SlideShare uma empresa Scribd logo
1 de 64
Baixar para ler offline
APPROACH TO A CASE OF 
EXTRACORPUSCULAR HEMOLYTIC 
ANEMIA 
Dr. Adrija Pathak
A.IMMUNE HEMOLYTIC ANEMIA 
1.AUTOIMMUNE HEMOLYTIC ANEMIA 
Warm antibodies 
Cold anti bodies 
2.ALLOIMMMUNE HEMOLYTIC 
ANEMIA 
Hemolytic disease of newborn 
Incompatible blood transfusion 
3. DRUG INDUCED 
B.NONIMMUNE HEMOLYTIC ANEMIA 
Microangiopathic hemolytic anemias(DIC 
,TTP,HUS) 
Trauma:prosthetic cardiac valve, thermal, 
exercise 
Infection: malaria, babesia 
Chemical and drugs 
Animal venoms 
Malignant hypertension 
Plasma lipid abnormalities 
Hypersplenism 
EXTRA CORPUSCULAR DEFECT
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 etiology 
a. Hereditary anemias ( defects within RBC ) 
b. Acquired anemias ( external causes )
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
Increased Bone Marrow Production 
of Erythrocytes 
Increased Erythrocyte destruction 
COMMON LABORATORY FINDINGS IN HEMOLYTIC ANAEMIA 
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
EVALUATION OF ANEMIA 
Low Hgb/Hct 
Corr. Retic 
Ct >2% 
Corr. Retic 
Ct <2% 
Acute 
Blood Loss 
MCV>100 
MCV 80- 
100 
MCV<80 
EVALUATE & 
TREAT 
APPRO-PRIATELY 
Evaluate for 
Hemolytic 
Anemias 
Evaluate for 
microcytic 
anemias 
Evaluate for 
macrocytic 
anemias 
Evaluate for 
normocytic 
anemias
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 Hct 
Retic 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)
 Reticulocyte production index(RPI) =corrected reticulocyte 
count/reticulocyte maturation time(days) 
Hct Maturation time (days) 
.35 1.5 
.25 2 
.15 2.5 
>2RPI appropriate bone marrow response 
Eg -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
ROLE OF PBS 
Hemolytic Anemia (CRC>2% + no blood loss) 
1 Sickled 
cells 
Bite 
cells 
Schisto-cytes 
Acantho-cytes 
Sphero-cytes 
Target 
cells 
parasite 
inclusions 
DAT 
(+) 
DAT 
(-) 
Hgb electro-phoresis 
G6PD 
level 
PT/PTT 
Crea 
platelets 
Auto- 
Immune 
Hemo-lytic 
Anemia 
Heredi-tary 
Sphero-cytosis 
Sickle 
Cell 
Ds 
G6PD 
Deficient 
Vs 
Unstable 
Hgbs 
Thalas-semias 
Hemo-globino-pathy 
Liver Ds 
Liver 
Ds 
Malaria 
Babe-siosis 
Barto-nella 
TTP-HUS 
DIC 
Prosthe-tic 
Valve 
Malignant 
HTN
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
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
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
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
Extravascular Hemolysis 
Destruction of red cells by 
reticuloendothelial cells in the 
liver, spleen, and bone 
marrow 
Significant lab finding: 
•Inc in expired carbon 
monoxide 
•Carboxyhemoglobin 
•Unconjugated bilirubin 
•Urine and fecal urobilinogen 
•Dec haptoglobin in severe 
hemolysis
Intravascular hemolysis Extravascular hemolysis 
 Activation of 
complement- PNH, 
PCH, transfusion rxn 
 MAHA 
 Physical/ mechanical 
trauma 
 Toxic 
microenvironment 
 Hemoglobinopathies 
 Enzymopathies 
 Membrane defects 
 Megaloblastic anemia 
 AIHA 
 Drug induced
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.
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.
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
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
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
 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.
 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.
 If there is a cold antibody: 
a. Has the antibody any specificity 
b. 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
 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.
AIHA CLASSIFICATION 
Warm autoimmune hemolytic anemia 
 Idiopathic, Secondary 
 (Lymphoproliferative disorders, autoimmune diseases-SLE,RA, 
viral, neoplastic) 
 Cold autoimmune hemolytic anemia 
 Cold agglutinin syndrome 
 (Idiopathic, Secondary- mycoplasma, infectious mono, LPD) 
 Paroxysmal cold hemoglobinuria 
 (Idiopathic, Secondary- measles, mumps, syphilis)
CHARACTERISTICS OF AGGLUTININS 
Warm Reacting Ab Cold Reacting Ab 
 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
LABORATORY IDENTIFICATION OF 
SENSITIZED RBC 
Agglutination of test sera and appropriate rbc suspended 
in saline- detect antibodies of IgM class
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
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
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.
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.
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
PBS- normocytic normochromic anemia, 
polychromasia, nucleated rbc, 
spherocytes
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
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
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
o Increased bilirubin 
o Decresed haptoglobin 
o Hemoglobinemia, hemoglobinuria in acute 
hemolysis 
o Hemosiderinuria in chronic hemolysis 
o 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
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
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
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)
DONATH-LANDSTEINER(D-L) TEST 
FOR DETECTING THE PRESENCE OF 
D-L ANTIBODIES 
Patient’s Whole Blood Control Test 
Incubate for 30 min at 
37 C 
Incubate for 30 min at 
37 C 
4 C 
37 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
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
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 
Autoantibody induced mechanism 
The 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
Serological features of the different types of drug-induced 
haemolytic anaemia of immunological origin 
Mechanism 
Prototype 
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
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 Hemoglobinemia 
Positive DAT (transient) 
Positive DAT 
Antibody in elute
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
 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
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
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
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
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
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
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.
LABORATORY FINDINGS 
Rh incompatibility ABO incompatibility 
 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
 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.
MICROANGIOPTHIC 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
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
DIC 
 PBS-schistiocyes 
 Thrombocytopenia 
 Abnormal 
coagulation test 
 Prolonged 
PT,APTT,TT 
 Elevated D-dimer 
test 
 Incresed fibrin 
degraded product 
(FDP) 
 Decrese fibrinogen
BABEIOSIS AND MALARIA
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.
REFERENCES 
•Dacie and lewis practical haematology 
•McKenzie- clinical laboratory hematology 
•Makroo -Compendium of transfusion 
medicine
Extracorpuscular hemolytic anemia

