WEBVTT 1 00:00:09.633 --> 00:00:11.633 Welcome to this presentation. 2 00:00:11.633 --> 00:00:16.400 Back to basics helpful hints for evaluating peripheral blood cells. 3 00:00:16.400 --> 00:00:25.400 I'm Karen Brown, adjunct professor in the Medical Laboratory Sciences division and with Arup Laboratories at the University of Utah. 4 00:00:26.200 --> 00:00:32.933 I'm a long time educator, not only in hematology, but also phlebotomy and education courses. 5 00:00:33.700 --> 00:00:40.933 I have also been a medical laboratory scientist, medical laboratory technician, program director, and clinical coordinator. 6 00:00:42.600 --> 00:00:49.766 During this presentation, I hope that you will be able to describe the staining properties of the following cell structures. 7 00:00:50.100 --> 00:00:58.833 Nucleus, nucleolus, cytoplasm, mitochondria, Golgi body, and RNA containing organelles. 8 00:00:59.600 --> 00:01:08.000 Discuss morphologic features that identify mature and immature leukocytes, and finally develop strategies 9 00:01:08.133 --> 00:01:16.166 for distinguishing morphologic lookalikes, such as blasts from various lymphocytes, reactive lymphocytes, and monocytes. 10 00:01:16.800 --> 00:01:25.033 Segmented neutrophils and band or hyper segmented cells and selected erythrocyte shape variations. 11 00:01:26.133 --> 00:01:30.066 But before we get started, I want to share a story with you. 12 00:01:30.066 --> 00:01:38.600 And one of the aspects of hematology that really excites me is the connections that we can see between hematology in history. 13 00:01:38.966 --> 00:01:42.966 So I'd like to share with you the story of not. 14 00:01:42.966 --> 00:01:51.166 I think many of us are familiar with the mummification process used by ancient Egyptians 15 00:01:51.533 --> 00:01:58.200 to preserve their dead, but working class families could not afford this process. 16 00:01:58.566 --> 00:02:04.566 So poorer people actually place their dead in caskets and bury them in shallow sand graves. 17 00:02:05.033 --> 00:02:11.233 As it turns out, drying and desiccation actually could occur naturally under these conditions. 18 00:02:13.133 --> 00:02:20.500 Not was a 16 year old Egyptian weaver from the 20th dynasty, which was about 1150 BC. 19 00:02:21.133 --> 00:02:24.933 His body had been naturally dehydrated, 20 00:02:24.933 --> 00:02:29.933 and it was remarkably preserved and showed evidence suggestive of anemia. 21 00:02:31.266 --> 00:02:32.966 His remains were discovered 22 00:02:32.966 --> 00:02:40.666 in the late 1970s by a group of researchers from the University of Toronto, and an autopsy was performed. 23 00:02:40.800 --> 00:02:43.933 And this is where this information was revealed. 24 00:02:43.933 --> 00:02:51.900 And not only did NOx abdominal cavity show evidence of a parasitic infection, which turned out to be schistosomiasis 25 00:02:51.900 --> 00:02:59.800 and could explain the anemia, but the bladder of this young 16 year old contained intact red blood cells. 26 00:03:00.533 --> 00:03:04.500 And I just found that incredibly fascinating. 27 00:03:04.500 --> 00:03:11.266 And here is an electron micrograph of what it's over 3200 year old red blood cells. 28 00:03:11.566 --> 00:03:16.333 And these are believed to be the oldest red blood cells ever discovered. 29 00:03:16.333 --> 00:03:23.100 And just for comparison, I've included a schematic of modern, if you will, red blood cells. 30 00:03:23.200 --> 00:03:26.800 Just for comparison. 31 00:03:26.800 --> 00:03:32.566 So, as we can see, not much has changed in red blood cells over all of these many, many years. 32 00:03:33.733 --> 00:03:39.733 So while red blood cells have not changed in thousands of years, technology has. 33 00:03:39.900 --> 00:03:45.933 And today we have all kinds of instrumentation that can help us in hematology. 34 00:03:48.033 --> 00:03:54.500 We have instruments that are capable of classifying white blood cells across predetermined categories. 35 00:03:55.333 --> 00:03:59.666 These instruments can provide several parameters of useful information. 36 00:03:59.666 --> 00:04:05.666 They also allow characterization of red blood cells and platelet morphology. 37 00:04:05.933 --> 00:04:09.533 But not all laboratories have these instruments. 38 00:04:09.533 --> 00:04:12.866 And there can be a variety of reasons for this. 39 00:04:12.866 --> 00:04:17.233 Perhaps cost is one of those. 40 00:04:17.233 --> 00:04:23.233 So there are many reasons to support the microscopic review of peripheral blood cells. 41 00:04:23.366 --> 00:04:29.400 This review may be needed if the instrument data is suppressed because of specific error codes or flags. 42 00:04:30.333 --> 00:04:32.700 A smear review 43 00:04:32.700 --> 00:04:38.700 can provide information that may guide diagnosis or monitoring for various patient conditions. 44 00:04:39.200 --> 00:04:43.166 Can guide selection and monitoring of therapy. 45 00:04:43.166 --> 00:04:50.000 Can indicate if the therapy is effective, and can also indicate if there might be adverse effects from the treatment. 46 00:04:51.900 --> 00:04:52.233 There are a 47 00:04:52.233 --> 00:05:01.566 number of conditions in which the Hema gram or the CBC will be normal, but the peripheral blood smear may show some abnormalities. 48 00:05:01.966 --> 00:05:06.033 And I've just listed a few examples. 49 00:05:06.033 --> 00:05:10.666 So for instance, in hereditary astrocyte ptosis we may see 50 00:05:10.666 --> 00:05:17.266 several sites on the peripheral last year and polychrome measure in heterozygous beta thalassemia. 51 00:05:17.400 --> 00:05:21.200 We might see target cells hyperkalemia 52 00:05:21.200 --> 00:05:22.100 lead poisoning. 53 00:05:22.100 --> 00:05:26.300 We might see basically like stippling multiple myeloma. 54 00:05:26.300 --> 00:05:29.700 We might see Rouleau or plasma cells. 55 00:05:29.700 --> 00:05:34.133 Malaria. Of course, we could see the parasites in the red blood cells. 56 00:05:34.133 --> 00:05:39.600 And in infectious mono and other viral conditions, we might see reactive lymphocytes. 57 00:05:39.600 --> 00:05:43.200 So again the CBC parameters may be normal. 58 00:05:43.200 --> 00:05:47.000 But if we look at the peripheral blood smear we can see some of these abnormalities. 59 00:05:50.166 --> 00:05:54.200 So therefore looking at the smear again can be very important. 60 00:05:54.200 --> 00:05:57.800 But let's review some of the basics that are needed. 61 00:05:57.800 --> 00:06:03.100 As we're going to look at a peripheral blood smear. 62 00:06:03.100 --> 00:06:07.666 Certainly the quality of the smear is a good place to start. 63 00:06:07.666 --> 00:06:14.066 The quality of our review can only be as good as the quality of the peripheral blood smear we are going to examine. 64 00:06:14.733 --> 00:06:20.733 We have to have a peripheral blood smear that is well made and properly stained. 65 00:06:21.833 --> 00:06:26.100 Keep in mind that cells don't always appear picture perfect. 66 00:06:26.100 --> 00:06:28.333 They can be some variations. 67 00:06:28.333 --> 00:06:35.133 The actual staining of the color of the cells may vary from what we see in textbooks, or even in some images. 68 00:06:35.766 --> 00:06:41.900 And it, again, is also important to begin examining the smear in a good area of the smear. 69 00:06:43.500 --> 00:06:44.700 So let's talk a little bit 70 00:06:44.700 --> 00:06:50.700 about what it means to have a well prepared smear and a good stain. 71 00:06:51.000 --> 00:06:56.100 So the image on the right is showing a good peripheral blood smear. 72 00:06:56.100 --> 00:07:02.100 So the length of the smear itself should be about one half to three quarters the length of the slide, 73 00:07:02.300 --> 00:07:05.966 or about an inch to an inch and a quarter in length. 74 00:07:05.966 --> 00:07:11.966 It should be about 0.5 to a bit under of an inch wide. 75 00:07:12.300 --> 00:07:17.666 It should be free of scratches, streaks, holes or bubbles. 76 00:07:17.666 --> 00:07:23.833 It should be smooth and a thick area on the left on this slide where you can see the little circle. 77 00:07:24.200 --> 00:07:30.200 It is thicker and it progresses to a thin area and eventually what is called the feathered edge. 78 00:07:31.066 --> 00:07:35.700 Macroscopically, the slide should look a nice deep purple. 