Lecture about complete blood count CBC for students to know its importance, normal range & uses in diagnosis many diseases

1. Dr. Mohamed Shaheen
MBBCh, MSc, MD, IPCD, HHMD
Lecturer of Clinical Pathology- Al-Azhar University
Chief of Infection Control team - El-Hussien hospital

2. Topics
 Introduction
- Indications of CBC.
- Components of CBC
- Reference ranges of CBC.
 Pre-analytical: Precaution, Sampling & Storage.
 Analytical:
- Methods: Manual & Automated CBC.
- Blood film (Smear + Stain)
- CBC parameters: WBCs + RBCs + Platelets.
- Development & maturation of Blood cells
- Analytical errors.
 Post-analytical: Interpretation
 Others: Effect of smoking & pregnancy.

3. Introduction
Components, Indications & RR

4. Basic terminology
Normal Values
Decrease Normal Increase
WBCs Leucopenia 4,000-11,000/cmm Leucocytosis
RBCs Anemia 4-6 m/cmm Polycythemia
PLTs Thrombocytopenia 150-400/cmm Thrombocytosis
An-Iso-cytosis
Un equal RBCs size
Microcytosis 80-100 fl Macrocytosis
Poikilocytosis Different RBCs shape Biconcave, circular Sphero, acantho,
pencil,….
Neutrophils Hyposegmented
< 3 lobes
3-5 lobes Hyper segmented
Nuclear Lobes > 5

5. CBC Components:
• White Blood Count (WBCs):
- Granulocytes: Neutrophil, Eosinophil, Basophil,
- Agranulocytes: Monocyte, Lymphocyte
• Red Blood Count (RBCs)
- Hb, HCT,
- Indices: MCV, MCH, MCHC, RDW.
• Platelets:
- Platelet Count,
- Indices: MPV, PDW.

6. Indications of CBC
 Check up.
 One of the routine investigations.
 Preoperative, postoperative.
 Monitoring of treatment.
 Anemia: as pallor, blurring of vision, easily fatigability, ..
 Bleeding: bruises, melena, hemoptysis, hematemesis,
 Infection: fever, redness, hotness, tenderness,
 Suspicion of leukemia, lymphoma, ….

7. Reference ranges of CBC
According to Age & Sex
WBCs: Count & Differential
RBCs: Count, Hb, HCT & indices.
Platelets: Count & indices.

8. WBCs 4 – 10 × 103/cmm
Platelet count 150 - 410 ×103/ul
Red cell lifespan 120 ± 30 days
Serum iron
Men and Women 60–160 mg/dl)
Total iron-binding capacity 250–400 mg/dl)
Relative %
Absolute count
Differential WBCs
(40–80%) ~60%
2.0–7.0 × 103/ul
Neutrophils
(20–40%) ~30%
1.0–3.0 × 103/ul
Lymphocytes
(2–10%) ~ 6%
0.2–1.0 × 103/ul
Monocytes
(1–6%) ~ 3%
0.02–0.5 ×103/ul
Eosinophils
(<1–2%) ~ 1%
0.02–0.1 ×103/ul
Basophils

9. RBCs & their indices (Adults)
Female
Male
Parameter
4.3 ± 0.5
3.8 – 4.8 × 106/cmm
5 ± 0.5
[4.5-5.5 × 106/cmm]
RBCs
13.5 ±1.5
12 -15 g/dl
15 ± 2
13-17 g/dl
Hb
41 ± 5
[36-46%]
45±5
[40-50%]
HCT
90±10 [80-100] fl
MCV
(30 ± 3) [27-33] pg
MCH
33 ± 2 [31-35] g/l
MCHC
CV=12 -14% SD = 42.5 ± 3.5 fl
RDW
0.5–2.5% Absolute: 50–100 × 109/l
Reticulocyte count

11. Normal values of children

12. Analytical issues
Methods
How to
prepare
blood smear
Blood cell
maturation
WBCs RBCs Platelets

13. Method
Manual
RBCs
WBCs
Platelets
Automated
3 Parts (N, L, MIX)
Electrical Impedance
5 Parts (N, L, M, E, B)
Electrical Imp + FCM
7 Parts (5+MC,IMC)
Electrical Imp + FCM

