Physio A _ Lab Pracs Lontabo, Jasmine A.

| 1D
HEMATOLOGY

Hematology

RBC Profile | Immunohematology | Coagulation Profile | WBC Profile

RBC PROFILE

RBC Count
Hemoglobin
Hematocrit
Red Cell Indices
Osmotic fragility test
Reticulocyte count
Erythrocyte Sedimentation

EDTA (ethylenediaminetetraacetic acid) | Tally Counter | Haemocytometer | RBC pipet

Notes

Draw blood first to 0.5 mark

Wipe off the blood with tissue paper
Draw diluting fluid to 101 mark
Count cells within medium squares

RBC ct- # of cells x area factor (5) x depth Reference Values: Male: 5.5-6.5 x 1012/ L
factor (10) x dilution factor (200) Female: 4.5-5.5 x 1012/ L

1|Page
Physio A _ Lab Pracs Lontabo, Jasmine A. | 1D
HEMATOLOGY

Hemoglobin Determination
SAHLI HELLIGE METHOD

N HCL
Distilled Water

Compara or block Sahli andard

Stand for 10
minutes! Reference Value: Male: 14-16g/dL
Female: 12-14 g/dL

MICROHEMATOCRIT

2|Page
Physio A _ Lab Pracs Lontabo, Jasmine A. | 1D
HEMATOLOGY

Microhematocrit visual estimation of volume of white cells and platelets that

constitute the buffy coat.

Sample- Capillary blood/ Venous blood comcentration EDTA capillet tubes (red
capillary; blue- venous)
Clay paraffin wax
¾ full
Centrifuge for 5 mins at 10,000 rpm

Reference Values: Male: 0.42-0.52 (42-52%)

Female: 0.37-0.47 (37-47%)

3|Page
 Physio A _ Lab Pracs Lontabo, Jasmine A. | 1D
HEMATOLOGY

ESR venous blood concentration oxalate or EDTA

Method: WINTROBE method

Spx: Venous blood with oxalate (EDTA may be used)

P i ci le Abili f RBC agg ega e a d ea ed b he a e f fall ed bl d cell i a

c l i hi a ecified e i d f i e

This picture shows a rack holding
Wintrobe tubes, in which
anticoagulated whole blood has
just been added.
Wintrobe method:
The Wintrobe method is performed similarly except that the Wintrobe
tube is smaller in diameter than the Westergren tube and only 100
mm long. EDTA anticoagulated blood without extra diluent is drawn
into the tube, and the rate of fall of red blood cells is measured in
millimeters after 1 hour. The shorter column makes this method less
sensitive than the Westergren method because the maximal possible
abnormal value is lower. However, this method is more practical for
demonstration purposes.

Reticulocyte count
Youngest form of erythrocyte that can be seen
in peripheral blood

4|Page
Physio A _ Lab Pracs Lontabo, Jasmine A. | 1D
HEMATOLOGY

Wha f Deg ee f e ic l c i a i a e he RATE OF RBC

d c i i he b e a
Supravital stain: Brilliant Cresyl Blue/ New methylene blue
RNA appears dark blue granules arranged in a network.
Oil immersion
Known Cell Preparations
Cross Matching Set up
Osmotic Fragility Set-Up helpful in determining cause of anemia
Oil Immersion for Differential Count
Clorox- disinfection of surfaces and/or hazardous waste (NAOCl/ Sodium
hypochlorite)
Puncture- resistant hazardous waste container for sharps, color is yellow, signifies
hazardous clinical waste.
Osmotic fragility test helpful in determining cause of anemia
Reticulocyte count but is not helpful in determining etiology of anemia
ESR venous blood conc. EDTA

Black ESR Anticoagulant Na Citrate (1-4)

Blue: coagulation determination (PT & PTT)

Red:Blood chemicals (BUN CREA, lipid test, b1 &b2) Anticoagulant: none

Violet: CBC Anticoagulant: EDTA

Gray: Glucose determination ; Anticoagulant: Na Fluoride & K Oxalate

Green: Arterial Blood Gas (ABG) Anticoagulant: Heparin

Yellow: Blood culture

Needle of vacutainer
o Other end of needle is sealed to avoid contamination
Blood lancet:
o Used tp prick
o Capillary blood samples
o Puncture site: mid or ring finger, earlobe (adult) heel (infant)
Hemocytometer (Neubauer)
o Cover slip
o Depth : 0.1mm

