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APPROACH TO A CASE OF EXTRACORPUSCULAR HEMOLYTIC ANEMIA Dr. Adrija Pathak

Extracorpuscular hemolytic anemia

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Page 1: Extracorpuscular hemolytic anemia

APPROACH TO A CASE OF EXTRACORPUSCULAR HEMOLYTIC ANEMIA

Dr. Adrija Pathak

Page 2: Extracorpuscular hemolytic anemia

A.IMMUNE HEMOLYTIC ANEMIA

1.AUTOIMMUNE HEMOLYTIC ANEMIAWarm antibodiesCold anti bodies

2.ALLOIMMMUNE HEMOLYTIC ANEMIAHemolytic disease of newbornIncompatible blood transfusion

3. DRUG INDUCED

B.NONIMMUNE HEMOLYTIC ANEMIA

Microangiopathic hemolytic anemias(DIC ,TTP,HUS)Trauma:prosthetic cardiac valve, thermal, exerciseInfection: malaria, babesiaChemical and drugsAnimal venomsMalignant hypertensionPlasma lipid abnormalitiesHypersplenism

EXTRA CORPUSCULAR DEFECT

Page 3: Extracorpuscular hemolytic anemia

HOW IS HEMOLYTIC ANEMIA DIAGNOSED?

Two main principles

One is to confirm that it is hemolysis Identify general diagnostic findings of

hemolytic anemia

Two is to determine the etiologya. Hereditary anemias ( defects within RBC )b. Acquired anemias ( external causes )

Page 4: Extracorpuscular hemolytic anemia

CLINICAL MANIFESTATIONS

Compensated or Symptomatic anemia Weakness, dizziness Fever, weight loss, fatigue Pallor Jaundice Dark urine Gall stone Splenomegaly Thinning of cortical bone Extramedullary hematopoetic masses

Page 5: Extracorpuscular hemolytic anemia

COMMON LABORATORY FINDINGS IN HEMOLYTIC ANAEMIA

Increased Bone Marrow Production of Erythrocytes

Increased Erythrocyte destruction

Reticulocytosis (RPI >2) anemia

Increased IRF Presence of spherocytes , schistocytes and/or other poililocytes

Nucleated erythrocytes in peripheral blood

haptoglobulin & hemopexin & glycosalated Hb

Polychromaisa of erythrocytes on romanowsky stained blood smears

bilirubin ( unconjugated) fecal & urine urobilinogen

Leucocytosis Hemoglbinemia, Hemoglobinuria, Hemosiderinuria, Methhemoglobinemia

Normoblastic erythroid hyperplasia in bone marrow

serum LD & expired CO

positive DAT

Page 6: Extracorpuscular hemolytic anemia

EVALUATION OF ANEMIALow Hgb/Hct

Low Hgb/Hct

Corr. Retic Ct >2%

Corr. Retic Ct >2%

Corr. Retic Ct <2%

Corr. Retic Ct <2%

Acute Blood Loss Acute Blood Loss MCV>100

MCV>100MCV 80-100

MCV 80-100

MCV<80

MCV<80

EVALUATE & TREAT APPRO-

PRIATELY

EVALUATE & TREAT APPRO-

PRIATELY

Evaluate for

Hemolytic Anemias

Evaluate for

Hemolytic Anemias

Evaluate for

microcytic anemias

Evaluate for

microcytic anemias

Evaluate for

macrocytic anemias

Evaluate for

macrocytic anemias

Evaluate for

normocytic anemias

Evaluate for

normocytic anemias

NOYES

Page 7: Extracorpuscular hemolytic anemia

RETICULOCYTE COUNTING

Reticulocyte % (0.5-2.5%) no. of retic (in n field) X100 total rbc (in n field)

Absolute reticulocyte (X10⁹/L) = RBC Count (X10¹²/L) X retic%

(18-158 X10⁹)

Corrected Reticulocyte Count= % Retic x Pt’s Hct normal HctRetic count: 10%

Pt’s Hct 29

Control Hct 45

Corrected Reticulocyte Count = 10% x 29/ 45 = 7.73 %

( > 2% if no blood loss Indicates hemolysis)

Page 8: Extracorpuscular hemolytic anemia

Reticulocyte production index(RPI) =corrected reticulocyte count/reticulocyte maturation time(days)

