Pantipa Tonsawan ,MD
Hemolytic anemia
Major mechanism : defined as a reduction of
RBC life span to less than normal range of
100-120 days
Classification
1.Abnormalities of RBC interior
a.enzyme defect
Intracorpuscular
b.hemoglobinopathies
hereditary
2.RBC membrane abnormality
a.hereditary spherocytosis,etc
b.PNH
c.spur cell anemia
3.Extrinsic factors
a.hyperslenism
Extracorpuscular
b.antibody : immune hemolysis
c.microangiopathic hemolysis
d.infection or toxin
acquired
Classification of autoimmune
hemolytic anemia
1.warm-reactive (IgG) antibody
2.cold-reactive IgM antibody
(cold agglutinin disease)
(paroxysmal cold hemoglobinuria)
3.drug-depent antibody
drug adsorption(haptene) type
drug-dependent antibody (immune complex)
autoimmune induction type
nonimmunological adsorption of protein
Incidence of immune hemolytic anemia
No. of
patients
Percent of
total
Warm AIHA
Cold agglutinin
sydrome
244
54
70.3
5.6
Paroxysmal cold
hemoglobinuria
Drug-induce
6
1.7
43
12.4
Pet LDGarry:Acquired Immune Hemolytic anemia,New york:Churchill Livinestone
ANTIGLOBULIN TESTING
•The antiglobulin test, referred to anti-human globulin test (AHG)
or the Coombs test : detect significant unexpected antibodies
coated cells either in vivo or in vitro.
•Types of Antiglobulin Tests
☺ Direct Antiglobulin Test (DAT) - Detects antibodies or
complement coating patient's cells in vivo.
☺ Indirect Antiglobulin Test (IAT) - Uses a 37oC incubation
step so antibodies in serum can react with antigens on cells in vitro
Principle of Antiglobulin Test
•Red cells coated with complement or IgG antibodies do not agglutinate
directly when centrifuged.
•Call : sensitized cell with IgG or complement.
Antibody molecule represents the anti-globulin reagent that binds
with the Fc portion of the IgG antibody attached to the red blood cells.
Antibody molecule represents the anti-globulin reagent
that binds with the complement attached to the red blood cells.
Direct antiglubulin test
Negative Antiglobulin Test
Antibodies are not
attached to the
antigens during
incubation.
Wash the cells 3 times to remove any unattached antibodies.
Add Anti-human globulin
No visible agglutination and therefore a negative test
Positive Antiglobulin Test
•Wash cells three times to remove unbound Ab
•Only antibody attached to the cells remain
Add Anti-Human Globulin
Visible Agglutination in the test tube
Clinical Causes of Positive DAT
•Warm-acting Autoimmune disease, lead to patient antibodies coating their own
cells. ,cold-acting autoimmune hemolytic anemia would be due to IgM antibodies
that in turn activate complement. The complement-coated cells would then be
detected by the antiglobulin reagent.
•Hemolytic disease of the newborn is due to the mother's IgG antibodies crossing
the placenta
•Complement on the red cells may be the result of antigen-antibody reactions which
may involve red cells
•Passive transfer of antibody from donor units of plasma or platelets may attach to
the patient's red cells since recipients are given ABO compatible blood but other
unexpected red cell antibodies may not have been detected.
•Sensitization of red cells due to medications like penicillin and cephalosporins
•Normal patient with unexplainable reasons for a positive DAT
Warm-reactive (IgG) antibody
•Due to the presence of warm agglutinins is almost
always due to IgG antibodies
•React with protein antigens on the RBC surface at
body temp. (37°c)
•Exclude drug induced autoantibody
•Destroy RBC by extravascular hemolysis in spleen
•Primary or idiopathic Vs secondary
ETIOLOGY :
idiopathic
•No underlying disorder or direct cause can be found
•Occur any age group
•Female predominate ratio: 2:1
•Review of Warm type 19 of 33 pt that label as
idiopathic could be link underlying immune-mediated
disorder with careful history & follow up
Conley CL :immunologic precusors of autoimmune hematologic disorder.Autoimmune hemolytic
observations withparticular reference to their prognotic value A survey of 57 cases ,Johns Hopkins
Med J1981;149:101-109
ETIOLOGY: Secondary cause
• Autoimmune disorder
Systemic lupus erythematosus , RA ,scleroderma
ulcerative colitis, antiphospholipid syndrome
•Malignancy associated AIHA
hematologic: non-Hodgkin's lymphoma, CLL ,AML ,MM
Waldenstorm macroglobulinemia
tumor :ovarian dermoid cyst , teratoma ,Kaposi sarcoma
•Infections: viral (usually in children) ,EBV ,hepatitis C infection
•Acquired immunodeficiency virus infection
•Prior allogenic blood transfusion or hematopoietic cell
transplantation
Clinical manifestation
•Signs and symptoms of AIHA are nonspecific
• AIHA developing symptoms due to anemia depend on
severity
rapidity with which the anemia develops
concurrent illness ( underlying cardiac disease).
•In healthy resting humans, normal O2 delivery can be
maintained by HB conc of 8 to 9 g/dL
•Compensation of increases in SV and HR (and therefore CO)
are included, O2 delivery can be maintained at a Hb as low as 5
g/dL (equivalent to a hematocrit of 15 percent)
Weiskopf, RB, Viele, MK, Feiner, J, et al. Human cardiovascular and metabolic response to acute,
severe isovolemic anemia. JAMA 1998; 279:217.
Common presenting Sign & symptom
Symptoms
Weakness
Dizziness
Fever
Bleeding
Dyspnea
angina
confusion
frequency(%)
88
50
30
10
9
2
2
Sign
Splenomegaly
Hepatomegaly
Lymphadenopathy
Jaundice
Cardiac failure
pallor
82
45
34
21
5
4
Modified from Pirosky B.clinical aspect of autoimmune hemolytic anemia. Semin Hematol 1976;13:251-265
Laboratory findings
•CBC: HB range 7 - 10 g/dL ,WBC slightly increase ,Plt typical normal
•RBC indicies: MCV increase ;young RBC ,relative folate deficiency
•Blood smear: microspherocytosis
•reticulocyte count : elevated, decrease in early course or BM
shutdown : malignant invasion , parvovirus B-19 infection
•Total bilirubin : elevated rarely above 5 mg/dl : indirect predominate
•serum LDH increase & serum haptoglobin markedly reduce
•combination of an increased serum LDH and reduced haptoglobin is
90 percent specific for diagnosing hemolysis,
•combination of a normal serum LDH and a serum haptoglobin greater
than 25 mg/dL is 92 percent sensitive for ruling out hemolysis
•Marchand, A, Galen, RS, Van Lente, F. The predictive value of serum haptoglobin in hemolytic disease. JAMA 1980; 243:1909.
•Galen, RS. Application of the predictive value model in the analysis of test effectiveness. Clin Lab Med
1982; 2:685.
Laboratory findings
•Increase in erythropoietin production induced by anemia should
raise the reticulocyte percentage above 4 to 5 percent;
•Biological false-positive : common in syphilis ,other that reported
antithyroid AB ,rheumatoid factor & anticardiolipin AB
•Direct Coombs' test — The diagnosis of warm agglutinin AIHA is
based upon detection of antibody on the surface of the RBC, usually
by the direct antiglobulin (Coombs') test
•Over 95 % warm AIHA that DAT positive , 5% DAT negative
•Hemolytic DAT-negative : malnutrition, protein loss, PROZONE
phenomenon , Low affinity of IgG AB, IgA mediated hemolysis
Technique testing,
normal
microsphrerocyte
Approximate RBC destruction rate as predicted
from serum lactic dehydrogenase levels
LDH (u/l)
< 200
200-500
500-1000
>1000
Approximate RBC
destruction rate (x normal)
1 (range0.5-1.5)
2(range1.5-2.5)
3(range2.0-4.0)
>4
Modifed from Myhre E,Rasmussen K,Andersen A:serum LDH activity in patient with
prosthetic heart vlves :A parameter of intravascular hemolysis:Am Heart J
1997;80:463-468
Reticulocyte Production index ;
absolute reticulocyte count /RMI
45%
35%
25%
15%
1.0
3.5
1.5
3.0
2.0
2.5
1.5
Marrow normoblast &
reticulocyte
maturation time(days)
2.5
Blood reticulocyte
maturation time (days)
BPI : rate of erythropoiesis relative to normal
worsening anemia and increasing erythropoietin stimulation, bone
marrow reticulocytes (left) leave the marrow
Adapted from Hillman, RS, Ault, KA (Eds). Normal erythropoiesis, in: Hematology in Clinical
Practice, McGraw-Hill, New York, p. 29.
