BLOOD
Objectives
1. Introductory
| Choose your lab partners and lab station. | |
| Obtain your microscope and slide sets. | |
| Understand how to set up your microscope optimally. | |
| Observe how maladjustment of your microscope affects viewing quality. | |
| Appreciate the importance of scanning a slide to find the optimal viewing area. | |
| Become acquainted with the computer program. |
2. Examination of the cells of blood.
| Distinguish the five types of white blood cells. | |
| Distinguish small from large lymphocytes. | |
| Observe platelets. | |
| Carry out a differential white blood count. | |
| Use the oil immersion objective of your microscope. | |
| Visit
the demonstrations and the demonstration microscopes in the back room |
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Blue Histology |
Slide to examine
| D-29 | Blood smear |
Prologue
This initial laboratory session is introductory. We will look at a single microscope slide, a blood smear. Blood is one of the simplest tissues histologically. Being a fluid it has minimal organization. To prepare slide D-29 a small drop of blood was smeared across a glass slide and dried. It was stained with "Wright's stain", a variant of H & E, widely used for blood smears. Like H & E staining, acidophilic structures appear reddish and basophilic components blue.
Smears differ from the usual sections cut from blocks of fixed tissue in several important ways (You can see blood cells in a tissue section on slide D-76 and in the demonstration room). First, living cells tend to flatten out on a clean glass surface and therefore look twice as big as they would in a section. This is an advantage for identifying and studying blood cells but expect them to look smaller on your other slides. Also, the thickness of the meniscus of blood varies from one part of the slide to another. You can verify this by holding the slide up to the light and noting the changes in intensity of color across the slide, reflecting the numbers of cells at various places. Where cells jammed together viewing will not be optimal. Also, where the film of blood was too thin the surface tension will have ruptured the larger cells. Therefore choose a place where the cells are close together but not packed.
On some parts of your slide the cells will be poorly preserved. These bad regions are easy to discern by looking at the abundant red blood cells. The cells should be round, smooth, uniform and not schrunched together. If the red blood cells are distorted, look elsewhere for white blood cells . In particular, avoid the very edge of the slide where various artifacts can confuse you. Most of the problems that you will encounter in identifying the various types of white blood cells will come from looking at torn or badly preserved cells. Save yourself the hassle.
Blood contains about five million red blood cells or erythrocytes per cubic mm. This huge concentration gives blood its red color. However, an isolated unstained erythrocyte under the microscope will have only a straw color. With the Wright's stain these cells become pink. In addition to erythrocytes (erythro = red) five types of leukocytes (leuko = white) circulate in blood. These cells are nucleated and therefore very easy to spot as you scan across the slide, even at low power. Wright's stain colors DNA (and RNA) purple. Some leukocytes have numerous stainable granules in their cytoplasm (granulocytes). Others (agranulocytes) do not. The granulocytes are dead-end cells incapable of further cell division. As one aspect of this differentiation their nuclei are distorted into characteristic morphologies. The combination of appearance of the nucleus, amount of cytoplasm and presence of particular types of granules distinguishes the various categories of cell types. Your exercise for today is to find examples of at least four of these nucleated cell types and to become acquainted with their relative abundance
As an introductory general remark, your textbook will be a useful guide to these cells. We suggest that you read the short sections on each cell type in your text as you look at that cell. This is a painless and efficient way to read the text and will relate the factual information about the cell to its histological appearance. In fact, we recommend that you routinely read the appropriate parts of your text as you go through each lab. If you do so you can read most of the text in the time allotted for the lab. Of course we can only offer this avuncular advice. As the old saying goes, "you can lead a horse to drink but you can't make him water".
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Slide description
D-29 Blood smear (Wright's stain)
Hold your slide up and choose the general area to examine. Place it under the microscope and focus on that part with the lowest power objective. You will just be able to make out the individual erythrocytes. The leukocytes will barely be visible as blackish specks. If the pavement of erythrocytes is not even, move to a different region of the slide. In fact, it might be a good idea to scan over the entire slide at this minimal power before choosing an area for higher power examination. Spend a minute with the 10X objective just to note that as you scan around you can see that many of the purple staining nuclei are irregular in shape. Then go to the 40X objective. Most of your examination can be made with this lens. It, in combination with the 10-fold magnification from your eyepiece lens, give a total magnifying power of 400 power.
Start off examining the erythrocytes (red blood cells). Note their color. In appropriately stained areas of your slide they should appear pale pink. They have this simple color because they contain lots of hemoglobin protein but no nucleic acid. As a final step in their maturation the cells discard their nuclei. Keep in mind as you search for the rarer types of leukocytes to look only in areas where the preservation has been good enough to keep the erythrocytes round in shape.
If you look closely with your 40X objective you can see that erythrocytes stain more darkly around their rim than in the middle. This is because they are bi-concave disks. With this shape no hemoglobin molecule is more than about a micron from the cell membrane.
Erythrocytes are a useful yardstick because they are a uniform 7-7.5 microns in diameter and can be found in just about any tissue section. They are somewhat larger on your smear slide as mentioned above.
Neutrophils: The first leukocyte that you will come across probably will be a neutrophil since they account for about 70% of the nucleated cells. Neutrophils are easily recognized because their nuclei are partitioned into 3-5 lobes connected by thin strands of chromatin. These cells are sometimes called polymorphonuclear cells or simply polymorphs. Look at half a dozen examples to realize that you can routinely pick out neutrophils. These are the main cells of the body for phagocytizing bacteria. They contain thousands of very small pinkish granules which can be seen only under the highest power, and even then are often difficult to see. Sometime today look at several well preserved neutrophils with the oil immersion (100 X) objective to see if you can see these granules. To do this find a neutrophil using the 40 X objective. If your lenses are clean and all is set up right you should see a brilliant, sharp image. If not, try to clean the lenses with lens paper or call over one of the instructors. Now, with a good looking neutrophil in focus at 400 X rotate out the 40 X objective and place a small drop of immersion oil on the spot of the slide that you were looking at (it will be at the center of the circle that your light source illuminates). Next, rotate the 100 X objective into the oil. (Do not rotate the 40X objective past the drop of oil because, if you do, you will get oil on that lens. Big pain worth avoiding: You will have to carefully clean the oily lens using Windex and lens paper.) Look. The slide should be almost in focus because the lenses should be "par focal". Carefully adjust the focus so you do not grind the lens into the slide. Again you should see a brilliant image enlarged to show considerably more detail. If something is wrong check the adjustment of the diaphragm and condenser lens, and call an instructor over if you are still unhappy. It might be a good idea to ask an instructor to check that your view is as good as your microscope is capapble of giving, since, starting out, you may not really know what to expect.
The granules of neutrophils are about 1/2 micron across so that they are near the limit of resolution of your microscope. If everything is ideal you will be able to see that the cytoplasm is packed with these small structures and you should feel good about having your microscope operating optimally. If not, look at several other cells. (I'll have to admit that I rarely can discern these wretchedly small granules when I look at blood slides through your microscopes). Did you read what the granules are and what their function is? Good! Now wipe the oil off of your slide and lens and go back to your 40 power objective. This magnification should be adequate for examining the other cells today.
Another interesting feature of neutrophils is that they can show whether a blood sample is from a male or female person. Females have two copies of the X chromosome. Males have only one X plus, a minute Y chromosome. The second X chromosome of a female is heterochromatic. That is, it is highly compacted, darkly staining and genetically silent. This chromosome often forms its own small lobe, called a Barr body, or ,"drumstick" in neutrophils. The Y chromosome is also heterochromatic but too small to form a visible lobe. Maybe 5% of the neutrophils from a female show a drumstick. IQ question: Did we bleed a male or female dental student for this slide?
Lymphocytes are the next most abundant cells of blood, accounting for about a quarter of the leukocytes. Their nuclei are round (with maybe a dimple on one side) and they have almost no cytoplasm visible. That whole dark structure that you see is the nucleus. We speak of "lymphocyte" as a visual category of cells but actually it includes a number of distinct cell types with different functions. These cells are lumped together because they look similar. If you examine five or ten lymphocytes you will notice some variation variation. A minority show substantially more cytoplasm (although still only a skimpy amount). These are called "large lymphocytes" in distinction to "small lymphocytes" with a rim of cytoplasm usually only along one side of the nucleus. "Medium sized" lymphocytes have an intermediate appearance. Some large lymphocytes have a few large, obvious granules in their cytoplasm (but they are still considered to be agranulocytes, illustration). These large lymphocytes are natural killer cells that survey the body for incipient cancer cells. The small lymphocytes are mainly T cells ("T lymphocytes") with about 10% B cells. We will discuss the functions and developmental origins of lymphocytes in details later in the course.
Monocytes are the next most abundant cells in blood. They are larger than the largest lymphocyte. Their nucleus is also larger and stains more palely. It usually is bean shaped although, as the colored plate in your textbook indicates, it may acquire a very distinctive horseshoe shape. To find monocytes you will have to scan around. I suggest using the 40X objective, although you dudes with sharp eyes may be able to scan fast with the 10X objective to find the cell you want. When you nail a monocyte look at it under 40 X or even oil and verify that your text describes it correctly. Here are a number of monocytes from your slides. You may want to compare them with the lymphocytes shown above.
Acidophils: While you are scanning keep your eyes peeled for acidophils (Why are they also called eosinophils?) They have very obvious large reddish granules in their cytoplasm. Since they make up only 2-5% of leukocytes, examine any that you come across, even if you were looking for monocytes.
Basophils are rarer yet and most of you will not come across one. They have inky purple granules. Call out if you find the elusive basophil and the people next to you may come over for a peek and to congratulate you. It will be a good idea to read the brief descriptions of acidophils and basophils in your text (extra examples).
Platelets: Most of you will already have seen platelets but ignored them as just specks. They are membrane-surrounded fragments pinched off of marrow cells called megakaryocytes and are about 2 microns across (erythrocytes are about 7). Some platelets are scattered singly but 5, 10 or fifty might clump together. Read about platelets in your text. They barely show up on your slides but they have very important and complex functions.
The various types of leukocytes occur in blood in characteristic frequencies. These numbers are changed in particular ways by various pathological conditions. Therefore a "differential white blood count" is a common and important clinical procedure. Making an elementary hematological count on your slide will give you a reason to systematically look at examples of these cells and identify what you see. To do so, classify the first 100 leukocytes you see according to cell type, thereby estimating what percentage of the total white cell population each cell type constitutes. Just skip over cells that are broken apart and unidentifiable. If you have time to count only 50 or 25 cells that is OK. Compare your results with the typical frequencies below:
| Expected % | Your tally | Your % | ||
| neutrophils |
65-75 | _________________________ | _____ | |
| eosinophils | 2-4 | _________________________ | _____ | |
| basophils | 0.5-1.0 | _________________________ | _____ | |
| lymphocytes |
20-30 | _________________________ | _____ | |
| monocytes | 3-8 | _________________________ | _____ |
When you go to put your microscope away, you will appreciate it later if you carefully wipe the oil off of your slide and objective lens.
As a parting comment, you will have an opportunity to come back to this slide later (when we examine the formation of blood in bone marrow) so do not fret if you did not get as far today as you had hoped. You survived. See ya tomorrow.