Laboratory Examination and

Work-Up for Hematologic Disorders

Dr. Arifoel hajat,SpPK (K)

How to learn laboratory
diagnosis?

 the methods used in laboratory diagnosis

are more automatic and computerized.
 the aim is to train for clinical application .
 For each test, referential values and
clinical significance always should be
known very well.
Complete Blood Count ( CBC )
 Complete Blood Count includes:
 White blood count (WBC)
 Red blood count (RBC)
 Hemoglobin (Hgb)
 Hematocrit (Hct)
 Mean corpuscular volume (MCV)
 Mean corpuscular hemoglobin (MCH)
 Mean corpuscular hemoglobin content (MCHC)
 Platelet count
 Red cell distribution width (RDW)
 Referential values of CBC
 Referential values for blood routine test in different
age and sex
___________________________________
male female newborn
___________________________________
 RBC 4.0 - 5.5 3.5 - 5.0 6.0 - 7.0
 HB 12 - 16 11 - 15 17 - 20
 WBC 4 - 10 4 - 10 15 - 20
 PLT 150 - 45 150 - 450

___________________________________
Referential values of differential count
___________________________________
type percent value (%) absolute value
___________________________________
 N stab 1-5 0.04-0.5
 N seg 50-70 2-7
 LYM 20-40 0.8-4
 Mo 3-8 0.12-0.8
 Eo 0.5-5 0.02-0.5
 Ba 0-1 0-0.1
________________________________
 Eo / Ba / Stab / Seg / Li / Mo___
Morphology of blood cells (pheripheral)

A. ERITHROCYTE
B. LARGE LIMPHOCYTE
C. SEGMENTED
NEUTROPHYL
D. EOSINOPHYL
E. SEGMENTED
NEUTROPHYL
F. MONOCYTE
G. THROMBOCYTE
H. SMALL LYMPHOCYTE
I. BAND NEUTROPHYL
J. BASOPHYL
Complete Blood Count
 Most automated instruments directly read the
WBC, RBC, hemoglobin and MCV directly
 Hematocrits are a calculated parameter
 Correlation checks between the hemoglobin
and hematocrits are a significant part of quality
assurance
 “Rule of Three.” The formulas for correlation
checks/rule of three are as follows:
 Hgb x3=Hct ± 3 or RBC x3 =Hgb.
Complete Blood Count
 Automated machines present a pictorial
representation of the data registered as
either a histogram or a scatterplot
 Most machines also offer automated
reticulocyte count
NORMAL

DEF FE

THALAS

AIHA

PANSITO

AML

AMOL

CML

ALL

MM
Normal Pattern Abnormal Pattern
I. Erythrocyte Index :
Mean corpuscular volume (MCV):
MCV = Hct/RBC 82-95fl
Mean corpuscular hemoglobin (MCH):
MCH = Hb/RBC 27-31pg
Mean corpuscular hemoglobin concentration
MCHC = Hb/Hct 320-360g/L
Morphological Classifications of
Anemia
 There are three morphologic classification of
anemia:
 Normochromic normocytic anemia
 MCV 80-100 fL
 MCHC 31-36%
 Microcytic, hypochromic anemia
 MCV < 80fL
 MCHC < 30%
 Red cells are microcytic, smaller and lacking hemoglobin
 Macrocytic normochromic anemia
 MCV >100 fL
 Red cells are larger than 8 µ
 Normal hemoglobin
tes slide bma 16
tes slide bma 17
tes slide bma 18
 Morphological classification of anemia
Classification MCV MCH MCHC diseases
Normocytic 82-95 27-31 320-360 AA, HA, leukemia
Macrocytic >100 > 31 320-360 MA, pernicious
anemia
Microcytic < 80 < 27 320-260 infection, tumor,
uremia
Microcytic < 80 < 27 < 320 IDA, thalassemia
Hypochromic sideroblastic
anemia
Calculating Red Cell Indices and Their Role in
Sample Integrity
 MCV is one of the most stable parameters in the CBC
 Used to evaluate pre-analytical and analytical specimen integrity

 MCV=Hematocrit / Red cell count x 100

 Normal value is between 80-100 Fl
 Implies a red cell that has a size of 6-8 microns

 Shift in MCV caused by:

 Cold agglutinins
 Transfusion therapy
 Reticulocytosis
 Pre-analytical changes in MCV caused by:
 Contamination by drawing through the IV lines or in-dwelling catheters
 Specimens from hyperglycemic patients
 Patients on some chemotherapy drugs

Hematocrit = PCV
Calculating Red Cell Indices and
Their Role in Sample Integrity
 Mean Corpuscular Hemoglobin and Mean Corpuscular
Hemoglobin Content provide information concerning red
cell hemoglobinization

