AIHA, Comlements, PNH

B12/Folate-Megaloblastic
anaemia
 Immune Haemolytic Anaemias &
Paroxysmal Nocturnal Haemoglobinuria
Immune Haemolytic Anaemias
• Definition: IHA are Ab mediated R C destruction
• Types
• Autoimmune : Autoantibodies against Px’s own own RC
• Alloimmune: Abs produced by px against foreign RBs e.g bld trx and drug induced
haemolysis
classificatuon
• Warm AIHA
• Abs active invitro at 370c
• Polyclonal IgG
• May be detected in serum at 370c
• 10-15% show specificity for Rh Ags ( Anti e, c and anti D)
• Cold Antibody Type
• Predominantly IgM
• Most actively bound to Ag in the cold
• (40c)
• Mainly specific for I Ag and
rarely i in assoc. with EBV
• Warm Autoimmune H A
• CAuses

• Idiopathic
• Associated with
• Autoimmune dxs e.g SLE, R A etc
• Lymphoproliferative disorders e.g B -cell CLL, B –cell NHL
• Infections –viral infections in children
• Cancers e.g ovarian c a
• Drugs e.g penicillin, methyldopa
• Evans syndrome (AIHA + ITP)
• Cold Antibody type
• Cold agglutinin syndromes
• Cold haemaglutinin dx
• (Idiopathic)
• Proxysmal cold haemoglobinuria
Autoimmune H A
• Definition
• A grp of anaemias xterised by:
• Shortened R C survival
• Presence of autoantibodies directed against autologous RCs
• +ve direct antiglobulin test(coomb’s test)
• Degree of anaemia depends on
• Rate of RC destruction’
• Capacity of marrow to compensate
pathogenesis
• WAHA is mainly extravascular, cell mediated and complement
mediated
• Macro. have Fc receptors for IgG1&IgG3
• IgG coated RBCs are destroyed in the spleen by phagocytosis
In partial phagocytosis, only portions of the RBCs are removed and they
re-enter circulation as spherocytes
In the liver RCs are destroyed when coated with c3
• In cold AIHA, IGM agglutinins bind RBCS in the PR circulation
• Here the temp. may be as low as 10-200 c
• Complement activation occurs and lead to c3b and c3d expression on
RBCs surface. Macro. have receptors for CR1 and CR3).
• Hence Macro. Destruction of RCs is the main mech. of haemolysis in
IgM coated cells
• In cold AIHA syndromes intravascular Haemolysis may be ppted by
cold
Factors influencing RC dest. and production
• 1.BM function may be impaired by:
• AutoAbs against reticulocytes & erytro.
• Folate def. 20 to incr. demand
• 2. RES Fnx. severity of cellular immune RC dest. Depends on macro.
Fnx eg SLE(↓RE. Fnx due to clearance of Immune compl) also
methyldopa↓clearance of IgG coated RCs hence px may have
strongly +ve DCT with little haemolysis
• 3. Hypocomplementaemia e.g SLE and chronic haemagglutinin
disease(CHAD)
Clinical Features
• Presentation is variable depending on marrow’s ability to compensate
& the underlying dx
• Female preponderance
• Insidious onset
• Heart failure in severe anaemia
• Mild splenomegaly abt 2-3cm
Lab. features
• Pbf
• Spherocytes, Macrocytes
• Circulating nucleated RCs
• Red cell agglutination
• Moderately elevated Bilirubin
• Inc. LDH
• +ve DAT
Treatment

