HEMATOLOGY: PRELIMINARIES
Romie Solacito, MLS3C
BLOOD CHARACTERISTICS
 Red in color; due to haemoglobin
 Think and viscous; due to plasma proteins: a.
albumin, b. globulin (antibodies), c. fibrinogen
 Fluid in vivo; due to heparin (from basophil origin
from liver).
 Blood clot; due to the presence of coagulation
factors (platelet and fibrinogen), and absence of
heparin.
 Salty; due to electrolytes: Sodium, Potassium,
Magnesium, HCO3, Calcium, Chloride.
 Metallic due to the presence of Fe (Iron) in the
haemoglobin use for binding oxygen.
BLOOD AN OVERVIEW
 Fluid state in vivo
 If blood is removed from the circulatory system, it
will clot
 Red in color
 pH: 7.35-7.45; due to buffers
 Specific gravity: 1.055 (1.045 – 1.065) due to cells,
and electrolytes.
 Thick and viscous: 3.5-4.5 thicker than the water
 HCO3 (bicarbonate) – basic & H2CO3/CO2 – acidic.
Ratio 20:1
Composition of Blood
 Centrifuged blood layers:
1. Fats
2. Plasma
3. Buffy Coat – WBC and Platelet
4. Red Blood Cells
 Liquid portion = 55%
o 55% of the White Blood Cells
o Composition:
 90% Water
 10% Electrolytes: Proteins, Carbohydrates,
and Lipids
o Plasma – transluscent, yellowish, somewhat
viscous; liquid portion unclotted blood contain
fibrinogen: carbohydrates, hormones, and
enzymes
o Serum - liquid portion of the clotted blood;
devoid of fibrinogen; lacks Coagulation Factor I
(Fibrinogen), II (Thrombin “heparin inhibits”), V,
& VIII
 Formed element = 45%
o Solid portion 42-47%
o Can be obtained only from Whole Blood
o Made up of formed elements/Hemocytes:
1. Red Blood Cells: anucleated packed with
oxygen carrying protein; under normal
conditions they never leave the circulation.
2. White Blood Cells: Chief defense against
infection; migrates to tissue and display their
function
3. Platelets: anucleated disk-like fragments;
participating in vessel healing when there is
an injury.
FUNCTION OF BLOOD
 Carrier: of gases, nutrients, hormones, and waste
products
 Regulator: of temperature, Acid-Base Balance, and
water content
 Defense
 Coagulation
Total Blood – Sum of the Red Blood Cells and White
Blood Cells
 Hypervolemia – increase blood volume
o Loss of Whole Blood
o Loss of Red Blood Cells
o Loss of Plasma
o Loss of Blood Fluid or Water
o Surgical shock
o Nephrotic Syndrome
 Hypovolemia – decrease blood volume
o Increase in take of fluid
o IV Fluid Injection
o Blood Transfusion
o Pregnancy – caused by hormonal changes;
Ecclamsia – high blood pressure of pregnant
women.
Mechanism for maintaining blood volume
 Capillary Fluid Shift Mechanism – inward and
outward movement of fluids inside the blood vessels
 o Nephrotic Syndrome (outward) – podocytes is  Sample misidentification
destroyed and RBC and Albumin will be in urine  Improper timing
which the water will go to the tissues that leads  Improper fasting
to Edema.  Meal within 2 hours
o Dehydration (inward) – epithelial cells in the  Smoking
blood will become more permeable to fluids, so  Physical activity within 20mins
that fluids in the tissue will go inside the blood  Stress
vessels 2. COLLECTION:
 Kidney Regulation Mechanism  Considerations in the Collection:
o Gives hormone – erythropoietin, acts as the  Consider fasting time for fasting sample
signal for RBC production in the bone marrow.  Collection should be done at exact time for
o RAAS – Three Outputs, to lessen the fluid loss timed collection (serial collection)
1. Constriction of tubules  Proper disinfection
2. Aldosterone – retain sodium  Venous blood should be used (except for blood
3. ADH/Anti-Diuretic Hormone – gas sample)
water retention  Follow standardized procedures to avoid
SPECIMEN COLLECTION: Safety in Hematology misleading results
 Provision for healthcare worker:  Always remember that sample are potentially
o Universal Precaution – only blood consider infectious.
