complete blood count analysis and blood disease investigation

How to read
a CBC?
Ahmad Darwish, MD
Assistant professor of Pediatric
Hematology/Oncology & BMT
Mansoura University

Agenda
 Introduction
 CBC Parameters
 RBC Parameters & Histograms
 Platelet Parameters & Histograms
 WBC Parameters & Histograms

Introduction
 Automated hematology analyzer is a cost- effective strategy in resource-
limited settings for diagnosis of common blood disorders.
 Moreover, given the difficulty of blood sampling in the pediatric age group,
extracting the maximum possible information from each investigation is
essential.
 Modern-day automated hematology analyzers, with their constantly
expanding assessment parameters provide a good, quick overview of the
patients’ blood picture.
Xie et al, J Biophotonics. 2018; Gounari et al, Cytometry B Clin Cytom. 2016.
CBC by Ahmad Darwish

CBC
 A complete blood count is a series of tests used to evaluate the
composition of the various cellular components of blood.
 It is a basic test.
 The most informative single investigation.
 It consists of:
1. RBC Parameters (Hb, RBCs, Hct, MCV, MCH, MCHC, RDW, etc.)
2. Platelet Parameters (PLT, MPV, PDW, P-LCR, Pct, etc.)
3. WBC Parameters
4. Histogram of RBC, PLT, WBC

Why CBC?
 CBC is an inexpensive tool and powerful tool which provide
information about:
1. Blood
2. Marrow
3. Health or disease state of other body organs
 CBC is the first investigation routinely performed in both in-
patient and out-patient settings.

CBC uses
 To diagnose:
1. Anemia
2. Haemoglobinopathies
3. Bone marrow aplasia
4. Nutritional deficiencies
5. Thrombocytopenia
6. Autoimmune conditions
7. Infections & parasitemia
8. Malignancies, response to drugs, chemotherapy etc.

INTERPRETATION OF CBC PARAMETERS
 Most automated hematology analyzers generate numerical and graphical data.
 Numerical data is in the form of measurement of various WBC, RBC and platelet
parameters.
 The automated analyzers also provide RBC, WBC, and platelet histograms
derived by plotting each cell’s size on the X-axis and their relative number on the
Y-axis.
 Interpretation of these CBC parameters and histograms together helps us assess
the presentation and etiology of anemia without requiring other expensive
investigations.
Xie et al, J Biophotonics. 2018; Gounari et al, Cytometry B Clin Cytom. 2016.

RBC Parameters
 Automated hematology analyzers provide information on several RBC parameters
that are used to assess the type of anemia, the treatment response & long-term
follow-up of patients like:
Hemoglobin (Hb)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Red cell distribution width (RDW)
RBC count
Hematocrit (HCT)
Kujovich, Obstet Gynecol Clin North Am. 2016.
Nathan et al, Nathan and Oski’s hematology and oncology of infancy and childhood. Elsevier. 2015 CBC by Ahmad Darwish

Increased RBCs
Polycythemia
High altitude
Pulmonary hypertension
Hypoventilation syndrome
Congestive heart failure
Obstructive sleep apnea
Poor blood flow to the kidneys

Decreased RBCs
Increased destruction
Impaired production
Blood loss
Intra-corpuscular
▪Hereditary spherocytosis
▪Sickle cell anemia
▪G6PD
▪Pyruvate kinase deficiency
▪PNH
Extra-corpuscular
▪Autoimmune Haemolytic
disease
▪Mismatch transfusion
▪Microangiopatic hemolytic
anemia TTP, HUS
▪DIC
▪Infection
▪Pure red cell aplasia
▪Pernicious anemia
▪Thalassemia
▪Anemia of prematurity
▪Anemia of chronic disorder
▪Trauma
▪surgery
▪Gl bleed
▪Menorrhagia

Blood indices
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
❖ Anemias are classified based on MCV & morphology as:
normocytic, microcytic and macrocytic.
Kujovich, Obstet Gynecol Clin North Am. 2016.
Nathan et al, Nathan and Oski’s hematology and oncology of infancy and childhood. Elsevier. 2015 CBC by Ahmad Darwish

