MTLE - HEMA 1.pdf

MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW – CLINICAL HEMATOLOGY HEMATOLOGY 1 – RBCs & WBCs Lecturer: Rene Jesus Alfredo Dinglasan Notes by: Xiao - The Conqueror of Demons, The Vigilant Yaksha, & Alatu...

MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW – CLINICAL HEMATOLOGY HEMATOLOGY 1 – RBCs & WBCs Lecturer: Rene Jesus Alfredo Dinglasan Notes by: Xiao - The Conqueror of Demons, The Vigilant Yaksha, & Alatus, the Golden-Winged King INTRODUCTION TO HEMATOLOGY COMMON SUFFIXES FROM GREEK AND LATIN USED IN THE - Hematology: from the Greek words haima (blood) and logos VOCABULARY OF HEMATOLOGY (study/science) SUFFIX MEANING - Blood: red liquid circulating in the heart, veins, arteries, and capillaries -algia pain along a nerve -ase an enzyme TYPES OF BLOOD VESSELS -cide the killer of 1. ARTERIES - distributing blood vessels that leave the heart -crit to separate (4 mm) - have the thickest walls of the vascular system -cyte cell 2. VEINS - collecting blood vessels that return to the heart -ectomy incision and removal (5 mm) - largest; have a more irregular lumen than arteries -emia blood 3. CAPILLARIES - blood passes from the arterial to the venous system -itis inflammation (8 um) via the capillaries -lysis destruction or dissolving - capillaries are the smallest, thinnest walled, and -oma swelling or tumor most numerous of the blood vessels. -opathy disease - Functions of the blood: -osis (1) abnormal increase; (2) disease o Respiratory -penia deficiency, decreased o Nutritional -phil(ic) attracted to, affinity for o Excretory -plasia(-plastic) cell production or repair o Buffering action -poiesis cell production, formation, and development o Maintenance of body temperature -poietin stimulates production o Transport of hormones o Defense mechanism BLOOD COLLECTION BLOOD COMPOSITION Liquid portion - Plasma: liquid portion of unclotted blood specimen I. PATENT IDENTIFICATION - Serum: liquid portion of clotted blood specimen - Identify patient by asking him to state and spell his full name, age, gender Solid portion - Red blood cells: a.k.a. erythrocytes, erythroplastids, and birthdate (cellular elements or akaryocyte (cell without nucleus) o The patient must be the one to identify himself hemocytes) - White blood cells: a.k.a. leukocytes, leukoplastids o If the patient cannot identify himself, the physician or nurse may identify o Granular WBC: Basophils, Eosinophils, him/her Neutrophils - Most critical step in blood collection o Agranular WBC: Lymphocytes, Monocytes - “mortal sin” of a phlebotomist – mislabeling - Platelets: a.k.a. thrombocytes, thromboplastids Gaseous portion - Oxygen, carbon dioxide, etc. Note: A phlebotomist should maintain good health and hygiene, always have clean clothes, clean hair, and clean, short fingernails. Standard precautions CHARACTERISTICS OF THE BLOOD: should always be followed, with special consideration to the proper use of gloves 1. Fluid (in vivo) and hand washing. 2. Red 3. Slightly alkaline 4. Average specific gravity of 1.055 II. SOME PHYSIOLOGIC FACTORS AFFECTING TEST RESULT 5. Thick and viscous (3.5 to 4.5 times thicker than water) 6. Makes up 75 to 85 mL blood per kilogram body weight FACTORS EFFECTS 7. There are about 20 grams of solids per 100 mL of blood Posture - Shift in posture from supine (lying) to a sitting or standing position may increase the levels of: COMMON PREFIXES FROM GREEK AND LATIN USED IN THE o Protein VOCABULARY OF HEMATOLOGY o Cholesterol PREFIX MEANING o Iron a-/an- lack, without absent, decreased - Note: Their concentration increases because these large aniso- unequal, dissimilar molecules cannot filter