Mais conteúdo relacionado

Mais procurados

Haemolytic anaemia
Haemolytic anaemiaHaemolytic anaemia
Haemolytic anaemiaRam Negi
 
Benign White blood cell (WBC) Disorders
Benign White blood cell (WBC) DisordersBenign White blood cell (WBC) Disorders
Benign White blood cell (WBC) DisordersDr. Varughese George
 
Approach to hemolytic anemia naglaa
Approach to hemolytic anemia naglaaApproach to hemolytic anemia naglaa
Approach to hemolytic anemia naglaaNaglaa Makram
 
Sickle cell disease
Sickle cell diseaseSickle cell disease
Sickle cell diseaseYara Mostafa
 
Autoimmune haemolytic anaemia
Autoimmune haemolytic anaemiaAutoimmune haemolytic anaemia
Autoimmune haemolytic anaemiaAfra Fathima
 
Thalassaemia hemoglobinopathies dr.neela-feb_2012
Thalassaemia hemoglobinopathies  dr.neela-feb_2012Thalassaemia hemoglobinopathies  dr.neela-feb_2012
Thalassaemia hemoglobinopathies dr.neela-feb_2012tareq chowdhury
 
Hemolytic anemia
Hemolytic anemiaHemolytic anemia
Hemolytic anemiaFatima Avci
 
Hemolytic Anemia Classification - By Thejus K. Thilak
Hemolytic Anemia  Classification - By Thejus K. Thilak Hemolytic Anemia  Classification - By Thejus K. Thilak
Hemolytic Anemia Classification - By Thejus K. Thilak Schin Dler
 
