February

9, 2016

GEN CAMATO

HEMATOLOGY 2 LABORATORY
HEMATOLOGY 2

DONATH LANDSTEINER COLD HEMOLYSIN TEST

Get sample from a patient in the control (the control
should come from an healthy individual without the
presence of cold hemolysin antibody)
Get 10 mL of blood from the patient and another 10
mL for the control
Each 10 mL should be divided into 2, to be
transferred in a 5mL tube

HAMS ACIDIFIED SERUM TEST

Pa#ent

Control

C1

C2

To evaluate for the presence of Paroxysmal

Nocturnal Hemoglobinuria
Procedure:

Prepare 2 test tube labeled Patient and

Control

0.5 mL of 0.2 N HCl (it should not hemolyse

RBC)

0.5 mL of Serum sample & drop of 50% RBC

suspension

Spin & observe for hemolysis: if (+) to

hemolysis then it is (+) to PNH

SUGAR WATER TEST

5mL

5mL

st

1 Set

5mL

5mL

nd

2 Set

P1 and C1
P2 and C2
Should incubate at Room
Incubate at Cold
temperature (19-22) aircon temperature (4) ref
* (both) Read for only 20 minutes
s After incubation we need to incubate both set at 37C
for 1 hour (Since our body is 37C so we need to look if there will be

RETICULOCYTE

Retics using Supravital stain.
It has Fine Reticulum of RNA

any changes or hemolysis of blood if it will return to normal blood

temperature)

s Centrifuge for 5 minutes

s Observe for the presence of Hemolysis
NOTES:
Observe presence of hemolysis in Serum
Remember! Either you will incubate it in Cold
temperature or Room temperature
If it returns to normal body temperature (37C) it should
be stable, no presence of hemolysis.
But in patient with Paroxysmal Cold Hemoglobinuria
(PCH) the antibody becomes reactive when it is
incubated at cold temp.
For example, (Patient with PCH) from our normal temp
then we go out our body changes its temperature thats
the time Hemolysis happens, so when you go inside to
urinate, you will have a tea colored urine sample
If Normal temp, you will not have a tea colored urine
PCH

Young form of RBC that matures within 2-4 days

It has reticulum network which are RNA and
protophorpyrin remnants
Normal Value: 0.5 1.5% in normal adult (20-60,000/mm )
Newborn: 2.5 6.5% *Since they are developing child
Normally their RBC, WBC & also Retics count are increased.

Physiologic increase of Retics:
1) Among new born or at birth
2) Menstruating women *To compensate for the loss of
3

3)

Screening test for PNH

If using this, you need a confirmatory test using a
Sucrose Hemolysis test
The difference from HAT is it uses 0.85 mL of
Sucrose
We still use serum sample & RBC suspension

whole blood due menstrual Period. If we are releasing a lot of blood,

our Bone marrow will compensate for that loss not only the Bone
Marrow but also the Kidneys since it is very sensitive to Hypoxia.
Therefore, releasing a lot of erythropoietin and then giving signal to
hasten in releasing the production and the maturation of RBC,
therefore, releasing of RBC not only mature but also the Retics,
specifically the Reticulocyte. Even Retics is not fully mature, it
should be increase released by the bone marrow just to compensate
for that loss of blood due to menstruation.

Among pregnant women (increase req.

because of the fetus) *because it increase in the

performance of the fetus with the blood. So in the Follicular aside

from the blood specifically Folic Acid

GEN CAMATO

HEMATOLOGY 2 LABORATORY
HEMATOLOGY 2

Abnormally increase retics

1) Patient with hemolytic anemia *Increase destruction
of RBC thereby, compensating for that loss of RBC (abnormal)
2) Lead poisoning *can be seen in patient suffering from
3)
4)

ERYTHROCYTE OSMOTIC FRAGILITY TEST (EOFT)

*Test used to differentiate Resistant cell (Sickle cell) from Fragile cell (Spherocytes)
* Uses Hypotonic NaCl solution in EOFT

Basophilic Stippling; this may lead to Pernicious Anemia in Adults

and the Pica eating disorder

Patient with Leukemia

Patient with malaria

Follows the law of Osmosis

Checking for the stability of the RBC
Isotonic solution: *Usually used

0.85 0.9 % NaCL (No change in the

morphology of RBC)
Hypotonic solution:

< 0.85% NaCl (swell)

Hypertonic solution:

> 0.85% NaCl (shrink)

Abnormally decrease retics

1) Idiopathic Aplastic Anemia brought a decrease
reticulocyte *In AA, ALL forms of blood cells are decrease
2) Acute Benzol poisoning causes suppression of
RBC production

A. Wet Method
1. New Methylene Blue Method Primary Use
2. Cook, Meyer, Tureen Method Brilliant Cresyl
Blue
3. Seivards method BCB *also categorized as Dry mtd

Methods of EOFT

B. Dry Method
1. Schillings rapid method BCB
2. Seiverds Method BCB
3. Osgood Wilhery Methylene Blue
4. Sabins Method Janus Green and Neutral
Red

1.

Sanford method

Principle: It test the stability of RBC under

different concentration of hypotonic NaCl
solution

Preparation: 12 test tube numbered with 25-14

Solution: 0.50 % NaCl

Specimen use: Fresh blood sample or

heparinized blood

*Dry method - drop reagent in slide (thin film of rgt in slide), then drop of blood, cover
slip. Stand for 10min. focus under Oil Immersion Objective. Dry because it is thin smear.

*Most commonly used is the Wet method specifically the New Methylene Blue Method

*Stand for 2 hours J

*Most commonly used Sanford method

Formula:

No.
of TT

#
% =


Normal value: 20 60 x 10

/mm3

%
=

Normal value: 24 84 x 10

24

23

22

21

20

19

18

17

16

15

14

D.
H2O

10

11

Bld.

0.36

0.34

0.32

0.30

0.28

Stand for 2 hours

Conc

/L

0.50

0.48

0.46

0.44

0.42

0.40

0.38

Interpretation of Results

*in 1 field there should be 250 RBCs; count usually in equal distribution. We dont need
to count 1 by one for rbc count.

= # .
Interpretation:

No hemolysis

*ARC Absolute Reticulocyte Count

250

250

250

250

Initial hemolysis

Complete hemolysis
2

25

.50%
NaCl

2 Types of cell in EOFT

1. Sickle cell
Resistant cell, presence of Hemoglobin S
2. Spherocytes *Observe in patient with Hemolytic anemia
Fragile cell

Methods of Reticulocyte

Tube with compact sediment and clear

supernate
st
1 tube from the left that shows not so
compact sediment and with slight pink
supernate
st
1 tube from the left that shows no
sediment and with dark red supernate

GEN CAMATO

HEMATOLOGY 2 LABORATORY
HEMATOLOGY 2

Take note of the tube number and the complete

hemolysis
Initial hemolysis normally in tube number 22 or 21
Complete hemolysis normally in tube 16 or 17
Increase EOFT normally in tube 24
Complete EOFT tube 19
Decrease EOFT tube 19 initial
Decrease EOFT tube 15 - complete

Eosinophil count

2.
3.
4.

Modified Sanford *Uses mL instead of drops; (2.5 mL)

Giffin and Sanford
Dacies method blood is diluted in hypotonic NaCl
solution, buffers 7.4 pH added with PO4
Fragillograph method automated method of EOFT

5.

Increase OFI
1. Hereditary spherocytosis
incase of Hemolytic anemia

Refers to the number of eosinophil per cubic mm of

blood
3
Normal value: 150-300/mm
Primarily detects parasitic, allergic and normal
adrenal function

Decrease OFI
1. Sickle cell
2. Sideroblastic anemia
3. Thalassemia *Target cells
4. Jaundice

*To differentiate, look for the colors of GRANULES; COARSE GRANULES

10-12um in diameter, slightly larger than RBC diameter
Red-orange granules using Wrights stain

*Platelets cytoplasmic fragments of Megakaryocyte

Precautions in doing EOFT

1)
2)
3)
4)

Methods of Eosinophil

The blood should be obtain with minimum stasis and

trauma
The test procedure should be set up ASAP
The capillary pipet must be held approximately same
angle
The blood should directly fall in the saline solution

Pilot method *Most commonly used

Friedmans method
Randolphs method
PRINCIPLE: A sample of blood is diluted with a
solution that selectively stains eosinophil and
eliminates all other leukocytes from view.

Reagents
1)
2)
3)
4)

Propylene glycol *Used to hemolyzed RBC

Distilled water *Diluting reagent
NaCO3 make other cell invisible and enhances
eosinophil granules
Phloxine or Eosin*used to stain eosinophil granules

GEN CAMATO

HEMATOLOGY 2 LABORATORY
HEMATOLOGY 2

Counting Basophil

Procedure

Using WBC pipet, suck blood up to 1 mark

Suck diluting fluid up to 11 mark
Mix within 10-15 minutes
Charge in 9 large square in counting chamber

Formula
=

Thorn test

*aka Eosinophil Depression Test

Use to detect normal adrenal function

Hyperadrenalism

Cushings disease
(decrease eosinophil)
* adrenal gland (above of kidney);
responsible for releasing Aldosterone
substance. Require for Sodium reabsorption;
associated with Hypercorticolism

Hypoadrenalism

Addisons disease
(increase eosinophil, but there is
an absence of eosinophilic
response)

Thorn procedure

12 hours fasting
Collect blood and get the base line eosinophil count
Inject the 25mg of Adrenocorticotrophic hormone
(ACTH) *Stimulate by Pituitary gland, then wait for 4 hours
After 4 hours, collect the blood and count again the
number of eosinophil

Interpretation

50% from base line Eosinophil = Normal

3
Value from the base line is 0 30mm after 4 hours,
a patient has Hyperadrenalism
If the patients value remains increase or no change
from baseline, it is Hypoadrenalism

Absolute basophil count

Reagent: Neutral Red staining, added with
Formaldehyde and Saponin Reagent
Basophil stain with Neutral red = Bright brick red
color
Eosinophil granules is Yellow reaction