
unstained non-hemoglobin–containing cell membrane.
 Hemighosts appear only when hemolysis is brisk.

Diagnosis of Hemolysis-Drug induced-HA
 Just as with idiopathic AIHA, anemia with
reticulocytosis and a positive DAT are hallmarks of
the condition.
 Elevated indirect bilirubin and LDH are common
findings.
 Rampant RBC destruction leads to hemoglobinemia,
hemoglobinuria, and elevated creatinine levels.
 One can differentiate the
 neoantigen mechanism from cold autoantibodies by
the absence of high-titer cold agglutinins or D–L
antibodies in the druginduced cases

Diagnosis of Hemolysis-Metals
 Copper Toxicity:
 Accidental exposure to toxic amounts of copper sulfate.
 Hemolytic episodes after hemodialysis due to faulty copper
tubing and increased levels of the metal in dialysis fluid.
 The release of inorganic copper into the circulation accounts for
the hemolytic anemia that is observed in patients with Wilson
disease (hepatolenticular degeneration).(defects in the copper
transporting intracellular ATPase ATP7B, which leads to a
deficiency of ceruloplasmin, the plasma copper transport
protein.)

 Arsenic poisoning:
 Industrial processes such as galvanizing, soldering
etching, and lead plating, transistor industry
 Manifestations of poisoning appear 2 to 24 hours after
exposure
 Abdominal pain, nausea, and vomiting; the passage of dark-
red urine; jaundice; anemia; reticulocytosis; leukocytosis;
and other signs of acute hemolytic anemia
 acute, oliguric renal failure may ensue.
 The antiglobulin test result is negative.
 Mortality 20%.

 Lead Toxicity:
 Manufacturing of batteries, paint or pigments, mining, or
smelting; the primary route of assimilation is by
inhalation. Environmental exposures in children occur by
ingestion of lead from ceramics, paint, gasoline, or water
from lead pipes, or having a parent who works in a high-
lead environment. Lead poisoning through use of herbal or
traditional medicines has also been reported.
 The peripheral smear shows extensive coarse basophilic
stippling and reticulocytosis.
 The diagnosis is confirmed by measuring the blood and
urine lead levels.

Diagnosis of Hemolysis-Misc
 Water (Osmotic Hemolysis):
 Water or hypotnic solution irrigation
 Near drowning
 Lab: Hemoglobinemia, hemoglobinurea.
 Spider Bites
 Snake bites
 Bee Stings

Diagnosis of Hemolysis-Misc
 Hemolysis with Thermal Injury:
 Acute hemolytic anemia has been observed after extensive thermal
burn. Signs of intravascular hemolysis are associated with
schistocytes, spherocytes, and echinocytes in the blood along with
increased osmotic and mechanical fragility of the erythrocytes.
 Heated Fluids and Blood
 Cardiac and Large Vessel Abnormalities
 March Hemolysis
 Hypophosphatemia
 Giant hemangiomas and heamangioendothelioma

4.Tests for Hemolytic Disorders Associated with
Heinz Body Formation
 Special supravital stains
 G6PD deficiency
 splenectomized individuals with unstable Hb disease

Diagnostic Strategy for the Patient
with Hemolytic Anemia
 Establishing the Presence of Hemolytic Anemia
 Conditions Sometimes Mistaken for Hemolytic
Anemia
 Determining the Specific Cause of Hemolysis

Determining the Specific Cause of
Hemolysis
 1. Those patients in whom the diagnosis is clear because of medical history
such as obvious exposure to infectious, chemical, or physical agents.
 Look for other causes that can precipitate the disorder
 2. Those patients with a positive direct antiglobulin test.
 Search for nature of antibody by serology.
 3. Those patients with antiglobulin-negative, spherocytic hemolytic anemia.
 Probable hereditary sperocytosis-OF, Family.
 Immune hemolytic anemia associated with Sperocytosis.
 4. Those patients with other specific morphologic abnormalities of
erythrocytes.
 Obvious on peripheral smear
 5. Those with no specific morphologic abnormalities and a negative reaction
to the antiglobulin test.
 Hb electrophoresis
 heat denaturation test
 Red cell enzymes
 screening test for paroxysmal nocturnal hemoglobinuria

References
 Wintrobe’s Clinical hematology-13th edition
 Harrison text of Hematology and Oncology
 Robbins and Cotran pathologic basis of disease
(2015)
 Wikipedia
 Medscape
 Web sources

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