HEMOLYTIC

ANEMIAS:
Laboratory
Diagnosis
Hemolytic anemias
The hemolytic anemia is the disease condition
of short erythrocyte survival partially offset
by increased activity of the bone marrow
erythropoiesis.
 The hemolytic
disease
hemolytic and
anemia
With optimal marrow compensation, the survival of red cells in
the circulation can decrease from the normal 120 days to as
few as 15 to 20 days without anemia developing.

Such an increase in both When red cell survival is

destruction and production of so short that anemia
erythrocytes can result in a develops despite a
compensated hemolytic state vigorous
erythropoietic response,
without anemia being however, the term hemolytic
present, so-called anemia is appropriate.
hemolytic
compensateddisease.
PATHOGENESIS AND CLASSIFICATION OF
HEMOLYTIC ANEMIAS

The course of the acute chronic

disease
The place of intravascular extravascular
RBC distraction
The whence acquired inherited
 Lab findings differ ,depending on :

The site of blood destruction

The amount of destroyed blood
The rate of destruction
 LABORATORY
MANIFESTATIONS
 Test findings
1. Related to the increase in erythrocyte
destruction,
2. Related to the compensatory increase in the rate
of erythropoiesis,
3. Found only in particular varieties of hemolytic
anemia, which therefore are useful in differential
diagnosis.
 I. Signs of Excessive Red Cell
Destruction
1. Erythrocyte Survival
2. Catabolism of Heme
3. Lactate Dehydrogenase increase
4. Disappearance / Decrease of Haptoglobin
5. Glycosylated Hemoglobin decrease
6. Signs of Intravascular Hemolysis
7. Signs of intracellular Hemolysis
 I. Signs of Excessive Red Cell
Destruction
1. Erythrocyte Survival
The life span of the red cell can be measured by
random labeling with 51 chromium.
Erythrocyte life span determinations are time-
consuming and
expensive.
This test is rarely necessary because the approximate amount
of red cell destruction usually can be determined by the
serial observations of the degree of anemia,
reticulocytosis, and jaundice.
For these reasons, determination of red cell survival
should be reserved for use in evaluating patients with
especially difficult diagnostic problems.
 I. Signs of Excessive Red Cell
Destruction
2. Catabolism of Heme
Serum Bilirubin unconjugated (indirect-reacting) ,
The increased serum bilirubin level in hemolysis almost
always consists of the unconjugated (indirect-reacting)
pigment. The conjugated fraction remains within
normal limits, and no bilirubin is evident in the urine.
Rate of Heme Catabolism. endogenous carbon monoxide
fecal urobilinogen excretion
 Bilirubin
Bilirubin
 Absence of hyperbilirubinemia does not exclude HA
 Unconjugated bilirubin also corrected for anemia

Upper limit of normal = 1.0 mg/dL X patient’s Hct/45

 Serum bilirubin 1 - 4 mg/dL especially if

unconjugated implies hemolysis or Gilbert’s syndrome or
ineffective erythropoeisis such as megaloblastic anemia;
serum bilirubin
> 4 mg/dL hemolysis plus liver dysfunction
 I. Signs of Excessive Red Cell
Destruction
3. Lactate Dehydrogenase increase.
Serum LD activity often is increased in patients
with hemolytic anemia, as in megaloblastic anemia.
Of the LDH, LDH-2 isozymes predominates in
hemolytic anemia, LDH-1 predominates in megaloblastic
conditions.
The increase in LDH probably results from liberation of
the erythrocyte enzyme into the plasma during
hemolysis.
is limited by this lack of specificity.
 The usefulness
LDH: of61%;
specificity LDH values in thevalue
predictive detection
40% of
hemolysis
 LDH + haptoglobin: specificity 92%
 I. Signs of Excessive Red Cell

•Destruction
•4. Disappearance of Haptoglobin

•A low haptoglobin level indicated an 87% probability of

hemolytic disease. When hemoglobin enters the plasma, it binds to
haptoglobin, and the hepatocyte removes the complex.
•Haptoglobin tends to disappear from the plasma in individuals
with hemolytic disease with the intravascular site and with
predominantly extravascular hemolysis, such as sickle cell anemia,
hereditary spherocytosis, hereditary elliptocytosis, and pyruvate
kinase deficiency.
•In hereditary spherocytosis, the characteristically reduced
haptoglobin levels are restored after splenectomy.
Serum Haptoglobin
Reduced both with intra and extravascular
hemolysis

