HEMA1 LAB (PRELIM PRACTICALS)

LESSON 1
SAFETY IN HEMATOLOGY LABORATORY
OSHA STANDARDS
 OSHA provides standards to maintain safe work
LABORATORY HAZARDS
environment
 The following practices are enforced inside the
Biological Hazard laboratory:
1. Handwashing
2. Food, drink and medications not allowed
Sharp Hazard 3. Applying cosmetics are prohibited
4. Fomites or any surfaces must be kept away from
mouth and all mucous membranes
5. Contaminated sharps must be disposed properly
Chemical Hazard 6. Personal Protective Equipment must be worn at all
times following the proper donning
7. Equipment should be check and maintained

Radiation Hazard

Electrical Hazard

Fire Hazard

Physical Hazard

OCCUPATIONAL HAZARD
1. Fire Hazard
 Enforcement of a non-smoking policy
Other Hazards  Placement of fire extinguishers every 75 feet,
checked monthly and maintained annually
 Placement of fire detection system and manual fire
alarm near exit doors which is less than 200 ft
away and should be tested every three months
 Written fire prevention and response procedures
and fire drills
BIOSAFETY LEVEL

SAFETY IN HEMATOLOGY LABORATORY

SAFETY PRECAUTIONS
 Exposure to blood and body fluids is the most common
risk associated in hematology laboratory
 Bloodborne pathogens are pathogenic microorganisms
present in blood causing infection or diseases
HEMA1 LAB (PRELIM PRACTICALS)

3. Electrical Hazard
 Use of adapters, gang plugs and extension cords
are prohibited.
 Stepping on cords, rolling heavy equipment over
cords should be prohibited.
 Before repair or adjustment of electrical
equipment, unplug first the equipment making
sure that the hand is dry and no jewelry should be
present.

4. Needle Puncture
 Containers should be puncture proof
 Improper disposal is the major cause of needle
prick incident
 Replaced once the container is ¾ full

PHYSICAL HAZARD
An agent, factor, or circumstance that can cause harm
without contact.
It includes:
2. Chemical Hazard  Ergonomic Hazard
 Labelling of all chemicals properly.  Vibration Hazard
 Follow handling, storage requirements  Noise Hazard
 Use adequate ventilation.
 Spill response procedures should be included in LABORATORY WASTE MANAGEMENT SEGREGATION
the safety procedures. i. BIODEGRADABLE
 MSDS should be available and reviewed by ii. NON- BIODEGRADABLE
laboratory personnel. iii. HAZARDOUS WASTE
 Special waste
 Biological waste
 Chemical waste
 Radioactive Wast

WASTE DISPOSAL STANDARD

Blood Containing Waste
 Objects contaminated with blood should be autoclaved
before disposal
 Blood should be treated before disposal; treatment
involves the use of aldehyde, chlorine compounds,
phenolic compounds or thru autoclaving before pouring
down the sink with running water

Standard Waste Protocol (PHIL)

LABORATORY WASTE MANAGEMENT DISPOSAL

 Flushing Down the Drain to the Sewer System
 Incineration
 Landfill Burial
HEMA1 LAB (PRELIM PRACTICALS)
 Recycling

OCCUPATIONAL SAFETY AND HEALTH ACT

 Public Law 11058
 ”An Act Strengthening Compliance with Occupational
Safety and Health Standards and Providing Penalties for
Violations Thereof”
 GOAL: Provide all employees (clinical laboratory
personnel included) with a safe work environment.
GOVERNMENT REGULATORY AGENCIES
1) Department of Labor: 29 Code of Federal Regulations
Parts 1900-1910
 Hazard Communication Standard
 Hazardous Waste Operations
 Occupational Exposure to bloodborne pathogens
Standards
2) Department of the Interior, Environmental Protection
Agency: 40 Code of Federal Regulations Parts 200-399
 Clean Air Act and Clean Water Act
 Toxic Substances Control Act
 Comprehensive Environmental Response,
Compensation and Liability Act (CERCLA)
3) Voluntary Agencies/Accrediting Agencies
 The Joint Commission
 College of American Pathologist
 Centers for Disease Control and Prevention (CDC)
 Clinical and Laboratory Standards Institutes
LESSON 2
BLOOD COLLECTION
 Blood = most commonly submitted and processed
specimen in laboratory
a. Arterial puncture
 Obtaining arterial blood
 Not performed by Medical Technologist
Arterial Puncture
 Collected from an artery, primarily to determine arterial
gases.
 Artery = where oxygenated blood takes place
 If we want to measure the totally of gases in our
body, giving importance with oxygen, carbon
dioxide etc, it is ideal to utilize an arterial blood.
 If the concern of the diagnosis of the disease by
the doctor related to blood gases (Ex. respiratory
diseases), it would be more appropriate to use an
arterial blood.
 Performed by: Respiratory therapists and physicians are
the only ones allowed
 Medtechs are not trained to do this (so do not
decide to perform this method because it is very
dangerous and complicated)
 Can be obtained either through a catheter placed in an
artery, or by using a needle syringe (standard) to
puncture an artery.
PUNCTURE SITES
a) Radial artery
 Most preferred
 Easily accessible
 Because of it’s small size, the use of this artery
requires special skills and extensive training
 Complications may arise due to improper puncture
(nerve damages, spasms etc.)
 thumb side of wrist (where pulse located)
 No need to apply torniquet, there’s already a
pressure because of the pulse.
 Do not apply pressure in pulling the plunger, hence
guide the plunger because blood will naturally
come out because of the pressure.
 In between the fingers used in locating the site,
separate them and in between you make the
puncture.

b. Skin puncture
 Obtaining a peripheral blood or capillary blood
(combination of capillary, arterial, venous, and
tissue fluids)

c. Venipuncture
 Obtaining venous blood
 The most commonly performed way of collecting
b) Brachial artery
blood
c) Femoral artery
The following are the main concern factors in collecting
*In Brachial and Fomoral arteries, arterial blood maybe
blood:
harder to collect because these two arteries are harder to
1. The uses of blood sample collected from a certain
locate than radial artery. Less superficial. Surrounded by a lot
technique.
of structures that could be damage because of improper
2. Different sites to be used.
techniques.
3. Different sites to be avoided.
4. Different adverse effects or complications during and
after collection.
COMPLICATIONS
HEMA1 LAB (PRELIM PRACTICALS)
Arteriospasm  If after with certain abnormalities affecting the
- involuntary contraction of the artery WBC or histiocytes
- it may happen whenever patient is stressed or anxious
before or during the collection (try to build rapport) c) <1 year old: lateral portion of the plantar surface of the
heel/toe
Hematoma  Puncture patient, once only especially newborns.
- or excessive bleeding
- prevented by applying pressure on the patient’s puncture SITES TO AVOID
site  Inflamed and pallor areas
 Cold and cyanotic areas
Nerve damage  Congested and edematous areas
- prevented by choosing an appropriate sampling site and  Scarred and heavily calloused areas
avoiding redirection of the needle.
- commonly encountered in searching artery.

Fainting
- prevented by ensuring that the patient is supine with feet
elevated before beginning the blood draw

INDICATIONS FOR SKIN PUNCTURE:

Question: Why should I perform skin puncture than

venipuncture? When we should perform this?
Answer: Patient is a newborn; Among geatric patient, this
procedure is advisable; Test that only requires small amount
of blood.

ADVANTAGES OF SKIN PUNCTURE

Tests requires small amount of blood: Blood typing, POCT
testings like glucose testing, Blood smear preparation etc.
*The angle of the needle towards the site of puncture should
o
always be adjusted, not always 90 The blood you collected from skin puncture can be
transferred to different tubes, we use Microtubes.
Skin Puncture  Some will require 0.1 or 0.5 mL of blood

 A mixture of capillary, venous and arterial blood with ORDER OF DRAW

interstitial (tissue) fluid and intracellular fluid.
 More painful than venipuncture because in this site,
there is a lot of nerve endings.
 A lot easier to do and perform
 To prevent any possible contamination of tissue juices
on sample, discard the first 2 drops of blood (not
applicable always).
 3-4mm deep (ideal depth to hit the capillary bed to
obtain ample amount of blood)
*Excessive collection blood = loss of blood resulting to anemia,
specifically iatrogenic anemia (applicable also in venipuncture
and arterial puncture)
1. Those tubes used for hematology (particularly EDTA).
 For cell counting
 Remember, as long as you have injury, the
platelets will then travel to that site and start to
aggregate.
st
 If the 1 drop of blood is not used for cell counting
and hematology, expect that platelets will then
clumping. Platelets already aggregated then you
will not obtain accurate result.
2. Those tubes containing anticoagulant or designed for
chemistry, bloodbanking etc.
*capillary tube = used for hematocrit testing

PUNCTURE SITES
a) Finger
rd th
 3 or 4 finger are commonly used
 Non-dominant hand (less callous)

b) Earlobe
HEMA1 LAB (PRELIM PRACTICALS)
 Never ever mention their name.
 Then check the name tags.
 Torniquet application
 Should be applied for maximum 1 minute only (to
avoid hemoconcentration that will then lead to
hemolysis)
 3-4 inches (or 10-15cm) away from the site of
puncture
 Disinfection
 70% alcohol
 inner to outer in circular motion
 Angle of needle insertion
 Varies
o
 Commonly 35-45
 If the vein is very prominent, no need to use a very
high angle.
 In needle insertion, it should be “hit the skin first
then into vein” or “skin going to the vein” (to
prevent blood spurts or blood spillage; prevent
hematoma)
 After needle removal, apply pressure to avoid
COMPLICATIONS hematoma
 Collapse of veins if the tibial artery is lacerated from  Bevel up
puncturing the medial aspect of the heel.  Needle length
 Osteomyelitis of the heel bone (calcaneus).  1-1.5 inches
 Nerve damage if the fingers of neonates are punctured.  Gauge/Bore = depend on the integrity of the vein
 Haematoma and loss of access to the venous branch  Green = 21
used.  Black = 22
 Scarring  Blue = 23
 Localized or generalized necrosis  25 = promote hemolysis
 Skin breakdown from repeated use of adhesive strips.  The higher the number, the smaller the bore.
(treatment: apply preassure to puncture site)  Position of the patient
 Label
Venipuncture  Only label the tube after blood has been
transferred.
 Manner of inserting a needle attached to syringe to a  No “pre-labeling” of tubes.
palpable vein to collect blood for laboratory testing.  Name, Age, Gender, Room/Bed number, Date.
 Specimen collected: Venous blood Time, Initials of the phlebotomist
 Most widely used blood sample in all laboratory tests.  Disposal
 Routinely performed in the laboratory  Puncture-resistant bottles = sharps
 Most common and ideal way of collecting blood.  Yellow bags = infectious materials
 In a single puncture alone, you will obtain large amount
of blood. (can be subjected to different testing or METHOD OF COLLECTION
procedures in the laboratory)  Single collection = syringe method and the blood
collected will be transferred in a single tube only.
Three (3) factors that affect or lead to a successful  Multiple collection = blood collected will then transfer
collection: in multiple tubes; ETS
A. Patient (accesibility of puncture sites).
B. Accesibility of materials/equipment.
C. Phlebotomist himself (most important; the readiness,
knowledge and skill of the Medtech)

THINGS TO REMEMBER!
 Proper identification of patient (the most important
and critical)
 Ask the patient about his/her complete name, let
his/her to state this
 Let the patient identify herself/himself for you.
 If patient is in coma, ask the relative. If the relative
is not around ask the nurse.
HEMA1 LAB (PRELIM PRACTICALS)
 Popliteal vein
 Ankle vein
*Antecubital fossa (RECOMMENDED)

 “Winged blood collection” = make use of butterfly

infusion attached to a syringe or vacutainer set.

 3 years old to adult life

 Wrist vein
 Dorsal vein of hand
 Dorsal vein of ankle
*Antecubital fossa (RECOMMENDED)

ANTECUBITAL FOSSA
 Two (2) patterns of vein:
i. “H” pattern
st
 Median capital vein (1 choice)
nd
 Cephalic vein (2 choice)
rd
 Basilic vein (3 choice; movable, more painful,
rolls, more likely to form hematoma)
ii. “M” pattern
 Median
 Accessory cephalic vein
 Basilic vein

SITES TO AVOID
 Sites with hematoma
 Occluded veins (impaired blood circulation due to
thrombus or clot, used for multiple puncture)
 Edematous area (tissue fluids will obtain)
 Sites with burns, scar, tattoo
 Sites with fistula
 IV fluid sites
 Let the nurse stop the IV infusion before you
collect blood.
 Stop it for 2-5 minutes.
 The first 5mL should be discarded

*those who undergo mastectomy, blood can’t be collected

here, find another site.
PUNCTURE SITES
 Newborn up to 18 months ADVERSE EVENTS
 External Jugular Vein
 Temporal vein
*Antecubital fossa (RECOMMENDED)

 Older children (18 months to 3 years old)

 Femoral vein
 Long saphenous vein
HEMA1 LAB (PRELIM PRACTICALS)
*to address hematoma: apply pressure (do not advice patient
not to bind his/her arm) HEMACYTOMETER
 “Heart of manual cell count”
COMPLICATIONS  Do not go back to the same field again.
 Hematoma  Contains lines and squares.
 Pain  Different Types
 Syncope and fainting  Improved Neubauer
 Iatrogenic anemia  Neubauer
 Infections  Fuchs – Rosenthal
 Edema  Speirs – Levy
 Allergies  Tuerk’s
 Petechiae  Bass - Jones

LESSON 3
RED BLOOD CELL COUNT
RBC Count
 The number of RBCs in 1 liter or 1 microliter of blood.
 Manual RBC counts are rarely performed because of the
inaccuracy of the count and questionable necessity.
 To have an idea how well our bodies in transporting
oxygen.
 Help us later on to diagnose a disease (specifically the
values that we’ll be obtain).
 Measures the RBC of the whole blood.
 Is just a part of the different test used in diagnosing
diseases. ( much better to be accompanied by
hematocrit, hemoglobin etc.)
 Subjective and tedious than automated RBC count.
 ↑in the Morning, ↓ in the Evening
 ↑ in myeloproliferative disorder such as Polycythemia.
 ↓ in Anemic cases, bleeding
 Specimen : EDTA blood
 Diluting fluid : Isotonic
 Dilution factor : 1:200
12
 SI unit : x 0.000001 (x 10 /L)

Materials Needed (Manual)

 Hemacytometer
 Thoma Pipette : mix up blood and diluting fluid;
contains 10 lines in stem that has 0.1 mark in each (total
volume = 1)
i. RBC pipette = has a red bead inside the bulb; can
contain up to 101 units of volume
 In each Hemacytometer, you have two (2) counting
ii. WBC pipette = has a white bead; can contain up to
chambers or area (red colored).
11 units of volume; o
 Primary square or 1 (biggest)
 Suction device
 In every primary square, contains 9 secondary
 Thick coverslip
squares.
 Cell Counter o
 Secondary square or 2
 Diluting fluids
 For every secondary square, it is equal to
 In RBC, this should be ISOTONIC (to preserve RBCs) 2
1mmx1mm or 1mm
 inHypertonic or Hypotonic solution, red cells are 2
 The entire secondary square is equal to 9mm
lysed and crenated.
 The four (4) corners are the WBC squares.
 WBC = Hypotonic
*you are still allowed to count RBC in WBC
 Platelets = Isotonic
squares, its just that the area factor will change.
*In each WBC square, there is 16 tertiary squares
*RBC, WBC and platelets have the same manner of counting, o
or 3 .
the are differentiated in what diluting fluid should be use.
HEMA1 LAB (PRELIM PRACTICALS)
 In the middle or center part, you will find the RBC *if this procedure will not be performed, the
square (it is only one). solution will dried up and you will no longer able
*RBC square has 25 tertiary squares. to count the cells accurately. Cells are damaged.
*In each tertiary square, there is 16 quarternary *after incubation, count the cells only under HPO.
o
squares or 4 .
 Other four (4) squares (vertical and horizontal)
 serves as guide or marker
 indicates that you reach the end, you need to stop

RBC DILUTING FLUIDS

 Dacies/Formol citrate (BEST TO USE;preserves red
cell best)
 Normal Saline Solution (NSS)
 Hayem’s
 3.8% Na Citrate
 Toisson’s
 Bethell’s
 Gower’s

***ISOTONIC
 Easy to prepare
 Cheap
 Has buffer action
 Does not promote growth of molds.
 Not contaminated  In RBC cell counting, out of 25 just count 5 squares.
*4 corners and the central square
Procedure  “L” rule
 all cells that will touch your left lines and lower
lines are included in the count
 All cells that will touch your upper lines and right
lines are not included in the count.

