Agus Susanto Kosasih

Laboratorium Patologi Klinik

RS Kanker Dharmais
1
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Definition of anaemia
Anaemia is defined as a reduction in the
haemoglobin concentration of the blood
This results in a decreased oxygen
carrying capacity
PATHOLOGY, SYMPTOMS, AND SIGNS OF ANEMIA
Normal values for peripheral blood

Female Male
Erythrocytes (per l) 4.80.6x106 5.40.8x106
Hemoglobin (g/dl) 14 2 16 2
Hematocrit (%) 42 5 47 5
Reticulocytes (%) 1 1
___________________________________________
Mean corpuscular volume (MCV; m3) 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
 8

Classification of Anemia
Classification of Anemia
 Morphologic classification of anemias

Type MCV MCHC Common cause

________________________________________________________
Macrocytic anemia increased normal Vitamin B12 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 B12 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
THALASSEMIAS:
Defects of Hb synthesis

Hemoglobin molecule

heme

heme
Thalassemia-: -globin chain synthesis or ()
Thalassemia-: -globin chain synthesis or ()
 18

Globin chain synthesis: developmental phases

 GEN PENYANDI SINTESIS RANTAI GLOBIN DAN

2 2 1
Gen & sejenis Kromosom 16

G A
Gen & sejenis Kromosom 11

Hb Gower 1
(2 2)
Hemoglobins Hb Gower 2 HbF HbA2 HbA
(22) (22) (2 2) (2 2)
Hb Portland
(22)

Masa Embrio Janin Dewasa

Perkembangan
 20

Human Hemoglobins

embryonic fetal adult

Hb Gower 1 (22) Hb F (22) Hb A (22): 95-97%
Hb Gower 2 (22) Hb A2 (22): 4%
Hb Portland 1 (22) Hb F (22): <1%
Hb Portland 2 (22)

Hb switches: 2 major

birth: fetal to adult

 21

Normal Hb F levels

newborn 70-90%

6 months old 2-13%

1 year 1-5%

2 years <2%

information on age of patient is important:

interpretation of Hb levels
premature: delay in Hb switch (fetal to adult)
 22

How is Thalassemia Classified?

Thalassemia






thalasemia hemoglobinopathies
 Genotypes to phenotypes of thalassemia

Molecular defects (mutations)

-globin gene -globin gene

(-thalassemia) (-thalassemia)

-globin chains /- -globin chains /-

Excess of Excess of
-globin chains (fetus) -globin chains
-globin chains (adult)
 24

Pathophysiology of thalassemia syndromes

globin chain imbalance

(excess or chains)
excess chains precipitate in RBC

RBC damage

ineffective erythropoiesis
peripheral hemolysis
anemia
 25

Clinical aspects: thalassemia syndromes

syndrome clinical age of need for

presentation BT

trait asymptomatic any age none

(normal
Hb)
thal- moderately after age 2 or none, occ
intermedia severe later some
(7-10 gm/dl)
thal- (0/0) severe 1-2 regular
major
(<7 gm/dl) (0/0)) fatal in-utero / birth
 26

Beta-thal syndromes

trait : + or 0

thalassemia-intermedia
/
+ +

/
+ 0

thalassemia-major: / 0 0
Identification of classical beta-thalassemia trait
in an adult

hallmark for classical beta-thalassaemia trait:

raised Hb A2

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Hb subtypes in the newborn

information on age of patient is important:

full-term or premature
Hb subtypes in normal newborn
F(22): 70-90%
A(22): 10-30%
*A2(22): 0% (not measurable by HPLC)
 THALASSEMIA-

HETEROZIGOT HOMOZIGOT o HOMOZIGOT + dan +

Asimptomatik Anemia berat sejak usia < 1 Anemia ringan sampai berat
Anemia ringan s/d berat tahun Hati dan limpa normal atau
Hb 8-15 g/dL (rata-rata 12) Perlu transfusi rutin membesar
MCV < 80 fl Hati dan limpa membesar Masih mempunyai HbA
HbA2 > 3.5% MCV < 70 fl HbF dari 10 s/d > 90%
HbF 1% Jenis Hb hanya HbA2 and HbF
HbF > 90%
 30
Clinical severity of -thal syndrome: related to number of
functional -globin genes

functional globin genes

trait
mild (+) -thal 2 (-/) 3
severe (0) -thal 1(--/) 2

Hb H disease
moderate 0 / + (--/-) 1
more severe 0 / T (--/ T) 1

Hb Barts hydrops 0 / 0 (--/--) 0

fetalis
 31

0 or -thal 1 trait

H inclusion test positive:

