BD Vacutainer Order of Draw

for Multiple Tube Collections

Designed for Your Safety Reflects change in CLSI recommended
Order of Draw (H3-A5, Vol 23, No 32, 8.10.2)

Closure Color Collection Tube Mix by Inverting

BD Vacutainer Blood Collection Tubes (glass or plastic)
®

• Blood Cultures - SPS 8 to 10 times

* When using a winged

blood collection set • Citrate Tube* 3 to 4 times
for venipuncture
and a coagulation or • BD Vacutainer SST
® ™
5 times
(citrate) tube is the Gel Separator Tube
first specimen tube to • Serum Tube 5 times (plastic)
be drawn, a discard (glass or plastic) none (glass)
tube should be drawn
• BD Vacutainer Rapid
®

first. The discard tube 5 to 6 times

Serum Tube (RST)
must be used to fill
the blood collection or • BD Vacutainer PST
® ™
8 to 10 times
set tubing’s “dead Gel Separator Tube
space” with blood With Heparin
but the discard tube
• Heparin Tube 8 to 10 times
does not need to be
completely filled. This
important step will or • EDTA Tube 8 to 10 times
ensure proper blood-
• BD Vacutainer PPT ™
®

to-additive ratio. The 8 to 10 times

discard tube should
Separator Tube
K2EDTA with Gel
be a nonadditive or
coagulation tube.
• Fluoride (glucose) Tube 8 to 10 times

Note: Always follow

your facility’s protocol
for order of draw BD Technical Services
Handle all biologic samples and blood collection 1.800.631.0174
“sharps” (lancets, needles, luer adapters and
blood collection sets) according to the policies BD Customer Service
and procedures of your facility. Obtain appropriate
medical attention in the event of any exposure
to biologic samples (for example, through a
puncture injury) since they may transmit viral
1.888.237.2762
hepatitis, HIV (AIDS), or other infectious diseases.
Utilize any built-in used needle protector if the
www.bd.com/vacutainer
blood collection device provides one. BD does
not recommend reshielding used needles, but
the policies and procedures of your facility may
differ and must always be followed. Discard any
1 Becton Drive
Franklin Lakes, NJ 07417
blood collection “sharps” in biohazard containers
approved for their disposal.
= 1 inversion
www.bd.com/vacutainer BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2010 BD
Franklin Lakes, NJ, 07417 1/10 VS5729-6
BD Vacutainer Venous Blood Collection®

Tube Guide
For the full array of BD Vacutainer Blood Collection Tubes, visit www.bd.com/vacutainer.
®

Many are available in a variety of sizes and draw volumes (for pediatric applications). Refer to our website for full descriptions.
® ®
BD Vacutainer Tubes BD Vacutainer Tubes Inversions
with with at Blood Your Lab’s
™
BD Hemogard Closure Conventional Stopper Additive Collection* Laboratory Use Draw Volume/Remarks
• Clot activator and gel 5 For serum determinations in chemistry.
for serum separation May be used for routine blood donor
Red/ screening and diagnostic testing of serum
Gold
Gray for infectious disease.** Tube inversions
ensure mixing of clot activator with blood.
Blood clotting time: 30 minutes.
• Lithium heparin 8 For plasma determinations in chemistry.
Light Green/ and gel for plasma Tube inversions ensure mixing of anticoagulant
Green Gray separation (heparin) with blood to prevent clotting.

• Silicone coated (glass) 0 For serum determinations in chemistry.

• Clot activator, Silicone 5 May be used for routine blood donor
coated (plastic) screening and diagnostic testing of serum
Red Red
for infectious disease.** Tube inversions
ensure mixing of clot activator with blood.
Blood clotting time: 60 minutes.
• Thrombin-based clot 5 to 6 For stat serum determinations in chemistry.
activator with gel for Tube inversions ensure mixing of clot activator
Orange serum separation with blood. Blood clotting time: 5 minutes.