into the tissues at a time when the ante- before body water transfers from the inside of the blood vessels to crena- wrinkled the interstitial spaces. cyt- cell Diurnal dys- abnormal, difficult bad In the morning In the afternoon erythro- red rhythm ↑ Cortisol ↓ Cortisol ferr- iron ↑ TSH ↓ TSH hemo-(hemato-) pertaining to blood ↑ Iron ↓ Iron hypo- beneath, under, deficient, decreased ↓ Eosinophil count ↑ Eosinophil count hyper- above, beyond, extreme iso- equal, alike, same Stress - ↑ WBCs, ↑ Fibrinogen group (a.k.a. Thrombin-sensitive leuk(o)- white group: Factors I, V, VIII, XIII) macro- large, long Exercise - Exercise can increase different blood constituents. mega- large, giant Examples are: meta- (1) after, next (2) change o Creatinine micro- small o Total protein myel(o)- (1) from bone marrow (2) spinal cord o Creatine kinase (CK) pan- all, overall, all-inclusive o Myoglobin phleb- vein phago- ingest o Aspartate aminotransferase (AST) poikilo- varied, irregular o HDL – cholesterol poly- many o Lactate dehydrogenase pre- or pro- before o WBC count pykno- dense o Platelet count reticulo- netlike Diet - After a fatty meal: schis- split o Falsely ↑ hemoglobin level scler- hard o ↑ Alkaline phosphatase (intestinal isoenzyme) sidero- iron Smoking - ↑ WBCs and Cortisol splen- spleen thromb(o)- clot, thrombus xanth- yellow  III. SKIN PUNCTURE IV. VENIPUNCTURE - Technique of choice to obtain blood specimen from the following: - Most common sites for venipuncture: o Newborns and pediatric patients o Superficial veins of the antecubital fossa (area in front of the elbow) o Severely burned patients - Most common skin antiseptic: 70% isopropyl alcohol o Patents whose veins are reserved for therapeutic purposes o NOTE: Clean the site using a circular motion, starting in the center and o Extremely obese patients working outward o Elderly patients with fragile veins - TWO anatomical patterns: o A: “H” pattern of veins PUNCTURE SITES: o B: “M” pattern of veins For infants (1 y.o.) and adults: Plantar surface of the Palmar surface of the non-dominant medial or lateral side of the heel hand (3rd or 4th finger, distal portion) Unshaded parts/areas are Puncture should be done across the prohibited sites fingerprints (perpendicular to the grooves in the whorls of the “H” pattern “M” pattern fingerprint) Veins to be used Veins to be used RECOMMENDED DEPTH OF SKIN PUNCTURE: (in order of preference): (in order of preference): For infants and small children For adults - Median cubital vein - Median vein 28 mm/hr has poorer prognosis  Sickle cells o In prostate cancer: ESR > 37 mm/hr has higher incidence of disease  Spherocytosis progression and death  Thalassemia o In Coronary artery disease: ESR >22 mm/hr in white men had high White blood  Leukemia  Leukocytosis (marked) risk of CAD cells o In patients with known cancer: when value exceeds 100 mm/h, Drugs  Dextran  Adrenocorticotropic  Heparin hormone (Corticotropin) metastases are usually present  Penicillamine  Cortisone o ESR - indicated in establishing the diagnosis and in monitoring  Procainamide  Ethambutol polymyalgia rheumatica and temporal arteritis (rate typically exceeds  Theophylline  Quinine 90 mm/hr)  Vitamin A  Salicylates o Moderate elevations - common in active inflammatory disease like Clinical  Inflammatory conditions  Cachexia rheumatoid arthritis, chronic infections, collagen disease, and conditions (associated with increased  Congestive heart failure neoplastic disease (NOTE: ESR has little diagnostic value in these plasma proteins, particularly,  Newborn status fibrinogen, alpha globulins, disorders, but can be useful in monitoring disease activity) and beta globulins) - Reminders:  Infections (acute and o ESR is prone to technical errors. chronic) o ESR has low specificity and sensitivity. Therefore, it is NOT  Subacute bacterial recommended as a screening test to detect inflammatory conditions endocarditis in asymptomatic individuals.  