WEBVTT 00:00:08.100 --> 00:00:09.233 Welcome to our video 00:00:09.233 --> 00:00:12.266 lecture entitled update in Red Blood Cell 00:00:12.266 --> 00:00:16.266 Membrane Disorders presented by Doctor Archana Agarwal. 00:00:16.300 --> 00:00:19.400 Doctor Archana Agarwal is the medical director 00:00:19.400 --> 00:00:23.333 of Hematology and Special Genetics at Air Laboratories. 00:00:23.666 --> 00:00:26.633 Additionally, she is an associate professor 00:00:26.633 --> 00:00:29.633 at the University of Utah School of Medicine. 00:00:29.766 --> 00:00:33.333 She obtained her medical degree from Delhi University. 00:00:33.866 --> 00:00:38.700 Doctor Agarwal completed her anatomic and clinical pathology residency, 00:00:39.066 --> 00:00:42.666 a fellowship in hematology and a Molecular genetic 00:00:42.666 --> 00:00:46.800 pathology fellowship at the University of Utah School of Medicine. 00:00:47.333 --> 00:00:51.166 Her research interests include red cell and the McCarthys, 00:00:51.533 --> 00:00:55.633 hemoglobin ofthese and molecular kinematic pathology. 00:00:55.700 --> 00:00:59.366 Doctor Agarwal is board certified in anatomic and clinical 00:00:59.366 --> 00:01:03.233 pathology, hematology, and molecular genetic pathology. 00:01:03.766 --> 00:01:06.833 I'll now turn our lecture over to Doctor Agarwal. 00:01:08.700 --> 00:01:10.266 Thank you very much, Brianna. 00:01:10.266 --> 00:01:13.033 That was a really nice introduction. 00:01:13.033 --> 00:01:16.166 So first of all, I like to 00:01:16.466 --> 00:01:19.466 welcome everyone, to this webinar. 00:01:19.566 --> 00:01:23.566 And the topic of today's presentation is update 00:01:23.566 --> 00:01:25.733 in Red Blood Cell Membrane Disorders. 00:01:26.866 --> 00:01:29.866 And these are the program objectives. 00:01:30.300 --> 00:01:34.633 So I'll go over different types of red blood cell membrane defects. 00:01:35.166 --> 00:01:40.700 And go also go over the different tests available for these 00:01:41.466 --> 00:01:46.566 and also try to give some correlative results. 00:01:46.700 --> 00:01:50.233 We'll go over some cases where we can look into 00:01:50.233 --> 00:01:53.433 how to correlate results of these laboratory test 00:01:53.766 --> 00:01:56.766 with these specific red blood cell membrane defects. 00:01:58.066 --> 00:02:00.400 We'll also try to go over 00:02:00.400 --> 00:02:03.400 some of the different technologies used for these, 00:02:04.200 --> 00:02:07.266 these testing that Geologies and last 00:02:07.266 --> 00:02:12.533 but not the least, I'll go over the utility of next generation 00:02:12.533 --> 00:02:15.533 sequencing or molecular diagnosis in, 00:02:16.133 --> 00:02:18.600 these disorders. 00:02:18.600 --> 00:02:21.366 So starting with very basic 00:02:21.366 --> 00:02:24.366 what the definition of anemia 00:02:24.433 --> 00:02:27.600 comes from a Greek word meaning without blood. 00:02:27.900 --> 00:02:33.300 So anemia is basically a condition where the capacity of blood 00:02:33.600 --> 00:02:36.933 to transport oxygen to tissues is reduced. 00:02:37.200 --> 00:02:41.333 So it's like it's the manifestations there could be the 00:02:41.400 --> 00:02:44.233 there they're usually very different. 00:02:44.233 --> 00:02:48.233 Pathophysiology which goes into anemia. 00:02:48.233 --> 00:02:51.800 And anemia has been described into different 00:02:52.533 --> 00:02:57.100 based on so many different parameters into many different types. 00:02:57.366 --> 00:03:02.400 And I won't go into detail about, classification of anemia. 00:03:03.100 --> 00:03:07.666 But I'll talk a little bit about hemolytic anemia, because 00:03:07.933 --> 00:03:10.666 the anemia seen in red blood cell membrane 00:03:10.666 --> 00:03:13.766 defect is a type of hemolytic anemia. 00:03:15.100 --> 00:03:18.766 And the treatment of anemia depends on, 00:03:18.766 --> 00:03:24.866 first of all, trying to understand what's what is the underlying pathophysiology. 00:03:24.866 --> 00:03:30.966 Because it could as I said, it could range from hemolytic process 00:03:30.966 --> 00:03:35.233 in the periphery to nutritional deficiency like iron efficiency, 00:03:35.500 --> 00:03:38.300 vitamin B12 deficiency or something, 00:03:38.300 --> 00:03:42.933 which is a problematic due to bone marrow itself. 00:03:43.200 --> 00:03:47.466 So the treatment ultimately would depend on the diagnosis. 00:03:47.466 --> 00:03:49.800 What's the underlying pathophysiology. 00:03:49.800 --> 00:03:52.800 And basically taking care of that problem. 00:03:54.000 --> 00:03:58.333 So hemolytic anemia is a type of anemia 00:03:58.333 --> 00:04:01.333 which is seen in red blood cell membrane defect. 00:04:01.633 --> 00:04:05.700 And it's characterized by premature destruction of red blood cells. 00:04:06.533 --> 00:04:10.600 And initially when there is premature destruction of red blood 00:04:10.600 --> 00:04:14.700 cells, bone marrow would try to compensate for it. 00:04:14.700 --> 00:04:17.700 And it does a really good job in compensating. 00:04:17.900 --> 00:04:21.900 And initially, if it's like a long standing chronic 00:04:21.900 --> 00:04:26.166 hemolytic process, the bone marrow would do a pretty good job 00:04:26.166 --> 00:04:32.100 in compensation, and patients might just present with mild, anemia. 00:04:34.200 --> 00:04:38.133 However, after some time or due to some pre 00:04:38.766 --> 00:04:42.933 predisposing condition or some kind of some other, 00:04:43.466 --> 00:04:46.466 condition like, sudden blood loss 00:04:46.633 --> 00:04:50.033 or maybe nutritional deficiency associated with this, 00:04:50.033 --> 00:04:54.166 the anemia would become really, kind of evident. 00:04:54.566 --> 00:04:57.933 And that's when we basically noticed anemia. 00:04:58.133 --> 00:05:03.533 If it's like a mild compensatory process, we might not even notice that anemia per 00:05:03.533 --> 00:05:06.766 se for the diagnosis of hemolytic 00:05:06.766 --> 00:05:09.766 anemia, there are a lot of different, 00:05:10.000 --> 00:05:12.033 parameters 00:05:12.033 --> 00:05:13.466 which are used. 00:05:13.466 --> 00:05:16.866 And one of the important parameter is the reticular site count, 00:05:16.866 --> 00:05:22.033 which basically tells that if tells us if the bone marrow is functioning or not. 00:05:22.133 --> 00:05:25.533 So usually in cases of hemolytic anemia, 00:05:25.533 --> 00:05:28.933 the bone marrow as the bone marrow will try to compensate for it. 00:05:29.200 --> 00:05:33.300 We would see increased in dramatic count in the periphery 00:05:33.300 --> 00:05:36.300 because these are the immature red blood cells. 00:05:36.300 --> 00:05:39.966 So bone marrow will try to compensate and it will basically 00:05:40.233 --> 00:05:43.366 will be hyper functioning and churn out 00:05:43.366 --> 00:05:46.366 immature red blood cells into the peripheral circulation. 00:05:46.566 --> 00:05:50.300 And then we also would see signs of red blood cell destruction. 00:05:50.300 --> 00:05:55.733 So like increase LDH, increase uncon conjugated bilirubin, 00:05:55.733 --> 00:05:58.800 which are all the byproduct, of red blood 00:05:58.800 --> 00:06:02.400 cells are ultimately some of the binding 00:06:02.800 --> 00:06:06.200 like Hep two globulin is a protein which usually binds to 00:06:07.433 --> 00:06:09.600 the red blood. 00:06:09.600 --> 00:06:11.733 The bilirubin. 00:06:11.733 --> 00:06:14.066 And then basically we will see, 00:06:14.066 --> 00:06:17.266 in cases of hemolytic process, we will see, 00:06:18.866 --> 00:06:21.633 the binding would be really high. 00:06:21.633 --> 00:06:24.633 So we will see a decrease in hep two globin as such. 00:06:24.633 --> 00:06:28.233 So these are some of the parameters which can be used to 00:06:28.233 --> 00:06:32.933 to look for hemolysis per se for the diagnosis of hemolytic anemia. 