THE RETICULOCYTE COUNT

INTRODUCTION:

Reticulocytes are immature red cells, which had lost their nuclei but had retained aggregates of ribonucleic acid RNA. They show reticulum when
stained supravitally. The younger the reticulocyte, the amount of RNA decreases as the reticulocyte matures, the more filamentous and plentiful is the reticulum.
Reticulocyte stay in the bone marrow for about 2-3 days and in the circulating blood for another 1 day. They are slightly bigger than mature red cells. A cell could be
considered a reticulocyte if it possesses 2 or more reticular dots.
INTRODUCTION: (continuation...)

The reticulocyte count is used as an index for evaluating the effectiveness of erythropoiesis. It is sometimes called as the “poor man’s bone marrow”
study. The importance of the reticulocyte count are:
o To confirm diagnosis of pernicious anemia by therapeutic response o To determine response of pernicious anemia patient to Vitamin B12 therapy o Aids in
diagnosis of lead poisoning and hemolytic anemia o To determine whether there is normal RBC regeneration o To aid in the prognosis of acute hemorrhage. An
increase in reticulocyte count indicates rbc regeneration.
CLINICAL SIGNIFICANCE:
RETICULOCYTOSIS - Increase in reticulocytes in the blood. 5. Blood intoxication
This is found in the following conditions: 1. Hemolytic anemia 6. Kala-azar
2. Lead poisoning 7. Erythroblastic anemia
3. Malaria 8. Sickle cell anemia
4. Parasitic infestations 9. Relapsing fever10. Leukemia
11. Splenic tumor
CLINICAL SIGNIFICANCE:
RETICULOCYTES IS DECREASED PHYSIOLOGIC INCREASE IS
IN: OBSERVED IN:
1. Aplastic anemia 1. Pregnancy
2. Acute benzol poisoning 2. At birth
3. Chronic infections 3. Menstruation
4. Anaplastic crisis of hemolytic 4. High altitudes anemia
SOURCES OF TECHNICAL ERROR IN RETICULOCYTE COUNT:

1. Failure to mix the blood and stain completely

2. Presence of refractile artifacts
3. Increased blood glucose level
4. Presence of pappenheimer bodies, heinz bodies and howell-jolly bodies
OBJECTIVES:

At the end of the exercise, the student should be able to:

1. To be knowledgeable of the different pre-analytical, analytical and post-analytical factors affecting reticulocyte counts.
2. To develop skill in performing the reticulocyte count using the routine light microscope and calibrated miller dish method.
3. To value the importance of proper techniques to minimize error in reticulocyte count results.
4. To correlate the clinical significance of reticulocyte counts not within the reference range.
PRINCIPLE:
Supravital stains, such as new methylene blue N or brilliant cresyl blue, bind,
neutralize, and cross-link RNA. These stains cause the ribosomal and residual RNA to
coprecipitate with the few remaining mitochondria and ferritin masses in living
young erythrocytes to form microscopically visible dark-blue clusters and filaments
(reticulum). An erythrocyte still possessing RNA is referred to as a reticulocyte (see
Fig. 26.2). The enumeration of reticulocytes is important in assessing the status of
erythrocyte production in the bone marrow (erythropoiesis).
MATERIALS:

Specimen: EDTA-anticoagulated blood or capillary blood

• EDTA evacuated tube
• Cotton balls
• 70% isopropyl alcohol
• Binocular microscope
• Calibrated miller disk
• New methylene blue or Brilliant cresyl blue stain
• Glass slide Microscopic slides
• Cedar wood oil
PROCEDURE: DRY METHOD

1. Mix equal volumes of blood and freshly filtered stain in a tube. Allow this mixture
to stand at room temperature for 15-30 minutes.
2. Remix the preparation and prepare smears. Allow the smears to dry.
3. Examine under OIO.
 Note: Mature RBCs stain gray blue while reticulocytes are identified by the presence of deep blue filamentous web or granules within the cells. Any cell
that contains two or more particles of bluestained materials is classified as reticulocyte.
4. Count the reticulocytes seen in 10 successive fields of vision while enumerating 1000 mature RBCs.
PROCEDURE: DRY METHOD

