Midterm Topics Hema 1 Lec
Midterm Topics Hema 1 Lec
Platelets
Hematopoiesis I Page 1 of 4
POST NATAL DEVELOPMENT LIVER
Hematopoietic tissue is involved in the proliferation and Liver serves as the major site of blood cell production
maturation of blood cells during the second trimester of fetal development
Organs involved: Functions
o Bone marrow o Protein synthesis and degradation
o Liver o Coagulation factor synthesis carbohydrate
o Spleen and lipid metabolism
o Lymph nodes o Drug and toxin clearance
o Thymus o Iron recycling and storage
o Hemoglobin degradation
BONE MARROW Capable of extramedullary hematopoiesis
Major function is the proliferation and production of SPLEEN
blood cells
Largest lymphoid organ in the body
Two Major Components:
It is located directly beneath the diaphragm behind the
1. Red Marrow fundus of the stomach in the upper left quadrant of the
- Hematopoietically active marrow consisting of the abdomen
developing blood cells and their progenitors It is vital but not essential for life
- Seen in: flat bones of the skull, clavicle, sternum, ribs, Functions:
vertebrae, pelvis o Indiscriminate filter of the circulating blood
- 4 Major Functions: o Serves as a storage site for platelets
a. Production of blood cells 3 Regions
b. Destruction of erythrocytes o White Pulp: consists of scattered follicles
c. Iron storage from hemoglobin breakdown with germinal centers that contains
d. Central organ for B lymphocyte development lymphocytes, macrophages and dendritic
cells
2. Yellow Marrow o Red Pulp: vascular sinusoids and sinuses
- Abundant: 5 and 7 years of age, where they occupy separated
the spaces in the long bones previously dominated by o Marginal Zone: surrounds the white pulp and
our red marrow forms reticular measure containing blood
- Hematopoietically inactive marrow composed vessels, macrophages and specialized B cells
primarily of adipocytes and (fat cells), with 2 Methods for Removing Senescent or Abnormal
undifferentiated mesencyhmal cells and macrophages RBCs from Circulation
o Culling: involved action of phagocytosis.
HEMATOPOIETIC MICROENVIRONMENT Cells being phagocytized with subsequent
degradation of the cell organelles. Whole cells
It is responsible for supplying semifluid matrix
degraded. Removal of old or abnormal
(stroma) that serves as an anchor for the developing
RBCs that is within the cords of billroth
hematopoietic cells o Pitting: splenic macrophages removed
Composition of Stroma; inclusions or damaged surface membrane
1. Endothelial cells – regulate flow of particles from the circulating RBCs. Removal of the
entering and living in hematopoietic spaces unwanted intracellular elements (e.g.,
2. Adipocytes – secretes various steroids and Heinz bodies, Howell-jolly bodies,
influences erythropoiesis and maintains bone Pappenheimer bodies)
integrity. Regulates the volume of the marrow
3. Macrophages – phagocytosis. Secretes various LYMPH NODES
cytokines that regulate hematopoiesis
4. Osteoblasts – bone forming cells. Termed as Are members of lymphatic system located along the
“water bug” or “comet appearance” lymphatic capillaries
5. Osteoclasts – bone resorbing cells or destroying Bean shaped structures, 1-5mm diameter
cells 3 Functions:
6. Reticular cells (fibroblasts) – supports the vascular o Play a role in the formation of new
sinuses and developing hematopoietic cells lymphocytes from germinal centers
Composition of Extracellular Matrix of Bone o Involved in the processing of specific Ig
Marrow o Involved in the filtration of particulate
1. Collagen - matter, debris and bacteria entering the
2. Fibronectin lymph node via the lymph
3. Thrombospondin Regions;
4. Laminin o Cortex: outer region. Contains follicles of B
5. Proteoglycans – hyaluronate, chondroitin cells. Proliferation centers termed as
sulphate, heparan sulphate germinal centers
Hematopoiesis I Page 2 of 4
o Paracortex: in between the cortex and CONTROL OF HEMATOPOIESIS
medulla. Contains predominantly T cells and
numerous macrophages The entry of mature blood cells into the intravascular
o Medulla: inner region that consists of T space relies upon:
lymphcoytes and plasma cells o Multiplication of developing cells
o Gradual maturation
THYMUS o Orderly release of cell from bone marrow
STEM CELLS
Hematopoiesis I Page 3 of 4
Hematopoiesis I Page 4 of 4
HEM 311: CLINICAL HEMATOLOGY 1
RMT LECTURE 3: LINEAGE SPECIFIC HEMATOPOIESIS
Transcribed by: Surell, Riyoma
OUTLINE Cytoplasm
Lineage-Specific Hematopoiesis o Dark blue (concentration of ribosomes)
General Characteristic of Blast Location
Hematopoietic System o Present only in the bone marrow in healthy
o Erythropoiesis states
o Granulopoiesis Cellular Activity
o Monocyte Development o Begins to accumulate the components
o Lymphocyte Development
necessary for hemoglobin production
o Megakaryopoiesis
o The proteins and enzymes necessary for iron
uptake and protoporphyrin synthesis are
LINEAGE SPECIFIC HEMATOPOIESIS produced
o Lasts 24 hours
Principle of Normal Blood Cell Maturation
(Synchronistic Maturation) 2. Basophilic Normoblast (Prorubricyte)
o Cytoplasmic Changes Nucleus
o Cytoplasmic Granules o Chromatin begins to condense
o Nuclear Changes o N:C ratio decreases to about 6:1
o Reduction in Cell Size o The chromatin stains deep purple-red
Principle of Abnormal Cell Maturation o Nucleoli may be present early in the stage but
(Asynchronistic Maturation) disappear later
o Abnormal Cytoplasmic Differentiation Cytoplasm
o Abnormal Nuclear Maturation o When stained it is deeper, richer blue in color
o Abnormal Size than in the pronormoblast
Location
GENERAL CHARACTERISTIC OF BLAST
o Present only in the bone marrow in healthy
Size: large cell with high N:C ratio states
Cytoplasm: very dark blue and small in amount in Cellular Activity
comparison to the size of the nucleus. No granular is o Detectable hemoglobin synthesis occurs,
present but many cytoplasmic organelles, including
Nucleus: large in size as compared to the size of ribosomes and a substantial amount of
cytoplasm messenger ribonucleic acid (chiefly for
o Chromatin which is reddish purple and hemoglobin production only), completely
indicates predominance of DNA mask the minute amount of hemoglobin
pigmentation
HEMATOPOIETIC SYSTEM
3. Polychromatic (Polychromatophilic) Normoblast or
Erythropoiesis Rubricyte
Granulopoiesis Division
Monopoiesis o This is the last stage in which the cell is
Lymphoiesis capable of undergoing mitosis
Megakaryopoiesis Location
o Present only in the bone marrow in healthy
ERYTHROPOIESIS states
Cellular Activity
A process by which erythroid precursor cells
o Hemoglobin synthesis increases and the
differentiates to become mature
accumulation begins to be visible in the color
Primary Regulator: Erythropoietin
of the cytoplasm
Length of Time
1. Pronormoblast (Rubriblast)
o 30 hours
Nucleus
o Takes up much of the cell (N:C ratio of 8:1)
o Round to oval
o Contains 1 or 2 nucleoli
o Purple red chromatin is open and contains