• Thrombin-based clot 8 For stat serum determinations in chemistry.

activator Tube inversions ensure mixing of clot activator
Orange with blood. Blood clotting time: 5 minutes.

• Clot activator 8 For trace-element, toxicology, and

(plastic serum) nutritional-chemistry determinations.
• K2EDTA (plastic) 8 Special stopper formulation provides
Royal
low levels of trace elements
Blue
(see package insert). Tube inversions ensure
mixing of either clot activator or anticoagulant
(EDTA) with blood.

• Sodium heparin 8 For plasma determinations in chemistry.

• Lithium heparin 8 Tube inversions ensure mixing of anticoagulant
Green Green (heparin) with blood to prevent clotting.

• Potassium oxalate/ 8 For glucose determinations. Oxalate and

sodium fluoride EDTA anticoagulants will give plasma
• Sodium fluoride/Na2 EDTA 8 samples. Sodium fluoride is the
Gray Gray
• Sodium fluoride 8 antiglycolytic agent. Tube inversions
(serum tube) ensure proper mixing of additive with blood.

• K2EDTA (plastic) 8 For lead determinations. This tube is certified

to contain less than .01 µg/mL(ppm) lead.
Tan Tube inversions prevent clotting.

• Sodium 8 SPS for blood culture specimen collections

polyanethol sulfonate (SPS) in microbiology.
• Acid citrate dextrose
additives (ACD): ACD for use in blood bank studies, HLA
Solution A - 8 phenotyping, and DNA and paternity testing.
Yellow
22.0 g/L trisodium citrate,
8.0 g/L citric acid, 24.5 g/L Tube inversions ensure mixing of anticoagulant
dextrose with blood to prevent clotting.
Solution B - 8
13.2 g/L trisodium citrate,
4.8 g/L citric acid, 14.7 g/L
dextrose
• Liquid K3EDTA (glass) 8 K2EDTA and K3EDTA for whole blood
• Spray-coated K2EDTA 8 hematology determinations. K2EDTA may be
(plastic) used for routine immunohematology testing,
Lavender Lavender
and blood donor screening.***
Tube inversions ensure mixing of anticoagulant
(EDTA) with blood to prevent clotting.
• K2EDTA and gel for 8 For use in molecular diagnostic test methods
plasma separation (such as, but not limited to, polymerase chain
reaction [PCR] and/or branched DNA [bDNA]
White
amplification techniques.) Tube inversions
ensure mixing of anticoagulant (EDTA) with
blood to prevent clotting.
• Spray-coated K2EDTA 8 For whole blood hematology determinations.
(plastic) May be used for routine immunohematology
testing and blood donor screening.***
Pink Pink
Designed with special cross-match label for
patient information required by the AABB.
Tube inversions prevent clotting.
• Buffered sodium citrate 3-4 For coagulation determinations. CTAD for
0.105 M (≈3.2%) glass selected platelet function assays and routine
Light Light 0.109 M (3.2%) plastic coagulation determination. Tube inversions
Blue Blue ensure mixing of anticoagulant (citrate) to
• Citrate, theophylline, 3-4
adenosine, dipyridamole prevent clotting.
(CTAD)

Clear

• None (plastic) 0 For use as a discard tube or secondary

New specimen tube.
Clear
Red/
Light Gray

®
Note: BD Vacutainer Tubes for pediatric and partial draw applications can be found on our website.
BD Diagnostics BD Global Technical Services: 1.800.631.0174 * Invert gently, do not shake
** The performance characteristics of these tubes have not been established for infectious disease testing in general; therefore, users must
Preanalytical Systems BD Customer Service: 1.888.237.2762 validate the use of these tubes for their specific assay-instrument/reagent system combinations and specimen storage conditions.
1 Becton Drive www.bd.com/vacutainer *** The performance characteristics of these tubes have not been established for immunohematology testing in general; therefore, users must
Franklin Lakes, NJ 07417 USA validate the use of these tubes for their specific assay-instrument/reagent system combinations and specimen storage conditions.

BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2010 BD Printed in USA 07/10 VS5229-13
 Module 2: Pre-collection Variables; Peripheral Blood Film; Blood Specimen
Collection
Far Eastern University, Manila – Medical Technology

PRECOLLECTION VARIABLES Purine- Rich Diet: show an increased urate value

FASTING:
Fat- rich food
Fasting Specimens:
1. Potassium
1. FBS 2. ALP
2. GTT 3. TG ELEVATED
3. Lipids 4. 5-HIAA
4. Lipoproteins
5. Gastrin
Stool Occult Blood Test
6. Insulin 1. Meat Cause false
2. Fish
Basal State Collection positive
3. Iron
1. Glucose 4. Horseradish reaction
2. Cholesterol
3. Lipids
Hyperchylomicronemia: Physiological changes;
4. Lipoproteins Increasing turbidity of the serum or plasma and
5. Gastrin potentially interfering with instrument readings.
6. Insulin Serotonin- Rich Food
Fasting for 48 hours 1. Bananas
2. Pineapple
 Increase serum bilirubin
3. Tomato
Fasting for 72 hours 4. Avocado

 in plasma TAG, glycerol, and free fatty acids Long- time vegetarian Diet
with no significant change in plasma
1. LDLS
cholesterol (men)
2. VLDLS
3. Total lipids Decrease
 of plasma glucose to 45 mg/dL – 2.5 mmol/L
4. Phospholipids concentration
(healthy women)
5. Cholesterol
6. Triglycerides

DIET Vitamin B12 Deficiency – also occur, unless

supplements are taken
 Individual’s diet greatly affects laboratory test
results High Unsaturated-to-saturated fatty acid
 Effect is transient and easily controlled ratio: may show decreased serum cholesterol

High Meat Diet Obesity

1. Urea  Serum concentrations of cholesterol,

2. Ammonia ELEVATED triglyceride, and apo B lipoproteins are
3. Urate correlated with obesity
 Plasma insulin concentration is also
Atkins Diet: greatly increase ketones in the urine and increased, but glucose tolerance is impaired.
increase the serum blood urea nitrogen  In obese men, testosterone concentration is
reduced.
Post-Absorptive Hormone Effects
Obese Persons
1. Glucose
2. Lipids 1. Glucose
3. Catecholamines 2. Cortisol
ELEVATED
3. TAG
Caffeine: elevate plasma and free fatty acids and 4. LDL
cause catecholamine release from the adrenal medulla
and brain tissue

| ESPINAS, YANDOC, 2021

 Module 2: Pre-collection Variables; Peripheral Blood Film; Blood Specimen
Collection
Far Eastern University, Manila – Medical Technology

EXERCISE Changing from standing to supine position:

Transient Changes 1. Cholesterol

2. Triglyceride DECREASED
1. Free fatty acids 3. Lipoprotein
2. Lactate
Posture or Position
Elevated
 Significant elevation of potassium after 30
1. CK minutes of standing is due to release of
2. AST potassium from muscles.
3. LD
Prolonged Bed Rest: decreased plasma albumin
Activation due to fluid retention