>2RPI appropriate bone marrow responseEg -Retic count: 10%

Pt’s Hct 29

Corrected retic 7.73

Immature reticulocyte fraction(IRF)- some automated instrument assess the maturity of reticulocyte by intensity of staining

Hct Maturation time (days)

.35 1.5

.25 2

.15 2.5

Page 9: Extracorpuscular hemolytic anemia

ROLE OF PBS

1 Sickled cells

Bite cells

Schisto-cytes

Acantho-cytes

Sphero-cytes

Target cells

parasiteinclusions

DAT(+)

DAT(-)

Hgb electro-phoresis

G6PDlevel

PT/PTTCrea

platelets

Auto-ImmuneHemo-lytic

Anemia

Heredi-tary

Sphero-cytosis

Sickle CellDs

G6PDDeficient

VsUnstable

Hgbs

Thalas-semiasHemo-

globino-pathy

Liver Ds

LiverDs

MalariaBabe-siosisBarto-nella

TTP-HUSDIC

Prosthe-tic Valve

MalignantHTN

Hemolytic Anemia (CRC>2% + no blood loss)

Page 10: Extracorpuscular hemolytic anemia

Finding on pbs Type of aquired hemolytic anemia suggested

Schistocytes Fragmentation syndromes including microangiopathic haemolytic anaemia

Spherocytes Autoimmune, alloimmune or drug-induced immune haemolytic anaemia, paroxysmal cold haemoglobinuria, burns, Clostridium perfringens sepsisa and mechanical haemolytic anaemia

Microspherocytes Burns, fragmentation syndromes

Irregularly contracted cells

Oxidant damage, Zieve’s syndrome

Ghost cells, suspicion of Heinz bodies

Acute oxidant damage

Marked red cell agglutination

Cold-antibody-induced haemolytic anaemia

Erythrophagocytosis

Paroxysmal cold haemoglobinuria

Page 11: Extracorpuscular hemolytic anemia

Hypochromia, microcytosis and basophilic stippling

Lead poisoning

Atypical lymphocytes Cold-antibody-induced haemolytic anaemia associated with infectious mononucleosis or, less often, other infections

Thrombocytopenia Autoimmune haemolytic anaemia (Evans’ syndrome), thrombotic thrombocytopenic purpura, microangiopathic haemolytic anaemia associated with disseminated intravascular coagulation, paroxysmal nocturnal haemoglobinuria

Neutropenia Paroxysmal cold haemoglobinuria

Page 12: Extracorpuscular hemolytic anemia

Intravascular Hemolysis

RBC LYSIS

HBG in plasma

HAPTOGLOBIN

REMOVED BY LIVER

HEMOGLOBINEMIA

HEMOGLOBINURIA

HBG TAKEN UP BY RENAL TUBULAR CELLS

HEMOSIDERIN

CELLS SLOUGHED IN

URINE 1 WEEK LATER

Page 13: Extracorpuscular hemolytic anemia

Features specific to intravascular haemolysis:• Absent haptoglobin and haemopexin• Haemoglobinaemia• Haemoglobinuria.• Methaemoglobinaemia. • Methemalbumin which is not excreted in urine but circulates in blood detected by Schumm’s test• Haemosiderinuria. • LDH

Page 14: Extracorpuscular hemolytic anemia

Extravascular Hemolysis

Destruction of red cells by reticuloendothelial cells in the liver, spleen, and bone marrow

•Inc in expired carbon monoxide•Carboxyhemoglobin•Unconjugated bilirubin•Urine and fecal urobilinogen•Dec haptoglobin in severe hemolysis

Significant lab finding:

Page 15: Extracorpuscular hemolytic anemia

Activation of complement- PNH, PCH, transfusion rxn

MAHA Physical/ mechanical

trauma Toxic

microenvironment

Hemoglobinopathies Enzymopathies Membrane defects Megaloblastic

anemia AIHA Drug induced

Intravascular hemolysisExtravascular hemolysis

Page 16: Extracorpuscular hemolytic anemia

WHAT IS THE PRECISE DIAGNOSIS?

1.If a hereditary haemolytic anaemia is suspected:

Osmotic-fragility glucose-6-phosphate dehydrogenase (G6PD)

assay electrophoresis or high-performance liquid

chromatography for abnormal Hb; tests for sickling; Examination of the proteins of the red cell

membrane and cytoskeleton (e.g. spectrin) by gel electrophoresis and by specific radioimmunoassay.