00:06:33.400 --> 00:06:37.333 So moving on to the classification of hemolytic anemia. 00:06:38.566 --> 00:06:41.033 So it could be due to intrinsic 00:06:41.033 --> 00:06:44.033 defect of red blood cells. 00:06:44.100 --> 00:06:46.900 Or it could be completely unrelated 00:06:46.900 --> 00:06:49.933 to red blood cell membrane or red blood cells per se. 00:06:50.066 --> 00:06:53.266 So completely extrinsic to red blood cells. 00:06:53.466 --> 00:06:56.666 So the intrinsic defect would be red blood cell membrane. 00:06:56.900 --> 00:07:00.333 That could be genetic defect in the red blood cell membrane per se. 00:07:00.633 --> 00:07:03.333 Or there could be problem with the red blood 00:07:03.333 --> 00:07:06.333 cell enzymes like G6 p d deficiency. 00:07:06.566 --> 00:07:10.700 Or it could be problem with the hemoglobin production 00:07:10.700 --> 00:07:14.966 as such, like we see in thalassemia and or hemoglobin properties, 00:07:15.600 --> 00:07:18.900 the extrinsic defect usually are due to some kind of 00:07:18.900 --> 00:07:22.566 autoimmune mediated conditions like autoimmune hemolytic anemia. 00:07:23.033 --> 00:07:25.766 It could also be due to some drugs and toxins 00:07:25.766 --> 00:07:29.366 which are very commonly seen er causing hemolytic process, 00:07:29.700 --> 00:07:34.666 or some kind of a mechanical disruption like valves or thermal injury. 00:07:34.833 --> 00:07:37.300 So these these are the extrinsic, 00:07:38.400 --> 00:07:40.500 causes of hemolytic anemia. 00:07:40.500 --> 00:07:44.800 For today's talk, we'll be focusing on the extrinsic defect 00:07:44.800 --> 00:07:47.800 which causes, which includes the 00:07:48.633 --> 00:07:51.066 genetic defect, 00:07:51.066 --> 00:07:53.600 causing red blood cell membrane defects. 00:07:53.600 --> 00:07:57.400 So this again, this is another way to look, look at it. 00:07:57.533 --> 00:08:01.633 So the majority of the intrinsic defect, 00:08:02.100 --> 00:08:05.733 of red blood cell membrane, like red blood cell membrane itself 00:08:05.733 --> 00:08:09.566 or enzyme deficiency or thalassemia is hemoglobin properties. 00:08:09.566 --> 00:08:10.933 These are headed to Turkey. 00:08:10.933 --> 00:08:13.733 So a person is born with it. 00:08:13.733 --> 00:08:16.700 So these are genetic defect acquired ones 00:08:16.700 --> 00:08:20.233 are usually these immune mediated 00:08:20.233 --> 00:08:24.733 or valve mechanical damage or physical thermal damage. 00:08:24.900 --> 00:08:27.900 So these are usually acquired. 00:08:28.200 --> 00:08:32.700 So let's go into a little bit detail on the red blood cell membrane defect. 00:08:32.700 --> 00:08:34.666 What are these. 00:08:34.666 --> 00:08:36.500 So it's 00:08:36.500 --> 00:08:40.100 broadly it is divided into two categories. 00:08:40.300 --> 00:08:46.000 One is intrinsic problems or structural problems. 00:08:47.000 --> 00:08:48.000 Intrinsic to red 00:08:48.000 --> 00:08:51.100 blood cell membrane like hereditary status, 00:08:51.100 --> 00:08:54.900 say ptosis or electro side ptosis and particular cytokines. 00:08:54.900 --> 00:08:57.900 So we'll I'll talk in a little bit detail. 00:08:58.266 --> 00:09:03.200 And there could be problem associated with red blood cells. 00:09:03.200 --> 00:09:05.733 So the membrane is more or less okay. 00:09:05.733 --> 00:09:08.233 There's no structural problem. 00:09:08.233 --> 00:09:11.233 However there is a problem in 00:09:12.000 --> 00:09:15.566 cation cation exchange leading to 00:09:16.700 --> 00:09:20.100 a defect in the hydration of red blood cells. 00:09:20.500 --> 00:09:23.500 These are relatively rare defect, but, 00:09:24.366 --> 00:09:26.400 it it can lead to either 00:09:26.400 --> 00:09:30.033 over hydrated or dehydrated stomata status. 00:09:30.166 --> 00:09:35.000 So this is, this is what, red blood cells, it looks like, stoma. 00:09:35.066 --> 00:09:37.033 So there is a mouth. 00:09:37.033 --> 00:09:40.466 And I'll go into a little bit more detail later on. 00:09:40.800 --> 00:09:48.000 So basically these are two major way to look into red blood cell membrane defect. 00:09:48.000 --> 00:09:53.366 One would be the structural problem or another would be the volume defect. 00:09:55.333 --> 00:09:57.200 So the most common 00:09:57.200 --> 00:10:00.966 red blood cell membrane defect is headed to this photo say ptosis. 00:10:00.966 --> 00:10:03.533 And I'm pretty sure everyone, 00:10:03.533 --> 00:10:04.633 has heard about this. 00:10:04.633 --> 00:10:07.633 This is a relatively common disorder, 00:10:08.733 --> 00:10:11.100 and it occurs due to alteration 00:10:11.100 --> 00:10:15.000 of one of the five genes which encode for proteins 00:10:15.300 --> 00:10:19.266 involved into the vertical association of red blood cell membrane. 00:10:19.266 --> 00:10:22.200 We'll look into that in the next slide. 00:10:22.200 --> 00:10:24.366 It occurs in all racial age. 00:10:24.366 --> 00:10:27.433 Racial groups, however, is particularly common 00:10:27.433 --> 00:10:30.433 in individuals of northern European ancestry, 00:10:31.200 --> 00:10:33.600 and it's seen anywhere 00:10:33.600 --> 00:10:36.666 from 1 in 1000 to 1 in 3000. 00:10:36.666 --> 00:10:43.333 So if we look into the genetic defect as such, like if you look if we look 00:10:43.333 --> 00:10:48.700 into the hereditary types of hemolytic anemia, this is not that uncommon. 00:10:49.233 --> 00:10:51.300 We see that, 00:10:51.300 --> 00:10:56.100 quite frequently, this is a relatively simplified 00:10:56.100 --> 00:10:58.900 diagram of red blood cell membrane structure. 00:10:58.900 --> 00:11:02.633 And I'm pretty sure everyone must have seen it at some point of time. 00:11:04.500 --> 00:11:06.000 This illustrates 00:11:06.000 --> 00:11:10.000 the kind of complex nature of that blood cell membrane. 00:11:10.166 --> 00:11:13.166 So this is the phospholipid bilayer. 00:11:13.433 --> 00:11:16.433 And these proteins. 00:11:17.066 --> 00:11:19.266 This is the band three anchoring 00:11:19.266 --> 00:11:22.266 protein 4.2, protein 4.1 00:11:22.833 --> 00:11:25.833 and which is anchored through this 00:11:27.033 --> 00:11:29.633 alpha and beta spectrum. 00:11:29.633 --> 00:11:31.633 So there are two different spectrum forms. 00:11:31.633 --> 00:11:34.566 This is the alpha spectrum. This is the beta spectrum. 00:11:34.566 --> 00:11:37.300 And they basically form tetra armors. 00:11:37.300 --> 00:11:43.966 And this is this supports the interaction of anchoring band three, 00:11:43.966 --> 00:11:48.766 which is the predominant protein, two the phospholipid bilayer. 00:11:49.366 --> 00:11:54.566 And due to this complex nature of these protein interaction 00:11:54.566 --> 00:11:58.733 between all these proteins in red blood cell membrane, 00:11:59.166 --> 00:12:02.200 it really gives the red blood 00:12:02.200 --> 00:12:05.200 cell the flexibility 00:12:05.366 --> 00:12:08.366 to change kind of the shape. 00:12:09.133 --> 00:12:11.300 The red blood cell is is a 00:12:11.300 --> 00:12:14.300 bi concave shaped structure. 00:12:14.400 --> 00:12:17.533 And it can it's a very flexible, 00:12:19.166 --> 00:12:20.066 structure. 00:12:20.066 --> 00:12:25.500 And because of these interaction and because of these proteins, 00:12:25.500 --> 00:12:30.866 the way it's kind of interacts with each other, it easily changes 00:12:30.866 --> 00:12:36.066 the configuration and can pass through the small splenic kind of venules. 00:12:37.166 --> 00:12:39.733 On the arterials in the spleen. 00:12:39.733 --> 00:12:44.133 And if there is, if anything happens to the, 00:12:44.400 --> 00:12:48.833 the complex kind of interaction of these proteins to each other, 00:12:49.400 --> 00:12:53.766 it loses it somehow loses the ability 00:12:53.766 --> 00:12:57.633 to basically traverse through these small capillaries. 