Εργαστηριακή διερεύνηση αναιμίας Αιματοκρίτης Αιμοσφαιρίνη MCV MCH (mean cellular hemoglobin) Αριθμός δικτυοερυθροκυττάρων Αμεση και έμμεση Coombs

Υπόχρωμη μικροκυτταρική Aναιμία Μακροκυτταρική Υπόχρωμη μικροκυτταρική Ορθόχρωμη Β12 φυλλικό ΔΕΚ Αλκοόλ Ηπατοπάθεια Απλαστική Χημειοθεραπεία Φερριτίνη Χαμηλή Φυσιολογική Ελατωμμένα Αυξημένα Σιδηροπενική Αιμολυτική Απλαστική Ηλεκτροφόρηση Hb Coombs Χρονίας νόσου Σιδηροβλαστική Θαλασσαιμία G6PD Αιμοσφαιρινοπάθειες Μεμβρανοπάθειες Μικροαγγειοπαθητικές + Αυτοάνοση

Περίπτωση 1 Γυναίκα 37 χρονών παρουσιάζεται στα εξωτερικά ιατρεία λόγω αναιμίας Υγιής, φυσιολογική δίαιατα ελαμβανε αντιβίωση για φαρυγγίτιδα Αδυναμία και δεκαδική πυρετική κίνηση

- Hemoglobin – 7.9 gr% - MCV – 100 - RDW – 17 - WBC – φυσιολογικά - Platelets - φυσιολογικά

ΔΕΚ Μακροκυτταρική αναιμία - Μεγαλοβλαστική -Αιμολυτική -Μυελοδυσπλαστικό/Απλαστική ΔΕΚ

- Reticulocytes – 14% - Corrected reticulocyte count: /% Retics X (measured to the expected Hb ratio)/ 14% x 7.9/15 = 7.4%

Μακροκυτταρική αναιμία - Μεγαλοβλαστική ΌΧΙ -Μυελοδυσπλαστικό/Απλαστική ΟΧΙ Αιμολυτική ΝΑΙ

Για την επιβεβαίωση χρειαζόμαστε - LDH – υψηλή - Εμμεση χολερυθρίνη υψηλή - Απτοσφαιρίνη χαμηλή Επιβεβαίωση διάγνωσης Αιμολυτική αναιμία

ΔΔ αιμολυτικής αναιμίας: - Εσωτερικό του ερυθροκυττάρου (αιμοσφαιρινοπάθεια, ενζυμοπάθεια) Μεμβράνη του ερυθροκυττάρου Εξωτερικό του ερυθροκυττάρου (υπερσπληνισμός, μηχανική αιμόλυση, άνοση αιμόλυση, τοξικοί/ φλεγμονώδεις παράγοντες)

Περιφερικό επίχρισμα - Ανισοκυττάρωση (high RDW) - Πολυχρωματοφιλία (ΔΕΚ) - Σφαιροκύτταρα? Ελλιπτοκύτταρα? Ακανθοκύτταρα? Σχιστοκύτταρα? Δρεπανοκύτταρα?

: - Αμεση (Coombs) test – IgG +3

Αυτοάνοση Αιμολυτική Αναιμία Μακροκυτταρική Αυξημένα δικτυερυθροκύτταρα Σφαιροκύτταρα Θετική Άμεση Coombs Αυτοάνοση Αιμολυτική Αναιμία

ΕΡΩΤΗΣΕΙΣ 1. MCV? 2. ΔΕΚ (λειτουργία μυελού) 3. Συμμετοχή άλλων σειρών

Περίπτωση 2 Ανδρας 63 ετώνπαρουσιάζεται στο ιατρείο με αδυναμία και αναιμία. Δεν λαμβάνει φάρμακα . Απύρετος Δεν εχει ηπατοσπληνομεγαλία ή λεμφαδενοπάθεια Πορφύρα στα κάτω άκρα

Περίπτωση 2 Laboratory evaluation - Set I: Ht 25 Hb 7.8 MCV 102 Περίπτωση 2 Laboratory evaluation - Set I: Ht 25 Hb 7.8 MCV 102 PLTs 62.000 ΔΕΚ1.2%

Αναιμία Μακροκυττάρωση Χαμηλά ΔΕΚ Θρομβοπενία

Υψηλό MCV, Χαμηλά ΔΕΚ Μεγαλοβλαστική Μυελοδυσπλαστικά Απλαστική Φάρμακα Αλκοολισμός

- B12 και φυλλικό φυσιολογικά Από το ιστορικό αλκοόλ, φάρμακα ΌΧΙ

Ringed Sideroblast Iron

- Μακροκυτταρική Θρομβοπενία Χαμηλά ΔΕΚ Δυσπλασία Φυσιολογικό Β12, φυλλικό Μυελοδυσπλαστικό