Page 17: Extracorpuscular hemolytic anemia

2.If acquired haemolytic anaemia is suspected: Direct antiglobulin test tests for autoantibodies in the patient’s serum titration of cold agglutinins Donath–Landsteiner test demonstration of thermal range of autoantibodies tests for agglutination and/or lysis of enzyme-

treated cells by autoantibodies history of autoimmune disease, recent blood transfusion,

recent infection, exposure to drugs or toxins the presence of a cardiac prosthesis and risk of malaria. Previous clinical history and laboratory results will help to

establish that the disorder is acquired.

Page 18: Extracorpuscular hemolytic anemia

3.If the haemolytic anaemia is suspected of being drug induced:

Screening test for red cell G6PD; glutathione stability test; staining for Heinz bodies; identification of methaemoglobin (Hi) and sulphaemoglobin (SHb); tests for drug-dependent antibodies.

4.If mechanical stress is suspected: Red cell morphology; platelet count; renal function tests;

coagulation screen; fibrinogen assay; test for fibrinogen/fibrin degradation products

5.In obscure cases: Investigations for paroxysmal nocturnal haemoglobinuria

(PNH) (e.g. acidified serum test [Ham’s test], sucrose lysis test, flow cytometric immunophenotyping for erythrocyte and neutrophil antigens)

Measurement of lifespan of patient’s red cells If splenectomy is contemplated, determination of sites of

haemolysis by radionuclide imaging

Page 19: Extracorpuscular hemolytic anemia

IMMUNE HEMOLYTIC ANEMIA

Immune Hemolysis is mediated by the antibodies and/or complement that bind to the RBC surface and initiate destruction

RBC destruction may be intravascular or extravascular

Classified as autoimmune, alloimmune, drug induced

Page 20: Extracorpuscular hemolytic anemia

SCHEME FOR SEROLOGICAL INVESTIGATION OF HAEMOLYTIC ANAEMIA SUSPECTED TO BE OF IMMUNOLOGICAL ORIGIN

Are the patient’s red cells ‘coated’ by immunoglobulins or complement (indicating an antigen–antibody reaction)?

Perform a DAT using a polyspecific ‘broad-spectrum’ reagent, which contains both anti-IgG and anti-C′. (If the DAT is negative, it is unlikely, although not impossible, that the diagnosis is AIHA.)

If the DAT is positive, are immunoglobulins or complement adsorbed to the red cells?

Repeat the DAT using monospecific sera (i.e. anti-IgG and anti-C3d).

If immunoglobulins are present on the red cells, is there antibody specificity?

Prepare eluates from the patient’s red cells. Test these later

Page 21: Extracorpuscular hemolytic anemia

What is the patient’s blood group? Determine the patient’s ABO and RhD and Kell

type. The Rh phenotype is particularly important in warm-type AIHA; other antigens must be determined if alloantibodies are to be differentiated from autoantibodies

Is there free antibody in the serum? Is there any underlying alloantibody present?

Screen the serum with two or three red cell suspensions suitable for routine pretransfusion antibody screening looking for agglutination and lysis at 37°C by the IAT. If positive,  identify the antibody using an antibody identification panel.

If an alloantibody is identified, blood lacking the corresponding antigen must be selected for transfusion.

Page 22: Extracorpuscular hemolytic anemia

If the autoantibody is pan-reacting antibody adsorption tests are needed to remove the autoantibody so as to identify any underlying alloantibody.

If there is a warm/cold autoantibody, what is the specificity of the autoantibody?

Test the serum also at 20°C against antibody-screening cells to show whether cold or warm antibodies or a mixture of the two, are present in the serum.

Test the eluate against the antibody identification panel of red cells by IAT.

Titration of autoantibody may be useful in the presence of a strong alloantibody.

Page 23: Extracorpuscular hemolytic anemia

If there is a cold antibody:a. Has the antibody any specificityb. What is the titre/thermal range of the

antibody? Test the serum/plasma against a panel of O cells,

O cord cells and patient’s own cells at 20°C. If an autoantibody is found, titrate at 4°C with

ABO-compatible adult (I) cells, cord blood (i) cells and the patient’s cells

Determine the highest temperature at which autoagglutination of the patient’s whole blood takes place

If PCH is suspected, carry out the direct and two-stage indirect Donath–Landsteiner tests

Page 24: Extracorpuscular hemolytic anemia

Is a drug suspected as the cause of the haemolytic anaemia?

