MODULE 1: INTRODUCTION TO HEMATOLOGY
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OUTLINE
INTRODUCTION
A. Definition
BLOOD COLLECTION
A. Venipuncture
B. Skin Puncture
C. Order of Draw
OVERVIEW OF HEMATOLOGY
A. Functions of Blood
B. Common Hematologic Tests
C. Physical Characteristics of Blood
D. Composition of Blood
E. Differentiation between Plasma and Serum
F. Common Prefixes used in Hematology Vocabulary
G. Common Suffixes used in Hematology Vocabulary
DEFINITION OF TERMS
SPECIMEN CONSIDERATIONS ON HEMATOLOGY
A. Patient Identification
B. Physiologic factors affecting test results
C. Sources of blood for Hematology testing
D. Additives
E. Order of draw
F. Other blood collection tubes
G. Sample preparations used in the Hematology Laboratory
H. Specimen Collection
I.
Pre-collection Variables
J. Specimen Collection
K. Order of draw (2.0)
L. Sources of blood for Hematology testing (2.0)
M. Indications of venipuncture
N. Patients’ considerations during venipuncture
O. Complications of venipuncture
ROUTINE VENIPUNCTURE AND SPECIMEN HANDLING
A. Venipuncture Procedure
B. Order Form/Requisition
C. Labelling Sample
D. Equipment
E. Order of Draw
F. Procedural Issues
G. Phlebotomy Procedure Illustration
H. Additional Considerations
I.
Reasons for Canceling a Laboratory Test
J. Safety and Infection Control
K. Troubleshooting Guidelines
L. Blood Collection on Babies
M. Pediatric Phlebotomy
N. Collection Tubes for Phlebotomy
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Diseases involving red blood cells (RBC’s)
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Anemia
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Iron-related diseases
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Iron is one of the main components of RBC
(hemoglobin is the iron containing protein).
There are diseases affecting all three: RBC’s, WBC’s
and platelet.
BLOOD COLLECTION
VENIPUNCTURE (VIDEO)
2 PROCEDURES FOR COLLECTING
SAMPLES
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BLOOD
Venipuncture
Phlebotomy
SUPPLIES
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Tourniquet (3 to 4 inches)
70% alcohol wipes
Cotton or gauge (21, 22, or 23 gauge)
Tape
Vacutainer tube(s)
Multi-sample straight needles
Additional:
o Lab order
o Specimen
o Hand sanitizer
o Disinfectant wipes
PPE
3 CONSIDERATIONS
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Safety
Patient experience
Specimen viability
Most appropriate vein to draw
o Inner elbow area called “anti-cubital space”
MOST PREFERRED VEIN:
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Median cubital vein (1st choice)
o Middle
Cephalic vein (2nd choice)
o Palm facing down
Basilic vein (3rd choice)
o Above brachial artery
INTRODUCTION
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The field of Clinical Hematology deals with the study of the
cellular components (red blood cells, white blood cells, and
platelets) and the hemostatic elements (pro-clotting factors
and inhibitors of coagulation) in order to aid in the diagnosis
and monitoring of diseases of the blood or other conditions
that affect these different components. Hematological tests,
primarily the Complete Blood Count, offers a wealth of
information to the clinician regarding various physiological
processes.
In this module, the students will be introduced to the field of
Hematology. Basic knowledge of hematology and
hematological tests will be laid out as the foundation for
future lessons.
Clinical Application of Hematology
3 D’S OF VEIN ASSESSMENT:
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Depth
Diameter
Direction
o 15 to 30° angle
o C method to press on the tube
o Invert the tube 2 to 3 times
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Mostly 8 to 10 inversions are needed
SKIN PUNCTURE (CAPILLARY BLOOD
COLLECTION VIDEO)
MOSTLY INDICATIONS IN ADULT PATIENTS:
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Severe burns
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Module 1: Introduction to Hematology
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the line of the
fingerprint.
Extreme obesity
Hypercoagulability
Geriatric or fragile veins
To preserve veins for therapy
Home testing
Apprehensive patients
Points of care testing
PROCEDURE:
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Warming
o Warm the puncture site as warming can increase blood
flow by seven fold a warm moist towel or warming
device is used.
o Heated higher than 42° centigrade
o Given time is 3 to 5 minutes
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Site Cleansing
TEST PERFORMED ON CAPILLARY BLOOD:
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Blood smear for:
o Microscopic assessment of cell morphology
o Preparations for malarial parasites
o Rough estimations of platelet number
o WBC morphology
Rapid tests:
o Dried blood spot for early infant diagnosis
o Blood glucose monitoring with glucometer
o Pre-blood donation (Hb, Blood group)
Biochemistry tests:
o Bilirubin and thyroid functions tests
Bleeding time:
o IV’s method Blood
Blood culture shouldn’t be performed on capillary blood
equipment and supplies.
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EQUIOMENTS AND MATERIALS:
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Filter paper for Dry Blood Spot (DBS)
Capillary tube
Skin Puncture Finger Prick
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Inform grown up patient about imminent pain
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Hold the finger and firmly place a new sterile lancet
at the selected site on the finger
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Self-retracting safety lancet
Skin Puncture Heel-prick
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90° angle length of the foot
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Discard the 1st drop of blood to avoid specimen
dilution by the tissue fluid, the excess of tissue fluid
would dilute the sample and reduce the
concentration of all analytes that will affect the
results.
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Never milk of scrape the puncture site
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Collect the sample with the 2nd drop
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Downward – micro collection tubes, collect EDTA
tube before serum tube mixed with anticoagulant.
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Do not use adhesive bandages on children with
the age of 2 years old and below
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Ensure proper guidelines
TEST SPECIFIC EQUIPMENT:
HEEL PUNCTURE PRECAUTIONS:
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Blood smear slides
Rapid test devices
Micro-collection tube
LANCETS
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Manual Lancets
o Single use lancet is available but not in depth of
penetration can’t be controlled.
Safety Lancets
o Single use lancet for different depths is available
o Activate when it is positions and pressed against the
skin
o Advantage
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Depth will not exceed to 2.4 mm
PUNCTURE DEPTHS
DEPTH
< 2.0 mm
< 1.5 mm
< 2.0 mm
< 2.4 mm
TYPE OF
PUNCTURE
Heel puncture
Finger puncture
Finger puncture
Finger puncture
AGE
Infant up to 6 months old
12 months and above
8 years old and above
For adults
SITE FOR CAPILLARY BLOOD COLLECTION:
CONDITIONS
Age
Weight
Recommended
finger
Placement
of
lancet
HEEL-PRICK
Birth up to 6
months
3 to 10 kg
N/A
FINGER PRICK
6 months and
above
10 kg and above
Second and third
On the medial or
lateral
plantar
surface
On the side of the
ball of the finger
perpendicular to
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Do not puncture deeper than 1.5 mm
Do not puncture previous puncture
Do not puncture outside the medial and lateral aspects of
the heel
Do not puncture posterior curvature of the heel
Do not puncture arch and other than the heel
COMPLICATIONS:
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Collapse of veins
o Tibial artery is lacerated from puncturing the medial
aspect of the heel
Osteomyelitis of the heel bone (Calcaneus)
Nerve damage
o Fingers of the neonates are punctured
Scarring
Localized or generalized necrosis
Skin breakdown from repeated use of adhesive strids
All of this could be avoided if there is sufficient pressure
applied to the puncture site is observed after the procedure.
ORDER OF DRAW (PHLEBOTOMY VIDEO)
BLOOD CULTURES “STERILE”
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Aerobic bottle
Anaerobic bottle
Maintain sterility
There is a couple of things inside the blood culture bottle
helping this process of being able to grow the bacteria and
properly identify things.
o First is there’s a little bit of film typically across the
bottom and this is primarily used to help identify growth
that’s taking place
o Inside, there’s going to be some sort of level of a
nutrient broth. It helps the bacteria to proliferate.
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Module 1: Introduction to Hematology
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Also, there is an anticoagulant and a chemical that reduce
the natural bacteriacidal action. It does this by stopping
complement and this slows down the phagocytosis that
really prevents the killing of the bacteria by the blood.
LIGHT BLUE TUBE “COAGULATION”
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PT/INR
PTT
Anti-Xa (anti-ten a)
Fibrinogen
D-dimer
TEG
Contains a set amount of sodium citrate
o It binds with calcium which plays an important role in
the clotting cascade.
o Helps to prevent the formation of a clot
o It is a truly a defined ratio of sodium citrate to blood
PINK TOP TUBE
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“Blood type” sample tube
Coated with EDTA
o High potassium
Used when sending a specimen to the blood bank
GREY TOP TUBE
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Used for:
o Checking lactate level
o Ethanol level
o Fasting glucose
Contains two different additives:
o Sodium fluoride
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Stops glycolysis
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Prevent bacterial growth
o Potassium oxalate
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Bind calcium to prevent clotting
RED TOP TUBE
ORDER OF DRAW
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Used for chemistry panels but not the one typically used
No gel or additive
o Sometimes have a silica clot activator
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Helps to form a clot inside the tube
GOLD/SST (SERUM SEPARATOR TUBE)
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Sometimes appear as tiger stripe top with the color of red
and black but it is basically the same
Used for chemistries but not typically used
A send out tubes for the checking of antigen or antibody
Has a gel that helps to separate the cells from the serum of
the blood
With or without clot activator
GREEN TOP TUBE
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For wide range of chemistry
Typically used to run chemistry tests in the hospital
draw for cardiac markers such as:
o troponin
there are a couple of tests that the tube ends up on ice
immediately and sent down to the laboratory
o ice helps in preserving the value of what is needed to
be checked within the blood sample
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ammonia levels
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ionized calcium
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lactate
sometimes referred as PST (plasma separating tube)
o has a gel at the bottom similar to SST
o contains fibrinogen
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separates out the plasma from the specimen
PURPLE TOP TUBE
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referred as “hematology tube”
used for:
o CBC
o ESR
o A1C
This tube comes sprayed right along the inside of the tube
with a chemical called “EDTA”
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Preserves the cell morphology
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Inhibits the clotting cascade by binding calcium to
prevent a clot
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It is very high in potassium
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Blood culture
o Sterile
Light blue
o Coagulation studies
Red top tube
o Not common chemistry tube
Gold/SST tube
o Not common chemistry tube
Green top tube
o Common chemistry tube
Purple top
o Hematology studies
Pink top
o Blood typing
Grey top tube
o Lactic acid
o Fasting glucose
o Ethanol
PROBLEMS WHEN THE ORDER OF DRAW WAS NOT
FOLLOWED
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If pink and purple tubes would be done first before the green
tube, the results may end up with high level of potassium.
ACRONYM
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Can help to memorize the order of Draw
Stop – Sterile tube
Light – light blue
Red – red
Stay – SST
Green
Power – purple
Light – light pink
Go – grey
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Hematology is defined as the study of blood, its
development, and diseases associated with it.
It includes the study of RBCs (erythrocytes), WBCs
(leukocytes), platelets (thrombocytes), and hemostatic
elements in plasma in order to provide a differential
diagnosis.
Thrombocytes are not true cells, these actually are the
fragments of the cytoplasm of it precursor.
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OVERVIEW OF HEMATOLOGY
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Module 1: Introduction to Hematology
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If the precursor will be the one to go out from the bone
marrow, it will be an indication of a problem or disease.
Hemostasis is a system within our body which allows blood
flow to a site of vessel insulin.
o This prevents excessive bleeding.
o Hemostatic components will work hand in hand to form
a platelet plug as well as a clot.
o Platelet plug will be formed first and a clot will form
around it.
o Has two parts.
Hemostatic elements:
o Proteins in the plasma
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Regulate or promote clot formation and
dissolution.
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Fibrinogen (involved in coagulation) is cleaved into
fibrin when activated. Those fibrin monomers,
when cross-linked with one another will
polymerase and for a fibrin clot.
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Within the fibrin clot, other cellular elements are
being trapped (RBCs and WBCs).
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Once you have the clot, it is abnormal for the clot
to remain there forever. It has to be removed for it
not to impede with the blood flow,
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IMMUNITY
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FUNTIONS OF THE BLOOD
RESPIRATORY
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Primary function of the blood, a function of red blood cells.
o RBCs deliver gases to and from the tissues.
o Oxygen will attach to hemoglobin, it is the oxygen
binding protein.
o While the RBCs travel throughout the body, they were
able to oxygenate parts of the body which are in need
of oxygen.
o Carbon dioxide can attach to hemoglobin however it
can also be dissolved in plasma.
Hemoglobin serves as a minor buffer system of the blood.
o It can help in maintaining the pH of the blood, a minor
role of accepting or releasing carbon dioxide.
refers to the delivery of gases to and from the tissues.
Oxygen attached to hemoglobin in RBCs for delivery from
the lungs to the peripheral tissues.
Carbon dioxide is also transported by the blood from the
tissues for elimination by the lungs
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refers to the regulation of certain balances in the body.
The blood is involved in a number of certain homeostatic
mechanisms such as pH homeostasis and temperature
regulation.
NUTRITIVE
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refers to the delivery of nutrients to the tissues. These
nutrients are dissolved in the plasma.
The nutrients will be absorbed through the gut and then
transported as they are dissolved in the plasma.
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refers to the delivery of waste products from the tissues to
the excretory organs for elimination from the body. Ex. Nonprotein nitrogenous wastes (NPNs) such as urea and
creatinine are dissolved in plasma and filtered by the
kidneys.
Waste products:
o Non-protein nitrogenous compounds.
the blood is involved in maintaining the water levels in the
body. Water is excreted or reabsorbed into the blood as a
function of plasma osmolality, which is the measure of
solute concentration of the blood. Increased plasma
osmolality promotes water reabsorption in the kidneys.
Decreased plasma osmolality promotes water excretion.
A system in the body acts as the regulator of the body to
determine if the body needs to conserve or discard water.
o Primarily a part in the endocrine system that detects
the increase and decrease in plasma osmolality is
called RAAS (Renin-Angiotensin-Aldosterone System)
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Increased plasma osmolality promotes water
reabsorption in the kidneys.
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Decreased plasma osmolality promotes water
excretion.
TRANSPORTATION OF HORMONES
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EXCRETORY
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Function of the WBCs in the blood.
refers to the body’s defense against infectious organisms
(Bacteria, Fungi, Parasites, or Viruses).
White blood cells populations in the blood are play a
protective role against a group of microorganisms.
o Neutrophils protect against most bacterial agents by
engulfing these bacteria, and releasing its granular
contents, which have anti-bacterial action.
o Monocytes protect against a wide range of infectious
agents by performing phagocytosis.
o Eosinophils protect against helminthic infections.
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Seen in people with allergies or helminth
infections.
o Lymphocytes are active against most viruses and its
subpopulation of B-lymphocytes differentiate into
plasma cells to produce antibodies.
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Natural immunity is provided by the actions by our
B cells (B-lymphocytes), particularly when B cells
differentiate into what we call “plasma cells”.
When blood cells are maturing, sometimes it goes in a
differentiation process.
WATER REGULATION
HOMEOSTASIS
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Urea and creatinine are primarily waste products
that should be excreted from the body. Its build up
in blood could be toxic to the body.
Urea is the breakdown products of proteins while
creatinine is the breakdown products of muscle
catabolism.
Analysis of their concentration in the blood are
primarily an indication of kidney function.
If the concentration is getting higher, there is an
indication of kidney disease.
Hormones are secreted by endocrine glands. The targets of
endocrine hormones are distant from its source. Thus, the
hormones need to be dissolved in the plasma to reach its
target tissue/organ.
o The only way for the hormone to travel its way to its
target is by being transported in the blood.
o Hormones always targets tissue/organ that is far from
the source of hormones.
HEMOSTASIS
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refers to the mechanism by which blood flow to an injured
blood vessel is arrested in order to prevent excessive blood
loss.
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Module 1: Introduction to Hematology
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This occurs as a function of platelets (forming a platelet
plug) and hemostatic elements in plasma (responsible for
clot formation and dissolution).
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COMMON HEMATOLOGIC TESTS
COMPLETE BLOOD COUNT (CBC)
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Composed of a panel of tests to evaluate the formed
elements in the blood.
o Cell count of RBCs, WBCs, and platelets.
o Morphology of blood cells as well as hemoglobin and
hematocrit.
the CBC results offer a wealth of information regarding the
health status of an individual.
o An increase or decrease within a cell count may reveal
a disease in an individual.
each component of CBC is associated with different
conditions (similar to the components of urinalysis).
Related to RBC count, hemoglobin determination is a
quantitative measure of hemoglobin concentration in blood.
o Hemoglobin is the protein in RBCs that is responsible
for binding with oxygen.
o If hemoglobin is low, there will be a problem with
oxygen delivery to the tissues.
o Hematocrit testing determines the amount or RBCs in
whole blood. Expresses RBCs as a percentage of
whole blood.
WBC differential count would determine the relative count
of each WBC population.
o Percentages of different WBC population.
o An increase or decrease in any of the WBC population
may imply different implications depending on the rage
of that specific WBC population.
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COMPONENTS OF CBC:
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Cell counts (RBC count, WBC count, Platelet count)
o determines the quantities of circulating blood cells
o increases or decreases in each of the cell counts may
reveal the presence of a disease process
Hemoglobin determination
o quantitative measure of hemoglobin, the protein in
RBCs responsible for binding oxygen. Low levels of
hemoglobin impair oxygen delivery to the tissues and
are most often associated with anemia.
Hematocrit testing
o determination of the amount of red blood cells in whole
blood (expressed as a relative quantity; ratio of red
blood cells to whole blood in a sample).
WBC differential count
o details the relative counts of each white blood cell
population (Neutrophils, Lymphocytes, Monocytes,
Eosinophils, and Basophils).
o each WBC population is expressed as the percentage
of white blood cells.
RBC indices
o values of these indices aid in making a specific
diagnosis of anemia.
o Mean Cell/Corpuscular Volume (MCV)–average
volume of red blood cells in a sample.
o Mean Cell/Corpuscular Hemoglobin (MCH) –average
amount of hemoglobin in each red blood cell in a
sample.
o Mean Cell/Corpuscular Hemoglobin Concentration
(MCHC) -average hemoglobin content for a specified
volume of red blood cells.
o Reticulocyte Distribution Width (RDW) –quantitates the
spread of RBC sizes in a sample. The greater the
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RDW, the greater is the difference in RBC sizes in the
sample.
Peripheral Blood Smear (PBS) Examination
o performed to analyze the morphologic characteristics
of RBCs, WBCs, and Platelets
o abnormal morphologic characteristics may indicate the
presence of disease processes
o Example: the presence of agranular platelets may be
associated with Gray Platelet Syndrome, which also
affects platelet function
Bleeding Time and Coagulation Tests
o These are tests used to evaluate hemostatic functions
of the blood
o Bleeding Time
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general
screening
test
for
hemostatic
abnormalities
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records the time before the bleeding stops after
skin puncture
o Prothrombin Time
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coagulation test used to evaluate the extrinsic
pathway
o (Activated) Partial Thromboplastin Time
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coagulation test used to evaluate the intrinsic
pathway
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APTT has an activator
o Thrombin Time and Reptilase Time
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coagulation tests used to evaluate the Fibrinogen
function
o Stypven Time / Dilute Russel Viper Venom Time
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coagulation test used to evaluate the common
pathway
Reticulocyte Count
o Reticulocytes are immature red blood cells. These are
normally found in the blood in small numbers.
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Reticulocytes and Mature RBCs have the same
appearance with Wright’s stain
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Supravital stain, the difference between RBCs and
reticulocytes.
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Reticulum is present within reticulocytes.
o Evaluation of its count reveals the bone marrow’s
response to hypoxia.
Erythrocyte Sedimentation Rate (ESR)
o This test is a measure of the rate of fall of red blood
cells within a packed column
o Traditionally, this test was used as a screening test for
the presence of inflammatory processes
o There are other correlations of ESR apart from
inflammation.
PHYSICAL CHARACTERISTICS OF BLOOD
Fluid in vivo (due to balance between pro-clotting factors
and anticoagulants)
o Thrombosis is promoted when pro-clotting factors is
higher than anticoagulants.
o On the other hand, if anticoagulant is higher than proclotting factor, excessive bleeding is promoted.
Volume: 4-6 L (normal adult volume) or 75-85mL per
kilogram of body weight
Viscosity: Viscous (3.5-4.5 times thicker than water)
COMPOSITIONS OF BLOOD
CELLULAR COMPONENTS (FORMED ELEMENTS)
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Red Blood Cells (Erythrocytes)
White Blood Cells (Leukocytes)
Platelets (Thrombocytes)
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Module 1: Introduction to Hematology
LIQUID COMPONENT (CALLED PLASMA OR SERUM)
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COMMON PREFIXES USED IN HEMATOLOGY
PREFIXES
a-/ ananisoantecrenacytdyserythroferrhemo- (hemato-)
hypo-
composed mostly of water
dissolved elements include carbohydrates, proteins,
lipids, non-protein nitrogenous compounds (NPNs),
vitamins, electrolytes, trace elements, hormones, and
gases.
Reaction: slightly alkaline
o Arterial blood pH: 7.35-7.45
o Venous blood pH: 7.38-7.48
Osmolality: 280-295 mOsm/kg
Average specific gravity: 1.055
Composition of Blood (image)
o Plasma – 55%
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Water (91.5%)
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Proteins (7%)
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Other solutes (1.5%)
o White blood cells and platelets – 4%
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“Buffy coat”
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White blood cells > Lymphocytes, Granulocytes,
Monocytes
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Granulocytes
>
Basophils,
Neutrophils,
Eosinophils
o Red blood cells – 41%
o
▪
hyperisoleuk(o)macromegametamicromyel(o)panphlebphagopoikiloPolyPre-(pro-)
PyknoReticuloSchisSclerSideroSpleenThromboXanth-
A blood sample after centrifugation
MEANING
lack, without, absent, decreased
unequal, dissimilar
before
wrinkled
cell
abnormal, difficult
red
Iron
pertaining to the blood
beneath,
under,
deficient,
decreased
above, beyond, extreme
equal, alike, same
white
large, long
large, giant
(1) after, next; (2) change
small
(1) from bone marrow; (2) spinal
cord
all, overall, all-inclusive
vein
Eat, ingest
Varied, irregular
Many
Before
Dense
Netlike
Split
Hard
Iron
Spleen
Clot, thrombus
Yellow
COMMON SUFFIXES USED IN HEMATOLOGY
SUFFIX
-algia
-ase
-cide
-crit
-cyte
-ectomy
-emia
-it is
-lysis
-oma
-opathy
-osis
o
DIFFERENTIATION BETWEEN PLASMA AND SERUM
PLASMA
Liquid part of
anticoagulated and
unclotted blood
Hazy
All Coagulation Factors
arepresent (Fresh
Plasma):FI, FII, FV, FVII,
FVIII:c, FIX, FX, FXI,
FXII, FXIII, PK, HMWK
SERUM
Liquid part of clotted blood
Clearer
Clotting Factors that are
consumed in Coagulation:
FI / I(Fibrinogen)
FV / V (Proaccelerin)
FVIII:c /VIII:c (Anti-Hemophilic
Factor A)
FXIII / XIII (Fibrin Stabilizing
Factor)
Only residual amount (less
than 20%) of FII / II
(Prothrombin) is present
-penia
-phil(ic)
-plasia (-plastic)
-poiesis
-poietin
MEANING
Pain along a nerve
An enzyme
The killer of
To separate
Cell
Incision and removal
Blood
Inflammation
Destruction or dissolving
Swelling or tumor
Disease
(1) Abnormal increase; (2)
disease
Deficiency, decreased
Attracted to, affinity for
Cell production or repair
Cell production, formation, and
development
Stimulates production
DEFINITION OF TERMS
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Hematology (Haematology)
o the study of blood. It involves the study of blood cells
and coagulation. It includes the study of diseases
associated with the blood, as well as the reaction of the
formed elements to the presence of disease.
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Module 1: Introduction to Hematology
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Agglutination
o clumping of cells
Aggregation
o in blood coagulation, it is the clumping of platelets
together in the formation of a platelet plug
Anisochromia
o variation in hemoglobin content of red blood cells
Anisocytosis
o increased variation in size of red blood cells
Anticoagulant
o substance that prevents clot formation; in vivo,
includes natural anticoagulants such as Heparin, Antithrombin III, and Proteins C and S; ex vivo, substances
that are added to a blood sample that inhibit clot
formation either by binding with calcium, precipitation
of calcium, inhibition of thrombin, or removal of fibrin.
Apoptosis
o programmed cell death; process of ordered removal of
organelles and cells
Arterial tap
o process of blood collection by accessing an artery
Basophil
o white blood cell morphologically characterized by
coarse blue-black granules that obscure the view of the
nucleus; involved mainly in mediating allergic
response.
Blast
o early stage of differentiation of a blood cell as it
transitions from stem cell to a mature cell. It is normally
confined to the bone marrow and is the earliest
recognizable stage of a blood cell using Light
Microscopy.
Blood Film
o aka Peripheral Blood Smear; a stained smear of a drop
of blood that, when viewed through a microscope,
produces additional morphologic information about the
blood cells
Bone Marrow
o soft tissue found inside hollow bones responsible for
production of blood cells.
Cellularity
o expression of the amount of blood cells within the bone
marrow
Chemotaxis
o movement of white blood cells toward or away from the
source of a chemical gradient
Clotting factors
o aka Pro-clotting Factors or Procoagulants; specialized
proteins that, when activated, form an interplay that
effects coagulation
Coagulation
o aka clot formation or clotting; process of clot formation
through the interaction of specialized proteins in
plasma culminating in the conversion of fibrinogen to
fibrin
Coagulopathy
o hereditary or acquired abnormality of blood coagulation
Complete Blood Count (CBC)
o test performed in the hematology laboratory that
determines red blood cell count, white blood cell count
and differential count, platelet count, hemoglobin
concentration and hematocrit of a patient
Cytopenia
o a reduction in number of one or more cell types in the
blood
Deoxyhemoglobin
o
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
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aka reduced hemoglobin; hemoglobin that is not
carrying oxygen
Dyserythropoiesis
o abnormal red blood cell development
Ecchymosis
o (pl: ecchymoses); bruising caused by leakage of blood
from blood vessels
EDTA
o ethylenediaminetetraacetate / -tetra acetic acid; most
common anticoagulant used for hematological studies,
especially for CBC
Embolus
o a blood clot that migrates through the bloodstream and
lodges into another vessel, causing blockage of blood
flow
Eosinophil
o white blood cells morphologically characterized by
bilobed nuclei and coarse orange granules; involved
mainly in anti-helminthic immune response and
regulating allergic response
Erythrocytes
o aka red blood cells; cells that contains hemoglobin and
carries oxygen through the blood
Erythropoietin
o glycoprotein hormone produced by the kidneys in
response to tissue hypoxia; targets red blood cell
precursors in the bone marrow to stimulate proliferation
and maturation
Fibrinolysis
o process of dissolution of the clot affected by the action
of plasmin (fibrinolysin)
Hematoma
o accumulation of blood in the tissues or cavities of the
body
Hematocrit
o relative expression of the amount of red blood cells in
relation to the amount of whole blood in a sample (in
vitro) or total body fluids (in vivo)
Hematopoiesis / Hemopoiesis
o involves the production, development, differentiation,
and maturation of blood cells in a blood-forming organ
or tissue
Hemoglobin
o oxygen-binding protein found within red blood cells
Hemolysis
o inappropriate destruction of red blood cells
Hemorrhage
o Excessive bleeding leading to leakage of blood from
the vessels to the surrounding tissues and brought
about by a breakdown of hemostasis
Hemostasis
o process of arresting blood flow to a site of vessel injury;
also, the system that keeps the blood in a fluid state
under normal conditions
Hypoxia
o lack of oxygen experienced by the tissues; physiologic
stimulus for production of red blood cells
Leukocytes
o aka white blood cells; blood cells that are involved in
the body’s immune response
Lymphocytes
o Smallest of the white blood cells; morphologically
characterized by a round nucleus and scanty sky blue
cytoplasm; involved in anti-viral immune response and
antibody production
BADONG, BONI, CENIZAL, DE GUZMAN, DE LIMA, DELOS REYES, ESPINOLA, MANGOHIG, PATAG, POSO, SALVADOR | 3D-MT
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Module 1: Introduction to Hematology
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•
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•
•
•
•
•
•
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•
•
•
•
•
•
•
•
•
•
Macrophages
o Phagocytes found in tissues
Monocytes
o white blood cells morphologically characterized by a
round or irregularly-shaped nucleus and agranular light
blue
cytoplasm; considered as professional
phagocytes of the blood; develop into macrophages in
the tissues
Neutrophils
o white blood cells morphologically characterized by
multilobed nuclei and neutral (lilac or purple) staining
of the cytoplasm; involved mainly in anti-bacterial
immune response and inflammatory response
Oxyhemoglobin
o hemoglobin with oxygen bound to it
Peripheral blood
o blood that is contained within the circulatory system
Peripheral puncture
o aka capillary puncture; process of blood collection via
skin puncture
Petechiae
red pinpoint-sized hemorrhages of small capillaries in
the skin or mucus membranes
Phagocytosis
o process of engulfment and destruction of foreign and
unwanted material (such as bacteria or senescent red
blood cells)
Phlebotomy
o process of blood collection
Plasma
o liquid portion of unclotted blood or anticoagulated
blood
Serum
o liquid portion of clotted blood
Supravital stain
o dyes employed in staining cellular elements while the
cell is in its living state (eg. New Methylene Blue,
Brilliant Cresyl Blue)
Thrombocytes
o aka platelets; cellular elements that are involved in
promoting hemostasis
Thrombosis
o inappropriate or pathological formation of a clot in an
artery or vein
Thrombus
o blood clot that usually develops in a deep vein of the
body
Venipuncture
o process of blood collection via intravenous access
Wright stain
o a type of Romanowsky stain; most common stain used
for studying blood cell morphology
SPECIMEN CONSIDERATIONS ON HEMATOLOGY
PATIENT IDENTIFICATION
considered as the most critical step in Specimen collection
Identify the patient by asking the patient to state his/her full
name (including middle name); if the patient cannot speak,
verify the patient’s identity from the relative, attending
physician, or nurse
Whenever possible, verify a 2ndpatient identifier (eg. Date
of Birth/ Social Security Number) for a positive patient
identification
PHYSIOLOGIC FACTORS AFFECTING TEST
RESULTS:
FACTORS
Posture
Diurnal Rhythm
Stress
Exercise
Diet
Smoking
EFFECTS
Shift in posture from supine to
erect may cause an increase in
the levels of Lipids, Enzymes,
and Proteins
In the afternoon, decreased
levels of ACTH and cortisol and
increased levels of eosinophils
may be seen
Causes an increase in WBC
counts, FI, FV, FVIII:c, and FXIII
Causes an increase in WBC and
platelet
counts,
Creatinine
Kinase (CK), and Lactate
Dehydrogenase (LD)
After a fatty meal, increased
Alkaline phosphatase (AP) may
be seen, false increase in
hemoglobin
using
the
cyanmethemoglobin method
Increases cortisol and WBC
counts; Chronic smoking may
increase
RBC
count,
hemoglobin, and hematocrit
SOURCES OF BLOOD FOR HEMATOLOGY TESTING
•
•
•
Skin Puncture/ Peripheral Puncture
o source of peripheral blood (contains a mixture of
capillary blood from the capillaries, venous blood from
the venules, arterial blood from the arteries, and
interstitial and intercellular fluids)
o used when only small quantities of blood sample are
required
Indicators of Skin Puncture
o Infants/neonates/pediatric patients
o Geriatric patients
o Adults who are/have
▪
Extremely obese
▪
A
history
of
thromboembolism,
stroke,
Disseminated
Intravascular
Coagulation
/Coagulopathy (DIC), or Transient Ischemic Attack
(TIA); or
▪
Extensive burns
o If the examination requested necessities skin puncture
(bleeding time, micro methods of clotting time)
Recommended sites for Skin Puncture:
o Distal portion on the palmar surface of the 3rd or 4th
digits of the non-dominant hand
▪
The middle and ring fingers are usually the least
used and are less calloused compared to the
thumb and index fingers
o For infants, the plantar surface of the foot/sole (heel
portion)
o
o
Traditionally, the free margin of the earlobe was used
since there was less tissue juice and there were less
nerve endings causing less pain. This is no longer
recommended due to less capillary access.
BADONG, BONI, CENIZAL, DE GUZMAN, DE LIMA, DELOS REYES, ESPINOLA, MANGOHIG, PATAG, POSO, SALVADOR | 3D-MT
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Module 1: Introduction to Hematology
o
•
Important considerations when performing skin
puncture:
▪
Depth of puncture should be 2-3mm for adults;
<2mm for infants and small children
▪
not milk or squeeze the site of puncture to avoid
contamination with interstitial fluids
▪
Do not puncture cyanotic, swollen, inflamed, or
edematous sites
▪
You may warm the site by massage, warm
compress, or warm water bath prior to disinfection
o Order of Draw for Skin Puncture:
▪
Tube for blood gas analysis
▪
Slides
▪
EDTA micro collection tubes (must be filled before
other micro collection tubes to ensure adequate
blood volume for testing)
▪
Other micro collection tubes with anticoagulants
▪
Serum micro collection tubes
Venipuncture
o source of venous blood; preferred when large volumes
of blood are required for testing.
o preferred sites of puncture are the veins in the
antecubital area: median cubital vein (vein of choice),
cephalic vein, and the basilic vein
o Important
considerations
when
performing
venipuncture:
▪
Angle between the skin and the needle:
15degrees
▪
Tourniquet application: Distance from the
venipuncture site should be 3-4inches (7.5-10cm);
Length of time should be at 1 minute (maximum)
▪
Prolonged
tourniquet
application
causes
hemoconcentration, hemolysis, and shortened
coagulation time.
▪
Release the tourniquet once blood enters the hub.
o A phlebotomist must never puncture the patient more
than twice. After the second attempt, the phlebotomist
must endorse to a more senior or more experienced
staff.
o
Standard needle for phlebotomy: 21G
o Most common needle lengths: 1.0 and 1.5 inches
o Common causes of hemolysis:
▪
Prolonged tourniquet application
▪
Moisture or contamination on blood collection
tubes
▪
Using needles with too small bores
▪
Excessive agitation during mixing of samples
▪
Introduction of bubbles (frothing) during blood
collection.
ADDITIVES
ADDITIVE
COLOR
NOTES
CODE
Antiglycolytic Agents (MOA: inhibits glycolysis)
Sodium Fluoride Gray Top Preserves glucose for up
to 3 days
For Blood glucose and
blood
alcohol
level
determinations
Anticoagulant
used:
Potassium Oxalate
Number of Inversions: 8
Lithium
Gray Top Preserves glucose for up
Iodoacetate
to 24 hours
For blood glucose and
blood
alcohol
determinations
Anticoagulant
used:
Potassium Heparin
Number of Inversions: 8
Clot Activators – accelerates clotting of samples
Glass tube or Red Top
Used for STAT serum
silica particles
determinations
Number of Inversions: 5
Thrombin
Orange
MOA: Cleaves Fibrinogen
Top
into Fibrin
Used for STAT serum
determinations
Number or Inversions: 5
Separator Gel – inert material that undergoes a
temporary change in viscosity during the centrifugation
process
Thixotropic Gel
Gold Top
Number of Inversions: 5
Anticoagulants – prevent the blood from clotting
EDTA
Lavender Optimal
anticoagulant
Top
concentration: 1.5-2.2mg /
mL of blood
MOA: chelates calcium
Used
for
routine
hematological
studies
(most commonly used
anticoagulant
for
Hematology testing)–CBC,
Reticulocyte
Count,
modified
Westergren
method of ESR Important
considerations:
•K2EDTA,
K3EDTA,
LiEDTA, or Na2EDTA can
be used (K2EDTA is the
most preferred) but never
use Na3EDTA
•Insufficient
EDTA
(overfilled
tube)-effect:
Presence of clots (or micro
clots)
•Excessive
EDTA
(underfilled tube) effects:
falsely
decreased
Hematocrit and ESR,
degenerative changes in
WBC,
and
falsely
increased MCHC and
platelet count
•When platelet clumping
(platelet aggregation) is
encountered
when
examining the smear,
recollect a blood sample
using 3.2% sodium citrate
and repeat the platelet
count
Number of inversions: 8
Heparin
Green
Optimal
anticoagulant
Top
concentration: 15-20 units/
mL of blood
MOA: inhibits thrombin
Anticoagulant of choice for
Osmotic Fragility Testing
and Blood Gas Analysis
Also used for STAT
Chemistry determinations
Three formulations:
1. Ammonium Heparin
BADONG, BONI, CENIZAL, DE GUZMAN, DE LIMA, DELOS REYES, ESPINOLA, MANGOHIG, PATAG, POSO, SALVADOR | 3D-MT
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Module 1: Introduction to Hematology
3.2%
citrate
sodium
Light blue
top
2. Sodium Heparin
3. Lithium Heparin causes
the least interference in
chemistry testing; most
commonly
used
anticoagulant for plasma
and whole blood chemistry
assays.
Important considerations:
•Lithium Heparin must not
be used for lithium
determinations
•Sodium Heparin must not
be used for sodium
determinations
or
electrolyte panel
•Ammonium Heparin must
not be used for ammonia
level determinations
•Heparin induces cellular
clumping, especially of
platelets, causing a false
increase in WBC count,
and falsely decreased
platelet count
•Not to be used for blood
smear
preparation
because
it
causes
morphologic distortion of
platelets and WBCs as
well
as
causes
background staining with
Romanowsky stains such
as Wright’s and Giemsa
•Not to be used for
coagulation
studies
because
it
inhibits
Thrombin
Number of inversions: 8
Critical ratio of blood to
anticoagulant: 9:1
MOA: binds to calcium in
an unionized form
Used for coagulation tests
(eg. PT and PTT/APTT)
Important consideration:
forceful
mixing
and
excessive inversions can
activate platelets and
shorten clotting times
Sodium Fluoride
Tubes
NUMBER OF
INVERSIONS
8
•
Tan
top
(certified
to
contain
less
than 0.01 ug/mL
of Lead)
Royal blue top
(contains very
low levels of
trace elements)
White Top
Black top
Pink top (with
special
crossmatch
label
approached by
AABB)
•
•
3-4
•
Light blue top
•
0
5
Heparin Tubes
EDTA Tube
COLOR CODE
Yellow top
8
8
Red – Glass (no
additive)
Red – Plastic (clot
activator)
Green top
Lavender top
Gray top
OTHER BLOOD COLLECTION TUBES
BLOOD
COLLECTION
TUBE
Yellow top
ORDER OF DRAW
BLOOD
COLLECTION
TUBE
Blood
Culture
Tubes (SPS)
Tubes
for
Coagulation
Testing
(3.2%
sodium acetate)
Serum Tubes
8
•
ANTICOAGULA
NT
Sodium
polyanethol
sulfonate
Acid Citrate
Dextrose
K2EDTA
USES
Blood culture
Blood bank tests,
paternity
testing,
HLA testing, DNA
testing
Lead determination
K2EDTA
Trace Elements
Nutritional
Chemistry
K2EDTA with Gel
separator
3.8% sodium
citrate
Molecular tests
K2EDTA
Westergren method
of
ESR
determination
Blood Bank Test
(crossmatching)
SAMPLE PREPARATIONS USED IN HEMATOLOGY
LABORATORY
Whole blood: prepared by collecting blood into a tube with
an anticoagulant. Whenever the tests require whole blood,
the sample must be well mixed prior to aliquoting. Used for
Osmotic Fragility Testing and ESR testing
o If the aliquot from the top part of the sample, it will be
plasma and if from the bottom, it is red blood cells.
Plasma: prepared by collecting blood into a tube containing
an anticoagulant, followed by centrifugation mixing
o Platelet-Poor Plasma (PPP): contains less than 10,000
platelets/uLand is prepared by a hard/heavy spin
(centrifugation at 1500-2000G for 10 minutes). Used
for coagulation tests.
▪
Prothrombin Time
▪
(Activated) Partial Thromboplastin Time
▪
Thrombin Time
▪
Reptilase Time
▪
Russell Viper Venom Clotting Time
Platelet-Rich Plasma (PRP): contains 200,000-300,000
platelets/uL and is prepared by a light/soft spin:
centrifugation at 50-100G for 10 minutes. Used for platelet
function tests (platelet aggregometry)
Serum: prepared by collecting blood into a tube containing
no anticoagulant. The blood is allowed to clot and then
centrifuged. The liquid portion is used for Prothrombin
consumption test.
Blood Smear: Prepared by spreading a drop of blood over
the surface of a slide. The smear is allowed to dry and
stained then examined microscopically. Used for Peripheral
blood smear examination, WBC Differential Count and
Reticulocyte Count.
BADONG, BONI, CENIZAL, DE GUZMAN, DE LIMA, DELOS REYES, ESPINOLA, MANGOHIG, PATAG, POSO, SALVADOR | 3D-MT
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Module 1: Introduction to Hematology
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•
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Buffy Coat Smear: Prepared in the same manner as a blood
smear but makes use of the buffy coat for smearing. Used
for WBC Differential counting in leukopenic patients (WBCs
are concentrated in the buffy coat), studies of abnormal cell
morphology (eg. Lupus Erythematosus cell or LE cell), and
diagnosis of blood-borne parasitic infections.
o Leukopenic patients have low WBC count.
o Buffy coat smear increase the sensitivity of WBC and
determined it easily in a test.
Defibrinated blood: Blood is collected in a flask with glass
beads or paper clips. The flask is continuously agitated until
a clot form on the surface of the beads or clips. The clot is
removed and the part that remains is defibrinated blood.
Used for special hematological tests.
o This will be mixed until the sound of the beads or clips
stop.
o The sound will stop because the clips are already
trapped within the fibrin clot.
o The liquid portion will be the defibrinated blood.
Fresh blood as it comes from skin puncture: used for
bleeding time and micro methods of clotting time.
•
•
SPECIMEN COLLECTION
Specimen collection is a pre-analytic variable that may have
serious implications in laboratory testing. According to
studies done in recent years, 32-75% of testing errors occur
during the pre-analytic phase.
•
PRE-COLLECTION VARIABLE
PHYSIOLOGIC FACTORS
•
Influence laboratory determinations. These includes:
o Diurnal variation: most commonly encountered when
testing for hormones, ACP, and urinary excretion of
electrolytes.
o Exercise: Physical activity may have both long-term
and transient effects on the patient. Exercise may
activate coagulation, fibrinolysis and platelets, but such
activation is due to increased metabolic activity and
returns to normal (pre-exercise) levels soon after
cessation of exercise.
o Diet: An individual’s diet may greatly affect laboratory
test
results,
notably
for
blood
chemistry
determinations.
o Stress: mental and physical stress has been
implicated in production of ACTH, cortisol, and
catecholamines. Total cholesterol has been
reported to increase with stress while HDL-chole has
been observed to decrease.
Hyperventilation
increases WBC count.
o Posture: supine or upright position of the patient during
specimen collection should be taken note of as this
could affect laboratory tests. Upright position of
the patient decreases plasma volume, thus there is an
increase in plasma concentrations of
proteins.
Hemoglobin
concentrations
of patients may
decrease after bed rest in the hospital.
o Age: Generally, four age groups have been
defined: Newborn, Childhood to Puberty, Adult, and
Elderly Adults. Most tests have different reference
values with regard to each age group. With some
tests, the differences are more pronounced with
each age group, compared to others.
o Gender: differences of reference values also exist
between men and women.
NON-PHYSIOLOGICAL INTERFERENCES
In vivo: Tobacco Smoking
o Tobacco smokers generally have high blood
concentrations
of carboxyhemoglobin, plasma
catecholamines, and cortisol.
These hormonal
changes also result in lower eosinophil counts and
higher neutrophil and monocyte counts. Chronic
smoking leads to increase Hgb, RBC count, MCV, and
WBC count.
In vitro (Collection-Associated Variables)
o Hemolysis: mechanical hemolysis may occur as a
result of:
▪
Difficult extraction
▪
Needle bore size too small
▪
Pulling a plunger too fast
▪
Improper transfer of blood to the tubes
▪
Vigorous shaking or mixing of tubes, or
▪
Performing venipuncture without allowing the
alcohol to dry.
o Hemoconcentration/Hemodilution: can occur due to
positional changes. Such problems can be avoided by
allowing the patient to sit in a supine position 15-20
minutes prior to drawing blood. Tourniquet application
must also not exceed one (1) minute to avoid
concentrating the blood. When using anticoagulants,
the order of draw, appropriate blood to anticoagulant
ratio, and adequate mixing should be observed,
In vitro (Non-Collection-Associated Variables)
o In vivo hemolysis: may be due to hemolytic anemias or
hemolytic transfusion reactions, among others. In vivo
hemolysis may be differentiated from collectionassociated hemolysis by observing the serum or
plasma from subsequent repeat collections.
o Lipemia: may be observe shortly after a high-fat meal
or due to high-fat diet.
o Icterus: may be observed in patients with high
concentrations of bilirubin (≥2.5 mg/dL)
SPECIMEN COLLECTION
ANTICOAGULANTS
USED
IN
HEMATOLOGY
LABORATORY
•
•
EDTA/versene/sequestrene: chelates calciumK2EDTA is
the preferred form due to its solubility, followed by K3EDTA
(or Li2EDTA), and Na2EDTA. Na3EDTA is not
recommended due to its high pH.1.5-2.2 mg EDTA per
mL of blood is the recommended ratio.
o Uses:
▪
Cell counts (RBC, WBC, Platelets)
▪
RBC parameters (Hgb, Hct)
▪
RBC indices (MCV, MCH, MCHC)
▪
ESR determination
▪
Peripheral blood smear*
▪
Differential count*
o Disadvantages:
▪
Not recommended for coagulation testing due to
interference of the anticoagulant with Fibrin
formation and instability of FV and FVIII:c in
EDTA.
▪
Using
old (>2
hours) blood
will
cause
morphological changes: such as Crenation of
RBCs, Vacuolization of WBCs, Formation
of
artifacts and crystals and phagocytosis of these
crystals, Nuclear changes in WBCs (eg.
separated PMN nuclei), and Disintegration of
platelets
Citrate: chelates calcium. May be 0.105M (3.13%) or
0.109M (3.2%) trisodium citrate (Na3C6H5O7•2H2O) in a
9:1
blood-to-anticoagulant
ratio.
0.129M
(3.8%)
BADONG, BONI, CENIZAL, DE GUZMAN, DE LIMA, DELOS REYES, ESPINOLA, MANGOHIG, PATAG, POSO, SALVADOR | 3D-MT
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Module 1: Introduction to Hematology
•
•
concentrations are no longer recommended because
it affects coagulation test results.
o Uses:
▪
Standard Westergren method of ESR: blood to
anticoagulant ration is 4:1
▪
Coagulation
testing (using
platelet
poor
plasma+)
▪
Modified Westergren method of ESR for blood
collected with EDTA
▪
Platelet aggregometry tests (using platelet rich
plasma++)
▪
Platelet counts when platelet satellitism or
clumping is encountered
Heparin: accelerates antithrombin action to inhibit thrombin
formation; inhibits thromboplastin. Use 15-30 units per mL
of blood.
o Uses:
▪
RBC count
▪
RBC parameters
▪
Osmotic fragility
o Disadvantages:
▪
Causes agglutination of WBC
▪
Causes aggregation of platelets
▪
Not recommended for coagulation tests
because it affects all stages of coagulation
▪
Produces a blue background with Wright’s stain
▪
Expensive
Oxalate: chelates calcium. May be used as Na2C2O4,
Li2C2O4, or a mixture of K2C2O4and (NH4)2C2O4. K2C2O4(NH4)2C2O4 mixture (in a 2:3 ratio) is also known as double
oxalate, balanced oxalate, Paul-Heller’s fluid, or wintrobe
fluid. It is prepared as a mixture because K2C2O4 causes
shrinkage of RBCs while (NH4)2C2O4 causes swelling of
RBCs.
o Uses:
▪
Coagulation Testing
▪
Lithium oxalate is used to prevent clotting of
bloody body fluids
▪
Double oxalate can be used for: RBC count, RBC
parameters, and ESR determination
▪
smear has to be prepared within 2 hours of
collection to prevent morphological changes in the
blood cells.
▪
+Platelet-poor plasma (PPP) contains <10,000
platelets/μL and is prepared by heavy spin:
centrifugation at 1500-2000g for 10 minutes.
▪
++Platelet-rich plasma (PRP) contains 200,000300,000 platelets/μL and is prepared by light spin:
centrifugation at 50-100gfor 10 minutes.
o Disadvantages:
▪
Causes agglutination of WBC
▪
Causes aggregation of platelets
▪
Produces the same morphologic changes seen
when using old EDTA-anticoagulated blood.
ORDER OF DRAW
•
•
•
•
•
•
blood culture tubes (yellow)
coagulation tubes ctg. citrate (blue)
serum tubes with or without gel separator
heparin tubes with or without gel separator (green)
EDTA tubes (lavender)
Fluoride tubes (gray)
SOURCES OF BLOOD FOR HEMATOLOGY TESTING
•
•
•
•
•
•
•
•
•
•
•
Skin/Peripheral puncture
o source of peripheral blood (contains a mixture of blood
from venules, arterioles capillaries, as well as
interstitial and intercellular fluids)
o Finger (ring or middle finger)
▪
least used
o Ear lobe (free margin): less nerve endings
▪
less tissue juices
▪
no longer recommended due to less capillary
access
o Heel: for newborns and infants
o Big toe: for infants
▪
depth of puncture should be 2-3 mm
▪
do not milk the site to avoid contamination with
tissue juice
▪
do not puncture cyanotic, swollen, inflamed, and
edematous sites.
▪
Warm the site by massage, warm compress, or
warm water bath
Venipuncture
o Source of venous blood
o Median cubital vein
o Cephalic cubital vein
o Basilic cubital vein
INDICATIONS OF SKIN PUNCTURE
all examinations except those that are indicated by skin
puncture, especially when a large amount of blood is
needed for testing
Infants/neonates/pediatric patients
Geriatric patients
Adults who are/have:
o Extremely obese
o History of thromboembolism, stroke, or DIC
o Extensive burns
Examination requested necessitates skin puncture
(bleeding time, micro methods of clotting time)
PATIENT CONSIDERATIONS DURING
VENIPUNCTURE
Identification
o Ask the patient to state his/her COMPLETE name. (Do
not volunteer the patient’s name)
o Verify the name of the patient with the wrist tag
o If the patient only speaks a foreign language or cannot
speak, verify the patient’s name with the companion,
nurse or the doctor.
Isolation restrictions
o Isolation
▪
for patients with contagious disease.
▪
Everything is left in the patient’s room after
obtaining the specimen (gloves, gown, mask,
etc.)
o Reverse Isolation
▪
for patients who are immunocompromised
▪
Nothing should be left in the patient’s room after
obtaining the specimen
Note the time of extraction.
o WBC count is noted to be increased in the morning
while RBC count, Hemoglobin, Hematocrit are
decreased.
If both arms are receiving intravenous (IV) infusion,
o Ask the doctor or nurse to stop infusion for at least 2
minutes. Discard the first tube of blood collected.
o Collect below the IV line. Discard the first tube
collected.
BADONG, BONI, CENIZAL, DE GUZMAN, DE LIMA, DELOS REYES, ESPINOLA, MANGOHIG, PATAG, POSO, SALVADOR | 3D-MT
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Module 1: Introduction to Hematology
o
•
Collect from the lower extremities, if and only if, the
patient is not diabetic.
Respect patient’s rights:
o Right to refuse extraction
o Right to confidentiality
▪
Confidentiality of results
▪
Confidentiality of identity
o Rights to be informed
▪
As to purpose of testing
▪
As to results of tests
o
ORDER FORM/REQUISITION
•
COMPLICATIONS OF VENIPUNCTURE
•
•
•
•
•
•
•
Hematoma
Hemoconcentration
Hemolysis
Syncope
ROUTINE VENIPUNCTURE AND SPECIMEN
HANDLING
VENIPUNCTURE PROCEDURE
The venipuncture procedure is complex, requiring both
knowledge and skill to perform. Each phlebotomist
generally establishes a routine that is comfortable for her or
him.
Phlebotomists are considered to have occupational
exposure to blood borne pathogens. The performance of
routine vascular access procedures by skilled
phlebotomists requires, at a minimum, the use of gloves to
prevent contact with blood. Laboratory coats or work
smocks are not typically needed as personal protective
equipment during routine venipuncture, but an employer
must assess the workplace to determine whether certain
tasks, workplace situations, or employee skill levels may
result in an employee's need for laboratory coats or other
personal protective equipment to prevent contact with
blood. It is an employer's responsibility to provide, clean,
repair, replace, and/or dispose of personal protective
equipment/clothing. As part of presenting a professional
appearance, an institutional dress code may include
wearing of a laboratory coat or smock.
Several essential steps are required for every successful
collection procedure:
o Patient comfort. Is the seating comfortable and has the
patient been seated for at least 5 minutes to avoid
being rushed or confused?
o Carry out hand hygiene before and after each patient
procedure, before putting on and after removing
gloves.
o Identify the patient using two different identifiers,
asking open ended questions such as, "What is your
name?" and "What is your date of birth?"
o Assess the patient's physical disposition (i.e. diet,
exercise, stress, basal state).
o Check the requisition form for requested tests, patient
information, and any special requirements.
o Label the collection tubes at the bedside or drawing
area.
o Select a suitable site for venipuncture.
o Prepare the equipment, the patient and the puncture
site.
o Perform the venipuncture, collecting the sample(s) in
the appropriate container(s).
o Recognize complications associated with the
phlebotomy procedure.
o Assess the need for sample recollection and/or
rejection.
Promptly send the specimens with the requisition to the
laboratory.
A requisition form must accompany each sample submitted
to the laboratory. This requisition form must contain the
proper information in order to process the specimen. The
essential elements of the requisition form are:
o Patient's surname, first name, and middle initial.
o Patient's ID number.
o Patient's date of birth and sex.
o Requesting physician's complete name.
o Source of specimen. This information must be given
when requesting microbiology, cytology, fluid analysis,
or other testing where analysis and reporting is site
specific.
o Date and time of collection.
o Initials of phlebotomist.
o Indicating the test(s) requested.
o An example of a simple requisition form with the
essential elements is shown below:
o
•
•
•
•
LABELLING THE SAMPLE
A properly labeled sample is essential so that the results of
the test match the patient. The key elements in labeling are:
o Patient's surname, first and middle.
o Patient's ID number.
o NOTE: Both of the above MUST match the same on
the requisition form.
o Date, time and initials of the phlebotomist must be on
the label of EACH tube.
Automated systems may include labels with bar codes.
Examples of labeled collection tubes are shown below:
EQUIPMENT
The following are needed for routine venipuncture:
o Evacuated Collection Tubes - The tubes are designed
to fill with a predetermined volume of blood by vacuum.
The rubber stoppers are color coded according to the
additive that the tube contains. Various sizes are
available. Blood should NEVER be poured from one
tube to another since the tubes can have different
additives or coatings (see illustrations at end).
o Needles - The gauge number indicates the bore size:
the larger the gauge number, the smaller the needle
BADONG, BONI, CENIZAL, DE GUZMAN, DE LIMA, DELOS REYES, ESPINOLA, MANGOHIG, PATAG, POSO, SALVADOR | 3D-MT
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Module 1: Introduction to Hematology
o
o
o
o
o
o
o
o
o
•
•
bore. Needles are available for evacuated systems and
for use with a syringe, single draw or butterfly system.
Holder/Adapter - use with the evacuated collection
system.
Tourniquet - Wipe off with alcohol and replace
frequently.
Alcohol Wipes - 70% isopropyl alcohol.
Povidone-iodine wipes/swabs - Used if blood culture is
to be drawn.
Gauze sponges - for application on the site from which
the needle is withdrawn.
Adhesive bandages / tape - protects the venipuncture
site after collection.
Needle disposal unit - needles should NEVER be
broken, bent, or recapped. Needles should be placed
in a proper disposal unit IMMEDIATELY after their use.
Gloves - can be made of latex, rubber, vinyl, etc.; worn
to protect the patient and the phlebotomist.
Syringes - may be used in place of the evacuated
collection tube for special circumstances.
•
•
•
•
ORDER OF DRAW
The phlebotomist's role requires a professional, courteous,
and understanding manner in all contacts with the patient.
Greet the patient and identify yourself and indicate the
procedure that will take place. Effective communication both verbal and nonverbal - is essential.
Proper patient identification MANDATORY. If an inpatient is
able to respond, ask for a full name and always check the
armband or bracelet for confirmation. DO NOT DRAW
BLOOD IF THE ARMBAND OR BRACELET IS MISSING.
For an inpatient the nursing staff can be contacted to aid in
identification prior to proceeding.
An outpatient must provide identification other than the
verbal statement of a name. Using the requisition for
reference, ask a patient to provide additional information
such as a surname or birthdate. A government issued photo
identification card such as a driver's license can aid in
resolving identification issues.
If possible, speak with the patient during the process. The
patient who is at ease will be less focused on the procedure.
Always thank the patient and excuse yourself courteously
when finished.
PATIENT'S BILL OF RIGHTS:
Blood collection tubes must be drawn in a specific order to
avoid cross-contamination of additives between tubes. The
recommended order of draw for plastic collection tubes is:
o First - blood culture bottle or tube (yellow or yellowblack top)
o Second - coagulation tube (light blue top). If just a
routine coagulation assay is the only test ordered, then
a single light blue top tube may be drawn. If there is a
concern regarding contamination by tissue fluids or
thromboplastins, then one may draw a non-additive
tube first, and then the light blue top tube.
o Third - non-additive tube (red top)
o Last draw - additive tubes in this order:
o SST (red-gray or gold top). Contains a gel separator
and clot activator.
o Sodium heparin (dark green top)
o PST (light green top). Contains lithium heparin
anticoagulant and a gel separator.
o EDTA (lavender top)
o ACDA or ACDB (pale yellow top). Contains acid citrate
dextrose.
o Oxalate/fluoride (light gray top)
NOTE: Tubes with additives must be thoroughly mixed.
Erroneous test results may be obtained when the blood is
not thoroughly mixed with the additive.
PROCEDURAL ISSUES
PATIENT RELATIONS AND IDENTIFICATION:
•
•
The Patient's Bill of Rights has been adopted by many
hospitals as declared by the Joint Commission on
Accreditation of Healthcare Organizations (JCAHO). The
basic patient rights endorsed by the JCAHO follow in
condensed form are given below.
The patient has the right to:
o Impartial access to treatment or accommodations that
are available or medically indicated, regardless of race,
creed, sex, national origin, or sources of payment for
care.
o Considerate, respectful care.
o Confidentiality of all communications and other records
pertaining to the patient's care.
o Expect that any discussion or consultation involving the
patient's case will be conducted discretely and that
individuals not directly involved in the case will not be
present without patient permission.
o Expect reasonable safety congruent with the hospital
practices and environment.
o Know the identity and professional status of individuals
providing service and to know which physician or other
practitioner is primarily responsible for his or her care.
o Obtain from the practitioner complete and current
information about diagnosis, treatment, and any known
prognosis, in terms the patient can reasonably be
expected to understand.
o Reasonable informed participation in decisions
involving the patient's health care. The patient shall be
informed if the hospital proposes to engage in or
perform
human
experimentation
or
other
research/educational profits affecting his or her care or
treatment. The patient has the right to refuse
participation in such activity.
o Consult a specialist at the patient's own request and
expense.
o Refuse treatment to the extent permitted by law.
o Regardless of the source of payment, request and
receive an itemized and detailed explanation of the
total bill for services rendered in the hospital.
o Be informed of the hospital rules and regulations
regarding patient conduct.
VENIOUNCTURE SITE SELECTION:
•
•
Although the larger and fuller median cubital and cephalic
veins of the arm are used most frequently, the basilic vein
on the dorsum of the arm or dorsal hand veins are also
acceptable for venipuncture. Foot veins are a last resort
because of the higher probability of complications.
Certain areas are to be avoided when choosing a site:
o Extensive scars from burns and surgery - it is difficult
to puncture the scar tissue and obtain a specimen.
o The upper extremity on the side of a previous
mastectomy - test results may be affected because of
lymphedema.
o Hematoma - may cause erroneous test results. If
another site is not available, collect the specimen distal
to the hematoma.
o Intravenous therapy (IV) / blood transfusions - fluid
may dilute the specimen, so collect from the opposite
arm if possible. Otherwise, satisfactory samples may
be drawn below the IV by following these procedures:
BADONG, BONI, CENIZAL, DE GUZMAN, DE LIMA, DELOS REYES, ESPINOLA, MANGOHIG, PATAG, POSO, SALVADOR | 3D-MT
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Module 1: Introduction to Hematology
▪
o
o
o
Turn off the IV for at least 2 minutes before
venipuncture.
▪
Apply the tourniquet below the IV site. Select a
vein other than the one with the IV.
▪
Perform the venipuncture. Draw 5 ml of blood and
discard before drawing the specimen tubes for
testing.
Lines - Drawing from an intravenous line may avoid a
difficult venipuncture, but introduces problems. The
line must be flushed first. When using a syringe
inserted into the line, blood must be withdrawn slowly
to avoid hemolysis.
Cannula/fistula/heparin lock - hospitals have special
policies regarding these devices. In general, blood
should not be drawn from an arm with a fistula or
cannula without consulting the attending physician.
Edematous extremities - tissue fluid accumulation
alters test results.
PROCEDURE FOR VEIN SELECTION:
•
•
Palpate and trace the path of veins with the index finger.
Arteries pulsate, are most elastic, and have a thick wall.
Thrombosed veins lack resilience, feel cord-like, and roll
easily.
If superficial veins are not readily apparent, you can force
blood into the vein by massaging the arm from wrist to
elbow, tap the site with index and second finger, apply a
warm, damp washcloth to the site for 5 minutes, or lower
the extremity over the bedside to allow the veins to fill.
•
•
•
•
•
•
•
When the last tube to be drawn is filling, remove the
tourniquet.
Remove the needle from the patient's arm using a swift
backward motion.
Press down on the gauze once the needle is out of the arm,
applying adequate pressure to avoid formation of a
hematoma.
Dispose of contaminated materials/supplies in designated
containers.
Mix and label all appropriate tubes at the patient bedside.
Deliver specimens promptly to the laboratory.
PHLEBOTOMY ILLUSTRATED:
Patient identification
o The first step is always to identify the patient.
Outpatient phlebotomy, as shown here, should take
place with the patient seated.
PERFORMANCE OF A VENIPUNCTURE
•
•
•
•
•
•
•
•
•
•
•
Approach the patient in a friendly, calm manner. Provide for
their comfort as much as possible, and gain the patient's
cooperation.
Identify the patient correctly.
Properly fill out appropriate requisition forms, indicating the
test(s) ordered.
Verify the patient's condition. Fasting, dietary restrictions,
medications, timing, and medical treatment are all of
concern and should be noted on the lab requisition.
Check for any allergies to antiseptics, adhesives, or latex
by observing for armbands and/or by asking the patient.
Position the patient. The patient should either sit in a chair,
lie down or sit up in bed. Hyperextend the patient's arm.
Apply the tourniquet 3-4 inches above the selected
puncture site. Do not place too tightly or leave on more than
2 minutes (and no more than a minute to avoid increasing
risk for hemoconcentration). Wait 2 minutes before
reapplying the tourniquet.
The patient should make a fist without pumping the hand.
Select the venipuncture site.
Prepare the patient's arm using an alcohol prep. Cleanse in
a circular fashion, beginning at the site and working
outward. Allow to air dry.
Grasp the patient's arm firmly using your thumb to draw the
skin taut and anchor the vein. The needle should form a 15
to 30 degree angle with the surface of the arm. Swiftly insert
the needle through the skin and into the lumen of the vein.
Avoid trauma and excessive probing.
•
•
•
•
•
•
•
o
Filling out the requisition
o The requisition form should be completely filled out,
and the requisition must indicate the tests ordered.
Equipment
o Here is the equipment for performing phlebotomy.
Barrier protection for the phlebotomist consists of the
latex gloves.
o
Apply tourniquet and palpate for vein
o The tourniquet is applied and the phlebotomist
palpates for a suitable vein for drawing blood.
Sterilize the site
o The area of skin is cleaned with a disinfectant, here an
alcohol swab.
Insert needle
o The vein is anchored and the needle is inserted.
Drawing the specimen
o The collection tube is depressed into the needle to
begin drawing blood.
Drawing the specimen
o Additional collection tubes can be utilized. Determine
what tests are ordered and what tubes will be
BADONG, BONI, CENIZAL, DE GUZMAN, DE LIMA, DELOS REYES, ESPINOLA, MANGOHIG, PATAG, POSO, SALVADOR | 3D-MT
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Module 1: Introduction to Hematology
•
•
•
•
•
necessary BEFORE you begin to draw blood, and
determine the order of draw for the tubes.
Releasing the tourniquet
o When the final tube is being drawn, release the
tourniquet. Then remove the tube, and remove the
needle.
Applying pressure over the vein
o After the needle is removed from the vein, apply firm
pressure over the site to achieve hemostasis.
Applying bandage
o Apply a bandage to the area.
Disposing needle into sharps
o Dispose of the needle into a sharps container that is
close by.
Labeling the specimens
o Label the tubes, checking the requisition for the proper
identification.
•
o
Proper location on finger
o The proper location on the 3rd or 4th finger of the nondominant hand for performing a fingerstick is outlined
here between the green lines. The puncture should be
made just off center and perpendicular to the
fingerprint ridges. (A puncture parallel to the ridges
tends to make the blood run down the ridges and
hamper collection.)
PERFORMANCE OF A FINGERSTICK
•
•
•
•
•
•
•
•
•
•
•
•
Follow the procedure as outlined above for greeting and
identifying the patient. As always, properly fill out
appropriate requisition forms, indicating the test(s) ordered.
Verify the patient's condition. Fasting, dietary restrictions,
medications, timing, and medical treatment are all of
concern and should be noted on the lab requisition.
Position the patient. The patient should either sit in a chair,
lie down or sit up in bed. Hyperextend the patient's arm.
The best locations for fingersticks are the 3rd (middle) and
4th (ring) fingers of the non-dominant hand. Do not use the
tip of the finger or the center of the finger. Avoid the side of
the finger where there is less soft tissue, where vessels and
nerves are located, and where the bone is closer to the
surface. The 2nd (index) finger tends to have thicker,
callused skin. The fifth finger tends to have less soft tissue
overlying the bone. Avoid puncturing a finger that is cold or
cyanotic, swollen, scarred, or covered with a rash.
Using a sterile lancet, make a skin puncture just off the
center of the finger pad. The puncture should be made
perpendicular to the ridges of the fingerprint so that the drop
of blood does not run down the ridges.
Wipe away the first drop of blood, which tends to contain
excess tissue fluid.
Collect drops of blood into the collection device by gently
massaging the finger. Avoid excessive pressure that may
squeeze tissue fluid into the drop of blood.
Cap, rotate and invert the collection device to mix the blood
collected.
Have the patient hold a small gauze pad over the puncture
site for a couple of minutes to stop the bleeding.
Dispose of contaminated materials/supplies in designated
containers.
Label all appropriate tubes at the patient bedside.
Deliver specimens promptly to the laboratory.
FINGERSTICK PROCEDURE ILLUSTRATED
•
Equipment
o Here is the equipment for fingersticks (heelsticks). The
lancets come in different lengths. There are several
standard small collection tubes utilized to collect
fingerstick (or baby heelstick) blood. The purple cap is
for hematology specimens and the green cap is for
chemistry specimens. The dark brown-red small
collection tube protects a neonatal bilirubin sample
from the light.
•
•
•
•
•
o
Puncture with lancet
o The lancet is placed over the proper location on the
finger and the puncture is made quickly.
Drop of blood
o A drop of blood appears at the puncture site.
Wipe first drop
o The first drop of blood that may contain tissue fluid is
wiped away.
Collecting the specimen
o The finger is gently massaged from base to tip and the
blood drops are collected into the proper collection
device.
Specimen container
o The blood is mixed in small collection tubes with an
additive.
o
ADDITIONAL CONSIDERATIONS
TO PREVENT HEMATOMA:
•
•
•
•
•
Puncture only the uppermost wall of the vein
Remove the tourniquet before removing the needle
Use the major superficial veins
Make sure the needle fully penetrates the upper most wall
of the vein. (Partial penetration may allow blood to leak into
the soft tissue surrounding the vein by way of the needle
bevel)
Apply pressure to the venipuncture site
TO PREVENT HEMOLYSIS (WHICH CAN INTERFERE
WITH MANY TESTS):
•
•
Mix tubes with anticoagulant additives gently 5-10 times
Avoid drawing blood from a hematoma
BADONG, BONI, CENIZAL, DE GUZMAN, DE LIMA, DELOS REYES, ESPINOLA, MANGOHIG, PATAG, POSO, SALVADOR | 3D-MT
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Module 1: Introduction to Hematology
•
•
•
•
Avoid drawing the plunger back too forcefully, if using a
needle and syringe, or too small a needle, and avoid
frothing of the sample
Make sure the venipuncture site is dry
Avoid a probing, traumatic venipuncture
Avoid prolonged tourniquet application or fist clenching.
INDWELLING LINES OR CATHETERS:
•
•
•
REASONS FOR CANCELING A LABORATORY TEST
•
•
Potential source of test error
Most lines are flushed with a solution of heparin to reduce
the risk of thrombosis
Discard a sample at least three times the volume of the line
before a specimen is obtained for analysis
HEMOCONCENTRATION:
•
•
•
•
•
An increased concentration of larger molecules and formed
elements in the blood may be due to several factors:
Prolonged tourniquet application (no more than 1 minute)
Massaging, squeezing, or probing a site
Long-term IV therapy
Sclerosed or occluded veins
A test that has been ordered may be cancelled due to
problems unrelated to drawing the specimen, and these are
the most common causes for cancellations:
o Duplicate test request
o Incorrect test ordered
o Test no longer needed
A test may be cancelled due to a technical problem in the
specimen collection process:
o Hemolysis of the specimen
o Clotted specimen
o Quantity of specimen not sufficient
o Collection of specimen in incorrect tube
o Contaminated specimen
o Identification of the specimen is suspect
o Delay in transport - specimen too old
SAFETY AND INFECTION CONTROL
•
Because of contacts with sick patients and their specimens,
it is important to follow safety and infection control
procedures.
PROTECT YOURSELF:
PROLONGED TOURNIQUET APPLICATION:
•
•
•
•
Primary effect is hemoconcentration of non-filterable
elements (i.e. proteins). The hydrostatic pressure causes
some water and filterable elements to leave the
extracellular space.
Significant increases can be found in total protein, aspartate
aminotransferase (AST), total lipids, cholesterol, iron
Affects packed cell volume and other cellular elements
Hemolysis may occur, with pseudohyperkalemia.
PATIENT PREPARATION FACTORS:
•
•
•
•
•
•
Therapeutic Drug Monitoring: different pharmacologic
agents have patterns of administration, body distribution,
metabolism, and elimination that affect the drug
concentration as measured in the blood. Many drugs will
have "peak" and "trough" levels that vary according to
dosage levels and intervals. Check for timing instructions
for drawing the appropriate samples.
Effects of Exercise: Muscular activity has both transient and
longer lasting effects. The creatine kinase (CK), aspartate
aminotransferase (AST), lactate dehydrogenase (LDH),
and platelet count may increase.
Stress: May cause transient elevation in white blood cells
(WBC's) and elevated adrenal hormone values (cortisol and
catecholamines). Anxiety that results in hyperventilation
may cause acid-base imbalances, and increased lactate.
Diurnal Rhythms: Diurnal rhythms are body fluid and
analyte fluctuations during the day. For example, serum
cortisol levels are highest in early morning but are
decreased in the afternoon. Serum iron levels tend to drop
during the day. You must check the timing of these
variations for the desired collection point.
Posture: Postural changes (supine to sitting etc.) are known
to vary lab results of some analytes. Certain larger
molecules are not filterable into the tissue, therefore they
are more concentrated in the blood. Enzymes, proteins,
lipids, iron, and calcium are significantly increased with
changes in position.
Other Factors: Age, gender, and pregnancy have an
influence on laboratory testing. Normal reference ranges
are often noted according to age.
•
•
•
•
Practice universal precautions:
o Wear gloves and a lab coat or gown when handling
blood/body fluids.
o Change gloves after each patient or when
contaminated.
o Wash hands frequently.
o Dispose of items in appropriate containers.
Dispose of needles immediately upon removal from the
patient's vein. Do not bend, break, recap, or resheath
needles to avoid accidental needle puncture or splashing of
contents.
Clean up any blood spills with a disinfectant such as freshly
made 10% bleach.
If you stick yourself with a contaminated needle:
o Remove your gloves and dispose of them properly.
o Squeeze puncture site to promote bleeding.
o Wash the area well with soap and water.
o Record the patient's name and ID number.
o Follow institution's guidelines regarding treatment and
follow-up.
o NOTE: The use of prophylactic zidovudine following
blood exposure to HIV has shown effectiveness (about
79%) in preventing seroconversion
PROTECT THE PATIENT:
•
•
Place blood collection equipment away from patients,
especially children and psychiatric patients.
Practice hygiene for the patient's protection. When wearing
gloves, change them between each patient and wash your
hands frequently. Always wear a clean lab coat or gown.
TROUBLESHOOTING GUIDELINES
IF AN INCOMPLETE COLLECTION OR NO BLOOD IS
OBTAINED:
•
Change the position of the needle. Move it forward (it may
not be in the lumen)
o
BADONG, BONI, CENIZAL, DE GUZMAN, DE LIMA, DELOS REYES, ESPINOLA, MANGOHIG, PATAG, POSO, SALVADOR | 3D-MT
17
Module 1: Introduction to Hematology
•
or move it backward (it may have penetrated too far).
•
•
•
•
•
•
•
•
o
Adjust the angle (the bevel may be against the vein wall).
o
Loosen the tourniquet. It may be obstructing blood flow.
Try another tube. Use a smaller tube with less vacuum.
There may be no vacuum in the tube being used.
Re-anchor the vein. Veins sometimes roll away from the
point of the needle and puncture site.
Have the patient make a fist and flex the arm, which helps
engorge muscles to fill veins.
Pre-warm the region of the vein to reduce vasoconstriction
and increase blood flow.
Have the patient drink fluids if dehydrated.
o
The blood is bright red (arterial) rather than venous. Apply
firm pressure for more than 5 minutes.
o
•
BLOOD COLLECTION ON BABIES:
The recommended location for blood collection on a
newborn baby or infant is the heel. The diagram below
indicates in green the proper area to use for heel punctures
for blood collection:
IF BLOOD STOPS FLOWING INTO THE TUBE:
•
•
The vein may have collapsed; resecure the tourniquet to
increase venous filling. If this is not successful, remove the
needle, take care of the puncture site, and redraw.
o
The needle may have pulled out of the vein when switching
tubes. Hold equipment firmly and place fingers against
patient's arm, using the flange for leverage when
withdrawing and inserting tubes.
PROBLEMS OTHER
COLLECTION:
•
THAN
AN
INCOMPLETE
A hematoma forms under the skin adjacent to the puncture
site - release the tourniquet immediately and withdraw the
needle. Apply firm pressure. Hematoma formation is a
problem in older patients.
o In older patients with thin, loose skin and less
subcutaneous tissue, the minor trauma of venipuncture
is more likely to produce local hemorrhage. Note the
small hematomas in the view above. In the view below,
there has been more extensive subcutaneous
hemorrhage, and even tearing of the skin from
adhesive tape applied with a bandage, in a patient on
corticosteroid therapy.
•
•
•
•
•
•
•
o
o
Prewarming the infant's heel (42 C for 3 to 5 minutes) is
important to obtain capillary blood gas samples and
warming also greatly increases the flow of blood for
collection of other specimens. However, do not use too high
a temperature warmer, because baby's skin is thin and
susceptible to thermal injury.
Clean the site to be punctured with an alcohol sponge. Dry
the cleaned area with a dry cotton sponge. Hold the baby's
foot firmly to avoid sudden movement.
Using a sterile blood lancet, puncture the side of the heel in
the appropriate regions shown above in green. Do not use
the central portion of the heel because you might injure the
underlying bone, which is close to the skin surface. Do not
use a previous puncture site. Make the cut across the
heelprint lines so that a drop of blood can well up and not
run down along the lines.
Wipe away the first drop of blood with a piece of clean, dry
cotton. Since newborns do not often bleed immediately, use
gentle pressure to produce a rounded drop of blood. Do not
use excessive pressure or heavy massaging because the
blood may become diluted with tissue fluid.
Fill the capillary tube(s) or micro collection device(s) as
needed.
When finished, elevate the heel, place a piece of clean, dry
cotton on the puncture site, and hold it in place until the
bleeding has stopped.
Be sure to dispose of the lancet in the appropriate sharps
container. Dispose of contaminated materials in appropriate
waste receptacles. Remove your gloves and wash your
hands.
HEELSTICK PROCEDURE ILLUSTRATED:
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Module 1: Introduction to Hematology
•
Two heelsticks have been performed on this baby. One of
them has been performed correctly. One was performed
improperly.
PLATELET POOR
PLASMA
The relative centrifugal force
(RCF) is higher in platelet
poor plasma.
The platelets will be on top of
the RBC.
The platelets will be pushed
on the buffy coat.
PLATELET RICH
PLASMA
The relative centrifugal force
(RCF) is lower in platelet rich
plasma.
There are platelets that’ll not
be pushed on the buffy coat.
