Hematology I | Midterms

Leukopoiesis - Cytoplasm:
- Pale, clear blue
REDLeukocytes
BLOOD CELLS - Contains RER
- Granulocytes: neutrophil, eosinophil, basophil - A developing Golgi apparatus
- Monocytes - No granules
- Lymphocytes - CD 33: myeloid marker
- Proliferation and maturation are influenced by: - CD 34: HSC marker
- Growth factors - Inclusion: Auer rods
- Interleukins - Needle-like crystals aggregates of
- Colony stimulating factors fused lysosomes may appear alone or in groups
Proliferative/Mitotic Pool Maturation/Storage Pool - Pathological/malignancy
- Maturational development and - Metamyelocyte, band and Promyelocyte
mitotic division from myeloblast, mature forms (neutrophil,
- N:C = 3:1
promyelocyte to myelocyte stage eosinophil, and basophil)
- Location: BM 2-5%
- Mature forms are known as
marrow reserve (4-8 days supply) - Duration: 24hrs
Peripheral blood - Size: 14-20µm
- Circulating pool: extracted during - Nucleus:
venipuncture; source of WBC - Round to oval
count - Nuclear chromatin begins to condense
- Marginating pool: adhere to - Nucleoli begin to fade
endothelium of blood vessels; a - Cytoplasm:
guard to immediately fight off - Basophilic
infection - Production of primary/azurophilic granules becomes
dominant
Myeloblast Myelocyte
- N:C = 4:1 - N:C = 2:1
- Location: BM 1-2% - Location: BM 5-19%
- Duration: 15hrs - Duration: 4.3 days
- Size: 15-20µm - Size: 12-18µm
- Nucleus: - Nucleus:
- Delicate round to oval to quadrangular with prominent - Round to oval
nucleoli - May have 1 flattened side near the well-developed Golgi apparatus
- Nuclear chromatin is finely reticular, uniform - Nucleoli is no longer available
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Navarro, Johanna A.
 - Chromatin is coarse and more condensed
- Last mitotic stage
- Cytoplasm:
- Slightly basophilic
- Primary granules are few to moderate;
secondary/specific granules are increasing
- Other granules can now be recognized and
differentiated Polymorphonuclear | Neutrophil
- Eosinophilic: reddish granules
- N:C = 1:1
- Neutrophilic: lilac
- Location: BM 3-11%; peripheral blood: 50-70%
Metamyelocyte - Duration: 7-10hrs in the circulation, several days in tissue
- N:C = 1:1 to 1.5:1 - Marker: CD13, CD15, CD16, CD11b
- Location: BM 13-22% - CD 63 is also found in the membrane of azurophilic granules
- Size: 10-15µm - Size: 10-15µm
- Nucleus: - Nucleus: 2-5 lobes connected by thin
- Indented, kidney bean-shaped filaments of coarse clumped chromatin
- No more nucleoli - Cytoplasm:
- Chromatin is coarse clumped - Pale blue to pink
- Cytoplasm: - Few primary granules; abundant
- Pale blue to pink secondary granules
- Few primary granules; predominantly - It has apoptotic genes programmed to die after 5.4 days (average lifespan
secondary granules of neutrophil; can be found in saliva, cough, urine)
- Basophil (pale blue) - Cancer cells: have anti-apoptotic genes
- Eosinophil (red-orange) - Neutrophil function: phagocytosis, bactericidal activity
- Neutrophil (metamyelocyte) - Phagocytosis: degranulation for bactericidal
Band | Stab Primary/ - Myeloperoxidase: It is a heme-containing
Azurophilic peroxidase expressed mainly in neutrophils and
- N:C = 1:1 granules to a lesser degree in monocytes; also used as a
- Location: BM 17-33%; peripheral blood: 0-5% marker of neutrophil infiltration into tissues.
- Size: 10-15µm - Chloroacetase esterase: it is a specific esterase
- Nucleus: that hydrolyzes chloroacetyl esters of
- C or S-shaped unsubstituted and substituted naphthols.