1. Coagulation Renin Plasma Level: higher when standing than

2. Fibrinolysis supine
3. Platelets
Postural Changes: drugs bound to proteins are
Long- term Effects affected
1. CK TOURNIQUET APPLICATION
2. Aldolase
3. AST  One minute application of tourniquet is
4. LD recommended.
 Effects of prolonged tourniquet
Chronic Aerobic Exercise
1.Hemoconcentration
 Muscle enzymes 2.Anaerobiosis
 Prolactin  Prolonged tourniquet application
 Serum gonadotropin 1. Potassium
2. Proteins
 Sex steroid
3. Enzymes ELEVATED
POSTURE OR POSITION 4. Lactate
5. Cholesterol
 Patient should be seated or supine for at least 6. Ammonia
15 to 20 minutes before blood collection to  Pressure from the tourniquet causes biological
prevent hemodilution or hemoconcentration. analytes to leak from the tissue cells into the
blood.
Preferred Position  Prolonged use of a tourniquet with fist exercise
can increase the serum potassium level by 1
 Upright position mmol/L
 Supine (lying)
Lactate Measurement
Changing from a supine to sitting or
standing  Tourniquet should not be applied, and the
patient should not clench his fist at the time of
1. Albumin the draw
2. Enzymes ELEVATED
3. Calcium Tourniquet application or muscular activity
Changing from sitting to supine position:  May decrease venous p02 and pH
1. Proteins
2. Lipids
TOBACCO SMOKING
3. BUN ELEVATED
4. Iron  It can cause elevated hormone levels such
5. Calcium as the catecholamines and cortisol

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 Module 2: Pre-collection Variables; Peripheral Blood Film; Blood Specimen
Collection
Far Eastern University, Manila – Medical Technology

Increased Levels  TDM – should be schedule according to

the time of the last dose.
1. Glucose
2. Growth hormone
 Hepatoxic drug – elevate liver function
3. Cholesterol enzymes.
4. Triglyceride  Diuretics – can cause decreased plasma
5. Ammonia sodium and potassium
6. Urea  Opiates – increases liver and pancreatic
7. Lactate enzymes.
8. Insulin  Ascorbic Acid – analytic methods based
9. Urinary 5-HIAA on oxidation-reduction reactions may be
influenced positively or negatively.
Decreased Plasma levels
TDM
1. Vitamin B12
 Tubes should not be collected in tubes with
ETHANOL INGESTION gel separator or serum separator tubes
 It causes hypoglycemia among patients with Gel absorbs
chronic alcoholism
1. Phenytoin
Increased levels 2. Phenobarbital
Falsely Low
3. Lidocaine
1. Lactate Result
4. Quinidine
2. Urate
5. Carbamazepine
3. Triglyceride
Gray Top:
Chronic alcohol abuse
 Fluoride oxalate – used for lactate sample
1. HDL cholesterol collection.
2. GGT  Sodium fluoride – used to collect ethanol
ELEVATED
3. Urate specimens.
4. MCV
STRESS BLOOD SPECIMEN COLLECTION
PATIENT IDENTIFICATION
Increased levels:
Conscious Patients:
1. Catecholamines
2. Cortisol  Ask their full names
3. ACTH
4. Prolactin
Sleeping Patients:
5. Albumin  Must be awakened before blood collection
6. Glucose
7. Lactate Unconscious:
Total Cholesterol – has been reported to increase  Asked the attending nurse or relative, ID
with mild stress, and HDL cholesterol to decrease by bracelet
as much as 15%.
Infants and Children:
Hyperventilation – affects acid-base balance and
elevates leukocyte counts, serum lactate, or free fatty  Ask the attending nurse or relative, ID
acids. bracelet

DRUGS Ambulatory:
 Ask their full names, address, or birth date
 Medications affecting plasma volume can
affect protein, BUN, iron, and calcium
concentrations

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 Module 2: Pre-collection Variables; Peripheral Blood Film; Blood Specimen
Collection
Far Eastern University, Manila – Medical Technology

SPECIMEN COLLECTION 2. Veins on the wrist and dorsal aspect of

Arterial Puncture:
 Blood is collected without a tourniquet
 USE: blood gas analysis and pH
measurement
 SITES:
1. Radial artery
2. Brachial artery
3. Femoral artery
4. Scalp artery
5. Umbilical artery
 Femoral Artery: large and easy to puncture
 Modified Allen’s Test:
the hand
3. Veins on the ankle
 CLSI standards: locate the median cubital
vein on both arms before considering the
alternate veins
 Median Cubital Vein:

 Major Complications:
1. Thrombosis
2. Hemorrhage
3. Possible infection
 Unacceptable Sites:
1. Irritated
2. Edematous
3. Near a wound
4. Fistula

