TRANS #38, FINAL EXAM

Phlebotomy, ESR, CBC

FARRAH FONTILLA-SANTIAGO, MD
08/22/2018
CLINICAL PATHOLOGY LAB

OUTLINE • NOTE: Apply the tourniquet in such a way that it will be easy to
I. Phlebotomy III. White Blood Differentiation remove after drawing blood.
A. Principles Count
B. Methodology A. Principles
C. Evacuated Collection Tubes B. Methodology
D. Results C. Normal Values
II. Erythrocyte Sedimentation D. Results
Rate Quick Review
A. Principles Review Questions
B. Methodology References
C. Results Appendix

I. PHLEBOTOMY
• Materials needed
→ 5 mL syringe
→ 10ml lavender-top test tube
→ Tourniquet
→ Dry cotton Figure 2. Applying a tourniquet. (A) Wrap the tourniquet around the arm
→ Alcohol swabs approximately 2-3 inches above the end of the elbow. (B) Cross the ends of the
→ Disposable gloves tourniquet at the point of your grasp. (C) Approximately 1 inch from the crossing
→ Micropore tape point, tuck a portion of the top length into the bottom length, forming a loop. (D)
The tourniquet should be snug but not so tight that the patient’s skin is pinched
(Fontilla-Santiago, 2018)
A. PRINCIPLES
• Phlebotomy is the process of taking blood samples from the patient
through a vein
→ Used to diagnose or treat diseases
→ Used for blood donations

B. METHODOLOGY
1. Introduce yourself and ask for the volunteer’s name.
2. Briefly inform the volunteer about the procedure.
3. Observe proper hand washing technique.
• If a fasting specimen or a dietary restriction is required, confirm if Figure 3. Tying a tourniquet (WHO, 2018).
patient has fasted or eliminated foods from diet as ordered by
physician.
4. Position the volunteer comfortably. Wear gloves.
5. Assemble equipment and supplies, including test tubes, tourniquet,
preparation for cleansing the area and syringes.
6. Instruct patient to make a fist without pumping the hand.
7. Select suitable vein for puncture. The median cubital and cephalic
veins are preferred.
• Palpation for a suitable vein can be done with or without a
tourniquet. If done with a tourniquet, remember to remove the
Figure 4. Several types of tourniquet (from top to bottom): buckle closure, latex
tourniquet after picking a vein of choice, before prepping the strap, nonlatex strap, and Velcro closure (Fontilla-Santiago, 2018).
area for puncture.
10. Anchor the vein firmly.
11. Insert needle in a 15-30 degree angle with bevel up.
• Insert the needle in a way that it follows the contour of the vein.
• Insert the needle fairly smoothly and rapidly to minimize
discomfort.

Figure 1. Best sites for venipuncture. (1) Cephalic vein, (2) Basilar vein, (3)
Median cubital vein (ResearchGate, 2018).

8. Cleanse the site with 70% isopropyl alcohol solution.

• Begin at puncture site and cleanse outward in circular motion, and
allow the area to dry. Do not touch cleansed area with unsterile
object.
→ Repeat antiseptic procedure if injection site comes into contact
with any unsterile area (e.g. ungloved hands, needle cap, hair).
→ Air-dry the area.
Figure 5. Traditional syringe system components (Fontilla-Santiago, 2018).
9. Apply tourniquet 2 inches above the site. Never leave the tourniquet
in place longer than 1 minute.
Trans #38 Group #11: Andaya, Cruz, Manahan, Ramos, Tenorio, Tinawin 1 of 6


Order of Draw (McPherson, 2011)
• Blood collection tubes must be drawn in a specific order to avoid
cross-contamination of additives between tubes. The recommended
order for plastic vacutainer tube:
1. Blood culture tubes (yellow)
2. Coagulation sodium citrate tube (blue stopper)
3. Serum tubes with or without clot activator or gel separator
4. Heparin tubes with or without gel (green stopper)
5. EDTA tubes (lavender stopper)
6. Glycolytic inhibitor tubes (gray stopper)
→ Glass or plastic tubes with additives, including gel tubes, are
drawn after the citrate tube (blue top) to avoid interference with
coagulation measurements.
→ Glass or plastic serum tubes, without a clot activator or gel
separator, may be drawn before coagulation tubes are drawn.
• NOTE: Tubes with additives must be thoroughly mixed. Erroneous
test results may be obtained when the blood is not thoroughly mixed
with the additive.

