WEBVTT 1 00:00:08.133 --> 00:00:11.219 Welcome everyone to this webinar on laboratory 2 00:00:11.219 --> 00:00:14.222 diagnosis of Hemoglobin and Thalassemia. 3 00:00:19.477 --> 00:00:22.397 So these are the learning objectives. 4 00:00:22.397 --> 00:00:26.609 I'll go over the basic pathophysiology of hemoglobin properties. 5 00:00:27.277 --> 00:00:30.280 We'll talk about the expected test results. 6 00:00:30.280 --> 00:00:34.743 And then try to go over some of the commonly used methodology 7 00:00:34.743 --> 00:00:36.369 testing methodology. 8 00:00:36.369 --> 00:00:39.330 Also focusing a little bit on the molecular 9 00:00:39.539 --> 00:00:43.418 methods using ARM to use to diagnose these disorders. 10 00:00:43.793 --> 00:00:46.796 And also point out some of the common 11 00:00:47.714 --> 00:00:50.049 or appropriate test 12 00:00:50.049 --> 00:00:52.969 which the physicians or the laboratory 13 00:00:52.969 --> 00:00:55.597 should be ordering for to diagnose 14 00:00:55.597 --> 00:00:58.600 these disorders. 15 00:00:58.933 --> 00:01:01.269 So to start with, really basic 16 00:01:01.269 --> 00:01:04.981 hemoglobin is a combination of heme and globin. 17 00:01:05.440 --> 00:01:08.401 Hemoglobin is a really important molecule. 18 00:01:08.693 --> 00:01:12.697 It transfers oxygen from the lungs to the tissues 19 00:01:12.697 --> 00:01:16.117 and brings back carbon dioxide to back to 20 00:01:16.951 --> 00:01:19.120 the lungs is a trauma 21 00:01:19.120 --> 00:01:22.207 composed of four globin molecules two alpha globin 22 00:01:22.499 --> 00:01:25.502 and two beta globin or beta like globin. 23 00:01:26.252 --> 00:01:29.339 The alpha globin is composed of about 141 24 00:01:29.339 --> 00:01:33.301 amino acids, and beta globin is about 146 amino acids. 25 00:01:33.843 --> 00:01:37.305 Each of these globin chain has its own heme molecule, 26 00:01:37.764 --> 00:01:43.269 and this key molecule is really the most the molecule which transfers oxygen. 27 00:01:44.854 --> 00:01:46.689 However, for the purpose of today's 28 00:01:46.689 --> 00:01:51.110 talk, I'll be focusing mostly on the globin part, not on the heme part. 29 00:01:51.527 --> 00:01:54.155 The globin part is the one which is responsible 30 00:01:54.155 --> 00:01:57.158 for the hemoglobin properties and thalassemia. 31 00:01:58.868 --> 00:02:00.745 This is the ribbon diagram, a simple, 32 00:02:00.745 --> 00:02:03.873 very simplified ribbon diagram showing the hemoglobin. 33 00:02:04.415 --> 00:02:07.418 So these four different colored ones, 34 00:02:08.336 --> 00:02:11.339 are the. 35 00:02:12.006 --> 00:02:17.512 Test the polypeptide subunits showing the 36 00:02:18.930 --> 00:02:21.933 the different globin chains 37 00:02:21.933 --> 00:02:27.230 with the red ones being the heme molecule in the center. 38 00:02:27.605 --> 00:02:30.608 And this, this, heme is the, 39 00:02:30.650 --> 00:02:33.653 where the oxygen binds. 40 00:02:35.405 --> 00:02:37.865 This is a very basic, 41 00:02:37.865 --> 00:02:40.868 diagram showing the genetics of globin genes. 42 00:02:41.244 --> 00:02:45.873 So there are four globin alpha globin genes 43 00:02:46.958 --> 00:02:49.961 located on chromosome 16. 44 00:02:50.712 --> 00:02:53.256 There are two beta globin 45 00:02:53.256 --> 00:02:56.259 genes located on chromosome 11. 46 00:02:56.634 --> 00:02:59.554 However, the beta globin locus also 47 00:02:59.554 --> 00:03:04.350 has this delta gene and gamma genes 48 00:03:05.852 --> 00:03:08.855 located on the same locus. 49 00:03:08.855 --> 00:03:13.026 The. As I mentioned earlier, the hemoglobin is 50 00:03:13.651 --> 00:03:15.611 is seen in a traditional form. 51 00:03:15.611 --> 00:03:18.573 So the combination of alpha 52 00:03:19.115 --> 00:03:22.827 and beta is the adult hemoglobin. 53 00:03:23.161 --> 00:03:28.791 The combination of alpha and delta is the hemoglobin A2, 54 00:03:28.791 --> 00:03:33.463 which I will talk about in a little bit more detail in my next few slides. 55 00:03:34.255 --> 00:03:37.341 The combination of alpha and gamma 56 00:03:37.633 --> 00:03:41.053 is the hemoglobin F or the fetal hemoglobin. 57 00:03:42.430 --> 00:03:45.433 So the next slide 58 00:03:46.225 --> 00:03:49.729 shows the the hemoglobin development. 59 00:03:49.896 --> 00:03:51.939 Switching. 60 00:03:51.939 --> 00:03:54.942 So as I alluded earlier, 61 00:03:55.067 --> 00:03:59.739 the the the the embryonic counterpart 62 00:04:00.156 --> 00:04:04.202 is the zeta and the epsilon for the alpha and beta globin. 63 00:04:04.660 --> 00:04:08.581 And once the zeta and epsilon, it's only expressed 64 00:04:08.581 --> 00:04:11.584 only for a few weeks of life. 65 00:04:12.376 --> 00:04:15.379 And after, 66 00:04:15.546 --> 00:04:18.132 the expression of zeta and epsilon goes 67 00:04:18.132 --> 00:04:21.344 away, the gamma comes over, 68 00:04:22.470 --> 00:04:23.888 and stage 69 00:04:23.888 --> 00:04:29.310 stays till like, about few weeks or maybe around 3 to 4 months after life. 70 00:04:29.310 --> 00:04:31.896 And then its expression goes down. 71 00:04:31.896 --> 00:04:34.982 However, once alpha alpha comes it 72 00:04:34.982 --> 00:04:37.985 it remains constant throughout life. 73 00:04:38.277 --> 00:04:40.780 As we can see here, 74 00:04:40.780 --> 00:04:42.031 the in a does. 75 00:04:42.031 --> 00:04:47.286 The predominant hemoglobin is the combination of alpha and beta. 76 00:04:47.870 --> 00:04:49.038 This is the predominant. 77 00:04:49.038 --> 00:04:52.750 It forms the stroma in kids or in neonates. 78 00:04:52.750 --> 00:04:58.005 The predominant hemoglobin is the combination of gamma 79 00:04:58.381 --> 00:05:01.384 and alpha, which is the fetal hemoglobin. 80 00:05:04.470 --> 00:05:06.514 This is the normal adult 81 00:05:06.514 --> 00:05:09.183 human hemoglobin composition. 82 00:05:09.183 --> 00:05:12.603 So predominant hemoglobin in an adult is the hemoglobin 83 00:05:12.603 --> 00:05:16.983 A, which is about 96 to 97% 84 00:05:16.983 --> 00:05:19.986 and is a tetra mode of alpha two and beta two. 85 00:05:21.445 --> 00:05:25.324 Hemoglobin A2, as I alluded to at about 86 00:05:25.324 --> 00:05:29.161 this earlier, is a two channel of alpha two and delta two, 87 00:05:29.620 --> 00:05:32.707 and it's seen in about a percentage of, 88 00:05:33.124 --> 00:05:36.085 anywhere from 2 to 3.7%. 89 00:05:36.085 --> 00:05:39.130 And it's dependent upon, upon the laboratories, 90 00:05:39.130 --> 00:05:43.884 the reference range of this, our reference cutoff is 3.7. 91 00:05:43.884 --> 00:05:47.680 So any time we see more than 3.7 we call it increased. 92 00:05:49.265 --> 00:05:52.268 Hemoglobin F is seen in about 1%. 93 00:05:52.727 --> 00:05:57.356 The percentage of hemoglobin A2 is really important for our diagnostic 94 00:05:57.356 --> 00:06:01.235 purposes, especially for the diagnosis of beta a similar trait. 95 00:06:01.652 --> 00:06:04.113 So as we can go back and look at this slide. 96 00:06:04.113 --> 00:06:08.743 So beta thalassemia is due to decrease in this beta globin gene. 97 00:06:09.160 --> 00:06:12.663 So what happens when there is a decrease in this beta globin gene. 98 00:06:12.955 --> 00:06:16.250 Since delta and gamma is on the same cluster, 99 00:06:16.542 --> 00:06:19.545 Delta and gamma tries to compensate for it 100 00:06:19.545 --> 00:06:22.548 by increasing its expression. 101 00:06:22.965 --> 00:06:25.968 So we see increased in hemoglobin A2 102 00:06:26.218 --> 00:06:29.472 which is a trauma of alpha two and delta two. 103 00:06:30.014 --> 00:06:33.601 And we can also see increase in hemoglobin F 104 00:06:33.893 --> 00:06:36.896 which is a stronger of alpha two and gamma. 105 00:06:36.937 --> 00:06:40.983 However, the increase in hemoglobin F is not that reliable. 106 00:06:40.983 --> 00:06:43.527 And it can be seen in many other conditions. 107 00:06:43.527 --> 00:06:47.990 And like some hematological malignancies drugs 108 00:06:48.407 --> 00:06:51.786 can also some of the drugs can also cause increase in hemoglobin F. 109 00:06:52.203 --> 00:06:57.249 However the total amount of alpha two and delta which is hemoglobin A2 110 00:06:57.500 --> 00:07:02.880 is really very specific for the diagnosis of beta thalassemia trait. 