If haemolysis induced by drugs is suspected, add the drug in solution to a mixture of the patient’s serum, normal cells and fresh normal serum. Look for agglutination of normal and enzyme-treated cells and use the IAT.

Are there any other serological abnormalities?

Consider carrying out the following tests: serum protein electrophoresis and quantitative estimation of immunoglobulins, estimation of complement, tests for antinuclear factor, a screening test for heterophile antibodies (infectious mononucleosis screening test) and a test for mycoplasma antibodies.

Page 25: Extracorpuscular hemolytic anemia

AIHA CLASSIFICATION

Warm autoimmune hemolytic anemia Idiopathic, Secondary

(Lymphoproliferative disorders, autoimmune diseases-SLE,RA, viral, neoplastic)

Cold autoimmune hemolytic anemiaCold agglutinin syndrome

(Idiopathic, Secondary- mycoplasma, infectious mono, LPD)

Paroxysmal cold hemoglobinuria (Idiopathic, Secondary- measles, mumps, syphilis)

Page 26: Extracorpuscular hemolytic anemia

CHARACTERISTICS OF AGGLUTININS

IgG IgM (rare),

IgA(usually withIgG) 37˚C Attachment of

membrane bound IgG or C3b to macrophage receptor (extravascular)

Broad specificity anti-Rh

IgM IgG(PCH only)

<30˚C, usually<10˚C Complement mediated

lysis (intravascular) or attachment of membrane bound C3b to macrophage receptor (extravascular)

Usually autoanti-I, occ autoanti-i, PCH- autoanti-P

Warm Reacting Ab Cold Reacting Ab

Page 27: Extracorpuscular hemolytic anemia

LABORATORY IDENTIFICATION OF SENSITIZED RBCAgglutination of test sera and appropriate rbc suspended in saline- detect antibodies of IgM class

Page 28: Extracorpuscular hemolytic anemia

ANTIHUMAN GLOBULIN TEST/COOMBS TEST

AHG is broad spectrum antisera produced in rabbits that reacts against human Ig and complement

Divalent antibodies attach to Fc region of IgG or complement component on two separate cell, briding the distance between cells→ agglutination

PRINCIPLE- RBC coated with incomplete antibody (IgG) or C3 component will be agglutinated by AHG reagent binding to the IgG antibodies coating the cells

Page 29: Extracorpuscular hemolytic anemia

APPLICATION OF ANTIGLOBULIN TEST

DAT-detect in vivo sensitization of RBC with IgG or C3d

Diagnosis of HDN Diagnosis of AIHA Investigation of drug induced sensitization Investigation of transfusion rxn

IAT-detect presence of incomplete Ab and complement binding ab in serum after coating red cell in vitro

Compatibility testing Screening and identification of unexpected ab Detection of red cell antigen using specific ab reacting

only in antiglobin test such as Fy,K,Jk Titration of Ab in unknown sera or amniotic fluid

Page 30: Extracorpuscular hemolytic anemia

DAT

A spin tube technique Make a 2–5% suspension of red cells that have

been washed four times in saline. Add 1 volume (drop) of the cell suspension to 2 volumes (drops) of antiglobulin reagent. Centrifuge for 10–60 s.

Examine for agglutination after gently resuspending the button of cells. A concave mirror and good light help in macroscopic readings. If the result appears to be negative, confirm this microscopically.

Check negative results by the addition of IgG-sensitized cells /complement-coated cells.

Page 31: Extracorpuscular hemolytic anemia

IAT

Reagent Red Cells Red Cell Suspensions- Normal ionic

strength saline/ Low ionic strength saline Sensitize red cells Wash the test cells Add antiglobulin reagent Read agglutination The addition of sensitized cells to all negative

tests.