- Coarse clumped chromatin - Lysosomal enzymes
- Cytoplasm: - Acid hydrolase: it hydrolyzes phosphoric acid
- Pale blue to pink esters and is important in the absorption and
- Few primary granules; abundant secondary granules metabolism of CHOs, nucleotides, and
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 phospholipids. It mediates in bacterial killing, - GM-CSF, IL-3: target early precursors
tissue damage, healing, and immune regulation - IL-5 promotes terminal maturation, functional activation, and prevention of
- Acid phosphatase: it catalyzes the hydrolysis of apoptosis of eosinophils
phosphate esters in an acidic environment - Nucleus: usually bilobed, rarely more than three
- B-glucoronidase: important lysosomal enzyme - Cytoplasm:
involved in the degradation of glucuronate- - Itself it colorless
containing glycosaminoglycan.
- Contains large, round-oval red-orange granules (major basic protein for
- Elastase: it is required for microbial clearance
toxic parasites and cells)
(Gram (-) bacteria)
- Arylsulfatase: arylsulfatase A – hydrolyzes - Function:
cerebroside sulfate - Anti-helminthic activity
- Cationic antibacterial proteins - Phagocytosis
Secondary/ - Lysozyme - Allergic response, asthma
Specific granules - Lactoferrin: chelates iron resulting in a - Modulate inflammatory reactions
bactericidal or bacteriostatic effect; it is an
indication of intravascular activation and
degranulation of neutrophils.
- Collagenase: an important mediator of tissue
destruction in inflammatory diseases (matrix
metalloproteinase 8; MMP-8)
- Plasminogen activator E. myelocyte E. metamyelocyte E. band Eosinophil
- Aminopeptidase: enhance vascular permeability
- E. myelocyte: earliest recognizable precursor
Tertiary granules - Gelatinase: allows penetration through the
- Organs: lungs, skin, intestine (for A. lumbricoides)
basement membrane of the endothelium
*All granules are identical in granulocytes EXC. lysozyme Eosinophil Specific Granules
Granulocytic Kinetics - Major basic protein: toxic parasites and cells, neutralizes heparin and
induces release of histamine from basophils
- The development, differentiation, maturation, and multiplication of
- Acid hydrolase
granulocytes
- Peroxidase, eosinophil cationic protein: anti-helminthic activity
- It takes about 14 days, wherein the last 6-7 days are spent in the
- Phospholipase: directly involved in the production of lipid mediators
maturation and storage pool during allergic inflammation
- PMN migrate from the circulating pool (source of WBC count) to the - Cathepsin: it has direct association with lung emphysema in COPD and
marginating pool, along the vessel walls and enters the tissues extracellular matrix degradation and remodeling
Eosinophil - Eosinophil-derived neurotoxin: major eosinophilic component of
human eosinophils that is also found in liver, lung, and spleen; a protein
- Size: average 13µm belonging to the ribonuclease A (RNase A) superfamily; biomarker for
- Location: peripheral blood 1-3% allergic DSEs such as asthma; encoded by the RNase2 gene (antiviral
- Duration: half-life: 18hrs in the blood before entering the tissues where it activity and immunomodulatory function)
can survive for 6 days
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Navarro, Johanna A.
