Erythrocyte Diagnosis & Kinetics PDF

Diagnosis of the production and destruction of erythrocytes At birth, all the bone marrow is hematopoietic, so it ensures: - erythropoiesis - granulocytopoiesis - thrombocytopoiesis Erythrokinetics is a global process that...

Diagnosis of the production and destruction of erythrocytes At birth, all the bone marrow is hematopoietic, so it ensures: - erythropoiesis - granulocytopoiesis - thrombocytopoiesis Erythrokinetics is a global process that derives from: production circulation and destruction of erythrocytes Destroyed erythrocytes, which no longer reach the peripheral blood, show the balance (or imbalance) between efficient and inefficient erythropoiesis.  Methods for evaluating global erythrokinetics Direct methods I. The number of cells in: a) peripheral blood + hemoglobin + hematocrit b) bone marrow no. of nucleated elements from the BM is achieved  no. hematocrits in the periphery allows knowing the proliferative state of the BM, but the results vary depending on: sample quality the functional region NV = 70,000 – 100,000 / l II. Morphological examination of: a) peripheral blood b) bone marrow = myelogram III. The lifespan of erythrocytes IV. Plasma Iron turnover V. UBG II. a) Normal aspects of the blood smear Size 1) anisocytosis = size difference  different ages normal to a certain degree according to the Prince – Jones curve (% distribution of red blood cells by size  peak → 7.2 m left – microcytosis right – macrocytosis 2) Microcytosis   7.2 m especially in anemias hypochromic hemolytic (congenital spherocytosis) 3) Macrocytosis  = 8 – 12 m  > 12 m → megalocytes or gigantocytes in megaloblastic anemia cirrhosis nutritional disorders intoxications with CO Tinctoriality 1. hypochromia​   hemoglobin; contains = annulocytes (hemoglobin only at the periphery) it is generally associated with microcytosis;​​  Hyperchromic real ; changes in the cell thickness – in macrocyte, spherocyte. 2. Anisochromia – Normal and hypochromic erythrocytes 3. Acromocytosis = very degenerated with a crescent appearance hemoglobin only stroma and membrane 4. Target cells (mexican hat) hemoglobin is distributed ≈ centrally and → hypochromic separation zone to the edges in thalassemia and other hemoglobinoses hypochromic anemia postsplenectomy 5. Basophilic erythrocytes  Erythroblasts that have lost their nucleus before loading with hemoglobin 6. Polychromatophilic erythrocytes big cells  erythroblasts that lost their nucleus in the young stage. white – violet; sign of cellular regeneration. NV = 0.5 – 1.5 % of erythrocytes 7. Basophilic punctations blue in panoptical coloring (MGG)  as dimension, in all cells greenish white – CH2 blue coloration have diagnostic value when no.  NV = 2-5‰ of erythrocytes 8. Jolly bodies = erythrocyte  nucleus remnants → round corpuscles with nucleus tinctoriality 9. Cabot rings = erythrocytes  a thin filament in the form of the number 8 or ≈ rings 10. Azurophilic granules (chromatin powder) = chromatin remnants red - purplish in all cells in severe anemias, megaloblastics Form​  1. Poikilocytosis  of form in anemias with serious hematopoietic disorders 2. Spherocytes ½ from the marrow ∙ Erythroblasts in the periphery → 10% ( > 10%  Biermer pernicious anemia, neoplasm)  Megaloblastic anemia  vitamin B12 deficiency Vitamin B 12 and folic acid indispensable for DNA synthesis  their deficiency determines a disorder in the maturation of rapidly renewing Vit B12 cobalamin derivative tetrapyrrolic nucleus centered on a cobalt atom, to which a group is attached: – adenosine type (and) – CN for cyano-cobalamin – OH for HO cobalamin active coenzymes​ CH3-cobalamin and 5 deoxy adenosyl cobalamin through animal (vegetable) food; absorption - through its connection to the intrinsic factor results in humans 2-5 mg at level liver m kidneys enough to cover the need for 4-5 years​​ IF glycoprotein synthesized by gastric parietal cells, under the influence - stimulation - histamine in parallel with HCl production - gastrin it is the antigenic substance  pathology​​  Antibodies fixed - a vitamin B12 molecule (a IF molecule) - in the stomach transport in the terminal ileum → fixation of the specific receptor for the vitamin B 12 – IF complex in the presence of Ca 2+ and Mg 2+ 80% is absorbed by an active mechanism, after which the protein-bound vitamin B12 transports into the plasma proteins = transcobalamins I, II, III TC II a distribution (after erythrocyte) → liver, where : - vit B12 is stored - and redistributed as needed →TC I and III, which bring it back into circulation TC I and III have common antigenic properties with FI are produced by granulocytes (TC II is produced by the hepatocyte only for the distribution of vitamin B12 to the tissues) excretion mainly through urine, faeces less - through epithelial cells that slough off losses / day = 0.05 – 0.1% of the total amount of vitamin B12 in the body BC – in humans Э 2 reactions dependent