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Band in Boston (Leukocytes #2)

Posted by Frederick Wasti
Nov 27 2013

[If you haven't yet seen my previous blog post, "And the Band Played On (Leukocytes #1)", please check it out first and then come back to this post, which is merely a continuation of it. Thanks.]

You might recall how my blood test results from 9/16 (from the Dana-Farber in Boston) looked, with their "odd" naming and counting of neutrophils -- well, here they are again, along with some more recent (and more "familiar" appearing) blood test results, as a comparison:

We saw, in my previous post, that the neutrophil count from 9/16, which (at first) seemed to be "missing", was indeed actually included, but that the neutrophils were (somehow) differentiated into Bands, Polys, and Metas. I still have to explain what Bands, Polys, and Metas represent, but first we have to go over how the five types of white cells are differentiated, and so that will be the subject of this post -- today you will see how we can visually tell the five leukocyte types apart.

[If you aren't familiar at all with the various types of blood cells, you might refer back to my post from 4/1/2012 on "Blood Cells 101".]

As mentioned in the last post, the five white cell types fall into two groups, "granulocytes" and "agranulocytes", the distinction being based upon whether the cells show specks or granules in the cytoplasm (i.e., the material within the cell that is outside the nucleus). Let's look at the granulocytes first:

If you look carefully at the above granulocyte drawings, you should notice visible reddish granules in the eosinophil and even more obvious bluish (or purplish) granules in the basophil, and perhaps, if you look really carefully, some faint, small reddish or pinkish granules in the neutrophil. (In contrast, the agranulocytes - lymphocytes and monocytes - do not show such granules in their cytoplasm.)

An erythrocyte (i.e., a red blood cell) is included in the above image to allow for estimating the size of the white cells. Since, when a blood smear is examined microscopically, the very numerous red cells will appear in every field of view, and since nearly all red cells are approximately the same size as each other, each red cell makes a useful "yardstick" for judging the relative sizes of white cells. You may have noticed above that the three granulocyte types are each somewhat larger than are red cells.

[This size estimation ability may not seem to be all that useful just yet, when looking at just the granulocytes, because each granulocyte is roughly the same size as every other granulocyte, but size estimation will seem to be more useful when we get to the agranulocytes, since lymphocytes are distinctly smaller than the granulocytes, while monocytes are often larger.]

Let's take a look specifically at the neutrophils first:

In the above microscopic image, there are four neutrophils. Note that they are distinctly larger than are the red cells, that their cytoplasm is "speckled" (i.e., granulated) (although not obviously so), and that their nuclei are multilobed (usually consisting of from two to five lobes, connected by thin filaments, not usually visible at this magnification). (You can also see that the more numerous red cells lack nuclei, and you should also be able to spot about a dozen or so very small platelets as well.)

Taking a closer look at another neutrophil, ...

... the small, faint, pinkish granules are (slightly) clearer, and the nuclear filaments can also be seen as well.

Of course, you already know that all these ~stained~ white cells no longer look white after staining, and it is, in fact, the nature of neutrophils and the stain that colors their granules that is the reason for their name. Blood stains generally are made of a mixture of several stains of varying pH values, and it is the ~neutral~ stain, and not the acidic or alkaline stains, that end up coloring their granules -- hence, they are called "neutro-phils" because they have an affinity for (or "love") the neutral stain in a blood stain mixture (the "phil" comes from the Greek word "philein", meaning "to love").

So, what do you have to remember about neutrophils, in order to be able to recognize them? Well, it really boils down to just three characteristics:

1. Neutrophils show faint reddish or pinkish granules in their cytoplasm.

2. Neutrophils show a multilobed nucleus (of two to five lobes). (In fact, if you spot a white cell with more than two lobes to its nucleus, it is likely to be a neutrophil.)

3. Neutrophils are "somewhat" larger (perhaps very roughly 1.5 to 2 times greater in diameter) than red cells, and are roughly the same size as eosinophils and basophils.