Interference in MCV
 Cold and warm antibodies
 Marked hyperglycemia
 Marked leukocytosis
 Marked reticulocytosis
 Methanol poisoning
Interference in MCH
 Lipemia
 Cold agglutinin
 Monoclonal protein in blood
Interference in MCHC
 Cold agglutinin
 Rouleaux

Normal blood smear
Basophil (B)
Monocyte (M)
Platelet (P)
Red cells (R)
Neutrophil (N)
Lymphocyte (L)
Eosinophil (E)

Anemia classification

 All macrocytic anemias are evaluated & treated for vit B12 & folate deficiency.
 Non-megaloblastic macrocytic anemia requires further evaluation of:
❖ Reticulocyte count
❖ Features of hemolysis
❖ Interpretation of bone marrow aspirate smears
 Reticulocyte count is necessary for evaluation of normocytic normochromic
anemias.
❑ megaloblastic anemia, liver disease and hypothyroidism, must be ruled out.
❑ further evaluation of WBC & platelet parameters followed by BM evaluation may be required.
RBC Parameters

Reticulocyte
 Normal value 0.5% - 1.5% (Hence 0.5% - 1.5% RBCs are replaced per day)
 Uses
1. To evaluate anemia
They have a role in diagnosis & monitoring of aplastic anemia.
2. Response to treatment of anemia
They provide information regarding treatment-response in conditions such as nutritional anemia.
 If the disease causing the anemia is inside the marrow, the RC is decreased
 If the disease causing the anemia is outside the marrow, the RC is increased

Methods
 Manual reticulocyte count using supravital stain
 Automated reticulocyte count by fluorescent method – gives immature reticulocyte
fraction (IRF) and removes errors like Howell-Jolly bodies, pappenheimer bodies
 Reticulocyte production index or corrected reticulocyte count:
an index corrected according to level of anemia.
 Reticulocyte index = reticulocyte count x patient’s haematocrit/ normal haematocrit

 Reticulocyte proliferation index: index is used to determine if a person’s bone
marrow is property responding to the body’s need for red blood cells.
 Shift correction factor: normal reticulocyte count survive 3.5 days in marrow and
1 day in peripheral circulation at normal PCV. In case of variation in PCV the
survival time is increased which is termed as shift correction factor
 Reticulocyte proliferation index = reticulocyte
Shift correction factor
maturation’s days = shift correction factor
PCV%
1
45
1.5
35
2
25
2.5
15

RBC Parameters

Decreased Reticulocyte count
Increased Reticulocyte count
Aplastic anemia
Haemolytic anemia
Megaloblastic anemia
Recent hemorrhage
Anemia of chronic disease
Thalassemia
Cirrhosis
Respond to treatment
Radiation
Leukemia
Decrease ACTH and pituitary
hormones

Reticulocyte haemoglobin measurement
(RET –HE)
 Reticulocyte haemoglobin measurement (RET –HE) is a direct assessment of
the incorporation of iron erythrocyte hemoglobin.
 It is a direct estimate of the recent functional availability of iron (2-3)days.
 Traditional chemistry tests used for iron assessment (serum iron, T sat, ferritin) are
indirect measurements.
 As a direct measurement, Ret-He may identify iron deficiency earlier than
traditional parameters.
 It is an established parameter used in KDOQI (kidney Disease Outcome Quality
initiative) guidelines for assessing iron status

 The conventional parameters are generated in all automated cell counters, while
the newer parameters are available in specific counters and need to be
customized to be generated as printouts.
 Additionally, there are several indicators that can be derived from the parameters
reported in the automated analyzers such as the Mentzer index (MCV/RBC) that
can also help differentiate between IDA and â-thalassemia.
RBC Parameters

What is the Mentzer index?
• MCV/RBC.
-This is one of the formulas that is used to distinguish the
hypochromic, microcytic anemias of the thalassemia
from iron deficiency.
-As a general rule, iron deficiency causes alterations
RBCs that tend to be variable, whereas thalassemia
generally results in more uniformly smaller cells.
trait
in
-In patients with the b-thalassemia trait, the Mentzer index
usually less than 13.
is
- in patients with iron deficiency, it is usually greater than 13.

