HEMOLYSISDr.Mansooreh Eslami

intrinsic hemolytic anemia:

due to a defect of the red cell itselfThese are usually hereditary and are commonlygrouped as membrane, metabolic, and hemoglobin defects.

extrinsic hemolytic anemia:

due to a factor outside the red cell andacting upon it

HEMOLYSIS—GENERAL

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

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.

Residual activity is an index ofthe intravascular life span of the labeled red cells.

The erythrocyte life span :the period duringwhich 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

Results of 51Cr erythrocyte survival curve

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.

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 enzyme-deficiency hemolytic anemias, sickle cell anemia, and PNH

Hemoglobin DestructionINtravascular:*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 clearedby the hepatic parenchymal cells.

*methemalbuminemia

Lactate dehydrogenase (LD)

• it is cleared more slowly than Hb

• In hemolytic anemias, reversal of the LDisoenzyme 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.

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 bilirubin3. An increase in urine urobilinogen or, more consistently, in fecalUrobilinogen

Examination of feces is more dependable thanexamination of urine because feces may show an increase when the urine shows none

Blood FilmThe 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 microangiopathichemolytic anemia

sickle cells, target cells, or crystals suggest a hemoglobinopathy. .

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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

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Bone Marrow

Normoblastic hyperplasia is present and may be striking in degree.

Storage iron is usually increased and sideroblasts are normal orincreased in number, reflecting the abundance of available iron forHb synthesis.

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

HS can be divided into the followingpathogenetic 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

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 solutionsis measured and plotted for each NaCl concentration.

Procedure

1- We will do this dilution:

Final conc )%( . D.W. )ml( 1%Nacl)ml( Test tube

1.00 0.0 10.0 10.85 1.5 8.5 20.75 2.5 7.5 3

0.65 3.5 6.5 40.60 4.0 6.0 50.55 4.0 5.5 60.50 5.0 5.0 70.45 5.50 4.5 80.40 6.0 4.0 90.35 6.0 3.5 100.30 7.0 3.0 110.20 8.0 2.0 120.10 9.0 1.0 130.00 10.0 0.0 14

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.

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Result:

• % of hemolysis = (Abs of tube / Abs of tube 14) * 100%

• Normal Range:– Hemolysis begins 0.45% and complete 0.35%

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 haveincreased osmotic fragility.

Conversely, cells that are hypochromic andflatter have a greater capacity to expand in hypotonic solutions, lyse at alower concentration than normal cells, and are said to have decreased osmotic fragility

Cells with increased surface/volumeratio are osmotic resistant.

iron deficiency, thalassemia,liver disease, and reticulocytosis

a, Thalassemia, showing a small fractionof 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.

Erythrocyte osmotic fragility

The osmotic fragility of freshly drawn blood is usually increased in HSbut may be normal in mildly affected patients. In blood that is incubatedat 37° C for 24 hours before the test is performed, the osmotic fragility isalmost 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.

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

Autohemolysis Test

Sterile, defibrinated blood is incubated at 37° C for 48 hours

. In normal blood, without addedglucose, 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 frequentlyand is probably no more sensitive than the incubated osmoticfragility test

autosomal dominant

weakening of the membrane skeleton and defective association ofproteins that hold the skeleton together

three groups: (1) common HE (including hereditary pyropoikilocytosis [HPP]), with elliptocytes thatmay be rod shaped, (2) spherocytic HE, and (3) Southeast Asian ovalocytosis.

Hereditary Elliptocytosis

The most commonly defined abnormality appears to be a defect inspectrin,. 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 adecreased spectrin/band 3 ratio

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, numberingapproximately 15%

whereas in normal individuals lessthan 5% of the red cells are elliptical

The deformity is increased in sealed, moist preparations.

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 neonatalperiod 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 4.1-actin interaction

Hereditary Pyropoikilocytosis

HPP is a severe congenital hemolytic anemia, which is

characterized bymicrocytosis, striking micropoikilocytosis and fragmentation,

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 fragmentat 45°–46° C.

Spherocytic HE

This subgroup accounts for 10% of cases. A mild to moderate hemolyticanemia and splenomegaly are present,

with both elliptocytes and spherocytes,

Abnormal osmotic fragilityAnd Autohemolysis tests.

Poikilocytes and fragments are usually absent.

The molecular basis of this subtype is unknown.

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 ridgesor 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

Hereditary ovalocytosis

Hereditary Stomatocytosis (Hereditary Hydrocytosis)

This is a rare, autosomally transmitted disorder

Heterozygotic individualshave no anemia, and 1%–25% of stomatocytes are seen on the blood film. In presumed homozygotic individuals, about one third of the red cells arestomatocytes, and there is a mild to moderate hemolytic anemia.

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.

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 complexesof complement.

MIRL is a protein that controls the membrane attack complex, C5b-9.

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 enzymessuch as acetyl cholinesterase and leukocyte alkaline phosphatase may bedeficient as well.

Recent work indicates that deficient proteins and enzymesare 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.

Proteins Deficient from PNH Blood Cells

CD59, CD90, CD109

CD55CD58CD59 CD48CD52PrPcCD16

CD24 CD55CD58 CD59 CD48 PrPC

CD73 CD108

CD55CD58CD59

CD109PrPC

GP500Gova/b

CD55CD58CD59PrPCAChE

JMH AgDombroch

HG Ag

CD55 CD58*CD59 CD14CD16 CD24

CD48 CD66bCD66c CD87

CD109 CD157LAPNB1 PrPC

p50-80 GPI-80ADP-RT NA1/NA2

CD14 CD55 CD58*CD59 CD48 CD52

CD87 CD109 CD157Group 8 PrPC GPI-80

CD16

CD55 CD58*CD59 CD48CD52 CD87 CD108 PrPcADP-RT CD73

CD90 CD109CD16*

Haematopoietic Stem Cell

Platelets

RBC

PMN

B cells

Monocytes

T cells

NK cells

QuickTime™ and aGIF decompressor

are needed to see this picture.)Courtesy of Lucio Luzzatto(

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

The blood usually shows a normocytic anemia with a reticulocytosisthat 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 intwo thirds of patients at some time during the course of disease,

so that pancytopenia is common.

The direct antiglobulin test is usually negative.

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, approximately40% of patients with AA have evidence of PNH clone at diagnosis

Thrombotic complications are common, occurring in approximately40% 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.

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 OriginThe principle of the test is that sucrose providesa medium of low ionic strength that promotes the binding of complement to the red cells.In PNH, a proportion of red cells are abnormally sensitiveto complement-mediated lysis.

Suspicious results can be seen in someother hematologic diseases, especially megaloblastic anemia and autoimmune hemolytic anemia. False-negative results occur if the serum lackscomplement activity. A simpler screening test, called the sugar water test,applies the same principle of mixing blood with sugar and observing forhemolysis.

Acidified Serum Test (Ham Test)

Definitive diagnosis of PNH

complement is activatedby 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,

A positive acidified serum test occurs in congenital dyserythropoietic anemia, type II (CDA-II), or HEM-PAS

In this situation, however, lysis does notoccur with the patient’s own serum, and occurs with only about 30% of normal sera.

Also, the sugar water screening test is negative in CDA-II.

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

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