*EDTA = preserves the morphological features of cells for

better and easier identification.
*If RBC pipette is not available, you can use WBC pipette (the
dilution factor will change as you compute for cell count) Depth factor
*Discard 3-5 drops to remove the fluid on the stem (contains  0.1mm
diluting fluid only).  Constant
*Charge the solution in both ends? (you need to count cells  Distance between cover slip and counting chamber
in both chambers to make sure that the distribution of cell is
equal) Dilution factor is 1:200 (VARIES)
 Improper dilution sample = unequal distribution of
cells Example:
 The difference in between the two chambers =  You aspirate 0.5 blood, the diluting fluid is 101.
should be 5-10%  101 minus 1 because of the liquid at the stem of the
 The fluid should only be confined in counting pipette
chamber!  0.5:100 = 1:200
*After charging the solution, incubate (5-10 mins) the
hemacytometer in petri dish with a wet gauze or tissue paper - routinely, we aspirate 0.5 blood.
 Incubation helps the cells to settle down -if patient has polycythemia vera, you need to lessen the
 Wet gauze or tissue paper = to make sure that the amount of blood.
solution kept at moist environment - if patient suffers from excessive bleeding, you need to
increase the amount of blood.
HEMA1 LAB (PRELIM PRACTICALS)
AREA: i. responsible for transporting oxygen from the lungs
 1° square = 9 mm
2 going into the tissue.
2 ii. Responsible of transporting carbon dioxide from
 2° square = 1 mm
2 the tissues to the lungs (for elimination of CO2
 3° square = 0.25 mm
 4° square = 0.04 mm
2 because it is a waste product)
*In short, it is a protein that carries oxygen to
Example: you counted cell in secondary RBC square… body organs and tissues and transports carbon
dioxide from organs and tissues back to the
1 (area)/25 = 0.04 x 5 (no. of squares you counted) = lungs.
0.2 area *Carries oxygen from the lungs to the tissues and
takes carbon dioxide.
Example computation: at rest: 250mL O2/min
st
1 chamber = 400  Hemoglobin also acts as a buffer (helps to balance
nd body’s pH)
2 chamber = 410
 It gives the red color of the blood.
Average = (400 + 410)/2 = 405  Presence of hemoglobin in plasma = HEMOGLOBINEMIA
 Felix Seyler (1862)
FORMULA:  Who identified the respiratory protein called
ave. # of RBC counted x Dilution (200) hemoglobin.
Area factor x Depth factor (0.1)  Proved that hemoglobin is the coloring matter of
the blood.
(405 x 200)/(0.1 x 0.2)  Hgb is slighlty soluble in water = 3mL O2/L of blood.
= 81000/0.02  a red globular protein weighing 68,000 Daltons,
= 4,050,000/uL x 0.000001 comprising 1/3 of the RBC’s weight
12
= 4.05x10 /L (this is what you will then include in CBC  each RBC contains 640 Million of Hgb molecules hemoglobin
report)  it is composed of 4 subunits, which each subunits
contains Heme & Globin (always come in pair):
Sources of Errors and Comments  1 Heme = 1 mole of O2
 Dust and fingerprints may cause difficulty in  1 Hgb = 4 heme = 4 moles of O2
distinguishing the cells.
 Diluting fluid should be free of contaminants. 1 Hemoglobin molecule
 If the count is low, a greater area may be counted.  1 GLOBIN = 4 HEME
 Chamber must be charged properly to ensure accurate
count. 1 Globin
 Allow cells to settle for 10mins before counting.  4 units of globin
 Use of other, more accurate manual RBC procedures, *Example: A1 contains 2 alpha and 2 beta
such as the microhematocrit and hemoglobin
concentration, is desirable when automation is not  Component:
available. 1. Globin
2. Protoporphyrin IX
Normal Values: 3. Iron
12
 Female: 3.6 – 5.6 x 10 /L
12
 Male: 4.2 – 6.0 x 10 /L *2,3-DPG (additional extra component of hgb)
12
 At Birth: 5.0 – 6.5 x 10 /L **Facilitates the affinity of oxygen to hgb.
*their bone marrow is 100% active
Protoporphyrin IX + Iron = HEME
MIDTERMS
 Through enzymatic reactions, will form HEME.
 Through bond connections, will form HEMOGLOBIN.
HEMOGLOBIN DETERMINATION Ch: 16 - alpha zeta
Ch: 11 - the rest/ all
Hemoglobin
 The primary function of hemoglobin within the red Hemoglobin Determination
blood cell is to carry oxygen to and carbon dioxide from
the tissues.  Hemoglobinometry
*The cyanmethemoglobin (hemoglobincyanide)  Measurement of concentration of Hemoglobin in blood
method for hemoglobin determination is the reference  uses Hemoglobinometer
method approved by the Clinical and Laboratory  Lyse the rbc first to obtain hemoglobin
Standards Institute.
 Hemoglobin (Hb or Hgb)is the main content of RBCs:
HEMA1 LAB (PRELIM PRACTICALS)
Methods
*Even if it is manual or automated techniques, lyse the
RBC first to obtain hemoglobin out from the cell
*In automated analyzers, you cannot count RBC and
hemoglobin at the same time.
**First, you’ll count RBC then lyse them for you to count
the hemoglobin.
**In automated analyzers. Hemoglobin and WBC count
are performed simultaneously.
 ACID HEMATIN
 COLORIMETRIC METHOD
 Lyse RBC here using 0.1N of HCl
 As soon as you lyse RBC, there is change in color of
solution (from red to brown if you mix with acid).
*Brown solution obtained will compare to the
comparator block (color standard) thus
SUBJECTIVE.
*Sahli pipette do not have a bulb and only has a SINGLE
MARK.
*Sahli graduated tube = where you will determine/identify
the hemoglobin content/value.
Additional note:
ALKALI HEMATIN
 not for Neonate/Newborns since Hgb-F is alkali
resistant.
 0.1 N of NaOH = for lysing RBC
Acid Hematin Procedure:
1) Dispense or introduced 0.1N HCl solution up to 2 mark
of Sahli graduated tube.
2) Using Sahli pipette, draw or aspirate blood up to 0.02cc
mark. Observe the same technique as in the use of
RBC/WBC pipette.
*wipe the tip of the pipette once you have aspirated
the desired amount of blood.
3) Dispense the measured volume of blood into graduated
tube. Gently draw up and down to rinse the pipette and
also to mix the resulting colloidal suspension.
*As you add that blood in Sahli graduated tube
containing HCl, and once its mixed up, you will then
aspirate the solution again then blow it down (do this
several times to make sure that your blood and HCl
will be mix properly)
4) Thoroughly mix with the use of stirrer and allow to
stand for 5mins (to allow full lysis of RBCs)
*After 5 mins, check the color (in WHITE
BACKGROUND).
*If you think that the color of your solution is the
SAME with comparator block, that’s it. This is the end.
*If you think that the color of your solution is NOT THE
SAME with comparator block, add a drop of distilled
water.
5) Add distilled water (H2O) drop by drop, stirring each
addition and compare with the comparator block.
*If the color of your solution is STILL NOT THE SAME
with comparator block, add another drop of distilled
water, stir, mix and check the color again.
*REPEAT this procedure again and again until you
obtain the same color with comparator block.
*The principle of adding distilled water drop by drop
will allow you to reach the color desired the same with
comparator block.
6) When the resulting solution compares with the color
standard in the comparator block, get the reading either
from the grams or percentage scale.
*Example grading: 14 g/dL (this is the hemoglobin
value)
*The value will be obtain when the sample has the
same color of the comparator block.
7) Write your result.
 CYANMETHEMOGLOBIN
 aka “Hemiglobincyanide”
 Of all the parameters we have in CBC or Hema, it is
the only standard.
 STANDARD approved by the Clinical and
Laboratory Standards Institute (CLSI)
*acceptable internationally
 Method of choice for Hemoglobin determination:
1. Stable
2. Standards are readily available
 All derivatives of hemoglobin can be measured
except Sulf-Hgb.
 INDIRECT/PHOTOELECTRIC METHOD
 Reagent: Drabkin
*added in an aliquot of whole blood
*After addition of Drabkin’s reagent, Hemoglobin iron
is later on converted from ferrous to ferric state (to
form methemoglobin because of potassium
ferricyanide).
Drabkin’s reagent
 POTASSIUM FERRICYANIDE: converts Hgb to Met-Hgb
 POTASSIUM CYANIDE: converts Met-Hgb to
Cyanmet-Hgb
 NON-IONIC DETERGENT: improves lysis of RBC
 DIHYDROGEN POTASSIUM PHOSPHATE: allow us to
measure the solution spectrophotometrically.
Principle
HEMA1 LAB (PRELIM PRACTICALS)
 Absorbance of cyanmethemoglobin at 540 nm *In automated analyzers/machines,
(spectrophotometer) is DIRECTLY PROPORTIONAL to cyanmethemoglobin employs commonly.
the hemoglobin concentration.
*the color intensity with absorbances compared with Hemoglobin continuation…
a known standard (provided by manufacturer)  ↑ in the Morning; ↓ in the Evening
 Uses a standard curve  ↑ at birth due to active Red Bone Marrow
*whatever values you will obtained in  ↑ in high altitudes (due to Hypoxia)
spectrophotometer, you should always compare it to  ↑in smokers due to formation of Carboxy-Hgb
standard curve.  ↑in strenuous muscular activity due to Androgen
*Example: you acquired 0.413 absorbance in spectro, hormone influence
this is not your final hemoglobin value yet, compare it  ↓when in lying position due to the dilution of
to standard curve to obtain the actual hemoglobin Interstitial fluid
value..  screening parameter/test for Anemia and may detect
RBC breakdown / Hemolytic Anemia.
Additional note:
This method avoids the necessity of sample dilution Low
and interference from turbidity.  Anemia
Another method that has been used in some  Blood loss
automated instruments involves the use of sodium  Iron, Folate and Vitamin B6, B12 deficiencies
lauryl sulfate (SLS) to convert hemoglobin to SLS
methemoglobin. This method does not generate toxic High
wastes compare to potassium ferricyanide.  Sickle cell anemia
 Thalassemia
Materials and Instrument  Transfusion reaction
 Anticoagulated blood (venous blood)  Hemolysis
 Test tubes  Dehydration
 Pipette  Polycythemia vera
 Spectrophotometer  High altitude
 Drabkin’s reagent
 Parafilm Normal Values:
 At birth/Newborn: 15-20 g/dL
Cyanmethemoglobin Procedure:  Women: 12-16 g/dL
1. Place 5cc of Drabkin’s reagent into a test tube.  Men: 13-18 g/dL
2. Using the Sahli pipette, add or draw blood to 0.02cc *may vary to locality, age, gender
mark. Make sure to wipe the outer wall of the pipette,
and then dispense the blood to the test tube. Sources of Error
3. Gently draw up and down to rinse the pipette and also  High WBC count & PLT count = Turbidity (Falsely
mix the resulting colloidal suspension. increased)
*The pipette should be rinsed thoroughly with the  Hemoglobin S & C
reagent to ensure that no blood remains.  Lipemic blood
4. Cover and mix well by inversion. Let is stand for 5
minutes. 1. Cyanmethemoglobin reagent is sensitive to light.
5. Transfer the mixture to a cuvette. *It should be stored in a brown bottle or in a dark
6. Set the Spectrophotomer to 100% transmittance at the place.
9
wavelength of 540nm, using a cyanmethemoglobin 2. A high WBC count (greater than 20x10 /L) or a high
9
reagent as a blank. platelet count (greater than 700x10 /L) can cause
7. Continue reading the patient’s sample, and record the turbidity and a falsely high result.
percentage transmittance. *In this case, the reagent-sample solution can be
8. The hemoglobin concentration is obtained from a centrifuged and the supernatant measured.
calibration curve prepared with the use of standards. 3. Lipemia also can cause turbidity and a falsely high
*Stable standard cyanmethemoglobin solution of result.
concentration representing 1:250 dilution of whole *It can be corrected by adding 0.01 mL of the patient’s
blood containing 5, 10, 15 grams of hemoglobin per plasma to 5 mL of the cyanmethemoglobin reagent
100mL are available commercially for calibration and using this solution as the reagent blank.
purposes and for periodic check-up on the accuracy of 4. Cells containing Hb S and Hb C may be resistant to
the colorimeter. hemolysis, causing turbidity; this can be corrected by
making a 1:2 dilution with distilled water (1 part diluted
Additional note: if you want to compute for sample plus 1 part water) and multiplying the results
hemoglobin value. Use a control and standard. from the standard curve by 2.
HEMA1 LAB (PRELIM PRACTICALS)
5. Abnormal globulins, such as those found in patients
with plasma cell myeloma or Waldenström
macroglobulinemia, may precipitate in the reagent.
*If this occurs, add 0.1 g of potassium carbonate to
the cyanmethemoglobin reagent.
*Commercially available cyanmethemoglobin reagent
has been modified to contain KH2PO4 salt, so this
problem is not likely to occur.
6. Carboxyhemoglobin takes 1 hour to convert to
cyanmethemoglobin and theoretically could cause
erroneous results in samples from heavy smokers.
*The degree of error is probably not clinically
significant, however.
7. Because the hemoglobin reagent contains cyanide, it is
highly toxic and must be used cautiously.
*Consult the safety data sheet supplied by the
manufacturer.
*Acidification of cyanide in the reagent releases highly
toxic hydrogen cyanide gas.
*A licensed waste disposal service should be
contracted to discard the reagent; reagent-sample
solutions should not be discarded into sinks.
8. Commercial absorbance standards kits are available to
calibrate spectrophotometers.
9. Handheld systems are commercially available to
measure the hemoglobin concentration
*An example is the HemoCue4,5 (HemoCue, Inc., Brea,
CA) in which hemoglobin is converted to
azidemethemoglobin and is read photometrically at
two wavelengths (570 nm and 880 nm).
Other Methods
GASOMETRIC METHOD
 also known as “Van Slyke Oxygen Capacity”
 1g of Hgb = 1.34mL of Oxygen
CHEMICAL METHOD
 also known as “Kennedy’s” or “Wong’s”
 1g of Hgb = 3.47mg of Iron
SPECIFIC GRAVITY METHOD
 also known as “Copper Sulfate Method”
 Specific Gravity = 1.053
 Change solution daily.
 Just 1 drop of blood should be added.
 If a drop of blood sinks and reached the bottom of the
beaker within 15 seconds, hemoglobin is normal.
 If a drop of blood just float within 15 seconds,
hemoglobin is decreased
*In case of blood banning, the donor is deferred or
rejected.
 30 mL container = 25 test (drop of blood and number of
patient to be tested)
HEMATOCRIT DETERMINATION
HEMATOCRIT
 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 graduated 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.
*Anemia can be tested using RBC Count, Hgb and Hct
determination
 The normal ratio approaches 50%.
HEMATOCRIT DETERMINATION
 The hematocrit is the volume of packed red blood cells
that occupies a given volume of whole blood.
*dealing with the amount of space occupied by red
cells alone in a given blood volume.
 Hematocrit is also called packed cell volume (PCV),
while the packed cells referring to RBCs.
 It is reported either as a percentage (e.g., 36%) or in
liters per liter (0.36 L/L).
*L/L (automatically discarded in reporting)
*Example: 45% = 0.45
LAYERS OF BLOOD AFTER CENTRIFUGATION:
 Centrifuge the whole blood first. Blood components are
separated to each other based on their density.
 Often one can see a light-colored layer between the
RBCs (RED) and plasma (YELLOW). This is the buffy coat
(WHITE) and contains WBCs and platelets, and it is
excluded from the hematocrit determination.
*Formed elements: Platelets, Monocytes and
Lymphocytes, Granulocytes, Reticulocytes or
immature/abnormal cells
*In lipemic patients, lipids are located at the top of
plasma.
 Packed cells contains RBCs alone (MOST DENSE because
it contains hemoglobin specifically oxygen)
 Only measure the Erythrocytes/RBC (45%), do not
include the Buffy coat.
*Manner of Reporting: PCV = 45% or 0.45
 If you include the buffy coat, hematocrit value will then
be falsely elevated.
HEMA1 LAB (PRELIM PRACTICALS)
MATERIALS AND INSTRUMENTS:
TWO (2) MANUAL METHODS:  Wintrobe tube with rubber caps
i. Micromethod – uses SMALLER amount of blood  Long stem pipette
(through skin puncture)  Centrifuge
*capillary tube  Anticoagulated blood (the procedure needs about
ii. Macromethod – uses LARGER amount of blood (through 3.0mL of whole blood)
venipuncture)  Cotton
*microtainer
PROCEDURE:
AUTOMATED machine 1. Mix the blood.
 hematocrit is just a computed value. 2. With the use of a long stem pipette, fill the wintrobe
 Based on MCV and RBC count tube with blood up to the 10 mark.
*NOTE: No air bubbles should be present in the
PRINCIPLE surface of the blood. It may be removed by touching it
 Whole blood is centrifuged to determine packing of red with a tissue paper or with a pipette. If the bubbles
blood cells. are present in the middle of the tube, Aspirate the
blood and proceed to step no. 2 again.
3. Place it inside a test tube with cottons/tissue paper in
1. MACROHEMATOCRIT METHOD between to make it stable.
 The venous blood obtained through venipuncture 4. Seal the Wintrobe tube with para film to avoid
should be transferred in EDTA tube. contaminants.
5. Centrifuge the blood at 2,000-3,000 RPM/GF for 30
 WINTROBE & LANDSBERG TUBE (double oxalate) minutes (light spin).
 Whole blood is transferred to a Wintrobe tube and 6. Remove the tubes and carefully place it in the Wintrobe
centrifuged for 30 minutes at 2000-3000 rpm tube stand.
 Anticoagulant: Oxalate 7. Read the height (in mm) of the packed red blood cells
 Disadvantage: on the scale at the right side of the tube, which is
i. Larger amount of blood is needed graduated from 0-10 upward.
ii. Time consuming 8. It is expressed in percentage (%)
*you will transfer blood from EDTA tube into
the Wintrobe tube using a pipette or syringe Example:
th
up to the 10 mark and centrifuge for 30 Value acquired in wintrobe tube stand: 4
mins (after this, you will check how much 4 x 10 = 40%
red cells has been packed)
iii. More prone to trapped plasma (amount of NOTE: There are times that after you centrifuge the
plasma that still remains in the RBC portion wintrobe tubes, packed cells are slanted. To resolve this,
after centrifugation) do not include in the measurement the slanted portion,
*Making hematocrit value falsely elevated include only the amount of packed cells that is parallel in
*Trapped in between RBCs. lines/markings.
 Might ask in board exam *If you included the slanted portion = FALSELY
 Length : 115 mm ELEVATED because of the presence of buffy coat.
 Diameter (bore) : 3 mm
 HCT marking (white) : 0-10 UPWARDS mark
2. MICROHEMATOCRIT METHOD
 ESR marking (red): 0-10 DOWNWARD mark
 Small amount of blood is needed (through skin
puncture)
 Advantages:
i. Faster
ii. More convenient
iii. Easier to do
 Much better than macromethod because of centrifugal
force applied (HIGHER compared to macromethod).
Cells properly packed if high centrifugal force is used.
 Specimen: either EDTA blood or capillary blood
*EDTA blood is obtained from venipuncture. Directly
*Reusable from EDTA tube using a capillary tube, you aspirate
the blood.
 Van Allen: sodium oxalate  Capillary tubes:
 Haden: sodium oxalate  Blue band = no anticoagulant
 Sanford-Magath: sodium oxalate *used if EDTA blood is obtained
 Bray: heparin  Red band = contains heparin
*used if capillary blood is obtained
HEMA1 LAB (PRELIM PRACTICALS)
**Length = 75 mm
**Bore (diameter) = 1 mm
MATERIALS AND INSTRUMENTS:
 Heparinized capillary tube
 Microhematocrit centrifuge
 Microhematocrit reader
 Lancet
 Cotton
 Alcohol
PROCEDURE:
1. After necessary preparations, make a capillary puncture
and produce a rounded drop of blood.
st
*skin puncture (wipe the 1 drop of blood)
2. In a horizontal position, put one end of the capillary
tube on the drop of blood and fill the tube about 2/3
full.
*or fill 2 capillary tubes (1/2 or 3/4 mark)
NOTE: the tube will be filled by capillary action. If
using tubes with a colored ring at one end, fill from
opposite end.
3. Seal one end of the capillary tube with the clay (3-4 mm)
by placing the dry end of the tube into the clay in a
vertical position..
4. After sealing the clay, seal it again with wax.
5. Assemble the tube in hematocrit centrifuge in such a
way that the unsealed end is nearest the center of the
centrifuge.
6. Spin at 10,000-15,000 RPM/GF for 5-10 minutes (heavy
spin).
7. Using the microhematocrit reading device, determine
the hematocrit.
NOTE: Buffy coat should not be included in the
reading.
8. The reading on the window of the hematocrit reader
corresponds to the hematocrit value.
Alternative procedure…
1. Fill two plain capillary tubes approximately three
quarters full with blood anticoagulated with EDTA or
heparin.
*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.
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 red blood cells. Do not use the brake to stop
the centrifuge.
5. Determine the hematocrit by using a microhematocrit
reading device. Read the level of red blood cell packing;
do not include the buffy coat (WBCs and platelets) when
taking the reading.
6. The values of the duplicate hematocrits should agree
within 1% (0.01 L/L)
NOTE: in measuring, do not include the clay and buffy coat.
*Diagonal line (ORANGE) = this is where you align the
top last (end) portion of plasma
*Horizontal line (YELLOW) = this is where you align the
bottom portion of packed red cell (start measurement at
zero “0”)
*Movable marker (PINK) = touch the last top portion of
packed cells.
**not all microhematocrit reader has movable
marker.
**the acceptable limit is ± 𝟏
***If the value obtained is 31%, the acceptable limit
value is ranging from 30-32%.
Determining Maximum Packing Time for Microhematocrit
 The time to obtain maximum packing of red blood cells
should be determined for each centrifuge.
 Duplicate microhematocrit determinations should be
made using fresh, well-mixed blood anticoagulated with
ethylenediaminetetraacetic acid (EDTA).
 Two specimens should be used, with one of the
specimens having a known hematocrit of 50% or higher.
 Starting at 2 minutes, centrifuge duplicates at 30-second
intervals and record results. When the hematocrit has
remained at the same value for two consecutive
readings, optimum packing has been achieved, and the
second time interval should be used for
microhematocrit determinations.
HEMA1 LAB (PRELIM PRACTICALS)

SOURCES OF ERRORS:
 A decreased or increased result may occur if the
specimen was not mixed properly.
 A decrease or increase in the readings may be seen if
the microhematocrit reader is not used properly.
 Hemoconcentration (prolonged torniquet application)
results to falsely increased hematocrit.

