ANEMIA

Basic principles of Anemia

Anemia :
Hb , Hematocrit is below lower limit of
95% ref.interval for individuals age, sex
and geographic location (altitude)
Anemia Absolute or Relative

ANEMIA symptoms / syndrome

Hb
PCV Hypoxia Brain , Muscles
RBC
Compensation :
- heart rate tachycardia flow rate
cardiomegaly heart failure
- blood flow priority (pallor)
- RBC 2,3-DPG content O2 dissoc.curve
shift to the right O2 release to the
tissues .
4

Diagnostic approach to anemic

patients :
History :
onset /bleeding tendency / routine medicinal /
occupation / hobby / travel history / family / diet /
GI symptoms / menstruation cycle / history of
previous pregnancy-delivery / alcohol
consumption , etc
Physical examinations :
conjunctiva / lips / mouth / tongue / gum , nails /
hair , jaundice , petechiae , liver & spleen ,
lymphenodes ,rectal / vaginal toucher , feet
(ulcer,arthritis)
5

Laboratory Examinations :
- Full blood count to confirm anemia (Hb,
PCV, RBC) & the type of anemia (MCV/ MCH/
MCHC)
- Reticulocyte count reflects marrows
responses .
- Iron status ( Serum Iron ,TIBC, % Transferrin
saturation , Iron storage )
- Blood chemistry ( direct/total bilirubin,LDH
and stool examination for occult blood test ) .
6

- Radiological examinations ( Chest X-ray,

USG , MRI )
- Cardiological examinations (EKG, Treadmill,
Echocardiography)

Notes ! :
- First confirm Anemia ( Hb , PCV , RBC )
- Classify the anemia (MCV, MCH, MCHC)
- Causes of anemia

CLASSIFICATIONs of ANEMIAS :
I.

Pathophysiologic classification :
- Impaired production of red cells :
a/ Disturbance of hemopoietic stem cells
proliferation & differentiation :
Anemia Aplastic
b/ Disturbance of DNA synthesis :
Anemia Megaloblastic
c/ Disturbance of Hb synthesis:
Iron Deficiency Anemia,
Thalassemia
10

d/ Disturbance of Erythropoietin synthesis :

Anemia of Chronic Renal Failure
e/ Multiple mechanisms :
Anemia of Chronic Disease, Anemia
associated with marrow infiltration,
Sideroblastic Anemia
Increased red cells destruction or loss :
Hemolytic Anemia
Blood loss

11

II. Morphologic Classification :

- Classification of anemia based on red blood cell

morphology ( Size and Color / pallor) under

microscopic examination , or by calculating Red
Cells Indices ( MCV, MCH, MCHC )
- Size criterion: Normocytic, microcytic,
macrocytic
- Color criterion : Normochromic, hypochromic

12

- How to revealed Red Cells size :

* comparing with lymphocytes nucleus :

equal size = normocytic
smaller = microcytic , bigger = macrocytic
* by calculating the MCV ( Mean Cell Volume )
MCV= PCV/Ery X 1000 (fL)
(1 fL=10-12L= 1m3)
N : adult= 80-100 fL , under 1 yr = 76- 86 fL
MCV : normocytic , microcytic , macrocytic
* red cells with abnormal size variation =
anisocytosis
13

Compare the red cells size with lymphocytes nucleus .

14

Revealed the red cells color :

- compare the diameter of central pallor(CP)
against the diameter of red blood cell .
- Normally, the red cells shape is like a flattened
bilaterally indented sphere (biconcave disk)
in fixed blood smears appears circular, 7-8
with area of central pallor corresponding to the
indented regions .
CP 1/3 Eri = normochromic
CP> Eri = hipochromic
15

16

Red cell with its central palor(CP) compare the

diameter of CP with red cells diameter

17

- Color is also revealed from MCH (Mean Cell Hb)

MCH= Hb/RBC (pg)

adult :MCH=27-32 pg , children : MCH=23-31 pg
(1pg=10-12g=1g)
Normal MCH normochromic
MCH < normal hypochromic
- MCHC(Mean Cell Hb Concentration) :

MCHC=Hb/PCV (g/dL)
Normal: MCHC = 32-36 g/dL

18

Morphologic Classification of Anemia

I. Hypochromic-Microcytic Anemia
II. Normochromic-Normocytic Anemia
III. Macrocytic Anemia

19

- Hypochromic-Microcytic :

20

- Normochromic normocytic :

21

- Macrocytic . Find oval-macrocyte ( Megaloblastic Anemia

is characterized by this cell ).