Calculation of the Mentzer index

Sometimes both conditions coexist
Thalassemia minor associated with IDA
A trial of oral iron is necessary
DD of Iron Deficiency Anemia

 Newer parameters, additionally, provide information that has made the detection
of type and cause of anemias easier and may, over time, reduce the dependence
on peripheral blood smear examination for all cases.
 They also help distinguish between the various etiologies of anemia such as:
❑ Iron deficiency anemia (IDA)
❑ Anemia of chronic disease (ACD)
❑ Anemia of inflammation (AI)
❑ Anemias due to inherited conditions such as thalassemia
Briggs ae al. Int J Lab Hemat.2009.
Kujovich et al. Obstet Gynecol Clin North Am. 2016.
Piva et al. Clin Lab Med. 2015.
Markoviæ et al. Scand J Clin Lab Invest. 2005.
Thomas et al. J Clin Diagn Res. 2017.
RBC Parameters

Newer RBC and Reticulocyte Parameters on Automated Analyzer
Significance in anemia
RBC parameter
• Along with RET-He helps in detecting onset of anemia
and also improvement in erythropoiesis
RBC hemoglobin equivalent (RBC-He): Hemoglobin
content of all mature RBCs
In detection of microangiopathies, DIC, infections, sepsis,
immune disorders, etc.
Fragmented red cell count (FRC): Fragmented
RBCs
• Low in IDA
• Can be used for IDA screening in pediatric population
Red cell size factor (RSf): Cellular hemoglobin
content of RBCs and reticulocytes
• Low in IDA
• Can be used for IDA screening in pediatric population
• Iron restricted erythropoiesis marker
Percentage hypochromic cells (%HC) or equivalent
low hemoglobin density (LHD%): hypochromic
RBCs (%)
Screening of thalassemia
Percentage unghosted cells: Target cells in
peripheral blood
ACD: anemia of chronic disease, AI: anemia of inflammation, DIC: disseminated intravascular
coagulation, EPO: erythropoietin, FID: functional iron deficiency, IDA: iron deficiency anemia.
Nathan et al. Nathan and Oski’s hematology and oncology of infancy and childhood. Elsevier. 2015.

Newer RBC and Reticulocyte Parameters on Automated Analyzer
RBC parameter
Can help distinguish between hemoglobinopathies & IDA.
RBC-Y: Size and contents of the RBCs
• Differentiate between IDA and FID
Reticulocyte hemoglobin equivalent (Ret-He) or Mean
Reticulocyte Hemoglobin Content (CHr): Mean content
of hemoglobin within reticulocytes
• Differentiate between IDA and FID
Low fluorescence reticulocyte (LFR), Medium
fluorescence reticulocyte (MFR), High fluorescence
reticulocyte (HFR): Maturity stages of reticulocytes
• Assesses effectiveness of erythropoiesis
• Assessment of response to iron or vitamin-B12/folate
supplementation in nutritional anemias
• Monitoring EPO therapy response
Immature reticulocyte fraction (IRF): Sum of HFR and
MFR
• Low in IDA and AI
Reticulocyte-Y (RET-Y): Size and contents of the
reticulocyte
ACD: anemia of chronic disease, AI: anemia of inflammation, DIC: disseminated intravascular
coagulation, EPO: erythropoietin, FID: functional iron deficiency, IDA: iron deficiency anemia.

 In RBC histograms, the cell-counters count RBCs between 25 and 250 femtoliter (fL).
 The histograms have two flexible discriminators that help differentiate RBC curves from
others:
❖ RBC lower discriminator (RL) that fluctuates between 25 and 75 fL and
❖ RBC upper discriminator (RU) that fluctuates between 200 and 250 fL.
 When the cell population is homogeneous, the curve shows a symmetrical bell-shaped
or Gaussian distribution.
 The area of the histogram’s peak (60 to 125 fL) helps to calculate MCV & RDW.
RBC Histograms

Interpretation of RBC Histograms:
(A) Normal RBC curve (B) Calculation of MCV
(C) Calculation of RDW