Computation:
% Reticulocyte = No. of reticulocytes x 100
1000
= No. of reticulocytes
10 Normal Values:
Adults: 0.5 % - 1.5 %
Babies: 2.0 % - 6.0 %
PROCEDURE: MICROSCOPIC METHOD

A. WET METHOD - Rapid method of Schilling, Osgood-Wilhelm, and Sabin:

1. Place a drop of freshly filtered stain on a clean dry glass slide.
2. Add an equal volume of blood. Mix by stirring.
3. Cover the mixture with a clean coverslip lined with Vaseline.
4. Proceed with counnting.
PROCEDURE: MICROSCOPIC METHOD

B. MILLER DISC METHOD - Miller disc is an optical aid inserted into the eyepiece of the
microscope. This allows more accurate count. The disc ruling consists of a center square
containing a secondary square ruled area that is 1/9 he area of the larger square.
PROCEDURE: MICROSCOPIC METHOD

B. MILLER DISC METHOD Computation:

% Reticulocyte = No. of reticulocytes in large square x 100 No. of RBC in

small square x 9

Normal Values:
Adults: 0.5 % - 1.5 %
Babies: 2.0 % - 6.0 %
 OSMOTIC FRAGILITY TEST
INTRODUCTION

Red blood cells are surrounded by a selectively permeable membrane that allows the exchange of gases and electrolytes. If the RBCs are placed in a
hypotonic saline solution, osmotic equilibrium will be established by drawing water into the cells, which then swell.
INTRODUCTION

Pathologically, increased permeability of the RBC membrane leads to the accumulation of water within the cell, and finally escape of hemoglobin
through the widened pores of the cell membrane.
Precautions must be taken in performing osmotic fragility test (OFT) such as:
1. The blood sample should be obtained with minimum stasis and trauma
2. The test should be set up as soon as possible
3. The sizes of the drops of blood must be uniform
4. Blood should fall directly into the saline solution and not on the sides of the tube

LEARNING OBJECTIVES

At the end of the activity, the student/s should be able to:

1. Perform the proper technique for OFT
2. Determine the pitfalls which may be encountered while performing the test
3. Identify the disease evaluated using the OFT
PRINCIPLE
In the osmotic fragility test, whole blood is added to varying concentrations of sodium chloride solution and allowed to incubate at room
temperature. The amount of hemolysis is then determined by examining
the supernatant fluid either visually or with a spectrophotometer.
If erythrocytes are placed in an isotonic solution (0.85%) of sodium chloride, water molecules will pass in and out of the membrane in equal amounts.
In hypotonic solutions, erythrocytes will hemolyze because more water molecules enter into the cell than leave. This net influx of water molecules eventually
ruptures the cell membrane.
The main factor in this procedure is the shape of the erythrocyte, which is dependent on the volume, surface area, and functional state of the
erythrocytic membrane.
A spherocytic erythrocyte ruptures much more quickly than normal erythrocytes or erythrocytes that have a large surface area per volume, such as
target or sickle cells.
The fragility of erythrocytes is increased when the rate of hemolysis is increased.
If the rate of hemolysis is decreased, the erythrocytic fragility is considered to be decreased. The clinical value of the procedure is in differentiating
various types of anemias.
MATERIALS

• 0.5 % sodium chloride (NaCl)

• Wassermann tubes (12)
• Test tube rack
• Gum label
• Pasteur pipette
• Parafilm
• Timer
1. Prepare 0.5% NaCl solution (0.5 gm dissolved in 100 mL distilled water)
2. Arrange 12 Wassermann test tubes in a rack. Label them down from 25 down to 14 (left to right)
3. Deliver 0.5% NaCl in each tube drop by drop using a Pasteur pipette. The number of drops should correspond to the number labeled on each tube
4. Use the similar pipette and add distilled water to each tube. The number of drops should bring the total volume of the solution in each tube to 25. Mix
thoroughly.
5. Deliver 1 drop of freshly drawn blood into each of the tubes. Mix gently.
6. Allow the tubes to stand for 2 hours at room temperature.
7. Examine the tubes for initial and complete hemolysis.
*Note: The cells will settle at the bottom of the tube and hemolysis will be recognized by the color of the supernatant fluid.
Hemolysis is partial if the fluid turns faintly pink.
It is complete when the fluid is red with no sediments.
PROCEDURE: GRIFFIN-SANFORD METHOD
Normally, hemolysis usually begins at tube 17.
Computation:
Tube number x 0.02 = %