Page 32: Extracorpuscular hemolytic anemia
Page 33: Extracorpuscular hemolytic anemia

WARM AUTOIMMUNE HEMOLYTIC ANEMIA

Most common form of AIHA Any age although incidence increases

after 40yrs Symptoms related to anemia in idiopathic In secondary AIHA symptoms of

underlying ds Mild to moderate splenomegaly

Page 34: Extracorpuscular hemolytic anemia

PBS- normocytic normochromic anemia, polychromasia, nucleated rbc, spherocytes

Page 35: Extracorpuscular hemolytic anemia

LABORATORY FINDINGS IN WAIHA PBS- normocytic normochromic anemia,

polychromasia, nucleated rbc, spherocytes Thrombocytopenia with WAIHA- Evan’s

syndrome Increased reticulocytes BM-erythroid h/p, erythrophagocytosis by

macrophages Positive DAT Presence of autoantibody in serum Positive antibody screen with all cells

incuding autocontrol Incompatible crossmatch with all donors Increased osmotic fragility

Page 36: Extracorpuscular hemolytic anemia

COLD AUTOIMMUNE HEMOLYTIC ANEMIA

Also called Cold Agglutinin Disease(CAD) >50yr, peak onset age>70yr Chronic hemolytic anemia with or without

jaundice In some hemolysis is episodic a/w chilling Acrocynosis Raynaud’s phenomenon Hemoglobinuria on exposure to cold splenomegaly

Page 37: Extracorpuscular hemolytic anemia

LABORATORY FINDINGS IN CAD

CBC- erythrocyte count inappropriately decreased for Hb content, false increase in MCV, MCH and MCHC

PBS- ncnc anemia,spherocytes, agglutinated rbcs, rouleaux, nrbc

Reticulocytosis Erythrophagocytosis in buffy coat BM- normoblastic h/p Decreased C3 and/or C4

Page 38: Extracorpuscular hemolytic anemia

o Increased bilirubino Decresed haptoglobino Hemoglobinemia, hemoglobinuria in acute

hemolysiso Hemosiderinuria in chronic hemolysiso Serological- DAT- positive with polyspecific AHG negative with anti IgG positive with anti C3 IAT- antibody showing characteristic

reactions at <25 ˚C Cold agglutinin titre >1000 at 4˚C

Page 39: Extracorpuscular hemolytic anemia

BENIGN COLD AGGLUTININ

Most normal individual when serum and cell incubated at 4 ˚C

Thermal amplitude and titre (<1:64) not high to cause problem

Cold agglutinin test when diagnosis of CAD is suspected

Pathological Ab agglutinates pt’s cell at 0-20˚C in saline and upto 32 ˚C in albumin

Page 40: Extracorpuscular hemolytic anemia

PAROXYSMAL COLD HEMOGLOBINURIA

Rare but cause of 30-40% of AIHA in children less than 5 ys

Biphasic complement fixing IgG Donath Landsteiner Ab specific for P antigen

Ab reacts with rbc in capillaries at temp <20˚C and bind to early acting complement

Upon warming to 37˚C Ab disperses from cell but MAC is activated causing lysis

Hemoglobinuria, fever,chill Raynaud’s phenomenon

Page 41: Extracorpuscular hemolytic anemia

LABORATORY FINDINGS Hb drops sharply to as low as 5g/dl Hemoglobinemia, methemalbuminemia and

hemoglobinuria Neutopenia with shift to left Reticulocytopenia and spherocytes Serum bilirubin, BUN and LD elevated Serum complement and haptoglobin

decreased Erythrophagocytosis invovling neutrophils Weakly positive DAT with anticompliment

antisera IAT can be positive if performed in cold D-L ab present in low titre (1:32)

Page 42: Extracorpuscular hemolytic anemia

DONATH-LANDSTEINER(D-L) TEST FOR DETECTING THE PRESENCE OF D-L ANTIBODIES

Patient’s Whole Blood

Control Test

Incubate for 30 min atIncubate for 30 min at

37 C37 C

4 C37 C

Centrifuge : Observe plasma for presence of hemolysis

Interpretation D-L antibodies present

No D-L antibodies present

No Hemolysis

No Hemolysis

Hemolysis

No Hemolysis

Page 43: Extracorpuscular hemolytic anemia

DRUG INDUCED HEMOLYTIC ANEMIA

Drug adsorption (hapten) mechanism• The drug binds nonspecifically to proteins on the RBC

membrane, antibodies are made (usually IgG), they bind to the drug and extravascular hemolysis occur

• High dose of iv pencillin

•Polyspecific AHG positive•Anti IgG positive•Anti C3 may be posive

Page 44: Extracorpuscular hemolytic anemia

Autoantibody induced mechanismThe drug adheres and alters cell membrane inducing formation of autoantibodies causing extravascular destruction.