Venipuncture:

 Blood is obtained from a patient’s vein

 SITES:
1. Antecubital fossa region

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 Module 2: Pre-collection Variables; Peripheral Blood Film; Blood Specimen
Collection
Far Eastern University, Manila – Medical Technology

SPECIAL SITUATION AND SITES TO BE

AVOIDED (PHLEBOTOMY)

Tourniquet:

 BLOOD PRESSURE CUFF: it is inflated 60

mmHg
RECOMMENDED ORDER FOR DRAWING
 ALLERGIC REACTION: ask the patient for
EVACUATED TUBES & FILLING TUBES FROM
latex sensitivity
A SYRINGE:
 PRELIMINARY VEIN SELECTION:
tourniquet should be released and reapplied
for 2 minutes.

NEEDLE SPECIFICATIONS:

 NEEDLE LENGTH:
 1 or 1.5 in – 21-23 gauge
 ½ - ¾ in – butterfly needle
 23-25 GAUGE NEEDLE: winged infusion set
(butterfly)
 21 GAUGE NEEDLE: standard for
venipuncture

BLOOD COLLECTION TUBES:

IMMEDIATE LOCAL COMPLICATION:
 SILICA PARTICLES: cause the blood to clot
within 15-30 minutes  HEMOCONCENTRATION: an increase in
 CLOT ACTIVATORS: approximately 30 the number formed elements in blood
resulting either from a decrease or increase in
minutes
plasma volume.
 THROMBIN PARTICLES: 5 minutes
 SYNCOPE (FAINTING): transient loss of
 PLAIN-RED TUBES: 60 minutes to clot
consciousness due to lack of oxygen in the
completely
brain and results in an inability to stay upright
position.

LATE LOCAL COMPLICATION:

 THROMBOSIS
 THROMBOPHLEBITIS

LATE GENERAL COMPLICATION:

1. Serum hepatitis
2. AIDS

HEMOLYSIS:
1. Using a needle that is too small
2. Pulling a syringe plunger back too fast

| ESPINAS, YANDOC, 2021

 Module 2: Pre-collection Variables; Peripheral Blood Film; Blood Specimen
Collection
Far Eastern University, Manila – Medical Technology

3. Expelling the blood vigorously into a tube OTHER BODY FLUIDS

4. Forcing the blood from a syringe into an
evacuated tube CEREBROSPINAL FLUID:
5. Shaking or mixing the tube vigorously
6. Performing blood collection before the alcohol  COLLECTION METHOD: Lumbar puncture
has dried at the collection site. (most common) between 3rd and 4; 4th and
5th
HEMATOMA:
1. Fragile and small veins
2. Needle penetrates all the way through the
vein
3. Needle is partly inserted into the vein
4. Needle is removed while tourniquet is still on
5. Excessive probing  OTHER METHODS:
6. Pressure is not adequately applied 1. Cisternal puncture
2. Lateral cervical
SKIN PUNCTURE:  VOLUME: 20mL of CSF
 ORDER OF DRAW:
 LENGTH OF LANCET: 1.75mm (preferred) 1. Chemistry
 DEPTH OF INCISION: 2. Microbiology
 <2.0 mm – infants 3. Hematology
 <2.5 mm – adults
 1.5-2.4 mm: distance from the skin surface DIURNAL VARIATION:
to bone or cartilage in the middle finger
 USEFUL IN ADULTS:
 Severely burned
 Extremely obese
 Thrombotic tendencies
 Fragile veins
 Veins (therapeutic purposes)
 PREFERED SITES:
1. Lateral plantar heel surface
2. Palmar surfaces of the fingers
3. Plantar surface of the big toe
4. Earlobes

MICROCOLLECTION TUBES:
1. Blood gases
ORDER OF FILING
2. EDTA
MICROCOLLECTION
3. Other additives tubes
TUBES
4. Serum tubes

| ESPINAS, YANDOC, 2021

 Module 2: Pre-collection Variables; Peripheral Blood Film; Blood Specimen
Collection
Far Eastern University, Manila – Medical Technology