few, fine clumps
Primary Granules
4. Neutrophil Metamyelocytes
o Charcot-Leyden Crystal Protein
Constitute 3% to 20% of nucleated marrow cells
Secondary Granules
The cells are no longer capable of division, and the
o Major basic protein (core)
major morphologic change is in the shape of the
o Eosinophil cationic protein (matrix)
nucleus
o Eosinophil-derived neurotoxin (matrix)
Nucleus
o Eosinophil peroxidase (matrix)
o Indented kidney bean or peanut shaped
o Lysozyme (matrix)
o Chromatin is increasingly clumped
o Catalase (core and matrix)
o Nucleoli: absent
o b-Glucoronidase (core and matrix)
o Cathepsin D (core and matrix)
5. Neutrophil Bands
o Interleukins 2,4 and 5 (core)
Make up 9% to 32% of nucleated marrow cells and
o Interleukin-6 (matrix)
0% to 5% of the nucleated peripheral blood cells
o Granulocyte-Macrophage Colony Stimualting
All evidence of RNA is absent Factor (core)
Tertiary granules continue to be formed during this Small Lysosomal Granules
stage o Acid phosphatase
Secretory granules (secretory vesicles) may begin to o Arylsulfatase B
be formed during this stage o Catalase
Nucleus o Cytochrome b558
o Highly clumped o Elastase
o Nuclear indentation that began in the o Eosinophil cationic protein
metamyelocyte stage now exceeds one half Lipid Bodies
the diameter of the nucleus o Cyclooxygenase
o 5-lipoxygenase
6. Segmented Neutrophils o 15-lipoxygenase
Make up 7% to 30% of nucleated cells in the bone o Leukotriene C4 synthase
marrow o Eosinophil peroxidase
Secretory granules continue to be formed during this o Esterase
stage Storage Vesicles
only morphologic difference between segmented o Carry proteins from secondary granules to be
neutrophils and bands is the presence of between 2 to released into the extracellular matrix
5 nuclear lobes that are connected by a thread-like
filaments BASOPHIL DEVELOPMENT
2. Prolymphocyte
Size: same with lymphoblast or smaller
Nucleus
o Round oval chromatin pattern is more
clumped
Cytoplasm
o Moderate to dark blue and usually non-
granular
MONOCYTE DEVELOPMENT N:C Ration: 4:1
MEGAKARYOPOIESIS
1. Megakaryoblast (Stage I)
Size: 14 to 18um in diameter
Cytoplasm
o Varying shades of blue, may have small blunt
pseudopods
o Narrow band around the nucleus
o As the cell matures the amount of cytoplasm
increases
Nucleus
o Round oval kidney shaped
N:C Ratio: 3:1
4. Platelets
Size: 2 to 4um in diameter
Erythrocytes I Page 2 of 4
HEXOSE MONOPHOSPHATE SHUNT LEUBERING-RAPOPORT PATHWAY
ERYTHROKINETICS
Erythrocytes I Page 3 of 4
Erythron o 10% of RBC undergo destruction
o Name given to the collection of all stages of intravascularly
erythrocytes throughout the body, developing
precursor in the bone marrow and the
circulating RBC in the peripheral blood
ERYTHROPOIETIN
Fragmentation Extravascular
o Loss of a portion of the erythrocytes o Lysis of erythrocytes outside the circulation
membrane Helps by reticuloendothelial system
o Accompanied by a loss of cellular contenets of the cell which are the liver and the
including Hgb spleen
Osmotic Lysis Usually happens through
o Passing of water into the red cell eventually phagocytosis and about 90% aged
burst the cell RBCs is destroyed extravascularly
Erythrophagocytosis o Little or no hemoglobin escapes into the
o Ingestion of the whole RBCs by a circulating circulation
monocytes or neutrophils or by macrophages o Decreased haptoglobin
(mononuclear phagocyte system) o Normal plasma hemoglobin
o Causes:
Complement Induced Cytolysis
Bacterial/ viral infections
o Complement attach itself to the cells and
Drug induced
induce lysis
Autoimmune
Hemoglobin Denaturation
Microangiopathy
o When Hgb is exposed to oxidant stress
Hemoglobinopathies
later on denature the Hgb
Membrane defects
o Happens when mechanism to protect the cell
Metabolic defects – G6PD
after exposure to stress fails to work and
deficiency
denatured Hgb later on precipitates forming
inclusion bodies (Heinz Bodies)
2 TYPES OF DESTRUCTION
Intravascular
o Usual outcome of sensitization of
erythrocytes with complement
o Lysis of erythrocytes which occurs within the
circulation through the classic pathway
Erythrocytes I Page 4 of 4
HEM 311: CLINICAL HEMATOLOGY 1
RMT LECTURE 5: HEMOGLOBIN
Transcribed by: Surell, Riyoma
OUTLINE
Lineage-Specific Hematopoiesis
General Characteristic of Blast
Hematopoietic System
o Erythropoiesis
o Granulopoiesis
o Monocyte Development
o Lymphocyte Development
o Megakaryopoiesis
HEMOGLOBIN
COMPONENTS
Globin
o Occurs in ribosomes
o Varied sequence of amino acids
o Difference in globin chains designation
relates both to the sequence and number of
amino acids
HgbA 4 heme groups and 4 polypeptide chains (574: total
Symbol Name # Amino Acids amino acid of the entire chain)
α Alpha 141
β Beta 146 Heme Production
γA Gamma A 146 (position 136: alanine) o Requires the formation of protoporphyrin IX
γG Gamma B 146 (position 136: glycine) and the availability of iron
𝛿 Delta 146 o The synthesis of heme begins in the
ε Epsilon 146 mitochondria with the formation of D-ALA
ζ Zeta 141 (Delta Aminolevunilic acid) from glycine and
θ Theta Unknown succinyl coenzyme A
Hemoglobin I Page 1 of 4
o Immature Red Cell with Ferritin:
sideroblasts
o Mature Red Cell: siderocytes
o Stain with: Prussian blue
Iron
o Most abundant transition metal in the body
o Iron uptake = controlled maintain iron
balance
E.g., Duodenum dietary 3 iron is
reduced to ferrous iron and taken up
from the intestinal lumen into the Normal Hemoglobin Variants
enterocytes. DMT1 (a protein called
divalent metal transporter 1
primarily responsible for transporting
ferrous iron to the enterocytes).
DMT1 is instrumental in the uptake
of iron stored in ferritin
(enterocytes) or exported into the
circulation by another transport
protein which is Ferroportin
o In the ferrous form is required to convert
protoporphyrin to become heme
o Doesn’t have mechanism of active excretion
o Body adjust iron levels by intestinal
absorption depending on our body needs
o Sources: dietary, recycled from senescent
erythrocytes
o Storage: ferritin and hemosiderin
o Transport: transferrin and ferroportin
Hemoglonin I Page 2 of 4
NORMAL HEMOGLOBIN FORMS
Oxyhemoglobin
o Hgb with ferrous iron and oxygen which is
seen in arterial circulation
Deoxyhemoglobin
o Hemoglobin with ferrous iron but no oxygen
which is seen in venous circulation
Regulator: phosphate regulating oxygen affinity
2,3-DPG (diphosphoglycerate) combines with the
beta chains of deoxyhemoglobin
Heme Regulation
o HEME: inhibits transcription of the ALA
synthase gene, ALA dehydrase and PBG
deaminase (negative feedback mechanism
stops the synthesis)
Globin Regulation
o Highly regulated balance of heme and
globin HEMOGLOBIN DERIVATIVES
o Controlled at the transcription level
Initiators: Dyshemoglobins dysfunctional no oxygen
Promoter DNA sequence transport formed and may accumulate to toxic levels
Key transcription factor (KLF-1 or after exposure to certain drugs, chemicals or gases
Kruppel-like factor 1) o Most are acquired