111 00:07:04.715 --> 00:07:09.929 So. Going back, to 112 00:07:10.721 --> 00:07:13.682 this slide, the hemoglobin ofthese 113 00:07:13.682 --> 00:07:16.185 for the purpose of definition, 114 00:07:16.185 --> 00:07:19.563 the hemoglobin properties are defined as 115 00:07:20.648 --> 00:07:23.651 structural problems. 116 00:07:23.692 --> 00:07:26.821 So here there are mutations in either 117 00:07:26.821 --> 00:07:30.908 alpha or beta globin gene that leads to change 118 00:07:30.908 --> 00:07:35.579 in the structure of these molecule, either alpha molecule or beta molecule. 119 00:07:35.871 --> 00:07:41.585 It can be due to any molecular change like substitution, deletion or insertions. 120 00:07:42.044 --> 00:07:46.215 The most common ones are sickle cell hemoglobin C, hemoglobin E, D, 121 00:07:46.298 --> 00:07:48.259 and all Arab. 122 00:07:48.259 --> 00:07:51.095 Majority of them are benign, however, and discovered 123 00:07:51.095 --> 00:07:54.974 incidentally and really do not cause any clinical problems. 124 00:07:55.474 --> 00:07:58.561 But as we know, many of these can cause, 125 00:07:59.645 --> 00:08:03.524 problems and caused severe hemolytic anemia due to the shape of their, 126 00:08:04.275 --> 00:08:05.526 due to change in the shape 127 00:08:05.526 --> 00:08:08.612 of the red blood cells like hemoglobin S or sickle cell disease. 128 00:08:09.446 --> 00:08:13.492 They can also form crystals like in hemoglobin C disease. 129 00:08:13.867 --> 00:08:17.079 Some of these, change in the structure 130 00:08:17.079 --> 00:08:22.209 can lead to unstable globin chains and can form 131 00:08:22.209 --> 00:08:25.421 Hanes bodies because of the unstable nature of the globin. 132 00:08:26.422 --> 00:08:27.339 Some of these can 133 00:08:27.339 --> 00:08:30.884 also cause change in the oxygen affinity 134 00:08:31.051 --> 00:08:34.263 and lead to high affinity or low affinity hemoglobin S, 135 00:08:34.305 --> 00:08:37.308 although those are rare. 136 00:08:39.476 --> 00:08:40.477 In contrast, 137 00:08:40.477 --> 00:08:44.940 the thalassemia are defined for definitive purposes are defined 138 00:08:44.940 --> 00:08:49.653 as quantitative reduction in either alpha or beta chain. 139 00:08:49.987 --> 00:08:55.159 So if there is decrease in alpha chain, which we will call it alpha thalassemia, 140 00:08:55.659 --> 00:08:58.954 if there is decrease in beta chain, we call it beta thalassemia. 141 00:09:00.372 --> 00:09:03.625 And the molecular change could be large 142 00:09:03.625 --> 00:09:06.754 deletions, point mutations, small insertions or deletion. 143 00:09:07.046 --> 00:09:11.759 One of the important thing which is important to remember for 144 00:09:12.760 --> 00:09:17.097 for the molecular change in beta thalassemia versus alpha thalassemia is 145 00:09:17.723 --> 00:09:21.393 beta thalassemia is majority of them are due to single 146 00:09:21.393 --> 00:09:24.688 point mutations, small insertions or deletions. 147 00:09:25.606 --> 00:09:29.401 In contrast, alpha thalassemia is majority of them. 148 00:09:29.401 --> 00:09:33.697 About 95% of them are due to large deletions in the alpha genes. 149 00:09:34.323 --> 00:09:38.202 And this molecular background becomes very important 150 00:09:38.202 --> 00:09:43.415 for our diagnostic purposes when we are, designing the molecular tests. 151 00:09:44.833 --> 00:09:46.919 Beta thalassemia is 152 00:09:46.919 --> 00:09:50.047 can be beta zero, which is like complete absence 153 00:09:50.047 --> 00:09:53.050 of beta production, or beta plus, 154 00:09:53.467 --> 00:09:56.512 which is, which can be quite variable 155 00:09:56.970 --> 00:10:00.099 depending upon the type of mutation a patient has. 156 00:10:00.307 --> 00:10:04.770 It could be really minor, mild, it can cause mild anemia 157 00:10:04.770 --> 00:10:08.107 or it can cause severe anemia depending upon 158 00:10:08.107 --> 00:10:11.110 the production of beta chain. 159 00:10:11.735 --> 00:10:16.031 One of the other important thing, which is important I want to point 160 00:10:16.031 --> 00:10:19.868 out, is the path of pathogenesis or pathophysiology. 161 00:10:20.619 --> 00:10:23.747 Regarding this thalassemia, beta thalassemia 162 00:10:23.789 --> 00:10:26.959 major is a major health issue and caused 163 00:10:26.959 --> 00:10:30.128 severe to severe hemolytic anemia. 164 00:10:30.587 --> 00:10:33.131 And majority of these patients are transfusion 165 00:10:33.131 --> 00:10:36.134 dependent and might need weekly transfusion. 166 00:10:36.510 --> 00:10:40.264 Because what happens when there is a beta thalassemia? 167 00:10:40.848 --> 00:10:43.642 There is decrease in beta production. 168 00:10:43.642 --> 00:10:46.186 So beta as we can recall, 169 00:10:46.186 --> 00:10:49.189 beta beta always binds to alpha. 170 00:10:49.439 --> 00:10:51.984 So when there is a decrease in beta production, 171 00:10:51.984 --> 00:10:55.737 there's free alpha subunits freely circulating. 172 00:10:56.530 --> 00:11:00.534 These free alpha sub subunits are really toxic 173 00:11:00.534 --> 00:11:04.830 to the red blood cell membrane and cause severe hemolytic anemia. 174 00:11:05.622 --> 00:11:09.751 So that's why beta thalassemia major is a major health problem and caused 175 00:11:09.751 --> 00:11:14.798 severe severe hemolytic anemia in comparison to beta plasma alpha color. 176 00:11:14.840 --> 00:11:17.551 Seniors. However, are not that, 177 00:11:18.760 --> 00:11:19.261 bad and 178 00:11:19.261 --> 00:11:22.264 doesn't really cause that much of severe hemolytic anemia. 179 00:11:22.431 --> 00:11:26.101 Even three alpha gene deletion would be, 180 00:11:26.852 --> 00:11:31.106 maybe moderately hemolytic and might not need transfusion 181 00:11:31.481 --> 00:11:36.445 because the free beta subunit seen in alpha thalassemia can form 182 00:11:36.445 --> 00:11:40.115 Tetra mode in itself and are not that toxic. 183 00:11:40.324 --> 00:11:42.909 To the red blood cell membrane. 184 00:11:42.909 --> 00:11:45.537 Moving on to the next slide. 185 00:11:45.537 --> 00:11:48.540 Demographics of thalassemia. 186 00:11:51.418 --> 00:11:53.045 Knowledge about the background 187 00:11:53.045 --> 00:11:56.214 ethnicity is really important for the thalassemia diagnosis. 188 00:11:56.423 --> 00:11:59.343 So in some of these disorders, thalassemia disorders 189 00:11:59.343 --> 00:12:04.056 are prevalent in certain ethnic population most commonly seen 190 00:12:04.056 --> 00:12:08.477 in Mediterranean region, African, Western and Southeast Asia, India and Burma. 191 00:12:09.311 --> 00:12:12.272 One of the other important thing which I want to point out 192 00:12:12.272 --> 00:12:16.318 is the distribution of these thalassemia parallels that of Plasmodium 193 00:12:16.318 --> 00:12:20.530 falciparum, or the one of the severe form of malarial infection. 194 00:12:21.531 --> 00:12:24.493 The theory is, 195 00:12:24.493 --> 00:12:28.705 the patients who had thalassemia, the, 196 00:12:29.748 --> 00:12:34.211 the complications of malarial infections were 197 00:12:34.669 --> 00:12:39.549 they were less severe than those patients who didn't have a severe. 198 00:12:39.758 --> 00:12:43.678 So these patients had been selected out in like thousands of years. 199 00:12:43.804 --> 00:12:46.807 So that's why we see distribution of these 200 00:12:46.890 --> 00:12:49.893 these disorders thalassemia, sickle cell anemia, 201 00:12:50.477 --> 00:12:54.856 d6, PD deficiency, paralleling with the Plasmodium 202 00:12:54.856 --> 00:12:57.859 falciparum infection. 203 00:12:58.819 --> 00:13:00.821 Moving on to alpha thalassemia. 204 00:13:00.821 --> 00:13:02.739 Classification and terminology. 205 00:13:02.739 --> 00:13:05.826 As we can recall there are four alpha genes. 206 00:13:05.826 --> 00:13:10.205 So the normal genotype is two alpha genes on both on in 207 00:13:10.956 --> 00:13:14.459 silent color state is deletion of one alpha gene 208 00:13:16.044 --> 00:13:19.381 minor or trait is deletion of two alpha genes. 