 MCH = Hemoglobin/red blood cell count x100

 Average weight of hemoglobin
 Normal value of 27-29

 MCHC = hemoglobin/hematocrit x10 and is expressed

in percentage
 Amount of hemoglobin per packed red cell
 Normal value is between 32-36%
Red Cell Distribution Width
 Red Cell Distribution Width (RDW)
 Mathematical calculation
 Gives insight into the amount of anisocytosis
(variation in size) and to some degree poikilocytosis
(variation in shape) in a peripheral smear
 RDW = Standard deviation of RBC volume
/mean MCV multiplied by 100
 Normal value is 11.5-14.5%
 Platelet count is also included in CBC
 Clinical significance
 Anomalies of RBC and HB

1. decrease of RBC and HB

 Anemia: When RBC and HB of individual is

lower than the referential values of the people
in same age , same sex and same area.
 Usually, if Hb of adult male and female is lower
than 11 g/dL or 10 g/dL respectively, they are
considered as anemia.
According to the level of Hb, anemia is
divided into four degrees in clinic

 mild anemia: Hb < 120g/L(male) or <

110g/L(female)
 medium anemia : Hb <90 g/L
 severe anemia : Hb <60g/L
 extreme severe anemia: Hb <30g/L
Pathological anemia.

 decrease in synthesis of red blood cell

in bone marrow
 increased destroy of red blood cell in
peripheral blood
 blood loss
Decrease in synthesis of red blood cell in bone
marrow

 disorder of hemopoiesis in bone marrow:

aplastic anemia
 infiltration of bone marrow by tumorous
cell: leukemia, multiple myeloma ,
lymphoma , metastatic tumor
 deficiency of hemopoietic materials or
factors: iron deficiency anemia (IDA),
megaloblastic anemia (folic acid
deficiency)
Destroy of RBC in peripheral blood --
hemolytic anemia

 Hereditory disease :
hereditory sphrocytosis (HS) ,
G6PD deficiency,
thalassemia , Hb disease
 Acquired anomalies :
hemolysis by immunological , physical,
chemical , biological and mechanical factors
Blood loss --
hemorrhagic anemia

 acute blood loss : acute upper digestive

tract bleeding , splenic rupture
 chronic blood loss : hemorrhoid,
hookworm disease, hypermenorrhea, GI
tumor
Thalassemia
beta
THALASSEMIA
Increase of RBC and Hb
• Comparative increase of RBC and Hb:
due to the decrease of volume of plasma--
severe dehydration: severe vomiting,
diarrhea, severe burn
• Absolute increase of RBC and Hb (ery-
throcytosis): polycythemia vera(PV),
chronic cardiopulmonary diseases: cor
pulmonale, obstructive emphysema,
scarcity of oxygen.
II. Anomalies of WBC and DC
•In most cases, increase and decrease of
WBC is chiefly depend on the numbers of
neutrophil granulocyte in the blood.
•Leukocytosis: WBC is high than 10 x109 /L
•Leukopenia: WBC is lower than 4 x109 /L
 1. Neutrophil granulocyte
A. granulocytosis: 5 causes leading to granulocytosis
• acute infection or inflammation: acute pyogenic
tonsilitis, acute appendicitis, hematosepsis

• damage or necrosis of tissue: severe burn, acute

myocardiac infarction
•acute blood loss: digestive tract bleeding,
splenic rupture
•Acute poisoning: uremia, ketoacidosis
•Malignant blood diseases and tumor:
leukemia, metastatic tumor
AML
An 9 th
ALL
A.Granulocytopenia:
• leukopenia: WBC < 4 x109 /L
• granulocytopenia:
neutrophil granulocyte < 1.5 x109 /L
• agranulocytopenia:
neutrophil granulocyte < 0.5 x109 /L
5 causes for granulocytopenia:
• Infection of bacteria and virus: typhoid, influenza,
measles
• some physical and chemical factors: drug
• autoimmune diseases:
systemic lupus erythematosis (SLE),
• malignant blood diseases: aplastic anemia,leukemia
•hypersplenism
Changes of nucleus
nucleus shift to left:
• mild shift to left: Nst > 6% only
• medium shift to left: Nst > 10% with meta-
myelocyte
• severe shift to left : Nst > 25% with more
immuture cell (leukemoid reaction )
nucleus shift to left can be seen in acute infection,
acute poisoning, acute hemolysis
 Nucleus shift to right: hyper-segmented N > 3%
3-lobed Nse is normal segmented neutrophil in BP.
Nucleus shift to right can be seen
• megaloblastic anemia
• administration of anti-metabolic drugs