OK, let's take a look at eosinophils ("ee-oh-sin-oh-fills") next. Eosinophils are granulocytes that have (when stained) large reddish granules in their cytoplasm. Compared to the neutrophils, the granulation is much more obvious. Most of the time the nucleus in an eosinophil is bilobed (i.e., having two lobes) in appearance. Take a look at the following image, of two eosinophils and a neutrophil, to see how these two types of granulocytes compare in size, in granulation, and in nucleation:

By now, you're probably trying to figure out the etymology of the name "eosinophil". You already know that the "-phil" means "loving" -- it's just the "eosino-" part of the name that might be confusing -- it turns out that the granules of eosinophils have an affinity for a particular ~acidic~ stain with the name of ~Eosin~. [Eosinophils could also, because of this, be referred to as "acidophils" (and sometimes they are), but that's a considerably less common name for these particular white cells, for some inconsistent reason as of yet unbeknownst to me.]

Anyway, what do you have to remember about eosinophils, in order to be able to recognize them? Well, once again, there are just three characteristics to look for:

1. Eosinophils show fairly obvious reddish granules in their cytoplasm (redder and more distinct than the pinker and much smaller granules in neutrophils).

2. Eosinophils usually show a bilobed nucleus.

3. Eosinophils are "somewhat" larger (perhaps very roughly 1.5 to 2 times greater in diameter) than red cells, and are roughly the same size as neutrophils and basophils.

So, then we have the third type of granulocyte, the basophil, to consider:

Basophils are somewhat like eosinophils, except that the granules are more purplish or bluish, rather than more reddish, as in eosinophils. Sometimes this difference may not be overly obvious, though, since the reddish granule color of eosinophils can sometimes seem a bit on the purplish side, while the bluish granule color of basophils can also tend toward purplish. However, sometimes the basophils can also be seen to have granules that are somewhat larger in size, or "coarser" in appearance, than those seen in eosinophils.

In the image above, the two basophils seem not too similar in appearance. Part of this is because the granules do differ in color somewhat between the two cells, but part of the difference is also because the nucleus is closer to the viewer in the upper cell, while the nucleus is farther from the viewer in the lower cell, so that, in the upper cell, the nucleus is hiding many of the granules behind it, while, in the bottom cell, the granules are doing a good job at hiding the nucleus behind them.

Basophils get their name because they and up reacting to the ~basic~ (or alkaline) stain in a typical blood stain mixture.

OK, so what do you have to remember about basophils, in order to be able to recognize them? Well, once again, there are just three characteristics to look for:

1. Basophils usually show obvious bluish or purplish granules in their cytoplasm (sometimes appearing a bit more pronounced than the granules of eosinophils, but always ~much~ more obvious than the faint granules of neutrophils).

2. Basophils usually show a bilobed or U-shaped nucleus.

3. Basophils are "somewhat" larger (perhaps very roughly 1.5 to 2 times greater in diameter) than red cells, and are roughly the same size as neutrophils and eosinophils.

Let's now turn our attention to the agranulocytes (a.k.a., the non-granulocytes), which include the lymphocytes and monocytes:

First, it should be clear (so to speak) that, as the name suggests, there are ~no~ granules in the cytoplasm of the ~non~-granulocytes (i.e., the agranulocytes). Second, it is apparent that there are size differences to be considered with the agranulocytes. And, finally, it should be noted that agranulocytes have a relatively simple nucleus, generally U-shaped or even cup-shaped in appearance.

Taking a look at lymphocytes first, ...

... it can be seen that lymphocytes are quite a bit smaller than any of the other white cells, with a size generally roughly the same as that of the red cells (or a bit larger). There is not too much cytoplasm to look at, because the nucleus of a lymphocyte, approximately round or oval in shape, seems to fill up most of the inside of the cell -- however, there are still no granules to be seen in the limited amount of cytoplasm that is visible.

So, to most easily distinguish a lymphocyte, look for these three characteristics:

1. Lymphocytes do not show much cytoplasm, but what cytoplasm they do show is relatively clear, with no obvious granules.

2. Lymphocytes show a round or oval nucleus that seems to almost fills the cell.

3. Lymphocytes are easily the smallest of the white cells, being very close to the red cells in size.

In contrast, monocytes, ...

... the other type of agranulocytes, are generally the largest of all the white cells, with a diameter of often approximately twice that of red blood cells. [The Golgi apparatus shown in the above monocytes, which is involved with cell secretion, is not usually too visible in most blood smear preparations.]

The nucleus of a monocyte is quite large (although it does not come close to filling up such a large cell) and is usually U-shaped or cup-shaped in appearance, although sometimes it can be irregular (even "blobby") in shape.

There are no granules in a monocyte's cytoplasm -- although the cytoplasm may not be totally transparent, ...

... there still is a lack of any colored granules to be seen in it.

Therefore, the three main characteristics to look for in trying to distinguish a monocyte are:

1. Monocytes, being agranulocytes, lack any visible colored granules in their cytoplasm.

2. Monocytes show a large nucleus that is U-shaped, cup-shaped, or irregular in shape.

3. Monocytes are significantly larger than any other white cells, often having a diameter twice (or even more than twice) that of the red blood cell "yardsticks".

Now, please do not be intimidated by all these characteristics. First, I have tried to limit the similarities and differences to just three characteristics for each of the five types of white cells -- you really have to look for only the granulation (if any) in the cytoplasm, the shape of the nucleus, and the relative size of the cell. It's that "simple". Really. Besides, you really don't have to be "good" at distinguishing these cell types -- I am merely trying to ~acquaint~ you with the morphology of the types of leukocytes. So, no pressure, right?

Well, er, um, maybe just a wee bit of pressure: (<grin>) How about a little quiz, where you can prove (to ~yourself~) that you can indeed do a pretty good job at telling one white cell type from another? You can grade it yourself, and you can score it on a "pass-pass" basis. And it's an "open book" quiz (or an "open blog" quiz anyway - you can look at all the characteristics provided above. So, just how hard can it be, right? Eh?

Just to help you succeed more easily, I'll even summarize the most useful things to look for before we begin, OK?

1. If the nucleus shows three or more lobes, it's likely to be a neutrophil.

2. If the cytoplasm shows obvious reddish granules, it's likely to be an eosinophil.

3. If the cytoplasm shows obvious bluish granules, it's likely to be a basophil.

4. If the cell is a very small cell, similar to a red cell in size, it's likely to be a lymphocyte.

5. If the nucleus seems to fill up most of the cell, leaving room for very little cytoplasm, it's likely to be a lymphocyte.

6. If the cell is a very large cell, at least twice as wide as a red cell, it's likely to be a monocyte.

Finally, I'll provide one more hint for the quiz: I'm going to show five images, and they are for the five types of white cells, one image for each type. Therefore, you might be able to come up with the answer for a "tougher" image by "the process of elimination". OK? (The bad news is that, if you guess one incorrectly, and are using "the process of elimination", you probably have at least two of 'em wrong - <grin>.) Of course, "in the real world", in a blood lab, the technicians can't use "the process of elimination" when counting cells.

So, grab a scrap of paper (perhaps the back of an envelope from a bill that you weren't going to pay anyhow, say), number it from 1 to 5, and try to put an 'N', an 'E', a 'B', a 'L', or an 'M' for each cell type next to each number. (See? -- you don't even have to spell out the names -- easy, eh?)

Ready? Here goes -- here are your "unknown" white cell types:

I'll provide the answers in the next blog entry -- so, please stay tuned. (And don't lose your "scrap of paper".)

[Please note: I did intend to include a little information on the background for the expression "Banned in Boston" as part of this entry, but, in order to get this blog entry "out the door", I will delay writing that until probably sometime after posting the third issue of this series (which will include the answers to today's little quiz), entitled "Band of Blood-ers (Leukocytes #3)" - as I said very recently, "please stay tuned".]

Categories: General, Leukemia

And the Band Played On (Leukocytes #1)

Posted by Frederick Wasti
Nov 04 2013

When I was at Dana-Farber in Boston for blood testing and treatment on 9/16, I was a bit surprised to see some of the results of that day's blood testing. Here's what I saw:

The ~Complete~ Blood Cell Count seemed to be more or less normal -- WBC Count = 7.4 (in K/UL, or thousands of cells per microliter), RBC Count = 4.59 (in M/UL, or millions of cells per microliter), Platelets = 120 (in K/UL, or thousands of cells per microliter), while the "REFERENCE" column showed the "normal range" of each measurement. [And, yes, my platelet count was a bit low, but that's pretty common for me nowadays, and it's been moderately low for some time now -- I guess, for me, it's "the new normal" -- but, in any event, the Complete Blood Cell Count numbers were not surprising.]

It was the ~Differential~ White Cell Count numbers that seemed a bit unusual (at first), at least in how they were expressed.

Now, if you're a regular reader of this blog, and/or you know at least a little bit about the different types of white blood cells (but, as with me, "maybe not too much"), the terminology used in the above report might be a little confusing and/or surprising. You might say "OK, I can figure out the abbreviations for Lymphocytes, Monocytes, Eosinophils, and Basophils, but where are the Neutrophils?", and/or "So, what the heck do Band, Poly, and Meta mean?". Of course, at that point you might have then guessed that Band, Poly, and Meta likely refer somehow to neutrophils, and you would indeed be quite correct (Congratulations!). You have maybe already noted that, if you add the Band, Poly, and Meta percentages together, you get 98%, which is about what my neutrophil count usually has been for many months now.

But, just exactly what ~are~ Bands, Metas, and Polys (as the plural of Poly is usually spelled in hematology, rather than "Polies"), and just how are they related to neutrophils, and what's with the "-A" and "-M" suffixes? Well, those questions are just what this blog entry, and the next two, are all about.

Let's take the easy question first - let's take a look at the "-A" and "-M" suffixes. (Maybe you've guessed correctly already, and this will just confirm your hunches). As it turns out, "-A" stands for "auto" or "automatic", while "-M" stands for "manual" - not as in automatic or manual transmission, of course, but as in automatic and manual methods of counting blood cells.

For an ~automatic~ blood "count", a beam of light is passed through a small sample of blood (in a very thin clear tube), and then a special light sensor and some nifty computer processing are used to estimate (with surprising accuracy) the number of cells of each type, without actually counting any cells at all. [If your response to this brief description is "So how dey doo dat?", well, it's actually quite clever and also (I think) pretty interesting just "how dey doo dat", and so it will be the subject of a future blog entry...]

In contrast, for a ~manual~ blood count, a trained technician examines a "blood smear" (a thin film of blood) under a microscope, looking at the first 100 white cells he/she finds, and records how many neutrophils, eosinophils, basophils, lymphocytes, and monocytes are seen -- then, the number found of each type, out of 100 cells counted, is considered to be the extrapolated percentage of each type in the blood.

So, looking back at the blood test results from 9/16 quoted above, it would seem that my differential white cell count for that day was performed manually. (There is nothing unusual about how my cells are counted on any particular Dana-Farber day - sometimes they're counted manually, and sometimes they're "counted" automatically.)

Of course, if my cells are to be counted manually, a blood smear has to be properly prepared to start with. Two microscope slides are generally employed to make a blood smear - one will have a drop of blood on it, and the edge of the other will be used to smear the drop into a thin film. Note that the smear involves ~pushing~ the drop away from the technician, and not sliding the drop towards him/her:

[Amongst my many jobs and "careers" (<grin>), I've taught both biology and human anatomy and physiology at the high school and college levels. Back in the 1970s, it was a normal biology lab activity for students to obtain drops of blood from their fingertips and to make blood smears to examine under the microscope (and to test their blood for blood type and Rh factor). Nowadays, it would be very unusual (and quite inadvisable) for high school students to be handling blood at all, but we weren't overly worried about AIDS (and Hepatitis, etc.) back then. Even after we did become more concerned about the hazards of blood-borne diseases (and stopped using "live" blood in high schools), making blood smears was still a normal lab activity for adults in college (with suitable safety precautions in place, of course). So, I've demonstrated such techniques to students and monitored how students performed them many, many times.]