Normal Value:
Initial Hemolysis 0.44 +/ - 0.02 %

Complete Hemolysis 0.32 +/ - 0.02 %

PROCEDURE: GRIFFIN-SANFORD METHOD
RESULTS OF THE TEST
PROCEDURE: GRIFFIN-SANFORD METHOD
PROCEDURE: GRIFFIN-SANFORD METHOD
Reference/s:

• Laboratory Manual in Hematology – Ma. Gina Sadang / Frederick Llanera

• Clinical Hematology 5th Edition - Turgeon

ERYTHROCYTE SEDIMENTATION RATE

ERYTHROCYTE SEDIMENTATION RATE

The erythrocyte sedimentation rate (ESR) is classical employed as an index of the presence of acute and chronic infection, inflammation and tissue
necrosis or infarction. Diseases like tuberculosis, tonsillitis, rheumatic fever, and rheumatic heart disease are often screened using this test. Results are affected by
factors other than that of the red cells and the plasma.
At present, more tests are being utilized to make diagnosis more effective and efficient. More reliable tests, such as, C-RP,SLE tests,R.A tests and
Streptozyme are now employed in the diagnosis of inflammatory conditions.
ERYTHROCYTE SEDIMENTATION RATE

The test depends on the fact that is the blood to which anticoagulant has been added, the red corpuscles sediment until they form a packed column in
the lower part of the tube or container.
The rate of this process depends on the number of factors like rouleaux formation, concentration of fibrinogen, alpha and beta globulin in the plasma,
etc. The test or the rate of fall of the red blood cells is reported in millimeters at the end of one hour.
PROCEDURE: GRIFFIN-SANFORD METHOD
ERYTHROCYTE SEDIMENTATION RATE

ESR is more constant in men than in women. In pregnancy, ESR begins to increase at the 3rd or 4th month and does not return to normal until the 3rd
or 4th week post partum. In newborn, ESR is reduced while older adults, it is high.
PRINCIPLE:

The erythrocyte sedimentation rate (ESR), also called the sedrate, measures the rate of settling of erythrocytes in diluted human plasma. This
phenomenon depends on an interrelationship of variables, such as the plasma protein composition, the concentration of erythrocytes, and the shape of the
erythrocytes.
The ESR value is determined by measuring the distance from the surface meniscus to the top of the erythrocyte sedimented in a special tube that is
placed perpendicular in a rack for 1 hour. The clinical value of this procedure is in the diagnosis and monitoring of inflammatory or infectious states.
OBJECTIVES:

At the end of the exercise, the student should be able to:

1. Explain and demonstrate the proper techniques in ESR determination.
2. Differentiate the intrinsic and extrinsic factors affecting ESR results.
3. Understand the different pre-analytical, analytical and postanalytical variables affecting the results in ESR.
4. Correlate the clinical implications when results vary from reference values.
MATERIALS:

• Double oxalate – anticoagulated blood

PROCEDURE: GRIFFIN-SANFORD METHOD
• EDTA – anticoagulated blood
• Wintrobe tube
• Westergren pipet
• Westergren pipet stand
• Wintrobe tube stand
• Long-tipped Pasteur pipet
• Rubber aspirator
PROCEDURE: GRIFFIN-SANFORD METHOD
MATERIALS:
PROCEDURE: GRIFFIN-SANFORD METHOD
PROCEDURE:

I. WINTROBE - LANDSBERG METHOD

1. Fill up the tube to the 0 mark with oxalated blood, using Pasteur pipette
with long stem and avoid bubble formation.
2. Allow to stand in a vertical position for one hour.
3. Take and record reading in mm/hour.
PROCEDURE:

WESTERGREN METHOD (MODIFIED)

1. Dilute EDTA-blood with NSS in a 4:1 ratio by adding 0.5 ml of NSS to 2.0 ml of
the blood sample.
2. Mix and fill the Westergren tube up to the “0” mark.
3. Allow to stand in a vertical position for one hour.
4. Avoid direct drafts, sunlight, and vibration.
5. Take and record reading in mm. If desired, reading could be taken after 2-
hour standing.

CLINICAL SIGNIFICANCE: