1. HEMOLYSIS
Dr.Mansooreh Eslami

2. HEMOLYSIS—GENERAL
intrinsic hemolytic anemia:
due to a defect of the red cell itself
These are usually hereditary and are commonly
grouped as membrane, metabolic, and hemoglobin defects.
extrinsic hemolytic anemia:
due to a factor outside the red cell and
acting upon it

3. Hemolytic anemias.
A shortened red cell survival
hemolysis is present
Erythrocyte Survival Studies
If the hemolytic process is mild or
obscure,
red cell survival studies may be necessary

4. Radioactive chromium (51Cr)
• Labeled chromate is added to a blood
sample in vitro and binds to β-chains of
Hb.
• The chromated red cells are injected
intravenously,
• their disappearance is measured by
counting blood
• every 1–2 days for 10–14 days
• Because 51Cr emits γ-rays, external
scanning can detect sites of red cell
destruction.

5. Residual activity is an index of
the intravascular life span of the
labeled red cells.
The erythrocyte life span :
the period during
which one half of the radioactivity
remains in the blood ,the T1/2 51Cr
● 28-35 days is considered normal half
time of survival for Cr-51 labeled
autologous RBC's

6. Results of 51Cr erythrocyte survival curve

7. autoimmune hemolytic anemia.
The mean cell life span (MCL) was 9–10 days and was recorded
at a period when 37% of cells were still circulating.
The time of 50% survival (T 1/2 Cr51 ) was 6–7 days.

9. Results of radioactive chromium (51Cr) erythrocyte
survival curve in a patient with hemolytic anemia containing
two cell populations.
40% of the cells have a mean life span of 100 days. Sixty
percent of cells have a mean life span of 5 days.
This type of curve has been seen in hereditary enzymedeficiency hemolytic anemias, sickle cell anemia, and PNH

10. Hemoglobin Destruction
INtravascular:
*hemoglobinemia
*when the plasma Hb level exceeds 50–200 mg/dL
(8–31 μmol/L)
hemoglobinuria
*Hemosiderinuria
*Plasma Hb not bound to haptoglobin or removed
by the kidney is oxidized to hemiglobin
*The oxidized heme groups (hemin)
bound
to hemopexin, a β-globulin
cleared
by the hepatic parenchymal cells.
*methemalbuminemia

11. Lactate dehydrogenase (LD)
• it is cleared more slowly than Hb
• In hemolytic anemias, reversal of the LD
isoenzyme pattern is seen, with LD1 exceeding LD2
• plasma Hb level:
• Normal:0.5–5 mg/dL (0.08–0.78 μmol/L)
• A rise to 10 mg/dL imparts to the plasma a yellow to
orange color
• With further increase, the color becomes pink.
•
Levels up to 25–30 mg/dL are common in hemolytic
anemia.

12. Extravascular:
no hemoglobinemia, hemoglobinuria,
or hemosiderinuria
products of heme catabolism:
1. An increase in CO expired, or in the blood
carboxyhemoglobin level.
2. An increase in indirect-reacting serum bilirubin
3. An increase in urine urobilinogen or, more
consistently, in fecal
Urobilinogen
Examination of feces is more dependable than
examination of urine because feces may show an
increase when the urine shows none

13. Blood Film
The anemia is normocytic or macrocytic
Macrocytosis is due to the presence of immature red
cells,
. Polychromasia is usually prominent; it may be
excessively basophilic normoblasts may be present,
Spherocytes suggest hereditary spherocytosis
(HS) or autoimmune hemolysis
schistocytes imply microangiopathic
hemolytic anemia
sickle cells, target cells, or crystals suggest a
hemoglobinopathy.
.

14. .
When hemolytic anemia is acute
increased numbers and younger forms of
leukocytes and platelets are often released
from the marrow, together with
erythrocytes.
The result is leukocytosis with a ―shift to
the left‖ and thrombocytosis with both
normal and giant platelets

16. .

17. .

18. .

19. .
Bone Marrow
Normoblastic hyperplasia is present and may
be striking in degree.
Storage iron is usually increased and
sideroblasts are normal or
increased in number, reflecting the
abundance of available iron for
Hb synthesis.

