Blood Smears, Staining and Differentials

Required reading

Hendrix, “Laboratory Procedures for Veterinary Technicians”, pages. 27-73

-Voigt. “Hematology Techniques and Concepts for Veterinary Technicians”, Chapter 5, pages 21 - 25 and Chapter 6 pages 27 - 44

Recommended Reading:

Sink and Feldman:  Section 3, pages 47 - 62

Required Website:

http://diaglab.vet.cornell.edu/clinpath/modules/

http://www.vet.uga.edu/vpp/ivcvm/1999/phillips/index.php

Objectives

After completing this unit you should be able to: 

1.                   Prepare a blood smear using the slide technique
2.                   Prepare a blood smear using the coverslip technique
3.                   Stain a slide of a blood smear
4.                   Recognize the following cellular components in a blood smear
                    a.        Erythrocytes
                    b.       Platelets
                    c.        Neutrophils
                    d.       Eosinophils
                    e.       Basophils
                    f.         Lymphocytes
                    g.        Monocytes
5.                   Perform a differential count

Introduction

Veterinary technicians frequently prepare smears of blood which are then stained and examined under the microscope.   A differential white blood cell count, also called a “diff”, is a relative count of the leukocyte types found in the blood smear.  A well prepared, and accurately described, blood smear is an important diagnostic tool and provides valuable information to the veterinarian.

Preparation of Blood Smear

Whenever possible, fresh blood should be used in preparing a blood smear in order to prevent the cellular distortion associated with the use of anticoagulants.  After collecting the blood specimen, discard the first few drops of blood and then make your smears before you use the sample for other tests.

The following steps are the most frequently used in making a blood smear: 

1. Lay a clean microscope slide on a flat surface.

2. Place a drop of blood about ˝ inch from the frosted end of the slide.  The drop of blood can be transferred from a blood tube by means of an applicator stick, a capillary tube or a needle.

3. Lightly balance another slide (called the “spreader slide”) on your fingertips and place the spreader slide at a 30 degree angle in front of the drop of blood

4. Pull the spreader slide back toward the blood droplet until you touch the droplet and blood spreads along the edge of the spreader slide.

5. Quickly push the spreader slide forward using a steady, even motion.   The weight of the slide is the only pressure applied to the smear during this procedure.Air dry the smear and stain within one hour.

A “good” blood smear should have the following appearance:

1. Should cover about half the length of the slide and be tapered, thin and feathered at the end  

2. Should not extend to the edges of the slide 

3. Should be uniform in consistency, i.e. without holes, scratches or ridges

The characteristics of a blood smear are affected by the size of the drop of blood, the angle at which the spreader slide is held and the speed at which the drop is spread.  These factors affect the amount of blood carried by the spreader slide which then affects the thickness of the smear.  It takes a lot of practice to consistently make a good blood smear.  If you are having problems, you should alter one or more of these factors to correct the problem.

Beside the technique described above, there are other acceptable methods of preparing blood smears.  An alternative, horizontal placement of the spreader slide on the smear may be easier for some technicians.  Blood smears can also be made on coverslips by placing a small drop of blood on a coverslip, placing another coverslip on the drop of blood and then pulling the coverslips apart.  Three methods of smear preparation are illustrated on pages 23 and 24 of Voigt.

Staining

Blood smears need to be stained in order to see the cytoplasmic and nuclear details of the cells.  The most frequently used stains in hematology are the Romanowsky stains, commonly called the "Diff- Quik" stain.  The new methylene blue stain is occasionally used as a supplemental stain to see reticulocytes.  Regardless of the stain technique, smears should be quickly air dried by brisk waving to preserve cell morphology.  DO NOT heat fix blood smears!!

Romanowsky stains (Diff-Quik and other quick Wright’s stains) are inexpensive, easy to prepare and to use.  They are excellent stains for microbiological organisms and for the cytoplasm of cells.   The staining system incorporates a red, acidic dye (usually eosin) that is attracted to the basic parts of a cell, and a dark-blue to purple, basic dye (usually methylene blue) that is attracted to the acid parts of a cell.

