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Pantipa Tonsawan ,MD
Hemolytic anemiaMajor 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
b.hemoglobinopathies
2.RBC membrane abnormality
a.hereditary spherocytosis,etc
b.PNH
c.spur cell anemia
3.Extrinsic factors
a.hyperslenism
b.antibody : immune hemolysis
c.microangiopathic hemolysis
d.infection or toxin
Intracorpuscular
Extracorpuscular
hereditary
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
Paroxysmal cold hemoglobinuria
Drug-induce
24454
6
43
70.35.6
1.7
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
Antibody molecule represents the anti-globulin reagent that binds with the complement attached to the red blood cells.
•Red cells coated with complement or IgG antibodies do not agglutinatedirectly 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.
Principle of Antiglobulin Test
Direct antiglubulin 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
Negative Antiglobulin Test
Positive Antiglobulin Test
Add Anti-Human Globulin
•Wash cells three times to remove unbound Ab•Only antibody attached to the cells remain
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 VsVs 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 disorderSystemic lupus erythematosus , RA ,scleroderma ulcerative colitis, antiphospholipid syndrome
•Malignancy associated AIHAhematologic: non-Hodgkin's lymphoma, CLL ,AML ,MM
Waldenstorm macroglobulinemiatumor :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.
Symptoms frequency(%)
Weakness
Dizziness
Fever
Bleeding
Dyspnea
angina
confusion
88
50
30
10
9
2
2
Sign
Splenomegaly
Hepatomegaly
Lymphadenopathy
Jaundice
Cardiac failure
pallor
82
45
34
21
5
4
Common presenting Sign & symptom
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,
microsphrerocyte
normal
Approximate RBC destruction rate as predicted from serum lactic dehydrogenase
levels
LDH (u/l) Approximate RBC destruction rate (x normal)
< 200
200-500
500-1000
>1000
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
Marrow normoblast & reticulocyte maturation time(days)
Blood reticulocyte maturation time (days)
45%
35%
25%
15%
1.0
1.5
2.0
2.5
BPI : rate of erythropoiesis relative to normal
Adapted from Hillman, RS, Ault, KA (Eds). Normal erythropoiesis, in: Hematology in Clinical Practice, McGraw-Hill, New York, p. 29.
worsening anemia and increasing erythropoietin stimulation, bone marrow reticulocytes (left) leave the marrow
3.5
3.0
2.5
1.5
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
PositiveDAT
NegativeDAT
Total
AIHANo AIHATotal
24
5
29
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
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.
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
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 syndromeDanazol: a limited experience ;report variable response
Cyclosporine and Mycophenolate mofetil
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.
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
Plasmapheresis :Limited success
Algorithm for treatment of immune hemolytic anemia
Access severity
Pred 60mg/d
response
watch Prednisolone
IVIg
splenectomy
minimal marked
moderate
2-3 wks
Reduce rapidly 20 mg/m2/d
Reduce slowly 5-10mg/wk
Discontinue if no sign of disease
yes
splenectomy no
No response
Cyclophosphamide 100mg/d
Azathioprine 150 mg/d
Rituximab 375 mg/m2 weekly
relapse
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
Transfusion strategy
≥10
8-10
6-8
6
Very lowLow
Moderate
high
AviodAvoidTry 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
•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.
•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.
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
TREATMENTPlasmapheresis :
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 documentedChronic 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
Target RBC antigen
IgM
Monoclonal
C3
Chronic ,mild
I
IgMMono/polyclonal
C3
Self-limited,mild to severe
I,i
IgG
Polyclonal
C3
Episodic, self-limtied: mild to severe
P
DRUG-RELATED IMMUNE HEMOLYSIS
☺ Hapten/Drug Adsorption mechanism
☺ Ternary 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 cell-antibody
•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
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 sequestrated
complement
Splenic sequestrated
No hemolysis
Difference of drug induce-hemoltsis mechanism
Drug causing IHA or positive DAT
antibiotic NSAID Anti
neoplastic
Anti
hypertensive
other
Ampho B
Penicillin
Cephalosporin
Sulfonamide
B-lactamase inhibitor
(sulbactam, tazobactam,
clavulonic acid)
Erythromicin
Rifampicin
INH
Diclofenac
Ibuprofen
Mefenamic acid
Carboplatin
Cisplatin
Melphalan
6-MP
Rituximab
5-FU
Interferon
Flutarabine
Hydralazine
Methyldopa
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
Extravascular Intravascular
Hematologic
Reticulocyte countBlood filmBM exam
PolychromatophiliaIncreasedErythroid hyperplasia
polychromatophiliaincreasedErythroid hyperplasia
Plasma or serum
BilirubinHaptoglobinPlasma hemoglobinLDH
↑Unconjugated↓Absent normal - ↑↑(variable)
↑ UnconjugatedAbsent ↑ ↑↑ ↑(variable)
Urine
BilirubinHemosiderin hemoglobin
000
0++ in severe
Laboratory evaluation of hemolysis
albuminHb
Hb
methemoalbumin
Hb-HP complex
Hb
haptoglobin
Hbhemosiderin
Intravascular RBC destruction
RES
renal
RBC