IMPORTANT KEY NOTES:
Serum tube with or without clot activator or gel
CLINICAL CHEMISTRY:
Multisampling collection
Phlebotomy
The phlebotomist uses standard precautions, which
include washing hands and applying gloves at the
beginning of the procedure and removing gloves and
washing hands at the end of the procedure. The
Clinical and Laboratory Standards Institute (CLSI)
recommends the following steps:
1. Prepare the accession (test request) order.
2. Greet the patient and identify the patient by
having the patient verbally state his or her full
name and confirm with the patient’s unique
identification number, address, and/ or birth date.
Ensure the same information is on the request
form.
3. Sanitize hands.
4. Verify that any dietary restrictions have been
met (e.g., fasting, if appropriate) and check for latex
sensitivity.
5. Assemble supplies and appropriate tubes for the
requested tests. Verify paperwork and tube
selection.
6. Reassure and position the patient.
7. If necessary to help locate a vein, request that
the patient clench his or her fist.
8. Apply the tourniquet and select an appropriate
venipuncture site, giving priority to the median
cubital or median vein. Ensure the tourniquet is on
for no longer that 1 minute.
9. Put on gloves.
10. Cleanse the venipuncture site with 70%
isopropyl alcohol using concentric circles from the
inside to outside. Allow skin to air-dry.
11. Inspect the equipment and needle tip for burrs
and bends.
12. Perform the venipuncture by anchoring the vein
with the thumb 1 to 2 inches below the site and
inserting the needle, bevel up, with an angle less
than 30 degrees between the needle and the skin.
Collect tubes using the correct order of draw, and
invert each tube containing any additive
immediately after collection. CLSI recommends a
particular order of draw when collecting blood in
multiple tubes from a single venipuncture. Its
purpose is to avoid possible test result error
because of cross-contamination from tube additives.
a. Blood culture tube (yellow stopper)
b. Coagulation tube (light blue stopper)
c.
(red, gold, red-gray marbled, orange, or yellow-gray
stopper)
d. Heparin tube (green or light green stopper) e.
EDTA tube (lavender or pink stopper)
f. Sodium fluoride tube with or without EDTA or
oxalate (gray stopper)
13. Release and remove the tourniquet as soon as
blood flow is established or after no longer than 1
minute.
14. Ensure that the patient’s hand is open.
15. Place gauze lightly over the puncture site
without pressing down.
16. After the last tube has been released from the
back of the multisample needle, remove the needle
and activate the safety device according to the
manufacturer’s directions.
17. Apply direct pressure to the puncture site using
a clean gauze pad.
18. Bandage the venipuncture site after checking to
ensure that bleeding has stopped.
19. If a syringe has been used, fill the evacuated
tubes using a syringe transfer device.
20. Dispose of the puncture equipment and other
biohazardous waste.
21. Label the tubes with the correct information.
The minimal amount of information that must be on
each tube is as follows:
a. Patient’s full name
b. Patient’s unique identification number
c. Date of collection
d. Time of collection (military time)
e. Collector’s initials or code number
NOTE: Compare the labeled tube with the patient’s
identification bracelet or have the patient verify
that the information on the labeled tube is correct
whenever possible.
ORDER OF THE DRAW
1. Blood culture tube (yellow stopper)
2. Coagulation tube (light blue stopper)
3. Serum tube with or without activator (red, gold,
red-gray marbled, orange, or yellow-gray stopper)
4. Heparin tube (green or light green stopper)
5. EDTA tube (lavender or pink stopper)
6. Sodium fluoride with or without EDTA or oxalate
(gray stopper)
Proper use of pipette
STEPS
1. Set the 250 ul
2. Press firmly to attach a tip.
3. Press and hold plunger at first stop.
4. Place tip in the sample liquid.
5. Slowly release the plunger.
6. Pause for 1 sec. Lift pipette straight up.
DELIVERY
1. Insert tip into the delivery tube.
2. Press the plunger to 2nd stop.
3. Pause for at least 1 second.
4. Remove the tip from the tube.
5. Slowly release the plunger.
6. Eject the tip.
Note: First Stop, Second Stop
Pag inaaspirate, isang kamay lang po ang hawak. Yung
kukuhanang container nakatayo, first stop, aspirate tas
make sure na nakalubog yung tip – dapat steady,
nakalubog at hindi din po dapat nakadikit sa ilalalim.
FIRST STOP, LUBOG SA LIQUID, ASPIRATE WAIT
MATAPOS. Aspirate slowly lalo na pag thick. Pag
pinunasan, hindi po dapat didikit sa tip kasi mag
cacapillary siya sa pamunas. Usually sa research lang po
ginagawa.
HEMATOLOGY
PHLEBOTOMY
CELL COUNTING
Using hemocytometer for WBC and RBC Count. Solve.
Whole blood anticoagulated with EDTA or blood from
a skin puncture is diluted with 1% buffered
ammonium oxalate or a weak acid solution (3%
acetic acid or 1% hydrochloric acid). The diluting
fluid lyses the nonnucleated RBCs in the specimen
to prevent their interference in the count. The
typical dilution of blood for the WBC count is 1:20.
A hemacytometer is charged (filled) with the well-
mixed dilution and placed under a microscope and
the number of cells in the four large corner squares
(4 mm2) is counted.
HEMATOCRIT
Perform the hematocrit test using an extracted sample
Hematocrit is the ratio of the volume of packed
RBCs to the volume of whole blood and is manually
determined by transferring blood to a plastic tube
with a uniform bore, centrifuging, measuring the
column of RBCs, and dividing by the total length of
the column of RBCs plus plasma.7 The normal ratio
approaches 50%.
Procedure:
1. Fill two plain capillary tubes approximately three-
quarters full with blood anticoagulated with EDTA or
heparin. Mylarwrapped tubes are recommended by
the National Institute for Occupational Safety and
Health to reduce the risk of capillary tube
injuries.14 Alternatively, blood may be collected into
heparinized capillary tubes by skin puncture. Wipe
any excess blood from the outside of the tube.
2. Seal the end of the tube with the colored ring
using nonabsorbent clay. Hold the filled tube
horizontally and seal by placing the dry end into the
tray with sealing compound at a 90-degree angle.
Rotate the tube slightly and remove it from the
tray. The plug should be at least 4 mm long.14
3. Balance the tubes in a microhematocrit
centrifuge with the clay ends facing the outside
away from the center, touching the rubber gasket.
4. Tighten the head cover on the centrifuge and
close the top. Centrifuge the tubes at 10,000 g to
15,000 g for the time that has been determined to
obtain maximum packing of RBCs, as detailed in
Box 11.2. Do not use the brake to stop the
centrifuge.
5. Determine the hematocrit by using a 
microhematocrit reading device (Figure 11.7). Read
the level of RBC packing; do not include the buffy
coat (WBCs and platelets) when taking the reading
(Figure 11.8).
6. The values of the duplicate hematocrits should
agree within 1% (0.01 L/L).14
NOTE: Capillary Tube with Anticoagulated Whole
Blood after Centrifugation. Notice the layers
containing plasma, the buffy coat (white blood cells
and platelets), and the red blood cells. - CLAY
Sources of Error and Comments
1. Improper sealing of the capillary tube causes a
decreased hematocrit reading as a result of leakage
of blood during centrifugation. A higher number of
RBCs are lost compared with plasma because of the
packing of the cells in the lower part of the tube
during centrifugation.
2. An increased concentration of anticoagulant
(short draw in an evacuated tube) decreases the
hematocrit reading as a result of RBC shrinkage.
3. A decreased or increased result may occur if the
specimen was not mixed properly.
4. The time and speed of the centrifugation and
the time when the results are read are important.
Insufficient centrifugation or a delay in reading
results after centrifugation causes hematocrit
readings to increase. Time for complete packing
should be determined for each centrifuge and
rechecked at regular intervals. When the
microhematocrit centrifuge is calibrated, one of the
specimens used must have a hematocrit of 50% or
higher.