209 00:13:20.382 --> 00:13:22.384 And the deletion of two alpha genes 210 00:13:22.384 --> 00:13:25.387 could be on the same allele 211 00:13:26.138 --> 00:13:29.141 or on the opposite ALS. 212 00:13:30.308 --> 00:13:33.687 The phenotype of both these patients would be exactly the same. 213 00:13:34.604 --> 00:13:37.816 They would they would have mild anemia all their life. 214 00:13:38.233 --> 00:13:42.487 However, it becomes really important for the genetic counseling purposes 215 00:13:42.863 --> 00:13:46.241 to, differentiate here between these two genotype. 216 00:13:46.867 --> 00:13:49.744 Since the patients who had a cyst 217 00:13:49.744 --> 00:13:52.747 deletion or deletion on the same old, 218 00:13:52.956 --> 00:13:56.167 if the spouse also has the same deletion, 219 00:13:56.543 --> 00:13:59.880 or even if the spouse has something like this one deletion, 220 00:14:00.213 --> 00:14:05.093 they have 1 in 4 chance of having this particular deletion 221 00:14:05.093 --> 00:14:09.931 or complete absence of alpha genes, or deletion of three alpha genes. 222 00:14:10.891 --> 00:14:11.766 So even though the 223 00:14:11.766 --> 00:14:14.769 phenotype of both these disorders are exactly the same, 224 00:14:15.145 --> 00:14:18.398 the genotype is really important for the genetic counseling 225 00:14:18.398 --> 00:14:21.401 purpose. 226 00:14:21.484 --> 00:14:22.152 Hemoglobin 227 00:14:22.152 --> 00:14:25.155 H disease is deletion of three alpha genes 228 00:14:25.989 --> 00:14:28.909 and Bart's or hydroxide analysis. 229 00:14:28.909 --> 00:14:31.912 Complete absence of alpha genes 230 00:14:32.495 --> 00:14:35.790 a little bit more about the clinical presentation of alpha thalassemia. 231 00:14:36.791 --> 00:14:42.047 A one gene deletion A single deletion is usually known as silent carriers state. 232 00:14:42.047 --> 00:14:45.383 And they they would not have any, any change 233 00:14:45.383 --> 00:14:48.511 in RBC morphology or hemoglobin concentration. 234 00:14:49.262 --> 00:14:52.265 Only thing not which can be noticed in these 235 00:14:52.265 --> 00:14:55.727 patients are slight decrease in their red blood cell size. 236 00:14:56.019 --> 00:14:59.648 So the MCV which is the main purpose color volume 237 00:14:59.856 --> 00:15:03.234 is on the lower side of normal in these patient 238 00:15:04.152 --> 00:15:06.571 two gene deletion, 239 00:15:06.571 --> 00:15:09.032 also known as alpha thalassemia minor. 240 00:15:09.032 --> 00:15:13.578 These patients would have mild micro acidic anemia all their life, 241 00:15:15.121 --> 00:15:16.164 and these patients are 242 00:15:16.164 --> 00:15:19.918 very commonly misdiagnosed as iron deficiency anemia. 243 00:15:20.251 --> 00:15:23.129 Since the CBC parameters 244 00:15:23.129 --> 00:15:26.675 can, have lot of overlap between these two disorders. 245 00:15:26.883 --> 00:15:29.886 And I'll talk a little bit more about that in detail. 246 00:15:30.011 --> 00:15:32.806 In my next slides, 247 00:15:32.806 --> 00:15:35.809 three gene deletion known as hemoglobin H disease. 248 00:15:37.060 --> 00:15:39.270 So what happens in hemoglobin H disease. 249 00:15:39.270 --> 00:15:43.066 We only have one remaining alpha gene 3 250 00:15:43.066 --> 00:15:46.319 or 3 or out of four alpha genes are deleted. 251 00:15:46.778 --> 00:15:50.407 So in these patients we have access of beta chains. 252 00:15:50.407 --> 00:15:53.910 Because obviously beta beta chains are normal 253 00:15:53.910 --> 00:15:57.122 and they do not have enough alpha chains to bind to. 254 00:15:57.539 --> 00:16:00.959 So they would form tetra merged or precipitated out. 255 00:16:01.292 --> 00:16:04.421 And the chamber of beta chains are known as hemoglobin H. 256 00:16:04.421 --> 00:16:09.551 So the in these patients hemoglobin H can be can be seen 257 00:16:10.969 --> 00:16:14.097 or can be basically identified on HPLC. 258 00:16:14.681 --> 00:16:19.477 These patients have moderate anemia marked micro ptosis. 259 00:16:19.853 --> 00:16:23.148 And because of the anemia they can also have spleen omega three 260 00:16:23.398 --> 00:16:26.860 and bone marrow increase in red blood cells in the bone marrow 261 00:16:26.943 --> 00:16:29.028 or precursors in the bone marrow. 262 00:16:29.028 --> 00:16:31.781 For gene deletion, also known as hydrops, fetal 263 00:16:31.781 --> 00:16:34.742 is usually not compatible with life 264 00:16:35.827 --> 00:16:38.830 unless there is a very early intervention. 265 00:16:39.706 --> 00:16:41.749 There have been recent case report or 266 00:16:41.749 --> 00:16:44.752 studies, showing 267 00:16:44.878 --> 00:16:47.589 that early in utero 268 00:16:47.589 --> 00:16:50.550 transplant in these patients can be effective 269 00:16:51.092 --> 00:16:54.095 and some of these patients can be saved. 270 00:16:54.512 --> 00:16:57.849 If, early in utero, a transplant can be done 271 00:16:58.391 --> 00:17:01.352 in these patients only 272 00:17:01.811 --> 00:17:05.648 globin chains are the gamma, sorry gamma, chains. 273 00:17:05.940 --> 00:17:08.526 And these gamma chains form tech jammers. 274 00:17:08.526 --> 00:17:11.529 And the gamma 40 jammers are known as birds. 275 00:17:11.613 --> 00:17:14.324 And these birds has very high oxygen 276 00:17:14.324 --> 00:17:17.452 affinity and is a poor oxygen transporter. 277 00:17:17.452 --> 00:17:21.206 So the problem happens because of the poor oxygen delivery to choose. 278 00:17:23.666 --> 00:17:24.250 So moving 279 00:17:24.250 --> 00:17:27.253 on to beta there are only two beta genes. 280 00:17:27.295 --> 00:17:30.298 So the normal is to one beta on both allele 281 00:17:31.090 --> 00:17:35.011 trait can be beta, beta zero or beta plus. 282 00:17:36.095 --> 00:17:38.014 Beta zero, as we can recall 283 00:17:38.014 --> 00:17:41.017 is complete absence of beta production. 284 00:17:41.017 --> 00:17:45.480 So even though there is complete absence of beta production from one allele, 285 00:17:46.230 --> 00:17:50.610 a normal beta from one is more or less sufficient, 286 00:17:50.944 --> 00:17:54.113 to have a normal kind of, 287 00:17:56.491 --> 00:17:57.116 not really 288 00:17:57.116 --> 00:18:01.079 normal as such, but these patients would not need transfusion 289 00:18:01.079 --> 00:18:04.958 and or would not have more moderate to severe hemolytic anemia. 290 00:18:05.708 --> 00:18:09.420 These patients would have mild, anemia all their life. 291 00:18:10.546 --> 00:18:12.215 Same thing goes with beta plus. 292 00:18:12.215 --> 00:18:15.218 So these patients are usually 293 00:18:15.218 --> 00:18:17.637 mild reduction in beta production 294 00:18:17.637 --> 00:18:20.807 and would have mild microscopic anemia all their life. 295 00:18:21.516 --> 00:18:25.561 However, beta major can be due to complete absence 296 00:18:25.561 --> 00:18:30.024 of beta production on both islet or beta plus beta plus. 297 00:18:31.734 --> 00:18:32.527 As is 298 00:18:32.527 --> 00:18:35.279 alluded to at earlier, beta plus beta plus 299 00:18:35.279 --> 00:18:38.408 could be both major or intermediate, 300 00:18:38.700 --> 00:18:42.453 depending upon the production of beta chain from these early. 301 00:18:42.870 --> 00:18:45.873 If the production of beta 302 00:18:47.208 --> 00:18:48.251 globin 303 00:18:48.251 --> 00:18:52.296 is really severely reduced, these patients would have moderate 304 00:18:52.296 --> 00:18:56.509 anemia all their life and would need, frequent transfusion. 305 00:18:56.509 --> 00:19:01.389 And if the beta production is are not that severely reduced, 306 00:19:01.389 --> 00:19:04.600 these patients would be something like intermediate. 307 00:19:05.435 --> 00:19:08.438 Intermediate is more or less a clinical diagnosis 308 00:19:08.688 --> 00:19:11.691 depending upon the transfusion need of the patients. 309 00:19:11.774 --> 00:19:16.028 So a patient could be, in, the genotype could be 310 00:19:16.028 --> 00:19:19.031 this something like this beta plus and beta plus. 311 00:19:19.282 --> 00:19:23.286 But phenotype could be beta thalassemia major or intermediate 312 00:19:23.286 --> 00:19:24.829 depending upon the production. 313 00:19:24.829 --> 00:19:27.832 All beta large globin. 314 00:19:30.835 --> 00:19:31.711 Beta thalassemia. 315 00:19:31.711 --> 00:19:32.044 A little bit 316 00:19:32.044 --> 00:19:35.923 more about the beta thalassemia, as I mentioned, might be a similar trait 317 00:19:35.923 --> 00:19:39.427 also known as heterozygous is usually clinically not. 318 00:19:39.969 --> 00:19:41.888 Clinically asymptomatic. 