HEMA 1A – 1ST SHIFTING

INTRODUCTION • Giulio Bizzozero (late 1800s)

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

PLASMA • Color
o Arterial: bright red (Hb is
• Plasma proteins (7%) saturated with O2; sat. Hb O2)
o Albumin (58%) o Venous: dark purplish red (Hb is
▪ Regulates osmotic pressure NOT saturated with O2)
▪ Carrier protein o IN PULMONARY ARTERIES &
o Globulin (38%) VEINS: reverse is true
▪ Antibodies and complement ▪ Hb with O2  purple
▪ Aka. Immunoglobulin
▪ Hb without O2  Blue
▪ Transport of hormones
▪ Blood clotting
o Fibrinogen (4%) FUNCTIONS OF THE BLOOD
▪ Factor 1 (FI) • Respiratory – most important
▪ Forms fibrin  blood clot • 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
THROMBOCYTES
Lymphocytes
• Pieces of a bone marrow cell known as a
• Big nucleus megakaryocyte
• Darker nucleus • Function to stop bleeding by forming a
o Chromatin pattern - clumped plug and also release coagulation factors
• Scanty cytoplasmic area - clear sky (controls hemostasis)
blue • Forms thrombus (clot)
• 20-40% of whole WBC • Capable of adhesion, aggregation,
• Increase in # - viral infection secretion
• B, T and NK cells o Adhesion - platelet to non-plt.
o B cells - humor immunity o Aggregation - platelet to platelet
o T cells - cell mediated immunity • 2-4 µ , oval, anucleated, slightly granular
o NK cells (Natural Killer cells) - • Reference Range: 150-450 K/cu.mm.
immune surveillance o Critical!: 50K/cu.mm.
▪ Suspicious cancer cells • Counted using manual or automated
• Big round nucleus, thin rim of cytoplasm • Automated: with MPV (6.8-10.2%)
• Slightly larger then RBCs • Mean Platelet Volume (MPV)
• Lymphocytosis: viral infection o a machine calculated
• Lymphopenia/Lymphocytopenia: long measurement of the average size of
term drug administration; platelets found in blood.
immunodeficiency o High MPV: regenerative BM
Monocytes response to platelet consumption
• Thrombocytosis: inflammation; trauma
• Loose chromatin pattern • Thrombocytopenia: consequence of
• Cytoplasmic area drug treatment
o Grainy
o Fine granules COMMON HEMATOLOGY
• 2-8% of whole WBC LABORATORY TESTS
• Macrophage in tissues
• Phagocytosis, presentation of epitopes • Check the integrity of the
o Antigen presentation specimen!!!!!
• Slightly larger than other WBCs • Short draws
• Monocytosis: hematologic disorder • Complete Blood Count (CBC) includes:
• Monocytopenia: theoretical o RBC count
o WBC count
o Hemoglobin

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HEMA 1A – 1ST SHIFTING

o Hematocrit o Examples include hemophilia,

o WBC Differential count sickle cell anemia, G6PD
• Automated: flags (when one of the results deficiency, thalassemias
is abnormal) – back up procedure • Secondary or acquired hematological
• Platelet count diseases
• Additional information on size and o Examples include hemolytic
appearance of blood cells (blood film disease due to renal pathologies or
examination) atypical lymphocytes due to viral
• Erythrocyte Sedimentation Rate infections
(ESR) o Examples include iron deficiency
• Reticulocyte Count anemia and certain leukemias
• Sickle Cell Testing
• Osmotic Fragility Test
• Coagulation Tests include: SPECIMEN COLLECTION AND
o Prothrombin Time (PT) PROCESSING
o Partial Thromboplastin Time
(PTT)
o Fibrinogen SAFE COLLECTION PRACTICES
o Factor Analysis • Understand equipment and handle
with care
• Wear gloves
ADVANCE HEMATOLOGY o For 1 use only
PROCEDURES o When removed no substances from
• BM Examination soiled gloves should be in contact
• Molecular assay with hand
• Cytochemical stains (differentiate • Wash hands
abnormal cells) o Use non-abrasive soap
• Flow cytometry o Substitutes:
▪ Hand sanitizer
Hematological Diseases (Diseases may ▪ Approx. 62% alc. And up
be classified in more than one category) • Dispose of sharps in puncture
resistant container
• Diseases of improper or insufficient
production
o Anemias – Examples include iron
PHYSIOLOGIC FACTORS
• Posture
deficiency anemia and aplastic o Supine - hemodiluted
anemia o Seating/standing -
o Leukemias – WBC production is
hemoconcentrated
affected and defective. Examples
• Diet
include acute and chronic leukemia o Fasting requirement for some tests
o Thrombocytopenia – Decreased
▪ Glucose, triglyceride
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

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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 Provide barrier to venous blood

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