5. The buffy coat of the specimen should not be
included in the hematocrit reading because this
falsely elevates the result. 6. A decrease or increase
in the readings may be seen if the microhematocrit
reader is not used properly.
SMEAR PREPARATION
Thick and Thin Smear
Malarial smear
Galing sa prick yung sample or needle
Thick Smear – hindi kinakalat, tas dalawang pair,
dinedehemoglobinized
THICK
 Place a small drop of blood in the center of the
pre-cleaned, labeled slide.
Using the corner of another slide or an
applicator stick, spread the drop in a circular
pattern until it is the size of a dime (1.5 cm2).
 A thick smear of proper density is one which, if
placed (wet) over newsprint, allows you to
barely read the words.
 Lay the slides flat and allow the smears to dry
thoroughly (protect from dust and insects!).
Insufficiently dried smears (and/or smears that
are too thick) can detach from the slides during
staining. The risk is increased in smears made
with anticoagulated blood. At room
temperature, drying can take several hours; 30
minutes is the minimum; in the latter case,
handle the smear very delicately during
staining. You can accelerate the drying by using
a fan or hair dryer (use cool setting). Protect
thick smears from hot environments to prevent
heat-fixing the smear.
 Do not fix thick smears with methanol or heat. If
there will be a delay in staining smears, dip the
thick smear briefly in water to hemolyse the
RBCs.
THIN
A thin smear being prepared.
 Place a small drop of blood on the pre-cleaned,
labeled slide, near its frosted end.
 Bring another slide at a 30-45° angle up to the
drop, allowing the drop to spread along the
contact line of the 2 slides.
 Quickly push the upper (spreader) slide toward
the unfrosted end of the lower slide.
 Make sure that the smears have a good
feathered edge. This is achieved by using the
correct amount of blood and spreading
technique.
 Allow the thin smears to dry. (They dry much
faster than the thick smears, and are less
subject to detachment because they will be
fixed.)
MICROSCOPE
Stained smear in all objectives
Proper Storage – Yung stage nasa lowest setting, yung
objective nasa lowest setting dahil para pag minove siya
hindi magbabangga at kunwari nilift up tas nakaatas
yung iba o kay anatapat sa oil, magagasgas yung
objective.
We find it first in scanner, yung area lumalaki tas pag
malinaw na imomove sa next objective tas move the
fine adjustment.
Wiping the first drop away prevents contamination of
the specimen with excess tissue fluid and removes
alcohol residue that could prevent formation of well-
rounded drops or hemolyze the specimen.
Step 14: Fill and mix the tubes/containers in order of
draw.
Mixing tubes immediately after they are filled is
necessary for proper additive function and to prevent
clotting in anticoagulant tubes.
Following the order of draw for capillary specimens
minimizes negative effects of clotting on hematology
specimens and other specimens collected in
anticoagulant tubes.

CAPILLARY PUNCTURE
2 Microhematocrit tubes, 1 smear for CBC

Step 1: Receive, review, and accession the test request.

Step 2: Approach, greet, and identify the patient.
Step 3: Explain the procedure and obtain consent.
Step 4: Verify collection requirements and identify
sensitivities and potential problems.
Step 5: Sanitize hands and put on gloves.
Step 6: Position the patient.
Step 7: Select the puncture site and warm the hand if
needed.
Step 8: Clean and air-dry the site.
Cleaning with 70% isopropyl alcohol removes or inhibits
skin flora that could infiltrate the puncture and cause
infection.
Step 9: Prepare equipment.
Step 10: Grasp the finger firmly. Grasping the finger
firmly helps prevent sudden movement by the patient.
Step 11: Position the lancet, puncture the site, and
discard the lancet.
Step 12: Lower the finger and apply gentle pressure
until a blood drop forms
Lowering the finger helps blood begin to flow freely.
Gentle pressure encourages blood flow without
compromising specimen integrity.
Step 13: Wipe away the first blood drop.