Systematic Approach in

Anemia Evaluation and Review

of Peripheral Smears

Dr.Muh Darwin Prenggono SpPD.KHOM

Subbagian Hematologi-Onkologi Medik,

Bagian Ilmu Penyakit Dalam, Fakultas Kedokteran
Universitas Lambung Mangkurat,
Banjarmasin
Objective

Recognize abnormal peripheral

blood smear
Review differentials through
systematic approach
 Components of Blood
55% plasma
45% cells
99% RBCs
< 1% WBCs and
platelets
 Recycling of Hemoglobin
Components

In macrophages of liver or spleen

globin portion broken down into amino acids & recycled
heme portion split into iron (Fe+3) and biliverdin (green pigment)
Iron Panel
Anemia of
Iron Chronic Sideroblastic
Deficiency Thalassemia Disease Anemia

Serum
Iron
Normal

TIBC
Normal Normal

Ferritin
Normal
 Hemolytic Anemia

Intravascular (typically extracorpuscular) fragmentation

hemolysis, direct toxic damage/destruction, complement
fixation/destruction
Extravascular macrophage-mediated in spleen and liver
(Igs, membrane defects)
There is a spectrum ie how much damage is done to the
cell determines where it is destroyed

Intravascular Extravascular
Peripheral smear schistocytes spherocytes
Haptoglobin decrease/absent mild decrease
Free hemoglobin Increased normal
Urine hemosiderin ++ negative
Urine hemoglobin ++ negative
Direct DAT usually negative ++++
LDH increase increase
Intravascular hemolysis
Hemosiderin Hemoglobin

ab-dimers + Haptoglobin Haptoglobin-Hb

Hepatic parenchymal
Free Hemoglobin
cells

Methemoglobin

Kidney
Globin
+ Hemopexin
Hb-uria Heme Hemopexin-Heme
Hemosiderinuria
Methemalbumin
+ Albumin + Hemopexin
Extravascular hemolysis
Hemoglobin

ab-dimers + Haptoglobin Haptoglobin-Hb

Hepatic parenchymal
Hemin Bilirubin direct
cells

Heme
Enterohepatic
Biliverdin Bilirubin Indirect
Fe
Urobilinogen Kidney

Stercobilin Urobilin
Stercobilinogen
Mesobilirubinogen
Basic Labs to Start

Repeat CBC w/ manual differential

(WBC, RBC, HCT, HGB, PLT, indices- MCH,
MCHC, MCV, RDW)
Peripheral Smear
Reticulocyte count
Reticulocyte count
Retic count = % immature RBC
Normal 0.5-1.5% (for non-anemic)
<1% Inadequate production
>=1% Increased production (? adequacy)
 Reticulocyte Correction
%Retic count frequently overestimates
Retic count should be compared to non-
anemic RBC count to assess adequacy of
response
Corrected Retic count = %Retic X HCT/45
Reticulocyte Production Index
Correction for left shift Retic lifespan is
increased in blood
RPI = % Retic X Hct/45 X 1/CF
Hct Correction factor (CF)
40-45 1.0
35-39 1.5
25-34 2.0
15-24 2.5
Normal RPI = 1 (for non-anemic pt)
RPI < 2 : hypoproliferative
RPI >=2 : hyperproliferative
Retic Production Index
Hypoproliferative Hyperproliferative

- Iron def. anemia - Hemolytic disease

- B12/folate def. - Hemoglobinopathy
(including thalassemia)
- Chronic disease
- Sideroblastic anemia
- Aplastic anemia
- Myeloproliferative
Penggolongan menurut Morfologi
volumetrik
Mikrositik Normositik
hipokrom normokrom Makrositik

MCV < 80 fL 80 100 fL > 100 fL

MCH 27 pg > 27 pg > 27 pg
MCHC < 30 g/dL 30 g/dL 30 g/dL

MCV= (Ht/Eritosit)x10 fL
MCH=(Hb/Eritrosit)x10 pg
MCHC=(Hb/Ht)x100 g/dL
 Peripheral smear

Optimal area for review

RBC morphology, WBC differential, PLT
(clumping?)
 RBC morphology
7-9 m with 1/3 central palor
Lifespan of 110-120 days
About the size of nucleus of normal
lymphocyte
Poikilocytosis & Anisocytosis
Basophilic stippling
(gambar titik- titk seperti basofil)