1 RBC cell per 5000 or 10,000 RBC
may be negative
cannot be done in newborn

`golf ball
inclusions
 32

Presumptive identification of -thalassemia

in newborns

anemia Hb
MCV<105 fl
MCH<27pg
Hb Barts >5%
 33
Levels of Hb Barts (4) in -thalassemia in
newborns

syndrome genotype Hb Barts (%)

normal / 0

trait -/ 0.5-3

--/ 4-8

-/- 9-13

Hb H disease --/- 19-27%

Hb Barts hydrops --/-- 97-100

fetalis
Pootrakul et al 1975
 34

BioRad Variant HPLC: -thal short program

FENOTIP THALASSEMIA - (CARRIER)
Normal -/- - /
/

Normal N/anemia ringan Normal
MCH : 27-31 pg < 25 pg 25-27pg
HbA2 : 2.5-3.5% normal or normal or
HbF : <1% atau (-) atau (-)
 FENOTIP THALASSEMIA - (PENDERITA)
Normal
/ --/--
- -/-

4=HbBart

4=HbH

Adult Fetus
MCH : < 26-32 pg Kematian janin HbH disease
HbA2 : 2.5-3.5% (28-32 minggu) mild to severe anemia
HbF : <1% Hydrops fetalis
HbBart hydrops fetalis Hb variant analysis

HbBart AFFECTED

HbH

Normal fetus
HbF

32 weeks gestational age HbA

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

Thalassemia Thalassemia
Normal Trait Mayor
Probability Thalassemia

Orang Tua
Orang Tua
Thalassemia
Thalassemia
Mayor
Trait

Thalassemia Thalassemia Thalassemia Thalassemia

Trait Mayor Trait Mayor
 Probability Thalassemia

Orang Tua Orang Tua

Thalassemia Normal
Trait

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

Cooleys face Hairy Skull

Pemeriksaan laboratorium (1)
Darah perifer:

- Hb rendah / normal
- MCV <80fl dan MCH <27pg,
- Retikulosit agak meningkat
- Jumlah leukosit normal
- Hitung jenis normal
 46

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

semakin besar variasi eritrosit

semakin besar RDW
 47

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 %
 Anemia
Iron Deficiency Anemia Suspected Thalassemia
Messwerte <RBC-Histogram> Measurement Data
Measurement Data
<RBC-Histogram> Messwerte
RBC + 5.97 x1012/L
RBC 4.48 x1012/L 12.7g/dl
HGB
HGB 8.8g/dl 41.1%
HCT
HCT 29.3%
MCV 68.8fl
MCV 65.4fl
MCH 21.3pg
MCH + 19.6pg
MCHC 30.9g/dl
MCHC 30.0g/dl 14.7%
RDW-CV
RDW-CV 18.2%
<PLT-Histogram>
<PLT-Histogram> Measurement Data

PLT 391 x109/L

Measurement Data PDW 12.0fl
Messwerte
MPV 10.3fl
PLT 235 x109/L
P-LCR 27.3%
PDW 11.7fl
MPV 9.4fl
P-LCR 21.7%

(x 1000) (x 1000)
 Anisocytosis
Case 1 Case 2
RBC-Histogramm Messwerte
<RBC-Histogram> Measurement Data <RBC-Histogram> Measurement Data
RBC-Histogramm Messwerte

RBC 4.15 x1012/L RBC 2.95 x1012/L

HGB 14.0g/dl HGB 9.9g/dl
HCT 40.8% HCT 28.7%
MCV 98.3fl MCV 97.3fl
MCH 33.7pg MCH 33.6pg
MCHC 34.3g/dl MCHC 34.5g/dl
+ 26.4%
RDW 22.7% RDW +

<PLT-Histogram> Measurement Data

<PLT-Histogram>
PLT PL* 98 x109/L
Messwerte
Measurement Data PDW DW ---.-fl
MPV PL ---.-fl
PLT 328 x109/L
P-LCR PL -.---%
PDW 12.4fl
MPV 10.2fl
P-LCR 26.5%

(x 1000) (x 1000)
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

Stomatocytosis Spheroscytosis
 Pemeriksaan laboratorium (3)

Elektroforesis Hb
Analisa Hb (HPLC)
DCIP

Analisis DNA
Elektroforesis Hb
 Diagram migrasi fraksi Hb hasil elektroforesa pada pH alkali & asam
H
Barts
+ Portland +
C
A
F Origin
H
S
S, D, G,
Lepore
A, A2, D, E, G, O
A2, E, C Lepore

Constant spring F, Barts, Portland

- Origin -

pH alkali (8,5) pH asam (6.0)

(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

S
F

C
A2
300 points with 15 zones Curve
Normal Hb Type
Beta thal heterozygote
Hb E heterozygoye

A2
Hb Constant Spring homozygote
Hb Barts
(Babys blood)
Hb Bart

Hb F

Hb A

Hb Barts
 Alpha thalassemia with Hb H

Hb
Hb A
H
Alk.

A2

Hb H
Anh.
Hb A2 Car.
N
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
 67

Limitations in new born

adult with thalassemia normal newborn

Blood counts Hb: N or High Hb (14-20 g/dl)

MCV<80 fl, MCH<27pg MCV fl>105 fl,
MCH>27pg

Blood films Hypochromia Effects of relative

Thalassemia picture hyposplenism

Hb subtypes Hb A2:4%-thal trait Hb F: 90-100%

Hb F(95-100%):-thal major Hb A: 5-20%
Hb A2: not measurable

H-inclusion test -thalassemia: + cannot be used

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
terima
kasih

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