5|Page
Physio A _ Lab Pracs Lontabo, Jasmine A. | 1D
HEMATOLOGY

o Counting chamber at middle

Eosinophil: Parasitic infection

Neutrophil: Bacterial
Leukocytes: viral

Osmotic Fragility Test

Assesses red cell membrane function

Hypertonic: Crenation
Hypotonic: Hemolysis
Varying NaCl: from 0.9%- 0.0%
Venous blood concentration EDTA
RBC membrane is fragile common concentration chronic anemia

IMMUNOHEMATOLOGY

Forward/ Cell typing - Ideally: 3-5% RBC suspension by diluting

blood in EDTA conc. saline
For screening: blood from capillary
Plasma or serum
a. Forward (using known anti-sera) colored anti-sera ANTIGEN

6|Page
 Physio A _ Lab Pracs Lontabo, Jasmine A. | 1D
HEMATOLOGY

b.Forward Typing (using KNOWN

SERUM) ANTIGEN

c. Reverse (Serum typing) (Using KNOWN RED CELLS/

INDIRECT) ANTIBODY | Plasma or Serum

CROSSMATCHING

P e PS
P -5% RC suspension (PC)
D -5% RC suspension
(DC)

PS- Patient Serum/ plasma DS- Donor Serum/plasma

DC- Donor red cells PS- Patient red cells
G
Grading in Gel Tube Method

7|Page
Physio A _ Lab Pracs Lontabo, Jasmine A. | 1D
HEMATOLOGY

COAGULATION PROFILE:
Bleedi g Ti e D ke
Clotting Time (Slide method)
Clot Retraction Time
Prothrombin Time
Activate Partial Thromboplastin Time

I. Bleedi g Ti e DUKE S METHOD capillary blood direct from puncture)

a. uses filter paper
b. 1-21 mins
c. Prick finger
d. End point: No more worked of blood
Note: Kapag nakakita ng BP CUFF sa bleeding time, hindi yun Duke’s method, Ivy’s method
(inflate BP cuff hanggang 40 mmHG) yun.

e. I Me h d
Different site of puncture
BP Cuff: inflate to 40mmHg
Clotting time (Slide method) - capillary blood direct from puncture)
o The surface of the glass tube initiates the clotting process. This test is
sensitive to the factors involved in the intrinsic pathway.
o The expected range for clotting time is 4-10min.
o (+) Result: FIBRIN CLOT Formation
o End point: Fibrin Strand
f. Clot Retration- 5ml whole blood red top blue
Activation of platelets, fibrinogen, and surface factors
Wire loop is placed in center of the tube and incubated for 24 hours.
% serum volume to the total blood volume is determined.
18-24 degree
To determine serum in blood volume
End point: jelly-like tip that is retarted serum, no anticoagulant
PET PITT

Measures EXTRINSIC PATHWAY of Measures INTRINSIC PATHWAY of Coagulation

Coagulant Fac

Factors I, II, V, VII, & X PITT reagent: Phospholipid, Activator (silica,

celite, kaolin, ellargic acid) and Calcium
PT reagent: Thromboplastin *partial Factor III not present
and Calcium 25-30secs
12,11,9,8
TI: Citrated Plasma

8|Page
Physio A _ Lab Pracs Lontabo, Jasmine A. | 1D
HEMATOLOGY

5ml venous blood

concentration citrate

WBC
I. WBC count- signifies infection
II. Differential count
III. PBS (Peripheral blood smear)

I. WBC Count

Notes
Draw blood first to 0.5 mark
Wipe off the blood with tissue paper
Draw diluting fluid to 11 mark
Count cells within 4 large square
WBC ct- # of cells x dilution
(20) x depth (10)
Area (4)

II. Differential count

SCHILLING S COUNT

III. Peripheral Blood Smear

blood test that gives information about the number and shape of blood cells. It is
often done as part of or along with a complete blood count (CBC).

9|Page
 PHYSIOLOGY A
Hematology Pracs
Guide for Apparatuses / Materials Used
RBC COUNT – MANUAL METHOD

RBC Pipette

EDTA
RBC Tally Counter
HEMOGLOBIN DETERMINATION

Hemometer Set Sahli-Hellige Pipette

 MICROHEMATOCRIT

Capillet / Capillary Tubes Clay / Paraffin Wax

 MICROHEMATOCRIT

Microhematocrit Centrifuge Microhematocrit Reader

FORWARD OR CELL TYPING

Anti-Sera A, B, AB
 CROSSMATCH

Anti-Sera D Antihuman Globulin Serological Centrifuge

*For other anti-sera, refer to previous slide/page.*

WBC Count
• Sample: Capilliary blood or venous blood with EDTA
WBC pipette

Hemocytometer
Sucking apparatus

Acetic acid (HOAc)

Cover slip

Microscope \
How to count WBC: Count the number of cells found in all 16 medium squares
for each large corner square. recodr the number for each large square. Add the
four results

Compute the number of WBC using the formula:

average# of cells counted X dilution (20) X depth (10) / Area (4)

reference value
\
5-10x109/L

Procedure note:
There should be a difference greater than 10 cells in each of the 4 squares
counted
Blood Smear/Film and stain
Sample: Capilliary blood or
venous blood with EDTA
Wright stain

Staining jars
Applicator stick

Buffer solution
\

2 glass slides

Distilled water
Procedural notes;
Features of an ideal smear:
• occupies 3/4 of the length of the slide
• has a thick and thin area
• has a feathery edge
\

• no holes or gaps present

Features of an ideal stain
• is indigo in color
• Color is uniform
• no vacuoles in blood ells
• RBC are pink
• Nuclei of WBC are purple
\

• Eosinophilic granules are red orange

• has minimal precipitates
Differentiate count
Leukocytes vary in size shape of the nucleus and
character of granules in the cytoplasm. the proportion
of each leukocyte type is of diagnostic importance for
certain disease (Neutrophilia in bacterial infection
Eosinophilia in parasitic infection). A differential count
gives the percentage of the various types of WBC on
on a stained smear
stained smear

Cedar wood oil \

Schillings counter

microscope
U e he Schilling co n er. A ign each finger for a
particular WBC
index finger neutrophil
middle finger Lymphocyte
ring finger monocyte
little finger eosinophil

Identify the white blood cells using the following

\
identification chart.
neutrophil-3 lobes-Lilac granules
Lymphocyte-round-no granules
monocyte- brain shaped- large amount, greyish
Eosinophil-2lobes-red granules
Basophil- 2 lobes-dark red granules
band- ribbon shaped- Red/blue/Liliac granules
neutrophil-3 lobes-Lilac granules

Lymphocyte-round-no granules
monocyte- brain shaped- large amount, greyish

Eosinophil-2lobes-red granules

Basophil- 2 lobes-dark red granules

Reference range
neutophil 0.55-0.65
Eosinophil 0.03-0.05
Lymphocyte 0.25-0.35
MOnocyte 0.02-0.08
Basophil 0.00-0.01
Procedural notes
• Abnormal cells ( smudge cells, basket like cells,
nucleated RBC, Endothelial cells) should not be
included in the count. However their presence
should be noted.
• Atypical Lymphocytes are included in differential
\

count under lymphocytes but with note

• Inclusions must also be noted
• an absolute count may be derived by multiplying
the WBC count by the percentage of each cell
type seen.
Peripheral blood smear
A peripheral smear (PBS) provides a description
of the cells seen on a stained smear. The
reading is usually done by the clinical
pathologist or the hematologist
Blood smear/ stained slide

oil \

microscope
Determine the WBC estimate using the table
2-4 4-7 X10 raise to 9/L
4-6 7-10
6-10 - 10-13
10-20 13-18
\

procedural note:
if smear was made directly from a finger prick, platelets
assume irregular shape are clumped making ti difficult to
evaluate.
 –
Transcribed by: Hernandez, Meg / MED 1-E (Batch 2021)

- Complete Blood Count (CBC) is one of the most sensitive and frequently requested hematologic test.
o Performed during annual physical exam and routine admission laboratory work up.
o It is a routine examination that includes: (RHHWDP)
1. RBC Count 4. WBC Count
2. Hemoglobin 5. Differential Count
3. Hematocrit 6. Platelet estimation
- The cells that circulate the bloodstream are the RBCs, WBCs, and platelets.