20. Hereditary Spherocytosis
• most often autosomal dominant
• In about 15%–30% of cases, however, neither parent
is affected
• The MCV is low-normal and the MCHC is often
increased

22. HS can be divided into the following
pathogenetic categories:
(1) isolated partial deficiency of spectrin,
(2) combined partial deficiency of spectrin
and ankyrin,
(3) partial deficiency of band 3 protein,
(4) deficiency of protein 4.2, and
(5) other, less common defects.
Most of these abnormalities are related to
the synthesis of abnormal protein, mostly
through point mutations or frameshift

23. Osmotic Fragility Test
Red cells are suspended in a series of tubes containing hypotonic
solutions of NaCl, varying from 0.9%–0.0%,
incubated at room temperature for 30 minutes, and centrifuged.
The percent hemolysis in the supernatant solutions
is measured and plotted for each NaCl concentration.

24. Procedure
1- We will do this dilution:
Test
tube
1
2
3
1%Nacl(ml)
D.W. (ml)
Final conc. (%)
10.0
8.5
7.5
0.0
1.5
2.5
1.00
0.85
0.75
4
5
6
7
8
9
10
11
12
13
14
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.0
1.0
0.0
3.5
4.0
4.0
5.0
5.50
6.0
6.0
7.0
8.0
9.0
10.0
0.65
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.20
0.10
0.00

25. Procedure
2- Then we divide every volume in 2 tubes so now
we get 28 tubes.
3- Add 50 micron of whole blood to every tube.
4- let the tubes at R.T for 30 min
5- Well mixing by using the vortex.
6- Centrifuge for 5 minutes at 2500 rpm.
7- Now we will measure the absorbance in the
tubes by using spectrophotometer (540 nm).
8- calculate the % of hemolysis.

26. Result:
• % of hemolysis =
(Abs of tube / Abs of tube 14) * 100%
• Normal Range
– Hemolysis begins 0.45% and complete 0.35%

27. Cells that are more spherical, with a decreased
surface/volume ratio, have a limited capacity to expand
in hypotonic solutions and lyse at a higher concentration
of NaCl than do normal biconcave red cells. They are
said to have
increased osmotic fragility.
Conversely, cells that are hypochromic and
flatter have a greater capacity to expand in hypotonic
solutions, lyse at a
lower concentration than normal cells, and are said to
have decreased osmotic fragility
Cells with increased surface/volume
ratio are osmotic resistant.
iron deficiency, thalassemia,
liver disease, and reticulocytosis

29. Erythrocyte osmotic fragility
a, Thalassemia, showing a small fraction
of cells with increased fragility (lower left) and a larger
fraction of cells with decreased fragility (upper right).
b, Normal curves fall in the shaded area.
c, Hereditary spherocytosis, showing increased osmotic
fragility.

30. The osmotic fragility of freshly drawn blood is usually
increased in HS
but may be normal in mildly affected patients.
In blood that is incubated
at 37° C for 24 hours before the test is performed, the
osmotic fragility is
almost always increased
A greater difference in median fragility (after
incubation from before incubation) occurs in HS cells
than in control normal cells; this is an important
diagnostic feature in HS.

31. Blood in HS characteristically shows a greater increase
in fragility with incubation than does normal blood or even blood
of acquired spherocytosis (e.g., autoimmune hemolytic anemia).

32. Autohemolysis Test
Sterile, defibrinated blood is incubated at 37° C for 48 hours
. In normal blood, without added
glucose, the amount of autohemolysis at 48 hours is 0.2%–2.0%. In
normal blood, incubated with added glucose, the amount of
autohemolysis is less—0%–0.9%
In HS, autohemolysis is virtually always increased; with glucose,
the lysis is diminished to a variable extent
This test is being used less frequently
and is probably no more sensitive than the incubated osmotic
fragility test

33. Hereditary Elliptocytosis
autosomal dominant
weakening of the membrane skeleton and
defective association of
proteins that hold the skeleton together
three groups: (1) common HE (including
hereditary pyropoikilocytosis [HPP]),
with elliptocytes that
may be rod shaped, (2) spherocytic HE,
and (3) Southeast Asian ovalocytosis.

34. The most commonly defined abnormality
appears to be a defect in
spectrin,. Other abnormalities include a
defect in protein 4.1 and deficiency of
glycophorin C
HPP is associated with two abnormalities: a
mutation in spectrin that disrupts spectrin
heterodimer self-association, and a partial
deficiency of spectrin that results in a
decreased spectrin/band 3 ratio

36. Common HE
Most persons with the common form of HE (˜90% of
cases) are nonanemic;
a minority of this group (perhaps 10%–20%) have mild
hemolysis
.
Nonhypochromic elliptocytes are abundant in the blood
film, numbering
approximately 15%
whereas in normal individuals less
than 5% of the red cells are elliptical
The deformity is increased in sealed, moist preparations.