Hypersegmented neutrophil : neutrophil with 5 or more lobes

2. lymphocyte
A . Lymphocytosis: DC: Lym > 40%
•Virus infection: infectious mononucleosis,
chickenpox,
•Lymphocytic leukemia: ALL, AA, lymphoma
•GVHD or GVHR after BMT
A. lymphocytopenia:
•administration of anti-tumor drugs, prednision
•radiation: x-ray, r-ray , isotope
3. monocytosis:
• malaria, black fever, TB, subacute bacterial
endocarditis(SBE)
• monocytic leukemia, malignant histocytosis,
3. eosinophilia
•allergic diseases, hypersensitive diseases
•parasites infection:
•Skin diseases: psoriasis
•blood diseases: CML, eosinophilic leukemia,
lymphoma
•Infectious diseas: scarlatina
•Hypereosinophilic syndrome
4. Basophil granulocyte
basophilia: CML, basophilic leukemia,
myelofibrosis
Other tests for red blood cell
1. Reticulocyte count (RC)
0.05-0.015 (0.5-1.5%)
24-84 x109 /L
 Reticulocytosis:
hemolytic anemia
acute hemorrhagic anemia
index of therapeutic effect:
IDA, megaloblastic anemia
Value of the Reticulocyte Count
 Reticulocyte count used in assessing red cell generation
or response to anemia
 Immature, un-nucleated red cell must be stained with
supra-vital stains like New Methylene Blue or Brilliant
Cresyl Blue
 On Wright’s stain, reticulocytes are seen as
polychromatophilic macrocytes, large, bluish cells
 Reticulocyte rate is 0.5-1.5 % in the adult and 2.0-6.0
%in the newborn
 Ineffective erythropoiesis - Failure to produce
reticulocytes during anemic stress
tes slide bma 48
tes slide bma 49
•Reticulocytopenia:
Aplastic anemia
leukemia
AIHA
Erythrocyte sedimentation rate ( ESR )
0-15mm/h (male), 0-20mm/h (female)
higher ESR:
• infection and inflammation: rheumatic
diseases,tuberculosis
• malignant tumor
• anemia
• damage or necrosis of tissue
• globulinemia, cholesterolemia
 5 mm

11 mm

READ THE RESULT

 Blood Smear
1. PERIPHERAL BLOOD SMEAR
EVALUATION:
EVALUATE ERYTHROCYTE, LEUCOCYTE &
THROMBOCYTE
2. LEUCOCYTE DIFFERENTIAL
COUNT:
COUNT A NUMBER OF EACH LEUCOCYTE
TYPE
NORMOCHROME NORMOCYTIC
 CIGAR
CELL

ANISOCYTOSIS-HYPOCHROME-MYCROCYTIC, CIGAR CELL (+)

24

ANISOCYTOSIS-HYPOCHROME-
MICROCYTIC
ANISO-POIKILOCYTOSIS-HYPOCHROME-MIKROCYTIC, POLYCHROMASI
TARGET CELL, TEAR DROP CELL, FRAGMENTOCYTE
 SPHEROCYTE

NORMOBLAST OVALOCYTE
AUTOAGLUTINATION
OTHERS
Gametocy Ring form
te

M
A
L
A
R
I
A

Gametocy Schizone
te
GAMETOCYTE
P. FALCIPARUM
 Bone Marrow Examination
1. Clinical application of marrow examination
 Diagnosis for hematopoietic system
diseases:
leukemia, myeloma, aplastic anemia, etc
 parasite infectious diseases: malaria, black
fever
 metabolic diseases: Gaucher disease.
Niemann-Pick disease
from
several cases of myelodysplastic syndrome showing dysplastic nuclear features seen
in
circulating granulocytes and nucleated RBCs. The right lower figure shows numerous
Pappenheimer bodies.

Dysplastic nuclear
feature in circulating
Cells from cases of
Myelodysplastic syn-
drome

Pappenheimer bodies

Wintrobe’s Atlas of Clinical Hematology , 2007

M.Y. 58 TH : BMA : mielosit
CML

Eo
Promielosit

Eo

Eo

stab

segmen

metamielosit
Limf

Normoblas
Polikrom.
Eo
mieloblas
CML
Granulocyte morphology: Nuclear features. A: Giant hypersegmented neutrophil
(arrow) from a case of vitamin B12 deficiency. B: Hypersegmented neutrophils from antifolate
chemotherapy. C: Dysplastic neutrophil (pseudopelgeroid cell) in case of myelodysplastic
syndrome. This neutrophil, which has two lobes connected by a thin filament, resembles the
neutrophils in the hereditary abnormality called Pelger-Huet anomaly. D: Degenerating neutrophil.