Περίπτωση 3 Ένας 66 χρονος άστεγος διακομίζεται λόγω γενικευμένης σύγχυσης , αναφέρεται πτώση από σκάλες Στη φυσική εξέταση υπάρχει ουλή λαπαροτομής στην κοιλιά . Ht 24 Hb 7.8 MCV 108 WBC 2.000 PLTs κ.φ

B12 and folate B12 is a known cofactor in 2 enzymatic reactions (1) B12 is a cofactor of methionine synthase which is required to regenerate folate within the cell

?? B12 63 pg/mL (211 – 911) Folate 13.2 ng/mL (>5.4) RBC Folate 805 ng/mL (293 – 809) ?? Μεβαλοβλαστική Ελλειψη Β12

B12 deficiency Decreased intake Impaired absorption Veganism Gastric Poor stomach acidity Gastrectomy Pernicious anemia Decreased secretion of intrinsic factor due to gastric atrophy Chronic pancreatitis Decreased digestive enzyme secretion Intestinal disease Ileal resection Ileal disease Chron’s disease Celiac sprue Fish tapeworm Diphyllobothrium latum Blind loop Intestinal bacterial overgrowth

Τι εγχείρηση ήταν η ουλή ? Μερική εντερεκτομή , με μέρος του ειλεού

Περίπτωση 4 Αγόρι 12 ετών εμφανίζεται στα Ε.Ι με ωχρότητα και αναιμία ενώ αναφέρει και κόκκινα ούρα Ηt 22 Hb 7.1 MCV 100.2 WBC ΔΕΚ 10.2

ΕλλειψηG-6PD

Θα μετρήσουμε το ένζυμο? ΌΧΙ δεν αξιολογείται στη φάση της αιμόλυσης γιατί παράγονται νέα ερυθρά ΔΕΚ που μπορεί να έχουν επαρκή ποσότητα

Περίπτωση 5 Γυναίκα την 7η εβδομάδα της κύησης hgb 6 , hct 20 , MCV 62, RDW 20, platelets 500K

Σίδηρος 23 Φερριτίνη 7

.

ΔΔ ετερόζυγη β-ΜΑ Η ηλεκτροφόρηση αιμοσφαιρίνης δεν αξιολογείται επί σιδηροπενίας

ΜΕΤΑΓΓΙΣΕΙΣ

Από τι πεθαίνουν οι ασθενείς με β-ΜΑ?

Tissue Iron Concentrations in Transfusion-dependent Thalassemia Patients Thyroid Fe = 1.6 – 6.8% d wt Heart Fe = 0.6 –1.3% d wt Liver Fe = 3.7-6.8% d wt Pancreas Fe = 1.4-3.9% d wt Adapted from Modell & Berdoukas, 1984

Τι έχει συμβεί στην ασθενή ? Σπληνεκτομή – Howell Jolie

Περίπτωση 7 άνδρας 28 ετών με HIVδιακομίζεται στα Ε.Ι λόγω αλλαγής του επιπέδου συνείδησηςΘερμοκρασία 39.2 wbc 4.2, ANC 2200 , hgb 7.2, mcv 80, rdw 18, platelets 52K, creatinine 3.2, LDH 4200, bili 2.2

ΤΤΡ Θροβοπενία Μ.Α.Α Νευρολογικά συμπτώματα (κεφαλαλγία,αδυναμία, παραισθήσεις, κώμα) Πυρετό Ευρήματα από τους νεφρούς.

Συσσώρευση ΑΜΠ Μικροθρόμβοι (Triggering event?) Μικροθρόμβοι Increased shear stress Unfolding of UL-VWF Καταστροφή ερυθρών Δυσλειτουργία οργάνων Αυτοαντίσωμα έναντι ADAMTS-13 Παραμονή UL-VWF στο αίμα Πλασμαφαίρεση απομακρύνει το αντίσωμα και τα UL-VWF αποκαθιστά τη ADAMTS-13 Το ενδοθήλιο εκκρίνει UL-VWF

Θρομβωτική θρομβοπενική πορφύρα

Copyright © 2005 Elsevier Inc. (USA) All rights reserved. Table 29-05 Copyright © 2005 Elsevier Inc. (USA) All rights reserved.

