HEMATOPOIETIC SYSTEM I: PERIPHERAL BLOOD

Histology Laboratory

WARNING:

TEMPORAL SEQUENCE

1. Perform the fingerstick.
2. Prepare your Blood Smear.
3. Determine your hematocrit.
4. Perform the differential.

PERFORMANCE OF FINGERSTICK

REAGENTS AND HARDWARE:

• alcohol wipe
• lancet
• 2 x 2 inch gauze
• 2 capillary blood collection tubes (heparinized)

PROCEDURE:

1. Choose a finger that is not cold, cyanotic or swollen.
2. Massage the finger 5 or 6 times from base to tip to ensure good blood flow.
3. With an alcohol swab, cleanse the ball of the finger.
4. Allow to air dry.
5. Remove the lancet from its protective wrapper without touching the tip.
6. Hold the finger firmly with one hand and make a swift, deep puncture with the lancet halfway between the center of the ball of the finger and its side.
7. The cut should be made across the fingerprints to produce a large round drop of blood.
8. Wipe the first drop of blood away with a clean gauze.
9. Massage the finger from base to tip to fill two hematocrit tubes about 2/3 full. Use one tube to make the blood smear.
10. Discard the specimens. Discard both capillary tubes when finished.

BLOOD SMEAR: PREPARATION AND STAINING

PRINCIPLE:

REAGENTS AND SUPPLIES:

1. Fixative Solution
2. Solution I
3. Solution II
4. Distilled water
5. Glass slides
6. Coplin jars

PROCEDURE:

1. Select one of the two hematocrit tubes. Make sure the same student name appears on the tubes and the glass slides.
2. Touch the end of the tube to the surface of a clean glass slide near the frosted end with the frosted end facing upward. A small drop of blood will be deposited.
3. Hold the slide between two fingers and the thumb of the left hand with the drop of blood on the upper surface toward your right. (Reverse for the left-handed individual).
4. Place an edge of the spreader slide on the first slide to left of the drop of blood and pull to the edge of the drop. The approximate angle between the two slides is 30 to 40 degrees.
5. Allow the drop of blood to bank evenly behind the spreader and then push to the left in a smooth, quick motion. The more rapid the motion, the shorter and thinner (better) the smear. The smear should cover approximately half the slide with a gradual transition from thick to thin. No ridges should be present and the end (called the "feather edge") should be smooth and even.
6. When the smear is completely dried, stain as follows:
   • Dip blood in Fixative solution 5 times, one second each time. Drain excess fixative.
   • Dip into Solution I, 5 times, one second each time. Drain excess solution.
   • Dip into Solution II, 5 times, one second each time. Drain excess solution.
   • Rinse slide with distilled water.
   • Allow slide to air dry.
   • Examine with oil immersion technique.
7. Expected results:

   Erythrocytes:

   Pink or yellowish red

   Lymphocytes:

   Nucleus, violet

   Cytoplasm, dark blue

   Neutrophils:

   Nucleus, dark blue

   Cytoplasm, pale pink

   Granules, pale reddish-lilac

   Basophils:

   Nucleus, purple to dark blue

   Granules, dark purple (almost black)

   Eosinophils:

   Nucleus, blue

   Cytoplasm, blue

   Granules, red to red-orange

   Monocytes:

   Nucleus, violet

   Cytoplasm, pale blue-clear

PERFORMANCE OF A HEMATOCRIT BY MICRO-HEMATOCRIT CENTRIFUGE

PRINCIPLE:

The hematocrit is a measure of the percent of total blood volume occupied by the erythrocytes, not the total blood cell volume. It is high in newborns, drops to its lowest point at about one year, then gradually climbs to adult levels.

Physiologic variations occur, the hematocrit tending to be higher in taller, heavier individuals. Acclimation to altitude is also a factor, the hematocrit being higher at high altitudes.

PROCEDURE:

1. Seal one end of each two heparinized capillary tubes with Critoseal.
2. Place the centrifuge tubes opposite each other into slots in the centrifuge. Note that each slot is numbered. Record the number of each of the two slots containing your tubes. If you were able to obtain only one tube containing blood, place an empty tube in the opposing slot (to balance the centrifuge).
3. Place the tubes such that the sealed ends are directed to the outside and against the rubber cushion.
4. Tighten the inside lid of the centrifuge on the top of the centrifuge.
5. Set the timer switch for the time indicated on the top of the centrifuge.
6. After the centrifuge stops, remove the hematocrit tubes.
7. Align the bottom of the packed cell layer of the hematocrit tube with the base mark near the center of the International Mcrocapillary reader. Move the tube to the left or right until the plasma-air interface crosses the upper line of the reader.
8. Read the percent packed cell volume. In cases of extremely high white cell count, care must be taken to read the top of the red cell layer, not the top of the buffy coat layer. Discard both tubes in lilly buckets.

NORMAL VALUES:

Male:

42-52%

Female:

37-47%

Newborns:

44-64%

Children (over 1 yr):

30.5-40.5%

Children (0-1 yr):

30-64%

DIFFERENTIAL COUNT

PRINCIPLE:

PROCEDURE:

1. Inspect the stained smear under low power. Choose a portion of the smear where there are no overlapping erythrocytes (the feathered edge).
2. Place a drop of immersion oil on the slide in the chosen area and shift to the 10OX oil immersion objective.
3. Move the slide from the extreme upper edge of the smear to the extreme lower edge, classifying each white cell in successive fields. Shift over one field and proceed to the upper edge of the slide, still classifying each white cell. Continued in this fashion until a total of 100 white cells have been counted.
4. Record the differential count.
5. What is the differential count of the WBC in normal peripheral blood? Discard slides in lilly buckets.

NORMAL RANGE: (Adults)

Type of Cells                             Percent of Peripheral White Calls
__________________________________________________________________________________________________
segmented neutrophils                     50-75% (3-5% of these are stab or band forms)
eosinophils                               1-3%
basophils                                 0.5-1%
monocytes                                 3-8%
lymphocytes                               20-50% (80% of these are T cells, 15% B cells, 5% nulls)

CLINICAL SIGNIFICANCE: