1) Slide 85: Normal Blood smear, Wrights stain.
Examine this smear with low power objective (10X or 20X) and select
an area with fairly uniform distribution of leukocytes and in which the
erythrocytes show little or no clumping or overlapping. The best areas
are generally just behind the feathered or tail end. Then place a drop
of oil on this area of the smear and switch to the oil immersion objective
with substage condenser and diaphragm adjusted for optimal illumination
and resolution.
Smear No. 85 is a normal blood smear showing normal red blood cell morphology, normal platelets and a normal population of white cells. Study this smear until you can readily recognize all normal formed elements of the blood. Learn the relative numbers of each type of leukocyte in normal blood.
2) Slide 86: Blood smear (Wrights stain) with increased Lymphocytes
and Monocytes:
This smear is from an infant with a viral infection. The white cell
count is elevated to 19,800. Monocytes are elevated to 20 25%. Lymphocytes
are plentiful. Band neutrophils are increased and there are occasional
Metamyelocytes and Myelocytes. Use this smear to reinforce your ability
to identify Monocytes and Lymphocytes.
3) Differential Count of leukocytes on either Slide 85 or 86.
A worksheet for your data is available in the labs. Compare your results
with the average normal values given two pages back. Turn in your completed
data sheet to your lab instructor at the next laboratory session.
Select a proper area where the red cell morphology appears good with low (1020X) power objective. Switch to oil immersion objective and count 100 200 consecutive white cells. The oil immersion lens must be used to insure accurate identification of cells. Examine the smears systematically so that the same cells are not counted twice. Gradually work from the thinner area toward the thicker, going back and forth to the long (side) edges of the smear including 2 4 side edge fields before proceeding back through the body of the smear to the opposite side edge. An example of the recommended route is illustrated.
Slide 88: NORMAL MARROW SMEAR (Wrights Giemsa): Examine this push type smear with low power objective, noting the differences in smear thickness and cellular distribution in the various areas. Select a good area for individual examination of the cells, generally found just behind the feathered or tail end. Switch to oil immersion. Find several examples of each stage of erythroblasts. Note the differences in cell size, N:C ratio, cytoplasmic color, nuclear chromatin pattern and color in the various stages.
Slide 89: NORMAL MARROW with INCREASED ERYTHROBLASTS and HISTIOCYTES (Wrights Giemsa stain): Use the same systematic approach in the examination of this aspirated marrow push smear. Note the abundance of erythroblasts in comparison with the number on smear 88.
Slide 91: BIOPSY SECTION OF MARROW H&E stain: Study the general types and arrangements of cells in red marrow. In what respects does it differ from yellow marrow?
Slide 87: RETlCULOCYTE PREPARATION:
This push smear preparation is made from a mixture of blood and New
Methylene Blue stain after incubation at room temperature for 10 minutes.
With oil immersion count the number of reticulated cells in 1000 erythrocytes,
selecting fields in which the RBC's are evenly distributed and well displayed.
Move from the thinner toward the thicker area on this push smear.
Precipitated reticulum appears as flaky, stringy, fine blue staining
network usually in the center of the cell and varies widely in amount from
several small dots to large masses or reticulum. All cells showing this
in any degree are counted as reticulocytes. Avoid confusion with artifacts
(such as precipitated stain granules, dust, dirt and platelets overlying
red cells) by careful focusing with the oil immersion objective.
| RETICULOCYTE
(New Methylene Blue Stain) |
POLYCHROMATOPHILIA:
(Wrights Stain) |
The count is expressed as percent erythrocytes containing reticulum.
Normal is approximately 1.0%.
This patient had a mild hemolytic process. Her compensating marrow
erythropoiesis is reflected by her elevated reticulocyte count.
The absolute number of reticulocytes per volume of blood is determined by multiplying the percent of red cells which are reticulocytes times the red cell count. The reticulocyte count is one of three essential observations to be made in any anemic subject, since it provides important information as to the pathogenesis of the anemia. An approximation of the number of reticulocytes present can be made by examining a well stained blood smear. The polychromatophilic cells (relatively large cells with slightly gray color) are the newly released erythrocytes which will stain as reticulocytes in vitally and supravitally stained preparations. These cells contain residual ribonucleic acid, which is diffusely scattered in the cytoplasm and adds a slight basophilic or polychromatophilic cast when dried blood is stained with a Romanovsky stain. Reticulocyte staining is a special procedure which makes these cells more easily visible by precipitating this basophilic material and staining it a bright blue.
2. Review other cells that may be found in a normal marrow smear, including lymphocytes, plasma cells, monocytes, megakaryocytes, and macrophages.
3. Examine slide 91: BIOPSY SECTION OF MARROW H&E stain:
again, this time looking for megakaryocytes.