Case 1 27 yo female with a history of lupus presents with a one week history of worsening dyspnea on exertion Reports dark urine Labs: hgb 8, mcv 101, rdw 19, total bili 1.9

Case 3 84 yo female living in an assisted living facility admitted for “failure to thrive” Admission labs: WBC 3.2 with normal diff, hgb 8.7, mcv 112, platelets 92K, LDH 1800

Case 4 68 yo white female with a 10 year history of poorly controlled type II DM, HTN, CAD presents for routine follow-up. CBC: wbc 7.2 with normal diff, hgb 9.8, hct 28, mcv 82, rdw 11, platelets 252K BMP: creatinine 3.4

Case 6 55 yo female with metastatic breast cancer reports worsening fatigue for a few weeks CBC: wbc 6.8 with 1% blasts, hgb 9.1,mcv 102, platelets 86K

Case 7 72 yo female with no significant PMH is brought for a routine physical by her daughter She has not seen a doctor in 20 years Daughter notes that mom is increasingly tired and has abdominal distension On exam, patient is pale and has marked splenomegaly CBC: wbc 4.2, hgb 7, hct 20, mcv 94, platelets 72K

(Reduced form) (Oxidised form)

Megaloblastic anemia: Morphology--Blood Megaloblastoid Normal Peripheral blood Macro-ovalocytes Neutrophil nuclear hypersegmentation Very sensitive and specific

Howell Jolly body. - Round, dark, refractile RBC inclusion Howell Jolly body - Round, dark, refractile RBC inclusion - Nuclear remnant - Most commonly seen in hyposplenism Basophilic stippling - Multiple small blue dots dispersed regularly throughout the RBC - RNA complexes Cabot ring - Ring shaped, thin - May be figure of 8 shaped - Microtubule, remnants of of the mitotic spindle - Rarely seen

Clinical Features: Acute drug induced hemolysis: Aspirin, primaquine, quinine, chloroquine, dapsone…. Chronic compensated hemolysis Infection/acute illness Neonatal jaundice Favism

Inv: e/o non-spherocytic intravascular hemolyis P. Smear: Bite cells, blister cells, irregular small cells, Heinz bodies, polychromasia G-6-PD level Treatment: Stop the precipitating drug or treat the infection Acute transfusions if required

2. Pyruvate Kinase Deficiency AR Deficient ATP production, Chronic hemolytic anemia Inv; P. Smear: Prickle cells Decreased enzyme activity Treatment: Transfusion may be required

Hemolobinopathies…

Autoimmune Hemolytic Anemia Result from RBC destruction due to RBC autoantibodies: Ig G, M, E, A Most commonly-idiopathic Classification Warm AI hemolysis:Ab binds at 37degree Celsius Cold AI Hemolysis: Ab binds at 4 degree Celsius

1.Warm AI Hemolysis: Can occurs at all age groups F > M Causes: 50% Idiopathic Rest - secondary causes: 1.Lymphoid neoplasm: CLL, Lymphoma, Myeloma 2.Solid Tumors: Lung, Colon, Kidney, Ovary, Thymoma 3.CTD: SLE,RA 4.Drugs: Alpha methyl DOPA, Penicillin , Quinine, Chloroquine 5.Misc: UC, HIV

IMMUNOHEMOLYTIC ANEMIA MACROCYTE SPHEROCYTE IMMUNOHEMOLYTIC ANEMIA

complement Direct antiglobulin test demonstrating the presence of autoantibodies (shown here) or complement on the surface of the red blood cell.

Inv: Treatment e/o hemolysis, MCV P Smear: Microspherocytosis, n-RBC Confirmation: Coomb’s Test / Antiglobulin test Treatment Correct the underlying cause Prednisolone 1mg/kg po until Hb reaches 10mg/dl then taper slowly and stop Transfusion: for life threatening problems If no response to steroids  Spleenectomy or, Immunosuppressive: Azathioprine, Cyclophosphamide

2. Cold AI Hemolysis Usually Ig M Acute or Chronic form Chronic: Inv: Elderly patients Cold , painful & often blue fingers, toes, ears, or nose ( Acrocyanosis) Inv: e/o hemolysis P Smear: Microspherocytosis Ig M with specificity to I or I Ag

Other causes of Cold Agglutination: Infection: Mycoplasma pneumonia, Infec Mononucleosis PCH : Rare cause seen in children in association with cong syphilis

Treatment: Treatment of the underlying cause Keep extremities warm Steroids treatment Blood transfusion

Non-Immune Acquired Hemolytic Anemia 1. Mechanical Trauma A). Mechanical heart valves, Arterial grafts: cause shear stress damage B).March hemoglobinuria: Red cell damage in capillaries of feet C). Thermal injury: burns D). Microangiopathic hemolytic anemia (MAHA): by passage of RBC through fibrin strands deposited in small vessels  disruption of RBC eg: DIC,PIH, Malignant HTN,TTP,HUS