 INCREASED in :
 Insuficient centrifugation
*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 samples used must have a
hematocrit of 50% or higher.
 Inclusion of buffy coat
 Dehydration
*The fluid loss associated with dehydration
causes a decrease in plasma volume and falsely
increases the hematocrit reading
 Disorders such as Macrocytic anemia,
Hypochromic anemia, and Sickle-cell anemia
*may cause plasma to be trapped in the red
blood cell layer even if the procedure is
performed properly.

 DECREASED in :
 Improper sealing of capillary tube (capillet)
*as a result of leakage (wash out) of blood during
centrifugation.
HEMA1 LAB (PRELIM PRACTICALS)
*A higher number of red blood cells are lost
compared with plasma due to the packing of the
cells in the lower part of the tube during
centrifugation.
 Increased concentration of Anticoagulants (ex.
EDTA)
*(short draw in an evacuated tube) decreases the
hematocrit reading as a result of red blood cell
shrinkage.
*Optimum concentration of EDTA: 1.5mg/mL
(1.5mg of EDTA x 5mL of blood = 7.5mg of EDTA)
*9:1
*excess EDTA (<5mL of blood) = decrease value of
hematocrit and ESR = false decrease result
(destruction of cells)
 Prolonged centrifugation
*damages RBCs
 Acute blood loss
*A temporarily low hematocrit reading may
result immediately after a blood loss because
plasma is replaced faster than are the red blood
cells.
 Improper specimen collection
*The introduction of interstitial fluid from a skin
puncture or the improper flushing of an
intravenous catheter causes decreased
hematocrit readings.
**TRAPPED PLASMA: amount of plasma that still remains in
the RBC portion after the microhematocrit has been spun.
 Making hematocrit value falsely elevated.
 Increased: macrocytic anemia, spherocytosis,
thalassemia, hypochromic anemia, sickle cell anemia
 Spun hematocrit is 1% to 3% higher than the hematocrit
from automated instrument due to plasma that is
trapped in erythrocytes.
 The trapping of the plasma causes the microhematocrit
to be 1% to 3% (0.01 to 0.03 L/L) higher than the value
obtained using automated instruments that calculate or
directly measure the hematocrit and are unaffected by
the trapped plasma.
↑ hematocrit in Polycythemia Vera, Macrocytic Anemia,
Hypochromic Anemia, Sickle-cell Anemia, Spherocytosis,
Thalassemia, Dehydration
*Anemia promote trapped plasma
↓ hematocrit in Anemia & after 50 years old
*condition in which you lack enough healthy red blood
cells to carry adequate oxygen to your body's tissues.
Polycythemia= increase in the number of red blood cells in
the body
Macrocytic Anemia= type of anemia that causes unusually
large red blood cells. (low hemoglobin)
Hypochromic Anemia = type of anemia in which the red
blood cells are paler than normal. (Hypo- refers to less, and
chromic means colour.)
Sickle-cell Anemia= inherited red blood cell disorder in which
there aren't enough healthy red blood cells to carry oxygen
throughout your body. crescent moons.
Spherocytosis = sphere-shaped rather than bi-concave disk
shaped as normal. Spherocytes are found in all hemolytic
anemias to some degree.
Thalassemia = blood disorder passed down through families
(inherited) in which the body makes an abnormal form or
inadequate amount of hemoglobin. results in large numbers
of red blood cells being destroyed, which leads to anemia.
NORMAL VALUES:
 Female = 36-48%
 Male = 40-55%
 Newborn = 45-60%
SI value of hematrocrit should be expressed in L/L.
ERYTHROCYTE SEDIMENTATION RATE
(ESR)
 aka “Sed rate”
 measures the rate of settling of RBCs.
 The erythrocyte sedimentation rate (ESR) is ordered
with other tests to detect and monitor the course of
inflammatory conditions such as, rheumatoid arthritis,
infections, or certain malignancies.
 It is also useful in the diagnosis of temporal arteritis and
polymyalgia rheumatica.
 The ESR, however, is not a specific test for inflammatory
diseases and is elevated in many other conditions such
as plasma cell myeloma, pregnancy, anemia, and older
age.
*NON-SPECIFIC measurement used to detect and
monitor an Inflammatory response to Tissue injury
(SCREENING TEST)
 It is also prone to technical errors that can falsely
elevate or decrease the sedimentation rate.
 Because of its LOW specificity and sensitivity, the ESR is
NOT RECOMMENDED as a screening test to detect
inflammatory conditions in ASYMPTOMATIC individuals.
 Can be perform in SYMPTOMATIC individuals.
 Other tests for inflammation, such as the C-reactive
protein level, may be a more predictable and reliable
alternative to monitor inflammation.
 Distance in millimeters at which the RBCs settle
down/sediment/fall in 1 hour.
*Determines how much of RBCs will settle down after
an hour.
HEMA1 LAB (PRELIM PRACTICALS)
*You need to complete 1 hour duration to complete
the stages involved in the sedimentation of RBCs.
*speed of fall of red blood cells
 Manner of reporting: mm/hour
proteins in plasma → push down in bottom → the ESR
elevated
PRINICPLE:
 When anticoagulated blood is allowed to stand at room
temperature undisturbed for a period of time, the red
blood cells settle toward the bottom of the tube.
Water = most abundant component of plasma
Proteins = most abundant and most dense (heaviest)
chemical constituent of plasma = NO. 1 FACTOR
*LIpids particularly cholesterol also contributes to ESR.
Acute phase reactants = proteins that elevates when
there is inflammation (Fibrinogen, Albumin)
*increase concentration of proteins in plasma, the
more it pushes down the RBCs thus the higher the ESR
will be.
↑ concentration of proteins = ↑ RBC pushed down thus
settle faster at the bottom = ↑ ESR (abnormally)
 The phenomenon depends on an interrelationship of
variables, such as the plasma protein synthesis, the
concentration of erythrocytes, and the shape of the
erythrocyte (poikilocyte).
*In Polycythemia vera, you have lots of RBC = less
plasma thus RBCs will not settle down affecting ESR
result.
*RBC has negative charge because of salicylic acid.
Because of negativity, RBCs repel one another (zeta
potential). The repulsive forces of RBCs are counteracted
(inhibited) if you have positive charge ions (such as
proteins).
 The ESR is affected by red blood cell, plasma, and
mechanical and technical factors.
 Red blood cells have a net negative surface charge
and tend to repel one another.
 The repulsive forces are partially or totally
counteracted if there are increased quantities of
positively charged plasma proteins.
 Under these conditions the red blood cells settle
more rapidly as a result of the formation of
rouleaux (stacking of red blood cells).
 Examples of macromolecules that can produce this
reaction are fibrinogen, b-globulins, and pathologic
immunoglobulins.
 Normal red blood cells have a relatively small mass and
settle slowly.
 Certain diseases can cause rouleaux formation, in which
the plasma fibrinogen and globulins are altered. This
alteration changes the red blood cell surface, which
leads to stacking of the red blood cells, increased red
blood cell mass, and a more rapid ESR.
 The ESR is directly proportional to the red blood cell
mass and inversely proportional to plasma viscosity.
 Several methods, both manual and automated, are
available for measuring the ESR.
STAGES OF SEDIMENTATION
1. INITIAL ROULEAUX FORMATION (10mins)
 Aggregation = Rouleaux
 Rouleaux formation (stacking of coins)
 As more proteins are present in plasma = the more
it initiate rouleaux formation = RBCs settles down
faster = ESR elevates
 Poikilocytes (RBCs varies in shape) do not form
rouleaux. Sickle cells is one of the example. ESR
DECREASES.
2. RAPID/FAST SETTLING OF RBCs (40mins)
3. SLOW/FINAL SEDIMENTATION OF RBCs (10mins)
>60 mins = some RBCs shrinks or destroyed (FALSELY
ELEVATED ESR)
<60 mins = RBCs will not totally settle down
METHODS FOR ESR:
1. STANDARD / ORIGINAL WESTERGREN
 Most sensitive method because of the tube used (longer)
but requires more blood (ADVANTAGE).
 It is the method recommended by the International
Council for Standardization in Hematology and the
Clinical and Laboratory Standards Institute.
 Disposable
 Ratio is 4 volume of blood to 1 volume of sodium citrate
(BLACK)
 Length : 300 mm
 Bore : 2.65 mm
 Anticoagulant: citrate
2. MODIFIED WESTERGREN
HEMA1 LAB (PRELIM PRACTICALS)
 EDTA anticoagulant is used, it must be diluted to the
ratio of 4 volume of blood to 1 volume of 0.9% sodium
chloride or sodium citrate.
PROCEDURE:
i. Mix the blood citrate or blood-EDTA-saline mixture
thoroughly.
ii. Aspirate a bubble-free specimen (blood) into a clean
and dry Westergren pipette. Fill to the zero (0) mark. Do
not pipette by mouth.
o o
iii. Place the pipette into a vertical rack at 20 C to 25 C in
an area free from vibrations, drafts, and direct sunlight.
iv. After 60 minutes (1 hour), read the distance in
millimeters from the bottom of the plasma meniscus to
the top of the sedimented RBCs or erythrocytes.
v. Record the value as mm in 1 hour.
Alternative procedure:
1) Use well-mixed blood collected in EDTA and dilute at
four parts blood to one part 3.8% sodium citrate or
0.85% sodium chloride (e.g., 2 mL blood and 0.5 mL
diluent). Alternatively, blood can be collected directly
into special sedimentation test tubes containing sodium
citrate. Standard coagulation test tubes are not
acceptable, because the dilution is nine parts blood to
one part sodium citrate.
2) Place the diluted sample in a 200-mm column with an
internal diameter of 2.55 mm or more.
3) Place the column into the rack and allow to stand
undisturbed for 60 minutes at room temperature (18 to
25° C). Ensure that the rack is level.
4) Record the number of millimeters the red blood cells
have fallen in 1 hour. The buffy coat should not be
included in the reading. Read the tube from the bottom
of the plasma layer to the top of the sedimented red
blood cells. Report the result as the ESR, 1 hour 5 x mm.
Normal Values:
 MALE <50yo = 0-15 mm
 MALE >50yo = 0-20 mm
 FEMALE <50yo = 0-20 mm
 FEMALE >50yo = 0-30 mm
 CHILDREN = 0-10 mm
3. WINTROBE-LANDSBERG METHOD
 require small amount of blood and requires no dilution
 also used in hematocrit
 ESR marking (red): 0-10 DOWNWARD mark
 Length : 115 mm
 Bore : 3 mm
 Anticoagulant: oxalate
 Anticoagulant: Oxalate (oxalate-anticoagulated whole
blood)
*But nowadays, we can also use EDTA or Citrate
(black)
*This was placed in a 100-mm column.
*Today, EDTA-treated or citrated (black) whole
blood is used with the shorter column.
*The shorter column height allows a somewhat
increased sensitivity in detecting mildly elevated ESRs
Differences in citrated tubes:
BLUE TOP TUBE: Blood Anticoagulant Ratio = 9:1 (use
for coagulation studies: buffered sodium citrate 3.8%)
*commonly used than black top
BLACK TOP TUBE: Blood Anticoagulant Ratio = 4:1
(use for ESR)
 Disadvantage:
iv. Larger amount of blood is needed
v. Time consuming
PROCEDURE:
1. Use fresh blood collected in EDTA anticoagulant. A
minimum of 2 mL of whole blood is needed.
2. After mixing the blood thoroughly, fill a Pasteur pipette
using a rubber pipette bulb.
3. Place the filled pipette into the Wintrobe tube until the
tip reaches the bottom of the tube.
4. Carefully squeeze the bulb and expel the blood into the
Wintrobe tube while pulling the Pasteur pipette up from
the bottom of the tube. There must be steady, even
pressure on the bulb to expel blood into the tube as
well as continuous movement of the pipette up the tube
to prevent the introduction of air bubbles into the
column of blood.
5. Fill the Wintrobe tube to the 0 mark.
6. Place the tube into a Wintrobe rack (tube holder) and
allow to stand undisturbed for 1 hour at room
temperature. The rack must be perfectly level and
placed in a draft-free room.
7. Record the number of millimeters the red blood cells
have fallen. Read the tube from the bottom of the
plasma meniscus to the top of the sedimented red cells.
The result is reported in millimeters per hour.
NORMAL VALUES:
 FEMALE = 0 – 20 mm
 MALE = 0 – 9 mm
 CHILDREN = 0 – 13 mm
Technically speaking, there is no “Low ESR” because values
starts at 0. So low ESR with value of 0 is still normal.
*Reference intervals according to sex and age
HEMA1 LAB (PRELIM PRACTICALS)

 In case of anemia, red cells are low = EASILY SETTLE

DOWN thus INCREASED ESR
0
Graduation = Total column heights  3 tilted ESR tube = cause 30% error in ESR (FALSELY
*taller column height allows the detection of highly elevated INCREASED
ESR.  Clotted specimen = fibrinogen is already consumed.

FACTORS AFFECTING ESR

Plasma
Composition
Technical
Erythrocytes (most
factors
important
factor)
↑Anemia ↑Fibrinogen Tilting: ↑ ESR
↑Macrocyte ↑α 1 globulin 3° = (30% error)
↓Microcyte ↑α 2 globulin ↑Vibration
↓Poikilocyte ↓Albumin ↑High temp
↓Polycythemia ↓Lecithin ↑Long tube
↑Large bore
↓Cold temp
↓ Short tube
↓Small bore
Acute phase reactant proteins = increased in cases of
inflammation.