79 00:07:35.700 --> 00:07:44.000 And if we hold the slide up to the light, we can often see a rainbow of color in that feathered edge 80 00:07:45.666 --> 00:07:49.000 microscopically in the image on the left. 81 00:07:49.000 --> 00:07:51.333 Red blood cells should look pink. 82 00:07:51.333 --> 00:07:58.133 The nuclei in white blood cells should be purple, and you can see the nice purple small cells that are white cells. 83 00:07:58.466 --> 00:08:01.000 And it's a little hard to see the cytoplasm. 84 00:08:01.000 --> 00:08:09.766 But the cytoplasm should be blue as in lymphocytes or in the segmented cells that are shown here can be slightly pink or tannish. 85 00:08:10.966 --> 00:08:15.766 And platelets of course will appear very small and purple. 86 00:08:15.766 --> 00:08:21.966 And there are some platelets in the middle of the smear to the light, to the slightly to the left. 87 00:08:23.066 --> 00:08:28.366 Now let's look at a couple images that are not well stained and actually small. 88 00:08:28.366 --> 00:08:32.200 Variations in stain, color and intensity are to be expected. 89 00:08:32.200 --> 00:08:39.500 And anybody that is an experienced evaluator of a peripheral blood smear can learn to adapt to changes in the stain 90 00:08:40.133 --> 00:08:42.133 where it becomes particularly problematic. 91 00:08:42.133 --> 00:08:46.066 Or for individuals that are new to looking at peripheral blood smears. 92 00:08:47.700 --> 00:08:52.266 So the cell on the left is really an eosinophil, 93 00:08:52.266 --> 00:08:58.966 but the stain has is too alkaline, the pH is too high, and so it is too bluish. 94 00:08:58.966 --> 00:09:02.800 And you can notice the and the red cells in particular look kind of blue. 95 00:09:02.800 --> 00:09:09.166 So there in turn our eosinophilic granules are also not looking the bright orange that we would expect. 96 00:09:09.966 --> 00:09:17.466 On the right side we have washed out basal fills and water in the methanol during the stain process 97 00:09:18.166 --> 00:09:24.633 can cause bubbles and can cause cells to the base of hills, in particular to look washed out. 98 00:09:24.633 --> 00:09:31.466 Because these basic solid granules are water soluble and the granules may leach or lose their color 99 00:09:31.466 --> 00:09:37.466 during the staining process, particularly during the the rinse phase. 100 00:09:38.733 --> 00:09:43.233 Here is a slide that shows again a nice peripheral, 101 00:09:43.233 --> 00:09:50.000 blood film, and it shows the head where the or where the drop of blood is placed and the body of the slide. 102 00:09:50.000 --> 00:09:57.866 And you can notice that the slide goes from a thick area out all the way to the zona morphology, or what I call the feathered edge. 103 00:10:00.333 --> 00:10:00.700 Now, you 104 00:10:00.700 --> 00:10:06.700 might wonder why I included a picture of a cat with its tongue sticking out. 105 00:10:06.700 --> 00:10:15.166 I had the opportunity to serve as a consultant to teach and review a medical laboratory science program in Vietnam, 106 00:10:15.833 --> 00:10:21.933 and my counterpart, hematology instructor in this Vietnamese program 107 00:10:22.366 --> 00:10:28.700 used a cat's tongue to show his students where on a peripheral blood slide, they should evaluate. 108 00:10:28.833 --> 00:10:33.533 And that is, they should start at the tip of the cat's tongue. 109 00:10:33.533 --> 00:10:39.533 So I just thought this was a really cool analogy, and I want to share it. 110 00:10:40.700 --> 00:10:44.233 Now let's talk some more about our area for examination. 111 00:10:44.233 --> 00:10:47.233 If we look at the image on the top left. 112 00:10:47.233 --> 00:10:50.566 This is a low power view of the feathered edge. 113 00:10:50.566 --> 00:10:53.800 And this is actually really beyond the feathered edge. 114 00:10:53.800 --> 00:10:57.100 And you can see all of these gaps in the cells. 115 00:10:57.100 --> 00:11:00.733 And it's very hard. We don't have really any consistency. 116 00:11:00.733 --> 00:11:05.700 Red cells are not in a nice even distribution. There's too much spread there. 117 00:11:05.700 --> 00:11:11.300 So that is not a good area to evaluate. And let's skip over to the far right. 118 00:11:11.300 --> 00:11:17.833 And in contrast this is more down towards the the placement of the drop of blood, the thick area on the slide. 119 00:11:18.433 --> 00:11:26.233 And in contrast, you can see that this is very thick and it's really too thick of an area to evaluate. 120 00:11:26.366 --> 00:11:29.833 So the image in the middle is really that nice. 121 00:11:29.833 --> 00:11:36.500 Good area to begin or evaluation where red cells are nicely spread out and we can see our white cells spread out. 122 00:11:36.533 --> 00:11:39.000 So this is a low power view. 123 00:11:39.000 --> 00:11:43.300 Now the bottom two images show a higher power view. 124 00:11:43.300 --> 00:11:50.400 And if we look at the left image first we can use red blood cells as our guide to help us find a good area. 125 00:11:50.866 --> 00:11:58.400 The red blood cells should be barely touching or overlapping, and you can see what I call see the whites of their eyes. 126 00:11:58.433 --> 00:12:01.500 So you see those areas of central pallor. 127 00:12:01.500 --> 00:12:07.500 In contrast, on the right correlates to the image just right above it where it's too thick. 128 00:12:07.500 --> 00:12:14.066 And then when you're in an area of the smear that is too thick, red cells are going to look like they were and were low in and changed. 129 00:12:14.233 --> 00:12:15.900 And that is not normal. 130 00:12:15.900 --> 00:12:18.133 And you can see a white blood cell in this image. 131 00:12:18.133 --> 00:12:24.933 But it's very contracted, very small and it's too hard sometimes to distinguish what type of white blood cell we have. 132 00:12:24.933 --> 00:12:28.633 So you don't want to be in too thick an area either. 133 00:12:31.133 --> 00:12:36.000 So during the presentation I'm going to be inserting what I call knowledge assessment. 134 00:12:36.000 --> 00:12:42.933 And so if you have a pen and paper handy or somewhere to make a note, I'd like you to identify 135 00:12:43.133 --> 00:12:50.200 at least three problems that may impair an accurate evaluation of this particular peripheral blood smear. 136 00:12:50.500 --> 00:12:51.300 So make a note. 137 00:12:51.300 --> 00:12:57.300 Jot down what you think might be at least three problems. 138 00:13:07.400 --> 00:13:14.133 Here is my assessment of what the problems are and if you have additional ones, that's great. 139 00:13:14.600 --> 00:13:17.233 But I think the stain is too blue. 140 00:13:17.233 --> 00:13:23.233 And that can be, indicated by the bluish tint to all our red cells. 141 00:13:23.766 --> 00:13:26.366 It is also not a good area of the slide. 142 00:13:26.366 --> 00:13:29.700 Notice the large gaps between the red blood cells. 143 00:13:29.700 --> 00:13:34.033 There are clumps of red blood cells, particularly in that lower left area. 144 00:13:35.500 --> 00:13:39.833 And many of the red blood cells have lost their central pallor. 145 00:13:39.833 --> 00:13:45.833 So these are reasons that this would not be a very good smear to evaluate. 146 00:13:48.066 --> 00:13:54.066 Now let's look specifically at some of our cellular structures and how they stain with the right stain. 147 00:13:54.600 --> 00:14:05.466 So a nucleus will stain dark purple nuclei only within a cell, stain almost a gray color cytoplasm in cells stains blue. 148 00:14:05.866 --> 00:14:11.333 And I say in most cells because in some cells like our granulocytes, we will see some granules. 149 00:14:11.333 --> 00:14:13.700 And we have lost that bluish background. 150 00:14:13.700 --> 00:14:18.700 But cells that stain blue have retained RNA in their cytoplasm. 151 00:14:18.700 --> 00:14:24.600 Primary granules that appear in cells stain various shades of purple. 152 00:14:24.600 --> 00:14:34.966 Secondary granules will stain various shades of orange, as in eosinophils, deep purple as in base of cells, or more pink or tan. 153 00:14:35.300 --> 00:14:38.100 In the case of a segmented neutrophil. 154 00:14:38.100 --> 00:14:44.500 But we also have some organelles the Golgi body, vacuoles and mitochondria, which do not stain. 155 00:14:48.266 --> 00:14:50.266 Let's look now at some of these 156 00:14:50.266 --> 00:14:56.266 cellular, staining features within, selected cells. 157 00:14:56.766 --> 00:14:59.700 So in this image 158 00:14:59.700 --> 00:15:01.700 we have 159 00:15:01.700 --> 00:15:06.800 a dark staining nucleus which the, red arrows pointing to. 160 00:15:06.800 --> 00:15:10.100 Nice purple color. 161 00:15:10.100 --> 00:15:16.433 The black arrow is pointing to a nucleolus, and there is actually another nucleolus just adjacent to that one. 162 00:15:16.433 --> 00:15:21.400 But notice how it's much, lighter than the purple, almost a gray color. 163 00:15:21.400 --> 00:15:25.766 And then the green area is pointing to the cytoplasm that is blue. 164 00:15:25.766 --> 00:15:27.600 And it might look a little bumpy. 165 00:15:27.600 --> 00:15:31.633 You can see a little white, but it's not granular. 166 00:15:31.633 --> 00:15:34.700 This is an image showing a pro-military site. 167 00:15:34.700 --> 00:15:38.400 And this site highlights the primary granules. 168 00:15:38.400 --> 00:15:45.400 And primary granules can also be called nonspecific granules lysosomal granules or as or filler granules. 169 00:15:45.600 --> 00:15:53.466 And I'll just comment that as are affiliate because it's actually somewhat of a misnomer, because the prefix azore means blue, 170 00:15:54.200 --> 00:16:00.200 and actually just means it picks up the blue, or the purple kind of dye within a right stain. 171 00:16:00.400 --> 00:16:04.766 And this is how the primary granules appear on a right stain. 172 00:16:06.266 --> 00:16:07.400 This image shows a 173 00:16:07.400 --> 00:16:13.400 metamodel site, and this minimalist site has a nice, darkly stained nucleus 174 00:16:13.533 --> 00:16:20.800 that's very a nice purple, but it also has the secondary granules or nonspecific granules. 175 00:16:21.000 --> 00:16:27.000 And you can see right under the arrow there's a nice cluster of these pinkish granules. 176 00:16:27.600 --> 00:16:31.400 This is an image of a basal fill. 177 00:16:31.400 --> 00:16:37.966 And it shows also some immature granular granules sites in this image and also some mature neutrophils. 178 00:16:38.400 --> 00:16:45.666 But the basal cell is recognizable because of its dark purple, almost black staining secondary granules. 179 00:16:46.033 --> 00:16:50.833 And many times these granules obscure the nucleus as well. 180 00:16:50.833 --> 00:16:56.833 But again, just showing some additional, examples of these specific granules. 181 00:16:58.333 --> 00:17:04.266 Here we have a monocyte and the two arrows, the blue arrows are pointing to vacuoles. 182 00:17:04.266 --> 00:17:10.266 And what I want to emphasize with this particular monocyte is that the vacuoles are not staining. 183 00:17:10.400 --> 00:17:14.400 And I didn't highlight all the vacuoles. You can see some other vacuoles. 184 00:17:14.400 --> 00:17:18.933 It also looks like there might be some vacuoles in the nucleus, but I don't think they're really vacuoles. 185 00:17:18.933 --> 00:17:24.033 I think they're just more open areas that did not stain within, the nucleus. 186 00:17:25.133 --> 00:17:27.766 And then finally we have a plasma cell. 187 00:17:27.766 --> 00:17:32.633 And the red arrow in this plasma cell is pointing to the Golgi body. 188 00:17:32.633 --> 00:17:33.700 Okay. 189 00:17:33.700 --> 00:17:42.300 The Golgi body is very significant in plasma cells because it packages up all the immunoglobulin that the plasma cell is 190 00:17:43.333 --> 00:17:44.200 is making. 191 00:17:44.200 --> 00:17:52.533 But again, I want to emphasize that the Golgi body also caught a called a host hof, the Golgi or the host areas. 192 00:17:52.533 --> 00:17:58.100 And cells do not stain. 193 00:17:58.100 --> 00:18:03.500 Next, let's discuss how red blood cells are evaluated on a peripheral blood smear. 194 00:18:03.500 --> 00:18:08.500 And they're basically several areas in which we want to evaluate our red blood cells. 195 00:18:08.500 --> 00:18:10.766 We want to look at their shape. 196 00:18:10.766 --> 00:18:16.766 And the orange arrow is identifying a red blood cell that looks like it has a little slit 197 00:18:16.933 --> 00:18:21.300 and area of central pallor, in contrast to the other areas. 198 00:18:21.300 --> 00:18:26.133 Notice, and the other red blood cells have that bigger, rounder, pale area. 199 00:18:26.133 --> 00:18:27.066 And I actually, 200 00:18:28.066 --> 00:18:29.700 think this is just an artifact. 201 00:18:29.700 --> 00:18:35.700 But if you see many of these cells with a slit like this, it could potentially be a stem out of sight. 202 00:18:35.733 --> 00:18:41.500 But this case, I think it's just artifact because I don't see anything else in that image. 203 00:18:41.500 --> 00:18:44.700 The red arrow is showing a microsite. 204 00:18:44.700 --> 00:18:49.000 So we also want to evaluate our red blood cells for their size. 205 00:18:49.000 --> 00:18:53.666 So we want to look at their shape. They should be round. 206 00:18:53.666 --> 00:18:55.200 We want to look at their size. 207 00:18:55.200 --> 00:18:57.766 And they should be 208 00:18:57.766 --> 00:19:04.366 normally about the same size as the nucleus of a small resting lymphocyte. 209 00:19:04.566 --> 00:19:07.233 And I included a resting lymphocyte in this image. 210 00:19:07.233 --> 00:19:14.433 And if we look around at many of the other red blood cells, they approximate that size of the lymphocyte nucleus. 211 00:19:15.733 --> 00:19:20.266 We also look at red blood cells for their hemoglobin content or their chromium city. 212 00:19:20.266 --> 00:19:23.700 So we're really trying to evaluate the area of central pallor. 213 00:19:23.700 --> 00:19:29.700 So that area of central pallor should be about a third of the diameter of the cell. 214 00:19:32.833 --> 00:19:37.066 We also look for their distribution of the red cells on the smear 215 00:19:37.066 --> 00:19:40.933 and the black arrows pointing just to a little bit of overlap in the red cells. 216 00:19:40.933 --> 00:19:43.533 And you always expect to see a little bit of that. 217 00:19:43.533 --> 00:19:48.300 So this is within normal expectations for peripheral blood smear. 218 00:19:48.300 --> 00:19:53.466 It's when we have lots of overlap or lots of low or agglutination red blood cells. 219 00:19:53.466 --> 00:19:59.266 It becomes problematic. And then we also identify whether or not red cells have inclusions. 220 00:19:59.266 --> 00:20:04.500 And this purple arrow arrow is not pointing to an inclusion. Actually it's just pointing to an artifact. 221 00:20:04.500 --> 00:20:13.600 But we do want to evaluate to make sure that our cells do not have inclusions such as how jolly bodies and so stippling and so forth. 222 00:20:15.233 --> 00:20:16.066 Okay. 223 00:20:16.066 --> 00:20:17.933 Time for knowledge assessment. 224 00:20:17.933 --> 00:20:21.966 Again. 225 00:20:21.966 --> 00:20:26.866 Looking at this image, I'd like I'd like you to 226 00:20:26.866 --> 00:20:34.633 find at least three red blood cell characteristics that indicate this is not a normal peripheral blood smear. 227 00:20:35.166 --> 00:20:40.100 So again jot down or make a mental note 228 00:20:40.100 --> 00:20:46.100 of some characteristics that tell us this is not a normal smear. 229 00:21:05.833 --> 00:21:09.633 Here again is my assessment of why this is not normal. 230 00:21:09.633 --> 00:21:14.133 So the blue arrow is showing that we have microsites. 231 00:21:14.133 --> 00:21:20.133 And again, if you look right in the middle of this image, you can see a nice resting lymphocyte. 232 00:21:20.333 --> 00:21:24.900 So that nucleus of this, this particular lymphocyte okay. 233 00:21:24.900 --> 00:21:30.433 And compare it to the blue arrow red. So we can see for sure this red sale's very small. 234 00:21:30.433 --> 00:21:35.433 We can see other microsites within this image as well. I did not identify them all. 235 00:21:35.433 --> 00:21:38.366 The orange arrow down in the bottom left 236 00:21:39.300 --> 00:21:42.366 is pointing to a hypo chromic red cell. 237 00:21:42.366 --> 00:21:47.600 So we can see that area of central pallor is leaving just a thin rim of hemoglobin. 238 00:21:47.600 --> 00:21:51.300 So it is greater than a third of the diameter of the cell. 239 00:21:51.300 --> 00:21:54.800 The red arrow is pointing to a target cell. 240 00:21:54.800 --> 00:21:58.300 Target cells can be considered an abnormal shape change. 241 00:21:58.300 --> 00:22:02.733 They really circulate as bells, or sometimes called Mexican hats. 242 00:22:02.733 --> 00:22:05.866 And when we make the peripheral blood smear that bell, 243 00:22:05.866 --> 00:22:13.400 the top part of the bell gets smooshed down on the slide and and makes that target within the red blood cell. 244 00:22:14.100 --> 00:22:20.466 We also have nucleated red blood cells present as identified by the yellow arrow arrow at the top left. 245 00:22:21.566 --> 00:22:22.633 So that is abnormal. 246 00:22:22.633 --> 00:22:27.933 We should not see nucleated red blood cells in a normal peripheral blood smear. 247 00:22:27.933 --> 00:22:33.900 Sometimes we can see them in the peripheral blood smear of a newborn, but we should not see them otherwise. 248 00:22:34.966 --> 00:22:39.900 The green arrow is pointing to just a side or a fragmented cell. 249 00:22:39.900 --> 00:22:42.900 And this is another abnormal shape change. 250 00:22:42.900 --> 00:22:48.900 And we can see other sister sites on the image that I did not identify. 251 00:22:49.866 --> 00:22:55.433 And then the black arrow is suggesting that maybe there is an inclusion. 252 00:22:55.433 --> 00:22:58.766 It's a little hard to tell, but I think it might be a jolly body. 253 00:22:58.766 --> 00:23:04.766 And of course, on still images like this, it's hard to identify if we really were looking at the smear 254 00:23:04.900 --> 00:23:12.666 on a microscope, we could focus back and forth, and any inclusion that is in a cell will focus with the cell. 255 00:23:13.200 --> 00:23:17.100 If it's an artifact, it will focus in a different plane from the cell. 256 00:23:17.100 --> 00:23:23.033 So that can help us make that identification. 257 00:23:23.033 --> 00:23:28.433 Just as with red cells, we have certain criteria we want to use to evaluate our red cells. 258 00:23:28.433 --> 00:23:31.900 We do have criteria to identify white blood cells. 259 00:23:31.900 --> 00:23:37.900 And I will say that some of these criteria and characteristics also apply to red blood cells. 260 00:23:38.333 --> 00:23:42.600 But in general as cells mature, they get smaller. 261 00:23:44.066 --> 00:23:48.600 We also see changes to the nuclei as the cell matures. 262 00:23:48.600 --> 00:23:54.600 So the nuclear to cytoplasmic ratio is going to decrease as the cell matures. 263 00:23:54.833 --> 00:24:02.333 In some cases, as is as with red blood cells, the nucleus is completely excluded or booted out of the cell. 264 00:24:02.933 --> 00:24:10.433 In other cases, like with white blood cells, we'll see in neutrophils, for example, we'll see the nuclear shape change. 265 00:24:10.500 --> 00:24:13.033 We see segmentations form 266 00:24:13.033 --> 00:24:20.900 the chromatin within the nucleus changes from loose an open structure to one that is more condensed and clumped. 267 00:24:22.766 --> 00:24:27.000 Nuclei only in immature cells will be visible. 268 00:24:27.000 --> 00:24:30.966 But as cells mature we do not any longer see nuclei. 269 00:24:30.966 --> 00:24:34.400 Only cytoplasm becomes more abundant. 270 00:24:34.400 --> 00:24:38.966 SSL matures again because that nucleus is becoming smaller. 271 00:24:38.966 --> 00:24:44.566 And in cases of red blood cells, that cytoplasmic color changes from a blue to red. 272 00:24:44.566 --> 00:24:48.233 And in the case of granulocytes, we lose the blue color. 273 00:24:48.233 --> 00:24:52.033 But we gain granules within the neutrophils. 274 00:24:52.033 --> 00:24:57.200 So this schematic just tries to illustrate what I've just mentioned. 275 00:24:57.200 --> 00:25:03.233 So the top image shows changes that occur to the nucleus as the cell matures. 276 00:25:03.433 --> 00:25:06.366 So as we look from left to right we can see that 277 00:25:06.366 --> 00:25:12.366 a loose open chromatin structure gives way to a chromatin structure that is more dense and clumped. 278 00:25:12.633 --> 00:25:19.500 And in this example showing neutrophil, we see lobes form as the cell matures. 279 00:25:20.133 --> 00:25:28.833 We also lose nucleus ly as illustrated in that far left image and the subsequent one sent to the fully mature cell. 280 00:25:28.933 --> 00:25:31.533 We see no, not no nuclear ly. 281 00:25:31.533 --> 00:25:37.500 The bottom image highlights cytoplasmic changes that include color changes as the cell matures 282 00:25:37.500 --> 00:25:43.500 from blue, in this case of granulocytes to the pinkish, and then also the appearance of granules. 283 00:25:43.500 --> 00:25:49.800 And I'll just emphasize the cell that is the third one from the left, because note 284 00:25:50.000 --> 00:25:55.200 that we can sometimes see an overlap between our primary and our secondary granule. 285 00:25:55.200 --> 00:25:57.600 So you'll notice two different colors of granules. 286 00:25:57.600 --> 00:26:02.233 Eventually we will no longer see primary granules or those darker granules. 287 00:26:02.233 --> 00:26:03.566 As the cell matures. 288 00:26:05.066 --> 00:26:07.066 These two images are illustrating 289 00:26:07.066 --> 00:26:13.400 immature cells on the left we have black cells versus more mature white blood cells. 290 00:26:13.400 --> 00:26:15.733 On the right, the left image. 291 00:26:15.733 --> 00:26:20.733 You'll notice that several of the cells have a high NC ratio. 292 00:26:20.733 --> 00:26:22.766 Nuclei to cytoplasmic ratio. 293 00:26:22.766 --> 00:26:29.966 The chromatin is very loose and open, and we have a nice blue cytoplasm on the right. 294 00:26:30.300 --> 00:26:34.933 We can see again a cell slightly off to the left of center. 295 00:26:34.933 --> 00:26:38.100 It's a pro minor site and it has primary granules. 296 00:26:38.100 --> 00:26:41.333 And then we can see above it a meta monocyte for example. 297 00:26:41.333 --> 00:26:46.900 But notice also some mature neutrophils. And the chromatin is dense and clumped. 298 00:26:46.900 --> 00:26:49.033 And then we see the segmentations. 299 00:26:49.033 --> 00:26:55.266 So again just emphasizing changes in cell size as a cell the cells mature 300 00:26:55.500 --> 00:27:01.500 changes in the nuclear to cytoplasmic ratio the chromatin structure and the appearance of granules. 301 00:27:05.466 --> 00:27:09.433 Time for another knowledge assessment. 302 00:27:09.433 --> 00:27:15.433 Is a cell likely a mature or immature white blood cell? 303 00:27:15.533 --> 00:27:19.666 And what morphology suggest to you one or the other? 304 00:27:19.666 --> 00:27:25.666 Maturation category. 305 00:27:34.933 --> 00:27:40.100 I'm sure you all identify this as an immature 306 00:27:40.100 --> 00:27:42.900 cell. This is a blast cell. 307 00:27:42.900 --> 00:27:47.400 It is a large scale as identified by the red area arrow. 308 00:27:47.400 --> 00:27:52.266 It has a high nuclear to cytoplasmic ratio. 309 00:27:52.266 --> 00:27:58.133 The chromatin is loose and open, as illustrated by the purple arrow. 310 00:27:58.133 --> 00:28:03.800 The two green arrows are pointing to two different nuclei only within this cell. 311 00:28:03.800 --> 00:28:06.400 So that helps us know it is immature. 312 00:28:06.400 --> 00:28:12.400 And then we have a blue a granular cytoplasm. 313 00:28:17.833 --> 00:28:19.900 Even though we have criteria 314 00:28:19.900 --> 00:28:26.666 that can help us identify, in this case white blood cells, many times we have morphological lookalikes. 315 00:28:28.200 --> 00:28:29.100 And one of the 316 00:28:29.100 --> 00:28:35.100 concerns can be sometimes with black cells versus lymphocytes. 317 00:28:35.200 --> 00:28:37.900 On the left we have blacks. 318 00:28:37.900 --> 00:28:42.733 Blacks are usually larger cells in contrast to that lymphocyte on the right. 319 00:28:42.733 --> 00:28:44.400 But not always. 320 00:28:44.400 --> 00:28:49.033 And likewise lymphocytes are highly variable in their size. 321 00:28:49.033 --> 00:28:52.500 They're not always small as the one indicated on the right. 322 00:28:52.500 --> 00:28:58.500 They can be more moderate, moderately large, or even larger cells. 323 00:28:58.766 --> 00:29:05.333 Both cells can have a high nuclear to cytoplasmic ratio, as illustrated by both of these examples. 324 00:29:05.800 --> 00:29:09.900 So it becomes important to look at the chromatin structure. 325 00:29:09.900 --> 00:29:14.166 And for nucleus I. 326 00:29:14.166 --> 00:29:17.566 When we look at the image on the left we can see nuclei. 327 00:29:17.566 --> 00:29:24.333 All I present in many of those cells the chromatin tends to be more open and loose and in contrast 328 00:29:24.833 --> 00:29:28.866 the lymphocyte on the right has more dense and clumped chromatin. 329 00:29:34.700 --> 00:29:36.033 A helpful hint as we're 330 00:29:36.033 --> 00:29:43.033 trying to distinguish blacks and lymphocytes, it's important to look at all characteristics of the cell. 331 00:29:43.266 --> 00:29:52.566 So we want to look at cell size, nuclear size and shape, chromatin structure, the cytoplasmic amount and color. 332 00:29:52.966 --> 00:29:56.600 So don't focus in on just one aspect of the cell. 333 00:29:56.600 --> 00:30:02.600 Look at all of the features. 334 00:30:05.700 --> 00:30:07.233 On this image 335 00:30:07.233 --> 00:30:13.233 we have on the left a reactive lymphocyte, and on the right a blast. 336 00:30:13.500 --> 00:30:18.266 So the orange arrows are telling us, well, both of these cells are large. 337 00:30:18.266 --> 00:30:22.833 So that feature is not really going to help us make a distinction. 338 00:30:22.833 --> 00:30:26.733 The red arrow is looking at chromatin structure. 339 00:30:26.733 --> 00:30:33.733 And now on the image on the right with the blast the chromatin is a little more open and loose than on the left. 340 00:30:34.100 --> 00:30:39.000 But it's important to note that reactive lymphocytes sometimes two, can have a little more open 341 00:30:40.266 --> 00:30:42.866 chromatin structure. 342 00:30:42.866 --> 00:30:49.566 The green arrow on the right is showing a nucleolus, and on the left that's not really a nucleolus. 343 00:30:49.566 --> 00:30:56.333 If we look throughout the cell, this is just areas of chromatin within this particular reactive lymphocyte. 344 00:30:57.466 --> 00:31:01.933 And the black arrow is showing the cytoplasm. 345 00:31:01.933 --> 00:31:07.800 And in cases of the blast on the right, first of all, the cytoplasmic margins tend to be more uniform. 346 00:31:07.800 --> 00:31:13.633 And regular reactive lymphocytes can have more irregular margins. 347 00:31:13.633 --> 00:31:21.633 And oftentimes the margins of the cell skirt around or scalp around red blood cells, as we see in this case. 348 00:31:22.233 --> 00:31:29.866 And we can see, the orange arrow on the left is also showing a little bit of hyper coloration 349 00:31:29.866 --> 00:31:36.000 within the reactive lymphocyte that often occurs when the cell bunches up against a red blood cell. 350 00:31:36.000 --> 00:31:39.066 It's like taking a rug and bunching it up under the door. 351 00:31:39.066 --> 00:31:43.233 So it's squishing up that cytoplasm against the red blood cell. 352 00:31:43.233 --> 00:31:47.166 So I just wanted to emphasize particularly that 353 00:31:48.200 --> 00:31:52.000 reactive lymphocytes and blast can often be confused, probably more 354 00:31:52.000 --> 00:31:58.000 so than maybe a normal lymphocyte, unless the normal lymphocyte is a larger cell. 355 00:31:59.700 --> 00:32:05.566 So again, helpful hints when we're trying to identify reactive lymphocytes in blast cells. 356 00:32:05.566 --> 00:32:12.000 Keep in mind that homogeneity is key in Re and in identifying 357 00:32:12.733 --> 00:32:18.733 some malignant cells, and the image on both images on the left are showing black cells. 358 00:32:19.400 --> 00:32:29.166 By homogeneity I mean we have these abnormal cells that have all arisen from an abnormal clone, a monoclonal progression. 359 00:32:29.466 --> 00:32:38.766 So there's been one primary stem cell that has undergone some kind of mutagenic event and given rise to malignant daughter cells. 360 00:32:38.900 --> 00:32:42.333 So all the daughter cells look like that mother cell. 361 00:32:42.333 --> 00:32:46.833 So there's homogeneity within the, smear. 362 00:32:46.833 --> 00:32:51.400 So the top image, you notice they're all about the same size. They're all looking very similar. 363 00:32:51.400 --> 00:32:59.500 And in the bottom we have some smaller cells and some larger cells, but they're all consistently, either one or the other. 364 00:32:59.500 --> 00:33:01.500 It's very homogeneous. 365 00:33:01.500 --> 00:33:06.566 Reactive lymphocytes on the other hand or originate from a heterogeneous morphology. 366 00:33:06.566 --> 00:33:08.733 So it's a polyclonal response. 367 00:33:08.733 --> 00:33:12.433 So there is many lymphocytes for example, that encounter an antigen. 368 00:33:12.433 --> 00:33:15.000 And they all react differently to that antigen. 369 00:33:15.000 --> 00:33:19.566 So we can see lots of morphology changes within reactive lymphocytes. 370 00:33:19.566 --> 00:33:21.100 The other thing to consider 371 00:33:21.100 --> 00:33:27.300 is that if we have a malignant population of cells, we're usually going to have a higher white blood cell count as well. 372 00:33:27.866 --> 00:33:32.766 We may also see anemia. We may also see thrombocytopenia near these malignant cells. 373 00:33:32.766 --> 00:33:39.633 And particularly in vision that the cells and the bottom image these cells overtake the bone marrow. 374 00:33:39.633 --> 00:33:42.233 And they make it hard for blood cells to produce. 375 00:33:42.233 --> 00:33:48.933 So, you know, mature normally makes it hard for mega sites to mature normally and release platelets 376 00:33:50.066 --> 00:33:54.000 when we have a reactive process, as with, viral infection, 377 00:33:54.000 --> 00:33:59.900 we are not going to see generally anemia and thrombocytopenia. 378 00:33:59.900 --> 00:34:07.400 Another way to look at some of this I like to say there is no typical atypical lymphocyte atypical off site. 379 00:34:07.566 --> 00:34:11.100 We used to use the term atypical 380 00:34:11.100 --> 00:34:14.833 to describe lymphocytes that are not normal morphology. 381 00:34:14.833 --> 00:34:19.766 We've tended to not use that term so much anymore because it implies a malignant process. 382 00:34:19.766 --> 00:34:22.800 So that's why we tend we use reactive now. 383 00:34:22.800 --> 00:34:28.800 But I still like to go back to the adage that no typical atypical lymphocyte, because it reminds me 384 00:34:28.866 --> 00:34:35.300 of all the changes that we can see in a reactive process and emphasizes that heterogeneity. 385 00:34:36.400 --> 00:34:42.400 So in reactive lymphocytes the nucleus may be lobular, oval, notched, elongated, 386 00:34:42.566 --> 00:34:52.166 the chromatin may be more fine or not, there can be evident chromatin or there can be a smeary appearance to the nuclei. 387 00:34:52.233 --> 00:34:56.300 And sometimes you may even see nuclei or Lyon reactive lymphocytes. 388 00:34:56.300 --> 00:35:00.600 Because they are reacting, they're ready to respond to an antigenic challenge. 389 00:35:01.800 --> 00:35:04.533 Cytoplasmic features may vary as well. 390 00:35:04.533 --> 00:35:09.333 We can see coloration changes, but we could also see increased base of philia. 391 00:35:09.333 --> 00:35:12.000 And if you'll notice that middle 392 00:35:12.000 --> 00:35:18.000 length, reactive lymphocyte has a much darker blue cytoplasm, then do the other two cells. 393 00:35:18.933 --> 00:35:25.266 As I mentioned we can see skirting or scalloping of the cytoplasmic margins around red blood cells. 394 00:35:25.666 --> 00:35:31.666 And that is a nice and often consistent characteristic of reactive lymphocytes. 395 00:35:32.066 --> 00:35:37.233 And keep in mind, reactive lenses sites are usually large. So lots of variety. 396 00:35:37.233 --> 00:35:43.233 So this image exemplifies that variety that we can see. 397 00:35:45.300 --> 00:35:51.633 I do want to make a comment about reactive lymphocytes and monocytes, which sometimes also can be confused 398 00:35:52.066 --> 00:35:58.433 because monocytes can be be large and typically are large as are reactive lymphocyte lymphocytes. 399 00:35:58.433 --> 00:35:59.466 Sometimes. 400 00:35:59.466 --> 00:36:06.000 But in a monocyte, typically we can see a horseshoe like shape that can be round, 401 00:36:06.000 --> 00:36:12.000 folded, indented, that can occur in reactive lymphocytes, but less consistently. 