Example ; male pt anemia Hct :25%
reticulocyte count : 15%

Absolute reticulocyte count :
=(Hctpt x reticulocte count)/ normal Hct
8%



Circulating RMI at Hct 25 % = 2
Production index :
=Absolute reticulocyte count /reticulocyte maturation time (days)
8/2 =4
Erythropoiesis 4 times of normal rate
Screening patient for
acquired immune hemolytic anemia
Positive
DAT
AIHA
No AIHA
Total
24
5
29
Total
Negative
DAT
1
70
71
25
75
100
Positive value of positive result = 89 %
Positive value of negative result =99%
Modified from Kaplan HS ,Garratty G:predictive value of DAT result:
Diagn Med 1995; 8;25 29-33
Treatment of Warm AIHA
REDUCTION IN ANTIBODY PRODUCTION
Corticosteroids
Immunosuppressive and cytotoxic agents
REDUCTION IN ANTIBODY EFFECTIVENESS
Splenectomy
Intravenous gamma globulin
RED BLOOD CELL TRANSFUSION
Corticosteroids
•First therapy for warm AIHA
•Dose : high 1 MKD of prednisone or its equivalent until 10 g/dl
then taper of steroid
•Rapidly dose reduced over 4-6 wks to 30 mg/d
•Tapering should be slowly over by 3-4 mo
•Then continue low dose 5mg EOD for prolong period to prevent
relapse
•No Data to support this practice
•Response(1-3 wk) : reticulocyte decrease ,Hct stable then
rising Hb/Hct 2-3 g/dl/week , if no improvement in 3wk :
steroid treatment failure
Corticosteroids
•80 % response to steroid
• not response to steroids, or require doses of corticosteroids
(15mg/d) to maintain their response ; suggest splenectomy
class2B
unwilling or unable to undergo splenectomy, suggest the
institution of immunosuppressive or cytotoxic agents (class 2C).
•DAT : positive although strength reaction decrease & clinical
remission
•Some case in long term remission pt : still positive DAT
Immunosuppressive and cytotoxic agents
• Indications : lack of response to or inability to tolerate prednisone
need for a maintenance prednisone dose >15 to 20 mg/day
Cyclophosphamide : very effective immunosoppressive agent
•doses of 100 mg/day oral, or 500 to 700 mg iv every 3 - 4 wks
•Response rate 40-60%
•numerous SE : hair loss, gonadal toxicity, bone marrow suppression
,hemolytic cystitis/bladder fibrosis ,secondary malignancies
Azathioprine
fewer side effects but less effective than cyclophosphamide
initial oral dose of 100 to 150 mg/day
generally does not reduce the reticulocyte count or other blood cell
counts by direct toxicity.
Cyclosporine and Mycophenolate mofetil
Cyclosporin A :complete &partial response in case report oral dose 5
to 10 MKD bid dose adjustment on hematologic response,BP, BUN, Cr, and
electrolytes)
Mycophenolate mofetil ; MMF, starting dose 500 to 1000 mg/d oral bid
increasing to 1000 to 2000 mg/day used in some cases of resistant
autoimmune disease.
Monoclonal antibodies : approved in lymphoma
•Multiple case reports have indicated success with use of the monoclonal
anti-CD20 antibody (rituximab) in patients with resistant AIHA ; dose identical
lymphoma 375mg/m2weekly * 4 weeks
•less experience is available with use of the monoclonal anti-CD52 antibody
Campath-1H (alemtuzumab):report response in AIHA with Evan ’s syndrome
Danazol: a limited experience ;report variable response
Plasmapheresis :Limited success
IVIG
•occasionally effective in Rx of refractory AIHA to conventional therapy
with prednisone and splenectomy
•initial regimen to control in pts with very severe disease
•Only about 40 percent of patients response
Splenectomy
•nearly as efficient as corticosteroids
•Mech: removal of major site of RBC sequestration+ destruction of
Warm AIHA due to IgG Ab & autoantibody
•60 to 70 % improve in the anemia, usually evident within 2 weeks
• about 1/2 of those that achieve remission, corticosteroids in lower
doses than necessary before splenectomy to maintain the
remission.