HIGH ESR LOW ESR

- Pregnancy - Polycythemia
- Menstruation - Poikilocytosis
- Nephrosis - Spherocyte
- Tuberculosis - Sickle cell
- Hepatitis - Hgb CC
- Inflammation - Hypo5brinogenemia
The following are just descriptions about the conditions that - Acute/Chronic infection - Conges)ve heart failure
may result to the failure of RBCs to settle down or factors - Hypo/Hyperthyroidism
that may contribute to the concentration of plasma: - Myocardial infarction
- Bacterial endocarditis
- Multiple Myeloma
- Rheumatic fever
- Rheumatic arthrtis
- Cryoglobulinemia
- Waldenstrom
Macroglobulinemia

Sources of Error and Comments

1) If the concentration of anticoagulant is increased, the
ESR will be falsely low as a result of sphering of the RBCs,
which inhibits rouleaux formation.
2) The anticoagulants sodium or potassium oxalate and
heparin cause the red blood cells to shrink and falsely
elevate the ESR.
HEMA1 LAB (PRELIM PRACTICALS)
3) A significant change in the temperature of the room
alters the ESR.
4) Even a slight tilt of the pipette causes the ESR to
increase.
5) Blood specimens must be analyzed within 4 hours of
collection if kept at room temperature (18 to 25°C). If
the specimen is allowed to sit at room temperature for
more than 4 hours, the red blood cells start to become
spherical, which may inhibit the formation of rouleaux.
Blood specimens may be stored at 4° C up to 24 hours
prior to testing, but must be rewarmed by holding the
specimen at ambient room temperature for at least 15
minutes prior to testing.
6) Bubbles in the column of blood invalidate the test
results.
7) The blood must be filled properly to the zero mark at
the beginning of the test.
8) A clotted specimen cannot be used.
9) The tubes must not be subjected to vibrations on the
lab bench which can falsely increase the ESR.
10) Hematologic disorders that prevent the formation of
rouleaux (e.g., the presence of sickle cells and
spherocytes) decrease the ESR.
11) The ESR of patients with severe anemia is of little
diagnostic value, because it will be falsely elevated.
Automated Erythrocyte Sedimentation Rate
 There are several automated ESR systems available
using the traditional Westergren and Wintrobe methods,
as well as alternate methods such as centrifugation.
 Ves-Matic system (Diesse, Inc., Hialeah, FL)
 is a bench-top analyzer designed to determine ESR
by use of an optoelectronic sensor, which
measures the change in opacity of a column of
blood as sedimentation of blood progresses.
 Blood is collected in special Ves-Tec or Vacu-Tec
tubes, which contain sodium citrate and are
compatible with the Vacutainer system.
 These tubes are used directly in the instrument.
 Acceleration of sedimentation is achieved by
positioning the tubes at an 18-degree angle in
relation to the vertical axis.
 Results comparable with Westergren 1-hour values
are obtained in 20 minutes.
 Sedimat 15 (Polymedco, Cortlandt Manor, NY)
 uses the principle of infrared measurement.
 It is capable of testing one to eight samples
randomly or simultaneously and provides results in
15 minutes.
 ESR STAT PLUS system (HemaTechnologies, Lebanon,
NJ)
 is based on centrifugation.
 The advantages of this method are a smaller
required sample volume and shorter testing time,
which makes it more suitable for a pediatric
patient population.
 The disadvantage of this method is the number of
exacting preanalytical steps that must be strictly
followed to prevent erroneous results.
 Compliance with these steps may be difficult to
achieve consistently in a busy hematology
laboratory.
DISADVANTAGE OF AUTOMATED: the number of exacting
pre-analytical steps that must be strictly followed are not
totally accomplished (specially the stages)
ADDITIONAL METHODS
 Additional manual and semi-automated methods are
included in other chapters that are relevant to their
clinical application.
 Examples include: the osmotic fragility test and
qualitative and quantitative assays for
glucose-6-phosphate dehydrogenase and pyruvate
kinase activity; the solubility test for Hb S, hemoglobin
electrophoresis (alkaline and acid pH), and unstable
hemoglobin test; and the vital stain for hemoglobin H
and the Kleihauer-Betke acid elution test for Hb F
distribution in the RBCs.
OSMOTIC FRAGILITY TESTING (OFT)
 Used to test RBC’s resistance to Hemolysis whenever
they are exposed to hypotonic solution.
 A measure of the ability of red cells to take up fluid
without lysing.
 Employed to help diagnose different types of anemia, in
which the physical properties of red cells are altered.
 Diagnostic test for Hereditary Spherocytosis (do not
have pallor area)
 It reflects the shape of Reticulocytes
 Sample/Specimen: heparinized or defibrinated blood
 Reagent : 0.5% NaCl
Principle:
 Red cells suspended in hypotonic solution of sodium
chloride take up water, swell, become spheroidal and
after reaching the critical volume eventually burst.
 Cells that are thicker than normal have a decreased
surface/volume ratio (FRAGILE).
 Thin or flat cells have an increased surface/volume ratio
(seems among hypochromic RBCs)
*We know in a fact that if RBCs are exposed to hypotonic
solution, they will be lysed.
--subjected to hypotonic solution--
Normal red cell = 1/3 pallor area = before they are lysed,
they will first resist and expand because of extra surface area
ND
(2 TO LYSE)
Hypochromic RBC = >1/3 pallor (low Hgb) = the more pallor
area, the more you have surface area thus the more volume
RD
(hypotonic solution) you can contain (3 TO LYSE)
Hyperchromic RBC = no pallor area (high Hgb) = little amount
ST
of hypotonic solution will easily cause lysis in RBCs. (1 TO
LYSE)
HEMA1 LAB (PRELIM PRACTICALS)
Normal Values:
Initial Hemolysis = Tube 21 or 22 (0.42%-0.44%)
Complete Hemolysis = Tube 16 or 17 (0.32%-0.34% )

MATERIALS:
 12 Kahn tubes or ordinary test tubes
 Test tube rack
 0.5% NaCl
 Distilled water
 Capillary pipette or Sahli pipette
 Anticoagulated blood (Heparinized blood)
INCREASED OFT (DECREASED resistance): found in hereditary
DETERMINATION: anemias (specially autoimmune or AIHA) and hereditary
1. GRIFFIN & SANDFORD METHOD spherocytosis whenever spherocytes are found. (YOU ARE
 tubes are numbered 14-25 FRAGILE).
 the no. of tubes correspond to the number of drops of
0.5% NaCl Test to differentiate hereditary anemias and hereditary
spherocytosis: Direct Antiglobulin Test or DAT or Coomb’s
PROCEDURE: Test = AIHA is positive that gives elevated OFT
1. Add drops of 0.5% NaCl or Saline solution indicated by
the # of tube. DECREASED OFT (INCREASED resistance): occurs following
2. Add drops of d.H2O required to bring the value of each splenectomy and in liver disease, sickle cell anemia, IDA and
tube to 25 drops. thalassemia. (YOU ARE NOT FRAGILE)
3. Draw blood (20cc mm) & dispense to each tube.
4. Mix by inverting tube once or twice
5. Stand for 2hrs. HIGH OFT LOW OFT
6. At the end of 2hrs, read the result. (↓ RESISTANCE) (↑ RESISTANCE)
- Hemolytic Anemia - Sickle cell anemia
- Hereditary Spherocytosis - Thalassemia
- Spherocyte- G6PD - IDA (Hypochromic)
- Old RBC - Splenectomy
- whenever spherocytes - Liver disease
are found - Reticulocytes
-megal

RETICULOCYTE
Reticulocytes
 “polychromatic erythrocytes”
 The last immature red blood cell stage that is capable of
synthesizing hemoglobin.
 Diagnostic test used to detect bone marrow’s effectivity
to produce RBC.
 Contain remnant cytoplasmic ribonucleic acid (RNA) and
organelles such as the mitochondria and ribosomes
 it is stained darkly with RNA remnants.
 Indicate the ability of the bone marrow to increase RBC
production in anemia due to blood loss or excessive RBC
destruction.
 ↑ in Hemolytic Anemia, Sideroblastic Anemia,
Thalassemia, IDA patients, Acute/Chronic Blood loss
 ↓ in Aplastic Anemia & BM not producing RBC

Reticulocyte/Diffusely Basophilic
Erythrocyte/Polychromatic Erythrocyte
 size: 7-10um
 takes 4-5days (or 3-5 days)
 no longer contain nucleus
HEMA1 LAB (PRELIM PRACTICALS)
 stay first in bone marrow in 1-2 days, then after it will
come out to become RBC.
 what makes different in RBC: in wright stain blood
smear: the cytoplasm is polychromatic (multiple color;
pinch of gray, pink ).
 special in reticulocyte:
*not contain nucleus but has dark area (remnants
of RNA) that keeps them capable of synthesizing
hemoglobin (1/3 of Hgb from reticulocyte, 2/3 from
polychomatophilic and ortho)
 last stage of capable of synthesizing hgb
 still immature, but can be normally present in the blood
(because of their size, similar to RBC)
 in counting reticulocyte using a blood smear: you need
special staining technique (supravital stain) Figure 14-9 Reticulocytes with new methylene blue vital stain
*supravital staining = means staining while they (peripheral blood 31000). Reticulocytes are nonnucleated red
are living blood cells with two or more blue-stained filaments or
*stains use: new methylene blue (NMB), particles.
brilliant-cresyl blue (BCB)
*before preparing the blood smear, supravital stain MATERIALS
is needed for us to preserve the life of the cell • SLIDES
(because methanol makes cells die) • ANTICOAGULATED BLOOD
 aside from RNA you are counting (in performing • SUPRAVITAL STAIN:
supravital stain), you may also recover the abnormalities  Brilliant Cresyl Blue
of cell, you need Heinz bodies and Hgb H  New Methylene Blue
 why should i count reticulocyte: if we want to know the • PIPETTE
activities of bone marrow in producing RBCs. • MICROSCOPE
*ex. patient has hemolytic anemia (red cells are • CEDARWOOD OIL
being lysed)
 as a response bone marrow generate more RBC. Dry method
 to know if the marrow is reacting or not in producing:  mix the stain and blood
count reticulocyte  should be in an equal amount (e. 1mL blood and 1mL
*don’t count RBC, because it dies in hemolytic Supravital stain)
anemia (lysing), result is false positive (low)  allow to stand for a couple of minutes
*because what hemolytic anemia damages is RBC  prepare usual a smear (wedge technique)
not reticulocyte.
*the more reticulocyte you have in Hemolytic Wet method
Anemia, the more it tells you that your bone  mix the stain and blood
marrow is effective and reacting  should be in an equal amount (e. 1mL blood and 1mL
*if reticulocyte is elevated, even without forms of Supravital stain)
anemia; then it is abnormal that also associated  allow to stand for a couple of minutes (5mins)
with Leukemia  add a drop of solution in the slide and cover it with
cover slip.
Reticulocyte Count
 An indicator of the rate of erythrocyte production  Either dry or wet method, read 1,000 red cells
 Use of supravital stains  Find a bluish strands in sight (remnants of RNA) =
 The count is expressed as a percentage of total RETICULOCYTES
erythrocytes
NORMAL VALUES
Principle Adult = 0.5% - 1.5 %
 Whole blood, anticoagulated with EDTA, is stained with Newborns = 2% - 6%
a supravital stain
 Any non-nucleated red blood cell that contains two or PROCEDURE
more particles of blue- stained granulofilamentous 1. Place three drops of reticulocytes stain in a small test
material after new methylene blue staining is tube.
defined/counted as a reticulocyte 2. Add three drops of well mixed whole blood to the tube
containing the stain.
3. Mix the tube and allow to stand at room temperature or
incubate at 37C for 15 minutes
4. After 15 minutes, mix the contents of the tube well and
prepare smears and allow it to dry.
HEMA1 LAB (PRELIM PRACTICALS)
5. Examine the smear under the oil immersion objective.
6. Count the total number of reticulocytes found per 1,000
RBCs

Alternative procedure:
1) Mix equal amounts of blood and new methylene blue
stain (2 to 3 drops, or approximately 50 mL each), and
allow to incubate at room temperature for 3 to 10
minutes.12
2) Remix the preparation.
3) Prepare two wedge films (Chapter 16).
4) In an area in which cells are close together but not
touching, count 1000 RBCs under the oil immersion
objective lens (10003 total magnification). Reticulocytes
are included in the total RBC count (i.e., a reticulocyte
counts as both an RBC and a reticulocyte).
5) To improve accuracy, have another laboratorian count
the other film; counts should agree within 20%.
6) Calculate the % reticulocyte count:
Example: There are 15 reticulocytes counted in 8 months old
infant.

Interpretation: NORMAL

Reference range
• Adult: 0.5-1.5%
• Newborn: 2.0-6.0%

Miller Disc
 Because large numbers of red blood cells should be
counted to obtain a more precise reticulocyte count, the
Miller disc was designed to reduce this labor-intensive
process.
 The disc is composed of two squares, with the area of
the smaller square measuring 1/9 the area of the larger
square. The disc is inserted into the eyepiece of the
microscope and the grid in Figure 14-10 is seen.
 RBCs are counted in the smaller square, and
reticulocytes are counted in the larger square. Selection
of the counting area is the same as described earlier.
 A minimum of 112 cells should be counted in the small
square, because this is equivalent to 1008 red cells in
the large square and satisfies the College of American
Pathologists (CAP) hematology standard for a manual
reticulocyte count based on at least 1000 red cells.
 The calculation formula for percent reticulocytes is:
HEMA1 LAB (PRELIM PRACTICALS)
Absolute Reticulocyte Count (ARC)
 the actual number of reticulocytes in 1 liter (L) or 1
microliter (mL) of blood
- Principle: actual number of reticulocytes in 1 L of whole
blood
- we have millions of red cells in a liter of blood
9
- Reference Range: 25 – 75 x10 /L

Equation Reference Interval

General reference intervals can be found on the inside front
cover of this text.

Sources of Errors
1. blood and stain are not mixed before the films are made
2. very anemic or polycythemic patient
3. moisture in the air, poor drying of the slide, or both
4. other red blood cell inclusions that stain supravitally
include Heinz, Howell-Jolly, and Pappenheimer bodies

a. If a patient is very anemic or polycythemic, the

proportion of dye to blood should be adjusted
accordingly.
b. An error may occur if the blood and stain are not mixed
before the films are made. The specific gravity of the
reticulocytes is lower than that of mature red blood
cells, and reticulocytes settle at the top of the mixture
during incubation.
c. Moisture in the air, poor drying of the slide, or both may
cause areas of the slide to appear refractile, and these
areas could be confused with reticulocytes. The RNA The absolute reticulocyte count can also be reported as the
remnants in a reticulocyte are not refractile. number of cells per mL. Using the example above, the RBC
d. Other red blood cell inclusions that stain supravitally count in mL (2.20 3 106/mL) is used in the formula, and the
include Heinz, Howell-Jolly, and Pappenheimer bodies ARC result is 44 3 103/mL.
(Table 19-3). Heinz bodies are precipitated hemoglobin,
usually appear round or oval, and tend to adhere to the Reference Interval
cell membrane (Figure 14-11). Howell-Jolly bodies are Values between 20 3 109/L and 115 3 109/L are within the
round nuclear fragments and are usually singular. reference interval for most populations.
Pappenheimer bodies are iron in the mitochondria
whose presence can be confirmed with an iron stain, Corrected Reticulocyte Count (CRC)
such as Prussian blue.  In specimens with a low hematocrit, the percentage of
e. If a Miller disc is used, it is important to heed the “edge reticulocytes may be falsely elevated because the whole
rule” as described in the WBC count procedure and blood contains fewer red blood cells.
illustrated in Figure 14-2. A significant bias is observed if  A correction factor is used, with the average normal
the rule is ignored. hematocrit considered to be 45%.
HEMA1 LAB (PRELIM PRACTICALS)
Reticulocyte Production Index (RPI)
 Measures erythropoietic activity when stress
reticulocytes are present
 What are “shift reticulocytes”?
 General indicator of erythrocyte production increase
above normal in anemias

 Reticulocytes that are released from the marrow

prematurely are called shift reticulocytes.
 These reticulocytes are “shifted” from the bone
marrow to the peripheral blood earlier than usual
to compensate for anemia.
 Instead of losing their reticulum in 1 day, as do
most normal circulating reticulocytes, these cells
take 2 to 3 days to lose their reticula.
 When erythropoiesis is evaluated, a correction
should be made for the presence of shift
reticulocytes if polychromasia is reported in the
red blood cell morphology.
 Most normal (nonshift) reticulocytes become
mature red blood cells within 1 day after entering
the bloodstream and thus represent 1 day’s
production of red blood cells in the bone marrow.
 Cells shifted to the peripheral blood prematurely stay
longer as reticulocytes and contribute to the
reticulocyte count for more than 1 day.
 For this reason, the reticulocyte count is falsely
increased when polychromasia is present, because
the count no longer represents the cells maturing
Reference Interval
in just 1 day.
Patients with a hematocrit of 35% should have an elevated
 On many automated instruments, this
corrected reticulocyte count of 2% to 3% to compensate for
mathematical adjustment of the reticulocyte count
the mild anemia. In patients with a hematocrit of less than
has been replaced by the measurement of
25%, the count should increase to 3% to 5% to compensate
immature reticulocyte fraction (Chapter 15)
for the moderate anemia. The corrected reticulocyte count
 The patient’s hematocrit is used to determine the
depends on the degree of anemia.
appropriate correction factor (reticulocyte
maturation time in days):

HEMA1 LAB (PRELIM PRACTICALS)
Reference Interval
 An adequate bone marrow response usually is indicated
by an RPI that is greater than 3. An inadequate
erythropoietic response is seen when the RPI is less
than 2.14.
Reticulocyte Control
 Several commercial controls are now available for
monitoring manual and automated reticulocyte counts
[e.g., Retic-Chex II, Streck Laboratories, Omaha, NE;
Liquichek Reticulocyte Control (A), Bio-Rad Laboratories,
Hercules, CA].
 Most of the controls are available at three levels. The
control samples are treated in the same manner as the
patient samples. The control can be used to verify the
laboratorian’s accuracy and precision when manual
counts are performed.
Automated Reticulocyte Count
 Analyzers evaluate reticulocytes using optical scatter or
fluorescence after the red blood cells are treated with
fluorescent dyes or nucleic acid stains to stain residual
RNA in the reticulocytes.
 The major instrument manufacturers offer are analyzers
that perform automated reticulocyte counts.
 All of the analyzers evaluate reticulocytes using optical
scatter or fluorescence after the red blood cells are
treated with fluorescent dyes or nucleic acid stains to
stain residual RNA in the reticulocytes.
 The percentage and the absolute count are
provided.These results are statistically more valid
because of the large number of cells counted.
 Other reticulocyte parameters that are offered on some
automated instruments include a maturation
index/immature reticulocyte fraction or IRF (reflecting
the proportion of the more immature reticulocytes in
the sample), the reticulocyte hemoglobin concentration,
and reticulocyte indices (such as the mean reticulocyte
volume and distribution width).
 The IRF may be especially useful in detecting early
erythropoietic activity after chemotherapy or
hematopoietic stem cell transplantation. The
reticulocyte hemoglobin is useful to detect early iron
deficiency.
Maturation Index:
• 36-45% = 1 day
• 26-35% = 1.5 day
• 16-25% = 2 days
• <15% = 2.5 days
>3 = bone marrow is responding (in cases of anemia)
<2 = bone marrow is not responding (not helping you)
RBC INDICES
 used to define the shape, size, & hemoglobin content of
the red blood cell
 aids in the diagnosis & differenting Anemia
 differentiates types of anemia
1. MEAN CELL VOLUME (MCV)
- indicates the average volume of RBC in femtoliter (fL) or
15
10 L
- directly proportional to size
- using a manual method: it is just a computed value
In automated: can compute the volume
- ↑ in Megaloblastic Anemia, Hemolytic Anemia with
Reticulocytosis, liver disease, & normal newborn
- ↓ in Iron deficiency Anemia, Sideroblastiic Anemia,
Thalassemia, & Lead poisoning
NORMAL VALUE = 150-400 x 10 /L
9
NORMAL RANGE (normocytic): 80 – 100 fL
MICROCYTIC : < 80 fL
MACROCYTIC : > 100 fL
Formula (manual method:
2. MEAN CELL HEMOGLOBIN (MCH)
- indicates the average weight of Hemoglobin in the RBC
expressed in picograms (pg)
- less valuable than MCHC and MCV.
- it is directly proportional to the size of RBC & concentration
of Hgb in the RBC
- ↑ in Macrocytic Anemia
- ↓in Microcytic, Hypochromic Anemia
-12
- Expressed in picograms (pg) or 10 g
HEMA1 LAB (PRELIM PRACTICALS)
- NORMAL RANGE: 26 – 32 pg