Interpretation of RBC Histograms:
(C) Rightward shift of curve as seen in the presence of macrocytic RBCs
(B) Leftward shift of curve as seen in the presence of microcytic RBCs
(A) Extension of lower end of curve as in case of normal MCV with flagging

 In a normal RBC histogram, RBCs are located between 55-125 fL.
 MCV is calculated using a perpendicular line between the base of the curve and its peak.
 RDW helps calculate the variation in RBC size & can be of 2 forms: RDW-SD & RDW-CV.
 RDW-SD is the standard deviation expressed as fL obtained by drawing a line of 20% on the y-axis.
Its normal range is between 35-45 fL.
 RDW-CV is the coefficient of variation percentage and lies within the range of 11.5% to 14.5%.
It is calculated as: RDW-CV = SD/MCV ×100.
RBC Histograms

RBC Histograms

RBC Parameters
It helps differentiate between pure micro/normocytic & mixed red cell populations.

 To avoid interference of aperture artifacts, giant platelets, RBC agglutinates, the
information <20% of the scale on the histogram is excluded.
 When RBCs are smaller than normal in size, as in microcytic anemia, the curve shifts
to the left
 When RBCs are larger than normal in size, as in macrocytic anemia, the curve shifts
to the right.
 The extension of the lower end of the scale helps in the detection of RBC fragments,
WBC fragments and platelets .
RBC Histograms

RBC Histograms Flags in Childhood Anemia
 Flags are signals that occur when automated hematology analyzers
detect an abnormal result.
 Any abnormal flag should always be correlated with the peripheral smear
findings.

Flags encountered in RBC histograms:

 RL Flag: This occurs due to abnormal height at lower discriminator
when it exceeds the preset height by >10%.
 This is seen in the presence of:
 Platelet clumps
 RBC fragments
 Extreme micro-erythrocytosis
 Giant platelets
 Micro-RBCs

 RU Flag: This occurs due to abnormal height at upper discriminator
when it exceeds the preset height by >5%.
 This is seen in the presence of:
 Nucleated RBCs
 RBC agglutination
 Cold agglutinins
(the flag disappears when the sample is incubated at 37ºC)

Variations in size and shape of red cells:-
• (A) Microcytic hypochromic red cells in iron deficiency
anemia;
(B) Oval macrocytes and a hypersegmented neutrophil in
megaloblastic anemia;
(C) Sickle cells in sickle cell anemia;
•
•

Variations in size and shape of red cells:-
(D) Spherocytes in hereditary spherocytosis
(E) schistocytes (Helmet cell ) as in HUS
(F) Target cells in iron deficiency or thalassemia trait

(G) Burr cells in chronic renal failure
(H) Tear drop red cells in myelofibrosis
(I) Bite cells & (J) Blister cell in G6PD def

Myeloproliferative Disorders
• Essential thrombocytosis
• Idiopathic Myelofibrosis
• Polycythaemia Vera
Transfer from extravascular pools into circulation
• Splenectomy (Over 70% of platelets stored in spleen)
Thrombocytosis • Exercise
• Epinephrine
Thrombocytosis Secondary to
• Iron deficiency anemia
• Acute blood loss
• Hemolysis
• Recovery from thrombocytopenia

Platelet Parameters
 Platelets may be abnormal in size or number in various anemias and may help
determine the etiology of anemia.
 Platelets are counted and represented between 2 and 20 fL in platelet histograms.
 At 20 fL, there may be interference in counting due to RBC & WBC fragments.
 At 2 fL, there may be interference in counting due to EDTA particles & air bubbles.
 Here, two flexible discriminators:
Lower discriminator (LD) or platelet lower discriminator (PL)
Upper discriminator (UD) or platelet upper discriminator (PU)
and a fixed discriminator at 12 fL

The platelet histogram curve
should lie between LD & UD
It starts & ends at the
baseline.
The platelet curve is
normally left skewed.