00:12:57.933 --> 00:13:01.700 And it gets kind of it becomes more redundant. 00:13:01.933 --> 00:13:03.700 So it's not that flexible. 00:13:03.700 --> 00:13:06.000 And it can break down easily. 00:13:06.000 --> 00:13:09.433 And that's what happens if there is a problem 00:13:09.900 --> 00:13:12.766 in any of these proteins. 00:13:12.766 --> 00:13:16.000 So I mentioned about in my previous slide, 00:13:16.000 --> 00:13:20.966 I mentioned that the headed towards photosynthesis happens 00:13:20.966 --> 00:13:26.000 due to problems in the proteins taking part in the vertical association. 00:13:26.233 --> 00:13:28.700 And this is what I mean by vertical association. 00:13:28.700 --> 00:13:32.400 So these proteins, the band three, which is a predominant protein 00:13:33.466 --> 00:13:36.100 that interacts with anchoring and spectra. 00:13:36.100 --> 00:13:39.700 And so these anchoring band three is a predominant protein 00:13:39.700 --> 00:13:42.566 which takes part in the vertical association. 00:13:42.566 --> 00:13:46.333 And most commonly we see mutations 00:13:46.333 --> 00:13:49.566 in the genes like in anchoring on band three. 00:13:50.733 --> 00:13:53.400 In this fellow say ptosis, 00:13:53.400 --> 00:13:58.366 the horizontal, horizontally positioned skeletal protein, it kind of maintains 00:13:58.366 --> 00:14:04.400 the shape like most commonly these proteins are defective in 00:14:04.400 --> 00:14:08.000 headed up to, say, ptosis and part of particular say ptosis. 00:14:08.400 --> 00:14:11.400 The look into that in a little bit more detail. 00:14:11.466 --> 00:14:15.433 So this is a peripheral smear of hereditary spiral statuses. 00:14:15.433 --> 00:14:20.300 So what happens if there is a problem with the red blood cell. 00:14:20.333 --> 00:14:22.000 Some of these proteins. 00:14:22.000 --> 00:14:27.333 So instead of forming these by concave shape with central pallor 00:14:27.666 --> 00:14:33.600 the red blood cells become smaller in size and it lacks the central pallor. 00:14:33.600 --> 00:14:37.500 And it becomes this small kind of condense to red blood cells. 00:14:37.500 --> 00:14:39.433 So these are this photo sites. 00:14:39.433 --> 00:14:43.000 And it, it basically it kinds of 00:14:43.000 --> 00:14:47.500 it has reduced surface to volume ratio as we can see 00:14:47.500 --> 00:14:51.900 there's kind of reduction in the red blood cell membrane structure. 00:14:52.566 --> 00:14:55.666 So it has reduced surface to volume 00:14:56.000 --> 00:14:59.000 ratio leading to spherical spherical shape. 00:14:59.466 --> 00:15:04.733 And it's, it's much more kind of, it's much less flexible. 00:15:04.733 --> 00:15:06.900 And it can break down easily. 00:15:06.900 --> 00:15:09.066 So these are these photo sites here. 00:15:09.066 --> 00:15:14.100 So these are, these are smaller in size and also lacks the central pallor. 00:15:15.266 --> 00:15:15.900 One thing which 00:15:15.900 --> 00:15:19.000 I need to mention here is this photo sites, 00:15:20.600 --> 00:15:23.900 although very commonly seen as inherited traits, farrokh 00:15:23.933 --> 00:15:28.200 ptosis is not specific for hey trace that will say ptosis. 00:15:29.033 --> 00:15:31.600 It can also be seen it easily 00:15:31.600 --> 00:15:34.633 commonly seen in autoimmune hemolytic process. 00:15:35.066 --> 00:15:39.533 So and those are much more commonly then headed towards photosynthesis. 00:15:39.733 --> 00:15:44.700 So if you're seeing as photo sites that first thing we need to question 00:15:44.833 --> 00:15:48.400 that does this patient have some kind of autoimmune process going on. 00:15:48.600 --> 00:15:51.600 Because the pathophysiology is kind of similar. 00:15:51.866 --> 00:15:56.200 When there is breakdown of red blood cells is takes it takes the shape 00:15:56.200 --> 00:15:59.200 of least resistance, which is the spherical shape. 00:16:00.233 --> 00:16:03.233 So it's seen in both autoimmune hemolytic process 00:16:03.266 --> 00:16:06.266 as well as head injuries ferocity ptosis. 00:16:07.633 --> 00:16:09.866 So headed to this fellow say ptosis 00:16:09.866 --> 00:16:12.866 because it's a hereditary form of hemolytic anemia. 00:16:13.266 --> 00:16:18.266 It's 75 to 80% of them are dominantly inherited. 00:16:19.666 --> 00:16:22.466 About 20% of them are recessive. 00:16:22.466 --> 00:16:25.466 Or denote that means, 00:16:26.100 --> 00:16:29.600 both the parents are carrying the gene, but they're not affected. 00:16:29.966 --> 00:16:33.000 De novo means the parents are pinched on. 00:16:33.000 --> 00:16:36.000 Parents are not affected. 00:16:36.000 --> 00:16:37.800 However, the, 00:16:37.800 --> 00:16:42.700 the genetic change happens only in the person who is affected. 00:16:43.200 --> 00:16:47.100 The clinical manifestations are quite variable, 00:16:47.400 --> 00:16:51.866 and it depends on what kind of genetic change the patient has. 00:16:51.866 --> 00:16:56.033 It can be completely, completely, mildly anemic. 00:16:56.033 --> 00:16:58.833 The patient can might not even know about it. 00:16:58.833 --> 00:17:01.833 So it could be mild hemolytic anemia all their life. 00:17:02.400 --> 00:17:05.766 And however, some patients could be moderately 00:17:05.766 --> 00:17:07.700 severe to transfusion dependent. 00:17:07.700 --> 00:17:10.700 So it has a wide, kind of, 00:17:10.733 --> 00:17:13.733 clinical presentation. 00:17:14.400 --> 00:17:17.233 Spectrum deficiency is often present 00:17:17.233 --> 00:17:20.333 in almost all cases of itches, 00:17:20.366 --> 00:17:25.000 as we can imagine, if we go back to the this picture, 00:17:26.133 --> 00:17:26.866 all these 00:17:26.866 --> 00:17:31.900 proteins, even though the commonly mutated gene is anchoring in band three, 00:17:32.200 --> 00:17:35.200 the all these proteins are linked together. 00:17:35.233 --> 00:17:38.233 So even even if, 00:17:38.333 --> 00:17:41.066 the mutation is in band three or anchoring 00:17:41.066 --> 00:17:46.266 or these are reduced in quantity, ultimately they are all linked together 00:17:46.266 --> 00:17:49.800 as a spectrum, which is the most, common kind of protein 00:17:50.133 --> 00:17:53.466 is commonly, reduced to. 00:17:55.600 --> 00:17:59.100 So, spectrum deficiency is seen 00:17:59.100 --> 00:18:02.533 in almost all these patients, including mutations. 00:18:02.533 --> 00:18:07.266 So the most common mutation in northern European population is incorrect, 00:18:08.366 --> 00:18:11.500 seen in approximately 50 to 60% of cases, 00:18:11.866 --> 00:18:15.300 but it's also ethnicity dependent about, 00:18:17.666 --> 00:18:18.400 it's only seen 00:18:18.400 --> 00:18:21.500 in about 5 to 10% of cases in Japan. 00:18:21.500 --> 00:18:24.933 So most common mutation in Japanese population is band three. 00:18:25.300 --> 00:18:29.366 So these mutations are also also ethnicity dependent. 00:18:30.466 --> 00:18:32.633 However, as I mentioned earlier, 00:18:32.633 --> 00:18:36.633 the it doesn't matter where the primary mutation is, 00:18:36.633 --> 00:18:39.633 and it could be in band three or anchoring. 00:18:39.866 --> 00:18:43.266 Ultimately, these proteins will lead on to 00:18:43.266 --> 00:18:46.700 the assembly to the spectrum and the spectrum deficiency 00:18:47.066 --> 00:18:50.966 also has been correlated with the the clinical severity. 00:18:52.933 --> 00:18:54.633 So moving on to headed to 00:18:54.633 --> 00:18:57.933 a lab to say ptosis or and say ptosis 00:18:58.466 --> 00:19:01.466 compared to x ray traced parasite ptosis, 00:19:01.466 --> 00:19:04.466 these are relatively uncommon, but still 00:19:05.433 --> 00:19:07.966 we see that 00:19:07.966 --> 00:19:11.500 again, these these are genetically and clinically 00:19:11.500 --> 00:19:15.400 heterogeneous disorder, in Aleppo, psychosis. 00:19:15.733 --> 00:19:18.733 The red blood cells are elliptical in shape. 00:19:19.200 --> 00:19:21.500 These are autism are 00:19:21.500 --> 00:19:24.500 autosomal dominantly inherited. 00:19:24.600 --> 00:19:28.566 And majority of these HIV patients are 00:19:29.933 --> 00:19:31.766 clinically more or less normal. 00:19:31.766 --> 00:19:34.766 They might have mild hemolytic anemia. 00:19:36.800 --> 00:19:37.766 It's again 00:19:37.766 --> 00:19:41.333 kind of ethnicity dependent is more common in malaria. 00:19:41.333 --> 00:19:45.566 Endemic reason with prevalence approaching up to 2% in West Africa. 00:19:46.500 --> 00:19:51.533 It has been shown initially I like to say ptosis and ptosis. 00:19:51.533 --> 00:19:54.900 They were supposed they were thought to be two different disorders. 00:19:55.266 --> 00:19:59.633 However, with the molecular understanding of these disorders. 00:19:59.633 --> 00:20:02.766 Now, we think that, the these 00:20:02.766 --> 00:20:05.800 both these disorders represent kind of 00:20:06.066 --> 00:20:10.200 on the same spectrum and lepto psychosis being the milder version. 00:20:10.200 --> 00:20:15.500 So lepto side lepto side ptosis patients usually carry one mutation. 00:20:16.033 --> 00:20:20.233 However, if these patients have another mutation 00:20:20.466 --> 00:20:25.133 that the patient was would present with a particular psychosis. 00:20:25.366 --> 00:20:27.200 So these these are basically 00:20:28.333 --> 00:20:30.833 these are kind of 00:20:30.833 --> 00:20:33.833 disorders with the completely 00:20:33.966 --> 00:20:36.466 asymptomatic to mild anemia. 00:20:36.466 --> 00:20:39.900 Two transfusion dependent marked hemolytic 00:20:39.900 --> 00:20:43.033 anemia leap to, say, ptosis being the milder form. 00:20:43.033 --> 00:20:48.100 And hatred or particular psychosis would be the moderate 00:20:48.100 --> 00:20:51.866 to severe hemolytic anemia and sometimes might need transfusion to. 00:20:52.600 --> 00:20:55.500 As I mentioned, this is, 00:20:55.500 --> 00:21:01.066 the the, the prevalence or of these disorders 00:21:01.066 --> 00:21:05.133 are difficult to know for sure because HIV patients 00:21:05.466 --> 00:21:08.466 might not even kind of diagnosed all the time 00:21:08.800 --> 00:21:11.800 because of the mild, really clinical phenotype. 00:21:13.500 --> 00:21:16.933 One of the one of the important thing which we need to remember, 00:21:16.933 --> 00:21:22.166 especially in HP patients, these usually present in units 00:21:22.400 --> 00:21:26.100 and because of the nature of the disease, because these are moderate 00:21:26.100 --> 00:21:29.266 to mark, these present with moderate to mark hemolytic anemia, 00:21:30.600 --> 00:21:34.033 the neonates can have significant, 00:21:34.266 --> 00:21:38.433 hemolysis and can also present with hyper bilirubin. 00:21:38.433 --> 00:21:42.200 Amia and clinical sequelae associated 00:21:42.200 --> 00:21:45.766 with like CNS manifestations associated with these 00:21:46.833 --> 00:21:49.133 a marked increase in bilirubin. 00:21:49.133 --> 00:21:52.133 So that's something to keep in mind. 00:21:52.300 --> 00:21:55.933 So this is what, peripheral smear of hereditary 00:21:55.933 --> 00:21:57.633 a lepto say ptosis would look like. 00:21:57.633 --> 00:22:00.400 So these are the lepto sites. 00:22:00.400 --> 00:22:03.633 These these are kind of elliptical in shape, 00:22:04.000 --> 00:22:06.833 and there's not a whole lot of 00:22:06.833 --> 00:22:09.833 and I suppose glossy ptosis here. 00:22:10.733 --> 00:22:14.066 But if you look at the glossy ptosis, 00:22:14.466 --> 00:22:18.300 which is the extreme form of kind of like itchy, 00:22:19.600 --> 00:22:21.766 they still see some lepto sites 00:22:21.766 --> 00:22:24.766 here, as we can, notice in this picture. 00:22:24.900 --> 00:22:28.000 But the peripheral smear, as we, as you can see 00:22:28.000 --> 00:22:31.133 here, is like marked and I suppose a glossy ptosis. 00:22:31.466 --> 00:22:34.633 But some of these red blood cells having the blurbs. 00:22:35.533 --> 00:22:37.733 So really bizarre 00:22:37.733 --> 00:22:41.466 looking red blood cells and this, these are the characteristic kind 00:22:41.466 --> 00:22:46.233 of finding on the periphery, smeared off, headed to this particular, say, ptosis. 00:22:46.566 --> 00:22:51.233 And the name, the pyro comes from the fact that usually red blood 00:22:51.233 --> 00:22:55.200 cells can withstand temperature up to 49°C. 00:22:55.500 --> 00:23:01.233 However, in this condition, they start to break down at around 44 45°C, 00:23:01.533 --> 00:23:06.766 and the peripheral smear of a born patient would look exactly like this smear. 00:23:06.966 --> 00:23:09.966 So that's why this is known as headed tree. 00:23:10.033 --> 00:23:13.866 This the name, the title comes from this, that nature. 00:23:14.666 --> 00:23:19.000 So there's extreme and I suppose closer to us with a lot of LPA sites 00:23:19.000 --> 00:23:22.266 in the background with red blood cells having bizarre shapes. 00:23:23.133 --> 00:23:27.500 Some microspheres sites like these can also be seen. 00:23:29.433 --> 00:23:32.800 So moving on to headed towards tomato psychosis. 00:23:32.800 --> 00:23:37.000 So the earlier ones like trace said 00:23:37.033 --> 00:23:41.400 okay, ketosis lepto psychosis and pineapple loci ptosis. 00:23:41.633 --> 00:23:45.233 Those were all problems like structural 00:23:45.233 --> 00:23:49.066 problems in red blood cell membrane and stormy to say 00:23:49.066 --> 00:23:53.866 ptosis are due to problems with the red blood cell hydration. 00:23:53.866 --> 00:23:56.866 So this leak is not really liquid as such. 00:23:56.866 --> 00:23:59.866 But there is a problem with the transfer 00:23:59.866 --> 00:24:02.866 of these cations. 00:24:03.133 --> 00:24:07.466 And because of the abnormal permeability, to the cation 00:24:07.466 --> 00:24:11.133 channel, there is change in the red blood cell hydration. 00:24:11.366 --> 00:24:15.333 So most common of these disorders are the dehydrated stomach 00:24:15.333 --> 00:24:18.800 or situs is also known as auditory zero say ptosis. 00:24:21.600 --> 00:24:23.100 Most of these patients 00:24:23.100 --> 00:24:26.300 these are not these are relatively rare disorders. 00:24:26.566 --> 00:24:30.100 However, many of these patients remain unrecognized 00:24:30.100 --> 00:24:33.033 or undiagnosed all throughout their life. 00:24:33.033 --> 00:24:35.933 These patients are present, usually present 00:24:35.933 --> 00:24:39.500 with high mean corpuscular hemoglobin concentration. 00:24:39.500 --> 00:24:43.233 Because of the dehydration and also because of the dehydration. 00:24:43.233 --> 00:24:45.366 They are resistant to osmotic fragility. 00:24:46.900 --> 00:24:49.500 I don't overload happens very commonly. 00:24:49.500 --> 00:24:52.666 And the etiology why that happens is not that clear. 00:24:54.566 --> 00:24:57.566 Most of them are due to the mutation in this P 00:24:57.600 --> 00:25:01.600 is only one gene which is responsible for the P is 00:25:01.700 --> 00:25:04.700 one proteins 00:25:04.766 --> 00:25:08.233 important for the mechanically activated cation channel. 00:25:08.233 --> 00:25:10.366 So there is a problem with the cation channels 00:25:10.366 --> 00:25:13.366 leading to dehydration of these red blood cells. 00:25:13.566 --> 00:25:16.433 One of the important things to remember for 00:25:16.433 --> 00:25:19.433 these tomato C tools is patients. 00:25:19.600 --> 00:25:21.233 Is splenectomy. 00:25:21.233 --> 00:25:24.733 Splenectomy, which is the treatment of choice 00:25:24.733 --> 00:25:29.433 in the earlier disorders like hydrous ferro ketosis and a little situs. 00:25:29.500 --> 00:25:32.500 Because if you take out the spleen in those disorders, 00:25:32.700 --> 00:25:37.800 the hemolytic process is much is kind of it becomes much lower. 00:25:38.000 --> 00:25:39.966 So the hemolysis decreases. 