Lesson 4: LABORATORY EVALUATION OF RBCS – PART 2

Topic: Blood Smearing and Staining, Test for Paroxysmal Nocturnal Hemoglobinuria,
and Test for G6PD Deficiency

Learning Outcomes: At the end of this module, you are expected to:

1. Describe the different techniques of smearing and various kinds of stains.

2. Perform correctly blood smearing, and staining.
3. Understand the principles of staining, Test for Paroxysmal Nocturnal Hemoglobinuria, and Test for
G6PD deficiency
LEARNING CONTENT

Introduction:

Anemia is one of the most common diseases in the Philippines. As a Medical Technologist, it is
recommended to study not only the cause of the anemia but also its clinical correlation.

In this module, different laboratory tests and diagnosis will be discussed in accordance to the
specific anemias. It covers Blood Smearing and Staining, Alkali Denaturation Test, Test for Paroxysmal
Nocturnal Hemoglobinuria, and Test for Paroxysmal Cold Hemoglobinuria.

MELS 1174 – Hematology 1 (Laboratory) | 1

Lesson Proper:

Laboratory Diagnosis of Anemias

Hematologic Examinations for Evaluation of anemia:

1. CBC
2. Examination of blood smear
3. Reticulocyte – measures effective erythropoiesis
4. Bone Marrow Examination
5. Red cell indices and Red cell survival Time
6. EPO level
7. Iron Studies (iron, TIBC, ferritin)

Parameters of CBC used to Diagnose Anemia:

1. Hemoglobin
NV: Males : 14-18 g/dL
Females: 12-16 g/dL
Moderate anemia: 7-10 g/dL
Severe anemia: <7 g/dL

2. Hematrocrit
NV: Males : 42-52%
Females: 37-47%
NB: the approximate relationship of the hemoglobin to the
hematocrit is 1:3. This may vary with the cause of the
anemia and the effect on the RBC indices, esp. the
MCV

3. RBC Indices:
MCV - Indicates the average size (volume) of the red cells
MCH - A measurement of the hgb content in RBC’s
MCHC – A measure of the concentration of hgb in the average
RBC

4. Examination of the RBC’s in the PBS :

a. Alteration in size (Anisocytosis)
b. Alteration in shape (Poikilocytosis)
c. An RBC with normal hgb content will appear hypochromic
(MCHC 32-36)
d. An RBC with decreased hgb content will appear hypochromic
(MCHC <32)
e. RBC size is designated as microcytic (MCV <80), normochromic
(MCV 80-100) or macrocytic (MCV >100)

5. Classification of anemia by RBC indices:

a. Normal size and color: NORMOCYTIC, NORMOCHROMIC
Causes: BM failure, Hemolytic anemia, chronic renal failure,
leukemia, metastatic malignancy
b. Increased size, normal color: MACROCYTIC,NORMOCHROMIC
Causes: Folate or B12 deficiency, liver disease
c. Decreased size, decreased color: MICROCYTIC,
HYPOCHROMIC
Causes: iron deficiency anemia, sideroblastic anemia,
thalassemia, chronic diseases

MELS 1174 – Hematology 1 (Laboratory) | 2

 6. RBC Inclusions - seen on Wright-stained smears in certain anemia

7. Reticulocyte (Adult normal : 0.5-1.5%)

a. Useful in determining the response to the anemia and the
potential on the BM to manufacture RBC’s.
b. Expressed as a percentage of RBC’s.
c. When anemia is present, it is useful to correct the retic using the
patient’s hct in order to assess the appropriate BM response

A supravital stain called New methylene Blue is used to stain retics.

On Wright’s stained smear, retics appear as bluish red cells. The term used for
retics on Wright’s stain is polychromasia.

Corrected retic (%) = % retic x Patient hct

Normal HCT*

Normal female Hct = 42%

Normal male Hct = 45%

d. Prematurely released retics remain in the blood and take from ½

to 1 ½ days longer to mature.

This will cause even the “corrected” retic to be elevated, so

calculation must be performed to correct this situation to obtain the reticulocyte
production index. A maturation time table is used for this calculation.

RPI = corrected retic

Maturation time* in days

BLOOD SMEAR PREPARATION and STAINING

The preparation of a blood smear may be conducted at the patient’s bedside or in the laboratory, if EDTA-
anticoagulated blood is used. Two of the most basic procedures conducted by the hematology technologist or
technician are the preparation and staining of blood smears.

A. THE PUSH-WEDGE METHOD

Specimen Either EDTA-anticoagulated whole blood or free-fl owing capillary blood can be used. If EDTA is
used, smears must be prepared within 1 hour of collection. Before preparing the smear, store the blood at
18°C to 25°C. Adequate mixing is necessary before blood smear preparation.

Supplies and Equipment

Clean glass slides (plain or with one frosted end), a No. 2 lead pencil, and (optional) a specially designed
pusher slide or a hemocytometer coverslip and pusher assembly.

Procedure

1. Place a small drop (0.05 mL) of well-mixed blood either directly from the freshly wiped fingertip puncture or
with an applicator stick approximately 0.5 inch from one end of the slide. If frosted slides are used, place the
blood near the frosted end of the slide.

MELS 1174 – Hematology 1 (Laboratory) | 3

2. Place the slide on a flat surface with the blood specimen to your right. Reverse this direction if you are left-
handed.

3. Using a second pusher slide, place this slide slightly in front of the drop of blood. The angle of this pusher
slide must be at approximately 45°.

4. Draw the pusher slide back toward the drop of blood. Allow the drop of blood to spread about three fourths
of the way across the bevel of the pusher slide. Do not allow the blood to spread to the edges. Quickly push
this slide forward (away from the drop). This forward movement must be smooth and continue to the end of the
slide.

5. Allow the smear to air dry before staining. The slides can be fanned in the air to dry them rapidly.

6. Label the slide using a No. 2 pencil. The labeling may be on the thick end (or frosted end) of the slide or on
one edge of the slide

B. COVERSLIP METHOD
 The coverslip method produces a good distribution of leukocytes in all areas of the preparation.
 Because of the smaller specimen amount, 50 leukocytes are counted per coverslip.
 Owing to the small size of the coverslip, it is usually mounted (attached) to a conventional glass
slide for staining.

Procedure

1. Hold two clean coverslips by their edges with the thumb and forefinger of each hand. Touch the center of
one coverslip to a small drop of blood.

2. Immediately place the second coverslip on top of a very small drop of blood in a diagonal position.
MELS 1174 – Hematology 1 (Laboratory) | 4
3. Allow the blood to spread by capillary action. Just before the spreading action has almost stopped, evenly
and smoothly pull the coverslips apart in the horizontal plane.

4. Place the smears in an upright position and allow to air dry before staining

Visual Evaluation of a Good Blood Smear An ideal smear has the following characteristics.

1. It progresses from being thick at the point of origin to thin with a uniform edge at the termination point.

2. It does not touch the outer borders of the slide or run off the sides or ends of the slide.

3. It appears smooth, without waves or gaps.

4. It does not have any streaks, ridges, or troughs, which indicate an increased number of leukocytes carried
to that area.

5. It is prepared with a proper amount of blood and spread to occupy approximately two thirds of the length of
the glass slide.

Causes of a Poor Blood Smear

1. Prolonged storage of anticoagulated whole blood specimens. This can produce cellular distortion.

2. A delay in smear preparation. It is important to perform the blood smear immediately after placing the drop
of blood on the slide. If this process is delayed, larger cells such as neutrophils and monocytes will be
disproportionately located at the feathered edge when examined microscopically.

3. Dirty or poor-quality slides. Slides should be free of dust and grease spots.

4. Inappropriate size of blood droplet. Too large a drop of blood will produce a thick, long smear. Too small a
drop of blood will produce a thin, short smear.

5. Improper angle of the pusher slide. The more the angle of the pusher slide is decreased, the longer the
smear. The greater the angle, the thicker the smear.

6. Improper speed of the pushing movement. Slowly pushing the drop of blood will produce irregularities and
affect the distribution of cells on the smear.

7. Improper pressure. The greater the pressure, the thinner the smear.

8. Humidity of the laboratory environment. High humidity can cause slides to dry too slowly. The prolonged
drying of slides will produce erythrocyte distortion on microscopic examination.

COUNTING METHODS

1. CROSS SECTIONAL OR CRENELLATION WBCs are counted in consecutive fields as the blood
film is moved from side to side
2. LONGITUDINAL METHOD WBCs are counted in consecutive fields from tail
toward the head of the smear
3. BATTLEMENT METHOD Uses a pattern of consecutive fields beginning near
the tail on a horizontal edge: count three consecutive
horizontal edge fields, count two fields towards the
MELS 1174 – Hematology 1 (Laboratory) | 5
 center of the smear, count two fields horizontally,
count two fields vertically to the edge

ROUTINE 100 CELL DIFFERENTIAL

 50 cells- Patient WBC <1.0 x 109/L

 200 cells- Over 10% eosinophils, Over 2% basophils, Over 11% monocytes, more lymphocytes
than neutrophils (except in children)

Routine Staining of Peripheral Blood Films

To examine cells on a blood smear in detail, it is necessary to stain the smear. The beginning student in
hematology should become familiar with the principles and practice of routine staining of a blood smear before
investigating specific characteristics of cells or performing other staining procedures. The most commonly used
stain in the hematology laboratory is a Romanowsky-type stain.

STAINING PRINCIPLES

 In 1891, Romanowsky and Malachowski first described the use of a stain that combined a polychrome
(oxidized) methylene blue solution with eosin as a blood stain. Ten years later, this stain was refined by
Leishman, who combined eosin with polychrome methylene blue, recovered the precipitate, and
dissolved this precipitate in methyl alcohol. Today, Romanowsky-type stains are prepared by use of this
modified technique.
 A Romanowsky stain is defined as any stain containing methylene blue and/or its products of oxidation
and a halogenated fl uorescein dye, usually eosin B or Y.
 Romanowskybased stains, such as Wright, Giemsa, or May-Grünwald stains, are alcoholic solutions
with basic and acidic components. Stains of this type are referred to as polychrome stains because
they can impart many colors and produce the Romanowsky effect. This effect imparts a typical color to
certain cell components and reflects the combined action of the dyes contained in the stain at a pH of
6.4 to 7.0. The characteristic colors are purple in the cell nucleus, blue and pink in the cytoplasm, and
various colors in specific granules.

Procedure

1. Place a thoroughly dried and labeled slide on a level staining rack with the smear side facing up.

2. Place freshly filtered stain slowly on the slide until the smear is completely covered. Do not add excess
stain. Staining times will vary. Commonly, 3 to 10 minutes may be needed for an acceptably stained blood
smear.

3. At the end of the staining time, gently add buffer (pH 6.4) to the slide without removing the stain. The buffer
should form a large bubble (convex shape) on the slide. Do not add excess buffer. Some technologists prefer
to use ordinary tap water in place of the buffer.

4. Mix the stain and buffer by gently blowing on the slide. A well-mixed slide will have a metallic green sheen
rise to the surface of the slide. The timing for this stage ranges from 2 to 5 minutes. If a Coplin jar is preferred
for staining, the slides are dipped into the stain and buffer solutions.

5. Wash the stain and buffer off the slide with a gentle flow of tap water. Pick the slide up by its edges and
wipe the back of the slide to remove any stain.

MELS 1174 – Hematology 1 (Laboratory) | 6

6. Allow the slide to air dry

 Thick films  Insufficient

 Prolonged staining
staining time  Prolonged
EXCESSIVELY BLUE  Inadequate EXCESSIVELY PINK washing time
STAIN washing STAIN  Mounting the
 Too high alkalinity coverslips before
of stain they are dry
 Diluent tends to  Too high acidity of
cause excessive stain
basophilia  Buffer may cause
excessive
acidophilia

TEST for PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

 Acquired intrinsic defect in the red cells that makes the cell more sensitive to lysis by heat labile serum
factors (complement)
 Defective membrane may be due to deficiency of decay accelerating factor (DAF) that normally inhibits
complement mediated lysis
 Disorder is characterized by intravascular hemolysis and hemoglobinuria during and following sleep in
the classic case

1. SUGAR WATER SCREENING TEST

 Simple screening procedure for PNH
 If positive, the sucrose hemolysis procedure should be performed before a diagnosis of PNH is
made
 Whole blood is mixed with a sugar water solution and incubated at room temperature
 Citrated blood
 Test should be performed within 2 hours of obtaining the specimen
 Use of defibrinated blood may cause FALSE POSITIVE result due to hemolysis of the
traumatized RBCs

2. SUCROSE HEMOLYSIS TEST

 Used as a confirmatory test for PNH when the sugar test is positive
 Result should correlate with the acid serum test
 Washed RBCs are incubated in an isotonic sucrose solution containing normal ABO compatible
serum
 Citrated blood

3. HAM’S ACIDIFIED SERUM TEST

 Reliable to diagnose PNH
 Whole blood defibrinated
 When patient has received blood transfusion, less lysis occurs because of the presence
of normal transfused red blood cells

MELS 1174 – Hematology 1 (Laboratory) | 7

TEST for G6PD DEFICIENCY

1. GLUCOSE-6-PHOSPHATE DEHYDROGENASE FLOURESCENT SCREENING

Normal: Maximum fluorescence at 10 minutes
Abnormal (G6PD deficiency): Little or no fluorescence

2. ASCORBATE CYANIDE SCREENING TEST

 Nonspecific procedure for detecting deficiencies in the pentose phosphate pathway: G6PD
deficiency (glutathione peroxidase, glutathione reductase)
 Normal: red
 Abnormal (enzyme deficiency): brown

3. AUTOHEMOLYSIS TEST
 In the past, this procedure was used in differentiating several types of congenital nonspherocytic
hemolytic anemias using glucose and ATP
 Test is positive in G6PD and pyruvate kinase deficiencies and in hereditary spherocytosis

**End of Lesson**

MELS 1174 – Hematology 1 (Laboratory) | 8