 Charcot-Leyden Crystals Monopoiesis
- Hexagonal, bipyramidal crystals REDMonoblast
BLOOD CELLS
composed of lysophospholipase
localized in the cytoplasm of - N:C = 4:1
eosinophils - Location: BM (low numbers)
- Seen in intense or prolonged - Size: 12-18µm
eosinophilic inflammatory reaction - Nucleus:
- Round to ovaleccentric
- 1-2 nucleoli
Basophil - Fine chromatin
- Cytoplasm: deeply basophilic
- Basophil myelocyte: earliest
- CD 33 and CD 34 positive: markers for
recognizable precursor
monoblastic leukemia
- Location: peripheral blood: 0-1%
- Weakly positive for CD 34 markers
- Marker: CD32
- Very similar to myeloblast (more cytoplasm than myeloblast and with rare
- Nucleus:
or absent granules); thus, impossible to distinguish (not reliable in
- Less segmented (usually indented or
microscopic)
partially lobulated)
- Obscured by the granules Promonocyte
- Cytoplasm: contains large, deep-purple - N:C = 2:1 to 3:1
granules - Location: BM 1%
- Function: immediate hypersensitivity - Size: 12-20µm
reactions - Nucleus:
- Irregularly shaped (elongated, folded,
indented)
- Nucleoli may/may not be visible (0-2)
- Fine chromatin
- Cytoplasm:
Basophil Specific Granules - Blue-gray w/ fine azurophilic granules
- Histamine: constricts smooth muscle - Vacuoles variable
- Heparin: basophils appear in more number during allergic reactions and - Similar in size to blast but may have some granulation; technically, it has
produce heparin to prevent blood clotting. granules (fine)
- Peroxidase - Can be motile and participate in phagocytosis but they lack the activity
- Eosinophilic chemotactic factor A: derived from mast cells; plays an seen in mature cells
important role in the recruitment of eosinophils to parasitic
- Undergoes 2-3 mitotic divisions in 2-2.5 days
inflammatory regions

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 Monocyte Lymphopoiesis
- N:C = 2:1 to 1:1 RED BLOOD
Primary/Central CELLS Organs
Lymphoid
- Location: BM 2%; peripheral blood: 3-11%
- Distribution: half-life is 8.4hrs then spend Bone Marrow
several months as tissue macrophages - Production of all lymphocyte stem cells (precursors, progenitor cells)
- Size: 12-20µm - Site where lymphocytes undergo antigen-independent lineage
- Nucleus: variable, maybe round, horseshoe or commitment
kidney-shaped, indented or curved with lacy - Site of the development of B-cells (production to maturation): pro-, pre-,
chromatin with small clumps or folds immature B-cell, mature B-cell
- Cytoplasm: - Bursa cell (in birds)
- Abundant blue-gray, may have pseudopods,
Thymus
maybe quite irregular
- Many fine granules giving ground glass - Located in the mediastinum
appearance - Site of migration to T-lymphoid stem cells (progenitor cells)
- Absent to numerous vacuoles - Site of development fr: pro-, pre-, immature T-cell, mature T-cell
- Great morphologic variability - Thymic education: they undergo antigen receptor genetic rearrangement
- Largest WBC in the normal blood to produce T-cell receptors that are unique to each cell
- Tends to marginate along vessel walls - To avoid making immune response against your own antigen or protein =
- Functions: autoimmune DSE
- Phagocytosis - Recognition between self-antigens and non-self antigens
- Antigen-presenting cell; antigen processing - Hindi gumagawa ng lymphocytes
- Cell-mediated immunity Secondary Lymphoid Organs
- Synthesis of bioactive molecules - Rich in B-cells and T-cells; acts as checkpoint/filters
- Macrophage/monocyte processes bilirubin - Site of migration and interaction of T-cells and B-cells
- Rich in iron: gives Fe to RBC developing cells - Provide a unique microenvironment for the initiation and development of
- In BM, it is also known as nurse cell immune responses; madalas nadadaanan ng pathogens
- Differential diagnoses: Secondary Lymphoid Organs
- AML: Acute Monocyte Leukemia OR Acute Myelomonocytic Leukemia - Spleen: kills bacteria with capsule
- Chronic Myelomonocytic Leukemia - Lymph nodes: lymphoma: solid tumor; kapag kumalat na sa kulani, it has
Monocyte Granules high chance na nagspread na rin sa dugo yung cancer
- Acid hydrolase - Tonsils
- Arylsulfatase - Mucosa-associated lymphoid tissue (MALT), Peyer’s patches
- Non-specific esterase - Skin-associated lymphoid tissue
