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
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:
Activation of complement- PNH, PCH, transfusion rxn
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 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
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.
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
LABORATORY IDENTIFICATION OF SENSITIZED RBCAgglutination 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
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.
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
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
Reticulocytosis Erythrophagocytosis in buffy coat BM- normoblastic h/p Decreased C3 and/or C4
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
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
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
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
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
No drug Serum + drug
Eluate + drug
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
HEMOLYTIC TRANSFUSION REACTION
Timing Immediate (within 24hrs)
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
HemoglobinemiaPositive DAT (transient)
Positive DATAntibody 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.
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
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
Severe CNS symptoms Mild CNS symptoms
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
PBS-schistiocyes Thrombocytopenia Abnormal
coagulation test Prolonged
PT,APTT,TT Elevated D-dimer
test Incresed fibrin
degraded product (FDP)
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.
•Dacie and lewis practical haematology•McKenzie- clinical laboratory hematology•Makroo -Compendium of transfusion medicine