Sensitive (reduced when hemoglobin
destruction exceeds 2-3 times normal) but may
require greater degrees of hemolysis in states of
increased synthesis (acute phase
reactant/inflammation, neoplasia and steroid
therapy). Low levels congenitally (rare) and
severe hepatocellular disease
Not usually decreased following transfusion
 I. Signs of Excessive Red Cell
Destruction
5. Glycosylated Hemoglobin decrease
The averaged 6.7% (range, 6.0 to 8.0%) normal
the average value fell to 3.9% (range, 2 to 5.5%) -
hemolytic anemias

6. Signs of Intravascular 7. Signs of intracellular

Hemolysis Hemolysis
Hemoglobinemia Splenomegaly
Hemoglobinuria
Urine Iron Excretion,
Hemosiderinuria
Hemoglobine
mia
Hemoglobinuria
Hemosiderinuria-urine sediment
Hemosiderinuria-urine
sediment
Iron stain
 II. Signs of Accelerated
Erythropoiesis
Blood
 Reticulocytosis (Polychromasia, Basophilic stippling)
 Macrocytosis
 Erythroblastosis (Nrbc)
 Leukocytosis and thrombocytosis
Bone marrow
 Erythroid hyperplasia (more then
20-25% normoblasts in BM aspirate)
Polychromasia
Polychromasia
Reticulocyte Counts
 Rule of thumb: uncorrected reticulocyte
count > 5% suspect hemolysis; > 10%
hemolysis very likely
 Differential diagnosis: blood loss and recent
treatment of a megaloblastic anemia
Erythroid Hyperplasia
 III. Laboratory Test Findings Useful in
Differential Diagnosis

1. Specific Morphologic Abnormalities of RBC

2. Hb Electrophoresis
3. The Antiglobulin (Coombs) Test
4.The Osmotic Fragility Test
4. Tests for Hemolic Disorders Associated with
Heinz-body Formation
5. Other Tests
Hereditary spherocytosis is characterized by
numerous spherocytes
 Hemolytic anemia

 The presence of spherocytes is not diagnostically

specific, since this may result from:
1. Hereditary spherocytosis,
2. Autoimmune hemolytic anemia, or
3. Alloimmune hemolytic anemia (e.g., hemolytic disease of the
newborn or a delayed transfusion reaction).

 Nevertheless, consideration of the clinical features, together with

the results of a direct antiglobulin test, in patients with
spherocytes will generally indicate the correct diagnosis.
Acute hemolysis in glucose-6-phosphate dehydrogenase
(G6PD) deficiency, with the
presence of a “bite” cell, or keratocyte (arrow).
"blister" cells
 Oxidized Hb
accumulates

blister
cell
 Hemolytic
anemia
 Microspherocytes (i.e., cells that are both hyperchromic
and significantly reduced in size and therefore in diameter)
may be present in low numbers in patients with a
spherocytic hemolytic anemia but are also characteristic of
burns and of microangiopathic hemolytic anemia.
 The detection of a microangiopathic hemolytic anemia is of
considerable clinical significance, since this type of anemia
may indicate pregnancy-associated hypertension,
disseminated cancer, chronic disseminated intravascular
coagulation, the hemolytic–uremic syndrome, or thrombotic
thrombocytopenic purpura;
 The latter two conditions both require urgent diagnosis so
that appropriate management can be initiated
 shows microangiopathic hemolytic anemia
resulting from

cyclosporine therapy, with numerous red-cell fragments.