402 00:36:12.366 --> 00:36:16.066 There are no nuclei that are present in monocytes. 403 00:36:16.066 --> 00:36:20.333 The chromatin can be lacy and open and more loose. 404 00:36:20.333 --> 00:36:23.433 Pure chromatin, however, is not often evident. 405 00:36:25.000 --> 00:36:30.066 Reactive lymphocytes do not usually have all the, 406 00:36:30.066 --> 00:36:32.200 vacuoles that we see in monocytes. 407 00:36:32.200 --> 00:36:34.500 So that's one thing to consider. 408 00:36:34.500 --> 00:36:42.733 The cytoplasm tends to be more blue or to varying shades within a reactive lymphocyte, where it's consistently a blue gray. 409 00:36:43.066 --> 00:36:45.633 In monocytes and tends to be grainy. 410 00:36:45.633 --> 00:36:53.133 Monocytes do not have granules, but their cytoplasm looks grainy or as if it has small sands or small grains of sand. 411 00:36:53.400 --> 00:36:57.700 And we don't tend to see that in reactive lymphocytes and monocytes. 412 00:36:57.700 --> 00:37:01.266 I like to think of as the bosses of the peripheral blood. 413 00:37:01.266 --> 00:37:05.866 And so they're just going to muscle their way and push red cells out of the way. 414 00:37:05.866 --> 00:37:11.433 Whereas really reactive lymphocytes tend to scallop or skirt around 415 00:37:11.433 --> 00:37:12.300 red blood cells. 416 00:37:12.300 --> 00:37:18.600 So just some other considerations when we're talking about reactive lymphocytes and other cells like monocytes. 417 00:37:20.700 --> 00:37:23.333 So again some helpful hints. 418 00:37:23.333 --> 00:37:26.333 Look for common features in each cell type. 419 00:37:26.333 --> 00:37:29.366 So the image on the left is showing some reactive lymphocytes. 420 00:37:29.366 --> 00:37:32.500 It's the smaller image. On the right is a monocytes. 421 00:37:32.500 --> 00:37:38.066 First of all they're both large cells. But then we want to try to find differences. 422 00:37:38.066 --> 00:37:43.866 So we notice that the cytoplasm in those reactive lymphocytes it's not a consistent color. 423 00:37:43.866 --> 00:37:46.633 There's some scalloping around the red cells. 424 00:37:46.633 --> 00:37:52.633 There's also some heavier blue at some of the interface where they have that blue gray 425 00:37:54.266 --> 00:37:56.400 cytoplasm in the monocyte. 426 00:37:56.400 --> 00:38:01.066 And now with the shape change in the monocyte versus the reactive lymphocytes. 427 00:38:01.066 --> 00:38:10.800 I also just want to make a comment that lymphocytes in particular are especially susceptible to any delay in preparing this slide and also 428 00:38:11.433 --> 00:38:12.400 in anticoagulant. 429 00:38:12.400 --> 00:38:14.633 So if we have an older blood sample 430 00:38:14.633 --> 00:38:22.700 we tend to see lymphocytes that do not look as nice and are harder to distinguish as either reactive or normal. 431 00:38:25.666 --> 00:38:30.300 Now let's shift and talk about some other morphological lookalikes. 432 00:38:30.300 --> 00:38:36.300 This time bands versus segmented neutrophils. 433 00:38:36.300 --> 00:38:40.933 So band cells as represented on the left 434 00:38:40.933 --> 00:38:45.366 have bridge of chromatin that separates the lobes. 435 00:38:45.366 --> 00:38:52.733 In contrast the nuclear lobes in a neutrophil segmented neutrophil as on the right connected by just a thin filament. 436 00:38:53.366 --> 00:38:55.866 And this schematic 437 00:38:55.866 --> 00:39:00.266 emphasizes what I'm trying to, describe for you. 438 00:39:00.266 --> 00:39:05.666 So the band cell again has a bridge of chromatin between the lobes. Chromatin. 439 00:39:05.666 --> 00:39:09.166 And in pair chromatin is visible within that bridge. 440 00:39:09.166 --> 00:39:16.200 And in contrast a stag or segmented neutrophil has lobes that are connected only by a filament. 441 00:39:16.400 --> 00:39:20.166 And you cannot really see any pair of chromatin within that filament. 442 00:39:20.166 --> 00:39:26.166 And it's a very thin strip. 443 00:39:26.966 --> 00:39:33.033 Even though we have criteria, it can still be very difficult to distinguish bands. 444 00:39:33.233 --> 00:39:40.266 There are some institutions that just put all our neutrophils together into one category. 445 00:39:40.266 --> 00:39:42.866 Don't do not try to separate band cells. 446 00:39:42.866 --> 00:39:48.800 But it's still important in some situations to actually count the band cells that are present. 447 00:39:50.266 --> 00:39:56.266 Sometimes bands can be confused with heterozygous Helga Hewett anomaly. 448 00:39:56.900 --> 00:40:01.666 And in the Pilger Hewett anomaly we have a nucleus that either 449 00:40:01.666 --> 00:40:08.400 consists of two symmetric, rounded lobes that are connected by a thin filament or 450 00:40:09.700 --> 00:40:18.433 the nucleus fails to segment at all and resembles a peanut or a dumbbell, and the cell on the right side shows more. 451 00:40:18.466 --> 00:40:20.866 The peanut formation. 452 00:40:20.866 --> 00:40:26.866 The chromatin generally is clumped and dense, for the most part because it is a mature cell. 453 00:40:29.700 --> 00:40:34.200 It is the cells that are connected by the thin filament 454 00:40:34.200 --> 00:40:42.600 that is often hard to distinguish from a true band cell, and I put a little image on the bottom. 455 00:40:43.766 --> 00:40:47.100 This is called a pince nez eyeglass. 456 00:40:47.100 --> 00:40:49.866 And this was very popular in the 1800s. 457 00:40:49.866 --> 00:40:53.666 So it just fits on the bridge of the nose. You don't have earpieces. 458 00:40:53.666 --> 00:41:01.066 And so sometimes the cells and it's not shown in with the white blood cells, but sometimes they're just connected by that thin strip. 459 00:41:02.533 --> 00:41:07.066 But it's more the dumbbell ones that can be confused with a band cell. 460 00:41:07.066 --> 00:41:09.966 So this image is actually showing a band. 461 00:41:09.966 --> 00:41:14.066 But we have to think about hue and anomaly and heterozygous power. 462 00:41:14.066 --> 00:41:19.500 Hewett is more common. The homozygous condition is is pretty rare. 463 00:41:19.500 --> 00:41:24.733 And we see that lobes tend to be just round singular ones. 464 00:41:24.733 --> 00:41:30.500 In heterozygous we can see a few round or single lobes, but most of them will be bi lobed. 465 00:41:30.500 --> 00:41:33.566 And all of the lobes nearly almost 100%. 466 00:41:33.566 --> 00:41:38.133 In heterozygous pale recue it will be either configuration. 467 00:41:38.133 --> 00:41:39.766 A unit lobe or bi lobe. 468 00:41:39.766 --> 00:41:44.866 So if you happen to see any cells with a nucleus with four or more lobes, you're not talking tail. 469 00:41:44.866 --> 00:41:46.933 Review it 470 00:41:46.933 --> 00:41:49.133 in Pilger. Hewett 471 00:41:49.133 --> 00:41:51.966 the lobes are going to be nice and symmetrical. 472 00:41:51.966 --> 00:41:56.166 Okay. In bands they're not so symmetrical on the lobes. 473 00:41:56.166 --> 00:42:01.933 Keep in mind that Pilger Hewitt is a benign condition and other hematologic parameters are going to be normal. 474 00:42:01.933 --> 00:42:05.733 So for example if we suspect we have a lot of bands, 475 00:42:05.733 --> 00:42:11.733 we would expect the white blood cell count to be increased, which could indicate to us we have an infection 476 00:42:11.933 --> 00:42:17.933 because if we have increase in bands, we could have potentially an infectious process. 477 00:42:17.933 --> 00:42:24.600 Not only would we see increase in bands as well, we might see other immature cells like metal monocytes. 478 00:42:24.900 --> 00:42:29.566 We could also see some toxic changes like dolly bodies or toxic granulation. 479 00:42:29.566 --> 00:42:33.600 If there is truly a left shift and an increase in 480 00:42:35.100 --> 00:42:38.033 band cells indicating an infection. 481 00:42:38.033 --> 00:42:43.933 And we don't see that in pale gray, Hewett. 482 00:42:43.933 --> 00:42:47.000 Another knowledge assessment 483 00:42:47.000 --> 00:42:56.066 when deciding if a cell is truly a band, focus on a the nucleus shape the color of the cytoplasm. 484 00:42:56.066 --> 00:43:02.433 Some c chromatin structure or D cell size, nucleus 485 00:43:03.133 --> 00:43:09.133 and cytoplasm. 486 00:43:13.733 --> 00:43:20.666 Well this again we want to focus on all of the features cell size nucleus and cytoplasm. 487 00:43:21.000 --> 00:43:27.366 And even though when we're trying to assess particular stage as with a band the cytoplasm may not be so important. 488 00:43:27.366 --> 00:43:34.333 You still want to evaluate that cytoplasm in this case it's really is the nucleus. 489 00:43:34.333 --> 00:43:39.000 But we will always look at all features before we make a decision. 490 00:43:39.000 --> 00:43:42.933 We don't focus on just one aspect and indicator. 491 00:43:42.933 --> 00:43:50.966 The tres here is just to remind us that we want to look at the entire forest as illustrated on the right, and not just one tree. 492 00:43:51.133 --> 00:43:55.300 Focus on all aspects of the information that is available to you. 493 00:43:57.300 --> 00:44:01.100 Now, I want to shift and look at some 494 00:44:01.100 --> 00:44:03.300 morphologic, 495 00:44:03.300 --> 00:44:09.100 confusions or lookalikes with red blood cells. 496 00:44:09.100 --> 00:44:14.600 We have partially circled red blood cells and oval sites that sometimes can be confused. 497 00:44:14.600 --> 00:44:21.166 Now, the images I selected here really aren't confusing, but but I wanted to pick the best ones to indicate to you 498 00:44:21.733 --> 00:44:25.200 the perfect morphology, if you will, for each cell type. 499 00:44:25.200 --> 00:44:32.433 Now partially circled red blood cells have repeatedly cycled through stages of cycling and non cycling. 500 00:44:32.933 --> 00:44:40.866 They circulate from an oxygen rich area to areas of the body that are lower in oxygen tension. 501 00:44:41.400 --> 00:44:47.400 And when that oxygen tension changes, red blood cells that are 502 00:44:47.400 --> 00:44:54.000 have the genetic mutation that the, for sickle cell will become a sickle cell. 503 00:44:54.366 --> 00:45:01.066 But when the cell circulates to an area of the body where oxygen tension is high, then they go back to the normal disk site. 504 00:45:01.733 --> 00:45:12.433 But these cells circulate millions of times through the the body, and eventually they can no longer become a classically shaped sickle cell. 505 00:45:12.433 --> 00:45:18.300 They're kind of stuck in between. And so we call them partially cycled or reversibly cycled cells. 506 00:45:18.300 --> 00:45:25.666 And so the black arrows identify some of these that are reversibly or partially cycled cells. 507 00:45:26.133 --> 00:45:31.633 Now the orange arrows, particularly the one on the left, looks more like an novelist site. 508 00:45:31.633 --> 00:45:38.400 But I want to emphasize that in the context of this mirror, with our other reversibly cycled or partially cycled cells, 509 00:45:38.533 --> 00:45:40.566 it is not is also a sickle cell. 510 00:45:40.566 --> 00:45:46.566 The, orange hair on the right looks more has more features suggestive of the sickle cell. 511 00:45:47.033 --> 00:45:52.800 Now on the right is someone that has her hereditary oval site, ptosis or hereditary ellipticity. 512 00:45:52.800 --> 00:45:53.466 Ptosis. 513 00:45:53.466 --> 00:45:58.800 So we can see, first of all, lots of ellipses are valid sites. 514 00:45:58.800 --> 00:46:01.233 And I just picked out a few of them with the blue arrows. 515 00:46:01.233 --> 00:46:08.033 So the sides are going to be parallel, the ends are rounded and we see a nice area of central power, 516 00:46:08.400 --> 00:46:15.700 but there's sometimes can be oval sites that are present that are not from someone that has a hereditary condition. 517 00:46:16.066 --> 00:46:21.000 Okay. So those sometimes can be interspersed or could be confused with 518 00:46:21.966 --> 00:46:27.200 well, particularly the partial or reversible sickle cells could be confused with those of our sites. 519 00:46:27.200 --> 00:46:33.133 So again these are showing classic conditions for each disorder to emphasize. 520 00:46:33.133 --> 00:46:36.600 However there can be changes. 521 00:46:36.600 --> 00:46:41.366 That could be confusing. 522 00:46:41.366 --> 00:46:48.366 So this image on the left is showing a nice classic, sickle cell anemia and nice classic classic sickle cells. 523 00:46:48.600 --> 00:46:54.166 So classic sickle cells do not have an area of central pallor. They have pointed ends. 524 00:46:54.166 --> 00:46:55.666 They're not rounded. 525 00:46:55.666 --> 00:47:00.166 Okay. So again is those partially or reversibly cycled cells. 526 00:47:00.166 --> 00:47:03.600 Sometimes that can be confused with a valid site. 527 00:47:03.600 --> 00:47:06.266 It's always important to review several fields. 528 00:47:06.266 --> 00:47:11.100 And I did not include multiple images in the context of this presentation. 529 00:47:11.100 --> 00:47:14.300 But keep in mind you do want to review several fields. 530 00:47:14.300 --> 00:47:21.266 Also, consider that anemia in sickle cell disease is very severe, so the hemoglobin can be very low. 531 00:47:21.566 --> 00:47:26.333 They could be slightly anemic, but not the severe anemia we see in sickle cell disease. 532 00:47:26.333 --> 00:47:28.433 Use other CBC parameters. 533 00:47:28.433 --> 00:47:33.533 As I mentioned, the hemoglobin, the hematocrit, the white count sometimes is slightly elevated. 534 00:47:33.533 --> 00:47:40.666 And somebody with sickle cell that we don't see in mitosis carefully evaluate red cell morphology 535 00:47:40.666 --> 00:47:46.800 through all of those characteristics of size, shape, chronicity distribution and inclusions. 536 00:47:48.233 --> 00:47:51.600 Patients with sickle cell anemia can have inclusions. 537 00:47:51.600 --> 00:47:56.366 And I think in a cell in the middle of this image, you can see a small how jolly body. 538 00:47:56.366 --> 00:47:59.833 We're not going to see inclusions like that in overall ptosis. 539 00:47:59.833 --> 00:48:03.166 We also see a little bit of polychrome on this slide. 540 00:48:03.166 --> 00:48:09.633 We could see some poly cremation of ALS eye ptosis, but generally not significant amounts of it. 541 00:48:09.833 --> 00:48:15.833 So keep in mind that looking at other parameters as well. 542 00:48:20.100 --> 00:48:20.400 Other 543 00:48:20.400 --> 00:48:26.533 red cells that can be confused and are morphologically possible lookalikes are acanthus 544 00:48:26.533 --> 00:48:33.333 sites, as illustrated on the left with yellow arrows, and then on the right it coincides with our black arrows. 545 00:48:35.166 --> 00:48:40.000 Acanthus sites have no area of central pallor. 546 00:48:40.000 --> 00:48:43.000 The projections are variable in the length. 547 00:48:43.000 --> 00:48:50.400 They're generally also less numerous than a can of sites, maybe only 3 to 12 projections. 548 00:48:51.700 --> 00:48:57.166 In contrast to the right, we do see areas of central pallor in a can of sites. 549 00:48:57.166 --> 00:49:01.133 The projections are more rounded and they're more numerous. 550 00:49:01.133 --> 00:49:04.200 In general, we can see 10 to 30. 551 00:49:04.200 --> 00:49:10.066 It kind of sites are most often seen as artifacts from the smear preparation, particularly 552 00:49:10.066 --> 00:49:17.300 if there's diffusion of basic substances from the glass slide into the cells during the smear preparation. 553 00:49:17.300 --> 00:49:23.300 That increases the and causes a kind of site formation. 554 00:49:24.966 --> 00:49:31.433 So hence, to help us distinguish acanthus sites and a kind of site, again, carefully review 555 00:49:31.433 --> 00:49:38.200 the smear and all aspects of red blood cell morphology, even though you think you're only dealing with a shape change. 556 00:49:38.400 --> 00:49:46.100 Evaluate all the features and be sure you're in an optimum area for evaluation of that smear. 557 00:49:50.800 --> 00:49:53.066 Another knowledge assessment. 558 00:49:53.066 --> 00:50:00.233 So which of the following is a useful strategy to distinguish irregular sickle cells from a valid site? 559 00:50:00.966 --> 00:50:04.000 Review 2 to 3 cells on the slide. 560 00:50:04.000 --> 00:50:07.333 Evaluate cells only for central pallor. 561 00:50:07.333 --> 00:50:11.033 Assess their regular cells for other abnormalities. 562 00:50:11.033 --> 00:50:15.033 Count the number of irregular cells in each field. 