Acute heavy metal - Methods of ESR Determination: poisoning  Collagen vascular o Westergren diseases  Results are determined after 1 hr. & after 2 hrs.  Diabetes mellitus  Anticoagulant used: 3.8% sodium citrate (black top tube)  End-stage renal failure  Hepatitis o Modified Westergren  Gout  Results are determined after 1 hr.  Malignancy  Most commonly used method  Menstruation  Recommended by the ICSH and CLSI  Multiple Myeloma  Waldenstrom's  ICSH - International Council for Standardization in macroglobulinemia Hematology  Myocardial infarction  CLSI - Clinical and Laboratory Standards Institute  Rheumatic fever  Anticoagulant used: Ethylenediaminetetraacetic acid (EDTA)  Rheumatoid arthritis  Diluents: 0.85% Sodium chloride or 3.8% sodium citrate  Syphilis  Diluted sample is placed in a 200-mm column with an internal  Temporal arteritis diameter of 2.55 mm or more.  Nephrosis  Tuberculosis  Adults over 60 years of age o Wintrobe (frequently have a slightly  results are determined after 1 hr. higher ESR value due  Anticoagulant used: double oxalate or EDTA primarily to decreased  Wintrobe tube concentrations of plasma  Left side (red) → 0 (top) and 10 cm (bottom) (for ESR) albumin)  Right side (white) → 10 cm (top) and 0 (bottom) (for Macro-  Pregnancy hematocrit). Technique  Refrigerated sample NOT  Clotted blood sample o Macrohematocrit determination is an obsolete manual returned to room  Delay in testing procedure temperature  Bubbles in ESR column  High room temperature  Low room temperature  Vibration  Narrow ESR column  Tilted ESR tube diameter o "A tilt of as little as 3o from the vertical can cause error of up to 30%." CASE STUDY GENERAL OVERVIEW OF ANEMIA - Because of lack of space in the laboratory: the rack of sedimentation rate - ANEMIA – defined as the decrease below normal of one or more of the tubes was placed directly on top of a small refrigerator. What are the three following: possible effects on the ESR results? o Number of red blood cells 1. A falsely decreased (↓) ESR - because of lower temperatures from o Hemoglobin air rushing out on opening the refrigerator or freezer o Volume of packed red cells (hematocrit) 2. A falsely increased (↑) ESR - because of vibrations from opening and - Anemia is not a disease but the consequence of having one or a closing the refrigerator and freezer doors manifestation of disease 3. A falsely increased (↑) ESR - because of heat released from the refrigerator motor MECHANISMS OF ANEMIA OSMOTIC FRAGILITY TEST (OFT) - HEMORRHAGE o Loss of erythrocytes through bleeding must always be the - Principle: Red Blood cells are diluted in 0 to 0.85% saline solutions and the FIRST/INITIAL FOCUS in any patient with anemia and must prompt amount of hemolysis at each concentration is determined by measuring the an evaluation of the hemostatic system. absorbance of the supernatant at 540nm. o Bleeding - may be secondary to trauma, surgery, or a disease - Anticoagulant to be used: HEPARIN (avoids adding more salts to the o Gastrointestinal tract - a common site for clinically significant bleeding blood) o Menstruation – a significant source of blood loss in women - Normal values: Hemolysis begins (initial hemolysis) at 0.45% and ends (complete hemolysis) at 0.30% - HEMOLYSIS o Destruction of erythrocytes prior to the end of their normal lifespan - Examples of Initial and