319 00:19:41.888 --> 00:19:45.683 They might have mild anemia, but they might not be diagnosed 320 00:19:45.725 --> 00:19:49.479 unless, unless these patients are pregnant or they need 321 00:19:51.731 --> 00:19:53.941 red blood cells. 322 00:19:53.941 --> 00:19:56.652 However, homozygous form of beta thalassemia is 323 00:19:56.652 --> 00:19:59.655 a severe disorder associated with transfusion. 324 00:20:00.740 --> 00:20:03.409 Sometimes weekly transfusion. 325 00:20:03.409 --> 00:20:08.497 Homozygous beta plus, as I alluded earlier, is a heterogeneous disorder 326 00:20:08.497 --> 00:20:13.711 and could be transfusion dependent or less severe depending 327 00:20:13.711 --> 00:20:16.839 upon the production of a hemoglobin 328 00:20:16.839 --> 00:20:19.842 A or beta chains a. 329 00:20:20.760 --> 00:20:23.554 Little bit about the, hemoglobin properties. 330 00:20:23.554 --> 00:20:28.351 I just want to, go over some of the common ones and just mention those. 331 00:20:28.351 --> 00:20:32.021 I won't have time to go in detail about all these disorders. 332 00:20:32.730 --> 00:20:35.816 So the sickle cell anemia is basically, 333 00:20:38.027 --> 00:20:42.365 is the change in valine instead of the glutamic acid 334 00:20:42.365 --> 00:20:46.911 at sixth position from the end, and terminus of the beta globin chain, 335 00:20:47.328 --> 00:20:50.915 it changes the shape of the red blood cells and deform 336 00:20:50.915 --> 00:20:52.416 ability of the red blood cells. 337 00:20:53.584 --> 00:20:56.921 If it's homozygous, it leads to an occlusive disease. 338 00:20:56.921 --> 00:21:00.341 Because of the change in the shape of the red blood cells 339 00:21:01.509 --> 00:21:05.429 and because of the change in the shape of the red blood cells, 340 00:21:05.429 --> 00:21:10.393 these red blood cells become less flexible and causes severe hemolysis. 341 00:21:10.935 --> 00:21:15.314 Heterozygous form of beta sickle cell anemia are usually these patients 342 00:21:15.314 --> 00:21:16.190 heterozygous form. 343 00:21:16.190 --> 00:21:21.404 So you sickle cell traits are usually normal unless the dude 344 00:21:21.862 --> 00:21:24.490 like extreme sports or starts 345 00:21:24.490 --> 00:21:27.994 to go to really high elevation suddenly. 346 00:21:27.994 --> 00:21:32.415 So majority 99% of the sickle cell trait patients are usually normal. 347 00:21:34.208 --> 00:21:37.545 This is a peripheral smear showing the, sickle cells. 348 00:21:37.545 --> 00:21:40.548 So these are the sickle cells 349 00:21:40.589 --> 00:21:43.384 in the peripheral 350 00:21:43.384 --> 00:21:45.177 circulation of a homozygous 351 00:21:45.177 --> 00:21:48.180 sickle cell patient. 352 00:21:48.973 --> 00:21:50.683 Hemoglobin E, 353 00:21:50.683 --> 00:21:54.270 I want to mention a little bit about hemoglobin E, because we have been seeing, 354 00:21:55.479 --> 00:21:56.856 quite often these 355 00:21:56.856 --> 00:21:59.859 patients now because of the migration pattern 356 00:22:00.693 --> 00:22:02.653 is very prevalent, is the second 357 00:22:02.653 --> 00:22:05.656 most prevalent hemoglobin variant worldwide. 358 00:22:05.823 --> 00:22:09.577 It's very common in Southeast Asian population, very, very common 359 00:22:10.369 --> 00:22:13.372 hemoglobin E trait or heterozygous 360 00:22:13.539 --> 00:22:16.500 mutation of the hemoglobin A 361 00:22:16.917 --> 00:22:18.753 would have no anemia. 362 00:22:18.753 --> 00:22:22.256 Only thing seen in these patients is microsite ptosis. 363 00:22:22.256 --> 00:22:24.967 Moderate microsite ptosis. 364 00:22:24.967 --> 00:22:29.805 So the red blood cell size of the red blood cells are really, really small. 365 00:22:30.306 --> 00:22:33.225 Normal mcvay's about 80 to 100 here 366 00:22:33.225 --> 00:22:36.228 the MCB is about 6065 367 00:22:36.520 --> 00:22:38.981 homozygous form of hemoglobin E disease. 368 00:22:38.981 --> 00:22:41.942 These patients also really don't have much anemia. 369 00:22:41.942 --> 00:22:45.905 They might have they might have minimal to mild anemia of their life. 370 00:22:46.447 --> 00:22:49.408 But if we look at the MCB, MCB 371 00:22:49.408 --> 00:22:52.411 is significantly reduced in these patients. 372 00:22:52.411 --> 00:22:56.248 It's not uncommon to see MCB of 60 or 59, 373 00:22:56.248 --> 00:22:58.792 in a homogeneous form of hemoglobin E disease. 374 00:23:00.252 --> 00:23:04.715 Both trait and hemoglobin E disease are 375 00:23:05.716 --> 00:23:09.678 usually not that, problematic in a patient. 376 00:23:09.970 --> 00:23:13.182 They might cause mild kind of borderline ish anemia. 377 00:23:13.682 --> 00:23:18.145 However, these patients again are important for the counseling purposes. 378 00:23:18.437 --> 00:23:21.524 If if this patient hemoglobin E patient or trait 379 00:23:21.607 --> 00:23:24.610 patient also has a beta thalassemia. 380 00:23:24.735 --> 00:23:27.738 These patients hemoglobin E and beta thalassemia 381 00:23:28.197 --> 00:23:31.533 have usually moderate to severe anemia all their life. 382 00:23:31.533 --> 00:23:34.995 So they become really important for the genetic counseling purposes. 383 00:23:36.288 --> 00:23:38.582 One of the other important thing to remember for 384 00:23:38.582 --> 00:23:43.170 about the hemoglobin E is on C, which is a common screening 385 00:23:43.212 --> 00:23:47.216 tool used for these, hemoglobin poverty diagnosis. 386 00:23:47.633 --> 00:23:50.636 The migration pattern of hemoglobin A2 387 00:23:51.845 --> 00:23:54.223 is similar to hemoglobin E. 388 00:23:54.223 --> 00:23:59.186 So it's not we cannot differentiate between hemoglobin E and hemoglobin E 389 00:23:59.478 --> 00:24:01.855 a on C. 390 00:24:01.855 --> 00:24:04.984 As we can recall hemoglobin A2 is really important 391 00:24:04.984 --> 00:24:07.653 for our beta thalassemia trait diagnosis. 392 00:24:07.653 --> 00:24:09.029 So it becomes important. 393 00:24:10.864 --> 00:24:12.700 To keep that in mind. 394 00:24:12.700 --> 00:24:16.453 In cases of beta thalassemia, patients who have both beta 395 00:24:17.079 --> 00:24:20.708 and E, it becomes, HPLC is really not, 396 00:24:20.958 --> 00:24:25.879 useful to differentiate between those two, since we cannot really differentiate 397 00:24:25.879 --> 00:24:28.882 between hemoglobin E and hemoglobin A2. 398 00:24:29.675 --> 00:24:32.970 However, capillary electrophoresis or the molecular test 399 00:24:32.970 --> 00:24:35.973 would differentiate between those two. 400 00:24:37.057 --> 00:24:37.683 Hemoglobin 401 00:24:37.683 --> 00:24:40.686 see a little bit about hemoglobin C. 402 00:24:41.020 --> 00:24:42.563 It's a beta mutation. 403 00:24:42.563 --> 00:24:46.817 The mutation is similar at the same position as sickle cell. 404 00:24:46.859 --> 00:24:49.862 However the amino acid change is different. 405 00:24:50.029 --> 00:24:53.157 Seen predominantly in black gene prevalence. 406 00:24:53.157 --> 00:24:56.160 In US black population is about 2 to 3%. 407 00:24:56.368 --> 00:24:59.788 This also done for malaria resistance. 408 00:24:59.997 --> 00:25:03.417 So as as we can see here many of the thalassemia 409 00:25:03.751 --> 00:25:06.670 sickle cell trait hemoglobin C trait 410 00:25:06.670 --> 00:25:09.631 these all give some kind of, 411 00:25:10.174 --> 00:25:11.467 resistance to malaria, 412 00:25:12.926 --> 00:25:13.427 which all three 413 00:25:13.427 --> 00:25:16.430 of these patients would be asymptomatic. 414 00:25:16.472 --> 00:25:20.434 Homozygous patients could have mild anemia and mild spleen. 415 00:25:20.434 --> 00:25:23.437 Omega on blood smear. 416 00:25:23.771 --> 00:25:26.190 It's very common to see target cells. 417 00:25:26.190 --> 00:25:29.193 Something like this with targeted appearance 418 00:25:30.360 --> 00:25:33.697 and homozygous forms can have intracellular crystals. 419 00:25:33.697 --> 00:25:36.700 Something like this. 420 00:25:37.326 --> 00:25:37.951 Again, 421 00:25:37.951 --> 00:25:42.080 both heterozygous and homozygous form of hemoglobin C 422 00:25:42.080 --> 00:25:46.710 only disease would not cause much problem in the patient per se. 423 00:25:47.169 --> 00:25:50.172 They might have mild anemia only. 424 00:25:50.506 --> 00:25:54.092 But again diagnosis or recognition of 425 00:25:54.092 --> 00:25:57.095 this is really important for the counseling purposes 426 00:25:57.304 --> 00:26:00.474 since a combination of sickle cell trait and C. 427 00:26:00.682 --> 00:26:05.646 So hemoglobin S C disease can cause severe moderate to severe anemia. 428 00:26:05.646 --> 00:26:07.397 All their life and hemolysis. 429 00:26:07.397 --> 00:26:11.109 So it's really important for the counseling purposes. 430 00:26:13.445 --> 00:26:16.448 So moving on to the diagnostic aspect. 431 00:26:16.532 --> 00:26:19.952 When do we suspect, hemoglobin diabetes or thalassemia 432 00:26:19.952 --> 00:26:23.247 is unexplained cause of hemolytic anemia. 433 00:26:24.164 --> 00:26:26.208 Family history of hemoglobin properties. 434 00:26:26.208 --> 00:26:27.251 And thalassemia is 435 00:26:28.418 --> 00:26:30.546 important for the genetic counseling purposes, 436 00:26:30.546 --> 00:26:33.215 especially if there is a background, 437 00:26:33.215 --> 00:26:37.553 of hemoglobin, diabetes or family history of hemoglobin ofthese certain 438 00:26:37.553 --> 00:26:42.307 ethnic population like Southeast Asian population, Mediterranean population, 439 00:26:42.307 --> 00:26:45.769 where the gene prevalence of these disorders are really high. 440 00:26:45.811 --> 00:26:49.565 Those patients should be definitely be, tested for these disorders. 441 00:26:51.191 --> 00:26:53.735 So the laboratory test where we should start 442 00:26:53.735 --> 00:26:57.572 is the CBC a complete blood count with facial smear. 443 00:26:59.574 --> 00:27:04.204 However, in cases of thalassemia trait or, 444 00:27:05.289 --> 00:27:07.708 like, simple, minor or trait, 445 00:27:07.708 --> 00:27:11.920 all these meat changes of, which I mentioned here, 446 00:27:12.921 --> 00:27:16.508 polychrome is here where we can see immature red blood cells 447 00:27:16.800 --> 00:27:19.761 as ferocity change in red blood cells, fragmented 448 00:27:19.761 --> 00:27:23.223 red blood cells, sickle cell, unstable hemoglobin. 449 00:27:23.557 --> 00:27:28.895 These all these changes we don't see these all these changes in a tree patient. 450 00:27:30.063 --> 00:27:31.940 These changes are only seen 451 00:27:31.940 --> 00:27:35.694 in B C major or homozygous sickle cell disease. 452 00:27:35.861 --> 00:27:37.946 So we should keep that in mind. 453 00:27:37.946 --> 00:27:41.950 So the patients who have like straight out trait 454 00:27:41.950 --> 00:27:45.495 beta thalassemia or alpha thalassemia trait or sickle cell trait, 455 00:27:45.746 --> 00:27:48.749 they might not have any finding on peripheral smear 456 00:27:49.708 --> 00:27:52.919 which are of these disorders, especially the structural. 457 00:27:53.336 --> 00:27:56.965 The changes can easily be diagnosed use 458 00:27:57.215 --> 00:28:02.304 using the commonly used HPLC, high performance liquid chromatography, 459 00:28:02.554 --> 00:28:06.516 or some kind of electrolytic technique, either capillary electrolytic 460 00:28:06.850 --> 00:28:09.811 or gel based electrolytic. 461 00:28:09.936 --> 00:28:14.066 However, some some of these can be missed and might need genetic testing. 462 00:28:14.524 --> 00:28:18.528 And we'll talk about I will talk about that in a little bit more detail. 463 00:28:20.614 --> 00:28:22.240 Before I talk more 464 00:28:22.240 --> 00:28:25.243 about the technical aspect of the, 465 00:28:25.994 --> 00:28:30.332 of the diagnosis of these disorders, I want to emphasize a little bit 466 00:28:30.332 --> 00:28:35.253 more about the importance of CBC, especially in thalassemia disorders. 467 00:28:36.755 --> 00:28:38.006 Red cell indices 468 00:28:38.006 --> 00:28:41.343 are critical to type for diagnosis of these disorders. 469 00:28:42.010 --> 00:28:45.430 So thalassemia all these patients 470 00:28:45.806 --> 00:28:48.850 present with hypo chromic microsatellite anemia. 471 00:28:48.850 --> 00:28:52.229 So microsatellite means smaller red blood cells size. 472 00:28:52.646 --> 00:28:55.732 And hypo chromic means less hemoglobin. 473 00:28:57.692 --> 00:29:00.487 So the major differential diagnosis for hypo 474 00:29:00.487 --> 00:29:04.407 chromic microsatellite anemia worldwide are two. 475 00:29:04.866 --> 00:29:08.411 One is iron deficiency anemia and thalassemia. 476 00:29:09.621 --> 00:29:12.123 And both these disorders are really common. 477 00:29:12.123 --> 00:29:16.503 Iron deficiency anemia is one of the most common nutritional deficiency worldwide, 478 00:29:16.962 --> 00:29:19.673 so we should be able to at least have 479 00:29:19.673 --> 00:29:22.676 some idea before ordering the test 480 00:29:22.801 --> 00:29:25.804 whether this is iron deficiency anemia or thalassemia. 481 00:29:26.763 --> 00:29:29.099 These three parameters, which I mentioned here 482 00:29:29.099 --> 00:29:32.102 can be helpful in, 483 00:29:32.477 --> 00:29:34.354 trying in 484 00:29:34.354 --> 00:29:37.357 differentiating between those two disorders. 485 00:29:37.440 --> 00:29:41.361 So Ncbi is the main purpose color volume that tells us about the 486 00:29:41.361 --> 00:29:43.029 size of the red blood cell 487 00:29:44.948 --> 00:29:46.366 RW Ratzel 488 00:29:46.366 --> 00:29:50.537 distribution, which is a parameter which basically measures 489 00:29:50.537 --> 00:29:53.540 the variation in red blood cell size 490 00:29:54.291 --> 00:29:57.294 and volume and red blood cell count. 491 00:29:59.754 --> 00:30:02.507 Red blood cell count is one of the important 492 00:30:02.507 --> 00:30:06.136 distinguishing factor between thalassemia and microsatellite anemia. 493 00:30:06.636 --> 00:30:09.639 So the next slide, 494 00:30:10.724 --> 00:30:12.934 is basically I feel I, 495 00:30:12.934 --> 00:30:16.980 mentioned the distinguishing features between iron deficiency and thalassemia. 496 00:30:18.231 --> 00:30:20.358 So what happened in thalassemia? 497 00:30:20.358 --> 00:30:22.402 Thalassemia is a genetic disorder. 498 00:30:22.402 --> 00:30:25.280 There's no nutritional deficiency 499 00:30:25.280 --> 00:30:26.531 in these disorders. 500 00:30:26.531 --> 00:30:31.328 In thalassemia disorders, the RBC count is either 501 00:30:31.786 --> 00:30:34.789 normal or in the higher range of normal. 502 00:30:35.248 --> 00:30:39.502 Since there's no nutritional deficiency in thalassemia, the bone marrow tries 503 00:30:39.502 --> 00:30:44.674 to compensate for it, for the anemia, and will increase the RBC count. 504 00:30:44.883 --> 00:30:48.303 So majority of the patient of thalassemia trait, 505 00:30:48.636 --> 00:30:53.266 we would see a slight increase on higher side of normal red blood cell. 506 00:30:53.975 --> 00:30:57.103 However, that never happens unless there is 507 00:30:57.103 --> 00:31:00.190 a co-existing thalassemia, an iron deficiency anemia. 508 00:31:00.815 --> 00:31:03.526 It usually would never happen in iron deficiency 509 00:31:03.526 --> 00:31:06.905 anemia, iron deficiency anemia is defined, by 510 00:31:08.073 --> 00:31:11.159 either normal, lower range of 511 00:31:11.159 --> 00:31:14.496 normal of red blood cell count or decreased red blood cell count. 512 00:31:15.330 --> 00:31:18.041 MCV is usually less than 70. 513 00:31:18.041 --> 00:31:21.878 In thalassemia, so talismanic patient would present 514 00:31:21.878 --> 00:31:24.881 with slightly higher 515 00:31:25.090 --> 00:31:27.550 red blood cell count with the MCB 516 00:31:27.550 --> 00:31:30.553 or maybe around 65 or 60, 517 00:31:31.221 --> 00:31:34.224 with a normal dead cell distribution with 518 00:31:34.599 --> 00:31:37.268 since it's a genetic disorder, 519 00:31:37.268 --> 00:31:40.563 red cell distribution, which is normal in these patients. 520 00:31:41.022 --> 00:31:43.942 But in cases of iron deficiency anemia, 521 00:31:43.942 --> 00:31:46.945 the this is a it's a nutritional deficiency. 522 00:31:47.070 --> 00:31:49.781 So red blood cells would be of one size. 523 00:31:49.781 --> 00:31:53.493 And when the red when the iron goes further down, 524 00:31:53.493 --> 00:31:56.913 the red blood cells become smaller in size and smaller in size. 525 00:31:57.247 --> 00:32:01.584 So the variation in red blood cell size is much, 526 00:32:01.584 --> 00:32:06.297 much higher in iron deficiency anemia compared to a thalassemia. 527 00:32:07.382 --> 00:32:08.466 So in common 528 00:32:08.466 --> 00:32:11.469 with combination of all these three parameters, 529 00:32:11.594 --> 00:32:15.306 in majority of the cases, I would say 90 to 95% of the cases 530 00:32:15.306 --> 00:32:18.518 we can have some idea whether it's iron deficiency anemia 531 00:32:18.518 --> 00:32:21.521 or thalassemia. 532 00:32:21.646 --> 00:32:23.982 So moving on to the diagnostic aspect, 533 00:32:23.982 --> 00:32:26.985 this is a picture of HPLC. 534 00:32:27.819 --> 00:32:29.028 So high pressure 535 00:32:29.028 --> 00:32:33.324 liquid chromatography is one of the commonly used screening 536 00:32:33.324 --> 00:32:36.953 technique used in majority of the bigger laboratories. 537 00:32:38.705 --> 00:32:40.498 We use a cation exchange 538 00:32:40.498 --> 00:32:43.501 chromatography technique, 539 00:32:43.668 --> 00:32:46.170 and it's based on the interaction 540 00:32:46.170 --> 00:32:49.173 between stationary phase and the, 541 00:32:50.091 --> 00:32:51.551 mobile phase. 542 00:32:51.551 --> 00:32:54.554 The an analytical cartridge 543 00:32:54.846 --> 00:32:57.932 acts as the stationary phase 544 00:32:58.266 --> 00:33:01.269 and contains the negatively charged silica. 545 00:33:02.103 --> 00:33:04.230 Himalaya is hemolysis. 546 00:33:04.230 --> 00:33:08.151 Is prepared and it contains the positively charged hemoglobin 547 00:33:08.943 --> 00:33:14.198 and the buffer acts as the mobile face is the phosphate buffer solutions. 548 00:33:15.783 --> 00:33:17.452 So the 549 00:33:17.452 --> 00:33:20.496 hemoglobin, the positively charged hemoglobin binds 550 00:33:20.496 --> 00:33:23.499 to the negatively charged silica at injection. 551 00:33:24.792 --> 00:33:26.544 The phosphate buffer concentration 552 00:33:26.544 --> 00:33:30.131 increases and separates the hemoglobin fragments from silicon. 553 00:33:30.131 --> 00:33:34.010 So that's that's how that's the in very basic nutshell, 554 00:33:34.052 --> 00:33:37.055 how the HPLC works. 555 00:33:37.555 --> 00:33:40.558 This is a normal, patient chromatogram. 556 00:33:41.768 --> 00:33:44.062 The first one is of a normal 557 00:33:44.062 --> 00:33:47.523 adult person, and this one is from a normal, 558 00:33:48.191 --> 00:33:52.403 neonate, as we can see here in in our normal adult, 559 00:33:52.403 --> 00:33:55.406 the predominant hemoglobin is the hemoglobin. 560 00:33:55.531 --> 00:33:59.994 Adult hemoglobin followed a little bit, followed by hemoglobin 561 00:33:59.994 --> 00:34:04.165 A2 and a little bit of F in UN unit. 562 00:34:04.165 --> 00:34:08.920 The predominant hemoglobin is hemoglobin F, followed by adult hemoglobin. 563 00:34:11.964 --> 00:34:15.301 So the advantages of HPLC is is fast. 564 00:34:15.301 --> 00:34:17.720 It takes only a few minutes 5 to 7 minutes. 565 00:34:17.720 --> 00:34:21.724 It requires very small quantity of sample few microliter 5 to 5. 566 00:34:22.350 --> 00:34:24.018 Microliter of sample. 567 00:34:24.018 --> 00:34:27.021 And it gives a accurate quantitation of hemoglobin A2, 568 00:34:27.021 --> 00:34:30.274 which is really important for our beta thalassemia diagnosis. 569 00:34:31.734 --> 00:34:33.820 However, there are some disadvantages. 570 00:34:33.820 --> 00:34:39.826 As of I alluded to it earlier, hemoglobin E cannot be separated from hemoglobin A2. 571 00:34:39.826 --> 00:34:42.370 They have the same retention time. 572 00:34:42.370 --> 00:34:44.705 However, if the percentage of hemoglobin A2, 573 00:34:44.705 --> 00:34:49.335 a normal percentage of hemoglobin A2 is usually not more than 8 to 10%, 574 00:34:49.710 --> 00:34:53.172 so any time we see more than 10% hemoglobin A2, 575 00:34:53.464 --> 00:34:56.884 we have a very high index of suspicion that this is hemoglobin E. 576 00:34:58.594 --> 00:35:02.014 Other caveat is hemoglobin H and bars which is seen in 577 00:35:02.014 --> 00:35:04.475 alpha thalassemia are two. 578 00:35:04.475 --> 00:35:05.810 They're really fast moving. 579 00:35:05.810 --> 00:35:08.771 And if you took quickly from column. 580 00:35:08.771 --> 00:35:11.732 So it might not be, identified. 581 00:35:13.234 --> 00:35:16.237 This is a picture of capillary electrophoresis. 582 00:35:16.571 --> 00:35:19.323 It's another 583 00:35:19.323 --> 00:35:23.286 method used for screening to, used as a screening technique. 584 00:35:23.828 --> 00:35:25.163 It works. 585 00:35:25.163 --> 00:35:29.333 On the interaction between the positive charge, 586 00:35:29.792 --> 00:35:35.047 from the buffer solution and, negative charge of the capillary was. 587 00:35:35.047 --> 00:35:40.928 So the interaction between these two, generates, electro 588 00:35:40.928 --> 00:35:45.099 osmotic flow, which is stronger than the electric field. And 589 00:35:46.601 --> 00:35:47.727 because of that, 590 00:35:47.727 --> 00:35:52.023 the all the proteins migrate towards the catalytic end of the capillary. 591 00:35:53.608 --> 00:35:55.776 This is, this is what a force is. 592 00:35:55.776 --> 00:35:59.614 Reports on look like this is on first one is a normal one 593 00:35:59.947 --> 00:36:02.950 and it's separated, in different zones. 594 00:36:02.950 --> 00:36:05.995 So this is the normal adult hemoglobin here. 595 00:36:05.995 --> 00:36:08.998 This is the abnormal one showing 596 00:36:09.040 --> 00:36:12.001 abnormal, radiant hemoglobin in zone two. 597 00:36:14.503 --> 00:36:17.340 Alkaline and acid gel electrophoresis. 598 00:36:17.340 --> 00:36:20.343 This is 599 00:36:20.676 --> 00:36:21.844 two different technique 600 00:36:21.844 --> 00:36:26.223 alkaline electrophoresis and the, acid gel electrophoresis. 601 00:36:27.308 --> 00:36:29.268 It's basically 602 00:36:29.268 --> 00:36:31.646 it's a slow and labor intensive. 603 00:36:31.646 --> 00:36:35.316 And one of the major problem with acid and alkaline electrophoresis 604 00:36:35.316 --> 00:36:38.319 is the inaccurate quantitation of, 605 00:36:38.611 --> 00:36:42.281 low concentration of hemoglobin variants, especially hemoglobin A2. 606 00:36:42.782 --> 00:36:47.286 It is very useful for the, confirmation of some of the variant hemoglobin 607 00:36:47.495 --> 00:36:50.498 seen by HPLC or capillary electrophoresis. 608 00:36:50.498 --> 00:36:55.628 However, it becomes, redundant or it's not that useful for the, 609 00:36:56.587 --> 00:36:58.589 accurate 610 00:36:58.589 --> 00:37:01.425 quantitation of hemoglobin A2, which is really important 611 00:37:01.425 --> 00:37:05.346 for beta thalassemia trait, diagnosis. 612 00:37:06.889 --> 00:37:08.891 Another 613 00:37:08.891 --> 00:37:12.311 commonly used method is isoelectric focus focusing, 614 00:37:12.603 --> 00:37:17.316 which is kind of similar to the alkaline electrophoresis with better, resolution. 615 00:37:17.858 --> 00:37:21.487 It is an electro fatigue technique with really excellent resolution, 616 00:37:21.487 --> 00:37:25.408 and it is an equilibrium process in which the hemoglobin migrates 617 00:37:25.408 --> 00:37:29.787 in a pH gradient to a position of zero negative charge. 618 00:37:31.539 --> 00:37:34.542 So a little bit about the molecular analysis 619 00:37:35.251 --> 00:37:38.087 for the alpha and beta thalassemia diagnosis. 620 00:37:38.087 --> 00:37:40.840 So for the alpha thalassemia 621 00:37:40.840 --> 00:37:45.553 again I want to emphasize on one of the important molecular 622 00:37:45.928 --> 00:37:50.975 background for alpha thalassemia is the, 95% of the alpha 623 00:37:50.975 --> 00:37:55.271 thalassemia are due to large deletions and large deletions. 624 00:37:55.813 --> 00:37:59.942 So in that scenario, alpha globin sequencing 625 00:38:00.651 --> 00:38:03.070 is kind of useless, 626 00:38:03.070 --> 00:38:07.616 because the alpha globin sequencing would basically would not have anything 627 00:38:07.616 --> 00:38:08.242 to bind to 628 00:38:08.242 --> 00:38:11.328 because of the large deletion, the primers would not have anything to bind to. 629 00:38:11.579 --> 00:38:16.250 So it will basically amplify the normal remaining alpha genes. 