563 00:50:15.033 --> 00:50:21.033 So make a note of what you think the answer is. 564 00:50:25.800 --> 00:50:27.300 The is our answer. 565 00:50:27.300 --> 00:50:32.666 We want to evaluate the cells in question for other abnormalities. 566 00:50:32.666 --> 00:50:39.766 And in general, look at the entire smear for other abnormalities as in inclusions okay. 567 00:50:40.366 --> 00:50:46.533 That we would see particularly in sickle cell anemia that we would not see in oval site. 568 00:50:50.066 --> 00:50:55.666 I'd like to finish out the presentation with a case study. 569 00:50:55.666 --> 00:50:59.933 We have a 25 year old female who presents to her physician 570 00:50:59.933 --> 00:51:06.900 for routine physical examination and laboratory evaluation, and the results of her CBC are listed. 571 00:51:07.866 --> 00:51:09.200 And just a note 572 00:51:09.200 --> 00:51:16.500 whenever I see a listing of the CBC values, I start at the top and my work my way down through all the parameters. 573 00:51:17.166 --> 00:51:19.933 And as I work my way through. 574 00:51:19.933 --> 00:51:25.100 It does not appear that there are any real abnormalities from the CBC. 575 00:51:25.100 --> 00:51:28.766 Everything looks pretty much within normal range. 576 00:51:28.766 --> 00:51:34.400 Maybe a slightly elevated RTW and maybe slightly low 577 00:51:34.400 --> 00:51:40.400 hemoglobin hemoglobin, but nothing that is significant. 578 00:51:40.966 --> 00:51:48.900 But results were flagged as a suspected left shift, so a slide was made right stained for manual review. 579 00:51:49.533 --> 00:51:55.933 There was a new employee in hematology who performed a manual diff, and the results are listed. 580 00:51:58.900 --> 00:52:01.700 As you look at these results, 581 00:52:01.700 --> 00:52:05.466 something should trigger 582 00:52:05.466 --> 00:52:11.433 to you that there's an abnormal value and it looks like bands are high. 583 00:52:11.433 --> 00:52:18.600 We can see some bands on a peripheral blood smear, but normally about 2 to 6% we're told. 584 00:52:18.600 --> 00:52:26.666 Also, red cells look good, platelets look good, and morphology was good with platelets two number and and morphology. 585 00:52:29.066 --> 00:52:33.700 And here's two representative images of what was seen. 586 00:52:33.700 --> 00:52:38.866 The new MLS felt uncomfortable reporting so many bands, even with a flag, 587 00:52:38.866 --> 00:52:44.866 and asked for a smear review by her supervisor. 588 00:52:45.533 --> 00:52:48.666 So here are some questions to consider. 589 00:52:48.666 --> 00:52:54.666 What's significant about the band morphology that the new employee overlooked? 590 00:52:58.500 --> 00:53:01.533 And here's the image. 591 00:53:01.533 --> 00:53:04.733 And we notice we have 592 00:53:04.733 --> 00:53:07.900 a cell with a thin filament and two lobes. 593 00:53:07.900 --> 00:53:13.900 Kind of looks like those eyeglasses I mentioned earlier. 594 00:53:15.600 --> 00:53:18.066 The lobes are fairly symmetrical, 595 00:53:18.066 --> 00:53:24.066 roughly the same size one on the left, maybe just a tinge bigger, but roughly 596 00:53:24.233 --> 00:53:26.800 roundish and same size. 597 00:53:26.800 --> 00:53:32.800 The chromatin is dense and clumped, indicating to us that we do have a mature cell. 598 00:53:35.366 --> 00:53:41.566 And between the images I showed previously and this, all the cells I'm showing 599 00:53:42.600 --> 00:53:47.500 appear to be by lobed. 600 00:53:47.500 --> 00:53:53.100 So what are some results from the CBC that was listed to help us know 601 00:53:53.100 --> 00:53:59.100 what the cause is, or the etiology of the questionable cells? 602 00:54:02.833 --> 00:54:06.500 Well, we would expect, if we really did have a left shift 603 00:54:06.500 --> 00:54:12.500 and an increase in bands, that we would have an elevated white count. 604 00:54:13.233 --> 00:54:18.466 We might also have seen other immature cells like mantle, monocytes. 605 00:54:18.466 --> 00:54:27.000 We may also have seen if there was truly an infectious process, toxic granulation, doli bodies within those neutrophils. 606 00:54:27.933 --> 00:54:33.233 So what is the cause? 607 00:54:33.233 --> 00:54:33.966 Well you're right. 608 00:54:33.966 --> 00:54:39.966 If you say this is a review, an anomaly and most likely heterozygous from what we're seeing. 609 00:54:42.300 --> 00:54:48.633 What are some of the implications for the patient if these questionable cells are misidentified. 610 00:54:48.933 --> 00:54:52.600 And what if 30% bands were actually reported. 611 00:54:56.900 --> 00:55:00.466 Well or people review it is a benign condition. 612 00:55:00.466 --> 00:55:03.000 The cells are completely functional. 613 00:55:03.000 --> 00:55:07.566 Patients are healthy. 614 00:55:07.566 --> 00:55:13.533 If this report was sent with 30% bands, 615 00:55:13.533 --> 00:55:19.533 it may have subjected the patient to additional and unnecessary testing. 616 00:55:22.966 --> 00:55:30.066 As we conclude today, I want you to think about something that you've learned, and maybe it's something that you learn new 617 00:55:30.100 --> 00:55:38.066 during the presentation, or maybe it's something that you remembered from today's presentation that you forgot about. 618 00:55:40.366 --> 00:55:42.000 Write it down. 619 00:55:42.000 --> 00:55:44.866 Keep it with you. Share it with colleagues. 620 00:55:44.866 --> 00:55:48.733 Use it as a reminder to yourself 621 00:55:48.733 --> 00:55:54.733 as you're looking at peripheral blood smears. 622 00:55:56.100 --> 00:55:59.333 In summary, here are some takeaways. 623 00:55:59.333 --> 00:56:05.333 There are still many reasons to support the microscopic review of a peripheral blood smear. 624 00:56:05.366 --> 00:56:10.866 Keep in mind, however, there are pitfalls and problems when we prepare a blood smear 625 00:56:12.633 --> 00:56:16.266 because of the stain and adequacy of the stain, 626 00:56:16.266 --> 00:56:21.600 we want to be sure that we are in an optimum area of the smear. 627 00:56:21.600 --> 00:56:27.066 We also keep in mind that cellular structures are identified based on 628 00:56:27.066 --> 00:56:33.066 whether or not the stain binds the location of the structure and its color. 629 00:56:33.133 --> 00:56:37.633 So stain is very important. 630 00:56:37.633 --> 00:56:43.366 We evaluate red cells for their size, shape, hemoglobin content, or 631 00:56:43.366 --> 00:56:48.000 their distribution, and whether or not there are inclusions. 632 00:56:48.000 --> 00:56:57.133 White blood cells are identified based on cell size, the nuclear to cytoplasmic ratio, other nuclear features such as chromatin structure, 633 00:56:57.133 --> 00:57:04.933 presence or absence of nuclear, holy and cytoplasmic characteristics that include color and whether or not there's granules present. 634 00:57:05.766 --> 00:57:10.166 There are many cells that have a morphological look like, 635 00:57:10.166 --> 00:57:12.866 so be sure to review several fields. 636 00:57:12.866 --> 00:57:18.866 Look at the forest as I indicate in the top right image and not just one tree. 637 00:57:18.866 --> 00:57:25.166 Consider other hematologic parameters as with the white blood count, hemoglobin platelet count. 638 00:57:25.933 --> 00:57:29.266 Now I also included an image of a monocyte. 639 00:57:30.466 --> 00:57:35.033 If we look just at the nucleus we might think this is a band. 640 00:57:35.033 --> 00:57:42.933 So just to remind you to look at all features cells, eyes, nuclear features and cytoplasm. 641 00:57:42.933 --> 00:57:48.100 And as we see here the cytoplasm is a blue gray color with vacuoles. 642 00:57:48.100 --> 00:57:52.966 It is not the pink or ten color we would expect to see in a band. 643 00:57:52.966 --> 00:57:58.833 So look at all the features of a cell. 644 00:57:58.833 --> 00:58:00.700 References 645 00:58:00.700 --> 00:58:03.533 for your further 646 00:58:03.533 --> 00:58:07.566 digestion and review. 647 00:58:07.566 --> 00:58:09.133 And I want to thank you. 648 00:58:09.133 --> 00:58:12.000 And just by way of a note, 649 00:58:12.000 --> 00:58:19.866 I live in Utah and this is an image of West Canyon, which is part of Snow Canyon State Park in southern Utah. 650 00:58:20.133 --> 00:58:24.666 One of the gems of our state. Thank you again.