00:25:39.966 --> 00:25:43.033 However, for some reason, which we don't understand very well, 00:25:43.633 --> 00:25:46.333 splenectomy is contraindicated in this. 00:25:47.366 --> 00:25:49.633 In this particular disorder 00:25:49.633 --> 00:25:53.466 due to an increased risk of thromboembolic complications. 00:25:54.833 --> 00:25:59.233 The other form of stomata, say ptosis, is the over hydrated form. 00:25:59.233 --> 00:26:04.233 So here the the red blood cells become really, hydrated. 00:26:04.233 --> 00:26:07.233 So there's a lot of fluid inside. 00:26:07.366 --> 00:26:10.500 And this is a classical form of stomata. 00:26:10.500 --> 00:26:12.900 Situs is where the name comes from. 00:26:12.900 --> 00:26:16.533 And it happens due to the mutation of the origin associated glycoproteins. 00:26:16.533 --> 00:26:19.733 Commonly this is relatively very, very rare. 00:26:19.733 --> 00:26:21.633 We don't see that very often. 00:26:21.633 --> 00:26:23.100 And this is what it would look like. 00:26:23.100 --> 00:26:28.500 So the classical form, which is the over hydrated stomata, say ptosis, 00:26:28.766 --> 00:26:32.266 would have this classic stoma like a mouth 00:26:32.266 --> 00:26:36.300 like appearance of red blood cells, few stoma like red 00:26:36.300 --> 00:26:39.900 blood cells like these are commonly seen in any peripheral smear. 00:26:39.900 --> 00:26:44.766 But to to have a suspicion of headed twist on it or say ptosis. 00:26:45.033 --> 00:26:48.066 Majority of the red blood cells should have something like this. 00:26:49.333 --> 00:26:51.166 The dehydrated forms, 00:26:51.166 --> 00:26:54.166 the peripheral smear findings are not really, 00:26:55.000 --> 00:26:57.200 very specific. 00:26:57.200 --> 00:27:02.266 We can see some target cells like this or some of these red blood cells 00:27:02.266 --> 00:27:07.066 with puddled hemoglobin in the, in the periphery, like this. 00:27:07.066 --> 00:27:11.233 Or this can be seen, however, the the peripheral 00:27:11.233 --> 00:27:14.866 smear findings are not really specific for dehydrated forms. 00:27:14.866 --> 00:27:17.866 So that also makes the diagnosis very difficult. 00:27:19.966 --> 00:27:22.533 These are the genetic findings 00:27:22.533 --> 00:27:26.266 or genetic mutations commonly seen in these disorders. 00:27:26.266 --> 00:27:30.433 Like as I mentioned earlier in headed to this video, say to say this, most 00:27:30.433 --> 00:27:36.000 commonly mutated genes are band three, also known as SLC four A1 00:27:36.300 --> 00:27:39.300 or anchoring 00:27:39.900 --> 00:27:42.633 followed by beta spectrum and alpha spectrum. 00:27:42.633 --> 00:27:46.200 Alpha spectrum mutations represent all causes. 00:27:46.200 --> 00:27:49.900 The autosomal recessive form of ptosis. 00:27:50.833 --> 00:27:53.833 Those are the 00:27:54.000 --> 00:27:58.266 more, more or less moderate to severe form of headdress, okay, ptosis. 00:27:58.266 --> 00:28:01.733 And since these are original recessive forms, the 00:28:02.100 --> 00:28:06.433 the parents are usually not really affected. And, 00:28:07.433 --> 00:28:08.666 the diagnosis can 00:28:08.666 --> 00:28:12.966 be very difficult because there is no family history as such. 00:28:14.166 --> 00:28:16.300 They like to say ptosis and pineapple. 00:28:16.300 --> 00:28:17.233 Closed ptosis. 00:28:17.233 --> 00:28:22.500 The most commonly mutated genes are spectrum genes for the dehydrated one. 00:28:22.500 --> 00:28:26.500 Peso one is the most commonly mutated gene, and for the over hydrated, 00:28:26.500 --> 00:28:29.933 the orange associated glycoproteins are commonly mutated. 00:28:31.633 --> 00:28:33.133 So let's move on 00:28:33.133 --> 00:28:36.133 to the diagnosis of these disorders. 00:28:36.266 --> 00:28:39.333 So family history because all these disorders are 00:28:39.600 --> 00:28:43.966 have a headed 2D kind of hereditary no inheritance. 00:28:44.500 --> 00:28:47.833 So family history, especially for original dominant ones 00:28:47.833 --> 00:28:50.833 becomes really, really important. 00:28:51.466 --> 00:28:52.900 Though for smear 00:28:52.900 --> 00:28:55.900 it's absolutely essential sometimes. 00:28:56.100 --> 00:29:00.333 Especially for ELP to say ptosis or loci ptosis, 00:29:00.333 --> 00:29:04.866 where we see Mark and I still particular say ptosis, purplish smear is 00:29:04.866 --> 00:29:08.500 would be the most important, diagnostic feature. 00:29:09.100 --> 00:29:10.200 Here. 00:29:10.200 --> 00:29:13.000 For some of these disorders headed towards fairer 00:29:13.000 --> 00:29:16.000 statuses, preference mirrors are also helpful. 00:29:16.066 --> 00:29:20.000 However, as I mentioned earlier, it might not be 100% 00:29:20.000 --> 00:29:22.033 specific as other conditions 00:29:22.033 --> 00:29:25.900 like autoimmune hemolytic process can also have several sites. 00:29:27.900 --> 00:29:30.900 CBC obviously is, important, 00:29:31.133 --> 00:29:33.833 including retcon and red blood cell indices 00:29:33.833 --> 00:29:36.833 for other hemolytic processes 00:29:37.633 --> 00:29:41.366 in etches because these cells are smaller in size, 00:29:42.066 --> 00:29:46.600 the mean corpuscular hemoglobin concentration of more than or equal 00:29:46.600 --> 00:29:50.866 to 36 are usually very supportive of a diagnosis. 00:29:50.866 --> 00:29:56.233 If there is a strong family history, usually there will be slightly low MCB 00:29:56.800 --> 00:30:01.166 in a lepto psychosis and situs is the red. 00:30:01.166 --> 00:30:04.900 Blood cells are really micro acidic with high MC. 00:30:04.966 --> 00:30:09.900 At C there can be mark and I suppose closer to six with RBC fragmentation. 00:30:10.933 --> 00:30:15.466 So both a smear and CBC would be really, 00:30:15.466 --> 00:30:18.866 really essential for the diagnosis of these disorders. 00:30:19.500 --> 00:30:24.566 Before we even think about moving on to some of the other specific testing. 00:30:24.566 --> 00:30:27.266 So these would be the first line, 00:30:27.266 --> 00:30:30.833 specially getting the family history, peripheral smear evaluation 00:30:31.200 --> 00:30:36.600 along with, correlation with CBC like mCAT c would be super helpful 00:30:36.833 --> 00:30:41.100 for etches diagnosis and peripheral smear evaluation. 00:30:41.700 --> 00:30:42.533 To look out for. 00:30:42.533 --> 00:30:46.933 Lepto sites are marked and I suppose close eye ptosis with fragmentation 00:30:46.933 --> 00:30:51.500 and bizarre red blood cells would be very helpful for the diagnosis of HPI. 00:30:53.366 --> 00:30:57.000 So after that, if the are suspicious of. 00:30:57.000 --> 00:30:59.866 Let's see etches based on the family history 00:30:59.866 --> 00:31:03.833 because majority of them are arterial dominant in nature. 00:31:04.533 --> 00:31:07.600 And there are further sites in the peripheral smear. 00:31:07.600 --> 00:31:12.200 And we think the mix is higher and we have a very high index 00:31:12.200 --> 00:31:16.600 of suspicion of status, very trace photo statuses. 00:31:16.600 --> 00:31:18.200 How can we confirm it? 00:31:18.200 --> 00:31:21.566 So there are two different test which we can, do. 00:31:21.566 --> 00:31:24.100 And these are relatively inexpensive. 00:31:24.100 --> 00:31:27.100 Most of the major laboratories offer these test. 00:31:27.966 --> 00:31:29.800 So first the preferred test 00:31:29.800 --> 00:31:32.800 would be a flow cytometry based test. 00:31:33.100 --> 00:31:37.700 Where we do look for the this dye 00:31:38.466 --> 00:31:40.866 using 5 million might dye 00:31:40.866 --> 00:31:43.866 binding to the intact red blood cells. 00:31:43.966 --> 00:31:47.866 And this dye binds specifically to the band three protein, 00:31:47.866 --> 00:31:51.233 which is, one of the most abundant protein and, 00:31:52.800 --> 00:31:54.900 here, basically we 00:31:54.900 --> 00:31:58.066 we look for the, decrease 00:31:58.066 --> 00:32:01.066 in fluorescence or binding by flow 00:32:01.200 --> 00:32:04.200 by using the flow cytometry method. 00:32:04.800 --> 00:32:07.700 If the flow cytometry is not available. 00:32:07.700 --> 00:32:11.400 Or this the also known as the EMA test the use in 5 million. 00:32:11.400 --> 00:32:14.100 My binding test is not available. 00:32:14.100 --> 00:32:17.100 Other test would be the osmotic fragility test 00:32:17.133 --> 00:32:20.133 is it's a relatively, 00:32:21.000 --> 00:32:24.000 inexpensive, easy to do test 00:32:25.300 --> 00:32:29.500 where basically the RBCs are incubated in hypertonic 00:32:29.500 --> 00:32:33.200 buffered salt solution of different osmolarity. 00:32:33.833 --> 00:32:37.633 And the test takes the advantage of the feature 00:32:37.633 --> 00:32:42.066 that these red blood cells, their specific red blood cells, would lyse easily. 00:32:42.666 --> 00:32:45.933 And the of the lysis of these 00:32:45.933 --> 00:32:48.933 red blood cells are measured by, 00:32:49.033 --> 00:32:52.033 a spectral photometric method. 00:32:52.700 --> 00:32:56.033 It's relatively I mean, it's not it's not a bad test. 00:32:56.033 --> 00:33:01.133 However, it's not a specific test compared to our EMR flow cytometry based test. 00:33:01.500 --> 00:33:04.633 So the the sensitivity and specificity of this flow 00:33:04.633 --> 00:33:08.366 cytometry based test is anywhere from 90 to 95%. 00:33:08.833 --> 00:33:13.666 However, the know the sensitivity is about 60 to 80% 00:33:13.666 --> 00:33:17.666 for the osmotic fragility test, so it would miss some of the milder 00:33:17.666 --> 00:33:23.400 forms of etches and also it's not specific. 00:33:23.766 --> 00:33:28.266 So that's one thing to remember because this this this test 00:33:28.300 --> 00:33:33.866 osmotic fragility test will diagnose any condition where we see several sites. 00:33:33.866 --> 00:33:37.000 So like it would be positive in both autoimmune 00:33:37.000 --> 00:33:40.300 hemolytic anemia as well as here trace photosynthesis 00:33:41.166 --> 00:33:45.166 compared to EMA tests which is relatively much more specific. 00:33:45.166 --> 00:33:50.400 It's it's it's not positive in autoimmune hemolytic process. 00:33:50.600 --> 00:33:55.066 There are only rare, scenarios where it can be falsely positive. 00:33:55.466 --> 00:33:57.366 So that's something to keep in mind. 00:33:58.466 --> 00:34:01.366 So the flow based tests, as I mentioned earlier, 00:34:01.366 --> 00:34:05.166 this dye specifically binds to the band triple protein. 00:34:05.166 --> 00:34:08.833 And we basically are looking at the reduction in the fluorescence. 00:34:11.433 --> 00:34:12.033 And it 00:34:12.033 --> 00:34:15.366 even though it binds specifically to band three protein, 00:34:15.366 --> 00:34:21.000 it will detect all problem with all sorts of membrane protein abnormalities. 00:34:21.000 --> 00:34:26.100 So it's not it will not only detect band three protein defect, it will detect 00:34:26.600 --> 00:34:31.100 a defect in spectra in or anchoring which are much more common. 00:34:32.000 --> 00:34:35.866 And sensitivity as I mentioned is almost about 95%. 00:34:35.866 --> 00:34:38.866 The specificity is more or less the same. 00:34:39.500 --> 00:34:41.533 It only requires a very small amount 00:34:41.533 --> 00:34:44.766 of blood, and the turnaround time is also very good. 00:34:44.766 --> 00:34:48.633 And the the dye is very, kind of stable. 00:34:48.633 --> 00:34:53.800 So the test can be done, for almost like 5 to 7 days old blood. 00:34:54.633 --> 00:34:59.300 So it, it works out pretty good for the diagnosis of, etches 00:35:00.566 --> 00:35:01.600 osmotic fragility. 00:35:01.600 --> 00:35:04.600 As I mentioned, if EMA test, 00:35:04.733 --> 00:35:08.633 is not available or if the EMA test is negative, 00:35:09.300 --> 00:35:13.333 then osmotic fragility test can be done. 00:35:13.600 --> 00:35:16.600 And basically it looks for the hemolysis. 00:35:17.066 --> 00:35:20.700 And in most patients the hemolysis would be markedly increased 00:35:20.700 --> 00:35:24.100 like something like this compared to, normal controls. 00:35:26.500 --> 00:35:28.266 There are other tests 00:35:28.266 --> 00:35:31.266 available for etches, however. 00:35:32.666 --> 00:35:35.666 On almost all of these tests 00:35:36.466 --> 00:35:39.733 are kind of only available in some of the esoteric, 00:35:41.033 --> 00:35:43.800 laboratories like osmotic gradient, actor 00:35:43.800 --> 00:35:47.400 cytometry test, which basically look for the 00:35:49.533 --> 00:35:51.600 RBC deform, 00:35:51.600 --> 00:35:54.800 deform ability under a defined shear stress. 00:35:54.800 --> 00:35:59.866 So it's a very complex, esoteric test and only off 00:35:59.866 --> 00:36:03.500 get only offered by 1 or 2 US laboratories. 00:36:06.600 --> 00:36:08.100 The, the other test, 00:36:08.100 --> 00:36:11.200 I'm mentioning these test because we hear 00:36:11.200 --> 00:36:14.800 about these tests and as mentioned in most of the books, however, these 00:36:14.800 --> 00:36:18.433 these tests are usually mostly not done in the US laboratories. 00:36:18.900 --> 00:36:19.900 These tests are 00:36:21.100 --> 00:36:24.100 offered in some of the European laboratories, 00:36:24.566 --> 00:36:27.966 where the incidence is very high. 00:36:28.466 --> 00:36:33.233 So glass glycerol lysis test, or acidified glycerol latest 00:36:33.266 --> 00:36:38.800 lysis test are kind of similar to osmotic fragility test, where the glycerol 00:36:38.800 --> 00:36:43.900 or glycerol plus the sodium phosphate is added to the hypotonic buffer solution. 00:36:44.266 --> 00:36:48.800 And RBCs are incubated with this in the major. 00:36:48.800 --> 00:36:51.400 The lysis of red blood cells. 00:36:51.400 --> 00:36:55.400 The cryo hemolysis tests here, the RBCs are suspended 00:36:55.400 --> 00:36:58.800 in hypertonic saline and then briefly heated 00:36:58.800 --> 00:37:01.800 and cooled down for ten minutes. 00:37:02.700 --> 00:37:06.000 Almost all of these tests, the sensitivity is 00:37:06.000 --> 00:37:11.500 our specificity is anywhere from 60 to 80 to 95%. 00:37:11.533 --> 00:37:11.966 None. 00:37:11.966 --> 00:37:17.100 None of these tests have a sensitivity and specificity of 100%. 00:37:17.366 --> 00:37:22.566 And it's recommended that in combination, like combination of two tests, 00:37:22.566 --> 00:37:26.133 like the flow cytometry based test or, 00:37:26.733 --> 00:37:30.700 osmotic fragility or flow cytometry or one of these tests, 00:37:32.000 --> 00:37:33.000 if both these 00:37:33.000 --> 00:37:36.466 tests, if two tests are done for the diagnosis, 00:37:36.833 --> 00:37:42.500 the sensitivity and specificity both increases to almost like 9,598%. 00:37:42.900 --> 00:37:47.800 So if one test comes back as negative, then it should, the end. 00:37:47.800 --> 00:37:51.800 If there is a high index of clinical suspicion, then another test 00:37:51.800 --> 00:37:55.700 should be done to rule out edges cases. 00:37:58.700 --> 00:38:02.700 So the confirmatory tests for H, E and HP. 00:38:02.700 --> 00:38:04.200 So for the h e. 00:38:04.200 --> 00:38:08.400 And I was talking mostly about the diagnosis where the EMA 00:38:08.400 --> 00:38:11.400 test and osmotic fragility tests are helpful 00:38:12.300 --> 00:38:14.700 for the H, E and HP. 00:38:14.700 --> 00:38:18.433 The the diagnosis is mostly based on the red blood cell. 00:38:19.300 --> 00:38:19.633 Sorry. 