630 00:38:16.792 --> 00:38:21.964 So the preferred method for to diagnose these large deletions are either multiplex 631 00:38:21.964 --> 00:38:25.301 ligation dependent probe amplification or also known 632 00:38:25.301 --> 00:38:28.262 as MLP or multiplex PCR. 633 00:38:29.555 --> 00:38:32.558 These are basically targeted 634 00:38:32.725 --> 00:38:35.728 to identify those large deletion. 635 00:38:35.769 --> 00:38:38.314 However, in contrast, beta thalassemia 636 00:38:38.314 --> 00:38:41.317 are usually 637 00:38:42.276 --> 00:38:43.694 due to single point 638 00:38:43.694 --> 00:38:46.697 mutation, small insertions or deletion. 639 00:38:46.989 --> 00:38:51.994 And in these in in this scenario, especially if it's point small point 640 00:38:52.119 --> 00:38:53.495 sorry single point mutation, 641 00:38:53.495 --> 00:38:57.291 small insertions and deletions, sequencing would work great. 642 00:38:57.666 --> 00:38:59.043 So the clinical sensitivity 643 00:38:59.043 --> 00:39:03.172 is about 97% based on that intensity for beta globin sequences. 644 00:39:03.464 --> 00:39:06.717 So for the beta thalassemia diagnosis beta globin sequencing 645 00:39:06.717 --> 00:39:09.720 would be the preferred method 646 00:39:10.054 --> 00:39:12.932 only like few percent of beta thalassemia. 647 00:39:12.932 --> 00:39:16.685 Maybe around 2 to 5% of thalassemia are due 648 00:39:16.685 --> 00:39:22.024 to large deletions and duplications, and those large deletions and duplications 649 00:39:22.024 --> 00:39:26.403 can be identified by the MLD, which is kind of the similar technique 650 00:39:26.403 --> 00:39:30.783 as the technique used for multiplex ligation dependent probe amplification 651 00:39:30.783 --> 00:39:32.618 for alpha thalassemia. Diagnosis. 652 00:39:35.162 --> 00:39:37.957 So coming back to the alpha 653 00:39:37.957 --> 00:39:40.960 thalassemia overall alpha thalassemia diagnosis, 654 00:39:41.919 --> 00:39:45.506 the hemoglobin gels, HPLC table 655 00:39:45.547 --> 00:39:50.260 electrophoresis is all these methods, all of these are screening 656 00:39:50.552 --> 00:39:55.557 tools are not that helpful if we have only 1 or 2 gene deletions. 657 00:39:55.599 --> 00:39:58.477 Basically in treat patients, 658 00:39:58.477 --> 00:40:02.231 only in patients where we have enough production or beta 659 00:40:02.231 --> 00:40:06.360 for like in three gene deletion or gamma for two traumas, 660 00:40:07.152 --> 00:40:10.781 these beta four and gamma four to tremors in hemoglobin 661 00:40:10.781 --> 00:40:16.161 H disease and bars can be identified by HPLC or electrophoresis. 662 00:40:16.704 --> 00:40:20.374 However, in the patients with 1 or 2 gene gene deletion, 663 00:40:20.374 --> 00:40:24.336 the production of these check tremors are not that enough, 664 00:40:25.087 --> 00:40:29.091 to be identified by the commonly used screening tools. 665 00:40:30.217 --> 00:40:32.553 So the point to remember in 666 00:40:32.553 --> 00:40:37.141 for alpha thalassemia diagnosis is if the Ccbc and ethnicity points 667 00:40:37.141 --> 00:40:41.687 towards alpha alpha thalassemia and the screening tools 668 00:40:41.687 --> 00:40:46.233 have come back as negative, we have not ruled out alpha thalassemia. 669 00:40:46.650 --> 00:40:51.155 To diagnose alpha thalassemia trait, we need to do molecular analysis, 670 00:40:51.447 --> 00:40:55.659 and the preferred molecular molecular analysis would be some kind of a multiplex 671 00:40:55.659 --> 00:41:00.164 PCR or MLP, which would identify all deletions and duplications. 672 00:41:01.957 --> 00:41:02.916 The advantage of 673 00:41:02.916 --> 00:41:06.879 MLP is that it will identify all deletions and duplications. 674 00:41:07.296 --> 00:41:10.382 The targeted multiplex PCR would identify 675 00:41:10.382 --> 00:41:13.385 only those targeted deletions for 676 00:41:14.219 --> 00:41:16.263 and the test we on we have 677 00:41:16.263 --> 00:41:20.225 is the multiplex PCR for seven common deletion. 678 00:41:20.434 --> 00:41:24.563 And the the seven common deletions would account for almost about 679 00:41:26.190 --> 00:41:29.985 9,590%, of the alpha thalassemia. 680 00:41:31.737 --> 00:41:36.116 Alpha globin sequencing would be helpful in those scenarios, 681 00:41:36.116 --> 00:41:39.620 like about 5% of our patients where we see, 682 00:41:41.413 --> 00:41:42.664 point mutations, 683 00:41:42.664 --> 00:41:45.667 small insertions or deletions. 684 00:41:46.126 --> 00:41:48.962 So the 685 00:41:48.962 --> 00:41:51.965 the molecular technique, 686 00:41:52.507 --> 00:41:54.426 useful for alpha thalassemia 687 00:41:54.426 --> 00:41:57.846 diagnosis would be either MLP or multiplex PCR, 688 00:41:58.347 --> 00:42:01.350 followed by alpha globin sequencing. 689 00:42:02.184 --> 00:42:04.561 For beta had a similar diagnosis. 690 00:42:04.561 --> 00:42:06.230 However, 691 00:42:06.230 --> 00:42:09.233 E As I alluded to earlier, 692 00:42:09.441 --> 00:42:12.444 the screening tools both HPLC 693 00:42:12.694 --> 00:42:15.697 or capillary electrophoresis, which what we are using, 694 00:42:16.990 --> 00:42:18.951 would be very helpful 695 00:42:18.951 --> 00:42:22.579 in, diagnosing the alpha thalassemia trait 696 00:42:22.579 --> 00:42:27.751 because the elevated hemoglobin A2 is seen in 697 00:42:28.877 --> 00:42:31.797 beta thalassemia trait can be identified 698 00:42:31.797 --> 00:42:35.050 by HPLC or capillary electrophoresis. 699 00:42:35.884 --> 00:42:38.887 So however, 700 00:42:39.304 --> 00:42:42.307 the elevated A2 is 701 00:42:42.641 --> 00:42:46.478 doesn't give us any idea about the underlying 702 00:42:47.229 --> 00:42:49.982 genotype, of the patient. 703 00:42:49.982 --> 00:42:54.486 So it becomes important to identify the, the genotype 704 00:42:54.486 --> 00:42:57.656 in these patients for especially for the genetic counseling purposes. 705 00:42:58.198 --> 00:43:01.994 So after HPLC or capillary electrophoresis, the 706 00:43:02.911 --> 00:43:06.248 the molecular method recommended for the beta thalassemia 707 00:43:06.248 --> 00:43:10.711 diagnosis would be beta globin sequencing, which would identify 708 00:43:11.253 --> 00:43:15.716 all sorry, all point mutations, single insert, 709 00:43:15.757 --> 00:43:18.927 a single point mutations, small insertions or deletions. 710 00:43:21.513 --> 00:43:22.556 Beta globin deletion 711 00:43:22.556 --> 00:43:25.600 and duplication testing can be useful in some cases. 712 00:43:25.600 --> 00:43:29.020 About 2 to 5% of the cases where beta thalassemia 713 00:43:29.104 --> 00:43:32.107 are due to large deletions. 714 00:43:34.401 --> 00:43:35.318 So that was about 715 00:43:35.318 --> 00:43:38.321 beta alpha and beta thalassemia diagnosis. 716 00:43:38.363 --> 00:43:41.616 Moving on to a little bit about the sickle cell disease diagnosis, 717 00:43:41.616 --> 00:43:44.703 because this is another common problem we see. 718 00:43:46.538 --> 00:43:47.497 So the screening 719 00:43:47.497 --> 00:43:52.002 tool used for sickle cell disease diagnosis is the sickle dex test 720 00:43:52.002 --> 00:43:56.214 which can be done which is can be used as a bedside test. 721 00:43:58.717 --> 00:44:01.511 The the solution used 722 00:44:01.511 --> 00:44:04.514 for this or this the test. 723 00:44:04.598 --> 00:44:06.808 Sorry. The kit, 724 00:44:06.808 --> 00:44:09.811 has be concentrated phosphate buffer solution. 725 00:44:10.896 --> 00:44:12.814 And a reducing agent. 726 00:44:12.814 --> 00:44:15.817 So the reducing agent, basically, 727 00:44:16.359 --> 00:44:18.195 causes, 728 00:44:18.195 --> 00:44:21.990 conversion of, hemoglobin S to deoxygenated form. 729 00:44:22.449 --> 00:44:27.287 And the deoxygenated form of hemoglobin S is insoluble in the phosphate 730 00:44:27.704 --> 00:44:31.333 phosphate buffer solution and forms a turbid suspension, 731 00:44:32.375 --> 00:44:35.378 something like this. 732 00:44:35.712 --> 00:44:36.796 Normal hemoglobin 733 00:44:36.796 --> 00:44:40.342 A and the other hemoglobin S, which are not sickle 734 00:44:40.342 --> 00:44:43.929 cell, would remain in solution and would not cause a turbidity. 735 00:44:45.805 --> 00:44:48.308 However, one of the important caveat, 736 00:44:48.308 --> 00:44:52.020 that needs to be remembered is it does not 737 00:44:52.020 --> 00:44:55.899 differentiate between sickle cell disease or sickle cell trait. 738 00:44:56.441 --> 00:44:59.569 So this cannot be used as a diagnostic tool. 739 00:44:59.569 --> 00:45:05.075 It's useful for a screening tool, but, it cannot be used as a diagnostic tool 740 00:45:05.408 --> 00:45:09.412 because both sickle cell trait as well as homozygous sickle cell 741 00:45:09.412 --> 00:45:13.917 would give rise to positive, sickle deaths and a Dex test. 742 00:45:15.293 --> 00:45:18.296 So the next test would would be either 743 00:45:18.546 --> 00:45:22.842 for confirmation would be either, some electrolytic technique. 744 00:45:22.842 --> 00:45:25.845 It could be gel based technique like this. 745 00:45:26.179 --> 00:45:28.348 Or it could be, HPLC. 746 00:45:28.348 --> 00:45:31.810 HPLC would be really helpful in differentiating and give, 747 00:45:32.185 --> 00:45:35.480 giving us an accurate quantitation of both hemoglobin S 748 00:45:35.855 --> 00:45:40.360 as well as hemoglobin A. So. 749 00:45:42.445 --> 00:45:45.949 Moving on to the simplified algorithm, 750 00:45:46.574 --> 00:45:50.537 for the diagnosis of hemoglobin, hemoglobin diabetes and thalassemia. 751 00:45:50.704 --> 00:45:53.707 And I want to spend a few minutes on this one. 752 00:45:53.915 --> 00:45:56.251 So any case of suspected 753 00:45:56.251 --> 00:45:59.254 hemoglobin, diabetes and thalassemia is 754 00:45:59.629 --> 00:46:02.632 the screening tool. 755 00:46:02.924 --> 00:46:07.303 And the suspicion could be based on the CBC 756 00:46:07.303 --> 00:46:10.306 ethnicity of the population or, 757 00:46:11.599 --> 00:46:14.602 or on a family history of thalassemia. 758 00:46:14.769 --> 00:46:17.772 These patients should have, 759 00:46:19.024 --> 00:46:22.027 some kind of screening methods. 760 00:46:22.485 --> 00:46:27.532 And, the, the, the sample should be done by some, some of the screening methods. 761 00:46:28.033 --> 00:46:30.910 The screening tool could be PLC 762 00:46:30.910 --> 00:46:34.414 or capillary electrophoresis or gel based electrophoresis. 763 00:46:34.414 --> 00:46:37.917 However, the gel based the problem with gel based electrophoresis, 764 00:46:38.251 --> 00:46:43.465 is that it does not really give us an accurate quantitation of hemoglobin A2, 765 00:46:43.882 --> 00:46:47.469 which is really important for the beta thalassemia trait. 766 00:46:48.636 --> 00:46:51.514 So majority of the big laboratories, including us, 767 00:46:51.514 --> 00:46:54.684 we use a combination of HPLC and capillary 768 00:46:54.934 --> 00:46:57.729 electrophoresis. 769 00:46:57.729 --> 00:47:01.649 So if the HPLC and capillary electrophoresis 770 00:47:01.649 --> 00:47:04.861 shows us increase in hemoglobin A2, 771 00:47:06.362 --> 00:47:08.281 that in pretty much 772 00:47:08.281 --> 00:47:13.745 I would say increase in hemoglobin A2 in about 99.9% of the cases 773 00:47:13.745 --> 00:47:18.917 would be suggest if all diagnostic of or orbital trait. 774 00:47:20.460 --> 00:47:24.380 So the next test for this 775 00:47:24.380 --> 00:47:28.760 these patients would be to do undergo beta globin sequencing. 776 00:47:30.303 --> 00:47:33.556 If the beta globin and again, majority of the patients 777 00:47:33.556 --> 00:47:37.352 would have single point mutations, small insertions and deletions 778 00:47:37.519 --> 00:47:40.897 and the beta globin sequencing would easily pick up those patients. 779 00:47:40.897 --> 00:47:44.943 So about 95 to 97% of the beta thalassemia 780 00:47:44.943 --> 00:47:47.946 patients would be diagnosed at this stage. 781 00:47:48.238 --> 00:47:50.573 The remaining 2 to 3% of the patients 782 00:47:50.573 --> 00:47:53.576 where there are large deletions 783 00:47:53.576 --> 00:47:57.997 accounting for beta thalassemia, those patients would need to 784 00:47:59.374 --> 00:47:59.916 undergo 785 00:47:59.916 --> 00:48:02.919 beta globin deletion and duplication. 786 00:48:03.002 --> 00:48:06.339 So, again, so 787 00:48:07.632 --> 00:48:10.635 increased hemoglobin A2 is very suggestive 788 00:48:10.802 --> 00:48:14.889 followed by beta globin sequencing and beta globin deletion and duplication. 789 00:48:16.266 --> 00:48:19.894 However, if the of the screening tools 790 00:48:20.395 --> 00:48:23.398 comes back as normal. 791 00:48:24.023 --> 00:48:26.651 Then we need to remember that 792 00:48:26.651 --> 00:48:31.573 we have not ruled out alpha thalassemia 1 or 2 gene deletion. 793 00:48:31.948 --> 00:48:35.910 Sometimes hemoglobin H disease also like the three gene 794 00:48:35.910 --> 00:48:38.913 deletion, might not give us enough hemoglobin H. 795 00:48:38.955 --> 00:48:43.126 And as we can recall, hemoglobin H is really a fast moving hemoglobin. 796 00:48:44.544 --> 00:48:48.172 We might not, identify that on our screening tool. 797 00:48:48.923 --> 00:48:51.926 So in those condition, 798 00:48:52.969 --> 00:48:55.888 we should be suspicious of alpha thalassemia. 799 00:48:55.888 --> 00:48:58.808 And the test which we need to use for alpha 800 00:48:58.808 --> 00:49:01.811 sigma is the alpha globin deletion and duplication. 801 00:49:02.520 --> 00:49:06.399 And or, we can also use seven common deletion tags, 802 00:49:06.399 --> 00:49:11.404 which is the multiplex PCR test if we know that the ethnicity of the population, 803 00:49:11.612 --> 00:49:16.909 because this test is designed to identify some of the common ethnic, deletions. 804 00:49:20.496 --> 00:49:22.457 If the, 805 00:49:22.457 --> 00:49:28.004 the deletion duplication or the common deletion test comes back as negative, 806 00:49:28.379 --> 00:49:31.924 which would I would say on only a few percent of the cases 807 00:49:31.924 --> 00:49:33.176 would come back as negative. 808 00:49:33.176 --> 00:49:38.264 If there is a patient has alpha thalassemia, about 3 to 5% of the cases, 809 00:49:38.765 --> 00:49:41.726 then the next test would be alpha globin sequencing. 810 00:49:41.726 --> 00:49:45.855 So in combination of in combination with these three, we should be able 811 00:49:45.855 --> 00:49:50.193 to identify pretty much 99% of the alpha thalassemia cases. 812 00:49:51.277 --> 00:49:53.571 The variant hemoglobin. 813 00:49:53.571 --> 00:49:56.783 But there is a change in the structure 814 00:49:57.492 --> 00:49:59.827 in the hemoglobin 815 00:49:59.827 --> 00:50:04.290 like hemoglobin S, C or that also change 816 00:50:04.290 --> 00:50:08.044 in the migration pattern or the protein structure of the hemoglobin. 817 00:50:08.461 --> 00:50:14.175 These can be easily identified by the other screening tool HPLC or Rep. 818 00:50:14.217 --> 00:50:16.385 References. 819 00:50:16.385 --> 00:50:19.639 So based upon the percentage of the variant hemoglobin, 820 00:50:19.639 --> 00:50:23.643 if it's about 40% 40 40% plus minus four, 821 00:50:24.227 --> 00:50:28.272 which is suggestive of beta variant, we can do better globin sequencing. 822 00:50:28.481 --> 00:50:32.735 Or if it's about 2,025%, then we can do alpha globin sequencing. 823 00:50:33.027 --> 00:50:36.614 So based upon the percentage of the variant hemoglobin 824 00:50:36.614 --> 00:50:41.285 we can decide either to do alpha globin sequencing or beta globin sequencing. 825 00:50:42.787 --> 00:50:43.579 So this is in 826 00:50:43.579 --> 00:50:46.958 nutshell a simplified algorithm to diagnose 827 00:50:47.750 --> 00:50:50.753 all forms of hemoglobin properties and thalassemia. 828 00:50:58.386 --> 00:51:00.221 So this is my last slide 829 00:51:00.221 --> 00:51:03.224 slide showing the references and acknowledgment. 830 00:51:05.560 --> 00:51:08.396 These are the references which I have used. 831 00:51:08.396 --> 00:51:13.025 And, I, I want to thank Doctor Carl and Dottie, 832 00:51:13.526 --> 00:51:16.529 which is really, they're really helpful in, 833 00:51:16.571 --> 00:51:18.864 in basically, 834 00:51:18.864 --> 00:51:23.202 providing us with providing me with some of the, the technical aspect 835 00:51:23.202 --> 00:51:26.706 and the clinical aspect of hemoglobin, diabetes and thalassemia. 836 00:51:30.084 --> 00:51:33.129 And, thank you very much. 837 00:51:33.129 --> 00:51:34.088 This is my last slide. 838 00:51:34.088 --> 00:51:37.383 Thank you very much for listening, to this webinar. 839 00:51:37.425 --> 00:51:37.967 Thank you.