Figure 6. (A) Correct needle insertion technique. (B) Bevel on upper vein wall. D. RESULTS
(C) Bevel on lower vein wall. (D) Needle inserted too far. (E) Needle partially
inserted. (F) Needle slipped beside the vein. (G) Collapsed vein prevents blood Hemolysis (2020 Trans)
flow (Fontilla-Santiago, 2018).
• Breaking of the red blood cells’ membrane, releasing free
hemoglobin into the circulating blood
12. Pull the plunger gently to withdraw blood smoothly.
→ Usually the result of mechanical damage due to poor technique
• Fill up the syringe or vial until the desired amount is reached.
• Can occur during phlebotomy or blood extraction and may alter
results
• Can lead to inaccurate results and repeat draws causing additional
pain, delaying treatment decisions, and increasing length of stay

Measures to Prevent Hemolysis (2020 Trans)

• Collect sample in the antecubital fossa
→ Site provides access to a large vein for drawing blood samples,
allowing easier access, the use of larger needles, and a lower
Figure 7. Inserting needle and drawing blood (Fontilla-Santiago, 2018).
likelihood of vessel collapse
13. Release tourniquet first, then withdraw the needle and apply • Ensure that the venipuncture site is dry
pressure with dry cotton. → Alcohol may cause hemolysis and contamination of the specimen
• Done to adequately stop bleeding and avoid hematoma. • Avoid trauma and excessive probing during venipuncture
14. Ask the volunteer to relax the fist, then apply tape over the wound. → A needle that passes through the vein and exit the other side will
15. Check the condition of the volunteer. cause excessive trauma in the vein
16. Put blood in the tube with the anticoagulant of choice → Can also cause a hematoma
• In the context of the phlebotomy experiment done in class, it is the • Use of new, straight needle venipuncture instead of existing
lavender-top test tube intravenous (IV) access
17. Thoroughly mix the blood and anticoagulant by gently inverting the • Larger bore (≤ 21 Gauge) needles may reduce hemolysis by reducing
tube at least 6 times, careful not to cause bubbles. the stress and/or turbulence for the red blood cells as the specimen
18. Label tube with the volunteer’s complete name, age, date and time is collected.
of collection. → Smaller gauge indicates a larger needle diameter
19. Put the test tube in the test tube rack. → Usually for adults, anything that is smaller than a 21-gauge needle
20. Dispose of the needle in the “sharps” disposal unit. (that is, a larger gauge number) makes blood withdrawal more
21. Dispose the used cotton, syringe, and gloves in a yellow disposal difficult
container. • Avoid drawing the plunger back too forcefully or too fast
• Mix the blood with the additives by gently inverting tubes a number
C. EVACUATED COLLECTION TUBES of times, depending on the anti-coagulants of choice
• Evacuated collection tubes are designed to be filled with a
predetermined volume of blood by vacuum Table 1. Number of inversions needed depending on what type of additive was
used (Fischbach, 2004).
• Various sizes are available (University of Utah, 2018)
Color of Number of
→ Collection tubes can vary in size for volume of blood drawn, Type of Additive
Stopper Inversions
appropriate to the tests ordered with sample size required, and
Sterile containing sodium
vary in the kind of additive for anticoagulation, separation of Yellow 8-10
polyanetholsulfonate
plasma, or preservation of analyte
Sodium citrate Light Blue 3-4
→ Larger tube sizes typically provide for collection of samples from
Plain (no additive) Red 5
6 to 10 mL
Heparin Tan 8-10
→ Smaller collection tubes for sample sizes of 2 mL or less may be
K3EDTA in liquid form Lavender 8-10
appropriate in situations where a smaller amount blood should be
drawn, as in pediatric patients, or to minimize hemolysis during Sodium fluoride, potassium oxalate Gray 8-10
collection, or to avoid insufficient sample volume in the collection
tube • Tourniquets should not be applied for more than 1 minute when
• Blood should never be poured from one tube to another collecting blood
• Blood collecting tubes have color-coded toppers that indicate the → Tourniquets constrict blood vessels and can result in hemolysis
presence/absence of specific anticoagulants or additives (see • Do not push down the syringe plunger
Appendix)
II. ERYTHROCYTE SEDIMENTATION RATE
• Materials needed
→ Westergren tube
Principles and Perspectives 01.38: PHLEBOTOMY, ESR, CBC 2 of 6

