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
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
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.
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
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
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