14. Automated CBC

15. 3 steps:
1- Make a blood smear
2- Fixation by Methanol
3- Stain blood smear
How to prepare a Blood film

16. 1- Making Blood Smear

17. Staining
1- Leishman stain 2- Rapid Stain

18. Good smear

19. Function.
Morphology.
- Maturation
- Differentiation:
Neutrophil, Eosinophil, Basophil, Monocytes, Lymphocytes.
Reference range.
White Blood Cells

20. WBCs Functions
 Neutrophil: common phagocytic cell (mainly anti-bacterial)
Monocytes: Large phagocytic cell (Bacterial, Viral & Fungal)
 Lymphocytes: Anti-viral
 Eosinophils: Anti-allergic
 Basophils: Anti-allergic
Neutrophil Monocytes Lymphocyte Eosinophils Basophils
Function common phagocytic
cell
APC
Phagocytosis
Cytokine: product
IL1, 12, TNF
Adaptive immunity
(Anti-viral)
B-cell: Ab product
T-cell: Memory,
helper, cytotoxic
NK cell
Cytotoxic
Allergic, parasite
Tissue repair
Ag bind to IgE bind
to basophil surface
degranulation and
release of histamin
Normal
- Relative
- Absolute
40-80%
2-7 x 109/L
2-10%
0.2-1 x109/L
20-40%
1-3 x109/L
2-4%
0.1-0.5 x109/L
0-1%
0-0.1 x109/L
Increase
1ry: (Malignant)
2ry: (Reactive)
Neutrophilia
CML, CMML,
Infect, inflame, stress
Monocytosis
AML-4,5, MPN, TB,
Brucella, $ SBE,
typhoid,
Lymphocytosis
ALL, CLL, NHL
Viral: CMV, HV
Bact: TB, Bruc, $
Eosinophilia
Allergy, Parasites
MPN, HL,
Basophilia
AML-M2E, HL
Allergy, 3X & DM
Decrease Neutropenia
Infection, BMF
Monocytopenia
Aplastic anemia
HCL, CLL
Cushing Sndrom
Corticosteroids
Lymphocytopeni
a
AIDS, COVID
Burn
Corticosteroids
Radiation
Eosinopenia
Acromegaly Acute
infection
Cushing syndrome
Corticosteroids
Basopenia
Urticaria or
anaphylaxis
Cushing Syndrom
Corticosteroid
Hyperthyroidism

21. WBCs Maturation & development

22. Granulocyte Development
Trends:
 Immature  Mature
 Large cell  Small cell
 No granules  Azurophilic (non-specific) granules  Cell-specific granules
 Round nucleus  indented nucleus  U-shaped  multilobed (specific for
cell type)

23. Lymphoblast, Myeloblast & Monoblast
Lymphoblast Myeloblast Monoblast
Size Smallest Moderate Largest
Shape Rounded mostly Round or not Many shape
Nucleus Large, Rounded,
may be clefted,
Moderate
No clefts
Largest
Irregular shape
Most fine
Nucleoli Not prominent
(but present)
Prominent
May be present or not
Bulged
N/C ratio 4:1
Chromatin Open fine, Immature
[most condensed]
Open fine, Immature
[moderate fine]
Open fine, Immature
[Most fine]
Cytoplasm
- Granules
- Medium blue color with
darker borders
- No granules
Types 3 types: ALL [L1, L2, L3]

24. Myeloblast
• Size:15-20 m
• Nucleus:
1. Shape: Large, round to oval,
2. Nucleoli= 2-5, Prominent
3. Chromatin: Fine
• Cytoplasm: basophilic cytoplasm with no
granules.
• N/C ratio: 4:1
• Present in: Only in BM &
can be seen in PB in certain leukemias
• Reference interval:
B.M. = 0- 1% PB= 0%