→ Rack for ESR determination

A. PRINCIPLES
• Erythrocyte Sedimentation Rate (ESR) determines the rate at which
erythrocytes settle at the bottom of a vertical tube of anti-
coagulated blood within a specified period
→ Measures distance red blood cells fall in the tube in mm/hr
• Also known as sedimentary rate
• Can reveal inflammatory activity in your body
→ Not a stand-alone diagnostic tool, but may aid in monitoring the
progress of inflammatory disease
• When your blood is placed in a tall, thin tube, erythrocytes gradually
settle to the bottom because:
→ RBCs repel each other due to the negative charges on their
surfaces
→ Large surface area-to-volume ratio of normal RBCs resists
settling Figure 10. ESR rack (Fontilla-Santiago, 2018).
• Sedimentation or settling occurs in three stages
→ Aggregation (first 10-15 mins): rouleaux formation C. RESULTS
§ When an inflammatory process is present, the high proportion
of fibrinogen in the blood causes the RBCs to stick to each Normal Values (McPherson, 2011)
other, forming the rouleaux • 0-50 years of age
§ Stacks or aggregation of RBCs form because of their unique → Male: 0-15 mm/hr
discoid shape
→ Female: 0-20 mm/hr
§ Most common promoter of rouleuax is increased plasma
• >50 years of age (2020 trans):
fibrinogen
→ Male: 0-20 mm/hr
→ Female: 0-30 mm/hr
• ESR increases with age

Results Outside Normal Range

• Values higher than the normal range indicate possibility of any of
the following (2020 trans):
→ Anemia
→ Rheumatic fever
→ Thyroid malfunction
Figure 8. Rouleaux formation showing the stacking of red blood cells (The
→ Kidney disease
Relevance of Rouleaux Formation, 2018).
→ Arthritis
→ Sedimentation (next 30-40 mins): when most of the settling or → Pregnancy
sedimentation occurs → Cancers
→ Packing (last 10 mins): sedimentation slows; packing of cells → Systemic infection
occur at the bottom of the tube • Values lower than the normal range indicate possibility of any of
• ESR is directly proportional to the weight of cell aggregates and the following (2020 trans):
inversely proportional to plasma viscosity. → Hyperviscosity
• The farther the red blood cells have descended, the greater the → Hypofibrinogenemia
inflammatory response of the immune system. → Congestive heart failure
• Values outside the normal range can reveal inflammatory activity in → Sickle cell anemia
your body → Polycythemia
→ Low plasma proteins
B. METHODOLOGY
1. Gently mix blood and anticoagulant in the tube by inverting a III. WHITE BLOOD CELL DIFFERENTIATION COUNT
minimum of 6 times. • Materials needed
2. Remove the cover of the lavender-top test tube. → Stained slide with peripheral smear
3. Insert the Westergren tube (vacuette tube) into the test tube, touching → Oil immersion
the bottom. → Microscope
• NOTE: The blood column on the vacuette tube must be free of
bubbles A. PRINCIPLES
4. Place the filled vacuette tube in a rack in an exactly vertical position • To identify the types of white blood cells in the peripheral blood, a
and note the time and room temperature. differential count is performed
5. After an hour, read the level to which the cells have settled on the → A differential WBC count determines a rough percentage of each
descending scale etched on the vacuette tube. Each mark equals 1.0 type of WBC in the peripheral blood
mm while each numbered mark equals 10mm (1cm). The • Clinical use (2019 trans)
measurement obtained is reported in mm per hour as the ESR.
→ Assesses the ability of the blood to respond to and eliminate
infection
→ Detects the severity of allergic and drug reactions, plus the
response to parasitic and other types of infection. It is essential in
evaluating the reaction to viral infections and response to
chemotherapy
→ Identify various stages of leukemia

Granulocytes
• Polymorphic nuclei
• Contains azurophilic or non-specific granules
Figure 9. Westergren method (How to Determine ESR, 2015).
Principles and Perspectives 01.38: PHLEBOTOMY, ESR, CBC 3 of 6