Spherocytes significance and differential diagnosis
Spherocytes  significance and differential diagnosisSpherocytes  significance and differential diagnosis
Spherocytes significance and differential diagnosisKasturba Medical College
 
Platelet immunohematology
Platelet immunohematologyPlatelet immunohematology
Platelet immunohematologyRafiq Ahmad
 
Autoimmune Hemolytic Anemia (AIHA)
Autoimmune Hemolytic Anemia (AIHA)Autoimmune Hemolytic Anemia (AIHA)
Autoimmune Hemolytic Anemia (AIHA)RGCL
 
Myelodysplastic syndrome according to WHO 2016
Myelodysplastic syndrome according to WHO 2016Myelodysplastic syndrome according to WHO 2016
Myelodysplastic syndrome according to WHO 2016Madhuri Reddy
 
Thalassaemias - By Sarasjothi
Thalassaemias - By SarasjothiThalassaemias - By Sarasjothi
Thalassaemias - By SarasjothiSchin Dler
 
Approach to Hemolytic Anemia
Approach to Hemolytic AnemiaApproach to Hemolytic Anemia
Approach to Hemolytic AnemiaAbdullah Ansari
 

Mais procurados (20)

Pancytopenia
PancytopeniaPancytopenia
Pancytopenia
 
Haemolytic anaemia
Haemolytic anaemiaHaemolytic anaemia
Haemolytic anaemia
 
Benign White blood cell (WBC) Disorders
Benign White blood cell (WBC) DisordersBenign White blood cell (WBC) Disorders
Benign White blood cell (WBC) Disorders
 
Approach to hemolytic anemia naglaa
Approach to hemolytic anemia naglaaApproach to hemolytic anemia naglaa
Approach to hemolytic anemia naglaa
 
Hemolyic Anemia ppt
Hemolyic   Anemia   pptHemolyic   Anemia   ppt
Hemolyic Anemia ppt
 
Sickle cell disease
Sickle cell diseaseSickle cell disease
Sickle cell disease
 
Autoimmune haemolytic anaemia
Autoimmune haemolytic anaemiaAutoimmune haemolytic anaemia
Autoimmune haemolytic anaemia
 
Thalassaemia hemoglobinopathies dr.neela-feb_2012
Thalassaemia hemoglobinopathies  dr.neela-feb_2012Thalassaemia hemoglobinopathies  dr.neela-feb_2012
Thalassaemia hemoglobinopathies dr.neela-feb_2012
 
Sickle cell Anemia
Sickle cell AnemiaSickle cell Anemia
Sickle cell Anemia
 
Hemolytic anemia
Hemolytic anemiaHemolytic anemia
Hemolytic anemia
 
Aplastic anemia
Aplastic anemiaAplastic anemia
Aplastic anemia
 
Hemolytic Anemia Classification - By Thejus K. Thilak
Hemolytic Anemia  Classification - By Thejus K. Thilak Hemolytic Anemia  Classification - By Thejus K. Thilak
Hemolytic Anemia Classification - By Thejus K. Thilak
 
Spherocytes significance and differential diagnosis
Spherocytes  significance and differential diagnosisSpherocytes  significance and differential diagnosis
Spherocytes significance and differential diagnosis
 
Hemolytic anemia; Harrison 19th edition
Hemolytic anemia; Harrison 19th editionHemolytic anemia; Harrison 19th edition
Hemolytic anemia; Harrison 19th edition
 
Hemolytic Anemia
Hemolytic AnemiaHemolytic Anemia
Hemolytic Anemia
 
Platelet immunohematology
Platelet immunohematologyPlatelet immunohematology
Platelet immunohematology
 
Autoimmune Hemolytic Anemia (AIHA)
Autoimmune Hemolytic Anemia (AIHA)Autoimmune Hemolytic Anemia (AIHA)
Autoimmune Hemolytic Anemia (AIHA)
 
Myelodysplastic syndrome according to WHO 2016
Myelodysplastic syndrome according to WHO 2016Myelodysplastic syndrome according to WHO 2016
Myelodysplastic syndrome according to WHO 2016
 