3. MEAN CORPUSCULAR HEMOGLOBIN

CONCENTRATION (MCHC)
- average concentration of hemoglobin (or with hematocrit)
values in the RBC expressed in grams per deciliter (g/dL)
- Expressed in g/dL
- ↑ in error in RBC, or presence of Spherocytes
- ↓in Hypochromic RBC, which is seen in Iron
Deficiency & Thalassemia RULE OF THREE:
- only applicaple for normocytic-normochromic RBC
NORMAL RANGE: 32 – 36 g/dL - in practice, we should not use this. Because it is only a
HYPOCHROMIC : <32 g/dL theoretical representation.
Incorrect calculation or presence of agglutinins : >37 g/dL - 3 x RBC = Hgb
Lipemia, presence of abnormal hemoglobin (S and C): <22 - 3 x Hgb = Hct
g/dL
Ex. 4x1012L
- 4 x 3 = 12g/dL (hgb) x 36% (hct)

WHITE BLOOD CELL (WBC) COUNT

 The WBC or leukocyte count is the number of WBCs in 1
liter (L) or 1 microliter (mL) of blood.
4. RBC DISTRIBUTION WIDTH (RDW)
 Utilized to indicate infection (by microorganisms).
- determined from the RBC Histogram
 A WBC count can detect hidden infection.
- reflects degree of Anisocytosis (variation in RBC size) &
 ↑ WBC count
coeficient of variation of the MCV
 Test that measures the number of WBC in the body.
- Increased RDW = Post-transfusion, Post-treatment,
 measures the TOTAL WBC component of the whole
Idiopathic
blood.
- Sideroblastic Anemia in presence of two concurrent
 Often included in CBC.
deficiencies
 Helps the doctor monitor the effectiveness of
NORMAL RANGE: 9.0-14.5%
chemotherapy or radiation treatment in cancer
patients.
 Microcytic/macrocytic – normal RDW + decrease or
 used to test the ability of the body to protect himself.
increase MCV
 same as RBC counting except for their diluting fluid.
 Anisocyte with average size within reference range =
 Important terms:
increase RDW + Normal MCV
 Leukocytosis
 Anisocyte with average size below or above the normal
 ↑ on WBC count
range= abnormal MCV and RDW
 Leukopenia
 ↓ on WBC count
Representative critical values:
 Immunocompromised patients
• Hemoglobin: less than 5.0 g/dL
• Hematocrit: less than 15%
MATERIALS:
• Platelet count: less than 30,000 per microliter
1. Hemacytometer
• WBC count: less than 2,500 per microliter and greater than
 Each of the four corner (WBC) squares is
30,000 per microliter
subdivided further into 16 squares.
 A hemacytometer is charged (filled) with the
well-mixed dilution and placed under a microscope
and the number of cells in the 4 large corner
2
squares (4 mm ) is counted.
2. Diluting fluids
 1-3% Acetic acid
 1% HCl
HEMA1 LAB (PRELIM PRACTICALS)
 Turk’s Diluting fluid: *Identifying cells in manual counting using
 Acetic acid hemacytometer = only done under HPO
 Gentian violet (makes WBCs appears purplish
in color)
 Distilled water

*Glitter cells = white cells in urine (brownian

movement),
*Artifacts = colorless, non-refractive, disappears
once fine adjustment knob moves
*WBCs = colorless, refractile, contains substances
inside and did not disappear

3. Thick coverslip
4. Thoma pipette (WBC pipette) = white bead, 11 unit of
volume
5. Suction device
6. Cell counter

 Specimen: 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).
How to Make a Moist Chamber
 Diluting fluid: HYPOTONIC (to lyse RBC) A moist chamber may be made by placing a piece of damp
 The diluting fluid lyses the non-nucleated red filter paper in the bottom of a petri dish. An applicator stick
blood cells in the sample to prevent their broken in half can serve as a support for the hemacytometer.
interference in the count.
 Dilution: 1:20
2
 Area factor: (4) 2° squares = 4mm
9
 SI unit: x 0.001 (x 10 /L)

NOTE: In manual WBC counting (using hemacytometer

and hypotonic solution, YOU CANNOT IDENTIFY WHICH
TYPE OF WBC IT IS. You just identify them as white cells.
*You can identify them by BLOOD SMEAR
PREPARATION.

PROCEDURE:
1) Aspirate blood using WBC pipette up to 0.5 mark.
2) Gently wipe the tip of WBC pipette, carefully not to
touch the mouth of the pipette.
3) Aspirate diluting fluid up to 11 mark. Wipe again to
remove excess fluid. There should no air bubbles or
spaces.
4) Gently mix the solution in a figure of 8 for 5-10 minutes
(to make sure that RBCs are lysed).
5) Discard the first 3-5 drops of the solution (it only
contains diluting fluid).
6) Charge both sides of Hemacytometer chamber with
coverslip.

*The fluid should stay in the chamber area only, there

should no excess fluid at the side or middle area of
hemacytometer.
*Presence of excess fluid = REPEAT the charging.
7) Incubate in a petri dish with wet gauze for 5mins.
8) Count RBC in microscope using HPO in (4) 2° squares.
Follow parfocal focusing.
--recall--
 In each Hemacytometer, you have two (2) counting
chambers or area (red colored).
o
 Primary square or 1 (biggest)
HEMA1 LAB (PRELIM PRACTICALS)
 In every primary square, contains 9 secondary
squares. Example Using the First Equation
o
 Secondary square or 2  When a 1:20 dilution is used, the four large squares on
 For every secondary square, it is equal to one side of the chamber yield counts of 23, 26, 22, and
2
1mmx1mm or 1mm 21. The total count is 92. The four large squares on the
2
 The entire secondary square is equal to 9mm . other side of the chamber yield counts of 28, 24, 22, and
 The four (4) corners are the WBC squares. 26. The total count is 100. The difference between sides
*you are still allowed to count RBC in WBC is less than 10%.
squares, its just that the area factor will change.  The average number of cells of the two sides of the
*In each WBC square, there is 16 tertiary squares chamber is 96.
o
or 3 .  Using the average in the formula:
*YOU NEED TO COUNT THE ENTIRE SQUARE of 4
2
secondary WBC square) = 4mm area
 In the middle or center part, you will find the RBC
square (it is only one).
*RBC square has 25 tertiary squares.
**5 squares should be counted (routinely).
**you can count WBC here, thus area factor
will changed during computation.
*In each tertiary square, there is 16 quaternary
o
squares or 4 . Convert to SI unit: multiplied by 0.001
 Other four (4) squares (vertical and horizontal)
 serves as guide or marker Alternately, a 1:100 dilution may be used counting the
 indicates that you reach the end, you need to stop number of cells in the entire counting area (9 large squares, 9
2
mm ) on both sides of the chamber.
“L” rule:
 Bottom and Left line is INCLUDED. As an example, if an average of 54 cells were counted in the
 Apply this on outer lines only. entire counting area on both sides of the chamber:
--end of recall--

General reference intervals for males and females in different

age groups can be found on the inside front cover of this text.
Reference intervals may vary slightly according to the
population tested and should be established for each
laboratory:

Sources of Error and Comments

1. The hemacytometer and coverslip should be cleaned
properly before they are used. Dust and fingerprints
may cause difficulty in distinguishing the cells.
2. The diluting fluid should be free of contaminants.
CALCULATION:

 Count the 4 square of both chambers then get the

average (to get the “cell counted”)
 Dilution factor (varies)
HEMA1 LAB (PRELIM PRACTICALS)

3. If the count is low, a greater area may be counted (e.g., 7. The accuracy of the manual WBC count can be assessed
2
9 mm ) to improve accuracy. by performing a WBC estimate on a Wright-stained
 Instead of counting of just 4 WBC squares, include peripheral blood film made from the same specimen.
RBC square.
4. The chamber must be charged properly to ensure an
accurate count. Uneven flow of the diluted blood into
the chamber results in an irregular distribution of cells.
If the chamber is overfilled or underfilled, the chamber
must be cleaned and recharged.
5. After the chamber is filled, allow the cells to settle for
10 minutes before counting.
6. Any nucleated red blood cells (NRBCs) present in the
sample are not lysed by the diluting fluid.
 The NRBCs are counted as WBCs because they are
indistinguishable when seen on the
hemacytometer (FALSE INCREASE in WBC count). HIV = viral infection = ↓ WBC count
 If five or more NRBCs per 100 WBCs are observed Psychological/Emotional stress = ↑ WBC count
on the differential count on a stained peripheral
blood film (for confirmation of the presence of
9
n-RBCs), the WBC count must be corrected for Normal values: 4 - 11 x10 /L
9
these cells. At birth: 10 - 30 x10 /L

Correct the WBC count if: Critical values:

9
i. Adult = there are >5 nRBC  >30 x10 /L = severely infected
9
ii. Newborns = there are >10 nRBC  <1 x10 /L = severely immunocompromised

 This is accomplished by using the following Most common opportunistic pathogens targeting patients
formula: with HIV:
 Pneumocystis jirovecii
 Pneumocystis carinii

 Kaposi's sarcoma (no. 1 cancer for HIV patients)

*Report the result as the “corrected” WBC count.

SMEAR PREPARATION AND

EXAMINATION
 A well-made, well-stained and well-examined blood
smear can provide valuable information about a
patient’s health.
HEMA1 LAB (PRELIM PRACTICALS)
 Blood smear gives us a picture of the true condition of
blood.
*whatever size, shape, appearance of cells, if there
are any unwanted or immature cells in the blood, it
can be identified by examining blood smears.
 By checking the morphologic feature of the cell, by
counting certain formed elements in the blood smear,
we can then be able to identify certain forms or certain
types of diseases.
 For us Medical Technologists, it’s very essential that we
know how to prepare and examine blood smear
properly.
When do we use of examine a blood smear?
Uses of Blood Smear Examination
 WBC differential count [what we will focus on today]
*part of routine CBC procedure
*not confirmatory but it still provides additional
information on how we can then arrive with proper
diagnosis of the patient
 Platelet Estimation
*Blood smears helps us In counting platelets
*Estimation = not accurate (because cell counting varies
with one another)
*In blood smear, distribution of cell will vary from one
another.
*Also WBC estimate
 Morphologic evaluation of formed elements.
*most taken for granted use of a blood smear.
*as you check WBC, you should also be mindful in
morphologic features of other cells
*recall: poikilocytes = cells that vary in shape (through
blood smear, you will then check if there is a variation
in shape etc.)
Specimen for Smear Examination
EDTA blood
 Disodium or Tripotassium
 More preferable: liquid type (specially liquid
tripotassium = easily mix with blood)
*powdered form EDTA = form artifacts in smear
 Preserves best the morphology of the cells.
*you have to make sure that you will extract
the optimum morphologic appearances of the
cells
 Ideally blood smears prepared within how many
hours after collection? 2-3 hours
*in some books (Turgeon) = within 1 hour
 Blood films in RT for more than 5 hours often have
unacceptable artifacts.
 Example of artifacts: formation of degraded
leukocytes, crenated RBCs
ADVANTAGES OF EDTA
 multiple slides can be made if necessary
*You should use EDTA to prepare multiple slides in a
single specimen (unlike with skin puncture, you should
add the drop of blood directly in the slide).
 generally prevents platelets from clumping on the glass
slide.
*use EDTA to avoid unwanted aggregation of
platelets.
 Some patients’ blood undergoes an in vitro
phenomenon called platelet satellitosis or Platelet
satellitism when anticoagulated with EDTA.
*Platelet satellitism commonly encounted in patients
with platelet autoantibodies (reacts in room
temperature that causes aggregation of platelets)
--try to imagine this scenario--
i. You have a neutrophil.
ii. Within the surface or cell membrane of neutrophil,it is
crowded or surrounded by platelets.
iii. If there is platelets satellitism and you counted platelets
using an automated machine, expect that your platelet
count will be falsely decreased (platelets have already
attached to neutrophils). Machine wont be able to
identify them.
iv. If on your machine, it reveal that your platelet count is
low, the next best thing to do is you should confirm it by
examining blood smears.
v. If you’ve notice platelet satelllitism in blood smears, you
should collect another blood.
*Sometimes platelets may aggregate with one
another. If this occurs, your count will also be
decreased. If they are clumping, it will falsely increase
your WBC count (because a clumped/aggregated
platelets will read by machine as WBC because of its
size).
 Spuriously low platelet count and falsely increased WBC
counts (pseudoleukocytosis) can result from
EDTA-induced platelet clumping.
 Correction in satellitosis: use of sodium citrate as
anticoagulant (instead of EDTA)
*only use citrate in counting WBC and PLATELETS
(but recommended is still EDTA)
*should not use in CBC and RBC count
Capillary blood sample
 finger and heel punctures
 Some platelet clumping must be expected
 Only a few films can be made directly from blood
from skin puncture
HEMA1 LAB (PRELIM PRACTICALS)
 Should be done bedside of the patient.
*After puncture, preparation of smear is the required
next step to do.
Limitations:
 some platelet clumping must be expected (unlike
when using EDTA, because here you will directly
drop the blood from puncture onto the slide for
smear preparation)
 only a few films can be made directly from blood
from a skin puncture
Peripheral blood smear preparation
Types of blood smear techniques:
1. Wedge technique (double slide/spreader slide/push
smear)
*most commonly used method
2. Coverslip technique (Ehrlich’s method)
3. Coverslip and slide technique (Beacom’s method)
4. Automated Slide Making (Spunner’s method)
*we have available automatic smear preparation and
stainer here
Wedge Technique
 The most convenient and commonly used method for
making peripheral blood films.
 Procedure: spread the drop of blood using the spreader
slide.
 One slide serves as the film/specimen slide, and the
other is the pusher or spreader slide
*in short, you will be using 2 different slides.
 Blood Drop (diameter): 2-3 mm
 Diff-Safe dispenser = safer way. You can obtain a
blood sample without opening the tube. You will
directly puncture the cover of the tube and it will
automatically let you obtain a drop of blood.
 Amount of blood: most important to consider
 >3mm, your smear will become thicker (falsely
elevated count of cells will occur).
 <2mm =your smear will become thinner (cell count
will be decrease)
 0.25 inch from the slide
*P (pressure), A (angle), S (Specimen), S (Speed)
WELL-MADE Peripheral Blood Smear:
- finger-shaped (not bullet shaped)
- Feathery edge
- No bubbles
- No holes/spaces, streaks
- 2/3 - 3/4 the length
o
- ideal angle : 30-45
- have thin to thick transition
Four (4) factors that will affect smear:
1. Pressure
2. Angle
3. Specimen
4. Speed
Causes of THICK smear: falsely-increased result (DARK RED)
- if the pressure you apply is low
o
- higher angle than 45
- specimen is larger than 3mm in diameter
- speed is too fast
Causes of THIN smear: falsely-decreased result (PALE)
-if the pressure you apply is high
o
- lower angle than 30
- specimen is smaller than 2mm in diameter
- speed is too slow
Take note:
 Hold the spreader slide using dominant hand.
*it’s up to you on how you hold the spreader slide
(angle varies)
 Before you let the spreader slide, come into contact
with the blood.
 Check first the consistency of the slide (smooth, no
rough edges or spaces).
 Once the consistency of the slide is already smooth,
touch the drop of blood using the spreader slide.
 Once the blood touches the spreader slide, let it spread
totally first from side to side.
 Once the blood is properly spread, you should quickly
but smoothly push forward the drop of blood to the end
of the slide.
 As much as possible, The drop of blood before you push
it forward, should not reach the end side of the slide.
(tendency is cells particularly WBC and platelets will
tend to accumulate in those area).
HEMA1 LAB (PRELIM PRACTICALS)
REMEMBER: in between the thick and thin area (transition) is
where you will count cells. In this area, cells are evenly
distributed. = HEEL area
Automated Slide Making and Staining
Automated slide making and staining system.
 The system adjusts the size of the drop of blood used
and the angle and speed of the spreader slide in making
a wedge preparation.
*This automated machine adjust everything BASED
ON THE HEMATOCRIT OF THE PATIENT
*for example: if the hematocrit is HIGH, its suggests
that RBCs are too much. So in preparing smear in
general you will expect that the smear is thick, but
here in automated smear machine, it will adjust
everything so that in a way the thickness of the smear
will lessen (and vice versa).
 The spreader slide is automatically cleaned and is ready
for the next blood film to be made.
 Films can be produced approximately every 30 seconds.
 Proper labeling of smear: Patient identification
information (printed on the slide)
 Name
 Number of specimen
 Date of specimen
Staining of Smears
Commonly used stains:
 Pure Wright stain
 Wright-Giemsa stain = use for recovery of possible
malarial parasite
*Purpose of staining: for you to be able to identify accurately,
visualize properly the cells and differentiate WBCs. (You will
be then identify morphologic changes in the cell, easily
visualize, characterize and count the cells).
*In general, staining is just adding of colors.
*before staining, fix the slide first.
 Purpose: make the cells more visible and to allow their
morphology to be evaluated
 Fixative: Methanol
*to preserve morphology of the cell and to make sure
that the cells are fixed or attached to the slide (so it
will not totally wipe out during staining process).
 pH: 6.4 – 6.8
 0.05M sodium phosphate buffer (6.4)
 Aged distilled water (6.4 - 6.8)
*Staining reactions are always pH DEPENDENT.
*Buffer is added to maintain the pH.
*Wright stain is a combination of alkaline and acidic stain.
*Eosinophil takes up acidic dye Eosin.
*Basophil takes up basic/alkaline dye Methylene blue (dark
purple granules)
*Neutrophils takes up both acidic and basic dyes because
of it’s neutral pH.
 Free methylene blue is a basic stain thus stains acidic
(and basophilic) cellular components of cell, such as
ribonucleic acid (RNA).
 Free eosin is an acidic stain thus stains basic (and
eosinophilic) components of cell, such as hemoglobin
and eosinophilic granules
2 methods:
i. Dip
ii. Flood
*either way, fix with methanol first.
Dipping method:
1) Fix with methanol (dip 2-3 times).
2) Allow to dry.
3) Dip in the stains (6-8 times).
HEMA1 LAB (PRELIM PRACTICALS)
4) Dip in the buffer (3-4 times).  Monocytes: gray ground glass appearance with
vacuoles inside
Flooding method:
1. Fix with methanol (leave for 2-3 mins). *Definitely the background of the smear is white. If
2. Allow to dry first. you use heparin, there is bluish discoloration of
3. Flood the smear with Wright’s stain (leave for 3 mins). background.
4. Add the buffer (leave for 3 mins)
*to adjust the appearance of the cell, adjust the timing
of staining processes. Staining Problems/Errors
Too Acidic Stain: Too Alkaline Stain:
Greenic metallic sheen coloration = normal appearance 1. Thin blood smear 1. Thick blood smear
(properly done) 2. Insufficient staining 2. Prolonged staining
time 3. Insufficient washing
5. When staining is already complete, gently wash the slide 3. Prolonged buffering or 4. Alkaline pH of stain
with distilled water. washing components
6. Wipe the back of the slide to remove stain residues. 4. Old stain
7. As you dry it, make sure that the slide is in vertical position
(to drain down the moisture and water components of the Correction: Correction:
slide) 1) Lengthen staining time 1) check pH
2) Check stain and buffer pH 2) shorten stain time
3) Shorten buffering or wash 3) prolong buffering time
time

Quick Stains
 Fast and easy which takes 1 minute.
*because of the quick staining process, there might be
color variations.
 Modified Wright or Wright-Giemsa stain
 Buffer: aged distilled water
 Stained slides are given a final rinse under a gentle
stream of tap water and allowed to air-dry.