Giant Hypersegmented
neutrophil

Pseudopelgeroid cell
Degenerating neutrophil

Wintrobe’s Atlas of Clinical Hematology , 2007

tes slide bma 69
thanks
Regulation of blood cell development

 Cytobody: from large to small,

Mega is an exception
 Cytoplasma: from less to more,
color from blue to pink or orange red,
granules from invisible to visible,
granules from non-specific to specific
Nucleus:
from large to small, Mega is an exception,
shape of nucleus from round to irregular,
chromatin patten of nucleus from fine to
coarse.
nucleolus from existance to nonexistance
Ration of nucleus to cytoplasma:
from bigger to smaller
5.Content and step of marrow
examination
 Myelogram:
 Low power (LP):
Wright stain of marrow film
plastic degree: 5 classification
Meg count : 7-35/1.5x3cm2
special cell: metastatic tumorous cell
•Oil immersion:
Nucleated cell count (%): 200-500
Ratio of myeloid to erythroid (M:E): 2-4:1
Morpholigical description of every series
Special cells and parasite
•Diagnosis
•Hemogram:
Low power(LP):
Oil immersion len: differential count 100
leukocyte (DC)
Immature cell (nucleated cell)
parasites
 6. Normal myelogram and
hemogram
1. Myelogram
 Normal cellularity: erythrocyte: nucleated cell
 M:E=2-4:1
 Granulocytic series is 40-60% in all nucleated cells
 Erythrocytic series is 20% in all nucleated cells
 Lymphocyte is 20% , monocyte is <4% in all nucleated c
 Meg count is 7-35, platelet is normal
 Other cell: plasmacyte and histiocyte are rare
 No specific cells and parasite
 Plastic degree of marrow
Plastic degree erythrocyte: nucleated diseases
Extreme hypercellularity 1:1 leukemia
Significant hyper- 10:1 leukemia, ITP,
Cellularity plastic anemia
Normal cellularity 20:1 anemia, normal
myelogram
Hypocellularity 50:1 AA,agranulocy-
topenia
Extreme hypocellularity 300:1 aplastic anemia
2. Hemogram:
•DC is normal
•No nucleated red blood cell
•No immature white cell
•Platelet is normal
7. Characters of common
blood diseases in microscope
 Iron deficiency anemia(IDA)
 Aplastic anemia(AA)
 Acute leukemia(AL)
 Chronic myelocytic leukemia(CML)
 Idiopathic thrombocytopenia purpura(ITP)
1. Iron Deficiency Anemia(IDA)
 Hemogram:
Hb , RBC , normal RC,
RBC hypochromia, exaggeration of
central pallor to from rings, anisocytosis
normal WBC and platelet
•Myelogram:
Significant hypercellularity
M:E
Erythroid hyperplasia with small normoblast
Pycnotic nuclei and scanty cytoplasma
irregular margin
2. Aplastic Anemia(AA)

 Hemogram:
pancytopenia: Hb ,RBC , WBC ,PLT
RC <0.005 or absolute value < 15 x109 /L
RBC is normocytic
•Myelogram:
Hypocellularity or extreme hypocellularity,
Hematopoietic cell decrease: erythroid, myeloid
cells , meg or absent, platelet rarely be
seen
Non-hematoietic cell increase: lymphocyte
Reticular cell , plasmacyte
3. Acute Leukemia(AL)
 FAB classification:
acute lymphocytic leukemia: L1-L3
acute non-lymphocytic leukemia: M0-M7
 Hemogram:
Hb, RBC
WBC uncertain: normal, increase,
decreas
platelet:
•Myelogram:
Extreme or significant hypercellularity
M:E
Hyperplasia of certain line :
Myeliod(M0,M1-3), monocyte(M4-5),
meg(M7), lymphocyte(L1-3) , erythroid(M6)
Depressed erythroid and meg line
4. Chronic Myelocytic
Leukemia(CML)

 Hemogram:
WBC increase rxtremly
DC: E0, Ba increas, myelocyte,
metamyelocyte, band cell increase
normal Hb, RBC or mild decrease
normal plt
•Myelogram:
Extreme hypercellularity
M:E increase
Extreme hyperplasia of granulocyte with
increas of myelocyte, metamyelocyte, band
cell, E0, Ba
Normal erythroid and Megakaryocyte
5. Idiopathic Thrombocytopenic
Purpura(ITP)

 Hemogram:
normal Hb, RBC
normal WBC and DC
PC decreas with abnormal morphology
•Myelogram:
Significant hypercellulerity or normal cellularity
Normal myeloid, erythroid line
Significant hyperplasia of Meg.
Impaired maturation of Meg:
Granular Meg or promeg increase
Platelet producting Meg absent
Platelet rarely be seen