So, the purpose of making a blood smear is often to determine the proportions of the types of white cells (i.e., leukocytes) -- that's what a ~Differential~ Blood Count consists of.

As you can see in the above image, the five white cell types fall into two groups, "granulocytes" and "non-granulocytes" (the latter often called "agranulocytes" instead, the prefix "a-" meaning "not"). The distinction is based upon whether the cells show specks or granules in the cytoplasm (i.e., the material within the cell that is outside the nucleus). If you look very carefully at the above cell images, you should notice visible reddish granules in the eosinophil and even more obvious blue granules in the basophil, and , if you really look carefully, some faint, small reddish granules in the neutrophil. In contrast, the lymphocyte and the monocyte do not show granules in their cytoplasm.

[If you aren't familiar at all with the various types of blood cells, you might refer back to my post from 4/1/2012 on "Blood Cells 101". Even if you did view that previously, you might still want to take a quick look at it once again anyway, as I very briefly covered the normal proportions and functions of each blood cell type there, and I won't be repeating that info again here, instead emphasizing the ~appearance~ of each white cell type here.]

You may also notice that the nucleus (the darker, more-or-less central area in each cell) can vary in shape quite a bit, and (probably surprisingly to you, if your only familiarity with the nucleus in a cell is from images of generalized cells from "way back" in high school biology) that the nucleus might even seem to be in two or more pieces -- in the granulocytes, the nucleus typically has two to five lobes, connected together by thin filaments (which are not generally visible without very high magnification).

However, I should point out that the colors seen in the granules and in the nuclei in the image above are not real -- in fact, if you looked in a microscope at a blood smear prepared just as described above, you would see something such as the following:

In the above image, there are several dozen red cells (that don't look particularly reddish), and a white cell (of some sort) in the center. And that's the problem involved in looking at such a simple blood smear - white cells really are quite white (or clear, actually), and don't naturally look like the cells in the "granulocytes" and "non-granulocytes" image further above -- so, how can you tell which type of white cell each is?

What is missing inside the white cell in the image just above is a stain that would color the granules and the nucleus so that the otherwise nearly invisible nucleus and granules could provide visual clues for telling which type it is. However, the stain must do more than just make structures visible - it must also act as a ~differential~ stain, one that colors cell structures differently for each of the different cell types. So, a suitable differential stain (which is generally a combination of two or more stains mixed together) is normally added to the blood smear, as shown by the lower smear preparation in the following image:

[Historically, the most commonly used blood stain (and certainly the most commonly used blood stain in science education) has been Wright's stain (named for James Homer Wright, who developed it in 1902), but there are others. (I do not know what stain is used at D-F for performing manual white cell counts.)]

Well, anyway, I've covered in this post how a blood smear is prepared, and why it has to be stained, in order to use it to perform either a Total or a Differential Blood Count. I've provided short answers to questions about the unusual notations from 9/16 (i.e., the Bands, Polys, and Metas are types of neutrophils, and the "-A" and "-M" abbreviations are for automatic and manual counting techniques). However, I haven't yet really got into how the white cell types are (more or less) easily distinguished, nor have I explained exactly what the Bands, Polys, and Metas really represent -- these will be the subjects of the next two blog entries ("Leukocytes #2" and "Leukocytes #3"). Please stay tuned.

In the meantime, I will include a couple of the latest graphs, showing how things are still pretty "boring":

Let the band(s) play on... Thank you for reading along.

Categories: General, Leukemia