Well-stained Blood Smear
 Macroscopically:
 Reddish brown : Wright’s stain(pink)
 Microscopically: Minimum precipitate with no artifacts
 Rbc: pink
 Nuclei of leukocytes: purple
 Eosinophilic granules: red orange
 Basophilic granules: dark purple
 Neutrophilic granules: rose pink
 Platelets: purplish or dark lilac
*If the stain is too alkaline, might as well to adjust it.
*Failed to rinse with water properly the smear after staining,
might as well rinse it more next time.
*Heparinized sample: Heparin is not an ideal anticoagulant
for blood smear preparation (gives discoloration =
BLUE/BLUISH and affect the appearance of the cell).
Macroscopic Examination
 Macroscopic examination sometimes can give us an
idea of possible abnormalities on your patient that
need checking and address.
HEMA1 LAB (PRELIM PRACTICALS)
 Bluer than normal: increased blood proteins and
possible rouleaux (RBCs formed stack of coins-like
appearance)
*in this case, it might possibly indicate that the
patient has a disease called Multiple Myeloma.
** increased production of immunoglobulins
 Grainy: RBC agglutination (aggregation)
*commonly encountered in Cold Agglutinin Disease
 Holes all over the smear: increased lipid levels
*maybe because of improper preparation of smear
* increased lipid levels = plasma is lipemic
 Blue specks at the feathery edge: markedly increased
WBC and platelet counts.
*there maybe an accumulation of WBCs or even
platelets and improper preparation of smear.
Microscopic Examination
 Main tool in examining blood smear.
 Highlight:Proper usage of objective lenses.
*Scanner and LPO = uses Coarse Adjustment Knob
*HPO and OIO = use Fine Adjustment Knob
*FOLLOW PARFOCAL FOCUSING without the need of
moving the stage (shifting of objectives with minimal
changes on adjustment knobs)
 Light from the illuminator should be properly centered
 Condenser should be almost all the way up and adjusted
correctly for the magnification used.
 Iris diaphragm should be opened to allow a comfortable
amount of light to the eye.
 Low Power Objective (LPO/10X)
 What you should check here: overall film/smear
quality
 *You will decide if the smear is good or not
 DO NOT IDENTIFY AND COUNT CELLS HERE.
 Check for:
 Color
 Distribution of cells (if there is Roleux
formation, Aggregation, Clumping)
 Possible to check for the presence of fibrin
strands (artifacts or clots)
 RBC distribution
 scanned quickly for any large abnormal cells
(might indicate immature cells)
 feather edge and lateral edges should be checked
quickly for WBC distribution.
 High Power Objective (HPO/40X)
 What you should check here: where we can decide
which specific portion of the smear is to used in
counting.
*Should not be in a thick and thin area
*it should be in between thick and thin area
(evenly spread)
 DO NOT IDENTIFY AND COUNT CELLS HERE ALSO.
 select the correct area of the film in which to begin
the differential count
 evaluate cellular morphology
 WBC estimate execute here BUT not
recommended to do so (not accurate)
 Oil Immersion Objective (OIO/100X)
 Where identifying, counting, differentiating and
checking the morphology of cells takes place.
 provides the highest magnification
 WBC differential count
 RBC, WBC, and platelet morphology evaluation
 platelet estimate
 segmented neutrophils can be readily
differentiated from bands
 RBC and WBC inclusions
 Reactive or abnormal cells enumeration
 n-RBC counting (if present)
*n-RBCs are abnormal cells
Optimal Assessment Area
 Part or area where cells are equally distributed.
 Between the thick area and the very thin feather edge
and also called as the HEEL.
*where you are allowed to count cells
 IDEAL:
 RBCs are uniformly and singly distributed, with few
touching or overlapping, and have their normal
biconcave appearance.
 200-250 RBCs per oil immersion field.
*not overlapping in one another
 Too thin area: red cells are distorted, flat and large
 Too thick area: distorted rbc (rouleaux); distorted wbc
Counting Methods
i. Cross-sectional or crenellation: From side to side.
ii. Longitudinal: From the tail toward the head of the
smear.
iii. Battlement: uses a pattern of consecutive fields
beginning near the tail on a horizontal edge.
*COMMONLY USED
*As you move from one filed to another, make sure
that you are still in a part where cells are evenly
distributed.
*whether of these 3, the main idea is, if you are through
counting a specific field, as much as possible DO NOT GO
BACK IN THE SAME FIELD AGAIN.
HEMA1 LAB (PRELIM PRACTICALS)

REMEMBER!
 Blood smear is a picture of the true condition of the
blood.

WBC DIFFERENTIAL COUNT

 Principle: A stained smear is examined to determine
the percentage of each type of leukocyte present and
assess the erythrocyte and platelet morphology.
 Increases in any of the normal leukocyte types and
the presence of immature leukocytes or
erythrocytes in peripheral blood are important
diagnostically in a wide variety of infl ammatory
disorders and leukemia.
 Erythrocyte abnormalities are clinically important
in various anemias. Platelet size irregularities are
suggestive of particular thrombocyte disorders.

 Fewer manual differentials are performed today

because of the superior accuracy of automated
differentials and because of cost and time constraints.
 When indicated, however, the manual differential
always should be performed in a systematic manner.
 When the correct area has been selected, use of a
back-and-forth serpentine, or “battlement,” track
pattern is preferred to minimize distribution errors and
ensure that each cell is counted only once.
 One hundred (100) WBCs are counted and classified
through the use of push-down button counters or
newer computer interfaced key pads.
 in manual counting, count 100 WBCs also.
 To increase accuracy, it is advisable to count at least
9
200 cells when the WBC count is higher than 40x10 /L.
9
 If the WBC count is 100x10 /L or greater, it would
be more precise and accurate to count 300 or 400
cells.
9
 If <5x10 /L, count 50 WBCs only.
 Results are reported as percentages (%) —for example,
54% segmented neutrophils, 6% bands, 28%
lymphocytes, 9% monocytes, 3% eosinophils.
 The evaluator always should check to ensure that the
sum of the percentages is 100.
HEMA1 LAB (PRELIM PRACTICALS)
 WBC differential count can determine the percentage of
each type of WBC present in blood.
 A differential count can detect immature and abnormal
WBC, both of which are signs of potential issues.
 Examined by smearing on a glass slide and staining the
sample with Wright’s Stain.
 Manner of reporting is by percentage or by decimal
point.
 Slides are kept for at least 1 year.
 differentiates based on nucleus, lobulation of nucleus,
color and appearance of granules.
 perform so that in a way we have an idea what infection
patient has
 Neutrophils (1) - bacterial (most likely),
inflammation
 Lymphocytes (2) - viral
 Monocytes (3) Photomicrographs of peripheral blood film with areas too
 Eosinophils (4) - parasitic, allergy thick to read.
 Basophils (5) - allergy
Number of WBCs:
 Never (most abundant)
 Let
 Monkey
 Eat
 Banana

 Routine/Manual : you’ll count 100 WBC

 Leukopenia : you’ll count 50 WBC only
 Leukocytosis (infection): you’ll count up to 200 WBC

-differential counting--
3 parameter machine = granulocyte, lymphocyte, monocyte
5 parameter machine = neutrophil, eosinophil, basophil,
lymphocyte, monocyte (multiple parameter) Laboratory differential tally counter.

Photomicrograph of good area of peripheral blood film.

Photomicrographs of peripheral blood film with areas too

thin to read

Computer interfaced key pad counter.

HEMA1 LAB (PRELIM PRACTICALS)
Performing 100-cell differentials on extremely low WBC
counts can become tedious and time consuming, even when
the 50X oil immersion objective is used.
 In some laboratories, the WBCs are concentrated by
centrifugation, and buffy coat smears are made. This
practice is helpful for examining the morphology of the
cells; however, it is not recommended for performing
differentials because of possible errors in cell
distribution from centrifugation.
 In other laboratories evaluators may perform a 50-cell
differential and multiply the results by 2 to get a
percentage. The accuracy of this practice is questionable,
and it should be avoided if possible.
 In some laboratories the buffy coat smear is examined
for the presence of blasts, but no differential is
performed.
 It is essential to include the side margins of the blood
film in any differential so that the larger cells, such as
monocytes, reactive lymphocytes, and immature cells,
are not missed.
In addition to counting the cells, the evaluator assesses
their appearance.
 If present, WBC abnormalities such as toxic granulation,
Döhle bodies, reactive lymphocytes, and Auer rods are
evaluated and reported.
 Unfortunately, the exact method by which these are
reported varies from laboratory to laboratory. Reactive
lymphocytes may be reported as a separate percentage
of the 100 cells, as a percentage of the total number of
lymphocytes, or semiquantitatively (“occasional” to
“many”).
 Toxic granulation generally is reported as “present” or
is sometimes reported semiquantititively (“slight” to
“marked,” or 1+ to 3+).
 Standardization of this process has been difficult, but
laboratorians must look to simplify blood film
morphology reports to help ensure better accuracy,
consistency, and clinical relevance.
 Simply reporting “present” is becoming preferable to
the older “semiquantitative” reporting of morphology
abnormalities.
 Regardless of reporting format, each laboratory should
establish criteria for reporting microscopic cell
morphology.
Because the differential alone provides only partial
information, reported in relative percentages, the absolute
cell counts are calculated for each cell type in some
laboratories.
 Automated differentials already include this
information.
Automation has also been applied to the processes of
microscopic cell location and identification.
 These automated systems are especially dependent on
the quality and consistency of the blood film and stain in
order for the digital systems to recognize and identify
the cells.
 Once cells are located, a digital image is made and the
cell is classified using sophisticated computerized visual
recognition systems.
 Digital images of the classified cells are presented to the
operator, who can override the instrument’s
identification, if needed, or add identifications for cells
that the instrument could not classify.
CellaVision DM96. CellaVision is an example of an automated
system for differential counting that locates blood cells on a
stained peripheral blood film, takes digital images, classifies
the cells, and presents the cells in the 100-cell differential
count to the operator on a computer screen. The operator
can override the instrument’s classification and identify cells
that the instrument could not classify.
Specimen
 Peripheral blood, bone marrow, or body fluid
sediments, such as spinal fluid, are appropriate
specimens.
 Whole blood smears may be made from
EDTA-anticoagulated blood or prepared from
free-flowing capillary blood.
 Smears should be made within 1 hour of blood
collection from EDTA specimens stored at room
temperature to avoid distortion of cell morphology.
HEMA1 LAB (PRELIM PRACTICALS)
 Unstained smears can be stored for indefinite periods,  Nucleated erythrocytes are not included in the
but stained smears gradually fade. total count but are noted per 100 white blood cells
(WBCs).
Reagents, Supplies, and Equipment  A total of at least 100 leukocytes should be
1. A manual cell counter designed for differential counts counted.
2. Microscope, immersion oil, and lens paper  Express the results as a percentage of total
leukocytes counted.
Quality Control
 Training and experience in examining immature and Reporting Results
abnormal cell morphology are essential.  Reference values, particularly the band neutrophil
 A set of reference slides with established parameters percentage, may vary.
should be established to assess the competence of an  Values for children differ from adult reference values.
individual to perform differential and morphological
identification of leukocytes and erythrocytes. Procedure Notes
 Participation in a quality assurance program continues  A well-made and well-stained smear is essential to the
to document the expertise of the hematologist in accuracy of the differential count.
microscopy.  The knowledge and ability of the cell morphologist are
 Questionable or abnormal smears should be referred to critical to high-quality results.
a supervisor for verification.  A minimum of 300 leukocytes must be within the
acceptable working area, when the total leukocyte
9
Procedure count is no less than 4 × 10 /L.
1. Begin the slide examination with a correctly prepared  The neutrophils, monocytes, and lymphocytes should
and stained smear. appear evenly distributed in the usable fields of the film.
2. Focus the microscope on the 10× objective (low  Less than 2% of the leukocytes should be disrupted or
power). nonidentifiable forms except in certain forms associated
 Scan the smear to check for cell distribution, with pathological states.
clumping, and abnormal cells. Add a drop of  If a disrupted cell is clearly identifiable, include it in the
immersion oil and switch to the 100× (oil differential count. Classify nonidentifiable disrupted
immersion) objective. cells (smudges or baskets) as “other” and note them on
 Begin the count by determining a suitable area. the report if more than a few are observed.
 Extend the examination from the area where
approximately half of the erythrocytes are barely The blood smear preparation techniques are commonly used
overlapping to an area where the erythrocytes in the laboratory for the preparation of blood smears. In
touch each other. certain circumstances, the preparation of a buffy coat
 It is important to examine cellular morphology and peripheral blood smear increases the accuracy of the
to count leukocytes in areas that are neither too leukocyte differential count.
thick nor too thin. In areas that are too thick,
cellular details such as nuclear chromatin patterns
are difficult to examine.
 In areas that are too thin, distortion of cells makes
it risky to identify a cell type.
3. Count the leukocytes using a tracking pattern.
 Each cell identified should be immediately tallied
as a neutrophil (band), neutrophil (segmented), or
polymorphonuclear neutrophil (PMN); lymphocyte;
monocyte; eosinophil; or basophil.
 A brief leukocyte morphology reference is included;
however, refer to specific chapters in the text for a
complete discussion of leukocyte and erythrocyte
cellular morphology.
4. Abnormalities of leukocytes, erythrocytes, and
platelets should be noted.
 Normally, 8 to 20 platelets are present in an oil
immersion field in a properly prepared smear
(where the RBCs barely touch each other).
 After examining at least 10 different fields, the
average number of platelets can be multiplied by a
factor of 20,000 to arrive at an approximate total
circulating platelet concentration.
LOBULATIONS:
HEMA1 LAB (PRELIM PRACTICALS)
 Neutrophils = 3-5 [rose pink granules] Total absolute count: 4.2 + 2.1 + 0.7 = 7
 Eosinophils = Bilobed [red-orangegranules]
 Monocyte = Very large cell with a kidney-bean shaped -make sure that the total absolute count should equal to
nucleus, there are vacuoles inside WBC count.
 Basophils = lobulation of nucleus (covered with darkly
stained purplish granules)
 Lymphocytes = mononuclear, same size with RBCs with
little portion of cytoplasm

Sometimes Basophils are confused with Lymphocytes

Band cell = cell with “S” or”C” shaped nucleus without any
lobulation

White Blood Cells Reference Range

Segmenters R: 47-77%
A: 1.8-7.8 x 109/L
Band R: 0-7%
A: 0.07 x 109/L
Lymphocytes R: 20-40%
A: 1.0-4.8 x 109/L
Monocytes R: 2-10%
A: 0.01-0.8 x 109/L
Eosinophils R: 0-6%
A: 0-0.6 x 109/L
Basophils R: 0-1% .
A: 0-0.2 x 109/L

Classification Neutrophil count COMPLETE BLOOD COUNT (CBC)

Mild 1.0 – 2.0 x 109/L
Moderate 0.5 – 1.0 x 109/L  a.k.a “Full Blood Count” or “Full Blood Exam”
Severe < 0.5 x 109/L  Most frequent blood test
 Basic screening test and one of the most
RELATIVE COUNT commonly ordered or requested laboratory
 Represents only 100 WBCs procedure.
 Screening test for most diseases.
 provides a detailed info about the cells in the blood.
ABSOLUTE COUNT
 One of the busiest section in the laboratory.
 more accurate than relative count  CBC provides a lot of vital informations of our
 the actual number of specific WBC in a L of blood body.