on vitamin B12 1. MET synthesis from homocysteine (by methylation) need – CH3 cobalamin = methyltransferase coenzime and CH3 tetrahydrofolate – CH3 donor 2. is a step in propionate catabolism: the conversion of methyl malonyl CoA  succinyl CoA Vitamin B12 deficiency​​  disrupt DNA and myelin synthesis DNA synthesis – 2 hypotheses the CH3 trap of folate the CH3 trap of folate DNA disruption  impairment of folate metabolism = CH3 FH4 → FH4 ↓ sequestered  deficiency of the coenzyme required for the synthesis of thymidinic acid conversion of ribonucleotides to deoxyribonucleotides the transfer of deoxyuridyl to thymidyrate – the essential step in DNA; alteration  megaloblastosis. Absence of vitamin B12  methylfolate accumulation  does not participate in the metabolic process 1. MET – needed for the production of methyl malonyl ~ CoA methylation of myelin basic protein 2. accumulation in tissues of CH3- malonyl ~ CoA  abnormal fatty acids and its precursor CH3-propionyl ~ CoA they will be incorporated into neuronal lipids  neurologic manifestation  urine excretion: CH3 malonate​ degradation of formiminoglutamic acid diagnostic test for folate deficiency​​ it accumulates in the urine role in pathology Role in DNA synthesis (especially thymidine) tetrahydrophyllates - in purine synthesis deficit  mature nuclear disorder (medullary megaloblastic)​​ maturation synchronism active erythropoiesis, ineffective cellular gigantism, especially erythrocytes Biermer's disease the most common form Characters : - megaloblastic changes in the cells of the BM and peripheral blood - and degenerative lesions at the level of the bone marrow of the digestive tract and the nervous system    absorption of B12 due to the lack of the intrinsic factor of a gastric atrophy installed probably through an autoimmune mechanism on a constitutional basis. Spreading variable; γ in Ol, T Scandinavian, England, USA γ  in E and S Europe very rare in the East and the black population Frequency​ maximum = 45 – 65 years rarely → 30 years exceptional in children sex - in some countrys; in others (USA, England) Hereditary the disease has a family incidence  genetic determinism in a generation, or "+" generation Э b 1. gene → forms FI ; dominant transmission individuals with a genetic defect have a susceptibility for the development of gastric atrophy 2. presents auto - antibodies in the majority of patients in serum – Antibodies FI – 50-55 % sick people → they are also in other autoimmune diseases (and) Ac parietal cells - 90% patients  histamine-refractory secretory insufficiency    gastric juice​​ disappear HCl peptic activity * bacterial population of the gastric cavity IF Some times : Biermer 's disease  genetic defect that determines immunological disorders =  immune tolerance for gastric epithelial cells​​ ➾ auto-antibodies Clinical - symptoms: precocious inappetence, disgust for meat gastric fullness, vomiting digestive burning on the tongue glossitis diarrhea / constipation fatigue, dizzy, palpitation pale with a yellow anemia​ edema cardiac murmurs splenomegaly grade I​​ the sensitivity of the membranes: tingling, numbness paresthesias nervous pain motor disorder​ mental disorder - rare There are also very rare touches - cranial / peripheral nerves - polyneurotic - psychopathic substrate - lesions skeletal muscles - posterior (and lateral) cord  destroy the myelin axon degeneration Blood - pancytopenia with prevalence of anemia Erythrocyte series Macrocytosis  ≥ 12  , oval (Oxyphilic macroovalocytosis) well loaded with Hb (no clear central area) Э and erythrocyte < 5  , some with irregular forms (poikilocytes) with basophilic punctations, some Cabott anisocytosis - highlight  wide Prince – Jones curve, peak → right DEM ≥ 8-9 m MCV ≥ 95-110 m3 → 140 m3 , but MCHC = N MCH = 33-38 pg → 50 pg Erythrocytes number = 1.5 mil E/mm3  Macrocytosis It is the corresponding small value for Hb and Ht ()  it is well tolerated reticulocytes < N inconstant - megaloblasts  important for diagnosis​​ megalocytosis Leukocyte series  moderate = 3000/mm3 – 3500/mm3  granulocytes Platelet series  moderate ; M (megalo) thrombocytes BM Hyperplasia (important) of erythrocyte precursors megaloblastic changes in all series with normal hypersegmentation, <  severe cases – ectopic foci of hematopoiesis - liver - spleen BC blood  bilirubin values - especially of medullary origin - with  elimination of bilirubin bodies in stool → 800 mg/day  enzymes - LDH (isoenzyme, of erythrocyte origin) - malic - 6 phosphogluconate dehydrogenase 2 hydroxybutyrate metabolic changes -​ protidic  elimination of AA  ESR  haptoglobin  hemolysis - lipidic  lipoproteins​ β  intestinal absorbtion of fatty acids disturbance of hemostasis (light)   platelets ↓ prothrombin action​ Iron metabolism​  sideremia  total transferrin binding capacity  intestinal resorption of iron  Urobilinogenuria  medullary hemosiderin Importance​ for diagnosis histamine anacidity - resistance