HEMA 1A – 1ST SHIFTING
INTRODUCTION
DEFINITION AND ETYMOLOGY
• Etymology
o Hema/Hemato --> Heimas - blood
o Logy --> Logos - study
• Definition
o Study of quality and quantity of
the cellular elements of the
peripheral blood & bone marrow
o Study of
disorders/abnormalities related
to/of the cellular elements of the
peripheral blood & bone marrow
o Study of the laboratory
procedure/techniques used to
examine the cellular elements of
the peripheral blood & bone
marrow
FUNCTIONS OF THE HEMATOLOGY
SECTION
• Study of formed elements of blood & blood
forming tissues
• Screen diagnosis & monitor hematologic
disease & also give a general idea of
overall health
o Establish a diagnosis or rule out
a diagnosis
o Confirm a physician’s clinical
impression of a possible
hematological disorder
o Detect an unsuspected disorder
o Monitor the effects of therapy
o Detect minimal residual disease
following therapy
HISTORY OF HEMATOLOGY
• Hippocrates (400 BC)
o 4 humors
▪ Black bile
▪ Yellow bile
▪ Phlegm
▪ Blood
o 4 humors must be in balance to
stay healthy
• Athanasius Kircher (1657)
o Platelets as "worms" in the blood
• Anton van Leeuwenhoek (1974)
o Accounts on RBCs
• Giulio Bizzozero (late 1800s)
o Platelets as "Petite plaques"
• Karl Vierondt (1852)
o Published the first quantitative
results of blood cell analysis
• James Homer-Wright (1902)
o Developed Wright's stain
▪ Acidic + basic dye
▪ Can differentiate WBCs
• Improvements in methods of blood
examination
• Modern hematologist
o Alterations in the components of
blood is a result of disease
BLOOD COMPOSITION
• Whole Blood
• Includes all elements of the blood
o RBC
o WBC
o Platelets
o Plasma
• Noncellular components
o Aka. Blood dust/ Hematoconia
o Chylomicron or Chyle
▪ Increased after eating
▪ Exogenous triglyceride
• Plasma
• Normally hazy & pale yellow
• Serum: normally clear
• Why does blood remain liquid inside the
body?
o Smooth lining of blood ve+ssel
o Negative charge of platelets
▪ Original (-) charge -->
repulsion
▪ Change of charge -->
attraction/ clumping
o Presence of heparin
▪ Natural anticoagulant
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HEMA 1A – 1ST SHIFTING

PLASMA
• Plasma proteins (7%)
o Albumin (58%)
▪ Regulates osmotic pressure
▪ Carrier protein
o Globulin (38%)
▪ Antibodies and complement
▪ Aka. Immunoglobulin
▪ Transport of hormones
▪ Blood clotting
o Fibrinogen (4%)
▪ Factor 1 (FI)
▪ Forms fibrin  blood clot
• Color
o Arterial: bright red (Hb is
saturated with O2; sat. Hb O2)
o Venous: dark purplish red (Hb is
NOT saturated with O2)
o IN PULMONARY ARTERIES &
VEINS: reverse is true
▪ Hb with O2  purple
▪ Hb without O2  Blue
FUNCTIONS OF THE BLOOD
• Respiratory – most important
• Nutritional
• Excretory
• Buffering Action
• Maintenance of constant body
temperature
• Transportation of hormones & other
endocrine secretions that regulate cell
function
• Body defense mechanisms

BLOOD HOMEOSTASIS
• Homeostasis is the body’s tendency to
move toward physiological stability. In
vitro testing of blood & other body fluids
must replicate exact environmental body
GENERAL CHARACTERISTICS OF conditions. These conditions should
BLOOD indicate the ff:
• In vivo: fluid o Osmotic Concentration
• 3.5-4.5% thicker than water o pH range
• Makes up 7 to 8% of the total body o Temperature
component or 75 to 85mL of blood per
kilogram body weight IMPORTANT HEMATOLOGY AFFIXES
• Approximately 20 grams solid per
100mL blood
• Total Blood Volume (TBV)
o Adult male -> 5-6 L
o Adult female -> 4-5 L
o Newborns -> 250-450 mL
• Blood pH:
o 7.35 to 7.45 (Average of 7.40)
o Venous 7.35
o Arterial 7.45
• Blood specific gravity: # of solutes
dissolved in bld
o Whole blood 1.045 – 1.066
o Serum 1.024 – 1.028
o Plasma 1.025 – 1.029
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HEMA 1A – 1ST SHIFTING

BLOOD PREPARATIONS FOR o Physiological condition

HEMATOLOGY TESTING ▪ Relative to:
• Age
• Whole blood • Gender
• Plasma • Altitude
• Serum
• Defibrinated blood placed in a container HEMOGLOBIN, HEMATOCRIT, & RBC
with beads INDICES
• Stained buffy coat smear • Hemoglobin (Hgb)
• Stained peripheral blood smear o Hgb determination uses
• Free flowing blood from a skin puncture Drabkin’s Reagent
▪ Sodium bicarbonate (NaHCO3)
FORMED ELEMENTS ▪ Potassium ferricyanide
• Red Blood Cells (RBC)/ Erythrocytes {K3[Fe(CN)6]}
• White Blood Cells (WBC)/ Leukocytes • Hemoglobin (Fe2+)
o Granulocytes • Forms methemoglobin
▪ Neutrophil (Fe3+)
▪ Basophil ▪ Potassium cyanide (KCN)
▪ Eosinophil • Stabilizer
• L-cyanmethemoglobin
o Agranulocytes
▪ Lysing agent
▪ Lymphocyte
▪ a = 540 nm
▪ Monocyte
• Platelets or Thrombocytes
o Automated methods uses Sodium
dodecyl sulfate
• Noncellular elements of blood: ▪ Aka. Sodium lauryl sulfate
chylomicrons, blood dust • Hematocrit (Hct)
(hematokonia, hemoconia) <1% o Ratio of the volume of RBCs to
volume of whole blood
ERYTHROCYTES o Packed Cell Volume (PCV)
o Normal ratio = 50% (estimated)
• 6-8 micrometer (diameter), biconcave disc
o To fit in capillaries o EVF: erythrocyte volume
o 1/3 central pallum fraction
• Non-nucleated , with hemoglobin • RBC indices
• Transport oxygen and carbon dioxide o Computed based on RBC ct, Hct
o ~ 4-6 million/µL : RBC Count and Hgb
• How to count: thru manual or automated o Mean Cell Volume (MCV): a
• Anemia measure of volume; RBC diameter
o Low O2 carrying capacity, Low o Mean Cell Hemoglobin
RBC count Concentration (MCHC): reflects
• Polycythemia RBC staining intensity or degree of
o Erythrocytosis, increased RBC Ct pallor
(Hct and Hgb also), increased RBC o Mean Cell Hemoglobin (MCH) :
mass, leads to hyperviscosity: expresses mass of hgb
result of chronic hypoxia o Red cell Distribution Width
o Polycythemia vera: (RDW): degree of anisocytosis
myeloproliferative neoplasm in w/c ▪ Anisocytosis - variation in
somatic mutation leads to marked size of RBCs
increase in RBC ct
o (hct and hgb, WBC ct and plt count
and total blood volume also)

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HEMA 1A – 1ST SHIFTING

RBC Formula Reference LEUKOCYTES

index Range • Hitch a ride in the blood
• Colorless in unstained smears
MCV (Hct / RBC ct) 80-100 fL
• ~ 4,500-11,500 /µL in circulating blood;
X 10
many more in tissues
MCH (Hgb/RBC ct) X 26-32 pg • Function in immunity and defense
10 against bacteria, viruses and all other
foreign materials (includes allergens like
MCHC (Hgb/Hct) X 32-36% pollen, etc.)
100 (g/dL in SI) • Leucopenia
• Leucocytosis - more clinically significant
RDW (Std Dev of • 11.5-
• Differentiated using Wright’s stain
MCV/MCV) X 14.5%
100 Types
• MCD: mean cell diameter; using a PBS • According to Nucleus
measure the RBC diameter using • Polymorphonuclear (PMN) - with
micrometer eyepiece segments
o *Neutrophils, Basophils,
• MCAT: mean cell average thickness; MCAT
Eosinophils
= MCV/ 3.14 (MCD/2)^2 3.14 (pi)
• Mononuclear - without segments
o *Lymphocytes, Monocytes
• According to cytoplasmic granules
• Granulocytes
o Neutrophils, Basophils,
Eosinophils
• Agranulocytes
o Lymphocytes, Monocytes

Neutrophils
• Neutral
o Acid dye (red) + basic dye (blue) =
violet color
• 3-5 lobes
o 4-5 lobes - hypersegmented (dying)
RETICULOCYTES • 50-70% of whole WBC
• Polychromatophilic RBCs • Increase in # - Bacterial infection
• About 1-2% • Neutrophilia: bacterial infection
• Newly released RBCs from the BM • Neutropenia: long term drug
• Contains RNA administration; viral infection
• To count: • Bands
• Supravital dye o Shift to the left - bacterial infection
o Brilliant Cresyl Blue
Eosinophils
o New Methylene Blue
o Manual (1000 RBCs) • Acidophilic
• Automation (TOA/Sysmex) o Red color or bright orange color
o Automated (32 000 RBCs) • With granules filled with antihistamine
• High amounts during massive loss of • 2-3 lobes
blood: Good Bone Marrow response • 2-5% of whole WBC

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HEMA 1A – 1ST SHIFTING
• Increase in # - Parasite infection,
Allergic rxn., Inflammation
• Eosinophilia: allergy; parasitism
• Eosinopenia: theoretical/ unused
Basophils
• Basophilic
o Blue-black
• Irregularly lobed
o Nucleus is hidden
• ~ 1% of whole WBC
• Increase in # - Allergic rxn.,
Inflammation
Lymphocytes
• Big nucleus
• Darker nucleus
o Chromatin pattern - clumped
• Scanty cytoplasmic area - clear sky
blue
• 20-40% of whole WBC
• Increase in # - viral infection
• B, T and NK cells
o B cells - humor immunity
o T cells - cell mediated immunity
o NK cells (Natural Killer cells) -
immune surveillance
▪ Suspicious cancer cells
• Big round nucleus, thin rim of cytoplasm
• Slightly larger then RBCs
• Lymphocytosis: viral infection
• Lymphopenia/Lymphocytopenia: long
term drug administration;
immunodeficiency
Monocytes
• Loose chromatin pattern
• Cytoplasmic area
o Grainy
o Fine granules
• 2-8% of whole WBC
• Macrophage in tissues
• Phagocytosis, presentation of epitopes
o Antigen presentation
• Slightly larger than other WBCs
• Monocytosis: hematologic disorder
• Monocytopenia: theoretical
THROMBOCYTES
• Pieces of a bone marrow cell known as a
megakaryocyte
• Function to stop bleeding by forming a
plug and also release coagulation factors
(controls hemostasis)
• Forms thrombus (clot)
• Capable of adhesion, aggregation,
secretion
o Adhesion - platelet to non-plt.
o Aggregation - platelet to platelet
• 2-4 µ , oval, anucleated, slightly granular
• Reference Range: 150-450 K/cu.mm.
o Critical!: 50K/cu.mm.
• Counted using manual or automated
• Automated: with MPV (6.8-10.2%)
• Mean Platelet Volume (MPV)
o a machine calculated
measurement of the average size of
platelets found in blood.
o High MPV: regenerative BM
response to platelet consumption
• Thrombocytosis: inflammation; trauma
• Thrombocytopenia: consequence of
drug treatment
COMMON HEMATOLOGY
LABORATORY TESTS
• Check the integrity of the
specimen!!!!!
• Short draws
• Complete Blood Count (CBC) includes:
o RBC count
o WBC count
o Hemoglobin
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HEMA 1A – 1ST SHIFTING
o Hematocrit
o WBC Differential count
• Automated: flags (when one of the results
is abnormal) – back up procedure
• Platelet count
• Additional information on size and
appearance of blood cells (blood film
examination)
• Erythrocyte Sedimentation Rate
(ESR)
• Reticulocyte Count
• Sickle Cell Testing
• Osmotic Fragility Test
• Coagulation Tests include:
o Prothrombin Time (PT)
o Partial Thromboplastin Time
(PTT)
o Fibrinogen
o Factor Analysis
ADVANCE HEMATOLOGY
PROCEDURES
• BM Examination
• Molecular assay
• Cytochemical stains (differentiate
abnormal cells)
• Flow cytometry
Hematological Diseases (Diseases may
be classified in more than one category)
• Diseases of improper or insufficient
production
o Anemias – Examples include iron
deficiency anemia and aplastic
anemia
o Leukemias – WBC production is
affected and defective. Examples
include acute and chronic leukemia
o Thrombocytopenia – Decreased
platelets. Patient may have
bleeding problems
• Diseases of defective cell function
o May be combination of improper
cell production and defective
function
o Examples include iron deficiency
anemia and certain leukemias
• Inherited hematological diseases
o Examples include hemophilia,
sickle cell anemia, G6PD
deficiency, thalassemias
• Secondary or acquired hematological
diseases
o Examples include hemolytic
disease due to renal pathologies or
atypical lymphocytes due to viral
infections
o Examples include iron deficiency
anemia and certain leukemias
SPECIMEN COLLECTION AND
PROCESSING
SAFE COLLECTION PRACTICES
• Understand equipment and handle
with care
• Wear gloves
o For 1 use only
o When removed no substances from
soiled gloves should be in contact
with hand
• Wash hands
o Use non-abrasive soap
o Substitutes:
▪ Hand sanitizer
▪ Approx. 62% alc. And up
• Dispose of sharps in puncture
resistant container
PHYSIOLOGIC FACTORS
• Posture
o Supine - hemodiluted
o Seating/standing -
hemoconcentrated
• Diet
o Fasting requirement for some tests
▪ Glucose, triglyceride
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HEMA 1A – 1ST SHIFTING