- Peroxidase - Bronchus-associated lymphoid tissue
- Acid-phosphatase - Thoracic duct

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Differences between LYMPHOCYTES from other leukocytes: - Capable of killing virus-infected cells and certain tumor cells (kahit sino
- Lymphocytes are not end cells: they still undergo mitosis to produce pinapatay)
memory cells and effector cells (adaptive immune system; specific) - Tumor immunology: cancer cells are also from our own body, it is innate
Parent T-cell → memory cells (same copies) and effector cells (soldier cells) Lymphocytic Development
- This is also why we vaccinate babies/start vaccination during infancy
- B and T lymphocytes are capable of rearranging antigen gene segments to RED BLOOD CELLS
Lymphoblast
produce wide varieties of antibodies and surface receptors - N:C = 4:1
3 Types of Lymphocytes - Size: 10-20µm
- Nucleus: round to oval, one or more nucleoli, fine
B-cell
chromatin, evenly stained
- 10-20% - Cytoplasm: scant, moderately
- Derived from the bone marrow
- Differentiation: pro-, pre, lymphoblast, B-cell, plasma cell (antibody)
- Involved in antibody production, humoral immunity Prolymphocyte
- Antibody-producing lymphocyte
- N:C = 3-4:1
T-cell
- Size: 9-18µm
- 60-80% - Nucleus: round to indented, fine chromatin to
- Arise from the thymus slightly clumped, intermediate between
- Differentiation: pro-, pre-, lymphoblast, T-cell lymphoblast and mature lymphocyte
- 2 major categories: CD4 and CD8 - Cytoplasm: light blue
- CD4+ cells are subdivided into:
Lymphocyte
- TH1: mediate immune responses against intracellular pathogens
- TH2: against extracellular parasites including helminths; also involved - N:C = 2-5:1
in the induction of asthma and other allergies (parang eosinophil) - Location: BM: 5-15%; peripheral blood: 20-
- TH17: involved against extracellular bacteria and fungi 40%Size: small: 9µm; medium: 14µm; large:
- T reg: regulates immune responses, tolerance 15µm
- CD8+ cells - Nucleus:
- Capable of killing target cells by secreting granules containing - Round to oval maybe slightly indented,
granzyme and perforin or by activating apoptotic pathways in the chromatin is condensed, clumped, blocky or
target cell; kills virus-infected cells slightly smudged
- Also known as cytotoxic cells - The chromatin stains purple/dark blue
Natural Killer Cell | NK - Cytoplasm: scant to moderate, sky blue, may
have few azurophilic granules and vacuoles; no
- <10% specific granules
- Develop in either in the bone marrow or thymus
- Involved in innate immunity
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Navarro, Johanna A.
 Plasma cell
- N:C = 1-2:1
- Size: 8-20µm
- Nucleus: round to oval, eccentric
- Chromatin: coarse
- Cytoplasm: basophilic often with perinuclear How to use Thoma pipettes:
clear zone (parang may halo) 1. Aspirate blood to 0.5 (most common) or 1.0 mark
- Mature B-cell producing antibodies 2. Dilute with diluting fluid to 101 for RBC or 11 for WBC
3. Mix using figure 8 motion.
CD Markers
4. Discard the volume in the stem (approx. 4 drops) since this contains
- B-lymphoid: CD10, CD19, CD20, CD21, CD22, CD24, CD28 diluting fluid.
- T-lymphoid: CD2, CD3, CD5, CD7 5. Use blood from the bulb in charging the hemocytometer.
- Tdt + (Terminal deoxynucleotidyl transferase): all pro-, pre- stages Calculating the dilutions for the pipette (DF: dilution factor)
- CD10 (CALLA): pre-B and immature B-cells bulb unit 100 10
= 𝐃𝐅 𝐑𝐁𝐂 = = 𝟐𝟎𝟎 𝐖𝐁𝐂 = = 𝟐𝟎
- Plasma cell: CD38 blood unit 5 5
Hemocytometry Diluting fluids
- Numerical evaluation of formed elements or the estimation of the number Characteristics of an IDEAL diluting fluid:
RED BLOOD CELLS
of blood cells in a known volume of blood - Isotonic: for RBC; hypotonic: for WBC; destroy RBC and single out WBC
- It is not done if the Spx is whole blood – too much blood cells! - Inexpensive/economical
- It is now used in other body fluids: CSF, sputum, serous, synovial, semen - Easy to prepare and secure
(fluids na wala dapat makitang blood cells) - With preservative action
Materials: Thoma pipettes, diluting fluids, counting chamber - Does not initiate the growth of molds
- With buffer action
Thoma pipettes
- Non-allergenic, non-corrosive
