Agus Susanto Kosasih

Laboratorium Patologi Klinik

RS Kanker Dharmais




1
Definition of anaemia
Anaemia is defined as a reduction in the
haemoglobin concentration of the blood
This results in a decreased oxygen
carrying capacity
2
PATHOLOGY, SYMPTOMS, AND SIGNS OF ANEMIA

Normal values for peripheral blood
Female Male
Erythrocytes (per l) 4.80.6x10
6
5.40.8x10
6
Hemoglobin (g/dl) 14 2 16 2
Hematocrit (%) 42 5 47 5
Reticulocytes (%) 1 1
___________________________________________
Mean corpuscular volume (MCV; m
3
) 82-92
Mean corpuscular hemoglobin (MCH; pg) 27-32
Mean corpuscular hemoglobin concentration (MCHC; %) 32-36





Etiologic classification of anemias (1)
I. Impaired red cell production
A. Disturbance of proliferation and differentiation of
stem cells ( aplastic anemia, pure red cell aplasia)
B. Disurbance of proliferation and maturation of
erythrocytes:
1.Defective DNA synthesis (megaloblastic anemias)
2.Defective Hb synthesis:
a/. Deficient heme synthesis (iron deficiency)
b/. Deficient globin synthesis (thalassemia)
3. Unknown or multiple mechanisms (anemia of chronic
disease, anemia of marrow replacement)
Etiologic classification of anemias (2)

II. Increased rate of destruction (hemolytic anemias)
A. Intrinsic abnormalities
Hereditary
1. Red cell membrane defects (hereditary spherocytosis,
hereditary eliptocytosis)
2. Red cell enzyme deficiencies
a/. Glycolytic enzymes: pyruvate kinase, hexokinase
b/. Enzymes of hexose monophosphate shunt:
G-6PD, glutathione synthetase
3. Disorders of globin synthesis
a/. Deficient globin synthesis (thalassemia)
b/. Structurally abnormal globin synthesis
(sickle cell anemia, unstable hemoglobins)
Acquired
1. Membrane defect: paroxysmal nocturnal hemoglobinuria


Etiologic classification of anemias (3)
B. Extrinsic abnormalities
1. Antibody mediated
a/. Autoantibodies (idiopathic, drug-associated,
SLE, malignancies)
b/. Isohemagglutinins (transfusion reactions,
erythroblastosis fetalis)
2. Mechanical trauma of RBC
a/. Microangiopathic hemolytic anemias (thrombotic
thrombocytopenic purpura, DIC)
b/. Cardiac traumatic hemolytic anemia
3. Chemicals and microorganisms
4. Sequestration in mononuclear phagocytic system
- hypersplenism
Classification of Anemia
8
Classification of Anemia
Morphologic classification of anemias
Type MCV MCHC Common cause
________________________________________________________
Macrocytic anemia increased normal Vitamin B
12
deficiency
Folic acid deficiency
Microcytic anemia
- hypochromic decreased decreased Iron deficiency

Thalassemia
- normochromic decreased normal Spherocytosis
or normal
Normocytic anemia normal normal Aplastic anemia
- normochromic Chronic renal failure
Some hemolytic anemia

Classification of Anaemia:
Microcytic Hypochromic
MCV <80fl
MCH <27pg
Iron deficiency

Microcytic anaemia
Ferritin >25ug/L
Thalassaemia
Sideroblastic anaemia (some cases)
Anaemia of chronic disease (some cases)
Lead poisoning

Classification of Anaemia:
Normocytic Normochromic
MCV 80-100fl
MCH >26pg
Often incidental finding in systemic disorders
May be first manifestation of a systemic
disorder
Many haemolytic anaemias
Anaemia of chronic disease (some cases)
After acute blood loss
Bone marrow failure, e.g. Post-chemotherapy,
infiltration by carcinoma etc


Classification of Anaemia:
Macrocytic
MCV >100fl
Megaloblastic: vitamin B
12
or folate
deficiency
Non-megaloblastic: alcohol, liver disease,
myelodysplasia, aplastic anaemia
Iron deficiency anaemia
Assess for
Dietary Iron deficiency
Malabsorption- coeliac
Chronic blood loss
Gastrointestinal
Menorrhagia

Iron deficiency anaemia
Thalassemia-o: o-globin chain synthesis + or ()

Thalassemia-|: |-globin chain synthesis + or ()

THALASSEMIAS:
Defects of Hb synthesis

heme
heme
Hemoglobin molecule
18
Globin chain synthesis: developmental phases
Gen o & sejenis Kromosom 16
Gen | & sejenis
Kromosom 11
,2 o2 o1
c
G