Other transcription factors o Methemoglobinemia: small fraction are
(GATA-1, Ikaros, TAL-1, p45-NF- hereditary
E2 and LDB1) Carboxyhemoglobin
Enhancer region of DNAse 1 o Carbon monoxide will bind with oxygen (240
hypersensitive nucleic acid times)
sequences (Locus Control o Shift to the left
Region) o Cannot bind and carry oxygen
o KLF1, GATA1, Ikaros, TAL1, p45-NF-E2, and o Gasoline motors, gas heaters, defective
LDB1 stoves, smoking
o Locus Control Region o It is also known as “silent killer” because it is
odorless and colorless
OXYHEMOGLOBIN DISSOCIATION CURVE o Patient may quickly become hypoxic
Methemoglobin
Readily bind oxygen molecules to the lung o Reversible oxidation of heme iron to ferric
Efficiently unload oxygen to the tissues (requires low state
oxygen affinity) o Decreased delivery of oxygen to tissue
During oxygenation each of the 4 heme iron atoms can o Levels of Methemoglobin:
reversibly bind oxygen (1.35mL of O2 = 1g of Hgb) <25% asymptomatic
Hemoglobin has the ability to bind large quantities of >30% cyanosis (bluish discoloration
O2, however, hemoglobin must be willing to release O2 of the skin and mucous membranes)
when needed and symptoms of hypoxia (dyspnea,
Shift to the left: lesser oxygen being released to the headache, vertigo and change in
tissue mental health status)
Shift to the right: oxygen given more to the tissues >50% coma and death
o Derivative of hemoglobin in which the ferrous
Shift to the Left Shift to the Right ions is oxidized to ferric state
DECREASED: INCREASED: o Chocolate brown discoloration of blood,
pCO2 pCO2
cyanosis and functional anemia
Hydrogen Ions Hydrogen Ions
2,3-DPG 2,3-DPG Sulfhemoglobin
Temperature Temperature o Mainly caused by:
INCREASED: DECREASED: drugs (sulfanilamides, phenacitin,
pH (alkalosis) pH (acidosis) nitrites and phenyl hydrogen)
Presence of Hgb F/HbF exposure to chemicals (industrial
**remember yung kwento ng pangangaliwa and environmental setting)
exposure to sulfur
Hemoglonin I Page 3 of 4
o Addition of sulfur atom to pyrrote ring of heme
(It has GREENISH pigment)
o Ineffective for oxygen transport
o Patients with elevated sulfhemoglobin may
exhibit cyanosis
o Mixture of oxidized, partially denatured forms
of hemoglobin that form during oxidative
hemolysis
o Associated with sufonamides administration,
severe constipation, Clostridium perfringens,
enterogenous cyanosis
o Cannot be reduced back to hemoglobin
Hemoglonin I Page 4 of 4
HEM 311: CLINICAL HEMATOLOGY 1
RMT LECTURE 6: RED BLOOD CELL ABNORMALITIES
Transcribed by: Surell, Riyoma
Other Poikilocytes
o Blister Cells
Red cell with single or multiple
POIKILOCYTES SECONDARY TO TRAUMA vacuoles or markedly thinned areas
at the periphery
Schistocytes
Characteristic of Heinz body
o Also known as Schizocyte, Fragmentocyte
mediated hemolysis
and “egg shell”
Appear to have a partially raised or
o Fragmentation produced by damage of RBC
blistered membrane
by fibrin, altered vessel walls, prosthetic heart
valves
o Schistocyte with Horn-like projections
Keratocytes/ Helmet cells
o Conditions Associated:
Disseminated intravascular
coagulation (DIC)
Thrombotic thrombocytopenic
purpura (TTP)
Burns
Microangiopathic haemolytic Degmacyte (Bite Cell)
anemia o Erythrocytes with irregular membrane results
from splenic macrophage mediated removal
of denatured hemoglobin molecule
o Drug-induced anemias
o G6PD Deficiency
o Thalassemia
o Unstable
o Hemoglobinopathies
Dacryocyte
o Also known as teardrop cells
o Squeezing and fragmentation during splenic