- Parts: stem and bulb - With high specific gravity
Characteristic RBC WBC - Stable
Size of the bulb Large Small RBC Diluting fluids
Color of the bead Red White Dacie’s | Formol Citrate - With formalin: acts as preservative
Volume of the bulb 100 10 Best diluting fluid - Keeps for a long time
Upper calibration/mark 101 11 - Preserves the morphology
- 40% solution of formaldehyde | 10mL
- 3% w/v trisodium citrate | 990mL
Hayem’s - Usually develops yeast colonies and produced
clumping of cells but can stand for long periods
of time; non-corrosive
- Mercuric chloride | 0.5g
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 - Sodium sulfate, crystalline anhydrous | 5g, - Distilled water | 100mL
2.65g Tuerk’s - Glacial acetic acid | 2mL
- Sodium chloride | 1g - Methyl violet | 1mL
- Distilled water | 200mL - `Distilled water | 100mL
Gower’s - Prevents rouleaux formation and precipitation - In other body fluids, WBC is counted in general
of protein
Counting chamber | Hemocytometer
- With glacial acetic acid
- Glacial acetic acid | 0.85g - Consist of a thick rectangular colorless glass slide
- Sodium sulfate | 6.25g - Center: ruled areas separated by moats from the
- Sodium chloride | 0.85g rest of the slide and two raised transverse barsThe
- Sodium sulfate | 40.5g ruled portion may be in the center of the central
- Glycerin | 33.3g area (single chamber) or there may be an upper and
- Distilled water | 100mL lower ruled portion (double chamber)
Toisson’s fluid - Has high SG Types: Fuchs-Rosenthal, Speirs-Levy, improved
- Stains WBC but supports growth of fungi Neubauer
- Sodium chloride | 1g
- Sodium sulfate | 8g Fuchs-Rosenthal
- Glycerin | 30g - The ruled area measures 4x4 mm & the depth
- Distilled water is 0.2 mm
- Methyl violet - The central ruling is divided into 16 smaller
Normal Saline Solution - Stable; acts as a preservative squares of 1 mm2
- Used in emergency cases
- Larger than the Neubauer
- Prevents rouleaux formation
- Used for low cell counts: eosinophil count,
- Sodium chloride | 0.85g
CSF, leukopenic counts
- Distilled water | 100mL
Bethel’s - Sodium sulfate, crystalline | 5g
- Sodium chloride | 1g
- Glycerin | 20g
- Sodium Merthiolate, 1:1000 | 2mL
- Distilled water | 200mL
3.8% Sodium citrate - Sodium citrate | 3.80g
Speirs-Levy
- Distilled water | 100mL
- Consist of 4 platforms per counting area
WBC Diluting fluids - Each ruled area consists of 10 squares each measuring 1x1 with a total area
1-3% Acetic acid with - Glacial acetic acid | 2g of 10mm2, arranged 2 horizontal rows of 5 squares which are further
Gentian violet - Gentian violet | 1mL subdivided into 16 squares
- Distilled water - Depth: 0.2mm
1% HCl - Hydrochloric acid | 1mL
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 or Cells counted x Dilution Factor x 10
Area (mm2)
RBC count Millions/mm3= Cells Counted
Area x Depth x Dilution Factor
(1/5) (0.1) (1/200)
or Cells Counted x 10000
Improved Neubauer or Cells Counted x 10 x 200 x 5, where 10 is the depth correction factor
- Has 2 identically ruled platforms with a raised ridge on both sides of the 2 200 is the dilution factor
platforms on which a cover glass is placed 5 is the area correction factor
3
- The space between the top of the platform and the cover glass is 0.1mm WBC count/mm = = Cells Counted_________
Area x Depth x Dilution Factor
(4) (0.1) (1/20)
or Cells Counted x 50
Correction of WBC count for Nucleated Red Blood Cells (NRBC)
- These cells contain nucleus & can be mistaken for WBC since they are
not lysed by the diluting fluids
Corrected WBC count= Uncorrected WBC________ x 100
100 + # NRBC/100 WBC
- The number of NRBC per 100 WBC is obtained in the differential count

- The middle large square is divided into 25 tertiary/intermediate squares,

each of these are further subdivided into 16 smaller squares
- The middle large has a volume of 0.1µL
- The volume of each 15 squares is 0.004µL for a total of 0.02µL for 5 squares
Counting of Cells
✓ Cells that touch the top and left lines should be counted
✕ Cells that touch the bottom and right lines should be ignored
Computation:
Total cell count= Cells counted x Dilution Factor
Area (mm2) x Depth (0.1 mm)

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Navarro, Johanna A.