 In thrombocytosis, the curve shifts upwards
 In thrombocytopenia, the curve shifts downwards

 Platelet histogram is used to calculate:
Mean platelet volume (MPV)
Platelet distribution width (PDW)
Platelet large cell ratio (P-LCR)

It is analogous to the MCV of RBCs.
It represents the average volume of the counted platelets.
It lies between 8 & 12 fL normally.
MPV (fL) = Plateletcrit (%) / platelet count (x 103/μL)
MPV, i.e., the range of platelet size, varies with platelet count.
In physiological conditions, MPV is inversely related to platelet count and is raised in
thrombocytopenia.
Dastjerdi et al. Hematology. 2006.
Korniluk et al. Mediators of Inflammation. 2019. CBC by Ahmad Darwish
MPV

IT is used to discriminate between
reactive (MPV normal) & malignant thrombocytosis (MPV raised).
It is increased in: splenectomy, CML, myelofibrosis, Bernard Soulier syndrome & ITP.
It is decreased in: hypersplenism, aplastic anemia, megaloblastic anemia, Wiskott
Aldrich syndrome & chemotherapy
Korniluk et al. Mediators of Inflammation. 2019. CBC by Ahmad Darwish
MPV

 It is a measure of the variation of platelet size.
 It is a coefficient of variation calculated as SD / MPV×100
 It has a reference range of 9 to 14%.
 It is expressed in the histogram by drawing an arbitrary line at the height of 20%.
 It is high in aplastic anemia, megaloblastic anemia, CML, chemotherapy.
 It is falsely high in the presence of platelet clumps, microcytic RBCs & fragments.
PDW

 It is the percentage of platelets that exceed the normal
value of platelet volume of 12 fL in the total platelet count.
 It is calculated as:
platelet cell concentration (PLCC) / platelet count
(where P-LCC refers to the platelets in the volume range of 12 to 30 fL).
 It is raised in the presence of platelet clumps, microcytic RBCs & giant platelets.
P-LCR

Platelet Histogram Flags
LD Flag (PL Flag):
 This occurs when LD exceeds preset height by 10%.
 This can occur due to the presence of:
1. A high blank value
2. Platelet aggregation
3. Cell fragments
4. Contaminated reagents
5. High numbers of bacteria

UD Flag (PU Flag):
 This occurs when there is abnormal height at UD
and it exceeds the preset height by >40 %.
 It can be seen in the presence of platelet clumps, giant platelets and
microcytic RBCs.

MP Flag:
 This occurs when there are multiple peaks present.
 It can be seen in cases of:
1. Platelet transfusion
2. Recovery from chemotherapy
3. Platelet aggregation

Platelet Parameters on Automated Analyzer
Significance in Anemia
Platelet parameters
❑ Raised when platelet anisocytosis present
Platelet volume distribution width (PDW):
Variation in platelet size
❑ Act as acute phase reactant;
(High in anemias associated with
myeloproliferative neoplasms & chronic disease,
e.g., type I diabetes mellitus)
Mean platelet volume (MPV):
Thrombocyte volume
❑ High in active stages of certain chronic diseases
(e.g., Crohn disease)
Plateletcrit (Pct):
Volume of circulating platelets in unit volume of blood
IDA: iron deficiency anemia

Platelet Parameters on Automated Analyzer
Significance in Anemia
Platelet parameters
❑ Peripheral destruction of platelets (autoimmune
conditions)
Reticulated platelets or immature platelet fraction (IPF):
Immature platelets
❑ Reactive thrombocytosis (IDA, viral infections)
❑ Peripheral destruction of platelets
Platelet large cell ratio (P-LCR):
Large circulating platelets
❑ Raised when variation in platelet shape is present
(e.g., giant platelets in reactive thrombocytosis)
Platelet component distribution width (PCDW)
❑ Raised in reactive thrombocytosis (IDA,
thalassemia)
Mean platelet mass (MPM) or
Mean platelet component (MPC)
IDA: iron deficiency anemia

WBC Parameters
 WBC differential indicates the chronicity of the disease and very high counts
may indicate severe infections and malignancies.
 The presence of leucopenia along with anemia, can point towards more specific
etiologies.