• Methyldopa, procainamide• Polyspecific AHG positive• Anti IgG positive• Anti C3 may be posive or negative

Immnune complex mechanism•Drug combines with plasma protein forming immune complex which adsorbs to cell membrane activating complement cascade causing intravascular lysis. •Quinidine, cephalosporin• Polyspecific AHG positive• Anti IgG negative• Anti C3 positive

Page 45: Extracorpuscular hemolytic anemia

MechanismPrototype drug

DAT IAT

No drug Serum + drug

Eluate + drug

Drug-dependent antibody

C′ activation Quin(id)ine C′ Neg C′a Neg

No C′ activation Penicillin IgG Neg IgG IgG

Autoantibody α-Methyldopa IgG IgG

 Serological features of the different types of drug-induced haemolytic anaemia of immunological origin

Page 46: Extracorpuscular hemolytic anemia

HEMOLYTIC TRANSFUSION REACTION

acute delayed

Timing Immediate (within 24hrs)

2-14 days

Underlying case Usually ABO antibodies

Other antibodies: often Kidd (anamestic response)

Hemolysis Intravascular Extravascular

Symptoms Fever, chills, back pain, hypotension, pain at site of infusion

Uncommon

Laboratory findings

HemoglobinemiaPositive DAT (transient)

Positive DATAntibody in elute

Page 47: Extracorpuscular hemolytic anemia

ACUTE INTRAVASCULAR HEMOLYSIS

Check for incompatibility Documentation check Repeat ABO group of pt pre-transfusion and post

transfusion and the donor unit Screen the pt for red cell ab pre-transfusion and

post transfusion Repeat cross match with pre-transfusion and post

transfusion sample DAT on pre-transfusion and post transfusion

samples Elute from pt’s red cell

Page 48: Extracorpuscular hemolytic anemia

Check for haemolysis Perform visual examination of patient’s plasma &

urine Blood film may show spherocytosis. Bilirubin and lactate dehydrogenase (LDH) levels.

Check for DIC- blood count and film, coagulation screen and FDP or d-dimer

Check for renal dysfunction- urea, creatinine and electrolytes

Check for bacterial infection- blood culture from pt and donor unit

Page 49: Extracorpuscular hemolytic anemia

DELAYED HEMOLYTIC TRANSFUSION REACTION

Hb falls more rapidly than would be expected after transfusion

Spherocytes Posive DAT Elution of ab aid identification or confirm

specificities in non-ABO incompatibility Uncojugated bilirubin raised

Page 50: Extracorpuscular hemolytic anemia

HEMOLYTIC DISEASE OF FETUS AND NEWBORN

Alloimmune ds a/w increased erythrocyte destruction during fetal/neonatal life

Fetomaternal blood group incompatibility ABO incompatibilty more common RhD incompatibility causes more serious ds Others include anti-K, anti-c, anti-C and anti-

E IgG crosses placenta

Page 51: Extracorpuscular hemolytic anemia

Antenatal Serology ABO and D Grouping and Antibody Screening-

early in pregnancy and again at 28 wk

Follow-Up Antibody Screening Pregnant women with anti-D, antibodies to

Kell-related antigens and anti-c should be tested monthly to 28 weeks and then every 2 weeks to delivery.

The tests should include antibody quantification or titration as well as testing for additional red cell antibodies.

It is now appreciated that an increasing titre rather than an individual level is more predictive of an affected fetus

Page 52: Extracorpuscular hemolytic anemia

Prediction of Fetal Blood Group Partner Testing

Testing Fetal DNA in the Maternal Circulation-using DNA amplification techniques

Fetal Blood Sampling Using ultrasound guidance, it is possible to take a sample

of fetal blood for blood grouping Contamination by maternal blood can hinder analysis of

the sample obtained, leading to false-negative results. In addition, the procedure itself can lead to fetomaternal

haemorrhage (FMH) and hence further sensitization to fetal antigens.

There is also a risk of miscarriage

Page 53: Extracorpuscular hemolytic anemia

Assessment of Fetal Anaemia Traditionally this was done using

amniocentesis to measure the optical density of the amniotic fluid (Lilley’s lines) using spectrophotometry.