37. In a subgroup of common HE, especially in black families,
affected neonates transiently have moderate poikilocytosis,
red cell fragmentation,
and budding, with hemolytic anemia; during the first year of
life,
hemolysis declines and typical HE emerges.
Worsening of hemolysis in the neonatal
period has been attributed to the presence of fetal
hemoglobin, which
binds poorly to 2,3-diphosphoglycerate (2,3-DPG).
Higher levels of the
latter exert a destabilizing effect on spectrin-protein 4.1actin interaction

38. Hereditary Pyropoikilocytosis
HPP is a severe congenital hemolytic anemia, which is
characterized by
microcytosis, striking micropoikilocytosis and fragmentation,

39. autosomal recessive inheritance.
HPP represents a subtype of
common HE.
In contrast to normal red cells,
which show budding and
fragmentation when heated to 49° C,
HPP red cells fragment
at 45°–46° C.

40. Spherocytic HE
This subgroup accounts for 10% of cases.
A mild to moderate hemolytic
anemia and splenomegaly are present,
with both elliptocytes and spherocytes,
Abnormal osmotic fragility
And Autohemolysis tests.
Poikilocytes and fragments are usually absent.
The molecular basis of this subtype is unknown.

41. Southeast Asian Ovalocytosis
Hemolysis is usually absent or mild.
The erythrocytes are less elongated, and some
have the appearance of stomatocytic ovalocytes
Many cells contain one or two transverse ridges
or a longitudinal slit.
This condition is associated with increased
resistance to malaria.
The underlying defect is related to a deletion of 27
bases from the band 3 gene

42. Hereditary ovalocytosis

44. Hereditary Stomatocytosis (Hereditary
Hydrocytosis)
This is a rare, autosomally transmitted disorder
Heterozygotic individuals
have no anemia, and 1%–25% of stomatocytes are
seen on the blood film.
In presumed homozygotic individuals, about one
third of the red cells are
stomatocytes, and there is a mild to moderate
hemolytic anemia.

46. The membrane abnormality results in increased
permeability of the membrane to Na +and K+ (and
therefore water), resulting in hydrated, macrocytic red
cells. The MCV may be as high as 150 fL
Osmotic fragility and autohemolysis are increased
Although the exact membrane defect is not known,
several reports indicate the absence of a membrane
protein located in the band 7 region called stomatin
2009).
Individuals with Rh deficiency syndrome, either absent
(Rh) or markedly reduced (Rh), usually have hemolytic
anemia with stomatocytosis.

47. Paroxysmal Nocturnal Hemoglobinuria
PNH is an acquired clonal stem cell disorder characterized by the
production of abnormal erythrocytes, granulocytes, and platelets
The red cell defect renders them more susceptible to
complementmediated intravascular lysis.
Several complement defense proteins are decreased or absent in
PNH.
These proteins include:
decay accelerating factor (DAF, CD55), membrane inhibitor of
reactive lysis (MIRL, CD59), and C8-binding protein (a homologous
restriction factor).
DAF is a glycoprotein that antagonizes the convertase complexes
of complement.
MIRL is a protein that controls the membrane attack complex,
C5b-9.

48. Other proteins that are deficient in PNH
include
CD58 (leukocyte function antigen 3),
CD14 (endotoxin-binding protein receptor),
CD24,
and CD16a (Fcγ receptor).
Membrane-associated enzymes
such as acetyl cholinesterase and leukocyte
alkaline phosphatase may be
deficient as well.

49. Recent work indicates that deficient
proteins and enzymes
are attached to the cell membrane by a
common glycolipid anchor called GPI.
Deficiency of GPI results in secondary
deficiency of the attached proteins.
Therefore, PNH can be redefined as partial
or complete lack of GPI-linked proteins on a
population of cells of the hematopoietic
system.

51. Proteins Deficient from PNH Blood Cells
CD55
CD58
CD59
PrPC
AChE
JMH Ag
Dombroch
HG Ag
CD55
CD58
CD59
CD109
PrPC
GP500
Gova/b
B cells
RBC
Haematopoietic
Stem Cell
CD24
CD58
CD48
CD73
T cells
CD59, CD90, CD109
CD55
CD59
PrPC
CD108
CD55
CD58*
CD59
CD48
CD52
CD87
CD108
PrPc
ADP-RT
CD73
CD90
CD109
CD16*
Platelets
NK cells
CD55
CD59
CD16
CD48
CD66c
CD109
LAPNB1
p50-80
ADP-RT
CD58*
CD14
CD24
CD66b
CD87
CD157
PrPC
GPI-80
NA1/NA2
Monocytes
PMN
CD14
CD59
CD87
Group 8
CD55
CD48
CD109
PrPC
CD16
CD58*
CD52
CD157
GPI-80
CD55
CD58
CD59
CD48
CD52
PrPc
CD16
(Courtesy of Lucio Luzzatto)
QuickTime™ and a
GIF decompressor
are needed to see this picture.

52. chronic intravascular hemolysis with or without obvious
hemoglobinuria.
However, hemosiderinuria is almost constantly present.
Typical nocturnal or sleep-related hemoglobinuria is present in a
minority of patients.
Bouts of hemolysis could be initiated by infection, surgery, whole
blood transfusion, injection of contrast dyes, or even severe
exercise.
The proposed relationship between mild drop in pH during sleep and
nocturnal hemoglobinuria has not been confirmed

53. The blood usually shows a normocytic anemia with a
reticulocytosis
that is often less than expected for the degree of anemia.
Hypochromic microcytic anemia is not uncommon, however,
and is due to loss of iron in the urine.
Neutropenia occurs in three fifths and thrombocytopenia
in
two thirds of patients at some time during the course of
disease,
so that pancytopenia is common.
The direct antiglobulin test is usually negative.

54. The marrow
may be hypercellular with erythroid hyperplasia,
but it may be hypocellular.
In some patients, marrow failure may occur
duringthe course of PNH;
in others, AA is the initial diagnosis, with signs
of PNH manifesting simultaneously or later.
As mentioned earlier, approximately
40% of patients with AA have evidence of PNH
clone at diagnosis

55. Thrombotic complications are common, occurring in approximately
40% of patients, and represent a major cause of mortality.
Thrombosis commonly occurs in hepatic, cerebral, and abdominal
veins.
The absence of CD59 on platelets results in externalization of
phosphatidylserine, a site for prothrombinase complexes, and
thus increases the propensity for thrombosis.
The disease may undergo partial remissions and exacerbations.
In more than half of patients, both the proportion of abnormal
cells and the clinical severity decrease with time
. Abnormal cytogenetics can be found in up to 20% of PNH
patients.
In approximately 3%–5% of PNH patients, the disease progresses
to acute leukemia.

56. Sucrose Hemolysis Test
This test should be performed whenever the diagnosis of PNH is
considered,
also in hypoplastic anemias and in any hemolytic anemia of
obscure Origin
The principle of the test is that sucrose provides
a medium of low ionic strength that promotes the binding of
complement to the red cells.
In PNH, a proportion of red cells are abnormally sensitive
to complement-mediated lysis.
Suspicious results can be seen in some
other hematologic diseases, especially megaloblastic anemia and
autoimmune hemolytic anemia
.
False-negative results occur if the serum lacks
complement activity.
A simpler screening test, called the sugar water test,
applies the same principle of mixing blood with sugar and
observing for
hemolysis.

57. Acidified Serum Test (Ham Test)
Definitive diagnosis of PNH
complement is activated
by the alternate pathway, binds to red cells, and lyses abnormal
PNH cells
The patient’s washed red cells are mixed with ABO compatible
normal serum (fresh or properly stored) and acid;
after an hour’s incubation at 37° C,
the PNH cells are lysed,
.
The patient’s own serum may or may not result in lysis,
depending on residual complement,
the other tubes provide controls.
If lysis also occurs with heat-inactivated serum, the test is not
positive,

59. A positive acidified serum test occurs in congenital
dyserythropoietic anemia, type II (CDA-II), or HEMPAS
In this situation, however, lysis does not
occur with the patient’s own serum, and occurs with only
about 30% of normal sera.
Also, the sugar water screening test is negative in CDAII.

60. Flow cytometry using immunofluorescent staining of red cells
with a monoclonal antibody against deficient proteins such as
CD55, CD58, and CD59.
Granulocytes provide excellent diagnostic targets for flow
cytometry.
A fluorescein-labeled proaerolysin variant (FLAER) is
increasingly being used for the diagnosis of PNH.
It binds selectively to the GPI anchor.
The gene responsible for the PNH phenotype has been
identified on the X chromosome and designated phosphatidyl
inositol glycan A

62. Thank you!

67. Thank you!