passage (one side is pointed resembling pear
shape)
o Associated Conditions:
Myeloid dysplasia
Hypersplenism
Thalassemia
Particular Variation:
Occasional: <1%
Few: 1 to 5%
Frequent: 5 to 10%
Many: >10%
INCLUSIONS
Heinz Bodies
Howell-Jolly Bodies
o Deep purple, irregularly shaped inclusions
o Dark blue purple, dense and round granule
o Requirement:
o 1% only
Supravital stains (crystal violet
o Associated Conditions:
dark or deep purple inclusions that
Megaloblastic anemia
are attached to inner RBC
Severe haemolytic process
membrane)
Thalassemia
o Associated Conditions:
Accelerated erythropoiesis
Defects in HMP
o Composition:
G6PD Deficiency
DNA (nuclear fragments)
Unstable Hb
Splenectomy
Thalassemia
o Composition:
Precipitated denatured hemoglobin
(can formed in patients with G6PD
deficiency)
Basophilic Stipplings
o Dark blue granules
o Blueberry bagel
o Associated Conditions:
Lead poisoning
Pyrimidine-5-nucleotidase
deficiency
Heavy metal poisoning
Hb H Inclusions
Thalassemia
o Inclusions represent precipitated Hb H
o Composition:
o Uses supravital stain (brilliant cresyl blue)
Precipitated RNA
o Hb H Disease:
Alpha thalassemia with moderate
hemolytic anemia
o Composition:
Precipitated B-globin chains of
hemoglobin
Cabot Ring
o Rings, loops, figure of eight, red to purple
(some are blue)
o Bell or tall hat shape on scanning EM
o Associated Conditions:
Megaloblastic anemia
Severe anemias
o Composition:
Mitotic spindle remnants
Malaria
o Protozoan transmitted by
mosquitoes
o Maturation Stages:
Rings
Trophozoites
Schizonts
Hb SC Crystals Gametocytes
o Finger-like, quartz-like crystal of dense
hemoglobin protruding from the RBC
membrane
o Dark-hued crystals of condensed Hb distort Babesia
the red cell membrane o Protozoan inclusion, tick
o Crystalline projection is often straight with bite
parallel sides and one blunt, pointed, o “Maltese cross” formation
protruding end
Others
o Agglutination:
Clumping of red cells
Red cell aggregation
o Rouleaux:
“Stack of coins” pattern of red cells
Increase plasma globulin
Ringed Sideroblast
o Nucleated RBC that contains non-heme iron
particles
o Excessive iron overload in mitochondria of
normoblasts
o Associated Conditions:
Sideroblastic anemia
MDS (Myelodysplastic Syndrome)
RBC MORPHOLOGY GRADING
MORPHOLOGY GRADE
Polychromatophilia 1+ = 1 to 5/field
Helmet cell, dacrocyte 2+ = 6 to 10/field
Spherocyte, acanthocyte, schistocyte 3+ = >10/field
Poikilocytosis 1+ = 3 to 10/field
Codocyte, Burr Cells 2+ = 11 to 20/field
Stomatocyte, Ovalocyte, Elliptocyte 3+ = >20/field
Rouleaux 1+ = 3 to 4 agg.
Pappenheimer Bodies
2+ = 5 to 10 agg.
o Dark staining iron granules that usually 3+ = numerous
clumped together at periphery of the cell agg.
o Associated Conditions: Sickle cells POSITIVE ONLY
Sideroblastic anemia Basophilic stippling
MDS Pappenheimer bodies
Thalassemia Howell-Jolly
Haemolytic anemia
Defective erythropoiesis
o Composition:
Iron
AUER RODS
CYTOPLASMIC ABNORMALITIES
CHEDIAK-HIGASHI GRANULES
ALDER-REILLY GRANULES
Giant red, blue to grayish round inclusions
Rare inherited disorder characterized by granulocytes These bodies are formed by aggregation and fusion
that are large, darkly staining cytoplasmic granules of the primary and secondary specific granules
Large purple-black coarse cytoplasmic granules Complications:
o Reilly Bodies o Neutropenia
Accumulation of degraded mucopolysaccharides o Thrombocytopenia
The primary defect is in special granules present in
