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