22

Hypochromic-Microcytic Anemia
- Every conditions that impairing Hb synthesis

will give hypochromic-microcytic appearance

.
- Iron Deficiency Anemia (IDA) is the most

common cause of anemia and

Hypochromic-Microcytic Anemia aware
about other causes (DD) before confirming
the diagnosis of IDA .

23

24

Body Iron Status :

Serum Iron = SI
Total Iron Binding Capacity (TIBC)
% Transferrin Saturation = SI/TIBCx100%
Iron Storage :
- Hemosiderin is water-insoluble ferritins
degradation product consists mostly of aggregates
of ferric oxyhydroxide crystals, FeOOH (in Liver and
bone marrow) detected by biopsy/aspiration
and iron staining ( invasive procedure !)
- Ferritin water-soluble complex of ferric salt
and apoferritin, in very small amounts in serum
(detected by Immunoassay method)
25

IDA :
Iron storage SI

Normal
+
Prelatent Def
Latent Def /-

+
+

Morph
Norm-Norm
Norm-Norm
Norm-Norm
Hypo -Norm

IDA

Hypo-Mikro

26

27

Body Iron content = 35-50 mg/kgBW:

80% - Functional Fe, as heme-Iron
(65% Hb, myoglobin, enzyme
heme : cytochrom-C,A,A3,B,
catalase , peroxidase)
- Non-heme-Fe (some)
20% - Iron storage (ferritin,
hemosiderin)
only 15% in female
0.2% - circulating (bound to Transferrin)
28

Type of Iron

% Total Iron

Iron content

Fe-Hb
Fe-storage
Fe-myoglobin
Fe-others

70%
25%
5%
< 1%

28.5 mg
14.2 mg
1.86 mg
< 1.3 mg

29

Iron Cycle in the body :

Fe-diet as heme-Fe (Hb, myoglobin,
enzyme-Fe), 5-35% adsorbed
from animal/meat sources ,
adsorbed easily .
as non-heme-Fe (vegetables ,
legumes), 90% of diet-Fe but
only 2-20% of it absorbed
depends on the iron-status and
the ratio of Enhancer:Inhibitor
30

Enhancers (substances that stimulate

absorption) :
Ascorbate, Cytrate, organic acids / other
amino acids , by reducing Fe3+ to Fe2+.

Inhibitors (substances that inhibit

absorption) :

Carbonate, Phytate, Tannins, Phosphate,

Oxalat chelate Non-heme-Fe unabsorbable

31

Food substances that inhibit absorption of

Non-heme Iron :
- Phytate (from legumes,vegetables)
- Tannin & Polyphenol (from
tea,coffee,wine, cocoa)
- Phosphate/phosphoprotein from eggyolk
- Minerals (Ca, Zn, Cd)
- Tetracycline reacted with Fe inhibit
absorption .
32

Meat absorption are every protein

stimulate absorption ? Not Right !
White-egg & cows milk protein inhibit
absorption , but
breast milk enhances non-heme-Fe
absorption

33

Bodys Iron Cycle :

Diets Iron duodenum / proximal jejunum .
Iron from gut released into circulation , bound to
transferin distributed to bodys organ / tissues( to bone
marrow as a part of heme / Hb ) circulate inside red blood
cells with blood flow

34

Fe absorption in gut :
Type of Fe absorbed :
- the type of Heme
( reduced as soluble Fe2+-salt )
Absorbed Heme enterocytes mucousmembrane (internalisation) processed by
DMT-1 aided by cytochrome-b into Fe2+, CO and
bilrubin-IXa .
Fe2+ enter Fe-pool, bind to mobilferrin and
paraferritin becoming Ferritin, or moved to
contralateral enterocytes surface , oxidized by
Ferroportin and Hephaestin , bound to
transferrin .
35

Nonheme-Fe absorption :
- Fe3+ + chelator (ascorbate) soluble-form
bound to binding-protein and change Fe3+
to Fe2+ Fe-binding protein complex fixed by
specific transporter (integrin ?) to be
absorbed by enterocytes mucous membrane
into enterocyte and joining mucosal Ferritin as
storage-Fe , or moved to contralateral
enterocytes surface and oxidized to bind
Transferrin (Fe-carrier protein)
36

Fe Absorption mechanism :
DMT-1 (Divalent Metal Transporter)
regulates Fe-transport from intestinal
surface to cells inside (aided by duodenal
cytochrome-b, which reduces Fe3+ to
Fe2+)
Fe-transport from inside cell to plasma
involves Ferroportin-1 and Hephaestin
37

Fe transport from duodenum to plasma :