infectious  Important things to do:
o Body Standard Isolation – all body fluids consider  Method to use: apparatus needed and
infectious but no hand washing sequence of draw
o Standard Precaution – all body fluids consider  Common errors:
infectious  Excessive negative pressure when drawing
 Proper disposal of waste: blood into the syringe
o Yellow – infectious  Short draw or wrong anticoagulant to blood
o Green – wet non-infectious ratio
o Black – dry non-infectious  Mixing problems/clot
o Orange – Radioactive  Wrong tubes/wrong anticoagulant
o Red – Sharps  Hemolysis/lypemic
 Sodium hypochlorite – use for disinfection (10% of  Diurnal variance
100 Water)  Posture
PHASES OF BLOOD COLLECTION:  Hemoconcentration
1. PRE-COLLECTION:  Sample used
 Main goal: to preserve the integrity of the 3. POST-COLLECTION:
specimen; to make considerations for various  Main goal: Proper patient care; Proper specimen
patients handling; Proper labelling
 Considerations in the Pre-collection:  Common errors:
 Stress - increase leukocytes  Delayed delivery to the laboratory
 Exercise – activates coagulation factors;  Processing errors; incomplete centrifugation
Increase platelets and leukocytes METHODS OF COLLECTION
 Smoking – increase leukocytes 1. SKIN PUNCTURE: Application of micro collection
 Meal within 2 hours – increases certain blood  Used for: new-borns, pediatrics (<6 months),
analytes patients with poor veins (extreme obesity,
 Common errors: severe burns, and geriatric patients, and patients
 Misidentification of patient with thrombotic tendencies)
 Capillary Blood – mixture of venous blood, b. Capillary tube(7.5cm/75mm):
arterial blood, and tissue fluid; generate slightly i. Caraway Pipet
different test result, specimens should be noted ii. Capillary Tube – bone (1mm-1.2mm),
as obtained by sin puncture. with anticoagulant heparin (red),
Parameters Comparison to Venous Blood without anticoagulant (blue), space
pH cell volume Slightly higher between blood and sealing clay
RBC Count Slightly higher (>5mm), depth of sealing clay (4-6mm).
Hemoglobin Slightly higher iii. Sarstedt Capillary - blood collection
WBC Count 15-20% higher system
platelet Count Lesser c. Cotton pods
 Collection Sites: Plantar heel, Earlobe (least site), d. 70% Isopropyl Alcohol
and Bog toe. e. Povidone Iodine (Betadine) – increase PUP
o NOTE: infant’s finger should not be (Potassium, Uric Acid, and Phosphorous)
punctured so as to avoid serious injury to the  Microsampling Equipments
bones o Microcontainters
o 1.5 to 2.4mm – skin and bone o Unopette
 Advantages of Skin Puncture: o Becton Dickinson
o Easily accessible to the MedTech  Puncturing Techniques
o Less intimidating o Warm the area before puncture
o Easy to manipulate o <42C for 2 to 5 minutes
o Ideal for peripheral blood smear o To increase blood flow
 Advantages of Earlobe Skin Puncture: o Depth of Incision
o Less painful a. Adult: <2.4mm (quick and firm)
o Less tissue juice contamination b. Child: not deeper than 1.6mm
o More free blood flow  No-No Puncturing Techniques
 Disadvantages of Skin Puncture: o Double Pricking with the same Lancet
o Less amount of blood can be obtained o Lock and Key
o Blood hemolysed easily 2. Phlebotomy – a process by which blood is obtained
o Additional and repeated tests cannot be from the vein; venous blood – darker
done  Factors we have to concider
 st
1 drop wipe: to remove alcohol, and tissue juice i. Phlebotomist
contamination ii. Patient and his/her veins
 No-No Areas: iii. Equipment Needed
o Cold and cyanotic areas  Different Methods
o Inflamed areas o Syringe Method – plunger, barrel, hub,
o Congested and edematous area needle, bevel
o Heavily callous area o Evacuated Method – evacuated tubes, multi
 Equipment to use: sample needle/two-way needle, needle
a. Puncturing device holder/adaptor
i. Needle – glover’s needle, 3 cornered o Butterfly Method – newborn, needle is
(short and stout needle) and Hagedorn introduced into vein.
needle (large needle with a cord at the  Vein Selection: Median, Cephalic, and Basilic
other end)  Alternative Vein Sites: Dorsal Veins, Wrist, Foot
ii. Scalpel – scalpel blade, and band parker  Site to Avoid:
blade 1 o Hematoma
iii. Lancet – 1.75mm o Burns
 o Scar Edema
o Where mastectomy was Perform
o Arm receiving an intravenous in fusion
 Stop the IV for 2 minutes
 Puncture blood below the IV line
 Discard 5mL of the first blood extract
 Equipment Needed
i. Tourniquet, should be applied 2 to 4 inches
above the puncture site; not longer than 1
minute (Seraket, Rubber, Velcro)
ii. Evacuated Tube, can be plastic or glass; with a
variety of premeasured additives; follow
proper order of draw
iii. Needles – length (1 to 1.5 inches); evacuated
system two way needle; gauges (18 to 25)
a. Pink – 18
b. Green – 21 (Adult)
c. Black – 22
d. Blue/Light Blue – 23
e. White – 16
f. For Children – 23 to 25
iv. Needle Holder/Adapter
v. Winged Infusion Sets/Butterfly – IV device
with short needle and a thin tibe attached to
plastic wings
vi. Disinfectant – 70%alcohol; alcohol testing –
Benzalfoniu Chloride (Zephiran); avoid provide
iodine for (PUP)
 Special Consideration for Coagulated Tubes
o Tubes must be filled correctly – blood to
anticoagulant ratio 9:1
o Consideration when using butterfly
 Other consideration:
o Obesity – blood pressure cuff can aid in
locating the vein; should not be inflated
above the diastolic pressure of the
patient; never do blind shooting.
o IV Theraphy
o Mastectomy Patient
 Complication
o Local Immediate – Hemoconcentration;
circulatory failure; syncope fainting
o Local Delayed – Hematoma; thrombosis;
thrombophlebitis
o Late General – Serum Hepatitis, AIDS
3. Arterial Puncture – process by where blood is obtain
from a patient’s arterial vein; to obtain blood gases
 Preferred Sites: Radial Artery, Brachial Artery,
Scalp Artery, Umbilical Artery, Femoral Artery
 Consideration
i. Cellulitis or other infection of
the radial
ii. Coagulation defect
iii. Absence of palpable radial
artery pulse
 Specimen Handling
o Proper Inversion – NO Shaking
o Transport in an upright position
o 45 minutes to 1 hour after collection –
separate within 2 hours
o Exposure to light – decrease: bilirubin,
carotene, RBC Folate, Urinary prophyrin
o Chilled Specimen – best for arterial blood
gas, ammonia, Lactic Acid, and certain
coagulation test
o Warm Specimen – best for Agguntinin Test
(Antibody) IgG
ANTICOAGULANT
 To prevent the clotting process by interfering in the
coagulation cascade
 To preserve certain analytes and cell morphology
prior to testing.
Types of Anticoagulant – “You Better Remember Girls
Love Gray”
 Yellow – SPS (Sodium Polyanethol Sulfunate); inhibit
cell lysis (complement pathway and phagocytosis)
 Blue – Sodium Citrate
 Red – No additives; for Clinical Chemistry
 Green – Heparin; for electrolytes and arterial blood
gases
 Lavender – EDTA; anticoagulant
 Gray – Sodium fluoride; inhibits glycolysis
Lavender Top Tube – EDTA
 Optimal concentration in the blood: 1.5mg/mL of
blood
 Mode of Action: removes ionized calcium through
the process of chelation.
 Calcium is actually a cofactor for other factors to
complete the coagulation process
 Anticoagulant of choice for Hematology because:
o Preserves cellular morphology
o Excellent for cell counting
o Blood is stable for 2 to 3 hours before smearing
o Dipotassium for 6 hours - cell swelling
o Tripotassium for 3 hours – cell shrinking
 Disadvantage: cause cell shrinkage in excess.