SOURCES OF BLOOD ▪ Distal portion of 3rd or 4th

• Skin/ Capillary finger
• Vein ▪ Accessible and easy to
manipulate
• Artery
▪ Ideal for peripheral smears
▪ Less intimidating
SKIN PUNCTURE o Ear lobe - Best site
• Aka. Dermal puncture, Pricking ▪ Less pain
▪ Less tissue juice
• Capillary blood or Peripheral blood ▪ Less contamination
o Contains: ▪ More free-flowing blood
▪ Venous blood ▪ Ideal when searching for
▪ Arterial blood abnormal sites (histiocytes in
▪ Tissue fluid/ juice bacterial endocarditis)
o Slightly different results (as ▪ Can be arteriolized
compared to venous blood) ▪ However, impractical
▪ Inc. RBC (15-20%)
▪ Inc. glucose (note if OGTT is • Skin puncture method
the test requested) o Alcohol only as skin prep
▪ Inc. platelet o Immobilize the finger, toe or heel
o Use blade (2.0mm) to avoid bone
• Skin puncture is for the ff: o Puncture with blade
o Newborns perpendicular to the fingerprint
o Children less than 1 year old lines
o Adults with difficult or reserved o Avoid swollen, bruised or
venous access previously punctured areas,
o Small amount of blood is needed edematous, cyanotic sites
o Microhematocrit Edematous
o Clotting time/Bleeding time ▪ Inc. plasma
(CT/BT) ▪ Hemodiluted
▪ Free-flowing blood ▪ Lower ct, - because of tissue
o Blood smear prep juice
▪ Non-anticoagulated blood Cold/cyanotic site
o Geriatrics ▪ Increased blood ct
▪ Less elastic skin, more ▪ Decreased in oxygen in the
moveable vein area
o Extreme burns ▪ Should be massaged first (not
o Obese too much)
▪ Should be warmed using water
o DIC, thromboembolism, bleeding
bath (hot compress; 40-42 C)
tendencies o Wipe away first drop of blood,
o For patients with Disseminated
collecting only when free flowing is
Intravascular Cardiomyopathy
achieved
(DIC) o Warming will increase blood
flow
• Capillary Puncture Sites
o Heel or Big toe
• Skin puncture in infants
▪ For less than 1 y/o
o Lateral or Medial plantar surface
o Adults: finger ▪ Lateral is more preferred
▪ Most common site for skin
(medial aspect of the foot has
puncture
the posterior tibial artery)
▪ Non-dominant finger
o Use shorter blade