00:38:19.633 --> 00:38:23.400 The peripheral smeared evaluation, the EMR binding test. 00:38:23.400 --> 00:38:27.833 The flow tests are mostly markedly decreased, 00:38:27.833 --> 00:38:32.966 so it won't distinguish between etching and HP just based on the, 00:38:35.066 --> 00:38:37.566 the, the test per se. 00:38:37.566 --> 00:38:39.866 However, if we look under the microscope, 00:38:39.866 --> 00:38:43.733 we would be able to easily distinguish whether it's HS or HCR. 00:38:43.800 --> 00:38:48.800 HP osmotic Gradient editor cytometry might be helpful in some of 00:38:48.833 --> 00:38:52.033 in some of the complex cases of E and NP 00:38:53.966 --> 00:38:56.266 or or basically 00:38:56.266 --> 00:39:00.133 giving us some idea about what kind of molecular testing might be needed 00:39:00.900 --> 00:39:03.900 that brings, that brings, 00:39:03.900 --> 00:39:06.533 the topic of molecular testing, 00:39:06.533 --> 00:39:11.000 why molecular testing would be helpful in these, in these scenarios. 00:39:11.333 --> 00:39:15.166 So let's go into a little bit more detail about the utility 00:39:15.166 --> 00:39:18.166 of molecular testing in this, in this, 00:39:19.433 --> 00:39:21.400 in these disorders and also 00:39:21.400 --> 00:39:24.633 the utility of this molecular testing, especially the next generation 00:39:24.633 --> 00:39:28.700 based next generation sequencing based testing in neonatal period. 00:39:30.000 --> 00:39:34.200 So, as we might recall, the headed to 00:39:34.200 --> 00:39:38.166 this philosophy tools is 70% of them are due to autosomal dominant. 00:39:38.166 --> 00:39:42.433 However about 20 to 25% of them are due to autosomal recessive. 00:39:43.500 --> 00:39:45.533 And as as we 00:39:45.533 --> 00:39:48.533 can't I mean, as we can imagine in these patients, 00:39:49.166 --> 00:39:52.166 with autosomal recessive, 00:39:52.200 --> 00:39:55.433 inheritance, the family members or the 00:39:56.500 --> 00:40:00.300 the parents of these patients would have a really milder 00:40:00.300 --> 00:40:03.700 or they might not even know if that they are affected. 00:40:04.233 --> 00:40:09.566 So there won't be any family history for these patients for etches 00:40:09.566 --> 00:40:13.833 or some of the NP, which is also a original recessive, disorder. 00:40:15.566 --> 00:40:18.466 Some of the screening tests which I just mentioned, 00:40:18.466 --> 00:40:25.200 like osmotic fragility, EMA and flow based test are not that useful during 00:40:25.200 --> 00:40:28.500 neonatal period because of increased hemoglobin 00:40:28.500 --> 00:40:31.500 F and hemoglobin F. 00:40:31.666 --> 00:40:33.633 It it because of the nature 00:40:33.633 --> 00:40:38.366 of the hemoglobin F D cells, the red blood cells with high level of hemoglobin 00:40:38.366 --> 00:40:42.633 F, they're not that they're not that kind of 00:40:43.066 --> 00:40:47.400 they don't get hemolysis compared to the specific red blood cells easily. 00:40:47.800 --> 00:40:52.133 So the some of these, these neonatal, some of these biochemical testing 00:40:52.133 --> 00:40:55.833 or screening tests are not that useful in the neonatal period. 00:40:57.366 --> 00:40:58.966 This I already mentioned this 00:40:58.966 --> 00:41:02.066 video sites are not really specific for etches. 00:41:02.100 --> 00:41:05.100 As such, one of the most important scenario, 00:41:05.100 --> 00:41:09.333 which we see, for the molecular testing request 00:41:09.333 --> 00:41:14.300 is some of these patients, especially with the original recessive variant, 00:41:14.633 --> 00:41:19.266 they require very frequent transfusion because of the nature of the disease. 00:41:19.266 --> 00:41:23.766 They are moderately to severely transfusion, severely hemolysis. 00:41:23.933 --> 00:41:25.833 And they require transfusion. 00:41:25.833 --> 00:41:30.200 So frequently that it becomes very difficult to get, sample 00:41:30.466 --> 00:41:32.433 in between the transfusion. 00:41:32.433 --> 00:41:35.233 And because of the transfusion, 00:41:35.233 --> 00:41:39.566 they will be getting normal red blood cells from the, donor, 00:41:40.233 --> 00:41:44.300 the test, the all these biochemical tests become really unreliable. 00:41:44.300 --> 00:41:46.766 And uninformative. 00:41:46.766 --> 00:41:51.233 So in these patients with really transfusion dependent anemia, 00:41:51.400 --> 00:41:55.466 only way to diagnose, diagnose these patient would be molecular tests. 00:41:56.400 --> 00:41:59.300 And also if you are looking into, 00:41:59.300 --> 00:42:03.466 multiple like complex interactions which is not that uncommon 00:42:03.466 --> 00:42:08.500 like d6, PD deficiency, but how to trace that or say ptosis 00:42:08.800 --> 00:42:11.800 or some of the bilirubin metabolism disorders 00:42:12.333 --> 00:42:16.500 and hemolytic anemia that would lead to really high level of bilirubin. 00:42:16.766 --> 00:42:20.300 So in these scenarios, molecular testing is the only way 00:42:20.733 --> 00:42:22.100 to diagnose these patients. 00:42:23.533 --> 00:42:27.333 So targeted next generation sequencing panels, 00:42:27.600 --> 00:42:31.300 have been around for almost like, 7 or 8 years now. 00:42:31.966 --> 00:42:36.200 And the gene panel varies anywhere from 28 to 70 genes. 00:42:36.200 --> 00:42:39.566 Now, most of the US, big laboratories offer 00:42:39.566 --> 00:42:42.900 some form of a targeted NGS panel. 00:42:43.233 --> 00:42:47.600 We also, we do offer a 28 gene NGS panel. 00:42:48.100 --> 00:42:51.100 And recently we've evaluated, 00:42:51.400 --> 00:42:55.233 like 268 patients, for last three years. 00:42:55.500 --> 00:42:58.366 Using this panel, 00:42:58.366 --> 00:43:02.233 and the age of the patients as can be anywhere from newborn 00:43:02.233 --> 00:43:04.500 to 62 years or later. 00:43:04.500 --> 00:43:08.366 So many of these patients as we can imagine, these remain undiagnosed. 00:43:08.633 --> 00:43:12.033 And when it comes to NGS panel or molecular panel, 00:43:12.233 --> 00:43:16.000 these patients are basically, the physicians are really trying 00:43:16.000 --> 00:43:22.066 hard to find a some kind of etiology or explanation to hemolytic process. 00:43:23.066 --> 00:43:23.733 These patients 00:43:23.733 --> 00:43:27.333 present with mildly uncompensated hemolytic anemia 00:43:27.633 --> 00:43:31.433 to severe hemolytic anemia with extreme hyper European Amia. 00:43:33.000 --> 00:43:36.433 And we identified pathogenic and likely pathogenic variants 00:43:36.433 --> 00:43:40.066 in about 25% of the patients that were clearly responsible 00:43:40.066 --> 00:43:43.833 for the disease phenotype from moderate to severe hemolytic anemia. 00:43:44.200 --> 00:43:48.800 And it might not seem like, okay, this is only the we can think that, okay, 00:43:48.800 --> 00:43:51.900 this is 25% is not really good enough, 00:43:52.900 --> 00:43:55.933 for analysis for molecular methods. 00:43:55.933 --> 00:44:00.233 But we need to remember that these, these patients, 00:44:00.500 --> 00:44:03.800 they have had all kind of workup done in these patients, 00:44:04.500 --> 00:44:07.500 all the biochemical testing and other testings, 00:44:07.833 --> 00:44:10.266 these patients are basically, 00:44:10.266 --> 00:44:13.933 the physicians are trying to find, some kind of, 00:44:14.133 --> 00:44:18.566 an explanation or etiology for the underlying hemolytic process. 00:44:19.333 --> 00:44:21.666 So even even if we found 20 00:44:21.666 --> 00:44:26.100 for only 25% of these patients with some, pathogenic variant, 00:44:26.333 --> 00:44:31.366 we did see a variant of unknown significance in a big chunk of patients, 00:44:32.133 --> 00:44:37.033 which is which could be causative, but we don't know exactly. 00:44:37.366 --> 00:44:40.800 Or many of these patients might have some, some, 00:44:41.300 --> 00:44:44.300 problem in genes which are not part of panel. 00:44:45.433 --> 00:44:47.566 So 25 to 30%, 00:44:47.566 --> 00:44:50.766 of these patients can have a diagnosis, 00:44:51.566 --> 00:44:54.900 based on the molecular testing and can get can get 00:44:55.266 --> 00:44:58.266 spleen splenectomy or some other, 00:44:59.400 --> 00:45:02.666 treatment based on these, molecular testing. 