Total WBC count: 4000 to 11,000/μL
Neutrophils(50-70%)
Monocytes(2-6%)
Lymphocytes
(20-40%)
Basophills(0-1%)
Eosinophils
(0-3%)

• Acute and chronic bacterial infections
• Viral infections
•
•
•
Allergy, especially severe allergic reactions
• Acute lymphocytic leukemia
Leucocytosis
• Acute myeloid leukemia (AML)
•
•
• Chronic myelogenous leukemia
•
•
•
•

leukemoid reaction
Acute leukaemia → (↑total leucocytic count mostly exceed
100,000+ Blastcells in peripheral blood +↓platelets +↓RBCs )
•
• leukemoid reaction (like = oid ) → ↑total leucocytic count more
than 50,000 (rarely exceed 50,000+ no blast cells + Bandeamia
+normal platelets + normal RBCs ) .
Normal leucocytes secrete leucocytic alkaline phosphatase but in
leukemic leucocytes never secrete so in leukemoid reaction
alkaline phosphatase is very ↑ ↑↑.
leukoerythroblastic reaction :- leucocytes & RBCs blasts appear
on Peripheral blood due to bone marrow infiltration (lymphoma -
leukaemia) .
•
•

• Chemotherapy and radiation therapy
• Sepsis
• Typhoid
•
•
Tuberculosis
•
Leucopenia
Enlargement of the spleen
•
Leukemia (as malignant cells overwhelm the bone marrow)
•
Folate deficiencies
•
•
Aplastic anemia (failure of bone marrow production)
•
HIV and AIDS
•
Influenza
• Systemic lupus erythematosus

Neutrophilia
Physiological: Stress, anxiety, seizure, exercise
Acute bacterial infection
Tissue injury and inflammation
Metabolic : DKA/Acute renal failure
Myeloproliferative disorders
Misc: Splenectomy, Hemolytic anemia

from circulation
Increased removal
•Immunological like SLE
•Hypersplenism
•Sepsis
Decreased or
ineffective
production
• Aplastic anemia
• Vit B12 & Folate deficiency
• MDS
Neutropenia

Mild (700–1500 per microliter)
• Allergic rhinitis, extrinsic asthma, mild drug reaction, long-term dialysis, immunodeficiency
Moderate (1500–5000 per microliter)
• Parasitic disease, intrinsic asthma, pulmonary eosinophilia syndrome
Marked (> 5000 per microliter)
Trichinella, hookworm, Toxocara canis, eosinophilic leukemia, severe drug reaction
Eosinophilia

• Usually related to increased circulating steroids
• Cushing’s syndrome
Eosinopenia • Drugs
• ACTH, epinephrine, thyroxine, exogenous steroids
• Acute bacterial infection
• Normal diurnal pattern.

• Hypothyroidism, myxedema
• Chronic myeloid leukemia
• Ulcerative colitis
Basophilia • Polycythemia vera
• Urticaria
• Hodgkin’s lymphoma/disease
• Splenectomy

• Viral infections
• Tuberculosis
• Subacute bacterial endocarditis
• Collagen diseases
Monocytosis • Infectious mononucleosis
• Sarcoidosis
• Crohn’s disease
• Rheumatoid disease
• Ulcerative colitis.

• Infectious mononucleosis
• Mycobacterium tuberculosis
• Acute lymphoblastic leukaemia
• Brucellosis
• Burkitt’s lymphoma
• Cytomegalovirus
Lymphocytosis
• Epstein-Barr virus
• Whooping cough
• Non-Hodgkin’s lymphoma
• Syphilis
• Toxoplasma

• Viral infection
• HIV
Lymphocytopenia
• Drugs (vinblastine, chloramphenicol,
Doxorubicin)
• Marrow suppression

WBC Histograms
 WBC histograms generated by automated hematology
analyzers plot the size of cells (in fL) on the X-axis and
the frequency of the cells on Y-axis.
 The counter here also sets a lower discriminator (LD or
WL) that fluctuates between 30 and 60 fL and an upper
discriminator (UD or WU) that is fixed at 300 fL.
 The number of cells between these two discriminators
is the total WBC count.

WBC Histograms
 A normal WBC histogram curve should be within the
discriminators and start and end at the baseline.
 WBC histograms consist of two troughs or valleys;
T1 lies between 78 & 114 fL and T2 <150 fL.
 These 2 troughs are detected by 2 inner discriminators
that separate the WBC populations into 3 groups,
based on the size of cells.