• Corticosteroids
• Prednisolone 1-2mg/kg/day x 3mths
• (H2 blocker to prevent PUD)
Azathioprine 1.5-2 mg/kg/day if poor response to pred. effect seen 4-
6wks
Cyclophosphamide 1.5-2mg/kg/day
Treat. cont.
• Cyclosporin 5mg/kg/day in 2 div. doses
• (Monitor renal and hepatic fnx)
• Response may not be seen untill 2-3 mths
• Blood trx (most grp compatible bld)
Splenectomy if no response to pred after 3mths (IgG coated cells )
Prognosis:
variable depends on:
Age, Assoc. dxs
Severity of haemolysis
Mortality-abt 10% at 5yrs, 40% at 7 yrs esp in >50 yrs
Children 5%
Cold Agglutinin syndromes
• Benign condition with distressing clinical symptoms
• Purplish discolouration of the skin maximal at extremities (acrocyanosis)
• Due to stasis in PR circ. 20 to Rc agglu.
• Colour returns upon warming the skin
• Mild icterus

• Spontaneous
agglu. Of RCs in the PBF
• Haemolysis in cold agglu. Dx may be seen after mycoplasma
pneumoniae infection
• Haemolysis usually abt 2-3wks after infection (self limiting
occasionally; fatal intra vascu Haem may occur)
• Infectious mononucleosis
• Toxoplasma infections
Treatment of CAD
• Avoidance of cold
• Bld trx tru a bld warmer
• F.acid
• Chlorambucil
• Plasma exchange
• Steroids have no role
Paroxysmal Cold Haemoglobinuria
• A rare syndrome
• Ist described by Donath, Landsteiner and Erlich in congenital and 30
syphlis
• Occurs in children following viral infections
Clinical features
• Haemoglobinuria
• Abdominal pains
• Prostration
• Cold Ab is IgG biphasic reacting with RCs below 200c in PR circ. and
causing lysis by complement activation as RCs are warmed to 370c in
the central vessels
• The Ab has P Ag specificty
•
trx
• Keep px warm
• Trx ABO and RH compatible bld tru a bld warmer
Drug induced H A
• Rare 3 main mech.s :
• Drug absorption (Hapten) mech eg penicillin, cephalosporins
• Form hapten carrier complexes with plasma pros which enhance drug specific Ab
production (IgG antipenicillin Abs)
• Attach to the drug bound to Rc surface- extravascular haemoliysis
• Menb. Modification mech.
• Eg cephalo drug modifies RC memb components, a variety of plasma proteins
including IgG and complem. Attach to Rc memb.
• Immune complex (Innocent Bystander)
• E.g rifampicin, quinine, hydrochlorothiazide etc
• Hapten-carrier complexes are formed b/w drugs and plasma pro
leading to production of drug specific Abs.
• once drug Ab s are present, re-intro of the drug causes immune more
Ab to form
• Immune Abs are absorbed on RC menb
• Complem is activated
• Haemol classically occurs on the 2nd or subsequent exposure to the
drug within mins or hrs of ingestion
• Severe intr vasc haemo may occur with fever, rigors, nausea and ARF
Diagnosis
• Dx of drug induced immune H A is made in 3 stages
• Dx of a +ve DAT HA
• Drug HX
• Serological demonstration of drug specific Ab which interacts with RCs
Paroxysmal Nocturnal Haemoglobinuria
• Def.
• PNH is an aquired HSC disor. Xterised by a chronic H A, thrombotic episodes
and often pancytopaenia
• It’s a clonal disor. Caused by somatic mutation of the X linked gene
phosphotidylinositor glycan A (PIG-A
Aetio pathog.
• Somatic mutation pIG protein A gene
is essential for formationof glyceryl phosphotidyl inositor (GPI)
• GPI is the anchor thru which a number of proteins are attached to the
cell surface
• In RCs memb 2 of these proteins protect:
• 1. The cells from lysis DAF (CD55) and
• 2. MIRL (CD 59) protects by activated complements
• Their absence lead to susceptibility to complement
lysis by several folds
• Absence causes lysis of RCs by acidified serum invitro
(Ham’s test)
• GPI anchor proteins are also missing on white cells and platelets
• Absence of CD 59 in platelets may account for the thrombosis seen in
pts
• Incidence & epidemiology
• Dx of young adults but children and elderly may also be affected
• Both sexes are equally affected
• Assoc. with aplastic anaemia & other forms of marrow failure
Clinical Fxs
• Classically red or dark urine particularly 1st samples after sleep clears gradually
• Budd-Chiari syndrome
• DVT, cerebral throb.
• Intermittent abd. pain
• Few pxs may dev. Marrow failure or acute leuk.
• Cause of death is thrombosis esp. Budd-Chiari syndrom
• Infections and Haemorhages rare
Lab fxs
• Anaemia (normo Normo)
• Leukopaenia
• Thrombocytopaenia
• Reticulocytosis but < deg. Of anaemia
• Inc. LDH
• Absent Haptoglobin
• BMA- Inc. cellularity esp in BMF
• NAP dec. ( a GPI anchor pro)
• Dec serun Fe & serum ferritin
• Definitive DX
• Acidified serum lysis test (HAM’s Test)
• Immunophenotyping CD 55 & CD 59
Rx
• Mainstay of RX:
• Correct anaemia
• Tx & prevention of throb
• Folic acid suppl
• Fe RX if there is def.
• Trx
• Leucocyte depleted bld
• Washed red cells
• Oral anticoagulant if throm
• Definitive - BMT
THE COMPLEMENT
SYSTEM
 Complement system