Transferrin

Fe-Ferroportin-1

BLOOD
---- Fe -----Hephaestin ----------------------------------- re-Oks Fe2+ Fe3+
- join Ferroportin-1

Fe mobilferrin
paraferritin

Ferritin
MUCOUS CELL

Fe2+

Fe-Transporter (DMT-1)
---------------

----------------------------------------------------Fe3+-reductase
LUMEN
(cytochrome-b)
38

Protein regulators for Fe :

Transferrin (Fe-transporter protein)
Transferrin-receptor , TrfR (accept Fe in
cell membrane)
Ferritin (Fe-storage)
TrfR and Ferritin regulate IRE-BP (FeResponsive Elements Binding Protein)
39

IRE-BP (Fe-Responsive Elements

Binding Protein)
= Fe-regulatory Factor
= Ferritin-repressive protein
= P90
coordinating the expression of TrfR and
intracellular-ferritin .
(IRE-BP also regulates the synthesis of
Apoferritin and Transferrin) .

40

Regulation of intracell-Fe metabolism :

Physiology :
- in Fe-sufficient cell synthesis of
apoferritin , and synthesis
of TrfR
- in Fe-deficient cell synthesis of TrfR
and synthesis of
apoferritin
41

if intracellular-Fe IRE-BP stimulates

and bind IRP (Fe-Regulatory Protein)
inhibit TrfR-mRNA digestion TrfR (and
also ALA-synthase) , and inhibit Ferritin-mRNA
translation Ferritin .
the amount of TrfRs mol.
reducing the Ferritin-trapping of Fe
entering the cell .
42

If intracell-Fe is sufficient IRE-BP is

not stimulated translation of TrfRs
mRNA < Apoferritins mRNA synthesis
TrfRs mol < ferritins mol.
(the amount of IRE-BP in TrfRs mRNA is
< IRE-BP in Apoferritins mRNA)

43

44

Transferrin
(Fe-saturated-Transferrin)

Transferrin-Receptor (TrfR)
TrfRs affinity to transferrin depends on
the cellular iron contents and pH .

45

46

47

48

Pathogenesis of Fe deficiency
3 pathogenetic factors :
- Impaired Hb synthesis (consequence of
reduced Fe supply)
Transf.saturation< 16% inadequate
Fe-supply to marrow Hb contents of
RBC hypochromic & microcytosis
- Generalized defect in cellular
proliferation .
49

- Fe-deficient oxidative damage to the

red cells membrane RBC deformability

RBC viability RBC destruction
especially in spleen reduced RBC
survival .

50

The development of IDA

* Stage-1 (prelatent Fe-deficient):
- progressive loss of storage-Fe
- bodys Fe reserve is still sufficient to
maintain both the transport and
functional
compartment , so RBC development is
still normal .
- peripheral blood picture is normal , no
symptoms of anemia , but ferritin is .
51

* Stage-2 (latent Fe-deficient)

- Exhaustion of storage-Fe , RBC
production is still normal , Ferritin
- Circulating-Fe (SI) begin , TransfReceptor .
* Stage-3 (Fe-Deficiency Anemia)
- Stadium of Iron Deficiency Anemia
52

Marrow
Ferritin
Transf-Sat
sTrfR
Retic Hb
content
Hb
MCV
Symptoms

Stage-1
(prelatent)

Stage-2
(latent)

Stage-3
(IDA)

N
N
N

(-)
<12ug/L
<16%

(-)
<12ug/L
<16%

N
N
fatigue

N
N
fatigue

<
<
pallor

53

Etiologic Factors in IDA :

Neg.Iron balance :
- Fe intake ( inadequate diet , impaired
absorption)
- Fe loss ( GI bleeding, excessive
menstrual flow, bleeding diathesis)
- demands ( infancy, pregnancy,
lactation)
54

Inadequate presentation to erythroid

precursors :
- atransferrinemia
- Anti TrfR Ab
Abnormal Fe balance :
- Aceruloplasminemia
- Autosomal dominant hemochromatosis
( mutations in ferroportin )
55

Transferrin :
- transport protein for Fe
-1/3 mol. Saturated with Fe(= %
transferrin saturation ) measured as
Serum Iron (SI)
- Transferrin is measured indirectly as
TIBC .
56

Transferrin

SI

Unsaturated Iron Binding

Capacity (UIBC)
SI + UIBC =
( TIBC )

57

Symptoms

Morphology

SI - TIBC

Ferritin

IDA

Anemia

Hypo
Micro

SI TIBC

A.C D

Anemia

Hypo
Micro

SI TIBC /N

N/

58

Diagnostic Approaches
to I D A
1. Anamnesis menstrual pattern, pregnancy /
delivery , bleeding tendency ,
chronic illnesses , diet ,
occupation , travel history