Thalassaemias - By Sarasjothi
Thalassaemias - By SarasjothiThalassaemias - By Sarasjothi
Thalassaemias - By Sarasjothi
 
Approach to Hemolytic Anemia
Approach to Hemolytic AnemiaApproach to Hemolytic Anemia
Approach to Hemolytic Anemia
 

Semelhante a Extracorpuscular hemolytic anemia

ACQUIRED HEMOLYTIC ANEMIA.ppt
ACQUIRED HEMOLYTIC ANEMIA.pptACQUIRED HEMOLYTIC ANEMIA.ppt
ACQUIRED HEMOLYTIC ANEMIA.pptJeenaRaj10
 
Hematology (1) The blood and bone marrow, abnormal blood count, anemias: an o...
Hematology (1) The blood and bone marrow, abnormal blood count, anemias: an o...Hematology (1) The blood and bone marrow, abnormal blood count, anemias: an o...
Hematology (1) The blood and bone marrow, abnormal blood count, anemias: an o...Ahmed Elshebiny
 
Lab diagnosis autoimmune disease
Lab diagnosis autoimmune diseaseLab diagnosis autoimmune disease
Lab diagnosis autoimmune diseaseAppy Akshay Agarwal
 
laboratory diagnosis of hemolytic anemia-190509145931.pptx
laboratory diagnosis of hemolytic anemia-190509145931.pptxlaboratory diagnosis of hemolytic anemia-190509145931.pptx
laboratory diagnosis of hemolytic anemia-190509145931.pptxDEEPA ANANTHA LAXMI N.V
 
Aiha presentation
Aiha presentationAiha presentation
Aiha presentationVika Bler
 
anaemia.pptx
anaemia.pptxanaemia.pptx
anaemia.pptxsamwel18
 
Blood banking and transfusion medicine i&amp;ii
Blood banking and transfusion medicine i&amp;iiBlood banking and transfusion medicine i&amp;ii
Blood banking and transfusion medicine i&amp;iiAbdulKaderSouid
 
Hemolytic anemia (1)
Hemolytic anemia (1)Hemolytic anemia (1)
Hemolytic anemia (1)vvyvi
 
Immune mediated haemolytic anemia in dogs
Immune mediated haemolytic anemia in dogsImmune mediated haemolytic anemia in dogs
Immune mediated haemolytic anemia in dogsRaaz Eve Mishra
 
Making the diagnosis in hematology
Making the diagnosis in hematologyMaking the diagnosis in hematology
Making the diagnosis in hematologyfracpractice
 
Hema practical 02 hematology
Hema practical 02 hematologyHema practical 02 hematology
Hema practical 02 hematologyMBBS IMS MSU
 
Approach to Autoimmune hemolytic anemia
Approach to Autoimmune hemolytic anemiaApproach to Autoimmune hemolytic anemia
Approach to Autoimmune hemolytic anemiaKumar Abhinav
 

Semelhante a Extracorpuscular hemolytic anemia (20)

Aiha
AihaAiha
Aiha
 
AIHA -Autoimmune Haemolytic Anaemias
AIHA -Autoimmune Haemolytic AnaemiasAIHA -Autoimmune Haemolytic Anaemias
AIHA -Autoimmune Haemolytic Anaemias
 
ACQUIRED HEMOLYTIC ANEMIA.ppt
ACQUIRED HEMOLYTIC ANEMIA.pptACQUIRED HEMOLYTIC ANEMIA.ppt
ACQUIRED HEMOLYTIC ANEMIA.ppt
 
Hematology (1) The blood and bone marrow, abnormal blood count, anemias: an o...
Hematology (1) The blood and bone marrow, abnormal blood count, anemias: an o...Hematology (1) The blood and bone marrow, abnormal blood count, anemias: an o...
Hematology (1) The blood and bone marrow, abnormal blood count, anemias: an o...
 