Precipitated RNA
lead or heavy
metal poisoning
Hemolytic anemia
Burr cells
(duri)

Altered lipid in cell

membrane
artifact
Uremia
Renal failure
gastric CA
transfused old blood
Howell Jolly body
(William Howel&Justin Jolly : butir2 dlm eritrosit ad sisa nukleus)

Nuclear remnant -
DNA
hemolytic anemia
absent or
hypofunction spleen
Schistocyte/helmet cells
(fragment sel darah merah/ helmet cell)

Fragmented
(mechanical or
phagocytosis)
DIC
TTP
HUS
Vasculitis
prosthetic heart valve
severe burns
 Anemia hemolitik ekstravaskuler

Pasien 34 thn prosthetic heart

valve , 3 bulan kmd dgn anemia
Hb 8 kemudian di tranfusi WBC
3 kolf, datang ke Rs Ulin dgn
Ikterik dan Hb 10 ?
 Sickle cells
(sabit)

Molecular aggregation
of Hgb-S
SS, SC, S-thal
rarely S-trait
Spherocyte
(kecil bulat hanya Hb tanpa warna pucat ditengah

Absent central
palor
look smaller
Hereditary
spherocytosis
immune hemolytic
anemia
 Stomatocyte
(seperti mulut)

Mouth like
Membrane defect
Smear artifact
Hereditary
stomatocytosis
Liver disease
 Target cells
(sasaran)

Increased redundancy
of membrane
hemoglobinopathies
thalassemia
liver disease
 Tear drop cells
(air mata)

Distorted drop shaped

Smear artifact
myelofibrosis
promyeloblastic
leukemia
space occupying
lesions of marrow
Gejala umum dan khusus anemia
Mekanisme System/organ Symptom
End arteries Pale (vasoconstriction)
Jantung Tachycardia, systolic ejection murmur, Forth gallop
Penurunan
Respiratory Tachypneu, Exertional dyspneu
kapasitas
Neuromuscular Faintness, Lack of concentration,
angkut oksigen
Hearing Tinnitus (ischemic)
Sighting Scotoma (edema papil)
Haid Menorhagia, Polymenorrhagia
Feses Melena, hematoschezia
Perdarahan
Hidung Epistaksis
Mulut Gusi berdarah
Atropi-Nekrosis Tongue Glossitis, hypotrophy papilla (B12, Folat, less in Fe deficient)
Mucosa Pharinx Angina Vincent/dysphagia (Fe deficient)
Neuritis Kaki/Tangan Foot/Hand gloves periveral neuropathy (B12 deficient)
Hemolisis Hemoglobinuria: Reddish urine (PNH, DD/hematuria)
Urin
Intravascular Hemosiderinuria: Blackish urine (Black water fever, Malaria)
Hemolisis Urin Urobilinogenuria with/without Brownish urine (bilirubinuria)
Extravascular Limpa Splenomegali
Extramedullar
Hati dan limpa Hepato-splenomegali
hemopoesis
 anemia

Koilonychia - Iron Deficiency

(kuku permukaan luar cekung

Sickle Cell Anemia

Pseudomacrocytosis-- (Hemolitik anemia)
Cold Agglutinins
Differentials

H&P
Indices (MCV, MCHC, RDW)
RBC Morphology
Retic response
Other labs as needed
 Anemia Differential Dx by Flow Chart
MCV/smear

Micro Normo Macro

Iron panel Retic Retic

Low High

Iron/B12/Folate Go to *Occult Blood Loss

**Normal Low

Bone Marrow Bx B12/Folate

Anemia of Chronic Dis. B12 Low

High Folate/Low

*Occult Blood Loss Normal

Yes MMA/Homocysteine

No MMA high B12 Low

Coombs (+) Homocysteine high Folate Low

Coombs (-) Normal Go to **

First use size (MCV) to sort the
Differential Dx

MCV

Micro Normo Macro

 Microcytic anemia
Get Iron panel- serum iron, TIBC, ferritin
Iron def. dec inc dec
anemia
Siderobla inc dec inc
-stic
anemia
Thalasse inc/nl dec/nl inc/nl
mia -mia
Chronic dec dec inc
disease
Iron def. Anemia

