Pantipa Tonsawan ,MD

1
Immune hemolytic anemia
Pantipa Tonsawan ,MD
2
Hemolytic anemia
Major mechanism defined as a reduction of RBC
life span to less than normal range of 100-120
days
3
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.infecti
on or toxin
hereditary
Intracorpuscular
acquired
Extracorpuscular
4
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 nonimmunologi
cal adsorption of protein
5
Incidence of immune hemolytic anemia
No. of patients Percent of total
Warm AIHA Cold agglutinin sydrome Paroxysmal cold hemoglobinuria Drug-induce 244 54 6 43 70.3 5.6 1.7 12.4
Pet LDGarryAcquired Immune Hemolytic anemia,New
yorkChurchill Livinestone
6
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

7
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. 
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Direct antiglubulin test
9
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
10
Add Anti-Human Globulin  
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
11
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

12
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. (37c)
• Exclude drug induced autoantibody
• Destroy RBC by extravascular hemolysis in spleen
• Primary or idiopathic Vs secondary

13
ETIOLOGY idiopathic

• No underlying disorder or direct cause can be
  found
• Occur any age group
• Female predominate ratio 21
• 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 J1981149101-109
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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

15
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 279217.
16
Common presenting Sign symptom
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
Modified from Pirosky B.clinical aspect of
autoimmune hemolytic anemia. Semin Hematol
197613251-265
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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 2431909.
• Galen, RS. Application of the predictive value
  model in the analysis of test effectiveness. Clin
  Lab Med
• 1982 2685.

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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,

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normal
microsphrerocyte
20
Approximate RBC destruction rate as predicted
from serum lactic dehydrogenase levels
LDH (u/l) Approximate RBC destruction rate (x normal)
lt 200 200-500 500-1000 gt1000 1 (range0.5-1.5) 2(range1.5-2.5) 3(range2.0-4.0) gt4
Modifed from Myhre E,Rasmussen K,Andersen Aserum
LDH activity in patient with prosthetic heart
vlves A parameter of intravascular hemolysisAm
Heart J 199780463-468
21
Reticulocyte Production index absolute
reticulocyte count /RMI
3.5
3.0
2.5
1.5
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.
22
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

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Screening patient for acquired immune
hemolytic anemia
Positive DAT Negative DAT Total
AIHA No AIHA Total 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 Gpredictive
value of DAT result Diagn Med 1995 825 29-33
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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
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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

26
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

27
Immunosuppressive and cytotoxic agents

• Indications lack of response to or inability
  to tolerate prednisone
• need for a maintenance prednisone dose gt15 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.
28
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

29
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.
30
Algorithm for treatment of immune hemolytic anemia
minimal
marked
Prednisolone IVIg splenectomy
Access severity
watch
moderate
Pred 60mg/d
2-3 wks
splenectomy
no
response
Reduce rapidly 20 mg/m2/d
yes
No response
Reduce slowly 5-10mg/wk
relapse
Cyclophosphamide 100mg/d Azathioprine 150
mg/d Rituximab 375 mg/m2 weekly
Discontinue if no sign of disease
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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 low Low Moderate high Aviod Avoid Try to aviod decrease act., transfusion Required transfusion
PetzLD blood transfusion in acquired hemolytic
anemia Clinical practice of tranfusion
Medicine3rd ed New york Churchill Livingstone
1996469-499
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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

33
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

34
CLINICAL MANIFESTATIONS

• Mild, chronic hemolytic anemia exacerbate in
  winter/ rule of CAD
• ,anemia (Rare Hb lt 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.
35
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.

36
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

37
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 / lt 3
  mg/dl
• Direct Coombs test positive with polyspecific
  anticomplement antisera

38
Blood smear
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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
40
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
41
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
42
Clinical manifestations

• Sudden onset fever ,back or leg pain
  hemoglubinuria after cold exposure
• Cold exposureonly 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

43
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

44
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 IgM Mono/polyclonal C3 Self-limited,mild to severe I,i IgG Polyclonal C3 Episodic, self-limtied mild to severe P
45
DRUG-RELATED IMMUNE HEMOLYSIS
? Hapten/Drug Adsorption mechanism ? Ternary
complex formation ? Autoantibody Binding ?
Nonimmunologic protein Adsorption
Hemolysis
46
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

47
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

48
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
  
  
  
  

49
Nonimmunologic protein Adsorption

• Most common in cephalosporin use
• lt 5 develop DAT positive
• Due to nonspecific adsorption of plasma protein
  on RBC
• Not cause RBC destruction

50
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 sequestrated complement Splenic sequestrated No hemolysis
51
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
52
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

53
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

54
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

55
THe end
56
Laboratory evaluation of hemolysis
Extravascular Intravascular
Hematologic Hematologic Hematologic
Reticulocyte count Blood film BM exam Polychromatophilia Increased Erythroid hyperplasia polychromatophilia increased Erythroid hyperplasia
Plasma or serum Plasma or serum Plasma or serum
Bilirubin Haptoglobin Plasma hemoglobin LDH ?Unconjugated ?Absent normal - ? ?(variable) ? Unconjugated Absent ? ? ? ?(variable)
Urine
Bilirubin Hemosiderin hemoglobin 0 0 0 0 in severe
57
Intravascular RBC destruction
RBC
haptoglobin
Hb
albumin
Hb
renal
Hb
Hb-HP complex
methemoalbumin
RES
hemosiderin
Hb