2. Physical exams systematic from bodys

surface to internal organs ( liver , spleen ,
lymphenodes )

59

3. Laboraty Hema (CBC,ESR , BSE, Retic)

- Serum (SI,TIBC,Ferritin, Bili)
- BMA (Bone Marrow Aspiration)
- Urine and Stools examinations
4. Supportive Radiology (EKG, USG,
Scanning)
- Endoscopy

60

SI
Normal

TIBC

N
(1/3 mol.Trsf)

IDA

An.of Chronic
Disease

N/

N/

Fe Overload

61

62

Laboratory Examinations
for I D A
1. CBC confirm Anemia & find hypochromic
microcytic picture from BSE and Red
Cells Indices ( Hb, PCV ,MCV , MCH ,
MCHC)
2. SI

Fe2+ released from Transferrin + ferrozine

(chromagen) measured colored
complex
TIBC serum + excess FeCl2 to fill all Transferrinbinding sites the excess Fe is fixed by Mgcarbonate Fe-saturated Transferrin is
measured with Ferrozine (= TIBC)
63

% Saturasi Transferrin = SI/TIBC X 100%

Erythropoeisis impaired when % Tf.Sat < 15%
3. Ferritin Serum :
Serum Ferritin level ~ Fe-storage
Ferritin <15 ug/L Definitive Fe-Deficient
N/ Ferritin in IDA , if :
- impaired liver function ( damaged
hepatocyte),
hemolysis, inflammation / infection /
malignancy ( Ferritin = acute-phase
protein )
64

4. Transferrin Serum :
measured by immunodiffusion methode
Normal value : 2-4 g/L
5. Bone Marrows Aspirate evaluation :
( using Perls or Prussian Blue stain )

65

Anemia of Chronic Infection

Clinical signs similar with IDA
Hematological pictures = IDA (An.Hypo-Micro, MCV,
MCH, SI) , but N/ TIBC and N/ Ferritin
Pathogenesis :
Fe storage // Transferrin

Tissues / RES
Theres very different treatments compare with IDA !

66

Causes of circ.Fe decreasing :

1. Impairment of Fe release from
macrophage in competing with
lactoferrin, phagocytes product , even
storage-Fe is still enough .
2. Inadequate EPO Respons towards
anemia (effects of cytokine production by
macrophage) .
67

Diagnosis of An. Of Chronic Diseases :

Hema lab :
- Hypochromic Microcytic Anemia
- SI , TIBC /N , Ferritin N/
( if Ferritin , Fe-deficiency confirmed )
- inflammation/infection (+) :
CRP and ESR
Problems : IDA with inflammation ferritin
(falsely diagnosed as ACD) ; it can be
differentiated by sTfR exam (serum transferrin
receptor) that in IDA but normal in ACD .
68

Sideroblastic Anemia
Defect of Heme synthesis Fe
accumulation in mitochondria Fe
degeneration Fe granules around
normoblasts nucleus forming ring-like
structure ( best seen with Perls stain )
Ringed Sideroblast (characteristic of
Sideroblastic Anemia)
Sideroblast can found normally in bone
marrow
69

Sideroblast and Ringed Sideroblast ( in

Sideroblastic Anemia )

70

71

Classification of Sideroblastic Anemia

1. Hereditary : X-linked, defect in hemesynthesis enzyme pathway
Fe absorption % of Transferrin
saturation and Ferritin level

72

2. Acquired :
- Primary :
Stem cell clonal mutations(MDS =
MyeloDysplastic Syndromes , RA-RS)
Normochromic-macrocytic anemia .
Marrow : erythroid hyperplasia with
dysplastic or megaloblastic appearance
- ringed sideroblast in normoblast .
73

-Secondary :
Abnormal metabolism of Vit.B6
(alcoholism, malabsorption) , impairment
of heme synthesis ( Pb intoxication) ,
Rhematoid Arthritis , or An.megaloblastik .
Usually related to myeloproliferative
diseases ( AML, Myelofibrosis,
Polycythemia or another types of MDS )
74

Macrocytic Anemia
Non-Megaloblastic Macrocytic Anemia :
Reticulocytosis
Liver disease / Alcoholism
Myelodysplastic Syndrome
Erythroleukemia (FAB-M6)
Megaloblastic Macrocytic Anemia
75

Megaloblastic Macrocytic Anemia :

macrocyte = erythrocyte with MCV > normal .
macrocyte/microcyte depend on the balance
between nuclei & cytoplasmic maturation .
(nuclear dividing stopped when intracellular Hb
production reach a proper level ) .
If nuclear maturation delayed ( in DNA
synthesiss defect ) or cytoplasmic maturation
( increase of EPOs activities ) critical level of
Hb achieved earlier Macrocyte
76

Megaloblast = bigger than normal

normoblast .
Megaloblastic changes = increased size of
hemopoietic precursor cells in bone
marrow ( not only in normoblast !)
Primary defect : Defect of DNA synthesis
( altered almost all active cells / organs i.e
: hemopoietic tissue, epithelial cells ,
mucous cells, etc )
77

Etiology of DNA synthesis defect :

deficiency of vit.B12 and folic acid
maturation dysharmony between nuclei &
cytoplasm (delayed nuclei maturation)
increased cels (megaloblastic changes)
marrows ineffective erythropoiesis
intramedullary hemolysis total/indirect
Bili and LDH .
78

Deficiency of Folic acid :

- Inadequate diet
(intake < / demand in pregnancy lactation , childs growth / malabsorption
in tropical sprue / bowel resection / small
intestine inflammation )
- Drugs effect (anti-epilepsi)
- FA loss (dialysis)
79

Deficiency of Vit.B12 :
- Inadequate diet :
Intake < in vegetarians , demand ,
impaired absorption caused by
decreased Intrinsic Factor
( gastrectomy , pernicious anemia )
Malabsorption (bowel infection , worms
/ blind loop syndr )
80

VITAMIN B12

ASAM FOLAT

-Food from animal products

-Heat stabile
-Storage : enough for 3 yrs
-Relatively low needs (only
1% of folate requirements)

-Limited sources (vegetable ,

CAUSE OF DEFICIENCy

CAUSE OF DEFICIENCY

-Vegetarian (seldom)
-Impaired Intrinsic Factor
(pernicious anemia)
-Gastrectomy
-Atropic Gastritis
-Anticonvulsant, alcoholism

-Nutrition (alcoholism, goats

milk diet)
-Prematurity
-Hemodyalisis
-Bowel resection
-Pregnancy
-Anticonvulsant , MTX

fruits)
-Heat labile
-Storage enough only for 3
mths
-Higher folate needs

81

The role of Folate & Vit. B12 in the

synthesis of DNA :
Deficiency of FA direct effect especially in
inhibiting d-UMP methylation dTMP ; also in
the generation of hyperhomocysteinemia
Deficiency of Vit.B12 indirectly inhibit the
production of THF (4H-Folate) from
5-me-THF increase of 5-me-THF (failed to
supply THF to folate cycle) and generate
hyperhomocysteinemia (homocystein cannot
changed to methionin)
82

Metabolisme B12 & Asam Folat

83

84

Pathogenesis of
Megaloblastic Anemia :
Megaloblastic changes
atrophy of tongue papilla & mucosal GI
glossitis , gastritis, nausea , constipation .
B12 defic demyelinisation of spinal
cord & peripheral nerve loss of foots
balance / sensory (Neuropatia)
FA defic hyperhomocysteinemia
thrombosis and vascular occlusion .
85

B12 Metabolism :
-Vit.B12 purine & pyrimidin synthesis
synthesis DNA & RNA mitosis and
maturation
-Vit.B12 made from microbiological source
because plants do not produce B12 ( meat ,
liver, eggs and milk are rich of Vit B12 )
-B12 content in the daily diet is 5-3ug , daily
requirement of B12 is 1-3 ug, and B12 bodys
storage is 2-5 mg (enough for 3 yrs)
86

Vit.B12 absorption :
B12 diet in gaster bind by IF (Intrinsic Factor)
produced by parietal cells IF-B12 complex ileum :
B12 absorbed , IF freed into the lumen
impaired IF : gastrectomy/gastritis/ Auto-Ab-antiIF or
Auto-Ab-antiparietal) no absorption of B12
impaired DNA synthesis (Pernicious Anemia with
Achlorhydria)
Pernicious Anemia = autoimmune disease auto-Ab
to parietal cells (Anti-IF or Anti-Parietal)

87

88

89

Hematological pictures of
Megaloblastic Anemia :
Bone Marrow :
- megaloblastosis
- ineffective erythropoiesis
Peripheral blood :
- Oval macrocytosis
- Hypersegmented neutrophil ( five 5-lobed
cells or one 6-lobed cell) or the mean lobes of
100 neutrophils is > 3.4

90

- Megaloblastic Anemia , find ovalMacrocyte cell and hypersegmented neutrophil .