→ Specialized lysosomes, darkly stained
• Contains specific granules
→ Binds to neutral, basic or acidic stains

Neutrophils
• Polymorphonuclear
→ Has a hyperlobulated nucleus, attached by chromatin
• Neutrophils can increase in response to bacterial infection or
inflammatory disease Figure 13. A normal basophil. The nucleus has three lobes. The cytoplasm is
→ Chemotaxis packed with large purple granules. The lower cell is a lymphocyte
§ Migrates to areas of bacterial products (Fontilla-Santiago, 2018).
→ Phagocytosis
§ Kills bacteria Agranulocytes
→ Pus formation • Rounded nucleus
§ Accumulation of dead neutrophils after mission • Azurophilic granules are present
→ Release of cytokines
→ Stimulation of additional neutrophils Lymphocytes
• Round, deeply staining nucleus
• Thin rim of blue cytoplasm
• Azurophilic, non-specific granules
• Sub-types
→ B Cells – for humoral immunity
→ T Cells – for cellular immunity
• Lymphocytes can increase in cases of viral infection, leukemia,
cancer of the bone marrow or radiation therapy

Figure 11. Neutrophils. Left: A normal neutrophil with a bi-lobed nucleus and
cytoplasm containing delicate lilac-staining granules. The other
nucleated cell is a small lymphocyte. Right: A normal neutrophil from a
female showing a nucleus with 4 lobes and a “drumstick” (Fontilla-
Santiago, 2018).

Eosinophils
• Large, pink, coarse granules
→ Bi-lobed nucleus that is less segmented than neutrophils Figure 14. A large lymphocyte with a less densely staining nucleus than occurs
in a small lymphocyte and more plentiful pale blue cytoplasm. A
• Primarily serves as defense against parasitic infection nucleolus is apparent, top left in the nucleus (Fontilla-Santiago, 2018).
• Damage control in allergic reactions
→ Take up and dispose antigen-antibody complexes formed in Monocytes
allergic conditions
• Nucleus is large and indented (kidney-shaped)
• Also participates in the inflammatory response as a phagocyte
• More cytoplasm than lymphocytes
• Eosinophils can increase in response to allergic disorders,
• Circulates for 12-100 hours in blood then enters connective tissue,
inflammation of the skin and parasitic infections
where it differentiates into macrophages and dendritic cells
• Mononuclear phagocyte system
→ Monocytes in the blood
→ Macrophages of connective tissue
→ Alveolar macrophages in the lungs
→ Kupffer cells in the liver
→ Osteoclasts of the bone
→ Langerhans cells in the skin
→ Microglial cells in the CNS
• Monocyte levels can increase in response to infection of all kinds,
Figure 12. A normal bi-lobed eosinophil. The granules are reddish-orange and as well as to inflammatory disorders
pack the cytoplasm (Fontilla-Santiago, 2018).

Basophils
• Nucleus has irregular bi-lobes, obscured by blue granules
• Same function as mast cells in releasing histamine
• Function
→ Anaphylaxis: immediate hypersensitivity reaction to migrating
tissue
→ Rapid degranulation can cause serious consequences:
§ Asthma attack Figure 15. A monocyte, showing a lobulated nucleus and voluminous, opaque
§ Urticaria (hives) cytoplasm containing very fine azurophilic granules. Several platelets
are also visible (Fontilla-Santiago, 2018).
§ Anaphylactic shock
• Basophils can increase in cases of leukemia, chronic
• Monocytes should not be confused with large granular lymphocytes
inflammation, the presence of a hypersensitivity reaction to food
(Fontilla-Santiago, 2018)
or radiation therapy
→ Lymphocytes have clear, pale blue cytoplasm and discrete,
sometimes prominent granules
→ Monocytes usually have more opaque, grey-blue cytoplasm with
very fine granules

Principles and Perspectives 01.38: PHLEBOTOMY, ESR, CBC 4 of 6

B. METHODOLOGY
1. Put a drop of oil on the thinner smear area (“feathery edge”).
2. Using the oil immersion objective (OIO), count the following white
blood cells until you reach a total count of 100 cells.
• Neutrophils/polymorphonuclear cells
• Lymphocytes
• Basophils
• Eosinophils
• Monocytes
3. For an efficient and systematic manner of viewing, start from one
corner, then proceed downwards. When cells are not visible
anymore, move the slide to the left or right, then start moving
upwards.
• Repeat this until total area of the slide has been viewed.
§ Rouleux formation
§ Most common promoter of rouleuax is increased plasma
fibrinogen.
§ When an inflammatory process is present, there is high
proportion of fibrinogen in the blood à RBCs to stick to each
other à rouleaux settle faster
→ Sedimentation
→ Packing
• ESR is directly proportional to the weight of cell aggregates and
inversely proportional to plasma viscosity.
• The farther the red blood cells have descended, the greater the
inflammatory response of the immune system.
• Values outside the normal range can reveal inflammatory activity in
your body
• Values higher than normal range indicates possibility of any of the
following: anemia, rheumatic fever. thyroid malfunction, kidney
disease, arthritis, pregnancy, cancers, systemic infection
• Values lower than normal range indicates possibility of any of the
following: hyperviscosity, hypofibrinogenemia, congestive heart
failure, sickle cell anemia, polycythemia, low plasma proteins

WBC Differential Count

• WBC Differential Count
→ A differential WBC count determines a rough percentage of each
type of WBC in the peripheral blood
→ Granulocytes
§ Polymorphic nuclei with either azurophilic/non-specific
Figure 16. Photomicrograph of a blood film showing ideal thickness for granules or specific granules
examination (Fontilla-Santiago, 2018). § Neutrophils
− Has a hyperlobulated nucleus, attached by chromatin
C. NORMAL VALUES − Increase in bacterial infection or inflammatory disease
• For Granulocytes: § Eosinophils
→ Eosinophils: 1-3% − Has a bilobed nucleus that is less segmented than
→ Neutrophils: 60% neutrophils
→ Basophils: 0-0.7% − Has large, pink, coarse granules
• For Agranulocytes: − Acts as a defense against parasitic infection, a control
→ Lymphocytes: 20-40% mechanism for damage in allergic reactions and a
→ Monocytes: 3-7% phagocyte in inflammatory response
− Increase in allergic disorders, skin inflammation and
parasitic infections
D. RESULTS § Basophils
• Leukocytosis (or a high number of WBCs) may be indicative of the − Has irregular bilobes
following:
− Same function as mast cells in releasing histamine
→ Infectious diseases
− Function in anaphylaxis
→ Inflammatory disease (such as rheumatoid arthritis or allergy)
− Increase in leukemia, chronic inflammation, presence of a
→ Leukemia hypersensitivity reaction to food or radiation therapy
→ Severe emotional or physical stress → Agranulocytes
→ Tissue damage (for example, burns) § Lymphocytes
• Leukopenia (or a decrease in WBC count) may be indicative of the − B cells – for humoral immunity
following: − T cells – for cellular immunity
→ Bone marrow failure (for example, due to granuloma, tumor, − Increase in viral infection, leukemia, bone marrow cancer or
fibrosis) radiation therapy
→ Presence of cytotoxic substance § Monocytes
→ Collagen-vascular diseases (such as lupus erythematosus) − Has a large, indented (kidney-shaped) nucleus
→ Disease of the liver or spleen − Monocyte phagocyte system
→ Radiation − Increase in infections and inflammatory disorders
→ Methodology
QUICK REVIEW § Oil immersion objective (OIO)
→ Normal Values
Phlebotomy § Eosinophils: 1-3%
• Phlebotomy – process extracting blood from a vessel § Neutrophils: 60%
→ Median Cubital vein – Ideal vein for extraction but other veins such § Basophils: 0-0.7%
as the Cephalic and Basilar can be used § Lymphocytes: 20-40%
→ Maintain proper aseptic technique throughout the procedure § Monocytes: 3-7%
(Hand washing, wearing gloves, cleaning site) → Results
→ Ensure patient comfort (Proper tourniquet application and needle § Leukocytosis (or a high number of WBCs)
insertion) § Leukopenia (or a decrease in WBC count)

Erythrocyte Sedimentation Rate REVIEW QUESTIONS

• Erythrocyte Sedimentation Rate (ESR) determines the rate at which 1. What is the right angle when inserting the needle in phlebotomy?
erythrocytes settle at the bottom of a vertical tube of anticoagulated a. 15 degrees
blood within 60 minutes or one hour. b. 45 degrees
• Sedimentation occurs in three stages: c. 90 degrees
→ Aggregation d. 120 degrees
Principles and Perspectives 01.38: PHLEBOTOMY, ESR, CBC 5 of 6