 Two forms:
o K2EDTA – Spray-coated; plastic tubes; will not
dilute the sample
o K3EDTA – liquid form; glass tubes; dilutes the
sample 1 to 2 %
Pink Top Tube – Spray-coated K2EDTA
 Used in blood banking and blood typing; Rh typing
and antibody screening
White Top Tube – EDTA with gel
 Used more often for molecular diagnostic testing.
Blue – Citrate
 Mode of Action: precipitates calcium into an
unusable form/non soluble complex; non-ionized
form
 Anticoagulant of choices for coagulation studies –
preserves the labile factory V and VIII better.
 Two Forms:
o Blue top – 0.105 or 3.2%; most commonly used;
blood to anticoagulant ratio – 9:1.
o Black top – 0.129 or 3.8%; buffered sodium
citrate; blood to anticoagulant ratio – 4:1; use for
Erythrocyte Sedimentation Rate using
Westergren Method.
Green Top Tube – Heparin
 Optimal concentration 15-20u/mL of blood
 Mode of Action: accelerating the action of anti-
thrombin III, neutralizing thrombin and preventing
the formation of fibrin.
 Isolated from liver cells and known to be the
naturally occurring anticoagulant
 Two Forms:
o Lithium Heparin – used for most chemistry test
except lithium and folate levels; for lithium
testing – royal blue sodium heparin can be used
instead.
o Sodium Heparin – Injectable form used for
anticoagulant therapy; recommended for trace
elements, lead and toxicology.
 Concentration – 0.2mg/mL of blood
 Anticoagulant of choice for: (1) blood gas analysis –
0.05mL/mL of blood (2) osmotic fragility testing (3)
Trace elements and toxicology.
 Does not affect levels of calcium
 preferred for potassium measurement
 Gives a blue background with Wright’s stain after 2
hours
Gray Top Tube – Fluoride
 Contains: (1) Sodium Fluoride – preserves glucose for
3 days. (2) Lithium Iodoacetate – preserves glucose
for 24hours.
 Mode of Action: forms weakly associated Calcium
components
 Two Forms:
o Sodium Fluoride
o Potassium Fluoride
 Prevents glycolysis because of fluoride forms and
ionic complex with magnesium, there by inhibiting
the Mg++ dependent enzyme enolase.
Oxalate
 Mode of Action: combines with calcium to
form an insoluble salt/complex
 Three Forms:
o Potassium Oxalate
o Ammonium Oxalate
o Double Oxalate – Paul Hellers
 Disadvantage: (1) distort cell morphology
(2) Potassium oxalate shrink RBC (3)
Ammonium oxalate swells RBC
Other Tubes
 Red/Gray or Gold Top Tube – generally called the
SST (Serum Separation Tubes) because they
contain clot activator and separation gel.
 Mostly used for chemistry test except for
therapeutic monitoring, blood dark and
immunologic reaction.
 Clotting Time:
o With Gel Separator: 30min
o With Clot Activator: 5min
o Plain Tubes: 60min
HEMATOPOEISIS 
Site of development: liver (up to 1 to 2 weeks
 Continuous, regulated process of blood cell after birth)
production that includes  Five to Seven Weeks
o Cell Renewal  PEAK of HEPATIC PHASE is 3rd month of fetal
o Proliferation life, declines after the sixth month
o Differentiation  Produces Developing Erythroblasts – cells
o Maturation more definitive and identifiable
 Types of Hematopoesis  Aside from liver, other organs that
o Synchronous (the nucleus and the cytoplasm contribute in blood cell production: Liver +
develops together at the same rate) vs. developing spleen, kidney, thymus and
Asynchronous (the development of the nucleus lymph nodes
and cytoplasm is not synchronized. i.e.  Characterized by recognizing cluster of
Megaloblastic Anemia – macrocytic; Iron developing erythroblast, granulocytes and
Deficiency Anemia - microcytic) monocytes.