25. Promyelocyte
Nucleoli
• Size:14-20 m
• Nucleus:
1. Shape: Large, round to oval,
2. Nucleoli= ≥ 3, Prominent
3. Chromatin: more courser than blast.
• Cytoplasm: basophilic cytoplasm.
• N/C ratio: 3:1
• Present in: Only in BM &
can be seen in PB in certain leukemias
• Reference interval:
B.M. = 2.5% PB= 0%
• First recognizable cell in granulopoiesis
• Largest cell in series
• Golgi Ghost
• Azurophilic (primary) granules in
cytoplasm, produced only at this stage

26. Myelocyte
 Spherical nucleus
 Becomes increasingly
heterochromatic
 Prominent Golgi apparatus
 Negative image
 Lots of azurophilic granules
 Formation of specific granules (2ry)
 Emerge from Golgi (cis face)
complex
 Characteristic staining reactions
for each line
 Last stage that can do mitosis
Late Myelocyte/ Early Metamyelocyte
Neutraphil
myelocyte
Eosinophil
myelocyte
golgi

27. Metamyelocyte
 First stage that is clearly divided
into separate lines
 Few hundred granules present
in the cytoplasm
 Specific granules outnumber
the azurophlic granules 4:1
 Nucleus
 Heterochromatic
 Indentation deepens to form
horse-shoe shape
Late neutraphilic
metamylocyte
Neutraphilic metamylocytes
Eosinophil
metamylocyte

28. Band Cells
 Last immature stage in
Neutrophilic series
 Sometimes seen in circulation
 Particularly during states of
chronic infection
 Nucleus is elongated and of
uniform width
 Nucleus constricts
 2-5 lobes are formed
 PMNs

29. Mature WBCs
[Agranulocytes + Granulocytes] ML+BEN
Never Let Monkey Eat Banana
Percent
Cell
60-80% (60%)
Neutrophil
20-40% (30%)
Lymphocyte
3-10% (6%)
Monocyte
1-3% (3%)
Eosinophil
0.02-1% (1%)
Basophil

30. Neutrophil
 PMNL:-Poly-morpho-nuclear Leucocytes.
 Percentage 40-80% TLC
 Appearance:
- Pink granules in cytoplasm,
- Nucleus has 3-4 lobes
- Nucleolus: No
 Function: Phagocytosis of bacteria.
 Azurophilic granules are "lysosomes of
PMNs", occur in all leukocytes

31. Eosinophil (Eos)
 Nucleus: Bi-lobed nucleus
 Percentage: 2-4% of TLC
 Action: Recruited to sites of
inflammation.
 Function: Involved in allergy, parasitic
infestations
 Contains: eosinophilic granules
 Granules contain: major basic protein
 Terminally differentiated
Azurophilic granuels

32. Basophil
 Circulating form of mast cells
 Percent: <1% WBC
 Contains: basophilic granules
 Granules contain: histamine and heparin
 Carry: IgE receptors
 Function: Involved in allergy
 Terminally differentiated

33. Monocyte/ Macrophage
Monocyte
 Percentage =3-8% TLC
 Circulating form (precursor) of tissue macrophages
 Recruited to sites of inflammation
Macrophages
 Function:
- Phagocytosis, bacterial killing,
- Antigen presentation cell (APC)
 Present in tissues:
- Peritoneal cavity: peritoneal macrophages
- Lung: alveolar macrophages
- Spleen: splenic macrophages
- Liver: Kupffer cells

34. Lymphocyte
•Size: small (nucleus same size as RBCs)
•Shape: rounded, little visible cytoplasm
•Cytoplasmic granules: NO specific granules
•Percent: 20-25% of WBC
•Types:
- T cells: Cell Mediated Immunity (for viral infections)
- B cells: humoral immunity (antibody)
- Natural Killer Cells
Reactive lymphocytes:
- Cytoplasm: Large abundant, basophilic periphery ± vacuolations.
- Scalloping margins, Skirting appearence
- Right lymphocyte has abundant cytoplasm with intensely basophilic
cytoplasmic & bleb formation.
- Left variant lymphocyte has vacuolated cytoplasm, skirting” appearance with
scalloped margins and indentations by the surrounding RBCs.
Reactive lymphocyte
Normal lymphocyte

35. WBCs reference range

36. Degree of CBC disorders
Leucocytosis
Leucopenia
11-20
4-2 X 103/ul
Mild
20-40
2-0.5 X 103/ul
Moderate
<40
>0.5 X 103/ul
Marked
Polycythemia
Anemia
16-18
13-10 X 106/ul
Mild
18-20
10-7 X 106/ul
Moderate
<20
> 7 X 106/ul
Marked
Thrombocytosis
Thrombocytopenia
450-600
150-100 X 103/ul
Mild
600-800
100-50 X 103/ul
Moderate
<800
> 50 X 103/ul
Marked

37. Red Blood Cells
Maturation
Morphology & Function
Measurement & Counting
-RBCs
-Hemoglobin (Hb)
-Hematocrit (HCT)
RBCs indices (MCV, MCH, MCHC, RDW)
Reference range.

38. Hb Measurement
Manual
Direct Visual
comparative
Sahli
WHO Hb color
scale
Sp. gravity
Photometric
Cyan-Hb
Oxy-Hb
Alkaline
Hematin
Automated
CBC
3 Parts
5 Parts
7 Parts
ABG POCT
Portable
Reagent less
Non invasive

39. Hematocrit/Packed Cell Volume (PCV)
 Definition: It is the volume % of RBCs in blood.
- Volume occupied by RBCs expressed as a fraction of the whole blood volume.
 Value:
- Simple screening test for anemia.
- Calculation of red cell indices.
- Reference method for calibrating automated blood count systems.
 Methods of measurement:
- Macro-method using wintrobe tube: not longer used.
- Micro-method
 Micro-haematocrit method:
 Principle:
- Packing of RBCs by centrifugation of whole blood in capillary tube at high
Relative centrifugal force (RCF).

41. Packed cell volume (PCV)
 Methods:
- Automated: PCV is calculated = RBCs X MCV = ‫عددهم‬ X ‫الخلية‬ ‫حجم‬
- Manual: PCV is measured
 Reference range:
- At birth: 42-60%
- Adult Male: 40-50%
- Adult Female: 36-46%
N.B:
- The normal ranges vary slightly depending upon the patient population.

42. Hematocrit abnormalities
Low HCT
 Hemorrhage
 Hemolysis
 BM failure (aplasia, hypoplasia, infiltration)
 Malnutrition
 Autoimmune: SLE, RA.
High HCT
 Dehydration
 High erythropoietin level
 Low O2 level/high altitude
 Congenital heart disease
 PRV
 Smoking

43. RBCs indices
 The RBCs indices are calculated using RBCs, Hb, Hct
Mean Cell Volume (MCV):
 Definition: It is the average volume/RBC in femto-liters (10-15 L)
 Methods:
- Automated: MCV is measured directly
- Manual: MCV is calculated
MCV= PCV (in %) x 10 = 45 x 10 = 90 fl
RBC count 5
 Reference range:
- At birth: 98-123 fl
- Adult: 80-100 fl

44. Classification according to size
MACROCYTIC CELL
>100 fl
MICROCYTIC CELL
< 80 fl
NORMOCYTIC CELL
80-100 FL

45. Anemia Algorithm
 Patient with anemia and decreased reticulocyte count-
What is the MCV ??
Microcytic Normocytic Macrocytic:
MCV ˃ 100 fl
•Megaloblastic an.
e Pancytopeni
B12 ± Folate↓
•Non Megaloblastic:
Not pancytopenia
•Reticulocytosis
•MDS
•Alcoholism
•Liver Disease
BM Diseases
•MDS
•Solid Tumor
•Myeloma
•Aplastic anemia
Systemic D
Renal D.
Liver D.
Endocrine D.
Anemia of chronic D.
MCV˂ 80 fl
IDA
Thalassemia
Sideroblastic anemia
Lead poisoning

46. MCV Interpretations
Microcytic Hypochromic
 IDA
 Thalassemia (Major or combined e
Hb-patheis)
 Lead poisoning
 Disorders of porphyria synthesis
Microcytic Normochromic
 Anemia of chronic diseases [1/3 pt]
 Heterozygous thalassemia & Hb-patheis.
False decrease value
 Hemolysis: fragments
False Increase value
 Antibodies: Cold agglutinins > Warm ab; clumps
 Marked reticulocytosis: larger
 Marked leukocytosis.
 Marked hyperglycemia 600mg/dl: pl.Osmolarity
 Rigid wall cells (Spherocyte, Sickle, …): flexibility

47.  Definition: It is the average Hb content/cell in picograms (pg= 10-12 g)
 Methods:
- Automated: MCH is calculated
- Manual: MCV is calculated
MCH= Hb (in g/dl) x 10 = 15 x 10 = 30 pg
RBCs count (million) 5
 Reference range:
- At birth: 31-37 pg
- Adult: 27-33 pg
 Importance: MCH can classify RBCs according to color as:
- Normochromic (27-33)
- Hypochromic (< 27)
- Hyperchromic (≥ 34), but RBCs don’t really have a higher than normal Hb, they just have ↓
amount of membrane (relative hyper).
Mean Cell hemoglobin (MCH)

48.  Definition: It is the average Hb concentration in g/dl (100ml/%) of packed RBCs.
 Methods:
- Automated: Hb/ [MCV x RBCs] X 100
- Manual: MCHC= Hb (in g/dl) x 100 = 15 x100 = 33%
HCT (in %) 45
 Reference range:
- At birth: 30-36 Adult: 31-37
 Importance:
 MCHC can classify RBCs according to color
 Assess control: it is affected by many factors, most valuable clue to artifacts.
- IDA: not accurate due to plasma trapping between RBCs. (↓Plasma, ↑HCT).
- Spherocytosis: Hyperchromic (> 37), but RBCs don’t really have a higher than normal Hb, but
as ↓ amount of membrane (relative hyper).
Mean Cell Hemoglobin Concentration (MCHC)

49. Classification according to color
HYPERCHROMIC CELL??
More 36
HYPOCHROMIC CELL
Less 33
NORMOCHROMIC CELL
33-36

50. ANISOCYTOSIS
Red cell distribution width (RDW) –
Definition: It is a measurement of the variation in RBCs size
Calculation: Standard deviation/mean MCV x 100
Normal range: is 11.5-14.5%
RDW > 14.5 → anisocytosis.
RDW < 11.5 → RBC population is more uniform in size than
normal.

51. POIKILOCYTOSIS

52. RBC MORPHOLOGY ON A PERIPHERAL SMEAR

53. DIAGNOSIS OF ANEMIA

54.  Introduction & functions
 Maturation & Differentiation.
 Morphology & structure.
 Measurement (counting) Methods: manual (staining)& automated.
 Reference range.
 Interpretation: low & high.
 Platelet disorders: ET, TTP, GPS, Bernard-Soulier Syndrome, May-Hegglin disease
 PLT indices: (MPV, PCT, PDW,P-LCR, IPF)
Platelets

55.  DEFINITION:.
- Platelets are mature non-nucleated disc shaped blood cells.
- Platelets means (a tiny plate), piece of very large cell (BM megakaryocytes).
 FUNCTION:
- Role of Platelets in Haemostasis: Activation of platelets refers to adhesion,
aggregation, and release reaction of platelets which occurs after platelet stimulation
(i.e. after vascular damage).
- Help form Blood clots to slow or stop bleeding (so called thrombocyte).
- Help wounds heal.
Introduction

56. Differentiation & Maturation

57.  Morphology:
- Diameter: 1-3 um. (large 4–8 µm & giant when wider ≥ RBC).
- Shape: By Romanowsky stains, appear as very small non-nucleated disc shape cells,
irregular with fine cytoplasmic processes. Cytoplasmic granules are often visible.
- Contain other organelles: which contain 3 types of granules alpha, dense & lysozymes
& organelles such as mitochondria & Golgi apparatus.
 Structure:
1. Peripheral zone: exterior coat (glycocalyx), cell membrane, open canalicular system
2. Sol-gel zone: microfilaments, circumferential microtubules, dense tubular system
3. Organelle zone: alpha granules, dense granules, mitochondria, lysosomes
Morphology & Structure