WBC Histograms
 The peak between LD and T1 represents a small cell
population, i.e., lymphocytes with their volume ranging
from 35 to 90 fL.
 The peak between T1and T2 represents medium cell
population comprising of eosinophils, basophils,
monocytes, promyelocytes and blasts with their
volume ranging between 90 and 160 fL.
 The peak between T2 and UD denotes neutrophils
with their size ranging from 160 to 300 fL.

Histograms showing abnormal distribution of neutrophils & lymphocytes
WBC Histograms

WBC Histogram Flags
WL flag (LD flag):
 This is an abnormal curve at the LD that occurs when the height of LD exceeds the
present 2% of the Y-axis.
 This may be seen in the presence of nucleated RBCs, clotted sample & cold agglutinins.

WBC Histogram Flags
WU flag (UD flag):
 This is an abnormal curve at the UD that occurs when the height of UD exceeds the
preset 10% of the Y-axis.
 This may be seen in the presence of inadequate WBC lysing, WBC aggregation & extreme
leukocytosis.

WBC Histogram Flags
T1 flag (LD flag):
 This is an abnormal curve at T1 level.
 T1 and T2 flags appear when discrimination between 3 populations is not possible.
 A T1 flag appears when differentiation between lymphocytes and medium-sized cell populations is
not possible, for example, in cases of CML & leukocytosis.

WBC Histogram Flags
T2 flag:
 This is an abnormal curve at T2 level.
 T2 flags appear when discrimination between 3 populations is not possible.
 A T2 flag appears when differentiation between mixed cells and neutrophils is not
possible,

 This occurs when the height of T1 surpasses the present limit of 40%.
 The F1 flag denotes that the discrimination between small cell and middle
cell populations is not an accurate example in ALL.
 An F2 flag occurs when the middle cell data is inaccurate.
 The T1 and T2 exceed the preset limits of 40% and 50%, respectively.
 Examples of F2 flags are eosinophilia, acute myeloid leukemia and
monocytosis.
 F3 flag occurs when the T2 exceeds the preset limit of 50%, denoting that
the large cells data is inaccurate.
WBC Histogram Flags
F1, F2 & F3 flags:

WBC parameters
Systemic inflammation, sepsis, hematological
disorders (MPN,
AML, bone marrow infiltrative disorder).
Immature granulocyte count (IMG): Immature
myeloid cells
Reactive lymphocytes, lymphoma cells, blasts;
Helps in sepsis monitoring.
High fluorescent lymphocytes (HFL) or Atypical
lymphocytes, ALY% or Large unstained cells,
%LUC
Raised in sepsis; Low in MDS or MDS/MPN.
Neutrophil granulation (NEUT-X/NEUT-Y):
Granularity/nucleic acid and protein content
More than 3.7 in the absence of a WBC peak in
malaria.
Malaria factor (Mf)
AML: acute myeloid leukemia, MDS: myelodysplastic syndrome, MDS/MPN: myelodysplastic
syndrome/myeloproliferative neoplasm.
1. Nathan D, Orkin S, Oski F. Nathan and Oski’s hematology and oncology of infancy and childhood. Elsevier. 2015.
White Blood Cell Parameters on Automated
Analyzer

Abnormal values in CBC reports

Quality control in
performance of a CBC
Pre-analytical factors
 Samples should not be left to stand for a long time
before processing
 Samples should be kept away from direct sunlight
 Insufficient samples should be rejected specially to
deter the effects of anticoagulant concentrations on
CBC results
 Sample collection should follow proper collection
procedures
 Proper labeling
 Right anticoagulant should be used (EDTA)

Sample Collection
EDTA
Trisodium citrate Heparin

 Automated hematology analyzers do not provide a complete answer regarding the
underlying etiology of anemia and may leave room for misinterpretation.
 Thus, further testing is required to make a confirmatory diagnosis.
 However, despite these pitfalls, it plays a role in early decision-making and can
help in reducing the time lag between clinical presentation and institution of
appropriate therapy.
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complete blood count analysis and blood disease investigation

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