Algorithm for treatment of immune hemolytic
anemia
minimal
marked
Access severity
watch
Prednisolone
IVIg
moderate
splenectomy
Pred 60mg/d
splenectomy
2-3 wks
no
response
yes
No
response
relapse
Reduce rapidly
20 mg/m2/d
Reduce slowly
5-10mg/wk
Cyclophosphamide 100mg/d
Azathioprine 150 mg/d
Rituximab 375 mg/m2 weekly
Discontinue if no
sign of disease
Blood transfusion
• required when the hemoglobin falls below a level tolerated by
the physiology of the patient.
• fully compatible blood ,aware side effect of blood transfusion
Hb level(g/dl) Probability of
significant
impairment
≥10
8-10
6-8
Very low
Low
Moderate
6
high
Transfusion
strategy
Aviod
Avoid
Try to aviod
:decrease act.,
transfusion
Required transfusion
PetzLD :blood transfusion in acquired hemolytic anemia ;Clinical practice of tranfusion
Medicine;3rd ed New york :Churchill Livingstone 1996:469-499
Cold-reactive IgM antibody
•Typical :direct against polysaccharide antigens on the RBC surface.
•RBC binding activity at 0 c°
•Less common than Warm AIHA ~ 20 %
•Two differenct clinical syndrome
Cold agglutinin syndrome (CAD)
Typical : associated with IgM Ab , adult ,may be primary or
secondary usually infection
Cold agglutinin attach to RBC in cooler peripheral circulation, back to
warmer circulation: Ab attach ,fixed complement response for destruction
Paroxysmal cold hemoglubinuria (PCH)
: Donath-Landsteiner Ab ,IgG hemolysis
Cold agglutinin syndrome (CAD)
Etiology
•Primary or idiopathic :older Pt ,peak around 70 yrs ,monoclonal IgM
•Secondary: young adult , transeint process ,self limited , polyclonal
IgM
Infection common : Mycoplasma pneumoniae (primary
atypical pneumonia) & infectious mononucleosis
Less common associated with other virus such as CMV and
varicella , One bacterial infection, a particular strain of Listeria
monocytogenes
Neoplasm : Waldenstorm macroglobulinemia
,Angioimmunoblastic lymphoma ,CLL , kaposi sarcoma , MGUS,
MM
CLINICAL MANIFESTATIONS
•Mild, chronic hemolytic anemia : exacerbate in winter/ rule of CAD
•,anemia (Rare Hb < 7 g/dl ) & jaundice:
•Some Pt: intermittent burst hemolysis with hemoglobinemia&
hemoglobinuria when expose to cold
•Patients with cold agglutinin AIHA may have symptoms related to both
the anemia and the agglutination of red blood cells.
Anemia — symptoms : DOE, dyspnea at rest, varying degrees of fatigue,
and signs and symptoms of the hyperdynamic state; depend on degree
and rapidity of the fall in Hct.
CLINICAL MANIFESTATIONS
•Changes on exposure to cold — symptoms related to the agglutination
of red cells in vivo upon exposure to cold temp.
Acrocyanosis common manifest :a dark, purple to gray discoloration of
the skin on the most acral parts — finger tips, toes, nose, and ears.
color disappears upon warming of the part and there is little or no reactive
hyperemia # (occurs in Raynaud phenomenon)
may be severe to cause ulceration of the skin.
may complain of pain and discomfort on swallowing cold food or liquids.
•other physical findings —mild enlarged spleen from the resulting hemolysis.
If larger or palpeble in secondary due to lymphoma or EBV infection
•if large lymph nodes are present, an underlying lymphoma should be
suspected.
CLINICAL MANIFESTATIONS
•Mycoplasma infection :
hemolysis begin when Pt recovering from pneumonia
= Titer for cold autoantibody at peak
resolved spontaneous within 1-3 wks
•Infectious mononucleosis
begin with onset of illness or within 3 wks
self limited
tend to affect younger
LABORATORY FINDINGS
•Anemia : mild / 5-6 g/dl
•Blood smear ; significant large agglutination & clumping/ not
prewarm specimen
•Dissolution with warm : suggest cold agglutination
•RBC indicies : MCV increase, RBC count decrease , high MCHC
reticulocyte count increase
•LDH level : high / complememt, haptoglobin level : low to absent
•Birirubin : unconjugate hyperbilirubinemia / < 3 mg/dl
•Direct Coomb’s test: positive with polyspecific & anticomplement
antisera
Blood smear
TREATMENT
in primary
Avoidance of cold — most useful single therapy :dress warmly
even in the summer. Warm shoes, stockings &gloves
Cytotoxic agents : cyclophosphamide and chlorambucil :reduce the
production of antibody. sometimes successful in combination with
corticosteroids : modality is not generally useful , underlying
lymphoma, appropriately aggressive chemotherapy is indicated
Rituximab —reports have indicated the usefulness of the
monoclonal anti-CD20 antibody rituximab in the treatment of subjects
with cold agglutinin disease and severe hemolysis not responding to
treatment with conventional therapy
TREATMENT
Plasmapheresis :
adjunctive treatment to remove the IgM Ab from the plasma,
to a reduction rate of hemolysis.
This procedure is effective ,if IgM, is confined to the
intravascular space.
effect of temporary ;difficult to use for chronic Rx.
used to reduce severe hemolysis, at initial present CAD due
to an infection
Splenectomy : ineffective ,not advice
Blood transfusion :Mild anemia : not need +to avoid blood transfusion
severe or cardiovascular compromised ; infuse RBC through
inline blood warmer at 37 °C /the best method
Secondary cold agglutinin : treat underlying disease ,infection –
self limited
Paroxysmal cold hemoglubinuria (PCH)
Dramatic presentation of intermittent hemolysis
Donath-Landsteiner antibody ; potent hemolysyn
Classical : Biphasic hemolysyn
cooler temp : Ab bind to RBC
warmer temp : complement activation
Three clinical syndrome :
1.Chronic PCH associated with late-stage or congenital syphilis
2.Acute transient PCH after infectious illness most common
3.Chronic idiopathic PCH
Clinical manifestations
•Sudden onset : fever ,back or leg pain & hemoglubinuria after cold
exposure
•Cold exposure:only a few minute & symptom may follow several hour
•Urine : dark red or black & typical clear in several hour
•Spleen :palpated during attack
•Vasomotor phenomena : cold urticaria, tingling of hands & feet
,cyanosis & Raynaud phenomenon
•Attack : associated with measles , Mumps, influenza A, adenovirus ,
EBV ,varicella ,mycoplasma pneumoniae : self limited
Laboratory feature
• PBS : sphererocytosis ,nucleated RBC ,polychromatophilia
•Urine : positive of hemoglobinuria, methemoglobiuria
•Donath-Landsteiner test :simple test; lysis positive
•Different of Cold agglutinin & D-L antibody include
specific & immunoglubulin class
classic age
Management
•Acute attack : supportive
•In severe case : steroid are usually given /benefit not documented
Chronic PCH :avoidance of cold & required any other theray
Different of Cold Autoantibodies
Primary cold
Agglutinin
disease
Secondary cold
autoantibodies
Paroxysmal cold
Hemoglobinuria
Ig
Clonality
DAT
Hemolysis
IgM
Monoclonal
C3
Chronic ,mild
IgM
IgG
Polyclonal
C3
Target RBC
antigen
I
Mono/polyclonal
C3
Self-limited,mild to
severe
I,i
Episodic, selflimtied: mild to
severe
P
DRUG-RELATED IMMUNE HEMOLYSIS
☺ Hapten/Drug
☺ Ternary
Adsorption mechanism
complex formation
☺ Autoantibody
Binding
☺ Nonimmunologic
protein Adsorption
Hemolysis
Hapten/Drug Adsorption mechanism