58. Platelets Life
 1/3 of platelets is trapped in the spleen and
 2/3 in peripheral blood. (1/3 fill endothelial gabs + 1/3 circulation)
 Normal count: 150,000 - 450,000/ul. (150-410)
 Abnormal count:
- < 20,000 → Risk of spontaneous bleeding.
- < 50,000 → bleeding occurs with cut or bruised.
- ≥ 500,000 or (million) → Risk of thrombosis.
 Normally the RBCs : Platelets ratio= 30 : 1
 Platelets usually present in blood film 7–20 /oil immersion field (100x lens).
 Production: is regulated by Thrombopoietin hormone (produced in liver)
 Apoptosis: They are removed by spleen when they are old or damaged.
 Lifespan: in blood about 9–12 days (10 days )

59.  Methods:
1- Manual:
- Diluent: Ammonium Oxalate 1%
- Dilution: 1: 20
- Procedure: Hemocytometer [RBCs squares]
2- Automated: CBC coulter, [3,5,7 diff]
- Diluent: cell Pack [Diluent, NaCl, antimicrobial agent)
- Dilution: 1: 20
- Procedure: RBCs chamber
Measurement/counting Methods

60. Reference Range

61. Platelet Indices
 PCT = Plateletcrit.
 MPV = Mean Platelet Volume.
 PDW = Platelet Distribution width.
 P-LCR= Platelet-Large Cell Ratio.
 IPF = Immature Platelet Fraction.

62. Variation in size

63. Summary
PLT indices Indicate Normal High Low
PDW PLT Variation 9-13 fl
Reactive
Thrombocytosis
Anisocytosis
MPN = ET
Megaloblastic an
Aplastic anemia
Fragmented RBCs
More uniform
in than
normal
MPV BM Production 7-11fl
MPV/Count
↑MPV + Normal count =
Early detection of
Megaloblastic an
Aplastic anemia (BMF)
↑MPV + ↓PLT count =
Activation
Consumption=DIC-TTP
Destruction= ITP
With ↓ Risk of bleeding
BMF
PCT PLT% to blood 0.11-0.30% Non significant
P-LCR Large PLT % 15-35 % ↑P-LCR + ↑PDW =
Destruction = ITP
IPF or RP Immature
Thrombopoiesis
Up to 12% Consumption=DIC-TTP
Destruction= ITP
Recovery after BMT
BMF

64.  The term (shift to the left) refers to
- A microscope adjustment - Immature cell forms in peripheral smear
- Increased red cell count - A trend on A levy – Jennings chart
- A calibration adjustment on an instrument
 Auer rods are most likely present in which of the following?
- CML - Myelofibrosis
- Erythroleukemia - AML - ALL
 Causes of microcytic anemia include:
- Pernicious anemia - Methotrexate
- Thalassemia - Vitamin C deficiency - Hyperthyroidism
 Anisocytosis refers to
- Un equal size of RBCs - Immature cell forms in RBCs.
- Abnormal shape of RBCs - Increase RBCs count above normal
 Normal count of platelets is which of the following?
- 150-450 ul/L - 100-150 ul/L.
- 4.000-11,000 ul/L - Non of the above

65.  Advanced Laboratory Methods in Hematology: R. Martin
 Essentials of Hematology: Shirish M.Kawthalkar, 3rd ed. 2020.
 Hoffbrand‘s Essential Hematology: A.vector Hoffbrand, 8th
ed.2020.
 Practical Hematology: V. Dacie & SM Lewis, 12th ed. 2017.
 The ABC of CBC Interpretation of Complete Blood Count and
Histograms: DP Lokwan, 1st ed. 2013.
References

66. ‫المحاضرة‬ ‫شرح‬ ‫رابط‬
 YouTube Channel ‫القناة‬ ‫رابط‬
 https://www.youtube.com/channel/UCrgIU3OIbph_MTL0aWzdJYw
 3rd year lecturers ‫الثالثة‬ ‫السنة‬ ‫محاضرات‬ ‫رابط‬
 https://www.youtube.com/playlist?list=PLQrhOyhVt5ia8VNMXRNR7M8JH8RgtFCLu
 CBC & Blood film lecture ( ‫شرح‬
‫المحاضرة‬ )
 https://www.youtube.com/watch?v=ezjO4CnEO1s&t=194s