00:45:03.900 --> 00:45:08.100 So I'll talk I'll go over a couple of cases, 00:45:08.433 --> 00:45:12.000 to summarize some of these, findings 00:45:13.100 --> 00:45:15.466 and then, 00:45:15.466 --> 00:45:19.333 and then at the end summarize the, overall, findings. 00:45:19.500 --> 00:45:24.133 So the first patient this patient was, 3.5 years of age 00:45:24.133 --> 00:45:28.200 with mild anemia associated with spleen, omega three. This 00:45:28.200 --> 00:45:31.200 this patient was of Caucasian ethnicity, lacked 00:45:31.200 --> 00:45:34.200 regular well-child visits. 00:45:34.200 --> 00:45:36.400 The peripheral smear was really striking. 00:45:36.400 --> 00:45:38.666 That marked erythrocyte particular status. 00:45:38.666 --> 00:45:41.700 So Mark and I supported closely to assess the patient also had 00:45:41.700 --> 00:45:44.700 hyper bilirubin in the newborn period. 00:45:45.333 --> 00:45:47.500 There was no family history. 00:45:47.500 --> 00:45:50.400 And he this patient didn't have regular child visits. 00:45:50.400 --> 00:45:53.133 So we didn't really know what was going on. 00:45:54.700 --> 00:45:57.700 Till like 3.4, three years of age, 00:45:58.233 --> 00:46:01.400 initially presented with mild anemia with spleen omega. 00:46:01.700 --> 00:46:05.433 However, later on, he turned to have severe, anemia 00:46:05.433 --> 00:46:07.200 with dilated cardiomyopathy. 00:46:07.200 --> 00:46:12.533 So because of the persistent anemia in this patient, the heart was dilated. 00:46:13.400 --> 00:46:16.800 So once the cardiomyopathy or dilatation 00:46:16.800 --> 00:46:20.466 of heart presented, then the physician ordered this genetic testing. 00:46:20.766 --> 00:46:24.066 And we found three different variants in the spectrum gene, 00:46:24.066 --> 00:46:28.066 including this novel mutation, which was not described earlier. 00:46:29.200 --> 00:46:31.500 And these mutations altogether 00:46:31.500 --> 00:46:34.666 would lead to had to close eight horses. 00:46:34.833 --> 00:46:37.833 So we made a diagnosis of hatred part of particular situs is 00:46:37.833 --> 00:46:41.400 based on the peripheral smear evaluation and the genetic testing. 00:46:42.166 --> 00:46:47.133 After after this molecular testing, the patient underwent splenic 00:46:47.400 --> 00:46:52.600 removal of spleen, and now the patient's hemoglobin is improved to almost like 15. 00:46:52.600 --> 00:46:56.100 And echocardiogram like heart changes are also resolving. 00:46:56.100 --> 00:47:00.733 So this was a positive, impact of molecular testing in this patient. 00:47:01.833 --> 00:47:06.000 So the second case also was kind of similar. 00:47:06.000 --> 00:47:09.933 So this two year old female presented with congenital hemolytic anemia. 00:47:09.933 --> 00:47:13.800 So so severe hemolytic anemia since both baseline 00:47:13.800 --> 00:47:16.800 hemoglobin was six grams per deciliter 00:47:17.666 --> 00:47:19.566 red was superduper high. 00:47:19.566 --> 00:47:23.500 So indicating that there is some kind of a hemolytic process going on. 00:47:23.500 --> 00:47:27.400 The patient was transfusion dependent with significant hepatic spleen, omega 00:47:27.933 --> 00:47:31.533 and iron overload due to this hemolytic process. 00:47:33.033 --> 00:47:35.233 The physician had done 00:47:35.233 --> 00:47:38.233 pretty much every test, 00:47:38.366 --> 00:47:41.366 for hemolytic anemia, including, 00:47:42.300 --> 00:47:45.433 osmotic fragility, briefly smeared evaluation, 00:47:45.700 --> 00:47:48.700 full red blood cell enzyme level, 00:47:48.900 --> 00:47:51.033 unstable all the hemoglobin poverty. 00:47:51.033 --> 00:47:55.500 Thalassemia testing GI and pulmonary evaluation. 00:47:55.500 --> 00:47:59.733 Extensive evaluation for occult bleeding because she was so anemic 00:48:00.200 --> 00:48:03.200 and all of these testing was negative. 00:48:04.300 --> 00:48:06.166 We did the, 00:48:06.166 --> 00:48:09.733 flow test, the EMA, the entry binding test. 00:48:09.733 --> 00:48:11.533 And that test was borderline, 00:48:13.200 --> 00:48:16.133 as I mentioned earlier, all these screening tests, 00:48:16.133 --> 00:48:19.900 as we can look into this, the asthmatic fragility test was normal to, 00:48:21.566 --> 00:48:24.533 and the EMA test was kind of borderline. 00:48:24.533 --> 00:48:29.500 However, however, we need to remember that this patient is transfusion dependent. 00:48:29.500 --> 00:48:34.866 And as I mentioned earlier, transfusion will completely change the, 00:48:35.400 --> 00:48:39.300 phenotype of the testing because obviously these patients 00:48:39.300 --> 00:48:43.000 with transfusion, frequent transfusion would have red blood cells from the donor. 00:48:44.100 --> 00:48:46.733 So we were on the EMA test, the flow test. 00:48:46.733 --> 00:48:50.600 We were seeing some of the donor red blood cells, but also we were seeing 00:48:50.600 --> 00:48:55.800 some of the decrease in fluorescence from the from the patient per se. 00:48:56.100 --> 00:49:00.700 So that made us suspicious of some kind of a diagnosis. 00:49:00.700 --> 00:49:02.933 Heydrich's ptosis diagnosis. 00:49:02.933 --> 00:49:04.500 And we did the NGS panel. 00:49:04.500 --> 00:49:07.733 Next one is in sequencing panel which picked up two mutations, 00:49:08.433 --> 00:49:10.800 the pathogenic mutations in anchoring gene. 00:49:10.800 --> 00:49:13.000 One of them was variant of unknown significance 00:49:13.000 --> 00:49:15.200 and one was the pathogenic variant. 00:49:15.200 --> 00:49:18.266 And we made a diagnosis of had tree ptosis. 00:49:20.866 --> 00:49:22.533 After that molecular diagnosis 00:49:22.533 --> 00:49:25.533 she also underwent splenectomy and 00:49:26.533 --> 00:49:31.500 and then since splenectomy, her baseline level is pretty good 10 to 11. 00:49:31.500 --> 00:49:34.866 And she has been doing, pretty well for the last six months. 00:49:35.100 --> 00:49:38.100 So this was another positive outcome of this patient. 00:49:38.566 --> 00:49:43.733 So that that were the two cases illustrating the importance of, 00:49:44.033 --> 00:49:47.033 molecular testing in these disorders. 00:49:48.000 --> 00:49:51.400 So to summarize, the red blood cell membrane 00:49:51.400 --> 00:49:55.500 defects are relatively common disorder with very different etiology 00:49:56.033 --> 00:50:00.000 can cause significant morbidity and mortality if not diagnosed. 00:50:00.000 --> 00:50:03.933 We see many patients in teenage or adult 00:50:04.333 --> 00:50:07.866 or older patients with mild to moderate, 00:50:08.666 --> 00:50:11.666 unexplained hemolytic anemia, undiagnosed 00:50:12.766 --> 00:50:13.633 in units. 00:50:13.633 --> 00:50:18.033 It can cause serious problems because of the hyper bilirubin Amia 00:50:18.533 --> 00:50:21.533 or increased in bilirubin 00:50:21.733 --> 00:50:26.833 that can also go through the blood brain barrier and cause significant CNS, impact. 00:50:27.866 --> 00:50:28.733 Diagnosis 00:50:28.733 --> 00:50:32.400 requires, a correlation with family history, 00:50:32.400 --> 00:50:35.700 complete blood count and peripheral smear evaluation. 00:50:36.500 --> 00:50:39.533 If the flow test and osmotic fragility test 00:50:39.533 --> 00:50:43.100 in combination can be very helpful for the diagnosis of HS 00:50:43.500 --> 00:50:47.400 for the H and diagnosis, peripheral smear evaluation, 00:50:47.600 --> 00:50:50.533 it's very helpful. 00:50:50.533 --> 00:50:52.533 Next generation sequencing, 00:50:52.533 --> 00:50:58.833 based on targeted panel can be helpful for the diagnosis of many of the unexplained 00:50:59.100 --> 00:51:02.166 or original recessive types of, hemolytic 00:51:02.166 --> 00:51:05.166 anemia of red blood cell membrane defect. 00:51:06.000 --> 00:51:06.833 That's about it.