Lab diagnosis autoimmune disease
Lab diagnosis autoimmune diseaseLab diagnosis autoimmune disease
Lab diagnosis autoimmune disease
 
laboratory diagnosis of hemolytic anemia-190509145931.pptx
laboratory diagnosis of hemolytic anemia-190509145931.pptxlaboratory diagnosis of hemolytic anemia-190509145931.pptx
laboratory diagnosis of hemolytic anemia-190509145931.pptx
 
Aiha presentation
Aiha presentationAiha presentation
Aiha presentation
 
anaemia.pptx
anaemia.pptxanaemia.pptx
anaemia.pptx
 
Haemolytic disorders
Haemolytic disordersHaemolytic disorders
Haemolytic disorders
 
Approach to anaemia
Approach to anaemiaApproach to anaemia
Approach to anaemia
 
Blood banking and transfusion medicine i&amp;ii
Blood banking and transfusion medicine i&amp;iiBlood banking and transfusion medicine i&amp;ii
Blood banking and transfusion medicine i&amp;ii
 
Aih
AihAih
Aih
 
Hemolytic anemia (1)
Hemolytic anemia (1)Hemolytic anemia (1)
Hemolytic anemia (1)
 
Noon conference 6_27_2018
Noon conference 6_27_2018Noon conference 6_27_2018
Noon conference 6_27_2018
 
Immune mediated haemolytic anemia in dogs
Immune mediated haemolytic anemia in dogsImmune mediated haemolytic anemia in dogs
Immune mediated haemolytic anemia in dogs
 
Hemolytic anemia
Hemolytic anemiaHemolytic anemia
Hemolytic anemia
 
Making the diagnosis in hematology
Making the diagnosis in hematologyMaking the diagnosis in hematology
Making the diagnosis in hematology
 
Hema practical 02 hematology
Hema practical 02 hematologyHema practical 02 hematology
Hema practical 02 hematology
 
Approach to Autoimmune hemolytic anemia
Approach to Autoimmune hemolytic anemiaApproach to Autoimmune hemolytic anemia
Approach to Autoimmune hemolytic anemia
 