91

Specific Diagnostic Tests :

Bone Marrow Evaluation :
- megaloblastosis & megaloblastic
changes, erythropoietic activitiy
( ineffective erythropoiesis)
Folate & B12 Assay (RIA)
Gastric juices Analysis (achlorhydria)
92

Schilling tests (for the diagnosis of

Pernicious Anemia):
- principle :
every time oral dose vit.B12 absorbed,
the excess of B12 (unused B12) will be
excreted via urine
so when B12 is not detected in
urine , it means that there is a
malabsorption .

93

- Steps of Schillings tests :

1. detection of malabsorption
- patient take Co57 B12 p.o
- after several hours , B12 is injected/i.m
(flushing dose) to saturate liver , so
B12-isotop is excreted into urine .
N: after 24 hrs B12-isotop is detected in
urine .
(how is the result if the flushing dose was
not given ?)
94

2. Step to decide if IF deficiency is the

cause of malabsorption ? :
- if there is malabsorption in step-1 , the
test followed with the addition of IF in
the oral dose of B12-isotop .
- if vit.B12-isotop is detected in urine
after adding IF , Pernicious Anemia is
confirmed .
( and whats the conclusion when after adding
IF, vit.B12-isotop is still not detected in urine
?)
95

Antibody Assay :
IF-AutoAb measured with ELISA method
and Parietal Cell-Ab with Indirect
Immunofluorescence method

96

Diagnosis of Megaloblastic Anemia :

Screening :
- CBC , Neutrophils lobe count
- Serum Indirect Bilirubin , LDH (lactate
dehydrogenase)
Spesific tests :
- Bone Marrow Aspiration
- Folate & Vit.B12 assay
- Gastric juice analysis
- Schilling Tests
- Antibody Assay
97

HEMOLYTIC ANEMIA
Normal red cells survival = 110-120 days
destructed by macrophage in marrow
and spleen .
When the survival are shortened EPO
production is stimulated (compensated)
no Hb changes anemia () .
If the destruction is acute or chronic with
very shortened life of red cells , there will
no compensation anemia (+) .
98

- Definition of Hemolytic Anemia :

anemia caused by shortened red cells
survival as a result of excessive
uncompensated destruction of red cells .
Hemolytic process = every process of
red cells destruction with still / without
compensated by bone marrow anemia
is not always present .
99

- Compensation ability of bone marrow :

Ability to red cells production ( 6-8 x
normal ) :
- survival shorten production 2x
- survival shorten production 4x
- survival shorten 1/6 production 6x
- survival shorten 1/8 production 8x
of production 6-8 x is maksimum .
If red cells live only 20 days anemia (+).
100

- Diagnostic approach in Hemolytic Anemia :

1. Confirm anemia (Hb/PCV/RBC)

an acute case usually acquired , and

chronic case is mostly hereditary .
2. To find the signs of hemolytic process .
3. Extra or Intravascular ?
4. Hereditary or acquired ?
5. The cause of hemolysis episodes .
101

The signs of Hemolytic process :

1. Increased of red cells destruction
- Unconjug.bilirubin serum jaundice
- Urobilinogenuria
- Hb-uria sign of intravascular hemolysis
- Abdom.pain splenomegaly, spleen infarction
- Legs Ulcer intrinsic defect of erythrocyte
- Haptoglobin serum /neg intravascular
hemolisys .

102

2. Red Cells destruction :

- Microspherocyte, Fragmentocyte, Poikilocyte
- Erythrocyte Osmotic Fragility
- Positive Autohemolysis test
- Shortened of red cells survival

3. Tanda Peningkatan Eritropoisis :

- Reticulocytosis
- Normoblastosis
- Erythropoietic Hyperplasia in bone marrow

103

104

105

106

107

108

109

110

- Classification of Hemolytic Anemia:

1. Hereditary Hemolytic Anemia
(Intracorpuscular)
- Membrane abnormality (hereditary
spherocytosis , hereditary ovalocytosis )
- defect of globin chain (Thalassemia, Hbpathia)
- enzyme defect ( G-6PD deficiency , PKdeficiency)

111

Hereditary Spherocytosis :

112

Hereditary Ovalocytosis :

113

2. Acquired Hemolytic Anemia

(extracorpuscular) :
- physical / chemical substances
- infections (bacteria, parasites, viruses,
fungi)
- mechanical trauma (prostetic heart valves)
- Immune mechanism (Alloimmune /
Autoimmune / Drug-Induced HA)