B12 metabolism blood concentration « 100pg/ml = critical level dosage – microbiological metabolism – radioisotopes intestinal absorption - Schilling test per os < quantity of B12 radioactive (0.5μCi) every 2 hours 1000 g of vitamin intramuscularly to prevent fixation of 60 CoB12  favors its elimination N – urine/day  10% radioactivity administered Biermer anemia – urine / day < 2% radioactive vitamin - absorption deficit  lack of IF, repeat test after 2 weeks - simultaneous administration of IF  correction; error factor - Renal failure and Heart failure, infectious diseases. rare - determines IF activity in gastric juice , or anti IF antibodies in serum – methods – serological radioisotopes parietal cells determine  LDH   lysis of megaloblasts evidence of the indirect effect of B12 deficiency - determination  urinary elimination of CH 3 - N = 1-7 mg/day Biermer anemia = tens-hundreds of mg/day  Other megaloblastic anemias with IF deficiency a) juvenile pernicious anemia b) postgastrectomy  iron deficiency hypochromic anemias c) primitive gastric diseases - occur very rarely; - iron deficiency anemia Other causes of vitamin B12 deficiency intestinal diseases​ vitamin B12 absorption disorders are important < disrupts the absorption of folic acid, with which it is associated are sprue gluten enteropathy​ ileum resections N secretarion of IF; HCl hyper inflammation gastric and intestinal mucosa bacterial proliferation​ botryocephalosis · Schilling test appears false (+)  vitamin D uptake by the parasite radioactive administration Megaloblastic anemia due to folic acid deficiency The structure of folic acid = pteroylmonoglutamic ∙ pteridinic ring (with 2-NH2 and 4-HO groups) ∙ remainder of pNH2 benzoic acid ∙ peptidic group  1 – 7 (10) glutamyl radicals linked together in the ɣ position ∙ for activation (as a coenzyme ) it must be reduced of dehydrofolate reductase in tetrahydrofolic acid (FH4) passing through the semi-reduced form (FH2) Folate = derivative of folic acid ∙ in food they are found as deconjugated polyglutamates to be absorbed and use as coenzymes of specific enzymes ("conjugases") body cells ∙ coenzyme (in the form of FH4) – role: transfers some units of "active C" which it easily fixes/gives up in the reactions of the metabolism of nucleic acids / some AA through interconversion reactions, they can be transferred into each other  biochemical circuits of folate, with the possibility of directing it to the most requested reactions Methylenetetrahydrofolate - donor of structural elements : uridylate (→ RNA) → thymidylate (→ RNA) conversion Dose of folates: method - bacteriologic: measure the growth of folate-dependent microorganisms l. casei S. faecalis for differential dosages P. cerevisiae of "radioisotopic dilution" Absorbtion in the duodenum proximal jejunum mechanism active transport ​ passive diffusion – cases of > quantitative administration​ it resorbs only monoglutamates polyglutamates - first deconjugated by intestinal conjugases (whose action is influenced by activated factor physiological pathological) inhibited​ in the course absorbtion methylation of folic acid occurs passing through the liver Plasma transport ∙ through the labile binding of globulins and β lipoproteins ∙ folic acid reduced is quickly taken up in the cell (at the level of FH 2 – reductase) and the latter is transformed slowly in active forms faster in a state of deficiency ∙ N serum folate = 6 – 20 mg/ml - > 90% as CH3 – FH4 folate stores = 6 – 10 mg – in the liver - the blood cells  160 – 640 ng/ml of erythrocyte mass ∙ hematopoiesis has a preferential regime in case of folic deficiency ∙ cellular folate mostly as CH3 – FH4 in conjugated form it is passed into active forms  necessary for the respective metabolism Excreted through ∙ kidneys ∙ stool - quantities > acquired 1. Membranopathies Hereditary spherocytosis Hereditary ovalocytosis Hereditary pyropoikilocytosis Hereditary acanthocytosis Hereditary stomatocytosis 2. Enzymes - deficiency of G-6-P dehydrogenase (aerobic cycle) PK (anaerobic cycle) 3. Hemoglobinopathies cantitative  imbalances produced between  chain  and non- of globin → thalassemias quality  affecting the primary structure of hemoglobin → hemoglobinopathies sickle cell through unstable hemoglobin and other​​ 4. Paroxysmal nocturnal hemoglobinuria (PNH)  Extrinsic (extracorpuscular) defects Erythrocytes are normal, but their living environment is normal all are acquired 1. mechanical agents marching hemoglobinuria marching hemolytic anemias​​ hemolytic​ anemias microangiopathy macro angiopathy change the area of the vessel metalic valvular prostheses gigantic hemangiomas vasculitis  Macroangiopathy 2. agents physical - fibrin (DIC) chemicals - drugs, toxins 3. infectious agents - septicemia with Clostridium 4. immune mechanism allo (iso) antibodies post transfusion reaction hemolytic diseases of the newborns auto antibodies hot / cold antibodies idiopathic hemolytic