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anemia dan permasalahannya
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Diagnosis Laboratorium Anemia dan Permasalahannya
Agus Susanto Kosasih
Laboratorium Patologi Klinik
RS Kanker Dharmais1
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
PATHOLOGY, SYMPTOMS, AND SIGNS OF ANEMIA
Normal values for peripheral blood
Female Male Erythrocytes (per µl) 4.8±0.6x106 5.4±0.8x106
Hemoglobin (g/dl) 14 ± 2 16 ±2Hematocrit (%) 42 ±5 47 ±5Reticulocytes (%) 1 1___________________________________________Mean corpuscular volume (MCV; µm3) 82-92Mean corpuscular hemoglobin (MCH; pg) 27-32Mean corpuscular hemoglobin concentration (MCHC; %) 32-
36
Etiologic classification of anemias (1)
I. Impaired red cell productionA. 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 synthetase3. Disorders of globin synthesis a/. Deficient globin synthesis (thalassemia) b/. Structurally abnormal globin synthesis (sickle cell anemia, unstable hemoglobins)
Acquired1. Membrane defect: paroxysmal nocturnal
hemoglobinuria
Etiologic classification of anemias (3)
B. Extrinsic abnormalities1. 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 anemia3. Chemicals and microorganisms4. 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 Spherocytosisor 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 anaemiaFerritin >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 anaemiaAssess for Dietary Iron deficiency Malabsorption- coeliac Chronic blood loss Gastrointestinal Menorrhagia
Iron deficiency anaemia
Thalassemia-: -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 & sejenis Kromosom 16
Gen & sejenis Kromosom 11
2 2 1
G A
Hemoglobins
MasaPerkembangan
Hb Gower 1 (2 2) Hb Gower 2 (22) Hb Portland (22)
Embrio
HbF(22)
Janin
HbA2 HbA(2 2) (2 2)
Dewasa
GEN PENYANDI SINTESIS RANTAI GLOBIN DAN
20
embryonic fetal adult
Hb Gower 1 (22)Hb Gower 2 (22)Hb Portland 1 (22)Hb Portland 2 (22)
Human Hemoglobins
Hb F (22) Hb A (22): 95-97% Hb A2 (22): 4%Hb F (22): <1%
Hb switches: 2 major•2 months gestation: embryo to fetus
•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 levelspremature: delay in Hb switch (fetal to adult)
How is Thalassemia Classified?
Thalassemia
thalasemia hemoglobinopathies
22
Genotypes to phenotypes of thalassemia
Molecular defects (mutations)
-globin gene(-thalassemia)
-globin gene(-thalassemia)
-globin chains /- -globin chains /-
Excess of-globin chains (fetus)-globin chains (adult)
Excess of -globin chains
24
Pathophysiology of thalassemia syndromes
globin chain imbalance (excess or chains)
RBC damage
•ineffective erythropoiesis•peripheral hemolysis
anemia
excess chains precipitate in RBC
Clinical aspects: thalassemia syndromes
syndrome clinical age of presentation
need forBT
trait(normal
Hb)
asymptomatic any age none
thal-intermedia
(7-10 gm/dl)
moderatelysevere
after age 2 or later
none, occsome
thal-major
(<7 gm/dl)
(0/0) severe
(0/0)) 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 A2
27
28
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)
Hb subtypes in the newborn
• Asimptomatik• Anemia ringan s/d
berat• Hb 8-15 g/dL (rata-rata
12)• MCV < 80 fl• HbA2 > 3.5%• HbF ≥ 1%
HETEROZIGOT HOMOZIGOT βo
THALASSEMIA-β
• Anemia berat sejak usia < 1 tahun
• Perlu transfusi rutin• Hati dan limpa membesar• MCV < 70 fl• Jenis Hb hanya HbA2 and
HbF• HbF > 90%
HOMOZIGOT β+ dan β+
• Anemia ringan sampai berat• Hati dan limpa normal atau membesar• Masih mempunyai HbA• HbF dari 10 s/d > 90%
30