FORMULA: NOTE: Complete hemogram includes a series of test

Absolute count = Percentage x WBC count which includes complete blood count (CBC, also known
as a complete blood cell count) along with Erythrocyte
Example: sedimentation rate (ESR). CBC is a test that provides
N = 60% information about blood cells like Red Blood Cells (RBC),
L = 30% White Blood Cells (WBC) and platelets.
M = 10%
9/
WBC = 7x10 L  The peripheral film evaluation is the capstone of a panel
of tests called the complete blood count (CBC) or
60% x 7 = 4.2 hemogram. The CBC includes enumeration of cellular
30% x 7 = 2.1 elements, quantitation of hemoglobin, and statistical
10% x 7 = 0.7 analyses that provide a snapshot of cell appearances.
 These results can be derived using the manual
Reported as: methods and calculations (subjective) or using the
9
4.2x10 /L automated instruments (objective) thus more
9
2.1x10 /L efficient to use.
9
0.7x10 /L  Regardless of method, the numerical values should
be consistent with the assessment derived by
HEMA1 LAB (PRELIM PRACTICALS)
examining the cells microscopically. Careful
examination of the data in a systematic way Complete Blood Count Measurements Generated
ensures that all relevant results are noted and
by Automated Hematology Profiling Instruments
taken into consideration in the diagnosis
RBC Parameters
 RBC ( red blood cell) count
 A complete blood count (CBC) is performed on
 HGB (hemoglobin)
automated hematology profiling instruments and
 HCT (hematocrit)
includes the RBC, WBC, and platelet measurements.
 MCV (mean cell volume)
 The medical laboratory professional may collect a blood
 MCH (mean cell hemoglobin)
specimen for the CBC, but often a phlebotomist, nurse,
 MCHC (mean cell hemoglobin concentration)
physician assistant, physician, or patient care technician
 RDW (RBC distribution width)
may also collect the specimen.
 RETIC (reticulocyte)
 No matter who collects, the medical laboratory
professional is responsible for the integrity of the
*RBC, Hemoglobin (part of RBC) and Hematocrit (deals
specimen and ensures that it is submitted in the
with examination of RBC)
appropriate anticoagulant and tube and is free of
*Blood Indices: MCV, MHC, MHCV (examine
clots and hemolysis (redtinted plasma indicating
morphologic features of RBC; size and color) and use to
RBC damage).
classify and differentiate types of anemia.
 The specimen must be of sufficient volume, as
“short draws” result in incorrect
anticoagulant-to-specimen ratios.
Platelet Parameters
 The specimen must be tested or prepared for
 PLT (platelet) count
storage within the appropriate time frame to
 MPV (mean platelet volume)
ensure accurate analysis and must be accurately
*volume = size
registered in the work list, a process known as
*counterpart is MCV
specimen accession. Accession may be automated,
 PDW (platelet Distribution Width)
relying on bar code or radiofrequency
identification technology, thus reducing instances
WBC Parameters
of identification error.
 WBC (white blood cell) count
 Although all laboratory scientists and technicians are
 NEUT (segmented neutrophil) count: % and absolute
equipped to perform visual RBC, WBC, and platelet
 LYMPH (lymphocyte) count: % and absolute
counts using dilution pipettes, hemacytometers, and
 MONO (monocyte) count: % and absolute
microscopes, most laboratories employ automated
 BASO (Basophil) and EO (eosinophil) counts: % and
profiling instruments to generate the CBC.
absolute
 Many profiling instruments also provide comments on
RBC, WBC, and platelet morphology.
*WBC count and differential counts (determine if there
 When one of the results from the profiling instrument is
is infection or your body is immunocompromised or has
abnormal, the instrument provides an indication of this,
weak immunity)
sometimes called a flag.
 In this case, a “reflex” blood film examination is
performed.
 Flags gives you alarm (either H (high) or L (low)) Reflects the degree of variation in size (anisocyte):
that indicates problem either in machine or  RDW = in RBC
equipment or the result of the patient is abnormal.  PDW = in platelets
 The blood film examination is a specialized, demanding,
and fundamental CBC activity. Nevertheless, if all Computed value by automated machine:
profiling instrument results are normal, the blood film  RDW
examination is usually omitted from the CBC. However,  PDW
physicians may request a blood film examination on the  Hematocit (can’t be measured by machine because
basis of clinical suspicion even when the profiling we don’t have centrifuge for this)
instrument results fall within their respective reference  MCH
intervals.  MCHC

 Sample: EDTA blood (presrves morphology of cells) Measured by automated machine:

 RBC count  RBC count
 Hemoglobin Oxygen
 WBC count
 Hematocrit  Platelet count
 WBC count
 WBC differential
 WBC Differential
 Hemoglobin
 Platelet
 Blood indices
 Blood Indices
HEMA1 LAB (PRELIM PRACTICALS)
 MPV
 Blood Indices (MCV, MHC, MCHC)
 examine morphologic features of RBC
*related to one another
 RBC, Hemoglobin (part of RBC) and Hematocrit (deals
with examination of RBC)
 WBC count and differential counts (determine if there is
infection or your body is immunocompromised or has
weak immunity)
 Blood Indices: MCV, MHC, MHCV (examine morphologic
features of RBC; size and color) and use to classify and
differentiate types of anemia.
--recall--
1. WBC Count (Leukocyte Count)
 Test that measures the number of WBC in the
body.
 Often included in CBC.
 A WBC count can detect hidden infection.
 Helps the doctor monitor the effectiveness of
chemotherapy or radiation treatment in cancer
patients.
2. RBC Count (Erythrocyte Count)
 Blood test used to find out how many RBCs you
have in the body.
 Important because RBCs contain Hemoglobin.
 If a hemoglobin test reveals that your
hemoglobin level is lower than normal, it
means you have a low red blood cell count
(anemia).
 Low RBC and Hgb = Anemia
 High RBC and Hgb = Polycythemia
3. Hemoglobin Determination (Hemoglobinometry)
 Measurement of concentration of Hemoglobin in
blood
 uses Hemoglobinometer
 Protein that carries oxygen to body organs and
tissues and transports carbon dioxide from organs
and tissues back to the lungs.
 Method: Cyanmethemoglobin Method
(colorimetric method)
4. Hematocrit Determination
 Volume of packed RBC that occupies a given
volume of whole blood.
 2 Methods:
 Micromethod – capillary tube
 Macromethod – microtainer
*Anemia can be tested using RBC Count, Hgb
and Hct determination
5. Differential Count
 WBC differential count can determine the
percentage of each type of WBC present in blood.
 A differential count can detect immature and
abnormal WBC, both of which are signs of
potential issues.
 Examined by smearing on a glass slide and staining
the sample with Wright’s Stain.
 Manner of reporting is by percentage or by
decimal point.
 Slides are kept for at least 1 year.
Special hematologic procedures in Hema1
A. Reticulocyte Count
 Reticulocytes - young or immature RBCs
 -eticulocyte count is used to assess erythropoietic
activity in the bone marrow. If bone marrow is
capable of producing enough RBC (the more
reticulocyte, the more it tells you that your bone
marrow is okay).
 -Whole blood, anticoagulation with EDTA is stained
with Supravital Stain.
 Supravital Stain – stains living cell
 New methylene blue
 Brilliant cresyl blue
 Pure azure blue (seldom used)
 Reference Value: 0.5 – 1.5%
 Neonates: 2 – 6%
 Blood:Stain Ratio: 1:1
 Has remnants of RNA which exhibits a blue/dark
blue/violet color
B. Erythrocyte Sedimentation Rate (ESR)
 identify presence of inflammation.
 rate at which RBCs sediment in a period of 1 hour.
 rate of fall of RBC
 common hematological test and is a nonspecific
measurement for infammation
 Rheumatoid Arthritis
 Pregnancy
 Cancer
 Tuberculosis
 2 Methods:
i. · Westergren – solely for ESR
ii. · Wintrobe – ESR and macromethod of Hct
--end of recall--
Methodology
 Current hematology analyzers use a combination of light
scatter, electrical impedance, fluorescence, light
absorption, and electrical conductivity methods to
produce complete red blood cell, platelet, and leukocyte
analyses.
 All the widely used automated instruments analyze cells
in flow and are essentially highly specialized flow
cytometers.
Fluorescence = analyze special structures of cell (DNA,
RNA) and also in cytochemistry
Automation
Two General Principles (most commonly used):
 Electronic resistance ( impedance) : single and multiple
parameter
 Coulter principle
HEMA1 LAB (PRELIM PRACTICALS)
 Measure the volume or the size of cell
 Identify and count cells based on the pulses
generated by cellular elements
 Utilizes non-conductive properties of blood cells.
 Cells are considered nonconductors of
electricity (as cells flows of passes through
electrical current (it will generate
pulses/spikes), the electrical flow stops)
 as blood cell passes through orifice of
aperture it displaces its own volume.
 generated pulses will tell you the size or
volume of the cell
 depending of the pulses will then be the
determinant on the identity of the cell
 small pulses = small cells (vice versa)
 RBCs and Platelets counted together, separated by
pulse heights.
 hydrodynamic focusing forces cells to pass single
file through sensing zone.
*Coulter*
A.
• RBC
• Platelets
(both of them utilizes isotonic fluids)
B.
• Cyanmethemoglobin
• Lymphocytes
• Monocytes
• Granulocytes
(lyse RBC first)
RBC and platelet (isotonic) counted simultaneously
first before WBC (hypotonic) and measure
hemoglobin due to the feature of diluting fluid
used.
Differential counting
 Based on the chemical dye/stain and light
scattering result
 3 part differential machine = granulocyte,
lymphocyte, monocyte
 5 part differential machine = neutrophil,
eosinophil, basophil, lymphocyte, monocyte
(multiple parameter)
 6 part differential machine = for nucleated RBCs
 Light scattering: multiple parameter
 Measures the volume, size and internal
characteristics of the cell or internal complexity of
cells
 Recommended
 Cells counted as passed through focused beam of
light (LASER)
 Sum of diffraction, refraction and reflection
 Multi angle polarized scatter separation (M.A.P.S.S)
 Paired with a lot of multiple principles:
cytochemistry, flow cytometry
 Light will pass into the cell (low angled light and
side scatter light) and as slide touches the cell it
will then be scattered. Scattered light measured by
photo detector. Photo detector will tell you the
size and internal complexity of cell.
o o
Low Angle Light (0 -2 )
o 0 o
i. Forward low-angle light scatter (0 to 2 / or 2 to
0
3)
 measures size or volume of the cell
0
ii. Orthogonal light scatter/Side scatter light (90 )
 Measures internal characteristics of the cell
or internal complexity of cells (nucleus and
cytoplasmic granules of the cell)
 Most important in WBC
0 0
iii. Forward high-angle 5 to 15
Manual Versus Automation
Limitations of manual cell counting
 Experience is needed to make technically adequate
smears consistently
 Non-uniform distribution of WBCs over the smear.
 Non-uniform distribution of red blood cells as well.
 It is subjective, labor-intensive, and statistically
unreliable
 It is imprecise.
 Cell identification errors in manual counting.
Advantages of the automated analyzers
 rapid ,objective, statistically significant
 not subject to the distributional bias of the manual
count.
 more efficient and cost effective
 The precision of the automated differential makes the
absolute leukocyte counts reliable and reproducible.
Disadvantages of the automated analyzers
 produce cell counts which are falsely increased or
decreased.
 Some analyzers check only the volume and number of
particles .
 Platelet clumps may be misclassified as leukocytes or
erythrocytes, and nucleated red blood cells can be
misclassified as leukocytes or, specifically, lymphocytes.
CBC Quality Control
 To make sure that the result from automated machine
are accurate and precise, we need to run control
testing.
 You don’t run controls to obtain normal values. You will
run control to know whether machines or reagents are
in good condition.
Commercial Controls:
 3 levels (low, normal, high)
 Normal control = the results should be normal
 Low control = the results should be low
 High control = the results should be high
HEMA1 LAB (PRELIM PRACTICALS)
 Values stored in instrument computer
 Levey-Jennings graph generated and stored for each
parameter
Mode to Mode QC:
 Most automated hematology instruments have a
primary and secondary mode of sample aspiration.
Controls must be run on BOTH and correlate.
 Primary = Automated or Closed = usually
employed in large automated analyzers of hema
 No need to remove the cover of tubes, you let
the machine aspirate the blood for you
 Secondary = Manual or Open
 Usually on back up machine
 You need to remove the cap of the tube
 Manually you will direct the tube near the
aspirator and let the machine aspirate the
blood
i. Routine machine : can run multiple samples at a
single time
*2 automated analyzers for CBC per hospital
ii. Back-up machine : Can run single sample at a given
time
Delta Checks
 When the Laboratory Information System (LIS) and the
instrument are interfaced (connected) delta checks are
conducted by the LIS on select parameters.
 compare results today from previous testing.
 To make sure what could be possible sources of errors
or there is any trends in QC.
 Autoloader Whole Blood =
 Closed Whole Blood = there is a random access testing
for STAT blood specimens (you can stop the machine
and give way for them)
 Closed pre diluted = test smaller amount of bloo (30-33
uL of blood), nut lesser amount for newborns (up to 20
uL of blood will do)
Throughput
 the performance of the analyzer
 How many of specimen will be tested in a given period
of time (per hour)
Since multiple specimens passes through the analyzers, there
should be cleansing.
 In every specimen tested, there is a cleansing solution
that passes through a tube.
 If you turn off the machine, you need to clean the entire
machine to make sure that there would be no
unwantted left overs in tubing.
Flow cytometry = to make sure cells are piling up.
Barcoding system = allows you to directly transmit data to
HIS system
In histogram, we can check the volume and number of cells.
Different Brands of Automated Machines (common):
MINDRAY BC-5390
 Impedance method for WBC/BAS, RBC and PLT counting
 Cyanide free reagent for hemoglobin test
 Flow Cytometry (FCM) + Laser scatter + Chemical dye
method for WBC differential analysis
SYSMEX
 Your machine can decide whether you need or prepare
or examine a blood smear or not based on differential
count at the same time you have your own power to
decide also.
SUMMARIZING COMPLETE BLOOD COUNT
 To this point, this chapter has focused on slide
preparation and performance of a differential cell count.
 The differential is only the capstone, however, of a
panel of tests collectively called the complete blood
count, or CBC, that includes many of the routine tests.
 Now that the testing for the component parts has been
described, interpretation of the results for the total
panel can be discussed.
 The CBC has evolved over time to the typical test panel
reported today, including assessment of WBCs, RBCs,
and platelets.
 The CBC provides information about the
hematopoietic system, but because abnormalities
of blood cells can be caused by diseases of other
organ systems, the CBC also plays a role in
screening of those organs for disease.
 The CBC provides such valuable information about
a patient’s health status that it is among the most
frequently ordered laboratory tests performed by
medical laboratory scientists and laboratory
technicians.
 The process of interpreting the CBC test results has two
phases.
 In phase 1, the numbers and descriptions
generated by the testing are summarized using
appropriate terminology.
 This summary provides a verbal picture of the
numbers that is easy to communicate to the
HEMA1 LAB (PRELIM PRACTICALS)
physician, other health care provider, or another
laboratorian.
 It is much more convenient to be able to say, “The
patient has a microcytic anemia” than to say, “The
hemoglobin was low, and the mean cell volume
was also low.”
 Phase 2 of interpretation is to recognize a pattern
of results consistent with various diseases and to
be able to narrow the diagnosis for the given
patient or perhaps even to pinpoint it so that
appropriate follow-up testing or treatment can be
HCT Parameter
recommended.
Interfering substances and Implications
--just for additional notes and information--
CBC PARAMETERS

MCV Parameter
Interfering substances and Implications

Hematocrit, MCH, and MCHC = are computed values

WBC Parameter
Interfering substances and Implications

RDW Parameter
Interfering substances and Implications

Nucleated RBCs = Falsely elevated

RBC Parameter
Interfering substances and Implications
Plt Parameter
Interfering substances and Implications

HGB Parameter
Interfering substances and Implications
HEMA1 LAB (PRELIM PRACTICALS)

WBC Differential Parameters

Clinical Implications in Adults

Use of a systematic approach to CBC interpretation ensures

that all valuable information is assessed and nothing is
overlooked.
HEMA1 LAB (PRELIM PRACTICALS)

FOMITES INDIRECT HAZARD

HEMA1 LAB (PRELIM PRACTICALS)

ALDEHYDE
CHLORINE
PHENOLIC COMPOUNDS

EXTREME RISK
HEMA1 LAB (PRELIM PRACTICALS)

ELECTRICAL HAZARD

SPECIMEN EXPOSURE
HEMA1 LAB (PRELIM PRACTICALS)

HEMOCONCENTRATION/hemolysis GLUCOSE/PHOSPHORUS
HEMA1 LAB (PRELIM PRACTICALS)
HEMA1 LAB (PRELIM PRACTICALS)
HEMA1 LAB (PRELIM PRACTICALS)

FISTULA FINGER
HEMA1 LAB (PRELIM PRACTICALS)
HEMA1 LAB (PRELIM PRACTICALS)
HEMA1 LAB (PRELIM PRACTICALS)
HEMA1 LAB (PRELIM PRACTICALS)
HEMA1 LAB (PRELIM PRACTICALS)
HEMA1 LAB (PRELIM PRACTICALS)
HEMA1 LAB (PRELIM PRACTICALS)
HEMA1 LAB (PRELIM PRACTICALS)
HEMA1 LAB (PRELIM PRACTICALS)

7.5
HEMA1 LAB (PRELIM PRACTICALS)
HEMA1 LAB (PRELIM PRACTICALS)

\
HEMA1 LAB (PRELIM PRACTICALS)

I, II, IV
HEMA1 LAB (PRELIM PRACTICALS)

PACKED CELLS
HEMA1 LAB (PRELIM PRACTICALS)

I, III, IV
HEMA1 LAB (PRELIM PRACTICALS)
HEMA1 LAB (PRELIM PRACTICALS)

1.88%

88.89fL 1
HEMA1 LAB (PRELIM PRACTICALS)
HEMA1 LAB (PRELIM PRACTICALS)

II,III,IV

IV
HEMA1 LAB (PRELIM PRACTICALS)

12
0.27x10 /L 33.75 = hematocrit
1.5 days

all
HEMA1 LAB (PRELIM PRACTICALS)

11 11.90

true hypotonic
HEMA1 LAB (PRELIM PRACTICALS)

8 ABC

hcl 4
HEMA1 LAB (PRELIM PRACTICALS)

1:20 BETWEEN THE THICK AND THIN

EDTA A, B
HEMA1 LAB (PRELIM PRACTICALS)

100 none

200 40x
HEMA1 LAB (PRELIM PRACTICALS)

30.375 monocyte

50
 OLFU WHITE BLOOD CELL COUNT
2021 – 2022
LAB 8
RMT 2023
CLINICAL HEMATOLOGY 1
1st Semester
Instructor: Prof. Antonio C. Pascua, Jr., RMT, MSMT
Date: December 7 , 2021 TRANS 8 HEMA311
LAB

Outline **Thoma pipette is also essential in manual cell counting procedures to

At the end of the session, the student must be able to learn: accurately dilute blood specimens with fluids needed for specific cell
A. WHITE BLOOD CELL COUNT counting procedures. A white cell pipet is the thoma pipet used in white
B. MATERIALS NEEDED (MANUAL) cell counting. A red cell pipet can contain 101units of volume whereas
a. Hemacytometer white cell pipet can contain 11units.
b. 1-3% Acetic Acid
c. 1 % HCL
d. Turk’s diluting fluid
C. PROCEDURE
D. POST LABORATORY
E. SOURCES OF ERRORS

I. WHTE BLOOD CELL

● The number of WBC’s in 1 liter or 1microliter of blood

(regardless if granulated or agranulated) WBC count
accounts for all the white cells. In manual counting it is
hard to identify which is which.
● Utilizes to indicate infection, during infection physiologically
normal na yung wbc count is increased good sign since your
body is helping you to eliminate infections.
● In manual white blood cell counting, you have to know the
terms, leukocytosis and leukopenia, which is the elevated
number of WBC above the normal range and the reduced
number of WBC, respectively. People with leukopenia has
higher risk of infection,.