114

- Hereditary Spherocytosis :
autosomal dominant
Spherocytosis, decreased membrane surface
area relative to cell volume osmotic fragility
test (OFT) among the family member .
The primary lesion is caused by membrane
protein defects (of spectrin) cytoskeleton
instability .
60% - chronic anemia , jaundice, splenomegaly,
20% without hemolysis / splenomegaly .
Bilirubin excretion ,causing bilestone in USG.
115

116

Thalassemia :
Defect of 1 or more globin-chain synthesis (the
amount = quantitatively) :
- deficiency of globin-chain -thalassemia
- deficiency of globin-chain -thalassemia
- deficiency of globin-chain -thalassemia
the primary defects in Hb-pathia is in the globin
amino acids structure (qualitatively)
117

118

119

-Thalassemia:
-Thalassemia = is caused by the impairment of
-globin chain production/synthesis .
-globin chain synthesis is directed by 2 pairs of
-gene (4 locus -gen) depending of the
number of defected locus 3 types of Thalassemia (-thal trait , HbH Disease, and
HbBarts Hydrops Fetalis)

120

- Clinical consequences in Thalassemia

Deficiency of -globin chain excess of
, chain since fetal life to form 4tetramers (HbH) or 4-tetramers (HbBart) .
Defect of 1-2 -Gen = -trait (clinically
good)
Defect of 3 -Gen = HbH disease ( Hb
10-11 g/dl) excess of -chain to form
4-tetramers (HbH) as intracellular
inclusion detected by BCB-stain .
121

- HbH-inclusion (4) in HbH Disease as shown in

BCB staining (compare with reticulocyte)

122

Defect of 4 -gene (HbBartshydrops

fetalis) clinically severe , stillborn baby
with hydrops fetalis ( severe hypoxia ) .
HbBarts = 4-tetramers (excess of chains that unable to form HbF ) .
HbBarts and HbH inclusions precipitated
in red cells membrane mechanical
trapping in spleen macrophagic
phagocytosis hemolysis .
123

124

125

- -Thalassemia :
Clinically consequences in -Thalassemia :
- No problems during fetal life because HbF
synthesis is normally produced
(normal and chains)
- When HbA is dominantly needed , the clinically
problems exist as incapability to synthesize
HbA (22) excess of -chain
compensated of and production HbA2
(in -Thalassemia minor) and HbF (in
-Thalassemia mayor)
126

-Thalassemia mayor :
- severe anemia repeated transfusion
is oftenly needed Fe
hemochromatosis
- chronic ineffective erythropoiesis medullary
hypertrophy in childhood facial
malformation :
* Frontal bossing
* Maxillary hypertrophy
* Hypertelorism (mongoloids eye)
127

- -chain deletion forms :

0-Thalassemia : no -chain production.
+Thalassemia : -chain production
<<
in heterozygous case : medium severe
in homozygous : severe (Cooleys anemia)

128

129

- Laboratory Diagnosis in Thalassemia :

1. CBC, Peripheral Blood Smear
2. Hb-Electrophoresis : in Celulose-Acetat

(pH 8.4) for thalassemia and Hb-pathia

screening
Using hemolysate formed bands of
different types of Hb ( normal : bands A,
F, and A2 , measured densitometrically)
130

131

3. HbA2 mesurement to diagnose Thalassemia trait using anion-exchange

resin column chromatography
in both HbELP and chromatography ,
HbC, HbE and HbO can interrupt the
conclusion because of the same band
location with HbA2 .

132

4. HbF determination :
- Alkali Denaturation Test
- Acid-elution (Kleihauer) test
- RID or ELISA methods

133

5. HbH Inclusion detection :

- Supravital staining using Brilliant
Cresyl Blue (BCB) or NewMethylene
Blue (NMB)
- HbH inclusion seen as dispersed bluegreen granules in red cells
(compare with reticulocyte as a filament)
- in HbH disease : HbH inclusion +++
- in Thalassemia--trait : HbH inclusion +
in 1: 10000 eritrosit .
134

- HbH inclusion (4) in HbH Disease as shown by

BCB staining (compare with reticulocyte)

135

G-6PD Deficiency :
- Oxidant produce H2O2 oxidizing
Hbs free sulfhydryl to form Sulf-Hb
aggregates that precipitated as Heinz
Bodies destructed in spleen .
- Oxidant / Sulf-Hb are controlled by
Reduced Glutathione (GSH)
136

137

- X-linked, 300 variants .

normal G-6PD genes : - type B (GdB)
- type A (GdA)
- Abnormal enzyme types :
1. GdA (type A)
2. Gd-Mediterranean (GdMed)
3. Gd-Canton : many in Asia
- G-6PD deficient red cells are resistent
to Plasmodium Falciparum .