TRAUMATIC HEMOLYSIS

Acquired hemolysis 2.Infection F. malaria: intravascular hemolysis: severe called ‘Blackwater fever’ Cl. perfringens septicemia 3.Chemical/Drugs: oxidant denaturation of hemoglobin Eg: Dapsone, sulphasalazine, Arsenic gas, Cu, Nitrates & Nitrobenzene

The direct antiglobulin test was positive for complement (C3d) (++), and IgG (++-). Also was positive for agglutinins of IgM type and had a titer of 1:1024. Case History 103

Serologies for human immunodeficiency virus, hepatitis B and C viruses, and Mycoplasma pneumoniae were negative. Rheumatoid factor and antinuclear antibodies were undetectable. Case History 104

Prednisone therapy was started at a dose of 1 mg/kg intravenously, daily. Hemoglobin level rose to 11 g/dL, concomitantly with the improvement of hemolytic signs. Case History 105

A reduction of positivity of both direct and indirect antiglobulin tests (polyvalent serum + ; C3d + ; IgG+ ), as well as a reduction of cold agglutinin titers (1:128), was observed 8 weeks after corticosteroid therapy. Case History 106

Three months later, corticosteroids were tapered to a maintenance dose of 25 mg daily. Hemolysis recurred again with the fall of hemoglobin to 7 g/dL. Case History 107

The direct antiglobulin test recurred positive for polyvalent serum (+++), complement (+++), and IgG (+++), while cold agglutinin titers again became strongly positive (1:256). Case History 108

Immunophenotyping of bone marrow cells showed that 10% of all the cells were CD20 and CD19 positive. Case History 109

CD20 is widely expressed on B-cells CD20 is widely expressed on B-cells. CD20 could play a role in Ca2+ influx across plasma membranes, maintaining intracellular Ca2+ concentration and allowing activation of B cells. Case History 110

Rituximab is a monoclonal antibody that binds to CD 20 Rituximab was started at the dose of 375 mg/mq once weekly, for a total of 4 doses Case History 111

Hemoglobin value reached 13 Hemoglobin value reached 13.5 g/dL just before the third dose, although biochemical signs of hemolysis remained substantially unaltered. Case History 112

At the end of therapy, the hemolytic signs disappeared, the direct and indirect antiglobulin tests became negative, and cold agglutinin titers fell to 1:32 Immunophenotyping of bone marrow cells showed the absence of CD20 and CD19 B cells. Case History 113

Summary of lecture Learning points

Approach to Anemia MCV: - Low: Iron deficiency Thalassemia trait Chronic disease Sideroblastic anemia - Normal: Iron deficiency (early) Renal failure

Approach to Anemia - High: Megaloblastic anemia Hemolytic anemia MDS / Aplastic anemia Drug-related Hypothyroidism Alcoholism Liver disease

Approach to Anemia Reticulocyte count: - Adequte (RPI>2): Hemolytic anemias (most) Acute blood loss Liver disease - Inadequate (RPI<2): Iron deficiency Megaloblastic anemia Anemia of chronic disease Bone marrow failure (malignancy, chemotherapy, MDS/Aplastic anemia)

Approach to Anemia Additional lineage(s) involvement: - Leukopenia: Malignancy Chemotherapy Hypersplenism Drug-related Megaloblastic anemia Aplastic anemia

Approach to Anemia - Thrombocytopenia: Malignancy Chemotherapy Hypersplenism Drug-related Megaloblastic anemia Aplastic anemia TTP DIC

Approach to Anemia In addition, blood smear always needs to be carefully assessed: Schistocytes? Spherocytes? Target cells? Normoblasts? Spur cells (acanthocytes)? Burr cells? Malignant cells (leukemia, lymphoma)? Malaria parasites? Sickle forms?

Anemia With Low MCV and Low Reticulocytes Iron deficiency Hereditary defects in hemoglobin synthesis (thalassemia trait) Defects in heme synthsis (sideroblastic anemia) Anemia of chronic disease

Anemia With Normal MCV and Low Reticulocytes Early or mild iron deficiency anemia Chemotherapy Anemia of chronic disease Anemia of renal failure

Anemia with High MCV and Low Reticulocytes Megaloblastic anemia Myelodysplastic syndrome / Aplastic anemia Drug-related anemia Hypothyroidism Alcoholism

Anemia with High Reticulocytes Low MCV: - Thalassemia Major (some cases) - Spherocytosis High MCV: - Sickle cell anemia - G-6PD deficiency - Immune hemolytic anemia - Malaria infection

Anemia with Leukopenia and/or Thrombocytopenia Low reticulocytes: - Aplastic anemia/Myelodysplastic syndrome - Chemotherapy - Alcoholism - Megaloblastic anemia High reticulocytes: - TTP / DIC - Hypersplenism

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