Direct fetal blood sampling by ultrasound-guided cordocentesis provides diagnostic information and a new approach to fetal therapy by direct fetal intravascular transfusion.

Carry the risk of miscarriage and further fetomaternal haemorrhage.

Non-invasive tests to determine fetal anaemia- middle cerebral artery Doppler studies have been very useful

Page 54: Extracorpuscular hemolytic anemia

Tests on Maternal and Cord Blood at Delivery

Cord blood (this is preferable to a sample from the baby because of the quantity of blood required)

ABO and D group and phenotype for the red cell antigen against which the antibody is directed

Direct antiglobulin test Haemoglobin concentration Bilirubin.

Maternal blood Repeat ABO and D group Repeat antibody screen.

Page 55: Extracorpuscular hemolytic anemia

LABORATORY FINDINGS

DAT positive Cord blood Hb<14g/dl-

indicator of anemia Macrocytic

normochromic ↑↑ reticulocyte Nrbc Mild to absent

poikilocytosis/ spherocytosis Bilirubin peaks on 3rd-4th day

Weakly positive DAT becomes negative within 12 hrs

PBS-nrbc, schistiocytes, spherocytes and polychromasia

Bilirubin not significantly elevated

Rh incompatibility ABO incompatibility

Page 56: Extracorpuscular hemolytic anemia

Anti-D Prophylaxis given routinely as soon as possible after delivery to

women who are D negative who deliver babies that are D positive. It should also be given at times during pregnancy when sensitization could occur, such as during medical or surgical therapeutic termination of pregnancy, chorionic villus sampling, amniocentesis and following any abdominal trauma

Measurement of Fetomaternal Haemorrhage Most commonly used is acid elution, also known as the

Kleihauer test, which depends on the Hb F in fetal cells resisting the acid elution to a greater extent than the Hb A in maternal cells.

The flow cytometry method uses a fluorochrome-labelled anti-D antibody to measure a minority of D positive cells in the maternal D negative blood and is recommended for confirmation of a positive acid elution test where the estimated FMH exceeds 2 ml.

Page 57: Extracorpuscular hemolytic anemia

MICROANGIOPTHIC HEMOLYTIC ANEMIA

Page 58: Extracorpuscular hemolytic anemia

HUS AND TTP

Thrombotic Thrombocytopenic Purpura

Hemolytic Uremic Syndrome

Adult ages 20-50 Children <5yrs

Hemolytic anemia with cell fragmentation

Hemolytic anemia with cell fragmentation

Mild to moderate Renal dysfunction

Acute renal faiure

Thrombocytopenia Thrombocytopenia

Severe CNS symptoms Mild CNS symptoms

Fever

Page 59: Extracorpuscular hemolytic anemia

LAB FINDINGS IN HUS AND TTP

Evidence of hemolysis-Hb, retic, schistiocytes, lecocytosis,inc bilirubin

Evidence of Intravascular hemolysis-hemoglobinemia,Hburia, dec haptoglobin.

Evidence of Thrombotic microangiopathy-thrombocytopenia, FDP, D-dimer, PT ,APTT

Urinanalysis Renal function Tests for verotoxin-secreting E. coli  If available, quantification of von Willebrand

factor-cleaving protease (ADAMTS13) is indicated in suspected TTP

Page 60: Extracorpuscular hemolytic anemia

DIC

PBS-schistiocyes Thrombocytopenia Abnormal

coagulation test Prolonged

PT,APTT,TT Elevated D-dimer

test Incresed fibrin

degraded product (FDP)

Decrese fibrinogen

Page 61: Extracorpuscular hemolytic anemia

BABEIOSIS AND MALARIA

Page 62: Extracorpuscular hemolytic anemia

Abnormal plasma lipid composition – note that these were also included in intracorpuscular problems, because they lead to intrinsic problems with the RBC.

Spur cell anemia – associated with severe hepatocellular disease which leads to increased serum lipoproteins, increased membrane cholesterol, decreased deformability and decreased survival

Abetalippoproteinemia – leads to an increased cholesterol/phospholipid ratio, acanthocytes, and decreased RBC survival.

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REFERENCES

•Dacie and lewis practical haematology•McKenzie- clinical laboratory hematology•Makroo -Compendium of transfusion medicine

Page 64: Extracorpuscular hemolytic anemia