A
o |
Hemoglobins




Masa
Perkembangan
Hb Gower 1
(,
2
c
2
)
Hb Gower 2
(o
2
c
2
)
Hb Portland
(,
2

2
)

Embrio

HbF
(o
2

2
)



Janin
HbA
2
HbA
(o
2
o
2
) (o
2
|
2
)



Dewasa
GEN PENYANDI SINTESIS RANTAI GLOBIN o DAN |
20
embryonic fetal adult
Hb Gower 1 (,
2
c
2
)
Hb Gower 2 (o
2
c
2
)
Hb Portland 1 (,
2

2
)
Hb Portland 2 (,
2
|
2
)
Human Hemoglobins
Hb F (o
2

2
) Hb A (o
2
|
2
): 95-97%
Hb A
2
(o
2
o
2
): 4%
Hb F (o
2

2
): <1%
Hb switches: 2 major
birth: fetal to adult
Normal Hb F levels
newborn 70-90%
6 months old 2-13%
1 year 1-5%
2 years <2%
21
information on age of patient is important:
interpretation of Hb levels
premature: delay in Hb switch (fetal to adult)
How is Thalassemia Classified?
Thalassemia
o
|
o

,
c
thalasemia hemoglobinopathies
22
Genotypes to phenotypes of thalassemia
Molecular defects (mutations)
|-globin gene
(|-thalassemia)
o-globin gene
(o-thalassemia)
o-globin chains +/- |-globin chains +/-
Excess of
-globin chains (fetus)
|-globin chains (adult)
Excess of
o-globin chains
24
Pathophysiology of thalassemia syndromes
globin chain imbalance
(excess o or | chains)
RBC damage
ineffective erythropoiesis
peripheral hemolysis
anemia
excess chains precipitate in RBC
Clinical aspects: thalassemia syndromes
syndrome clinical age of
presentation
need for
BT
trait
(normal
Hb)
asymptomatic any age none
thal-
intermedia
(7-10 gm/dl)
moderately
severe
after age 2 or
later
none, occ
some
thal-
major
(<7 gm/dl)
(|
0
/|
0
) severe

(o
0
/o
0
)) fatal
1-2

in-utero / birth
regular


25
Beta-thal syndromes
trait : |
+
or

|
0

thalassemia-intermedia
|
+
/ |
+

|
+
/ |
0

thalassemia-major: |
0
/ |
0
26
Identification of classical beta-thalassemia trait
in an adult
hallmark for classical beta-thalassaemia trait:
raised Hb A
2
27
28
information on age of patient is important:
full-term or premature
Hb subtypes in normal newborn
F(o
2

2
): 70-90%
A(o
2
|
2
): 10-30%
*A
2
(o
2
o
2
): 0% (not measurable by HPLC)



Hb subtypes in the newborn
|
o

o
Asimptomatik
Anemia ringan s/d berat
Hb 8-15 g/dL (rata-rata 12)
MCV < 80 fl
HbA
2
> 3.5%
HbF 1%
o

o
HETEROZIGOT
HOMOZIGOT
o
THALASSEMIA-
Anemia berat sejak usia < 1
tahun
Perlu transfusi rutin
Hati dan limpa membesar
MCV < 70 fl
Jenis Hb hanya HbA
2
and HbF
HbF > 90%
HOMOZIGOT
+
dan
+
o

o
|
Anemia ringan sampai berat
Hati dan limpa normal atau
membesar
Masih mempunyai HbA
HbF dari 10 s/d > 90%
30
functional o globin genes
trait
mild (o
+
)
severe (o
0
)

o-thal 2 (-o/oo)
o-thal 1(--/oo)

3
2
Hb H disease
moderate
more severe

o
0
/ o
+
(--/-o)
o
0
/ o
T
(--/ oo
T
)

1
1
Hb Barts hydrops
fetalis
o
0
/ o
0
(--/--) 0
Clinical severity of o-thal syndrome: related to number of
functional o-globin genes
o
0
or o-thal 1 trait

H inclusion test positive:
1 RBC cell per 5000 or 10,000 RBC
may be negative
cannot be done in newborn