functional globin genes
trait mild (+)
severe (0)-thal 2 (-/)-thal 1(--/)
32
Hb H diseasemoderate
more severe0 / + (--/-)0 / T (--/ T)
11
Hb Bart’s hydrops fetalis
0 / 0 (--/--) 0
Clinical severity of -thal syndrome: related to number of functional -globin genes
0 or -thal 1 trait
H inclusion test positive: 1 RBC cell per 5000 or 10,000 RBC
may be negativecannot be done in newborn
31
`golf ball inclusions’
Presumptive identification of -thalassemia in newborns
anemia Hb
MCV<105 fl
MCH<27pg
Hb Bart’s >5%
32
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 fetalis
--/-- 97-100
33
Pootrakul et al 1975
BioRad Variant HPLC: -thal short program
34
Normal/
FENOTIP THALASSEMIA - (CARRIER)
-/-
- /
N/anemia ringan< 25 pgnormal or ↓↓ atau (-)
Normal25-27pgnormal or ↓↓ atau (-)
NormalMCH : 27-31 pgHbA2 : 2.5-3.5%HbF : <1%
• Kematian janin (28-32 minggu)• Hydrops fetalis
• HbH disease• mild to severe anemia
--/--
4=HbBart
- -/-
4=HbH
MCH : < 26-32 pgHbA2 : 2.5-3.5%HbF : <1%
FENOTIP THALASSEMIA - (PENDERITA)
Normal/
Adult Fetus
HbBart
HbH
AFFECTED
Normal fetus
HbA
HbF
Hb variant analysisHbBart’ 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
ThalassemiaMayor
ThalassemiaTrait
Orang Tua Thalassemia
Trait
Orang Tua Thalassemia
Mayor
ThalassemiaMayor
ThalassemiaMayor
ThalassemiaTrait
ThalassemiaTrait
Probability Thalassemia
Probability Thalassemia
Orang Tua Thalassemia
Trait
Orang Tua Normal
ThalassemiaTraitNormal
Pemeriksaan fisik► Sangat bervariasi (ringanberat)► Pucat ► Gizi kurang► Pertumbuhan kurang/lambat► Facies mongoloid/ Cooley► Hepar dan limpa membesar► Fraktura patologis
Thalassemia Mayor
Hairy SkullCooley’s 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
RBCHGBHCTMCVMCHMCHCRDW-CV
4.48 x1012/L8.8g/dl29.3%65.4fl
19.6pg30.0g/dl
18.2%
–––
+
MesswertePLTPDWMPVP-LCR
235 x109/L11.7fl
9.4fl21.7%
(x 1000)
Suspected Thalassemia
Messwerte
RBCHGBHCTMCVMCHMCHCRDW-CV
5.97 x1012/L12.7g/dl
41.1%68.8fl
21.3pg30.9g/dl
14.7%
+
–––
PLTPDWMPVP-LCR
391 x109/L12.0fl10.3fl
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
RBCHGBHCTMCVMCHMCHCRDW
4.15 x1012/L
14.0g/dl40.8%98.3fl
33.7pg34.3g/dl
22.7%
+
Messwerte
PLTPDWMPVP-LCR
328 x109/L12.4fl10.2fl
26.5%
(x 1000)
RBC-Histogramm Messwerte
RBCHGBHCTMCVMCHMCHCRDW
2.95 x1012/L9.9g/dl28.7%97.3fl
33.6pg34.5g/dl
26.4%
+
PLTPDWMPVP-LCR
PL*DWPLPL
98 x109/L---.-fl---.-fl
-.---%
(x 1000)
<PLT-Histogram>
<PLT-Histogram> Measurement Data
Measurement Data
Measurement DataMeasurement 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
SpheroscytosisStomatocytosis
Pemeriksaan laboratorium (3)
Elektroforesis Hb
Analisa Hb (HPLC)
DCIP
Analisis DNA
Elektroforesis Hb
+
-
+
-
HBart’sPortland
AF
S, D, G,LeporeA2, E, C
Constant springOrigin
C
OriginHS
A, A2, D, E, G, OLepore
F, Bart’s, 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)
Analisis Hb HPLCDigunakan 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
FS
A2
C
300 points with 15 zones Curve
Normal Hb Type
Beta thal heterozygote
Hb E heterozygoye
E
A2
Hb Constant Spring homozygote
Hb Bart’s
(Baby’s blood)
Hb F
Hb A
Hb Bart’s
Hb Bart
Alpha thalassemia with Hb H
Hb A
Hb A2Hb H
Alk.
N
A
Anh. Car.
H
A2
Hb H
Badan Inklusi HbHHbH 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),
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 HypochromiaThalassemia picture
Effects of relative hyposplenism
Hb subtypes Hb A2:4%-thal trait
Hb F(95-100%):-thal major
Hb F: 90-100%Hb A: 5-20%Hb A2: not measurable
H-inclusion test -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