syndrome secondary hemolytic anemias 5. Hyperactivity of the monocyto-macrophagic system - hypersplenism Hereditary membranopathies Hereditary spherocytosis autosomal transmission  alteration of some protein components in the structure of the cellular skeleton MbE - bilipid layer (on the outside)  2 layers of phospholipids and cholesterol - protein network = cellular skeleton - integral proteins proteins and glycoproteins that fix the 2 layers between them from the EPH point of view → band 1, 2, 3 etc ▪ band 1 and 2 (chains  and ) combine with each other  spectrin 3 is a transfixing protein (= integral) has a canal in the center transporter of ions in cellular exchanges 4 subband 1 – ensures links between chains 5 = actin – contractile protein 2.1 binder – between spectrin dimers  spectrin tetramer of this band 3 tetramer glycophorins - especially 3 - specific to the red series cell  Functional links that ensure the elasticity and contractility of the membrane are between the chains​​  and  of spectrin => spectrin dimers that are "U" head-to-head with the help of ankyrin, which later makes a " U" with band 3 affect – spectrin – spectrin bond  alter the dimer associations between them with ankyrin ankyrin structure and function  alteration of dimer associations There are also connections that ensure the association of tetramers with each other  the network (the tetramer is linear) - with the help of proteins 4.1 and 5 Alteration of the erythrocyte membrane in Hereditary Spherocytosis 1) alteration of proteins in the cellular skeleton some of the injuries are due to: - spectrin  alteration of bonds with proteins 4.1 and 5  affects the elasticity of the membrane - ankyrins (lane 2.1)  alteration of spectrin dimer associations  their catabolism  ↓ no. tetramers   mebrane surface   S / V ratio - very important ratio for: - maintaining plasticity​​ erythrocytes and - resistance to loading with H2O -  vulnerable to H2O and Na inputs (especially) 2)  permeability for Na + and H2 O - it is not known why - it's functional alteration  Overload for the enzymatic requirement that contributes to the functioning of the cation pump   ATP consumption   need for glucose   the efficiency of the pump  loading the cell with H2O  her ballooning  spherocyte also from a functional point of view, it was observed that it binds more easily with Ca  stiffening of the membrane proteins   elasticity of the membrane 3) alteration of membrane lipids Electr. microscop: buds - on RBC series  excess fat, which curls - they are stripped, especially in the spleen   S !!! Erythrocytes that leaving BM are normal (and in BM, the shape of erythrocytes is N) The erythrocytes on the smear look like spherocytes  pass through the spleen Behind  with splenic macrophages, some of them are lesions – Pinched erythrocytes (E  mushrooms) In - the sinuses are few erythrocytes  blood drainage is fast and quasi-free cords – agglomeration of erythrocytes (dilated cords) After splenectomy -  Importance of the presence of spherocytes Clinical The vast majority is asymptomatic 1) First year from birth ; is >, severe < a small percentage make very severe neonates anemias - postnatal jaundice is very important, from the first 48 hours and with  hemoglobin !!! differential diagnosis hereditary spherocytosis - neonatal jaundice due to hemolytic anemia → difficult  microspherocytes appear in both the Coombs test makes a differential diagnosis, but sometimes there are non-specific changes that make it difficult to interpret the test and is summed up with particular aspects of the newborn - the spleen is not enlarged - does not have hemoglobin F - after birth reticulocytes decrease 2) jaundice So in this limited situation​  the parents are being investigated Hereditary spherocytosis - one of the parents has it, or Anti-erythrocyte antibodies to A part of children one year after birth (slightly, serum) evolve towards different girls from boys - easy – hemoglobin stabilizes at a threshold → equivalent to hemolysis (hemoglobin = 9 g%); quasi-normal aggravation of anemia - consumption of folic acid Diagnosis - clinical examination - splenomegaly (always exists  permanent activity a​ macrophages) - exceptional blood smear: normal spleen - severity - hemolysis is important in permanent - requires repeated transfusions (transfused erythrocytes have duration = N)  risk of hemochromatosis  Desferrioxamine (Desferal) = urinary Iron chelator - administered from the beginning Laboratory diagnosis​​ peripheral blood  Reticulocytes – expression of regeneration  hemolysis​  MCHC (? why?) MCV, MCH = N 2 extremes - macrocytes - microcytes among them normocytes Coombs test  Immune hemolytic anemia is more negative  Hereditary spherocytosis for confirmation osmotic resistance + +/- (i) autohemolysis (with glucose correction) the analysis of proteins from the erythrocyte membrane – Not for current use  very laborious, takes time It's not very specific  spectrin damage also appears in ovalocytosis !!! Microspherocyte - it is NOT pathognomonic; just a signal that announce hemolysis  appears in 1) Hereditary spherocytosis (the younger the patient, any age) 2) Immune Hem An - auto antibodies - allo antibodies - posttransfusion reaction​ - mother-fetus incompatibility 3) oxidizing aggressions - drugs, chemical substances - forces a metabolic pathway of cellular detoxification 4) hereditary elliptocytosis with spherocytosis 5) snake venom 6) bacterial endotoxins (sepsis with Clostridium welchii) 7) Hypersplenism From practical point of view I 4 as Coombs test Checking erythrocyte enzymes - especially G6PD Hereditary spherocytosis Osmotic fragility VN 4.6 all 3.9 isotone (7,4)  discocyte → hypotone (2) spherocyte plasticite    permeability for H2O​​ adjusts the membrane on the content   S Sometimes the osmotic resistance curve  normal value  the conditioned osmotic resistance is carried out, after incubation at 37 C for 24h So there are 2 tests without incubation with incubation Autohemolysis Normal erythrocytes in a hypo-osmotic environment/ in their plasma spontaneously lyse  senescent ones Incubating hereditary spherocytes - autohemolysis is very high  lack of glucose - correctable with glucose Complications of Hereditary Spherocytosis I. Crises ∙ hemolytic  incurable infections idiopathic   reticulocytes jaundice accent ∙ aplastic = the appearance of an anemia that occurs rapidly in a patient with stable hemoglobin +  reticulocytes (+) f  other cytopathies likewise, it does not modify the morphology erythrocytes  parvovirus B19 (  pure Ebl penia) ∙ megaloblastic the depth of anemias (+)  reticulocytes Morphological aspects – Macrocytes (Macroovalocytes) oxyphiles - the appearance of polysegmentates differential diagnosis - Coombs test BM - MANDATORY Hyperplasia of the red series - hemolytic crisis Erythroblastopenia - aplastic crisis megaloblasts – megaloblastic crisis quasi-constant   folic acid II. Biliary lithiasis - as  age III. Conditions induced by us – hemochromatosis diseases  - stunting, gonadal retardation, etc. (by hypoxia) - bone changes - leg ulcers - gout - oral masses of hematopoiesis ectopic (erythroid tissue) - splenectomy - myeloma "+" Splenectomy – does not cure the disease, but helps compensate for hemolysis !!!​ Hereditary elliptocytosis Spectrin damage, which makes it difficult for the dimers to associate with each other Mechanism - Mendelian, autosomal dominant Clinical - medium form (common) 80% from cases is very easy; no treatment required only folic acid and biliary drainage its variants sporadic hemolysis in 20% of cases they periodically undergo intense hemolysis  splenectomy combined form with Hereditary Spherocytosis in heterozygous blacks from the USA many fragmented erythrocytes and small is the pyknotic form of the disease (poikilocytosis transiting with pyknocytosis) it appears in infancy, lasts 6 → 12 months, then returns to the common form homozygous form – requires transfusions form with stomatocytosis – in the Pacific Ovalocytes are NOT pathogenic for diagnosis; in Hereditary Elliptocytosis - > 40% in smear, normal - < 15% other anemias < 35% 1. megaloblastics 2. thalassemias 3. enzymopenias 4. sickle cell disease - rare 5. microcytic hypochromic anemias In sickle cell disease, ovalocytes rarely appear on the smear  shaking the bottle, when oxygenation occurs !!! Diagnosis​  smear on the subject parents Tratament -  splenectomy​ Pyropoikilocytosis (PPK) ∙ severe, autosomal hemolysis ∙ mechanism: spectrin damage form  Hereditary elliptocytosis  disturb the association of dimers its exaggerated proteolysis thermal fragility blood smear number of poikilocytes, budded, , broken small and spherocytes ∙ pathognomonic - T ​ fragility ∙ for diagnosis : sample heated to​ 45  C 15 min  erythrocyte fragmentation 37  C 6 h  ↑ hemolysis​ ∙ treatment – does not exist  ENZYMOPATHIES Definition = hereditary conditions that determine hemolysis ∙ mechanism - disorders in the synthesis of erythrocyte enzymes  production​  functional activity​​ very quickly catabolized and inactivated ∙ the main enzyme metabolism is the glycolytic one  glucose is the source of energy through the production of ATP, NADH, NADPH being H+ donors 2 ways I. anaerobic it is catabolized > 95 % 1 mol glucose → 1 mol pyruvic acid 2 ATP molecules are formed which remain available for energy collate cycle, in II with ATP  2.3-DPG → affinity for O2 of hemoglobin II. aerobic 5% of glucose is catabolized I. 1) Luebering–Rapoport pathway Hb ⊃ 2.3-DPG   Hb affinity Hb without 2.3-DPG   Hb affinity affinity for 2.3 DPG verry  to HbA  HbF is more avid for O2 ( hardly gives it to the tissue )  for HbF​  HbF - inappropriate for atmospheric breathing 2) ATP → energy for membrane ion pumps ensure membrane integrity de novo synthesis of GSH 3) NADH – H+ donor for metHb reduction (thus maintaining Iron in Hemoglobin) II. The aerobic pathway ∙ main role – provides H2 to maintain the cellular storage of NADPH  reduction of oxidized bodies that appear in erythrocytes ∙ oxidized bodies cause different aggressions on erythrocytes  oxidation of Iron - Fe ++ → Fe +++ a superoxide anion is released (very high oxidation capacity) Fe+++ → metHb - the O2 greedy and non-functional form so the body will reduce Fe  NADH and NADPH it will get rid of the superoxide SOD H2O2 abnormal dissociation of oxyHb  drugs, infections  H2O2 for H2O2 → H2O – enzymes glutathione peroxidase catalase   damage to the membrane   the life span of erythrocytes ‡ Hb  Heinz bodies H2O2 → H2O ∙ GSH oxidation : GSH (reduced) releases H+ → H2O2 ∙ restoring the stock of GSH: NAPDH releases an H+ restore the NAPDH generation, by accepting an H+ ion ← G-6-P DH (V) disturbances can decrease glycolysis in one of 2 ways   life span of erythrocytes G-6-P DH enzymes (antitoxic antioxidant mechanical alteration) PK enzymes (alteration of ATP formation) Deficiency of G-6-P DH ∙ it affects 1/10 of the global population​ ∙ Э "+" isoenzymatic forms (> 300)  posttranslational molecular modifications ∙ the most common – form "A+" form only in black people "M" (mediterranean) in black and white ∙ disturbances  hemoglobin alteration → formation of metHb  changes in the color of erythrocytes weakening of the heme - globin bond ( heme detaches) ‡ globins  Heinz bodies (they are attached to the membrane) - one part from erythrocytes damaged in the spleen, or eliminate Heinz bodies  fragmented erythrocytes Erythrocytes from which Heinz bodies have been extracted (Pitting phenomenon)   Membrane synthesis  spherocytes Pitting phenomenon = phagocytosis limited by the extraction of area with Heinz bodies membrane alterations they are functional  intravascular hemolysis - has the largest share  in attacks of hemolysis it is observed more frequently, sometimes with hemoglobinuria  G-6-P DH has the gene on chromosome X  the enzyme deficiency is linked to sex → expression  hemophilia enzyme deficiency​   resistance to malaria; 2 types of enzyme deficiency​ a) "A-"only blacks  with "A+", but → >> unstable Reticulocytes leaving the BM have a normal amount of enzymes, but after 1 month the enzyme action disappears b) Mediterranean a)  the hemolytic crisis after medication resolves itself, even if the antigen does not stop, once the crisis occurs Reticulocytes  reticulocytes have normal enzymatic equipment​ If 5 days after the onset, we dose the enzymes  false „-“ ← reticulocyte crisis (must wait 1 month for reticulocytes to age) b) the Mediterranean type very unstable enzymes ➾ lack of enzymes in erythrocytes of (V) age cause oxidizing agent  gross hemolysis, self-powered blacks and whites Hemolytic crisis at A"-" it is autolimited ( only blacks) the Mediterranean type, self-powered serious, rapid deglobulization shock state Renal insufficiency laboratory - in crisis only accumulation of morphological changes µspherocytes schizocytes outside of crises, normal morphology Diagnosis​ clinical the triggering of the crisis in certain conditions intermittent appearance of seizures (have been before) ? black people (help) blood smear (not always) Positive diagnosis - quantitative determination of enzyme (!!! to "A-" after Rt crisis) by method: directs (modern) – best electrophoresis of enzymes indirect effects : 1) we cause oxidative stress that consumes NADPH we observe in UV - it is fluorescence; the absence of  the absence of enzymes 2) with ascorbic acid and cyanide oxidizing agent​ catalase inhibitor  lysis of enzyme G6-PDH the result is (+) in deficiency of Piruvate K​inase Hemoglobinopathies with unstable Hb 3) test for Heinz bodies – very limited  Heinz bodies only in hemolytic crisis also appears in thalasemia unstable Hb Treatment determining the defect identification of dangerous substances prophylaxis and treatment of infections PK deficiency  Nonspherocytic chronic hemolytic anemias autosomal  chronic hemolytic anemia – intrasplenic (extravascular)  splenomegaly​​ blood – modified morphology – spherocytes → group name​ ∙ PK is involved in an etiology of ATP generation ∙ blood sample in SF  autohemolysis is very important and uncorrectable with glucose – it is a presumptive test reasons for excluding spherocytosis and other hemolytic anemias  non- correctability with glucose is presumption  look for the deficit  HEMOGLOBINOPATHIES Definition = hereditary conditions, basic disorders in Hb synthesis  genetic anomalies in Hb synthesis​​ Clinical hemolytic anemia rare cyanosis or polyglobulia Hemoglobinopathies ∙ qualitative - abnormal Hb chemical structure - sickle cell disease ∙ quantitative – thalasemias defect in the rate of synthesis of 1 / "+" polypeptide chains of globin the structure of the affected chains remains​ normal The genes are on autosomal chromosome; Hb genotype α, β, ε, δ, γ- α, ε, β, δ, γ- Throughout ontogenetic development, the genotype remains unchanged ; phenotype changes during