31
`golf ball
inclusions
Presumptive identification of o-thalassemia
in newborns
anemia +Hb
MCV<105 fl
MCH<27pg
Hb Barts >5%
32
Levels of Hb Barts (4) in o-thalassemia in
newborns
syndrome genotype Hb Barts (%)
normal oo/oo 0
trait -o/oo 0.5-3
--/oo 4-8
-o/-o 9-13
Hb H disease --/-o 19-27%
Hb Barts hydrops
fetalis
--/-- 97-100
33
Pootrakul et al 1975
BioRad Variant HPLC: o-thal short program
34
o


|
o

Normal
oo/oo
o

o


|
FENOTIP THALASSEMIA - o (CARRIER)
-o/-o
o

o


|
- o/oo
N/anemia ringan
< 25 pg
normal or
atau (-)
Normal
25-27pg
normal or
atau (-)
Normal
MCH : 27-31 pg
HbA
2
: 2.5-3.5%
HbF : <1%
Kematian janin
(28-32 minggu)
Hydrops fetalis
HbH disease
mild to severe anemia

|

--/--

4
=HbBart
o

o


|
- -/-o
|
4
=HbH
MCH : < 26-32 pg
HbA
2
: 2.5-3.5%
HbF : <1%
FENOTIP THALASSEMIA - o (PENDERITA)
Normal
oo/oo
o
o


|
o

o
|

Adult Fetus
o
HbBart
HbH
AFFECTED
Normal fetus
HbA
HbF
Hb variant analysis
HbBart hydrops fetalis
32 weeks gestational age
Diagnosis Thalassemia

Anamnesis/pedigree
Pemeriksaan Fisik
Pemeriksaan laboratorium
Anamnesis/Pedigree

Pucat lama (kronik)
Riwayat keluarga dengan penyakit serupa
Ras
Anorexia
Gangguan Pertumbuhan
Probability Thalassemia
Orang Tua
Thalassemia Trait
Orang Tua
Thalassemia
Trait

Normal
Thalassemia
Mayor
Thalassemia
Trait
Orang Tua
Thalassemia
Trait
Orang Tua
Thalassemia
Mayor
Thalassemia
Mayor
Thalassemia
Mayor
Thalassemia
Trait
Thalassemia
Trait
Probability Thalassemia
Probability Thalassemia
Orang Tua
Thalassemia
Trait
Orang Tua
Normal
Thalassemia
Trait Normal
Pemeriksaan fisik
Sangat bervariasi (ringanberat)
Pucat
Gizi kurang
Pertumbuhan kurang/lambat
Facies mongoloid/ Cooley
Hepar dan limpa membesar
Fraktura patologis

Thalassemia Mayor
Hairy Skull
Cooleys face
Pemeriksaan laboratorium (1)
Darah perifer:

- Hb rendah / normal
- MCV <80fl dan MCH <27pg,
- Retikulosit agak meningkat
- Jumlah leukosit normal
- Hitung jenis normal

Pemeriksaan Hematologi
MCV : ukuran eritrosit dibandingkan dengan inti
limfosit kecil (Normal)
MCH : warna eritrosit atau content dari hemoglobin
RDW : perbedaan ukuran eritrosit.

46
semakin besar variasi eritrosit
semakin besar RDW


Pemeriksaan Hematologi
RDW-SD: mengukur lebar kurva. Bila kurva makin lebar
makan RDW SD semakin lebar
Nilai Normal :
perempuan: 36.4 46.3fL
Laki-laki: 35.1 43.9fL

RDW-CV: dihitung dengan formula:
RDW-CV = 1SD/MCV x 100 %
1SD merupakan variasi eritrosit sekitar mean ukuran eritrosit
Nilai Normal :
perempuan: 11.7 14.4 %
Laki-laki: 11.6 14.4 %

47
Iron Deficiency Anemia
Anemia








Messwerte
RBC
HGB
HCT
MCV
MCH
MCHC
RDW-CV
4.48 x10
12
/L
8.8g/dl
29.3%
65.4fl
19.6pg
30.0g/dl
18.2%






+
Messwerte
PLT
PDW
MPV
P-LCR
235 x10
9
/L
11.7fl
9.4fl
21.7%
(x 1000)
Suspected Thalassemia
Messwerte
RBC
HGB
HCT
MCV
MCH
MCHC
RDW-CV
5.97 x10
12
/L
12.7g/dl
41.1%
68.8fl
21.3pg
30.9g/dl
14.7%
+