Low Retic count

High RDW
Due to chronic blood
loss
Diet deficiency
 Thalassemia
Normal to inc. RPI
Normal RDW
Target cells
Mentzer index <13
=MCV/RBC
- sensitivity = 82%
- specificity = 80%
confirm w/ Hgb
electrophoresis
 Sideroblastic anemia

Accumulation of
mitochondrial iron in
erythroblasts
Hereditary
Drugs - INH, lead,
zinc, alcohol,
chloramphenicol,
cycloserine, plavix
Hypothermia
Confirm w/ BM Bx
Anemia of chronic disease
Infections: TB, osteo, Chronic inflammatory
chronic UTI or pyelo, disorders: RA, SLE,
fungal Sarcoid, collagen
vascular disease,
Malignancy: mets, polymyalgia
leukemia, lymphoma, rheumatica, chronic
myeloma hepatitis, decubitus
ulcer
Macrocytic anemia
Macro

RPI >= 2 RPI < 2

Check Occult Blood Loss Check B12 and folate

No

Coombs test

Yes
Macrocytic: RPI < 2

B12/Folate

B12 Low Normal Folate Low

MMA
Homocysteine
MMA High Homocysteine
High
Normal

Consider
Liver, Renal,
Thyroid,
Alcohol,
Chronic dis.

Consider
Bone Marrow
Bx
Macrocytic: RPI < 2
Megaloblastic Anemia
B12 Folate
Inadequate absorption Inadequate intake
Synthesized by Synthesized by plants
bacteria and micro-organism
Meat, fish, dairy (strict Fruits
vegans) Absorbed in jejunum
Absorbed as B12-IF
complex in ileum
(gastrectomy)
Ca++ and pH
dependant (PPI)
 Macrocytic: RPI < 2
Megaloblastic Anemia
Smear
Macro-ovalocytic
Polychromasia
Hypersegmented neutrophil
Other Labs
Homocysteine Folate def.
Methylmalonic acid B12 def.
Intrinsic Factor Ab test very
specific for pernicious anemia
but only 50% sensitive
Macrocytic: RPI < 2
Non-megaloblastic

Consider Liver, Renal,

Endocrine (thyroid),
alcohol, drugs
Consider anemia of
chronic disease
Get Bone Marrow
Biopsy
Myelodysplastic
Myeloproliferative -
Leukemia, Lymphoma,
Multiple Myeloma
Macrocytic: RPI < 2 continues
Aplastic Anemia

Fanconi anemia congenital

Direct stem cell destruction external
radiation
Drugs - chloramphenicol, gold, sulfonamides, felbamate
Other Toxins - Solvents, degreasing agents, pesticides
Viral infection - parvovirus B19, HIV, other
Idiopathic
Macrocytic: RPI >= 2
Occult Blood Loss?

Yes No

Investigate source Check for Hemolysis Peripheral smear

Coombs (DAT)
Macrocytic: RPI >= 2
Hemolytic Anemia
Other Lab Characteristics

RBC morphology
Serum haptoglobin
Serum LDH
Unconjugated bilirubin
Hemoglobinuria
Hemosiderinuria
 Macrocytic: RPI >= 2
Hemolytic Anemia

Coombs (DAT)

Positive Negative

Immune Hemolysis
Hemoglobinopathy, G6PD, PK,
Drug related Hemolysis
Spherocytosis, Eliptocytosis,
Transfusion, Infection, Cancer
PNH, TTP, DIC
Coombs positive with Spherocytes
Autoimmune hemolytic anemia

Warm AIHA Cold AIHA

Abrupt onset Insidious onset
IgG IgM, complement
Anti-Rh, e, C, c, LW, U Anti-I, I, Pr
Jaundice Cold agglutinin titer
Splenomegaly Absent jaundice
SLE, CLL, Lymphoma Mycoplasma
Drugs: methyl-dopa, Virus
mefenamic acid,
cimetidine, cefazolin
Coombs positive with Spherocytes
Other immune hemolytic anemia

Alloantibody hemolytic anemia

Transfusion reaction
Feto-maternal incompatibility (Kleihauer-Betke test)
Drug related Hemolytic anemia
Toxic immune complex (drug+Ab+C3)
- Quinine, Quinidine, Rifampin, INH, Sulfonamides,
Tetracyclin
Hapten formation (anti-IgG)
- PCN, methicillin, ampicillin
Coombs Negative
Hemolytic anemia

Episodic - G6PD def., PNH

Hemoglobinopathy
- Sickle, crystals or target cells
Elliptocytosis
Spherocytosis
DIC, TTP
Coombs Negative Hemolytic Anemia
Membrane Defects
Spherocytosis Elliptocytosis
Common among 90% with no clinically
Northern European significant hemolysis
Autosomal dominant Abnormal membrane
Decreased spectrin protein
Osmotic fragility test
Autohemolysis test
Coombs Negative Hemolytic Anemia
Deficiency of RBC Enzymes
G6PD Def. Pyruvate Kinase Def.

X-linked Severe anemia in

Mediterranean, newborns
African American, and Adults symptomatic
Asian
Jaundice
Oxidant drugs ASA,
quinine, primaquine, Splenomegaly
chloroquine, sulfacetamide,
sulfamethoxazole, Fluorescent screening
nitrofurantoin, chloramphenicol, test
procainamide, quinidine
Infections Quantitative test
Quantitative test
Coombs Negative Hemolytic Anemia
Hemoglobinopathy

HbS disease
Valine substitution for
Glutamic acid at the
6th position of b-chain
Sickle crises
Severe anemia
Screening test - Na
Metabisulfite solubility
Hgb electrophoresis
Coombs Negative Hemolytic Anemia
Hemoglobinopathy continues
HbC disease HbSC disease
Mild hemolysis Sickle and SC crystals
Splenomegaly Washington monument
Lysine substitution Less crises
HbC crystals bar of gold More retinopathy/aseptic
Hgb electrophoresis necrosis
Coombs Negative Hemolytic Anemia
Paroxysmal Nocturnal Hemoglobinuria

Rare chronic condition

Recurrent abdominal pain, vomiting,
headaches, eye pain, thrombophlebitis
Episodic Hgb in urine, Hemosiderinuria
Abnormal cell membrane - increased lysis
by complement
Screening - Sucrose hemolysis test
Confirm - Acid hemolysis test (Hams test)
Coombs Negative Hemolytic Anemia
Fragmented RBCs & Thrombocytopenia
TTP-HUS DIC
Thrombocytopenia Depletion of clotting factor
Microangiopathic (TTP normal)
hemolytic anemia Thrombocytopenia
Neurologic symptoms and Bleeding (64%)
signs Renal dysfunction (25%)
Renal failure Hepatic dysfunction (19%)
Fever Respiratory dysfunction
(16%)
Idiopathic - 37 %
Shock (14%)
Drug-associated - 13 %
Thromboemboli (7%)
Autoimmune disease - 13 %
Central nervous system
Sepsis - 9 % involvement (2%)
Pregnancy - 7 % Sepsis, trauma, malignancy
Bloody diarrhea - 6 %
Hematopoietic cell
transplantation - 4 %
TTP-HUS / DIC
 Normocytic Anemia
Hyperproliferative (RPI >= 2)

Use same flow chart as macrocytic

hyperproliferative

Occult Blood Loss?

Yes No

Investigate source Check for Hemolysis Peripheral smear

Coombs (DAT)
Normocytic Anemia
Hypoproliferative (RPI < 2)
1. Get iron panel (ferritin)/B12/folate
- some clue from RBC indices to check early
disease, high RDW, peripheral smear.

2. Consider liver, renal, drugs, toxin, endocrine

(thyroid), and anemia of chronic disease.

3. Get BM bx
- Leukopenia, thrombocytopenia,
- Aplastic anemia/pancytopenia
- Abnormal (immature) cells on smear
 Myelophthisic Anemia
BM Replacement BM failure:
Metastatic Carcinoma Most
Common
Destruction By Non-Neoplastic
Process is Less Common i.e.
Fibrosis, Infection
Peripheral Blood Cytopenias,
Immature Circulating Cells
Breast Cancer Replacing BM
Terima kasih
Terima kasih
 Summary Comparison of Iron
Chelators

Comparison of Iron Chelators

ICL670
Property Deferoxamine Deferiprone (deferasirox)

Chelator:iron 1:1 3:1 2:1

binding

Usual dosage 2540 mg/kg/d 75 mg/kg/d 2030 mg/kg/d

Route of Subcutaneous, Oral, 2-3 times daily Oral, once daily

administration intravenous

Half-life 20-30 minutes 3-4 hours 12-16 hours

Excretion Urinary, fecal Urinary Fecal

Adverse effects Local reactions, Gastrointestinal disturbances, Gastrointestinal

ophthalmologic, agranulocytosis1/ neutropenia2, disturbances, rash
auditory, bone arthralgia3, liver enzyme
abnormalities, elevations
pulmonary, allergic
reactions, neurologic

1 absence of Status
granulocyticLicensed Licensed
white blood cells which in Europe for patients In clinical trials
fight
bacterial infection; does not apply to lymphocytes who are unable to use
deferoxamine effectively
2 decreased number of granulocytic white blood cells
3 joint pain
Anemia tersering pada kehamilan

Makrositer (Normal Megaloblastik) akibat

defisiensi asam folat,
Suplemen asam folat 0,4 mg/hari dan
Suplemen vitamin B12 50 mg/hari mulai sejak
awal kehamilan.
Mikrositik hipokrom akibat defisiensi Fe,
Suplemen Fe 30-60 mg/hari mulai kehamilan
18-20 minggu.
Anemia makrositer
Diagnostik: MCV > 100 fL
Penyebab:
Ineffective metabolisme vitamin B12
dan/atau asam folat.
Hambatan:
Pasokan vit.B12 dan/atau asam folat,
Transport vit.B12 dan/atau asam folat, atau
Pengikatan vit.B12 dan/atau asam folat
oleh reseptornya (Defisiensi enzim).
Iron Gluconate Iron Sucrose
typical medium-sized and big no necroses
necroses

liver histotoxicity: microscopic pictures of mouse liver 4 hours after

injection
Geisser et al. (1992) Drug Research, 42: 1439-1452
 Rumus kebutuhan Iron sukrosa
intravena
Fase koreksi
Dosis terapheutic 3-4 mg/kg BB
BB X (tgt Hb actual Hb) X 0.24 + 500 mg
70 kg X(11-8) X 0.24 + 500 mg = 550 mg
BB > 35 kg : 1 level Hb setara dengan 2 ampul Venofer
Fase maintenance
100 mg/1 amp setiap HD selama 10 X HD
Dilanjutkan 200 mg / 2 ampl /bulan selama 3 bulan
 Pemberian Iron Sukrosa Venofer
per drip

5 ml (=100 mg besi) dilarutkan dalam 100 ml NaCl

0,9%
Test dose 20 ml (=25 mg) diberikan dalam 15 menit
(12 tetes/menit)
Sisanya 75 ml (=75 mg): Diberikan selama 45 menit
/ 32 tetes/menit
Dosis Max : 2 amp / hari
6 amp/ minggu
 Summary Comparison of Iron
Chelators

Comparison of Iron Chelators

ICL670
Property Deferoxamine Deferiprone (deferasirox)

Chelator:iron 1:1 3:1 2:1

binding

Usual dosage 2540 mg/kg/d 75 mg/kg/d 2030 mg/kg/d

Route of Subcutaneous, Oral, 2-3 times daily Oral, once daily

administration intravenous

Half-life 20-30 minutes 3-4 hours 12-16 hours

Excretion Urinary, fecal Urinary Fecal

Adverse effects Local reactions, Gastrointestinal disturbances, Gastrointestinal

ophthalmologic, agranulocytosis1/ neutropenia2, disturbances, rash
auditory, bone arthralgia3, liver enzyme
abnormalities, elevations
pulmonary, allergic
reactions, neurologic

1 absence of Status
granulocyticLicensed Licensed
white blood cells which in Europe for patients In clinical trials
fight
bacterial infection; does not apply to lymphocytes who are unable to use
deferoxamine effectively
2 decreased number of granulocytic white blood cells
3 joint pain