jenis anemia dlm diagnosis laboratorium

1. Agus Susanto Kosasih
Laboratorium Patologi Klinik
RS Kanker Dharmais
1

2. 2
Definition of anaemia
 Anaemia is defined as a reduction in the
haemoglobin concentration of the blood
 This results in a decreased oxygen
carrying capacity

3. PATHOLOGY, SYMPTOMS, AND SIGNS OF ANEMIA

4. Normal values for peripheral blood
Female Male
Erythrocytes (per µl) 4.8±0.6x106 5.4±0.8x106
Hemoglobin (g/dl) 14 ± 2 16 ±2
Hematocrit (%) 42 ±5 47 ±5
Reticulocytes (%) 1 1
___________________________________________
Mean corpuscular volume (MCV; µm3) 82-92
Mean corpuscular hemoglobin (MCH; pg) 27-32
Mean corpuscular hemoglobin concentration (MCHC; %) 32-36

5. 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)

6. 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

7. 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. 8
Classification of Anemia

9. Classification of Anemia

10. 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

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

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

13. 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

14. Classification of Anaemia:
Macrocytic
 MCV >100fl
 Megaloblastic: vitamin B12 or folate
deficiency
 Non-megaloblastic: alcohol, liver
disease, myelodysplasia, aplastic
anaemia

15. Iron deficiency anaemia
Assess for
 Dietary Iron deficiency
 Malabsorption- coeliac
 Chronic blood loss
 Gastrointestinal
 Menorrhagia

16. Iron deficiency anaemia

17. THALASSEMIAS:
Defects of Hb synthesis
Hemoglobin molecule
heme
heme
Thalassemia- : -globin chain synthesis or ( )
Thalassemia- : -globin chain synthesis or ( )

18. 18
Globin chain synthesis: developmental phases

19. 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
( 2 2) ( 2 2) ( 2 2) ( 2 2)
Hb Portland
( 2 2)
Masa Embrio Janin Dewasa
Perkembangan

20. 20
Human Hemoglobins
embryonic fetal adult
Hb Gower 1 ( 2 2) Hb F ( 2 2) Hb A ( 2 2): 95-97%
Hb Gower 2 ( 2 2) Hb A2 ( 2 2): 4%
Hb Portland 1 ( 2 2) Hb F ( 2 2): <1%
Hb Portland 2 ( 2 2)
Hb switches: 2 major
•birth: fetal to adult

21. 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. 22
How is Thalassemia Classified?
Thalassemia






thalasemia hemoglobinopathies

23. 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. 24
Pathophysiology of thalassemia syndromes
globin chain imbalance
(excess or chains)
excess chains precipitate in RBC
RBC damage
•ineffective erythropoiesis
•peripheral hemolysis
anemia

25. 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. 26
Beta-thal syndromes
trait : + or 0
thalassemia-intermedia
 / + +
 / + 0
thalassemia-major: / 0 0

27. Identification of classical beta-thalassemia trait
in an adult
hallmark for classical beta-thalassaemia trait:
raised Hb A2
27

28. 28
Hb subtypes in the newborn
information on age of patient is important:
full-term or premature
 Hb subtypes in normal newborn
 F( 2 2): 70-90%
 A( 2 2): 10-30%
*A2( 2 2): 0% (not measurable by HPLC)

29. THALASSEMIA-β
HETEROZIGOT HOMOZIGOT βo HOMOZIGOT β+ dan β+
• 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. 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 Bart’s hydrops 0 / 0 (--/--) 0
fetalis

31. 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. 32
Presumptive identification of -thalassemia
in newborns
 anemia Hb
 MCV<105 fl
 MCH<27pg
 Hb Bart’s >5%

33. 33
Levels of Hb Bart’s ( 4) in -thalassemia in newborns
syndrome genotype Hb Bart’s (%)
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. 34
BioRad Variant HPLC: -thal short program

35. 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 (-)

36. 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

37. HbBart’ hydrops fetalis Hb variant analysis
HbBart AFFECTED
HbH
Normal fetus
HbF
32 weeks gestational age HbA

38. Diagnosis Thalassemia
 Anamnesis/pedigree
 Pemeriksaan Fisik
 Pemeriksaan laboratorium

39. Anamnesis/Pedigree
 Pucat lama (kronik)
 Riwayat keluarga dengan penyakit serupa
 Ras
 Anorexia
 Gangguan Pertumbuhan

40. Probability Thalassemia
Orang Tua
Thalassemia Trait Orang Tua
Thalassemia
Trait
Thalassemia Thalassemia
Normal Trait Mayor

41. Probability Thalassemia
Orang Tua
Orang Tua
Thalassemia
Thalassemia
Mayor
Trait
Thalassemia Thalassemia Thalassemia Thalassemia
Trait Mayor Trait Mayor

42. Probability Thalassemia
Orang Tua Orang Tua
Thalassemia Normal
Trait
Thalassemia
Normal Trait

43. Pemeriksaan fisik
► Sangat bervariasi (ringan berat)
► Pucat
► Gizi kurang
► Pertumbuhan kurang/lambat
► Facies mongoloid/ Cooley
► Hepar dan limpa membesar
► Fraktura patologis

44. Thalassemia Mayor
Cooley’s face Hairy Skull

45. Pemeriksaan laboratorium (1)
Darah perifer:
- Hb rendah / normal
- MCV <80fl dan MCH <27pg,
- Retikulosit agak meningkat
- Jumlah leukosit normal
- Hitung jenis normal

46. 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. 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 %

48. 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)

49. 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)

50. Pemeriksaan laboratorium (2)
Sedian hapus darah tepi :
Khas, gambaran hemolitik kronik
- Mikrositik Hipokrom
- Anisositosis
- Poikilositosis
- Sel target
- Fragmentosit
- Eritrosit berinti (berat)

51. Gambaran Sediaan Hapus Thalassemia
Mikrositik Hipokrom Anisositosis Poikilositosis
Sel Target Fragmentosit Eritrosit berinti

52. Mikrositik Hipokrom Bukan Thalassemia
Anemia def besi Elliptocytosis
Stomatocytosis Spheroscytosis

53. Pemeriksaan laboratorium (3)
 Elektroforesis Hb
 Analisa Hb (HPLC)
 DCIP
 Analisis DNA

54. Elektroforesis Hb

55. Diagram migrasi fraksi Hb hasil elektroforesa pada pH alkali & asam
H
Bart’s
+ Portland +
C
A
F Origin
H
S
S, D, G,
Lepore
A, A2, D, E, G, O
A2, E, C Lepore
Constant spring F, Bart’s, Portland
- Origin -
pH alkali (8,5) pH asam (6.0)
(Pemeriksaan Laboratorium Klinik Thalassemia & Hemoglobin Varian; Riady Wirawan, Dr, SpPK
FKUI, 1997)

56. 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

57. A
S
F
C
A2

58. 300 points with 15 zones Curve

59. Normal Hb Type

60. Beta thal heterozygote

61. Hb E heterozygoye
E
A2

62. Hb Constant Spring homozygote

63. Hb Bart’s
(Baby’s blood)
Hb Bart
Hb F
Hb A
Hb Bart’s

64. Alpha thalassemia with Hb H
Hb
Hb A
H
Alk.
H
A
A2
Hb H
Anh.
Hb A2 Car.
N

65. 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, (1/100–1/10.000 eritrosit),

66. HbH disease HbH inclusion bodies

67. 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

68. 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
70