II. MATERIALS NEEDED (MANUAL)

**COMPLICATED AND SUBJECTIV
Hemacytometer
Thoma Pipet
Suction device Primary square: 2
Thick coverslip Each primary square has 9 SECONDARY SQUARE
Cell Counter The four corners of the secondary square is for WBC
Diluting fluids WBC secondary square has 16 TERTIARY SQUARE.
For RBC in the center has 25 TERTIARY SQUARE
1. HEMACYTOMETER Each 25 TERTIARY SQUARE has 16 QUATERNARY SQUARE.
More convenient, accurate, organize **each secondary square has 1mm x 1mm ➔ Total of 9mm2
•“Heart of manual cell count”
•Different Types
- Improved Neubauer 2. WBC DILUTING FLUID

- Neubauer • 1-3% ACETIC ACID

- Fuchs –Rosenthal
• 1% HCl

- Speirs–Levy -Tuerk’s
• TURK’S DILUTING FLUID IS MADE UP OF:
- Acetic acid
- Bass –Jones
- Distilled water
** The most commonly used counting chamber is the Levy chamber with
improved Neubauer ruling. - Gentian Violet Stain
***ISOTONIC solution (RBC)
***HYPOTONIC (WBC) red cells must be lysed

III. PROCEDURE

Largueza, J.M -- TRANSCRIBER

[HEMA311]3.01 White Blood Cell Count Prof. Antonio C. Pascua, Jr., RMT, MSMT

***use of Turk’s Diluting Fluid

You may use this as a supplement reference in the WHITE BLOOD CELL
COUNT Procedure

https://fankhauserblog.wordpress.com/1980/02/01/white-blood-cell-count/

V. SOURCES OF ERRORS AND COMMENTS

USE L RULE, count all WBC’s inside the 4 secondary squares
Dust and fingertips may cause difficulty in
distinguishing the cells
Diluting fluid should be free ofcontaminants
Of the count is low, a greater area may be counted
Chamber must be charged properlyto ensure accurate
count
Allow cell to settle for 10 mins before counting
***The accuracy of the manual WBC count can be assessed by
performing a WBC estimate on a Wright-stained peripheral blood film
made from the same specimen

N-RBC’s

Falsely counted as WBC

Not lysed by WBC diluting fluids

USE HPO to confirm the WBC Correct the WBC Count by dividing uncorrected WBC x100 by the
number of nRBC per 100 WBC + 100
IV. POST LABORATORY
VI. NORMAL VALUES

•4.0-11.0 x 109/L
•at birth: 10.0-30.0 x 109/L

EXAMPLE

Multiple to 0.001 to convert to the SI Unit

Largueza, J.M -- TRANSCRIBER

[HEMA311]3.01 White Blood Cell Count Prof. Antonio C. Pascua, Jr., RMT, MSMT

Leukocytosis Leukopenia

● Bacterial ● Viral infections(HIV)

infection ● Brucellosis
● Appendicitis ● Typhoid fever
● Leukemia ● Rheumatoid
● Pregnancy arthritis
● Uremia ● Cirrhosis
● Ulcers ● Typhoid and
paratyphoid
** in most cases sa infection, inc ● Anemia:
ang wbc.
- Aplastic anemia
● Exudates: - Megaloblastic
anemia
- Empyema /
pyothorax or ● Hypersplenism
purulent pleuritis ● Drugs:
- Tuberculosis - cytotoxic drugs
- Malignant ● Radiation
neoplasms ● Rarely
- Lymphoma leukemia
- Mesothelioma ● transudates:
- Pancreatitis - Congestive
heart failure
- Rheumatoid
arthritis - Cirrhosis with
ascites

ADDITIONAL NOTES

Whole blood anticoagulated with

ethylenediaminetetraacetic acid (EDTA) or blood from a
skin puncture is diluted with 1% bufferedammonium
oxalate or a weak acid solution (3% acetic acid or 1%
hydrochloric acid).

Alternately, a 1:100 dilution may be used counting the

number of cells inthe entire counting area (9 large squares,
9 mm2) on both sides of the chamber (Table 14-1). As an
example, if an average of 54 cells were counted in the
entire counting area on both sides of the chamber
- Cell count:
** 1% ammonium oxalate. Df is 1:20,area counted is
4mm2
** 1% ammonium oxalate. Df is1:100, area counted is
9mm2

- Extremely elevated total leukocyte(WBC) counts of 50.0 ×

109 /L or higher are consistent with a diagnosis of
empyema.

- In general, WBC counts less than

1.0 × 109 /L are associated withtransudates, and WBC
counts greater than 1,000 × 109 /L are associated with
exudates.

Largueza, J.M -- TRANSCRIBER

 OLFU WHITE BLOOD CELL DIFFERENTIAL
2021 – 2022
COUNT LAB 9
RMT 2023 CLINICAL HEMATOLOGY 1
1st Semester

Instructor: Prof. Antonio C. Pascua, Jr., RMT, MSMT

TRANS 9 HEMA311
LAB
Date: December 14 , 2021

Outline
At the end of the session, the student must be able to learn:
A. LEUKOCYTE DIFFERENTIAL COUNT 7. After staining, rinse the smear with neutral pH distilled
B. MATERIALS water
C. PROCEDURE IN SMEAR PREPARATION 8. Wipe the back of the slide. Air dry the slides
D. STAINING PROBLEMS
E. DIFFERENTIAL COUNT 9. As drying the stained smear, make sure it is in vertical
F. ABSOLUTE COUNT position

STAINING OF SMEARS

 Before staining, make sure that the slide is dry

A. LEUKOCYTE DIFFERENTIAL COUNT  Stains:
o Pure Wright Stain
• The white blood cell differential count determines the o Wright-Giemsa stain
number of each type of white blood cell, present in the
blood.  Purpose: make the cells more visible and to allow their
morphology tobe evaluated
USES OF BLOOD SMEAR EXAMINATION
 Fixative: Methanol
 pH: 6.4 – 6.8; staining is always pH dependent
 Used for WBC Differential Count
WEDGE TECHNIQUE: easiest way
 Can also count WBC but for estimation only
Too much blood may result to thick smear
 Platelet estimation >3 mm in diameter: thick smear : falsely elevated
Estimate means inaccurate value.

EDTA: specimen of choice because it preserves the cell THICK THIN

morphology
OTHER SPECIMEN: skin puncture is also applicable but for small Pressure Decrease increase
amounts of blood only.
FRESHLY COLLECTED BLOOD: within 2 hours, smears must be Angle (30 - 45 increase decrease
prepared. degree)

PLATELET SATELLITISM: platelets adhere at the surface of Speed Increase (short and decrease
neutrophils due to the used of EDTA and the antibodies may result dark red)
in adhesion
CITRATE: specimen of choice for platelet counting. Specimen >3mm increase decrease
Only used citrate if there is presence of satellitism
If there is presence of increase in Hct : thin smear should be
prepared
B. MATERIALS THICK AREA: cells are clumping
THIN AREA: cells are deteriorated
• Cell counter THICK TO THIN AREA SMEAR TRANSITION: cells are equally
• Glass slides distributed
• Microscope As much as possible do not allow the end of the blood touch
• Cedar wood oil the sides of the slide
• Spreader slides
• Wright’s stain

C. PROCEDURE IN SMEAR PREPARATION

STEPS IN PREPARING SMEAR
1. After preparing the smear, fix it with methanol for 2-3
minutes
2. Allow to dry
3. Proceed with staining, flood the smear with Wright or
Wright-Giemsastain for atleast 3 minutes
4. After 3 minutes, add the buffer.
5. Allow to buffer to stay for around 3 minutes
6. Mixing of the buffer and stain will produce green-metallic
sheen

Acuña J, Largueza, J.M -- TRANSCRIBER

 D. STAINING PROBLEMS
TOO ACIDIC STAIN TOO ALKALINE STAIN
1. Thin blood smear 1. Thick blood smear
2. Insufficient staining time 2. Prolonged staining
3. Prolonged buffering or 3. Insufficient washing
washing 4. Alkaline
4. Old stain pH of
stain
compone
nts

MACROSCOPIC EXAMINATION

HEPARINIZED BLOOD SAMPLE: imparts bluish discoloration in

smear background
GRAINY: RBC Agglutination
: Cold agglutinin: Anti-I Antibody
Figure 1-3: Examples of the Blood Smear
MICROSCOPIC EXAMINATION

Thick smear: overlapping of cells

High Power Objective (HPO) Field: specific area
: 10 HPO (white cells count estimation)

Computation
Average count cells x 2,000
SI unit : multiply to 0.001 x10^9/L

Oil Immersion Objective: used for white cells differential counting

: Platelet estimate: 10 OIO field

Figure 4: Observing direction of the blood smear. Computation

Average count x 20,000
BLOOD SMEARS UNDER MICROSCOPE Cells/uL x0.001 x10^9/L
Minimum white cell count must be 100 but due to some
condition and hindi naabot yung 100, you can lower it down to
50
Battlement technique : zigzag like : performed it on the part where
cells are equally distributed

E. DIFFERENTIAL COUNT

 One hundred WBCs are counted and classified through

FEATURES OF A WELL-MADE WEDGE PERIPHERAL BLOOD
FILM
1. The film is two thirds to three fourths the length of the slide
2. The film is finger shaped, very slightly rounded at the
feather edge, notbullet shaped; this provides the widest
area for examination
3. The lateral edges of the film are visible
4. The film is smooth without irregularities, holes or streaks
5. When the slide is held up to the light, the thin portion
(feather edge) ofthe film has a “rainbow” appearance.
6. The whole drop of blood is picked up and spread.
7. There is a transition from thick to thin portion (area
where cells areequally distributed
the use of push-down button counters
 Results are reported as percentage
 Principle: a stained smear is examined to determine the
percentage of each type of leukocyte present and assess
the erythrocyte and plateletmorphology.
 Manual : 100 WBC
 The more white cells counted, the more cells to be
differentiated
 Eosinopenia and Basopenia: technically they don’t
exist because basophil and eosinophil are normal in
low count
 MANNER OF REPORTING : % (percent) : relative WBC
differential count

Acuña J, Largueza, J.M -- TRANSCRIBER

[HEMA311]3.02 White Blood Cell Count Differential Count Prof. Antonio C. Pascua, Jr., RMT, MSMT

Figure 5: Comparison of Normal WBC in the blood

F. ABSOLUTE COUNT

Computation
% relative count X WBC count

Unit: x10^9/L

**** Sum of absolute count must be equal to WBC

count
Scanner and LPO: coarse adjustment
OIO and HPO: fine adjustment

Addition of oil must be in between OIO and HPO

objective (no need to bring down the film/stage

EXAMPLE ABSOLUTE BLOOD COUNT

 WBC absolute count:
o WBC count = 6 x 109/L
o Multiply the relative count with the
WBC count:
o Neutrophil: 50% x 6 = 3x109/L
o Lymphocyte: 34% x 6 = 2.04x109/L
o Monocyte: 10% x 6 = 0.6x109/L
o Eosinophil: 5% x 6 = 0.3x109/L
o Basophil: 1% x 6 = 0.06x109/L
 The sum of the values should and must be
equal to the value of the WBC count

NOTE: The Trans might be different from the

discussion of Sir Anton since no handouts was
uploaded in the Canvas. Reading the book or other
reference is highly recommended. Thank You and
Happy Holidays

Largueza, J.M -- TRANSCRIBER

Hematology Laboratory
Reticulocyte Count and Red Cell Indices

acpj
Reticulocytes
• The last immature red blood cell stage

• Contain remnant cytoplasmic ribonucleic acid (RNA) and organelles

such as the mitochondria and ribosomes

• “polychromatic erythrocytes” –increased retics in the blood

• Indicate the ability of the bone marrow to increase RBC production in

anemia due to blood loss or excessive RBC destruction
Reticulocyte Count
• An indicator of the rate of erythrocyte production

• Use of supravital stains: stains the living cell, used for the visualization of
the inner parts of the cell

• The count is expressed as a percentage of total erythrocytes

Principle
• Whole blood, anticoagulated with EDTA, is stained with a supravital
stain

• Any non-nucleated red blood cell that contains two or more particles
of blue- stained granulofilamentous material after new methylene
blue staining is defined/counted as a reticulocyte
Rodak’s Hematology 5th Edition
 MATERIALS

• SLIDES
• ANTICOAGULATED BLOOD
• SUPRAVITAL STAIN:
• Brilliant Cresyl Blue
• New Methylene Blue
• PIPETTE
• MICROSCOPE
• CEDARWOOD OIL
PROCEDURE
1. Place three drops of reticulocytes stain in a small
test tube.
2. Add three drops of well mixed whole blood to the
tube containing the stain. Add 3 drops of stain Add 3 drops of blood

3. Mix the tube and allow to stand at room

temperature or incubate at 37C for 15 minutes Mix the tube and
4. After 15 minutes, mix the contents of the tube well incubate for 15 minutes.
and prepare smears and allow it to dry.
5. Examine the smear under the oil immersion
objective.
6. Count the total number of reticulocytes found per
1,000 RBCs Read in oil immersion objective
Pipette small amount of blood
once dried and count the mixed with stain and make a
reticulocyte present smear and allow to dry
PROCEDURE
Example: There are 15 reticulocytes counted in 8 months old infant.
Retics count (%) = 15 reticulocytes / 1,000 = 0.015 x 100 = 1.5%
Interpretation: NORMAL

Reference range
• Adult: 0.5-1.5%
• Newborn: 2.0-6.0%
Miller Disc
• Designed to reduce the labor-intensive process of counting
reticulocytes

• Disc is inserted into the eyepiece of the microscope

• Minimum of 112 cells should be counted in the small square

Rodak’s Hematology 5th Edition
Sources of Errors
1. Blood and stain are not mixed before the films are made.
2. Very anemic or polycythemic patient
3. Moisture in the air, poor drying of the slide, or both
4. Other red blood cell inclusions that stain supravitally include
Heinz,Howell-Jolly, and Pappenheimer bodies
Absolute Reticulocyte Count (ARC)
• the actual number of reticulocytes in 1 liter (L) or 1 microliter (mL) of
blood
Corrected Reticulocyte Count (CRC)
• In specimens with a low hematocrit, the percentage of reticulocytes
may be falsely elevated because the whole blood contains fewer red
blood cells.

• A correction factor is used, with the average normal hematocrit

considered to be 45%.
 Rodak’s Hematology 5th Edition

Hematology by Turgeon 5th Edition

Reticulocyte Production Index (RPI)
• Measures erythropoietic activity when stress reticulocytes are
present

• What are “shift reticulocytes”? Retics were shifted prematurely from the
bone marrow. Happens in hemolytic anemia cases
Automated Reticulocyte Count

• Analyzers evaluate reticulocytes using optical scatter or fluorescence

after the red blood cells are treated with fluorescent dyes or nucleic
acid stains to stain residual RNA in the reticulocytes.
Red Blood Cell Indices
• Mean Cell Volume (MCV) –measured by a machine
• Mean Cell Hemoglobin (MCH) – has to be computed
• Mean Cell Hemoglobin Concentration (MCHC) - has to be computed
Mean Cell Volume
• Average volume of red blood cells expressed in femtoliters (fL)

• NV: 80-100 fL, greater than 80 microcytic RBC, less than 100 macrocytic
RBC
Mean Cell Hemoglobin (MCH)
• Average weight of hemoglobin in a red blood cell expressed in
picograms (pg)

• NV: 26-32 pg
Mean Cell Hemoglobin Concentration (MCHC)
• Average concentration of hemoglobin in each individual red blood cell
expressed in grams per deciliter (g/dL)

• NV: 32-36 g/dL greater than 32 hypochromic

Uses the Rule of 3 , but only in cases of RBC being normocytic (normal
sizes), normochromic (normal color)
References
• Rodak’s Hematology: Clinical Principles and Applications, 5th Edition
• Clinical Hematology: Theory and Procedures, 5th Edition, Turgeon,
M.L.
Thank you…
Hematology

acpj
Complete Blood Count
• Most frequent Blood test
• Provides a detailed information about the cells in the blood
• Screening test for most diseases
• Aka
• Full Blood Count
• Full Blood Exam
• Sample:
Complete Blood Count
• RBC count
• Hemoglobin
• Hematocrit
• WBC count
• WBC Differential
• Platelet
• Blood Indices
Methodology
• Current hematology analyzers use a combination of
light scatter, electrical impedance, fluorescence, light
absorption, and electrical conductivity methods to
produce complete red blood cell, platelet, and
leukocyte analyses. All the widely used automated
instruments analyze cells in flow and are essentially
highly specialized flow cytometers.
Automation
• Two General Principles

• Electronic resistance ( impedance) : single & multiple

parameter

• Light scattering: multiple parameter

Electronic Impedance
• Utilizes non-conductive properties of blood cells
• as blood cell passes through orifice of aperture it
displaces its own volume
• RBCs and Platelets counted together, separated by
pulse heights
• Hydrodynamic focusing forces cells to pass single
file through sensing zone

Image source: Rodaks Hematology 6th Edition

Image source: Rodaks Hematology 6th Edition
Optical Scatter
• A hydrodynamically focused sample stream is directed through a
quartz flow cell past a focused light source
• Cells counted as passed through focused beam of light (LASER)
• Sum of diffraction, refraction and reflection
• Multi angle polarized scatter separation (M.A.P.S.S)
• Forward-angle light scatter ( 00 )
• Forward low-angle light scatter ( 20 to 30).
• Forward high-angle 50 to 150.
• Orthogonal light scatter (900)
Image source: Rodaks Hematology 6th Edition
Radiofrequency
• Used in conjunction with electrical impedance
• Cell interior density is proportional to pulse height or change in the RF
signal

Image source: Rodaks Hematology 6th Edition

Manual Versus Automation

Limitations of manual cell counting

Ø Experience is needed to make technically
adequate smears consistently
Ø Non-uniform distribution of WBCs over the smear.
Ø Non-uniform distribution of red blood cells as well.
Ø It is subjective, labor-intensive, and statistically unreliable
Ø It is imprecise
Ø Cell identification errors in manual counting
Advantages of the automated analyzers
Ørapid ,objective, statistically significant
Ønot subject to the distributional bias of the manual count.
Ømore efficient and cost effective
Ø The precision of the automated differential makes the absolute
leukocyte counts reliable and reproducible.
CBC Quality Control
• Commercial Controls:
• 3 levels (low, normal, high)
• Values stored in instrument computer
• Levey-Jennings graph generated and stored for each parameter
• Mode to Mode QC:
• Most automated hematology instruments have a primary and
secondary mode of sample aspiration. Controls must be run on
BOTH and correlate.
• Primary=Automated or Closed
• Secondary=Manual or Open
• Delta Checks
• When the Laboratory Information System (LIS) and the instrument
are interfaced (connected) delta checks are conducted by the LIS
on select parameters.
References
• Rodak’s Hematology: Clinical Principles and Applications, 6th Edition
• Clinical Hematology: Theory and Procedures, 5th Edition, Turgeon,
M.L.
Thank you…