•Classic setting : very high dose penicillin therapy(10-30mu/d)
•Mech: bind firmly to protein RBC Mb , IgG antibody, gradual onset
•Develop substantial coating, not injurious
•IgG antipenicillin antibody bind to RBC –bound peniciilin :DAT positive
•Destruction : sequestration by splenic macrophage
•Not all pt : hemolytic anemia
•Typical duration : after receive the drug for
7-10 days & improve a few days to 2 wk
after discontinuting the drug
•Cephalosporin cross-reactivity with penicillin
•Example : penicillin ,cephalosorin
,tetracycline
Ternary complex formation
•Immune complex mechanism (or innocent
bystander ): drug- Mb binding target cellantibody
•Difference from Hapten/Drug Adsorption
mechanism
Exhibit only weak direct bind toRBC Mb
Small dose : trigger destructed of RBC
Hemolysis: sudden, severe,
Cellular injury : c’ activation
•Example ; quinine ,chlorpropamide
ampho B,diclofenac ,rifampicin
Autoantibody Binding
•Mech.: induce the formation of autoAb reactive with
autologous RBC
•Hemolytic : not depend on drug dose
•Hemolysis: mild to moderate, destruction by splenic sequestration
•DAT :positive 1 mo after drug use
•Ex. Methydopa ,levodopa ,
procanamide
• Rechallange does not produce an
anamnestic response but delay
in Ab
Nonimmunologic protein Adsorption
•Most common in
cephalosporin use
•< 5%: develop DAT positive
•Due to nonspecific
adsorption of plasma protein
on RBC
•Not cause RBC
destruction
Difference of drug induce-hemoltsis mechanism
Hapten
Ternary
complex
autoantibody
Non-immune
Prototype
drug
penicillin
Quinidine
methydopa
cephalosporin
Role of
drug
Bind RBC
protein
Ternary
complex
AB to RBC
antigen
Alter RBC mb
Drug affinity
to cell
Strong
weak
None
demonstrated
strong
AB to drug
Present
Present
Absent
Absent
AB class
IgG
IgG
IgG
Non IgG
Dose asso
DAT
High
Low
high
high
Mech.
destruction
Splenic
complement
sequestrated
Splenic
sequestrated
No hemolysis
Drug causing IHA or positive DAT
antibiotic
NSAID
Anti
neoplastic
other
Anti
hypertensive
Ampho B
Penicillin
Cephalosporin
Sulfonamide
B-lactamase
inhibitor
(sulbactam,
tazobactam,
clavulonic acid)
Diclofenac
Ibuprofen
Mefenamic
acid
Carboplatin
Cisplatin
Melphalan
6-MP
Rituximab
5-FU
Interferon
Flutarabine
Hydralazine
Methyldopa
Erythromicin
Rifampicin
INH
HCTZ
furosemide
Levodopa
Phenytoin
Quinidine
procainamide
Clinical feature
•Careful history of drug use all Pt with hemolytic anemia
•Severity depend on : rate of hemolysis
•Hapten/drug adsorption & autoimmune type: mild to
moderate RBC destruction
•Ternary mechanism : severe hemolysis
Lab investigation
•Similar in warm AIHA
•Thrombocytopenia & leukoplenia : due to Ternary
complex mechanism
Therapy ,course & prognosis
•Discontinue of drug use : only treatment
•High dose penicillin : change other ATB
•Taking methyldopa: DAT positive but not hemolysis :not stop
consider alternative antihypertensive drug
•Glucocorticoid: unnecessery Except : CLL with hemolysis
cause by purine analogs
•Prognosis :good in mild hemolysis
•Occasional severe ,renal failure, death due to ternary
complex ,purine analogs with CLL
conclusion







Clinical clue suggested : AIHA
Evidence support
Exclude other cause
Indication of blood transfusion risk/benefit
Medication/surgery
Response/non response ; follow & mornitoring
Treatment of underlying disease & precipitating
factor
Laboratory evaluation of hemolysis
Extravascular
Intravascular
Polychromatophilia
Increased
Erythroid hyperplasia
polychromatophilia
increased
Erythroid hyperplasia
↑Unconjugated
↓Absent
normal - ↑
↑(variable)
↑ Unconjugated
Absent
↑↑
↑ ↑(variable)
0
0
0
0
+
+ in severe
Hematologic
Reticulocyte count
Blood film
BM exam
Plasma or serum
Bilirubin
Haptoglobin
Plasma hemoglobin
LDH
Urine
Bilirubin
Hemosiderin
hemoglobin
Intravascular RBC destruction
RBC
haptoglobin
Hb
albumin
Hb
methemoalbumin
renal
Hb
Hb-HP complex
RES
Hb
hemosiderin