138

- Substances causing lysis in G-6PD

deficiency :
1. Antimalaria
2. Sulfonamides

6. Fava beans
7. Naphtalene

3. Vit.K, Vit.C
4. Lung Infection
(virus,bacteria)
5. Antipyreticum

8. Uremia
9. Antibiotics
(Penicilline ,
streptomycine

139

The highest G-6PD activity is in

reticulocyte .
G-6PD screening test :
Tests principle :
G-6PD
G-6P + NADP

6-PG + NADPH
UV
(fluorescence)

140

Acquired Hemolytic Anemia :

- Secondary Hemolytic Anemia caused by
infection / systemic disorders :

Malignancy Autoimmune-reacted
hemolysis , microangiopathy or
hypersplenisme , appearing Anemia of
chronic disease, bleeding tendencies, and
marrows suppression

141

Disseminated Intravascular
Coagulation (DIC):
Systemic intravascular coagulation fibrin
deposit intravascularly / endothelial damage
(microangiopathyi) caused by sepsis red cells
destruction .
Chronic Liver Disease : hemolysis caused by
hypersplenism .
Chronic Renal Disease: hemolysis caused by
microangiopathy
142

Acquired Hemolytic Anemia

(Extracorpuscular) :
Immune Hemolytic Anemia
Red cell membrane-bound Ab hemolysis .
The speed & hemolysis location depend
on IgG or IgM, and the ability to activate
complement .
Optimal temperature to bind Ab :
370C Warm-IgG-Type
<300C Cold-IgG-Type
143

Cell+IgG destructed by spleen

Cell+IgM enhance the activation of
complements cascade intravascular
hemolysis
Immune destruction often cause minimally
membrane damage shape change into
spherocyte .
144

Immune Hemolytic Anemia

classification :
1. Alloimmune : Transfusion Rx , Hemolytic
Disease of the Newborn (HDN)
2. Autoimmune : Warm/Cold AIHA,
Paroxysmal Cold Hb-uria (PCH)
3. Drug-induced HA : penicilline type,
aldomet, and stibophen type .
145

Hemolytic Disease of the Newborn (HDN)

Rh-neg mother , with Rh-Pos fetus , during I and second pregnancy

146

Antiglobulin Tests (Coombs) :

Direct Coombs Test (Direct Antiglobulin
Test/DAT) = Ab detection test (IgG and or
C3d /complement-bound red cells) .
Indirect Coombs Test = test for serum
free Ab .
DAT usually positive in AIHA .
147

Drug-Induced hemolytic anemia :

Penicilline type : drug as hapten binds red cell
membrane antigenic stimulate Ab production
against Drug in drug-red cell complex
Phenacetin/Quinidin type : Drug (hapten)
adsorbed protein stimulated-Ab binds drugprotein complex activate complement red
cell lysis.
Aldomet type : drug change red cell membranes
structure detected as foreign cell
Autoantibody production .
148

149

Aplastic (Hypoplastic?) Anemia

Severe & fatal Anemia because of red
cells/leucocytes/platelet production
(pancytopenia) caused by Stem Cells
impairment (radiation, chemicals, drugs, or
genetic matters)
Marrow aplasia / hypoplasia-causing substances
- radiation , benzene, cytostatics (6-MP,
busulfan), arsen, chloramphenicol,
anticonvulsant (phenytoin), analgetic
(phenylbutazone) , DDT, etc
150

- Symptoms & Lab.appearance of

Aplastic Anemia :
fatigue, palpitation, infections, bleeding tendency
Lab : - pancytopenia
- normochromic normocytic
- dry-tap marrow , hypocellularity
Prognosis :
- bad especially for < 40 yrs old patients
marrow transplantation .

151

- Treatment for Aplastic Anemia :

1. Avoid every toxic material
2. Avoid infections / bleeding tendency
3. Use Washed-Erythrocyte if transfusion is

needed or Plat.Concentrate (PC) for any

profuse bleeding ( give corticosteroid if
bleeding is minimal)
4. Marrow stimulants (androgenic hormon )
5. Marrow Transplantation
152

END

153

154

155

156

Anemia , MCV,oval makrositosis

Gejala neurologik +,hipersegmentasi netrofil

Assay Vit.B12
Defisiensi

Normal

Ab-Anti-FI
Pos
An.Pernisiosa
Terapi

Neg

Sum.Tulang

Megalobl

Tes Schilling
Pos

Asam Folat

Non-Megalobl

Neg

An.Pernisiosa
Defis.Vit.B12

Defis.As.Folat

MMA,homosistein

157