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HEMA 1A – 1ST SHIFTING
• Capillary collection devices
o Capillary tube
▪ Red - Heparinized
▪ Blue - no anti-coagulant
o Micro-collection tubes with or
without additives
• Order of collection
o Tube for blood gas analysis
o Slides (unless blood is collected in
EDTA)
o EDTA microtainers
o Other microtainers w/
anticoagulant
o Serum micro-collection tube
VENIPUNCTURE
• Venous blood or Deoxygenated blood
• Methods
o Syringe method
o Evacuated Tube System (ETS)/
Closed System
▪ Ideal for multiple draw
o Winged Infused set/ Butterfly
method
▪ Commonly used for children
• Collection equipment for venipuncture
o Evacuated Tube Systems (ETS)
▪ Tube, tubes contain
appropriate additives and are
sealed in a vacuum
▪ ADDITIVES
• Antiglycolytic
• Anticoagulant
• Clot activator
• Separator gel
• Tube holder (adapter)
• Needle - two way needle
• Needles (syringe method/ ETS)
o Sterile, disposable (one use)
o Adapt to tube holders or to a
syringe
o May be single or multi-sample
• Needle holders/ Adapters (for ETS/
Butterfly)
o Manufactured to fit specific
needles and tubes
o Usually disposable
• Tourniquet
o Provide barrier to venous blood
return
o Latex or latex-free
o Applied 2-4 inches above puncture
o Applied no longer than 1 minute
prior to puncture
• Syringes
o Useful for tiny fragile veins
o Pressure controlled by
phlebotomist
• Butterflies
o Short needle, IV tube, wings
o Attached to the tube adapter,
syringes or blood culture bottles
o Useful with pediatric patients,
long term studies (GTT)
• Solutions for skin preparation
o 70% isopropyl alcohol
o Work from puncture dry site
outward in a spiral
o Allow to air dry before puncture
• Sterile skin preparation
o Alcohol followed by iodine
o Chlorhexidine gluconate/ isopropyl
alcohol
o Benzalkonium chloride
• Selecting the venipuncture site
o Cephalic, basilic and median
cubital veins located in the
antecubital fossa
▪ Median cubital - best site
o Dorsal side of wrist/hand or
veins in the feet
▪ Secondary choice
▪ Avoid feet if patient is diabetic
o Avoid intravenous fluids
▪ Ask for the nurse to stop the
IV (don't even try)
▪ Wait for 2 minutes
▪ Tourniquet should be placed
below IV site
▪ Blood should be drawn below
the IV site
• Order of draw
o Sterile specimens - Blood culture
tubes (Yellow)
o Coagulation tubes (Light blue)
o Serum Separator Tube - (Gold or
marbled red or black)
o Serum (plain red)
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HEMA 1A – 1ST SHIFTING

o Green - with heparin o Hemolysis

o Light green o Burned, damaged, scarred,
o Lavender - with EDTA occluded veins
o Gray - w/ antiglycolitic agent o Seizures, tremors, vomiting,
• Complications encountered in choking
venipuncture o Allergies to skin
o Special challenges of pediatric prep/tourniquet
patients o Mastectomy px
o Echymoses (bruising) • Types of complications
▪ Small amount of blood seeping o Immediate local
under the skin o Late local
o Syncope (fainting) o Late general
o Hematoma • Other considerations
▪ Larger amount usually caused o 2 attempts
by passing through or not ▪ (1-3 attempts) depending on
completely into the lumen of the patient
the vein o Never flex the arm after
▪ Usually appears a few days
phlebotomy
after collection
o Never recap needles w/ bare hands
o w/ anticoagulant tube: mix by
inversion
o Deliver blood into tubes:
▪ Pull plunger
▪ Remove needle
▪ Deliver along sides
• Issues in phlebotomy
o The patient has the right to say no
o Document competency of staff
o Follow established procedure
o Respect the patient’s bill of rights
Image – treatment of Hematoma o Laboratory test results are only as
o Failure to obtain a sample good as the sample that is tested
▪ If needle if missed or change in
the tube
o Petechiae
▪ A pinpoint, nonraised,
perfectly round, purplish red
spot caused by intrademal or
submucous hemorrhage
o Edema
o Obesity
▪ Pressure cuff can be used
▪ Must not be longer than 1
minute
▪ Must not be higher than
diastolic pressure
o Intravenous Therapy
▪ CLSI recommends
discarding the first 5 mL of
blood collected
o Hemoconcentration

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