Apheresis.pdf
Apheresis.pdfApheresis.pdf
Apheresis.pdf
 

Extracorpuscular hemolytic anemia

  • 1. APPROACH TO A CASE OF EXTRACORPUSCULAR HEMOLYTIC ANEMIA Dr. Adrija Pathak
  • 2. A.IMMUNE HEMOLYTIC ANEMIA 1.AUTOIMMUNE HEMOLYTIC ANEMIA Warm antibodies Cold anti bodies 2.ALLOIMMMUNE HEMOLYTIC ANEMIA Hemolytic disease of newborn Incompatible blood transfusion 3. DRUG INDUCED B.NONIMMUNE HEMOLYTIC ANEMIA Microangiopathic hemolytic anemias(DIC ,TTP,HUS) Trauma:prosthetic cardiac valve, thermal, exercise Infection: malaria, babesia Chemical and drugs Animal venoms Malignant hypertension Plasma lipid abnormalities Hypersplenism EXTRA CORPUSCULAR DEFECT
  • 3. 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 etiology a. Hereditary anemias ( defects within RBC ) b. Acquired anemias ( external causes )
  • 4. 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
  • 5. Increased Bone Marrow Production of Erythrocytes Increased Erythrocyte destruction COMMON LABORATORY FINDINGS IN HEMOLYTIC ANAEMIA 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
  • 6. EVALUATION OF ANEMIA Low Hgb/Hct Corr. Retic Ct >2% Corr. Retic Ct <2% Acute Blood Loss MCV>100 MCV 80- 100 MCV<80 EVALUATE & TREAT APPRO-PRIATELY Evaluate for Hemolytic Anemias Evaluate for microcytic anemias Evaluate for macrocytic anemias Evaluate for normocytic anemias
  • 7. 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 Hct Retic 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)
  • 8.  Reticulocyte production index(RPI) =corrected reticulocyte count/reticulocyte maturation time(days) Hct Maturation time (days) .35 1.5 .25 2 .15 2.5 >2RPI appropriate bone marrow response Eg -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
  • 9. ROLE OF PBS Hemolytic Anemia (CRC>2% + no blood loss) 1 Sickled cells Bite cells Schisto-cytes Acantho-cytes Sphero-cytes Target cells parasite inclusions DAT (+) DAT (-) Hgb electro-phoresis G6PD level PT/PTT Crea platelets Auto- Immune Hemo-lytic Anemia Heredi-tary Sphero-cytosis Sickle Cell Ds G6PD Deficient Vs Unstable Hgbs Thalas-semias Hemo-globino-pathy Liver Ds Liver Ds Malaria Babe-siosis Barto-nella TTP-HUS DIC Prosthe-tic Valve Malignant HTN
  • 10. 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
  • 11. 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
  • 12. 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
  • 13. 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
  • 14. Extravascular Hemolysis Destruction of red cells by reticuloendothelial cells in the liver, spleen, and bone marrow Significant lab finding: •Inc in expired carbon monoxide •Carboxyhemoglobin •Unconjugated bilirubin •Urine and fecal urobilinogen •Dec haptoglobin in severe hemolysis
  • 15. Intravascular hemolysis Extravascular hemolysis  Activation of complement- PNH, PCH, transfusion rxn  MAHA  Physical/ mechanical trauma  Toxic microenvironment  Hemoglobinopathies  Enzymopathies  Membrane defects  Megaloblastic anemia  AIHA  Drug induced
  • 16. 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.
  • 17. 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.
  • 18. 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
  • 19. 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
  • 20. 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
  • 21.  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.
  • 22.  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.
  • 23.  If there is a cold antibody: a. Has the antibody any specificity b. 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
  • 24.  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.
  • 25. AIHA CLASSIFICATION Warm autoimmune hemolytic anemia  Idiopathic, Secondary  (Lymphoproliferative disorders, autoimmune diseases-SLE,RA, viral, neoplastic)  Cold autoimmune hemolytic anemia  Cold agglutinin syndrome  (Idiopathic, Secondary- mycoplasma, infectious mono, LPD)  Paroxysmal cold hemoglobinuria  (Idiopathic, Secondary- measles, mumps, syphilis)
  • 26. CHARACTERISTICS OF AGGLUTININS Warm Reacting Ab Cold Reacting Ab  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
  • 27. LABORATORY IDENTIFICATION OF SENSITIZED RBC Agglutination of test sera and appropriate rbc suspended in saline- detect antibodies of IgM class
  • 28. 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
  • 29. 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
  • 30. 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.
  • 31. 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.
  • 33. 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
  • 34. PBS- normocytic normochromic anemia, polychromasia, nucleated rbc, spherocytes
  • 35. 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
  • 36. 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
  • 37. 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
  • 38. o Increased bilirubin o Decresed haptoglobin o Hemoglobinemia, hemoglobinuria in acute hemolysis o Hemosiderinuria in chronic hemolysis o 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
  • 39. 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
  • 40. 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
  • 41. 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)
  • 42. DONATH-LANDSTEINER(D-L) TEST FOR DETECTING THE PRESENCE OF D-L ANTIBODIES Patient’s Whole Blood Control Test Incubate for 30 min at 37 C Incubate for 30 min at 37 C 4 C 37 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
  • 43. 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
  • 44. 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 Autoantibody induced mechanism The 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
  • 45. Serological features of the different types of drug-induced haemolytic anaemia of immunological origin Mechanism Prototype 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
  • 46. 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 Hemoglobinemia Positive DAT (transient) Positive DAT Antibody in elute
  • 47. 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
  • 48.  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
  • 49. 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
  • 50. 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
  • 51. 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
  • 52. 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
  • 53. 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
  • 54. 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.
  • 55. LABORATORY FINDINGS Rh incompatibility ABO incompatibility  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
  • 56.  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.
  • 58. 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
  • 59. 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
  • 60. DIC  PBS-schistiocyes  Thrombocytopenia  Abnormal coagulation test  Prolonged PT,APTT,TT  Elevated D-dimer test  Incresed fibrin degraded product (FDP)  Decrese fibrinogen
  • 62. 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.
  • 63. REFERENCES •Dacie and lewis practical haematology •McKenzie- clinical laboratory hematology •Makroo -Compendium of transfusion medicine