2. True or False. Erythrocyte Sedimentation Rate (ESR) is indirectly Answers: 1A, 2F, 3D, 4C
proportional to the weight of cell aggregates.
REFERENCES
3. Which of the following statements is TRUE? (1) Fontilla-Santiago, Farrah. August 22, 2018. Phlembotomy, ESR,
a. Leukocytosis refers to a decrease in the WBC count WBC Differential Count [Lecture slides].
b. Neutrophils and lymphocytes are examples of granulocytes (2) ASMPH Batch 2019. 2015. Trans Format.
c. B Lymphocytes are specifically for cell-mediated immunity (3) Que, Matthew. 2017. ASMPH Batch 2022 Header.
d. The WBC Differentiation Count procedure involves using the oil (4) Fischbach, F. T. (2004). A manual of laboratory and diagnostic tests
immersion objective (OIO) to view the cells th
(7 ed). Philadelphia: Lippincott Williams and Wikins.
(5) McPherson, R. A. (2011). Henry’s clinical diagnosis and
4. Which of the following procedures won’t help in preventing hemolysis management by laboratory methods (22
nd
ed). Philadelphia:
of blood samples? Saunders Elsevier.
a. Avoiding trauma and excessive probing during venipuncture (6) University of Utah. n.d. Blood Collection. Retrieved from
b. Using new, straight needle venipuncture instead of existing https://library.med.utah.edu/WebPath/TUTORIAL/PHLEB/PHLEB.ht
intravenous access ml
c. Ensure that the venipuncture site is still wet
d. Avoiding drawing the plunger back too forcefully or too quickly

APPENDIX
Table 2. Different collection tubes and their respective additives, mode of action and uses (McPherson, 2011)
Stopper color Anticoagulant/Additive Specimen Type/Use Mechanism of Action
Serum/chemistry, blood blank, and
Red (glass) None N/A
serology
Red (plastic/hemogard)
Clot activator Serum/chemistry Silica clot activator
Lavender (glass) K3EDTA in liquid form Whole blood/hematology Chelates (binds) calcium
Lavender (plastic) K2EDTA/spray-dried Whole blood/hematology Chelates (binds) calcium
Whole blood/blood bank and molecular
Pink Spray-dried K2EDTA Chelates (binds) calcium
diagnostics
White K2EDTA with gel Plasma/molecular diagnostics Chelates (binds) calcium
Sodium citrate Plasma/coagulation Chelates (binds) calcium
Light Blue
Thrombin and soybean trypsin
Plasma/coagulation Fibrin degradation products
inhibitor
Black Sodium citrate Plasma/sed rates-hematology Chelates (binds) calcium
Light green Lithium heparin and gel Plasma/chemistry Inhibits thrombin formation
Green Sodium heparin, lithium heparin Plasma/chemistry Inhibits thrombin formation
Heparin inhibits thrombin formation
Royal blue Sodium heparin, K2EDTA Plasma/chemistry/toxicology
Na2EDTA binds calcium
Sodium fluoride + potassium
Gray oxalate, Sodium fluoride + EDTA, Plasma/glucose testing Inhibits glycolysis
Sodium fluoride
Aids in bacterial recovery by inhibiting
Sterile containing sodium
Serum/microbiology culture complement, phagocytes, and certain
polyanetholesulfonate (SPS)
antibiotics
Yellow
Plasma/blood bank, HLA phenotyping,
Acid citrate dextrose (ACD) WBC preservative
and paternity testing
Tan (glass) Sodium heparin Plasma/lead testing Inhibits thrombin formation
Tan (plastic) K2EDTA Plasma/lead testing Chelates (binds) calcium
Yellow/gray and orange Thrombin Serum/chemistry Clot activator
Red/gray and gold Clot activator separation gel Serum/chemistry Silica clot activator
**(EDTA, Ethylenediaminetetraacetic acid; HLA, human leukocyte antigen; K2EDTA, dipotassium form of EDTA; K3EDTA, tripotassium form of EDTA; Na2EDTA, disodium
EDTA)

Figure 17. Different test tube tops for different tests (Fontilla-Santiago, 2018).

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