the development of the organism: egg → adult Phenotypic variability  regulatory system with the repression of certain genes it leaves free what is necessary during the period responsible for ontogenesis Abnormal Hb – genetic lesions at the level of structural genes  modifies the genetic message  alters the structural area of the corresponding polypeptidic chains. Mechanism​ 1) replacing AA – majority 2) lack of AA – VAL from β  Hb Freiburg 3) genetic fusion  abnormal crossing over that determines the synthesis of abnormal polypeptide chains 4) addition  Hb with elongated polypeptide chain – Hb Constance Spring (⊃ 172 AA instead of 147) 5) abnormal pairing of chains with normal structure Diagnosis​ ∙ the history suggests : AHC (+), onset in childhood ∙ jaundice (sub-jaundice) ∙ splenomegaly​ ∙ or cyanosis Laboratory ∙ anemia >, < ∙ hemolysis ∙ erythrocyte morphological changes ∙ special exams MANDATORY cyclization determination of Hb F (alkaline resistance) Heinz bodies thermal stability Hb electrophoresis– basic – although abnormal Hb exists with normal electrophoresis migration!!! AA analysis – specialized laboratory MetH hemoglobinosis M – by globin mutation Replacement of any HYS (from the  or β chain) proximal / terminal; with TYR Clinical – cyanosis  other methemoglobinemias they do not disappear after intravenous injections of albumin CH2/vit C Diagnosis – Hb electrophoresis Sickle cell disease Hemoglobinosis S = sickle cell anemia ∙ the most serious qualitative hemoglobinopathy form I. = disease - homozygous – only hemoglobin S - instead of hemoglobin A substitution of glutamic acid with VAL in position 6 of the β chain II. = heterozygous -S–A - in E Э HbS and Hb A III. double heterozygous S – β thalassemia or thalassemic variants S–C S – Hb F S–D S – Lepore S – O Arab Qualitative hemoglobinopathy, underlying disorders = intra-erythrocytic presence of HbS Characteristic Chronic hemolytic anemia vascular occlusions Pathophysiological - affinity for O2 is normal - the anomaly consists in the instability of Hb that precipitates in the hypoxic environment: ∙ lungs: Hb is soluble in erythrocytes ∙ tissue ( PO2) → Hb precipitates gradually, passing through many phases: nucleation elements, where many Hb molecules join and aggregate among themselves as the nucleation elements increase, Hb goes from solution to gel  viscosity ➾ nucleating elements to form fibers (very important polymerization) these fibers → bundles ➾ crystallization the Hb molecule becomes rigid, the membrane is molded on it  The erythrocyte becomes sickle-shaped, sickle cell, cyclocyte. - this cyclosite dictates the entire pathology of the disease: ◼ on the way from the lungs (a lot of O2) → peripheral (a little O2) ◼ undergoes a change in shape, elasticity ◼ if there is short, everything is reversible long, erythrocytes stiffen in small vessels, even microcircular obstruction  ischemia​  microinfarction If such an erythrocyte passes many times, the cyclization is reversible, but over time  alteration of the membrane → stiffening → irreversible silicification Hemolysis is predominantly extravascular and in the spleen​​ but possibly also intravascular  Erythrocytes are fragile, brittle  urine, plasma - pink Clinical - major disease 1 ) sickle cell disease ∙ hemolysis  anemia occurs in young children it is chronic and important  Cardiac overload  Heart failure biliary lithiasis  biliary crises growth and development delay ∙ vascular obstruction = clinical translation of some cyclizations at the tissue level in the spleen  microinfarcts (hardness with fibrous scarring  autosplenectomy  small spleen Jolly bodies infections with encapsulated bacteria (Pneumococcus, Salmonella) general immunological deficiency:  Serum complement;  Antibodies  low opsonization CNS – cerebral infarcts Bottom of eyes – retinitis Lung – pulmonary infarcts and the existence of pulmonary scars  Circulatory insufficiency Kidney hypoxia > particular pH changes, especially in the renal medulla (tubules) medullary infarctions  renal papillary necrosis → chronic renal failure and hyposthenuria Bone aseptic necrosis  osteomyelitis hardness at the level of: shoulders, chest 2) aplastic crisis ( parvovirus infections) 3) the cyclization crisis in the spleen - > URGENT - emergency splenectomy - blood transfusion  in the spleen there is a blockage and a brutal cyclization   rapid splenomegaly  shock state   death All these elements dominate in childhood; if they pass – aplastic crisis; hemolytic crisis; infarcts then the disease is tamed, but it is fatal - cardiac; renal; pulmonary. Smear Fragmented erythrocytes  fragments in circulation all that appears are  irrelevant changes  the bottle shakes cyclocites Polychromatophilic erythrocytes Diagnosis ∙ cyclization test – blood mixture with metabisulfite (+) 1-2 hours after initiation → 40 – 50% erythrocytes have a sickle appearance ∙ Hb electrophoresis: 30 – 40% HbS 55 – 65% HbA1 Evolution​ II. benign, compatible with normal survival I. chronic, progressive, often fatal in the second decade of life  Unstable haemoglobins Abnormal hemoglobin  dissociation and intraerythrocytic precipitate  Heinz bodies Clinical chronic hemolytic anemia with periods of exacerbation - jaundice - hyperchromic urine gravity according to shape mark - splenomegaly 10% of causes - cyanosis – causes tendency to methemoglobinization Laboratory normocytic normochromic anemia  smear – changes  thalassemia – very obvious erythrocyte dimorphism, hypochromia, ovalocytosis, red blood cells in the target  reticulocytes WBC, PLT – N; in crises -   Indirect bilirubin Heinz body test (+) – very important thermal stability test - (+) and pathognomonic - ‡ from test tubes with hemolysate held for 2-3 hours at 50C  Hb instability  Electrophoresis Hb - rarely isolated abnormal fractions - – migrates  Hb A1 autohemolysis test – can be (+) methemoglobin dosage – can be  Pathophysiology ‡ erythrocyte inclusions with the appearance of Heinz bodies – binds to methemoglobin through disulfide bonds  membrane alterations erythrocyte antibodies are retained in the spleen and destroyed   life span Evolution – common,  chronic hemoglobinopathies, depending on the type of form Treatment – only symptomatic Thalassemic sdr Definition =  genetic disorders whose phenotypic expression is an imbalance between the amount of α and non-α chains elaborated at the erythroblastic level. α2 β2 = A β4 = H a2  2 = F 4 = Bart's α2 δ2 = A2 ε4 = Gower I α2 ε2 = Gower II For each molecule of Hb: Э 4 heme units Σ chains α = Σ chain non  (2  and 2 non ) Synthesis disorders heme  iron deficiency anemia hypochromic anemia balanced of globin chains  unpaired chains remain. Genotype –  and non  chains have genes on separate chromosomes α on chromosome 16 – gene  is duplicated – 1 / 2  mandatory in all Hb  any disorders appear in fetal life non α on chromosome 11 – γ, δ, β and the γ gene is duplicated (codon = GLI is replaced by codon = ALA)  γ chains with the same function; the 2 AA are not functionally different The non-α genes come into play successively. genes  → Hb F  their activity → 0 to 1 year Hb F in adults ≤ 1% Hb β gene  activity → max at 1 year in adults HbA1 (α2β2) = 97 – 98% total Hb minor δ HbA2 gene (α2 δ2) in adults = 2 → max 3% (> 3%  pathology) Disorder of non-α chains may not occur in life β thalassemia ≈ at 6 months ←↓↓ HbF and must be replaced by HbA1 α thalassemia ➾ fatal death in many cases; the disease is present at birth. causes ↓ non α chains, the body compensates by ↑ other chains, but ∑ α ≠ ∑ non α ➾ remain uncoupled α chains that precipitate the erythrocyte inclusions  α – thalassemia I. does not have α chains  Hb β 4 (Bart's) suffering occurs in the fetus  Hb behaves like methemoglobin (does not give up O2)  acute circulatory failure  Severe Heart Failure  fetal hydrops (sudden death) / die immediately after birth. II. it has α chains, but only one is functional anemia is important; Hb β4 appears and HbA1 continues to decrease unpaired β chains remain Hb H is unstable → rapidly catalyzes → aggravation of anemia Clinical : maximum severity I. → minor forms (asymptomatic carriage) the excess of polypeptide chains  ‡ their erythrocyte inclusions   life span of Erythrocytes Hyperhemolysis  β - thalassemia autosomal transmission form major β chain  Hb A2 and F compensation = Cooley anemia – is the homozygous form minor – the heterozygous form intermediate Cooley's anemia onset in I ½ year severe and progressive anemia symptoms  anemia expansion of the medullary space of the bone skeleton  bone deformation hemosiderosis  (and) repeated blood transfusions hematological anemia is constant, severe and progressive Hb  < 5 g% (fast, no changes) hypochromic microcytes  iron deficiency   Hb >  erythrocytes number (2.5 – 3 mil.) MCV    MCH    MCHC     erythrocyte osmotic resistance  low hemoglobin load blood smear: ◼ high anisocytosis (  = 3 - 12µm) ◼ constant hypochromia ◼ absurd poikilocytosis (rod, crescent) ◼ annulocytes, erythrocytes in the target ◼ Jolly bodies ◼ erythroblasts reticulocytes - moderate growth  Inefficient erythropoiesis contrasting with erythroblastic hyperplasia of BM N WBC and PLT can  in cases of hypersplenism BM hypercellular marrow; Marked erythroblastosis  responds to hyperhemolysis Inefficient erythropoiesis  the number of Erythroblasts – constant; is eliminated by the differential diagnosis with iron deficiency anemia BC – sideremia  > 150  % maybe  - case added to Iron deficiency  saturation capacity of transferrin (the body is saturated with Iron) globular resistance   Indirect bilirubin, although it can be N Electrophoresis  HbF  = 20 → 90% Hb alkali-resistant Hb test HbA2  HbA1 – Э f homozygotes Isotopes Fe59  Inefficient erythropoiesis rapid fixation of Iron in the bone marrow (BM) incorporation into erythrocytes slow and  Cr 51   life span – the most important test for the diagnosis of hemolysis