skin pigment cells and certain white blood cells
Chediak-Higashi Syndrome is caused by mutations
in the LYST gene (specifically CHS I LYST
Lysosomal Trafficking Regulator Gene located in
chromosome 1q42.1 – 2 encodes for a protein that
regulates the morphology and function of the lysosome
related organelles)
o Rare autosomal recessive disease of immune
dysregulation
White Blood Cells Abnormalities I Page 2 of 5
TOXIC GRANULES
MAY-HEGGLIN ANOMALY
Early classification system of certain forms of variant Mononuclear cell with cerebriform (grooved like or
lymphocytes folded convoluted pattern, surrounds thin rim of
Type I cytoplasm) nucleus
o Turk’s irritation cell Cutaneous T cell lymphomas
o With block of chromatin o Leukemic phase is the Sezary syndrome
o Small cells with minimum cytoplasm Round lymph cell with nucleus that is grooved or
o Nucleus: indented convoluted
Type II Sezary Syndrome
o IM cells Infectious Mononucleosis Cells o Leukemic phase of T cell lymphomas
o Most common type o Characterized by exfoliative erythroderma,
o Large cells with abundant cytoplasm peripheral lymphadenopathy
o Round mass of chromatin o Can be seen in skin, lymph nodes and
o Ballerina skirt appearance peripheral blood
Type III o Mycosis fungoides
o Vacuolated
o Large moderated basophilic cytoplasm
o Nucleoli: apparent
o Swiss chief or moth eaten appearance
Natural artifacts
Represents the bare nuclei of leukocytes
Degenerated nucleus
Pressure in making smear
Increased number = Chronic Lymphoblastic
Leukemia (CLL)
GRAPE CELL
OUTLINE Hemacytometer
Complete Blood Count
o Manual Cell Count Also known as counting chamber
Differential Count Improved Neubauer/ Levy Chamber
o Absolute Count
o Rule of Three
Red Cell Indices
o MCV
o MCH
o MCHC
Red Cell Distribution Width
Reticulocyte Count
o Miller Disc
o Absolute Reticulocyte Count
o Corrected Reticulocyte Count
o Reticulocyte Production Index
o Automated Reticulocyte Count
Erythrocyte Sedimentation Rate (ESR)
Osmotic Fragility Test (OFT)
Point of Care Test
Automation
o Electronic Impedance Table 1: Manual Cell Counts with Most Common Dilutions,
o Optical Scatter Counting Areas
o Radiofrequency
Cells Diluting Fluid Dilutio Objecti Area
o CBC Parameters and Quality Control Counted n ve Counted
WBC 1% ammonium oxalate or 120 10x 4mm2
3% acetic acid or 1:100 10x 9mm2
COMPLETE BLOOD COUNT 1% hydrochloric acid
RBC Isotonic Saline 1:100 40x 0.2mm2 (5
small
Cell Counts squares of
o RBC Count center
o Platelet Count different request from the square)
Platelets 1% ammonium oxalate 1:100 40x 1mm2
physician, not included in CBC phase
o WBC Count **hayems fluid RBC much better result than isotonic saline
o Differential Count because it can cause formation of rouleaux
Hemoglobin
Hematocrit Calculations
Blood Cell Indices (MCV, MCH, MCHC)
The general formula for manual cell counts is as follows and can
MANUAL CELL COUNT be used to calculate any type of cell count: (1 chamber only
proceed directly to the computation but if 2 chamber counted
Red Blood Cell Count get the average and proceed to the formula below)
Platelet Count
𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
White Blood Cell Count Total Count =
𝑎𝑟𝑒𝑎 (𝑚𝑚2)𝑥 𝑑𝑒𝑝𝑡ℎ (0.1)
One hundred WBCs are counted and classified Blood is diluted using Drabkin Solution (potassium
through the use of push-down button counters ferricyanide, potassium cyanide, sodium bicarbonate,
Results are reported as percentages surfactant)
Principle: a stained smear is examined to determine Hemoglobin (Fe2+) + K3 Fe (CN)6 methemoglobin (Fe3+) +