• part of hummoral innate immune system

• group of serum and cell surface proteins

• important in antiinfectious and inflammatory immune response

• activated by a cascade of reactions

• during activation generates active components

Complement activation pathways

• Alternative pathway (pathogen surface)

• Mannan binding lectin pathway (pathogen surface)

• Classical pathway (antigen-antibody complexes)

Complement activation pathways
Primary complement deficiencies

1) Components of activation pathways

C1q, C1r,C1s,C4,C2,C3, MBL, D,B,C5,C6,C7,C8,C9

2) Control proteins
• soluble control proteins
C1 inhibitor, factor I, factor H,C4b binding protein,S protein,
SP-40,40
• membrane regulatory proteins
CD 55(DAF), MCP CD 46, CD 59, HRF/C8bp

3) Receptors for complement

C1q receptor, CR1(CD 35), CR2 (CD21), CR3 (CD11b/CD18), CR4 (CD11c-
CD18)
Primary complement deficiencies
clinical manifestation
• Increased susceptibility to infections
with systemic course - bacteremia+meningitis
• S. pneumoniae, S.pyogenes, H.influenzae
(early components , defect in opsonization)
• Neisseria meningitidis
(defect in terminal components )
• Autoimmune disorders
defective immune complex clearance
• Angioedema
Complement genes

• Complement system proteins – members of various gene

families
• Some groups of complement proteins are in close chromosomal
linkage
• chromosome 1
• chromosome 6
Primary complement deficiencies
Conclusions
• Relatively uncommon diagnosed group of diseases
• Can be the underlying disease in autoimmunity and recurrent
infections
• Laboratoty investigation of complement components is not easy, but
C3 and C4 is normal available
• Most frequent is C2 deficiency
• Angioedema in C1 inhibitor deficiency appears in more generations in
one family, differential diagnosis to allergy
 MEGALOBLASTIC ANAEMIA

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 Definition-Megaloblastic anaemias are a
group of disorders xterisd by:
*Distinctive morphologic appearance of
developing erythroid cells in the bone marrow
*Macrocytes on the blood film
*MCV in excess of 95fl
*Caused usually by Vit B12 or folate def

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 CAUSES
• Vit B12 Deficiency/abnormalities its metabolism
• Folate def/abnomalies of its metabolism
• Therapy with anti-folate drugs
• Independent of Vit B12 or folate; acutee leukaemias,
MDS, cytotoxic-AZT,ara-C HU, 6MP
• Orotic acidudia
• Lesch-Nyhan syndrome
• Alcohol (acquired enz def)

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 CHEMISTRY OF VITAMIN B12
- Planar
- Nucleotide
corrin corrin
- 5’ deoxyadenosyl (ado)
- Methylcobalamin Co
- Hydroxocobalamin +
corrin corrin