Mechanical Trauma - Schistocytes
Schistocytes
 Hereditary
Elliptocytosis
Stomatocytes
 Sickle
cell
anemia,PBS
Sickle cell anemia,PBS
Sickle cells
 Diagnostic recommendations regarding the
laboratory investigation of abnormal Hbs and
thalassemias

1. Initial tests:
Complete blood count (CBC),
Electrophoresis at pH 9.2,
Tests for solubility and
sickling,
& quantification of Hb A2
and Hb F.
2. If an abnormal Hb is identified on the
preliminary tests:
electrophoresis at pH 6.0–6.2,
Globin chain separation,
and isoelectric focusing
(IEF).
 The elements of one approach include

CBC
Hb H test
Ferritin
HPLC or capillary Elect for Hb A2 and F
quantification
 of
The use and detection
HPLC of any
or Capillary ElectHb variants
streamlines thefollowed
recommended preliminary and follow-up tests for the by
identification of hemoglobinopathies and thalassemias and
electrophoresis at both alkaline and acid pH.
provides for rapid and complete diagnostic work up in a
majority of cases.
Control
 A control sample containing Hb A, F, S, and C
should be applied to each strip containing the
unknown samples.
 With each analysis, slight variations in the migration
rate may be caused by slight current fluctuations,
different buffer lots, or variations in application.
 Participation in an inter laboratory trial or
proficiency check system is recommended.
 Results should occasionally be rechecked using
duplicate samples.
 Separation
 With every new lot of cellulose acetate, samples
containing a combination of Hbs A & F and of Hbs S
& F should be applied and tested for separation
properties.
 Distinct separation with a small, clear area between
these hemoglobins must be obtained.
 It may be necessary to reduce the concentration of
hemoglobin in the sample and also to adjust time &
voltage to obtain these distinct separations.
 Principle of capillary electrophoresis
Thermal bridge
Temperature controlled
by Peltier elements

Migratio
n
Detector Deuterium Lampe

High
voltage

Cathode - Anode +
Capillarys: advantages
Fully automated and very fast by application of very high
voltage
No support, free migration in liquid medium
Very good resolution and reproducibility
Very low analyzed sample volume (very good sensitivity)
No dye, direct measurement at 200 nm / 415 nm
Curves similar to those obtained by densitometry
Advanced software capabilities
SPECIALISTS

Technology
300 points with 16 zones Curve
Normal
Pattern
 III. Laboratory Test Findings Useful in
Differential Diagnosis
2. The Antiglobulin (Coombs) Test
Positive test results indicate that the red cells are
coated with IgG or complement components,
especially C3.
The test is usually satisfactory,
but 2 to 5% of patients with immunohemolytic disease
associated with warm complete agglutinins 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.
 Significance Positive DAT
 Incidence of positive DAT is hospitalized population is
7 – 8%
- 80% due to C3d with no IgG
-Most have no evidence of hemolysis even among
patients with anemia
- In most, eluates are non-reactive with RBCs and
positive DAT associated with elevated plasma gamma
globulins
- Predictive value positive DAT in random patient:
1.4%
-In patient with hemolytic anemia PPV positive
DAT 83%; NPV negative DAT is 99%
 Significance Positive DAT
 DAT-negative healthy individuals have 5 – 90
IgG and up to 560 C3d molecules per RBC
Distribution, physiological role?, RBC
senescence?, Complement receptors
 1:13,000 normal blood donors have positive
DAT
IgG, increasing age, may persist for years,
with sensitive testing possible to demonstrate
increased RBC turnover but clinically significant
hemolysis rare
DAT Immune Hemolysis
 Warm antibody AIHA
- 67% positive IgG and C3d
- 20% positive IgG and negative C3d
- 13% Positive C3d and negative IgG
 Cold Agglutinin Syndrome
- 100% positive C3d and negative IgG
 Paroxysmal Cold Hemoglobinuria
- 100% positive C3d and negative IgG
3. The Osmotic Fragility Test
The osmotic fragility test is a measure of the resistance
of erythrocytes to hemolysis by osmotic stress.
.
Increased osmotic fragility is observed in
conditions associated with spherocytosis.
OFT
 Osmotic Fragility Tests