RED BLOOD CELL PROFILE

- Consist of RBC Count, Hemoglobin, Hematocrit, and indices that are all part of the CBC.
- Tests that are helpful in determining anemia: reticulocyte count and osmotic fragility test.
Other tests: erythrocyte sedimentation rate (included in RBC profile, but is not helpful for determining anemia)
TEST MATERIAL
USED FOR PROCEDURE COMPUTATION REFERENCE VALUE
OR SAMPLE
Under LPO:
Find one central large square

25 medium squares

Under HPO: RBC Count (cu.Mm)

= (# of cells in med.
Ha em dil ing Female: 4.5 to 5.5
4 corners and 1 center of the Squares)(5)(10)(200)
fluid Male: 5.5 to 6.5
RBC Count Count the total number of medium squares
> Counting
(Manual Method) RBCs in the blood 5 area factor
chamber UNIT: x 1012/L
In each 5 med. squares 10 depth factor
> Tally counter
200 diution factor
16 smaller squares

Count cells that are lying on the

upper left lines

Ave. the cells in 5 medium squares

Add HCl in the tube

Add Blood 20mm using the Sahli-

Hellige pipette

Hemoglobin > Capillary blood HCl + Blood Female: 12 to 14

Convert to
determination (red tip) Male: 14 to 16
gm L e l
(Sahli Hellige > Hemometer set Add water until the hematin
mmol L
Method) > N HCl solution UNIT: g/dL

Matches the color standard in both

sides of the comparator block

Read on the gram scale of the tube

Capillet should be ¾ full

> Capillet tube

Visual estimation of the vol. Seal with clay then paraffin at the Female: 37 to 47
(red if capillary,
of WBC and platelets that periphery Male: 42 to 52
Microhematocrit blue if venous)
constitute the buffy coat b/w
> Clay/paraffin
plasma and RBC Balance with blank UNIT: % or decimal
wax

Centrifuge for 5mins at 10,000 rpm

RBC indices
- Indices are calculated from hematocrit, Hgb, and RBC count
- Used for the classification of anemia
- Composed of MCV, MCH, MCHC
Female and Male:
Describe the RBC size which
Mean Corpuscular Hema c i 82 to 92
can be: microcytic,
Volume (MCV) RBC c n
normocytic, and macrocytic
UNIT: femtoliters (fl)
 Female and Male:
Mean Corpuscular Approximate the Hgb gm 27 to 33
Hem gl bin
Hemoglobin content in RBC by describing dL
(MCH) the chromacity of the RBC RBC C n UNIT: picograms
(pg)
Mean Corpuscular Female and Male:
Degree of hemoglobinization gm
Hemoglobin Hem gl bin 32 to 38
And to describe chromacity dL
Concentration Hema c i
of RBC
(MCHC) UNIT: %
Erythrocyte Sedimentation Rate (Wintrobe Method)
- ability of the RBC to aggregate; Not diagnostic of any disease
- Measured by the rate of fall of RBC in a column within a specified period of time
- RBC e ling dependen on i abili o fo m rouleaux, which are clumps of RBCs joined by surface attraction only)
- Based on the fact that inflammatory and necrotic processes cause an alteration in the blood proteins, resulting in aggregation of RBC, which makes them heavier
and more likely to fall rapidly when placed in a special vertical test tube.
- fa e clo ing ESR
Erythrocyte To indicate that a disease
Sedimentation process is ongoing and must Female: 0-20
Venous blood
Rate be investigated. Male: 0-10
with oxalate or
(Wintrobe Useful in monitoring the
EDTA
Method) progression of inflammatory UNIT: mm/hr
diseases
Degree of reticulocytosis
Venous blood Adult: 0.5 to 1.5 %
Reticulocyte approximates the rate of RBC
with EDTA Or 50 x 109/L
production.
Initial hemolysis (1st
tube that has pink
supernatant): 0.42
Asses the RBC membrane to 0.44% of NaCl
function RBC are suspended in a series of
Osmotic fragility Venous blood
HYPERTONIC c ena ion tubes containing hypotonic Complete hemolysis
test with EDTA
HYPOTONIC ell solutions of NaCl (0.9 to 0.0%) (1st tube which there
hemolysis is red supernantant
and no cells at the
bottom): 0.32 to
0.34% of NaCl

MATERIAL
TEST USED FOR PRINCIPLE PROCEDURE INTERPRETATION
OR SAMPLE
Label slide with
pa ien name With agglutination = that is the blood type
SAMPLE: 3-5% RBC
Without agglutination = not the blood type
suspension by diluting
Capillary puncture
venous blood in EDTA
Example: A person had her blood type
This method uses with saline. But blood
2 drops of blood, checked, and in the cell typing. It showed (+)
Forward or Cell identify the ABO KNOWN ANTISERA from capillary
one inch apart agglutination for Anti-A and (-) for Anti-B. What
Typing antigen on the RBC to the ABO group puncture can be used
is her blood type?
to detect
+ Anti A on the 1st
MATERIAL: Anti-A
drop of blood Answer: She is Type A, because Anti-A contains
(blue) and Anti-B
antibody A, and her RBC contains antigen A.
(yellow)
+ Anti B on the 2nd In e ac ion of Ag A and Ab A aggl ina ion
drop of blood
With agglutination = not the blood type
Without agglutination = that is the blood type

Example: A person had her blood type

Tests for the SAMPLE: Plasma or
checked, and in the serum typing. It showed
presence of serum can be used. Label slides with
(+) agglutination for A cell and (-) for B cell.
naturally occurring However, serum is pa ien name
RBC with KNOWN What is her blood type?
Ab in a pe on preferred because it label A cell and B cell
Reverse (Serum) ABO Ag are mixed
blood against ABO. contains less
Typing pa ien e m Answer: She is Type B, because the unknown
interfering proteins. Place a drop of
to detect Ab serum contains Ab B, and her RBC contains
*should never be unknown serum
an igen B In e ac ion of Ag B and Ab B
performed in MATERIALS: Known A onto each slides
agglutination
neonates RBC and Known B RBC
NOTE: Hemolysis is also interpreted as (+). This
procedure should not be performed if sample
is already hemolysed.
 Performed prior to
blood transfusion. SAMPLE:
Pa ien e m PS
To know if the Pa ien o RBC
Preformed Ab
pa ien blood i suspension (PC)
Cross match present in the Major c o ma ch eac i e do no an f e
compatible with the Dono o RBC
pa ien e m 12 test tubes Mino c o ma ch eac i e an f e b
dono blood suspension (DC)
(ABO/Rh groups) will react with the with caution
dono RBC Ag
Detects Ab in MATERIALS: Anti A,
pa ien e m ha Anti B, Anti D, Known
will destroy the A, B, and O cells
dono blood

- Primary hemostasis: bleeding time and platelet count

- Secondary hemostasis: clotting time, prothrombin time, and partial prothrombin time

MATERIAL
TEST USED FOR PRINCIPLE PROCEDURE REFERENCE VALUE NOTES
OR SAMPLE
Capillary puncture

Do not wipe first

Upon puncture, drop
Capillary blood
capillaries bleed
from direct
until the defect is Record time of The series of blots
Bleeding time To check the function puncture
closed by puncture 1 to 4 minutes should gradually
D ke me hod and activity of platelets (earlobe,
vasoconstriction and decrease in size.
fingertips, and
aggregating Blot every 30 sec
sole of foot)
platelets
Until no blood is
seen on the filter
paper
Capillary puncture

Wipe first drop

Rarely requested and
Clotting time
has been replaced by PT
(Slide method)
and PTT
Platelets are best
counted with automated
hematology analyzer.
Manual counts are
extremely difficult an
Estimated Platelet
inaccurate. An indirect
Count
count using a stained
smear is a simple and
reliable way of
estimating platelet
number.
Clot Retraction
Time
Be careful not to let
Record appearance
the drop of blood
of second drop
The end point of dry out before the
Blood from
coagulation cascade formation of fibrin
capillary Run the tip of lancet 2 to 5 minutes
is the formation of strand.
puncture every 30 sec looking
the fibrin clot
for the formation of
View slide at eye
fibrin threads
level
Record time when
the fibrin threads
first appear
10 immersion fields
Count platelets, get Platelets cannot be
average and estimated if they are
Stained smear multiply by 20,000. in clumps and are
better visualized if
10 immersion fields staining is alkaline.
Count platelets, add
then place unit 109/L
Blood is placed in a
graduated cylinder
10 immersion fields 40 to 60%
Clot retraction
involves interaction 5ml whole
Wire loop is placed Red cell fall out is
of platelets, blood red top
in the center and usually <5% of the
fibrinogen, and blue
the tube is sealed original blood sample
surface factors.
volume
After 24 hours of
incubation
 PT or international
normalized ratio(INR) is a
measure of the extrinsic 5ml venous
PT 11 to 16 sec
Prothrombin Time pathway of coagulation blood with
INR 0.8 to 1.2
citrate
Measures factors
1 2 5 7 10
In order to activate
the intrinsic The time is
pathway, these are measured until a
aPTT is the performance
mixed in the sample: thrombus (clot)
indicator measuring the
Activated Partial phospholipid, an 5ml venous forms. The test is
efficacy of both the
Thromboplastin activator (silica, blood with 25 to 39 seconds termed partial due
intrinsic and the
Time celite, kaolin, ellagic citrate to the absence of
common coagulation
acid), and calcium tissue factor from
pathways
(to reverse the reaction
anticoagulant mixture.
effect).