In order to stain a slide with Diff-Quik, sequentially dip a dried blood smear slide five to six times in each of the following solutions:  fixative (methyl alcohol is the most commonly used), acidic dye, basic dye, water wash.  The slide must be drained between solutions.  The staining solutions should be occasionally filtered to remove debris, and replenished or replaced when necessary.  Keep the solutions tightly capped between uses.  Every technician develops a preferred staining technique.  Experiment with varying dipping times until you get the staining characteristics you desire.

                                

        Dipping slide in fixative               Draining slide between solutions             Stained and unstained slides

 

New methylene blue stain is an excellent dye for nuclear and nucleolar details.   It is also used to demonstrate erythrocyte alterations and inclusions, and some erythrocyte parasites, as well as to visualize reticulocytes.  The methylene blue is different from that in Romanowsky stains.   

In order to stain a slide with new methylene blue a drop of stain is placed on a coverslip.  The coverslip is then placed on an unfixed blood smear.  An alternative method is to mix some stain with fresh blood before a blood smear is made. 

Differentials

After a blood smear is stained, it is examined to be sure that the slide is a good slide for doing a differential count.  The slide is scanned under low power in order to find areas where the cells are one layer thick (monolayer).  Monolayers are usually toward the end of the smear and at the edges of the smear.  In the monolayer, the RBCs should just touch each other.

In doing a differential leukocyte count, a technician examines a smear with oil immersion, classifies and counts at least 100 white blood cells.  The counts are usually reported as percentage of the TOTAL RBC count.   The cells can be tallied with a hand counter or on paper.

    Cell Counter

In order to ensure a random sampling of cells, and to avoid counting the same cell more than once, you need to develop a pattern for examining slides.  The battlement method involves examining the edge of the smear for 3–5 fields, move in a short distance and examine for 3-5 fields, move back to the edge, etc., until 100 cells are counted.  The wandering method involves counting cells across the entire monolayer.  The two techniques are illustrated on page 41 of Voigt.

In this unit you will be examining blood smears that you prepare and will begin learning to identify the different kinds of cells in blood..  The pictures below will help you with these identifications.  In future units, you will be studying more about the structure and function of each of these cell types.

Below are some photos of red blood cells, platelets and white blood cells.  Use these to help you identify cells in the slides you prepare.  You will be spending time throughout the semester identifying these cells and will get a lot practice doing so.  The following website contains a slide with leukocytes.  You can click on a cell and it will tell you the type of leukoctye. www-micro.msb.le.ac.uk/MBchB/bloodmap/Blood.html

        Red blood cells and platelets

        Neutrophil

        Lymphocyte

       Monocyte

       Eosinophil - has red staining granules

      Basophil - has blue staining granules - very rare, you may not see one

Assignment: 

        1.                   Practice making blood smears from a healthy animal using the slide technique until you are able to make a “good” slide.  Try making a preparation using the coverslip technique. Some people who have had difficulties making good slides sitting down, will try it standing up with some success...so don't be afraid to experiment, and go with what works for you!

2.                   Stain the slide using a Romanowsky stain (ex. Diff-Quik)

3.                   Examine the slide under oil immersion.  Locate red blood cells, platelets, neutrophils, eosinophils, basophils, lymphocytes and monocytes.  Do a differential count on the smear and ask you mentor to check your results.

4.                   Report the results of your differential on Lab Report #4 and email it to Dr. Durham.  Send Dr. Durham the blood smear that you prepared.  Mail it to:  Dr. S. M. Durham, Division of Natural and Applied Science, Veterinary Technology, Loudoun Campus, Northern Virginia Community College, Sterling, VA 20164.  Your blood smear should be postmarked NO LATER than 10/01/07.