PERIPHERAL BLOOD FILM

Peripheral Blood Film

PBS EXAMINATION:
 Important component in the evaluation of an
anemia
 Gives particular attention of RBC diameter,
shape, color, and inclusion
 Serves as a quality control to verify results
produced by automated analyzers

PHYSIOLOGICAL FACTORS:

COMPLETE BLOOD COUNT

HEMOGRAM:
SPECIMEN REJECTION:  A capstone of a panel of tests in the peripheral
film evaluation
 Includes enumeration of cellular elements,
quantitation of hemoglobin, and statistical
analyses that provides a snapshot of cell
appearance.

BLOOD SPECIMEN COLLECTION

SKIN PUNCTURE:

 A drop of blood
 Finger, heel, or microhematocrit tube
 May be delivered using a Diff-Safe dispenser
that is inserted through the rubber stopper of
the EDTA tube
 Size of the drop of blood is important
 Thick film: too large drop
 Thin film: too small drop

SOURCES OF SPECIMENS

EDTA BLOOD:

 Disodium/ tripotassium EDTA

 Made within 2 to 3 hours of drawing the
specimen

ADVANTAGES:

1. Multiple slides

| ESPINAS, YANDOC, 2021

 Module 2: Pre-collection Variables; Peripheral Blood Film; Blood Specimen
Collection
Far Eastern University, Manila – Medical Technology

2. May be prepared at later time METHODS OF BLOOD FILM

3. Avoids platelet clumping PREPARATION:
EDTA BLOOD:
PUSH-TYPE WEDGE: It is called push because the
 EDTA tubes that remain at room temperature spreader slide is pulled into the drop of blood, and the
for more than 5 hours often have film is made by pushing the blood along the slide.
unacceptable blood artifacts
PULLED FILM: In this procedure, the spreader slide
BLOOD CELL ARTIFACTS: is pushed into the drop of blood and pulled along the
length of the slide to make the film.
1. Echinocytic RBCs
2. Spherocytes TWO-GLASS SLIDE METHOD:
3. Necrobiotic Leukocytes
4. Vacuolated Neutrophils  Manual Wedge Technique or aka Push-
Wedge Method
ANTICOAGULANT-FREE BLOOD:  Easiest to master; most convenient;
commonly used
 Best for evaluation of blood cell morphology  SLIDES BEING USED:
1. Pusher or Spreader slide
MAIN ADVANTAGES:
2. Film slide: 3-inch x 1-inch (75-mm x 25-
1. Made at the patient’s side mm)
2. Some artifacts may be prevented  ANGLE: 30-45 degree
 Too high – thicker smear
DISADVANTAGES:  Too low – thinner smear
 DROP OF BLOOD: 2-3 mm
1. Platelet clumping
 Large amount of blood → thicker or
2. Few films can be made
longer smear
 Small amount of blood → thinner or
EDTA BLOOD SMEAR
shorter smear
PLATELET SATELLITOSIS:  SPEED OF SPREADER:
 Too fast → thicker smear
 Platelets surround or adhere to the  Too slow → thinner smear
neutrophils  HEMATOCRIT OF PATIENT:
 EFFECTS:  Too high → angle is lowered as low as
1. PSEUDOTHROMBOCYTOPENIA 25˚
2. PSEUDOLEUKOCYTES  Too low → angle is raised
 CORRECTION: recollect blood sample using
3.2% Na citrate CHARACTERISTICS OF AN IDEAL
 DILUTION FACTOR: is the reciprocal of the BLOOD SMEAR (WEDGE METHOD)
dilution (i.e., 10/9 or 1.1)
1. Gradual transition from thick to thin area
EDTA INDUCED PLATELET CLUMPING: 2. 2/3rd to 3/4th the length of the film slide
3. Finger-shaped
 Platelets agglutinates are similar in size to 4. Visible lateral edges
WBC 5. Without irregularities, holes, or streaks
 EFFECTS: 6. Feather edge has rainbow appearance
3. PSEUDOTHROMBOCYTOPENIA 7. Whole blood is picked up and spread
4. PSEUDOLEUKOCYTES
 CORRECTION: recollect blood sample using WATER OR DRYING ARTIFACT
3.2% Na citrate
 DILUTION FACTOR: is the reciprocal of the A. - May appear as: moth-eaten look to the
dilution (i.e., 10/9 or 1.1) RBCs;
- Heavily demarcated central pallor;
- Refractive (shiny) blotches on the RBC
B. Manifest as echinocytes (crenation) seen in
the areas of the slide that dried most slowly
C. Causes: Humidity in the air as the slide dries;
films from extremely anemic patients because