PLT
PDW
MPV
P-LCR
391 x10
9
/L
12.0fl
10.3fl
27.3%
(x 1000)
<RBC-Histogram>
Measurement Data
<PLT-Histogram>
Measurement Data
<RBC-Histogram> Measurement Data
<PLT-Histogram> Measurement Data
Case 1 Case 2
Anisocytosis
RBC-Histogramm Messwerte
RBC
HGB
HCT
MCV
MCH
MCHC
RDW
4.15 x10
12
/L
14.0g/dl
40.8%
98.3fl
33.7pg
34.3g/dl
22.7%






+
Messwerte
PLT
PDW
MPV
P-LCR




328 x10
9
/L
12.4fl
10.2fl
26.5%
(x 1000)
RBC-Histogramm Messwerte
RBC
HGB
HCT
MCV
MCH
MCHC
RDW
2.95 x10
12
/L
9.9g/dl
28.7%
97.3fl
33.6pg
34.5g/dl
26.4%






+
PLT
PDW
MPV
P-LCR
PL*
DW
PL
PL
98 x10
9
/L
---.-fl
---.-fl
-.---%
(x 1000)
<PLT-Histogram>
<PLT-Histogram> Measurement Data
Measurement Data
Measurement Data Measurement Data
<RBC-Histogram>
<RBC-Histogram>
Pemeriksaan laboratorium (2)
Sedian hapus darah tepi :
Khas, gambaran hemolitik kronik
- Mikrositik Hipokrom
- Anisositosis
- Poikilositosis
- Sel target
- Fragmentosit
- Eritrosit berinti (berat)

Gambaran Sediaan Hapus Thalassemia
Mikrositik Hipokrom Anisositosis Poikilositosis
Sel Target
Fragmentosit
Eritrosit berinti
Mikrositik Hipokrom Bukan Thalassemia
Anemia def besi
Elliptocytosis
Spheroscytosis
Stomatocytosis
Pemeriksaan laboratorium (3)

Elektroforesis Hb
Analisa Hb (HPLC)
DCIP
Analisis DNA
Elektroforesis Hb
+
-
+
-
H
Barts
Portland


A
F

S, D, G,
Lepore
A2, E, C

Constant spring
Origin
C

Origin
H
S

A, A2, D, E, G, O
Lepore

F, Barts, Portland
pH alkali (8,5) pH asam (6.0)
Diagram migrasi fraksi Hb hasil elektroforesa pada pH alkali & asam
(Pemeriksaan Laboratorium Klinik Thalassemia & Hemoglobin Varian; Riady Wirawan, Dr, SpPK
FKUI, 1997)
Digunakan alat HPLC Variant
Tes-tes Hb yang dapat dilakukan di alat Variant :
Beta Thal Short
Sickle Cell Short
Alpha Thal Short
Globin Chains
HbA1c
A
F
S
A
2
C
300 points with 15 zones Curve
Normal Hb Type
Beta thal heterozygote
Hb E heterozygoye
E
A
2
Hb Constant Spring homozygote
Hb Barts
(Babys blood)
Hb F
Hb A
Hb Barts
Hb Bart
Alpha thalassemia with Hb H
Hb A
Hb A2
Hb H
Alk.
N
A

Anh.
Car.
H
A
2

Hb
H
Badan Inklusi HbH
HbH adalah hemoglobin yang tidak stabil akan
mengalami denaturasi oksidatif dan presipitasi
jika eritrosit terpapar dengan zat warna new
methylene blue atau brilliant cresyl blue dan
membentuk gambaran seperti bola golf

Badan inklusi HbH dijumpai pada eritrosit
penderita :
- HbH
- Thalassemia a-1 trait, (1/1001/10.000 eritrosit),


HbH disease HbH inclusion bodies
Limitations in new born
adult with thalassemia normal newborn
Blood counts Hb: N or +
MCV<80 fl, MCH<27pg
High Hb (14-20 g/dl)
MCV fl>105 fl,
MCH>27pg
Blood films Hypochromia
Thalassemia picture
Effects of relative
hyposplenism
Hb subtypes Hb A
2
:4%|-thal trait
Hb F(95-100%):|-thal major
Hb F: 90-100%
Hb A: 5-20%
Hb A
2
: not measurable
H-inclusion test o-thalassemia: + cannot be used
67
Thalassemia merupakan penyakit yang dapat
menurunkan kualitas hidup

Memerlukan biaya yang cukup besar untuk terapi
thalassemia (terutama pasien thalassemia yang
membutuhkan transfusi sepanjang hidupnya

Insiden thalassemia dapat diminimalkan melalui
pemeriksaan skrining yang optimal (Total Solution)

Meningkatkan kesadaran masyarakat mengenai
penyakit thalassemia dan akibatnya
70
terima
kasih