The platelets will remain
suspended in plasma.
o
PEDIATRIC PHLEBOTOMY
•
•
•
•
Children, particularly under the age of 10, may experience
pain and anxiety during the phlebotomy procedure.
A variety of techniques can be employed to reduce pain and
anxiety. Effective methods use distraction. These may
include listening to music or a story, watching a video,
playing with a toy, having a parent provide distraction with
talk or touch, using flash cards, and squeezing a rubber ball.
(Uman et al, 2013)
COLLECTION TUBES FOR PHLEBOTOMY
Collection tubes can vary in size for volume of blood drawn,
appropriate to the tests ordered with sample size required,
and vary in the kind of additive for anticoagulation,
separation of plasma, or preservation of analyte. Larger
tubes sizes typically provide for collection of samples 6 to
10 mL.
o
Smaller collection tubes for sample sizes of 2 mL or less
may be appropriate in situations where a smaller amount of
blood should be drawn, as in pediatric patients, or to
minimize hemolysis during collection, or to avoid insufficient
sample volume in the collection tube.
Figure 1. Platelet poor plasma and
platelet rich plasma.
Clotted blood
Plasma
Serum
Whole blood
Diluted blood
o A form of sample preparation
o Whole blood mixed with a diluent
o Used for cell counts:
▪
RBC’s, WBC’s and platelet
• Platelet is not a true cell. These are fragments
of the cytoplasm of its precursor
(megakaryocyte). From the bone marrow, the
fragments of the cytoplasm (of the
megakaryocyte) leaves, then forms into
platelets.
• RBC’s and WBC’s are true cells. Mature
RBC’s seen in the blood doesn’t contain
nucleus (anucleated). Nucleated RBC’s are
seen in the bone marrow.
•
•
Finger prick or skin puncture
Heel-stick puncture
o Babies are punctured in their heels
Used in micro-methods of clotting time
o Slide method
o Capillary tube method
▪
Dale-Laidlaw test
o
ADDITIONAL NOTES
PLASMA
PLATELET POOR PLASMA
•
•
Plasma with very minimal platelets
Used in coagulation tests
o Prothrombin Time (PT)
o Partial Thromboplastin Time (PTT)
PLATELET RICH PLASMA
•
•
Plasma with platelet
Used in Platelet Function Tests (PFT)
•
It is the difference in the relative centrifugal force (RCF)
when preparing the plasma.
A platelet poor plasma and platelet rich plasma is
dependent on the difference in the RCF.
FRESH BLOOD
•
MANNER OF PREPARATION
•
SAMPLES FOR HEMATOLOGY
•
•
•
•
•
•
•
BUFFY COAT SMEAR
Uses buffy coat
Used for WBC abnormal morphology
o Buffy coat is concentrated with WBC and platelets
BADONG, BONI, CENIZAL, DE GUZMAN, DE LIMA, DELOS REYES, ESPINOLA, MANGOHIG, PATAG, POSO, SALVADOR | 3D-MT
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Module 1: Introduction to Hematology
•
Traditionally, it is used in LE prep for SLE
o Uses LE as marker
o Low sensitivity
▪
Today, immunological tests are used for SLE for
its higher sensitivity for LE cells
TABLE OF DIFFERENT COLLECTION TUBES IS
PLACED IN THE END
REFERENCES
Notes from the discussion by
Centro Escolar University’s powerpoint presentation:
Keohane, E. M., Smith, L. J., and Walenga, J. M.
(Eds).(2016). Rodak’sHematology: Clinical Principles and
Applications (5thed.).SG: ElsevierPte Ltd.
McPherson, R. A. and Pincus, M. R. (Eds). (2012).
Henry’s Clinical Diagnosis and Management by Laboratory
Methods (22nded.). SG: ElsevierPte Ltd.
Moore, G. W., Knight, G., and Blann, A. D. (2010).
Fundamentals
of Biomedical
Science: Haematology.
Oxford, NY: Oxford University Press
Turgeon, M. L. (2012). Clinical Hematology: theory and
procedures (5thed.). Baltimore, MD: Lippincott Williams and
Wilkins
Stiene-Martin, E. A., Lotspeich-Steininger, C. A., and
Koepke, J. A. (1998). Clinical Hematology: principles,
procedures,
correlations(2nded.). Philadelphia,
PA:
Lippincott-Raven Publishers
Aceron, Z. B. (unpublished).Lecture notes
Bain, B.J. (2011). Collection and handling of
blood, InC. Jury, et. al. (Eds.) Dacie and Lewis’
Practical haematology(pp 1-9). China: Churchill
Livingstone
Benett, S.T., et. al. (Eds.). (2007.) Laboratory
hemostasis: A practical guide for pathologists. New
York, NY: Springer Science+Business Media, LLC
Dinglasan, R. J. A. R. (unpublished). Review notes
Henry, J.B. (2012). Preanalysis. In K.W. Sanford
&R.A. McPherson (Eds.) Clinical diagnosis and
management by laboratory methods (pp 24-36).
Singapore: Elsevier Pte. Ltd.
Hillyer, C.D., et. al. (2009). Transfusion medicine and
hemostasis: Clinical and laboratory aspects. New
York, NY: Elsevier
Rodak, B.F., et. al. (2012). Laboratory evaluation
of hemostasis. In G.A. Fritsma (Ed.)Hematology:
Clinical principles and correlations(pp 734-764)
Singapore: Elsevier Pte. Ltd.
Tricore Reference Laboratories (2011). Preparation of
platelet poor plasma for coagulation testing. Retrieved
from http://www.tricore.org/HealthcareProfessionals/Test-Information/TestingProtocols/Preparation-of-Platelet-Poor-Plasma-forCoagulatio
Turgeon, M.L. (2012). Clinical hematology: theory and
procedures. Baltimore, MD: Lippincott Williams and
Wilkins
Zhou, L. & Schmaier, A.H. (2005). Description of
procedureswith the aim to develop standards in the
field. AmericanJournal of ClinicalPathology,123, 172183. DOI: 10.1309/Y9EC63RW3XG1V313
Giavarina D, Lippi G. Blood venous sample collection:
Recommendations overview and a checklist to
improve quality. Clin Biochem. 2017;50(10-11):568573.
Kiechle FL. So You're Going to Collect a Blood
Specimen: An Introduction to Phlebotomy, 13th
Edition (2010), College of American Pathologists,
Northfield, IL.
Dalal BI, Brigden ML. Factitious biochemical
measurements resulting from hematologic conditions.
Am J Clin Pathol. 2009 Feb;131(2):195-204.
Lippi G, Salvagno GL, Montagnana M, Franchini M,
Guidi GC. Phlebotomy issues and quality
improvement in results of laboratory testing. Clin Lab.
2006;52(5-6):217-30.
Lippi G, Blanckaert N, Bonini P, Green S, Kitchen S,
Palicka V, Vassault AJ, Mattiuzzi C, Plebani M.
Causes, consequences, detection, and prevention of
identification errors in laboratory diagnostics. Clin
Chem Lab Med. 2009;47(2):143-53.
Occupational Safety and Health Administration,
United States Department of Labor.
https://www.osha.gov/pls/oshaweb/owadisp.show_do
cument?p_table=INTERPRETATIONS&p_id=25913
and
https://www.osha.gov/OshDoc/data_BloodborneFacts/
bbfact03.pdf (Accessed January 10, 2018).
Phelan MP, Reineks EZ, Berriochoa JP, Schold JD,
Hustey FM, Chamberlin J, Kovach A. Impact of Use of
Smaller Volume, Smaller Vacuum Blood Collection
Tubes on Hemolysis in Emergency Department Blood
Samples. Am J Clin Pathol. 2017;148(4):330-335.
Uman LS, Birnie KA, Noel M, et al. Psychological
interventions for needle-related procedural pain and
distress in children and adolescents. Cochrane
Database Syst Rev. 2013 Oct 10;(10):CD005179. doi:
10.1002/14651858.CD005179.pub3.
BADONG, BONI, CENIZAL, DE GUZMAN, DE LIMA, DELOS REYES, ESPINOLA, MANGOHIG, PATAG, POSO, SALVADOR | 3D-MT
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Module 1: Introduction to Hematology
Valenstein PN, Sirota RL. Identification errors in
pathology and laboratory medicine. Clin Lab Med.
2004;24(4):979-96, vii.
World Health Organization. WHO guidelines on
drawing blood: best practices in phlebotomy.
https://www.ncbi.nlm.nih.gov/books/NBK138650/pdf/B
ookshelf_NBK138650.pdf (Accessed January 10,
2018)
BADONG, BONI, CENIZAL, DE GUZMAN, DE LIMA, DELOS REYES, ESPINOLA, MANGOHIG, PATAG, POSO, SALVADOR | 3D-MT
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Module 1: Introduction to Hematology
T UB E
AD D IT I V E
RED TOP
None
GOLD TOP
None
LIGHT
GREEN TOP
Plasma
Separating Tube
(PST)
with
Lithium heparin
MO D E O F AC TI O N
Blood clots, and the serum
is
separated
by
centrifugation
Serum separator tube
(SST) contains a gel at the
bottom to separate blood
from
serum
on
centrifugation
Anticoagulates with lithium
heparin;
Plasma
is
separated with PST gel at
the bottom of the tube
USES
Chemistries, Immunology and
Serology,
Blood
Bank
(Crossmatch)
Chemistries,
Serology
Immunology
and
Chemistries
Hematology (CBC) and Blood
Bank (Crossmatch); requires full
draw - invert 8 times to prevent
clotting and platelet clumping
Coagulation tests (protime and
prothrombin
time), full
draw required
•
For lithium level: sodium
heparin
•
For ammonia level: sodium
or lithium heparin
Trace element testing (zinc,
copper, lead, mercury) and
toxicology
PURPLE TOP
EDTA
Forms calcium salts to
remove calcium
LIGHT BLUE
TOP
Sodium citrate
Forms calcium salts to
remove calcium
GREEN TOP
Sodium heparin
or lithium heparin
Inactivates thrombin and
thromboplastin
DARK
TOP
EDTA-
Tube is designed to contain
no contaminating metals
LIGHT GRAY
TOP
Sodium fluoride
and potassium
oxalate
Antiglycolytic
agent
preserves glucose up to 5
days
Glucoses, requires full draw (may
cause hemolysis if short draw)
YELLOW TOP
ACD
(acidcitrate-dextrose)
Complement inactivation
HLA tissue typing,
testing, DNA studies
paternity
YELLOWBLACK TOP
Broth mixture
Preserves
viability
microorganisms
Microbiology
anaerobes, fungi
aerobes,
BLACK TOP
Sodium
citrate
(buffered)
Forms calcium salts to
remove calcium
Westergren Sedimentation Rate;
requires full draw
ORANGE TOP
Thrombin
Quickly clots blood
STAT serum chemistries
LIGHT
BROWN TOP
Sodium heparin
Inactivates thrombin and
thromboplastin; contains
virtually no lead
Serum lead determination
PINK TOP
Potassium EDTA
Forms calcium salts
Immunohematology
WHITE TOP
Potassium EDTA
Forms calcium salts
Molecular/PCR and bDNA testing
BLUE
of
BADONG, BONI, CENIZAL, DE GUZMAN, DE LIMA, DELOS REYES, ESPINOLA, MANGOHIG, PATAG, POSO, SALVADOR | 3D-MT
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