| ESPINAS, YANDOC, 2021

 Module 2: Pre-collection Variables; Peripheral Blood Film; Blood Specimen
Collection
Far Eastern University, Manila – Medical Technology

of very high ratio of plasma to RBCS: Water  WBC nuclei: purple to blue
absorbed from the air into the alcohol-based  Cytoplasm of neutrophil: pink to tan with
stain violet or lilac granules
D. Prevention: drying the slide as quickly as  Eosinophil: bright orange refractile granules
possible help keeps moisture out; fix slides in
pure, anhydrous methanol before staining; BLOOD FILM STAINING
use 10% volume-to-volume methanol.
BLOOD FILM STAINING:
PROBLEMS ENCOUNTERED:
 PURPOSE: for evaluation of cellular
1. Uneven distribution of WBCs in various morphology
areas of slide  SOLUTIONS:
2. Feather edge and side edges: more 1. FIXATIVE: Methanol
segmented neutrophils, monocytes, and 2. STAIN: Romanowsky stain
eosinophils 3. BUFFER: Age dist. H2O
3. At the center of the film: more small
lymphocytes ROMANOWSKY-BASED STAINS

COVER SLIP TECHNIQUE:  ROMANOWSKY STAINS: defined as any

stain which contains methylene blue and a
 It is rarely used; sometimes used for bone halogenated fluorescein dye
marrow aspirate smear preparation  EXAMPLES:
 Excellent WBC distribution 1. Wright stain
 METHODS: 2. Giemsa stain
1. Beacom’s Method 3. May – Grunwald stain
2. Ehrlich’s Method  METHYLENE BLUE: a basic stain; it colors
the nucleus and some cytoplasmic structures
AUTOMATED METHOD: a blue or purple color
 EOSIN: an acidic stain; it colors some
MINIPREP: semi-automatic portable instrument; cytoplasmic an orange – red color
Stimulates manual-wedge technique of blood smear
preparation
CENTRIFUGAL TYPE: uses approximately 0.2 mL of
well mixed anticoagulated blood

SYSMEX SP-10:
1. An automated slide-making and staining
system
2. After a CBC for a specimen, a conveyor
moves the rack tube to the SP-10, where
barcode read
3. Based on the hematocrit reading, the system
adjusts the size of the drop of blood used in
the angle and speed of the spreader slide in
making a wedge preparation
4. Films can be produced approximately every
30 seconds
5. Stain and then buffer and rinse are added at MANUAL TECHNIQUE
designated times  Performed over a sink or pan with a staining
rack
WELL-STAINED PBS
 Stain may be filtered before use or poured
MACROSCOPIC: directly from the bottle through a filter onto the
slide
 Blood film: pink to purple  Stain should remain on the slide atleast 1 to
3 minutes
MICROSCOPIC:  Approximately equal amount of buffer is
 RBC: orange to salmon pink added to the slide:

| ESPINAS, YANDOC, 2021

 Module 2: Pre-collection Variables; Peripheral Blood Film; Blood Specimen
Collection
Far Eastern University, Manila – Medical Technology

o Surface tension: allows very little of the  Disadvantage: Quality is often a concern, but
buffer to run off. color quality can be acceptable.
o Shows metallic sheen on the slide if
mixing of buffer is correct IMPORTANT REMINDERS:
o Mixture is allowed to remain on the slide
for 3 minutes or more 1. Best staining results are obtained on fresh
 After staining, the slide is rinsed with of slides because the blood itself acts as a
neutral-pH water and air-dried in a vertical buffer in the staining process
position. 2. Slides stained after 1 week or longer turn-out
 This technique is desirable for staining too blue
peripheral blood films containing very high 3. Specimens that have increased levels of
WBC counts, such as the films from leukemic proteins produce bluer-staining blood films,
patients. even freshly stained
 Disadvantages: increased risk of spilling the
stain and the longer time required to complete
METHODS OF BLOOD FILM
the procedure. PREPARATION:
 This technique is best suited for low-volume
laboratories. SCANNING METHODS:
▪ METHODS
AUTOMATED SLIDE STAINERS
1. Cross-sectional or Crenellation
 This technique is best suited for high-volume
2. Longitudinal
laboratories
3. Battlement
 Takes about 5 to 10 minutes to stain a batch
of slides.
 Slides may be automatically dipped in stain
and then in buffer and a series of rinses
propelled along a platen surface by two
conveyor spirals
 Disadvantages of the dip-type batch strainers:
o Stat slides cannot be added to the batch
once the staining process has begun
o Working or aqueous solutions of stain are
stable for only 3 to 6 hours and need to
be made often.
EVALUATION OF PBS

HEMA-TEK DEVICE LOW POWER EXAMINATION:

 Used to assess distribution and the
 Stain, buffer, and rinse are pumped through
appropriate evaluation area for RBC
holes in the platen surface, flooding the slide
 Locate rare abnormal WBCs or other cells
at the appropriate time
which may be examined more closely under
 Stat sides can be added at any time to the
higher magnification
Hema-Tek Stainer, and stain packages are
 Feathered Edge: red cells appear
stable for about 6 months.
macrocytic, flattened, lack central pallor, and
QUICK STAINS white blood cells are often distorted
 Thick Part: red cells appear microcytic and
 are fast and easy; takes about 1 minute. may seem to form rouleaux
 Stain is a modified Wright or Wright-Giemsa
stain HIGH POWER EXAMINATION:
 required quantity can be filtered into a Coplin
 Used also to estimate total WBC count
jar or a staining dish
 Buffer: Aged distilled water NO. WBC PER ESTIMATED WBC COUNT
 Stained slides are given a final rinse under a HPF
gentle stream of tap water and allowed to air- 2 to 5 WBCs/ hpf 4000 to 7000 WBCs/uL
dry. 4 to 6 WBCs/ hpf 7,000 to 10,000 WBCs/uL
 This technique are convenient and cost- 6 to 10 WBCs/ hpf 10,000 to 13,000 WBCs/uL
effective for low-volume laboratories 10 to 20 WBCs/ hpf 13,000 to 18,000 WBCs/uL

| ESPINAS, YANDOC, 2021

 Module 2: Pre-collection Variables; Peripheral Blood Film; Blood Specimen
Collection
Far Eastern University, Manila – Medical Technology

2. Trypanosoma gambiense
3. Trypanosoma cruzi

OIL IMMERSION EXAMINATION:

 Used to examine the nuclear details of the
white blood cells
 Used also for the tabulation of the actual WBC
differential and estimation of platelet count
 Scan ten (10) oil immersion fields for the
number of platelets
 Estimated platelet count per uL

PARASITES FOUND IN PBS

MALARIA:

1. Plasmodium falciparum
2. Plasmodium malariae
3. Plasmodium vivax
4. Plasmodium ovale

FILARIA:

1. Wucheria bancrofti
2. Brugia malayi
3. Loa loa

TRYPANOSOMES:

1. Trypanosoma rhodiense

| ESPINAS, YANDOC, 2021