Nucleotide
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 DIETARY SOURCES AND REQUIREMENTS
*Kidney, liver shell fish, muscles, fish

*Normal daily dietary intake 7 –30 g

*Adult daily loss in urine and faeces 1 - 3g
*Daily requirement 1 - 2g
*Body store (2 – 3mg)

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 ABSORPTION-site is ilium
Intrinsic factor
R-binder
Cubilin-IF-B12
Enterohepatic circulation
0.5 – 5gm
Transport
Transcobalamin II(TC), I(Haptocorrin) and III

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PERNICIOUS ANAEMIA

• Autoimmune gastritis
• Histology- g. atrophy, plasma cells/lymphoid cells infilteration
• Helicobacteer pylori may be implicated
• M:F -1:1.6
• Associated: polyendocrine dx, ca. stomach, blue eyes, blood group A,
early greying

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 CHEMISTRY OF FOLATE
Pteroylglutamic acid
- Pteridine, para-amino benzoate and L-glutamic acid

Body stores and requirements

Total body folate = 10-12mg
Daily adult requirement = 100 - 150 g
Daily loss in urine 13 g
Absorption : SITE- Duodenum/jejunum
Enterohepatic circulation about 80-100 g

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 AETIOLOGY OF VIT B12 DEFICIENCY
-Nutritional
-Malabsorption
-Gastric causes
-Intestinal causes
AETIOLOGY OF FOLATE DEFICIENCY
-Dietary
-Malabsoprtion
-Excessive utilization or loss
Antifolate drugs
Mixed causes

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 EPIDEMOLOGY
United States of America
PA = 0.25 – 0.5cases/1000 persons in their 7th decade
International
PA = 100 – 130 cases per 100,000 of the population

RACE

AGE

MORTALITY AND MORBIDITY

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 BIOCHEMICAL BASIS OF MEGALOBLASTIC ANAEMIA
* Defect in DNA synthesis rapidly dividing cells

-Disparity in rate of synthesis of DNA precursors

-Nuleo-cytoplasmic asynchrony and
POSSIBLE EXPLANATION:
-‘Methyl folate trap’ hypothesis
-Formate –starvation hypothesis

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 CLINICAL FEATURES
-Onset is insiduous
-Anaemia
-GIT symptoms
-Sore tongue and canker sores
-Early neurological symptoms
-Purpura
-Melanin pigmentation

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 DIFFERENTIAL DAIGNOSIS
-Lesch – Nyhan syndrome
-Orotic aciduria
-Some cases of MDS and AML
-Therapy with some drugs
- Cytosine arabinoside
- Hydroxyurea
- Antifolate

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 DIFFERENTIAL DIAGNOSES OF MACROCYTOSIS

Macrocytosis Alcohol
Liver disease (especially alcoholic)
Reticulocytosis (haemolysis or haemorrhage)
Aplastic anaemia or red cell aplasia
Hypothyroidism
Myelodysplasia
Myeloma and macroglobulinaemia
Leucoerythroblastic anaemia
Myeloproliferative disease
Pregnancy
Newborn
Congenital dyserythropoietic anaemia (type II)
? Chronic respiratory failure
Hypersegmented neutrophils Renal failure
Congenital (familial)
? Iron deficiency

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 LABORATORY FINDINGS
-FBC
-PBF
-Bone Marrow Aspiration/Cytology
-LDH and indirect bilirubin assay
-Serum iron and ferritin
-Vit B12 specific test
- Therapeutic trial
- Deoxyuridine suppression test
- Serum B12 level
- TC blood levels, rbc/serum folate

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Megaloblasts/Hypercellular marrow
(aspirated cytology)
•A BB B

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Haemoglobinised megaloblasts

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Excess iron in the bone marrow

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 TREATMENT
-Cobalamin (1000 gm given parentally daily for 2 weeks, then weekly
until hematpcrit value is normal, monthly for life
-Folate (1 -5 mg) administered orally 4-6/12

RESPONSE TO THERAPY
Prophylactic therapy
Prognosis

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