the percentage of each type of leukocyte to present KCN cyanmethemoglobin
and assess the erythrocyte and platelet
morphology 1. Hemoglobin first oxidized to methemoglobin by
WBC Abnormalities are also reported potassium ferricyanide
o Reporting: 2. Converts methemoglobin to cyanmethemoglobin
Reactive Lymphocytes (measured by spectrophotometer)
Separate % as a % of total 3. Absorbance of cyanmethemoglobin will be read at
lymphocytes 540nm directly proportional to hemoglobin
Semi-quantitatively: concentration
occasional to many
Toxic Granulation Hematocrit
Present “packed cell volume”
Semi-quantitatively: Volume of packed RBCs that
slight to marked/ 1+ to 4+ occupies a given volume of
Specimen whole blood
o Peripheral blood Reported: percentage (%) or
o Bone marrow L/L
o Body fluid sediments Reference Range:
o Female: 36 to 45%
o Male: 41.5 to 50.4%
Applicable if red cells are normocytic-normochromic Table 2: Red Blood Cell Indices, Red Blood Cell Morphology,
The value of the haematocrit should be three times the and Disease States
value of the hemoglobin plus or minus 3
MCV MCHC RBC Found in
Hgb =12g/dL (fL) (g/dL) Morphology
<80 <32 Microcytic, IDA, Anemia of
HCT = 36% (0.36L/L) Hypochromic inflammation, thalassemia
Hb E disease and trait,
sideroblastic anemia
According to the rule of three,
80 to 32 to 36 Normocytic, haemolytic anemia,
100 Normochromic myelophthisic anemia, bone
Hgb (12) x 3 = HCT (36)
marrow failure, chronic renal
disease
An acceptable range for the haematocrit
>100 32 to 36 Macrocytic, Megaloblastic anemia,
would be 33% to 39%. These values conform Normochromic chronic liver disease, bone
to the rule of three marrow failure,
myelodysplastic syndrome
RED CELL INDICES
Used to define the size and hemoglobin content of the RED CELL DISTRIBUTION WIDTH
red blood cell
Aids in diagnosing and differentiating anemia It reflects the degree of red cell variation in size
o MCV mean cell volume Formula:
o MCH mean cell hemoglobin 𝑆𝐷
o MCHC mean cell hemoglobin RDW= 𝑥 100
𝑀𝑒𝑎𝑛 𝑆𝑖𝑧𝑒
concentration
Normal Value: 11.5 to 14.5%
MEAN CELL VOLUME (MCV) Increased RDW anisocytosis
Microcytic/ Macrocytic normal RDW + dec or inc
Average volume of red blood cells expressed in MCV
femtoliters (fL) Anisocyte wih size within reference range = increase
Formula: RDW + Normal MCV
𝐻𝐶𝑇 (%) 𝑥 10 Anisocyte with size below or above the normal range =
MCV = abnormal MCV and RDW
𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 (𝑥1012 /𝐿)
Designated to reduce the labor – intensive process of RETICULOCYTE PRODUCTION INDEX (RPI)
counting reticulocytes
Measures erythropoietic activity when stress
Disc is inserted into the eyepiece
reticulocytes are present
of the microscope
Shift Reticulocytes
Minimum of 112 cells should be
o Prematurely released reticulocytes from the
counted in the small square
marrow
o Smaller Square (B):
o Shifted from the bone marrow to the
1/9 of the larger square
peripheral blood
o 112 cells = 1,008 red
Acute bleeding (low levels of RBC
cells
count)
o Larger Square (A): reticulocytes
Anemia
Formula:
o Instead of 1 day 2 to 3 days in the
Reticulocytes % =
𝑛𝑢𝑚𝑏𝑒𝑟 𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 𝑖𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 (𝑙𝑎𝑟𝑔𝑒 𝑠𝑞𝑢𝑎𝑟𝑒)𝑥 100
peripheral blood
𝑛𝑢𝑚𝑏𝑒𝑟 𝑅𝐵𝐶𝑠 𝑖𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 𝐵 (𝑠𝑚𝑎𝑙𝑙 𝑠𝑞𝑢𝑎𝑟𝑒) 𝑥 9 o Larger and bluish color
CRC =
0.30 𝐿/𝐿
0.3 x = 0.02 = 2%
0.45 𝐿/𝐿 (𝑎𝑑𝑢𝑙𝑡 𝑚𝑎𝑙𝑒 𝑛𝑜𝑟𝑚𝑎𝑙 𝑣𝑎𝑙𝑢𝑒)