o Medullary (blood production occurs in the inner  Intravascular Alpha & Gamma
part of the bone marrow) vs. Extramedullary  Development of Erythroblast that produces
(blood production occurs outside the bone haemoglobin F, A1, and A2
marrow)  Thymus - Thymus: first fully developed
 Theories on Blood Formation organ (T cell production)
o Monophyletic Theory/Unitarian Theory - “One  Beginning of Megakaryocyte production
parent cell (Hemocytoblast) – give rise to all Type of Cell
blood cell types” Produced
o Polyphyletic Theory/ Dualistic Theory - “Two Developing Lymphoid cell (B 4th month
precursors (Myeloid&Lymphoid) – give rise to all kidney & Spleen cell)
blood cell types” Spleen Myelopoeisis 2nd to 5th
month
 Progenitor Cell
Spleen Monopoeisis 5th month
o Myeloblast
o Medullary Phase
o Immature Monocyte
 Bone cavities begin to form during 5th
o Megakaryocyte
month of gestation and begin to assume the
o Pronormoblast
responsibility of blood cell production.
 Phases of Hematopoeisis
 At 6th month, bone marrow will be the
o Mesoblastic Phase
primary site of blood cell production
 Begins at 19th day of gestation
 Myeloid : Erythroid ratio in the bone marrow
 HSCs arise from the aorta-gonad-
is 3:1.
mesonephros region of the Yolk Sac
 Measureable levels of erythropoietin, G-
 The remaining phase of the yolk sac are
CSF, GM-CSF, Hemoglobins A and F are
called ANGIOBLAST which form the future
detected.
blood vessels Occur Site Type of Type of Hemoglobin
 Produces Primitive Erythroblast (consider as Hematopoeis cells
is
the first primitive cell to be produced [2 to 8 Mesoblast 1st Yolk sac Intravascular Primitive Portland,
ic Phase trimest (AGM) erythrobla Gower I,
weeks]) that are mesodermal in origin er st Gower II
 Characterized by development of Primitive Hepatic 2nd LIVER Extramedulla Developin Predominan
Phase trimest ry g t: Hbg F
Erythroblast that produces haemoglobin: er erythrobla
st
Portland, Gower 1, and Gower 2 Medullary 3rd BONE Intramedullar Hgb F, Hgb
o Hepatic Phase Phase trimest MARRO y A2
er W Predominan
t: Hgb A1
 o Blast forms of RBCs, WBCs and
 Site of Red Bone Marrow Megakaryocytes: 4:1
o Child: Flat Bone – Clavicle Sternum, o Lymphocyte: 4:1-3:1
Skull, Ribs, Vertebrae, and Pelvis; Long o Other Leukocyte: 2:1-1:1
Bone  Cytoplasmic Characteristics/Features:
o Adult: Flat Bone – Proximal Ends of long o Staining color and intensity
bone; Yellow Marrow may be replaced o Granulation
by Red Bone Marrow when needed. o Shape
 Summary o Quantity of Cytoplasm
Age Site of Hematopoesis o Vacuolization
Embryo Yolk Sac o Inclusion Bodies
3 to 7 Months Spleen
4 to 5 Months Marrow Cavity especially
granulocytes & platelets
7 Month Marrow Cavity - Erythrocyte
Birth Mostly Bone Marrow; Spleen and
Liver when needed
Birth to Maturity Number of Active Sites in Bone
Marrow decreases but retain
ability for hematopoesis
Adult Bone Marrow of skull, sternum,
vertebral column, pelvis, proximal
ends of femurs
 STEM CELL MODEL
o Totipotential
o Pluripotential
o Multipotential
o Intermediate
o Mature
Stem cell lineage Regulation
Erythrocytes CFU-GEMM, CFU-E, Hypoxia,
BFU-E erythropoietin
Monocytes CFU-GEMM, CFU,
GM, CFU-M
Megakaryocyte CFU-GEMM Thrombopeitin
Granulocyte CFU,GEMM,CFU- Infection
GM,CFU-G
Eosinophil CFU-GEMM,CFU-Eo Parasitic infection
Basophil CFU-GEMM,CFU-Ba Hypersensitivity

Regulation of Lymphopoeisis
Lymphocyte CFU-L Interleukin IL-2, IL-3, IL-4,
IL-5)

 Blood Cell Characteristics: It is important to

observe its:
o Overall size
o Nuclear-Cytoplasmic ratio
 Nucleus : Cytoplasm ratio