NaClg/L 9 7.5 6.5 6 5.5 5 4 3.5 3 2 1

% hem cont-0 0% 0% 0% 0% 0.70% 1.60% 64% 93% 94% 97% 99%
%hem cont-24 0% 0.30% 0.40% 0.70% 5% 45% 82% 95% 96% 98% 99%
%hem pt -0 0.30% 0.30% 0.40% 0.45% 2.10% 46% 91.80%92.50% 95.60% 94.40% 99.40
%
%hem pt-24 1.10% 5.80% 35.10% 61.90% 74.40%80.60%91.60%94.10% 95.70% 96.70% 98.90
%
 : ‫ﻢﻧﺎﺧ‬
Osmograph ‫رﺎﻤﻴﺑ مﺎﻧ ﺮﺘﻛد‬
‫ ﻚﺷﺰﭘ مﺎﻧ‬: ‫يﺎﻗآ‬
100
% MCF(50% lysis), time 0 : 4.8 (RI: 4.0-4.45)
90 MCF(50% lysis), After 24 Hr.: 6.2 (RI: 4.65-5.9)
%

80
%

70
%
% Hemolysis

60
% Normal control,time:0
50% Normal control,after
50
% 24Hrs
Neda mi,time:0
Shahkara
Neda mi,after
40 Shahkara
24Hrs
%

30
%

20
%

10
% 0 1 2 3 4 5 6 7 8 9 1
0
NaCl conc. (g/L)
0%
 III. Laboratory Test Findings Useful in
Differential Diagnosis
4. Tests for Hemolic Associated with Heinz-body
Disorders Formation
Heinz Body
History
 They are named after Robert Heinz (1865-
1924), a German physician who in 1890
described these inclusions in connection with
cases of hemolytic anemia
 Heinz Body - Definition
• Refractile red cell inclusions of variable size(1-
3μm) and usually eccentrically located and
adhered to the red cell membrane.
• Seen only with supravital staining with crystal
violet, brilliant cresyl blue, methyl violet or on a
fresh, wet preparation of blood.
• Not seen on a Wright-Giemsa stain, but
spherocytosis of varying degree, depending on
the severity of the hemolysis, is usually
present, and bite cells may be seen.
Pathobiology
 The inclusions are composed of denatured
hemoglobin that occurs as a result of
oxidative injury to the red cell. Oxidative
injury to the red cell membrane also
occurs.
Differential diagnosis
 Red cell enzymopathies (usually a result of
oxidant drug exposure or infection)
 acute Heinz body hemolytic anemia, e.g. G-6PD
deficiency
 chemical poisoning, drug intoxication,
 Unstable hemoglobinopathies, e.g. Hb Gun-
Hill
 Thalassemias
 Heinz Body
Peripheral blood smear, BCB stain, 1000x
 Heinz bodies

Braza, J. ASH Image Bank 2007;2007:7-00003

Copyright ©2007 American Society of Hematology. Copyright restrictions may apply.

 Heinz Body
Peripheral blood smear,1000x
Reticulocytosis- Heinz bodies
Alpha Chain Precipitation
Alpha Chain Precipitation
In β-thalassaemia major, methyl violet
staining of the BM will demonstrate
precipitated α-chains.
These appear as large irregular inclusions in
late normoblasts, usually single and closely
adhering to the nucleus.
If such patients are splenectomized,
inclusions are also found in reticulocytes and
mature red blood cells.
Heinz body, wright stain
Hb H INCLUSION BODIES IN BCB STAIN
,RETICULOCYTE:5-10%
Golf Ball Appearing
RBC
 Hemoglobin H Inclusions
Mix together in a small tube as for staining
reticulocytes equal volumes of fresh blood or
EDTA-blood and 10 g/l brilliant cresyl blue or
20 g/l New methylene blue in iso-osmotic
phosphate buffer pH 7.4.
Leave the preparation at 37oC for 1-3 hours,
and make films at intervals during this time.
Haemoglobin H precipitates as multiple pale-
staining greenish-blue, almost spherical,
bodies of varying size
 5. Other Tests

Other important procedures for detecting and

differentiating the hemolytic anemias include
methods for identifying enzyme deficiency, for
detecting and defining abnormal hemoglobins,
the other serologic techniques for evaluating
immunohemolytic anemias, and tests for
paroxysmal nocturnal hemoglobinuria.
Ham's Test Sucrose
Hemolysis Test
DIAGNOSTIC APPROACH
A final diagnosis of one of the hemolytic
anemias is established by a two-step process:
First, demonstrating that a hemolytic anemia
is present and,
Second, determining the specific cause of the
condition.
Thank you for attention