complete hemolysis in different conditions: (abnormal hemolysis) CONDITIONS INITIAL HEMOLYSIS COMPLETE HEMOLYSIS o Shortened erythrocyte survival time NOT explained by bleeding (% NaCl) (% NaCl) o Average survival time of RBCs after leaving bone marrow: 120 days Normal 0.45 0.30 o May occur inside the blood vessel or inside the spleen o Normal hemolysis: destruction of erythrocytes (in the spleen) who has Hereditary 0.65 0.45 already consumed their lifespan spherocytosis Thalassemia 0.35 0.20 (characterized by - DECREASED PRODUCTION OF ERYTHROCYTES numerous target o Nutritional deficiencies (iron, vitamin B12, or folic acid) are common cells) and readily correctible causes of hypoproliferative anemia. Sickle cell anemia 0.35 0.20 o Kidney disease may be associated with decreased production of EPO - Increased (↑) OF seen in: Hereditary Spherocytosis and acquired hemolytic anemias with spherocytes CLASSIFICATION OF ANEMIAS - Decreased (↓) OF seen in: anemias where there are many target cells - Morphologic classification (basis: MCV and MCHC) (these cells are thin and are able to expand when water enters) - Physiologic classification (less commonly used; basis: mechanism of anemia) INTRAVASCULAR & EXTRAVASCULAR HEMOLYSIS MORPHOLOGIC CLASSIFICATION NORMAL HEMOLYSIS - Normocytic, normochromic - RBCs live approximately 120 days. - Microcytic, hypochromic - Normal catabolism of aged (senescent) RBCs is MOSTLY Extravascular. - Macrocytic, normochromic - Intravascular hemolysis accounts for a minor component of normal RBC destruction. NORMOCYTIC, NORMOCHROMIC ANEMIAS EXTRAVASCULAR HEMOLYSIS - Aplastic anemia - A.k.a. "Macrophage-mediated Hemolysis" - Renal disease - Macrophages involved: - Paroxysmal nocturnal hemoglobinuria - Paroxysmal cold hemoglobinuria Kupffer cells Splenic macrophages/ *MCV and MCHC are both normal (liver) Littoral cells (spleen) - Spleen: "graveyard" of RBCS Normal (N) or decreased (↓) Increased (↑) reticulocyte count reticulocyte count Process: Process: - Aplastic anemia - Paroxysmal nocturnal hemoglobinuria - RBCs with: C3b on surface - RBCs with: inclusion bodies - Anemia of renal disease - Paroxysmal cold hemoglobinuria - RBCs which are: severely damaged - RBCs which are: senescent - Sickle cell disease - Hemolytic anemia - Lab findings in excessive extravascular hemolysis: o Intrinsic Urine Serum Peripheral Blood Smear  Membrane defects  ↑ Urobilinogen  ↑ Total Bilirubin  Spherocytes  Hemoglobinopathies  ↑ B1  Enzyme deficiencies (ex.  Normal B2 G6PD, PK def.) o Extrinsic INTRAVASCULAR HEMOLYSIS  Immune causes - A.k.a. "Fragmentation Hemolysis”  Non-immune RBC injury  Microangiopathic - Lab findings:  Macroangiopathic Urine Serum Peripheral Blood Smear  Infectious agents  ↑ Urobilinogen  ↑ Total bilirubin  Schistocytes  Other injury: drugs,  Hemoglobinuria  ↑ B1  Chemicals, venoms,  (+) Prussian blue  Normal B2  Extensive burns reaction (urine  ↓ Haptoglobin sediments)  ↓ Hemopexin APLASTIC ANEMIA - Associated with chronic infections such as tuberculosis, chronic - Rare but potentially deadly bone marrow failure syndrome inflammatory conditions such as rheumatoid arthritis, and tumors - Characteristic features: - Central feature of ACI: o pancytopenia (marked ↓ in the number of RBCs, WBCs, and platelets o sideropenia (↓ serum iron) despite abundant iron stores in the blood) - Usually PBS shows normocytic, normochromic o reticulocytopenia o advanced states: microcytic, hypochromic o bone marrow hypocellularity - Acute phase reactants (APRs – increase during inflammation) that o depletion of hematopoietic stem cells contribute to ACI: o Ferritin May be classified as: o Lactoferrin o Hepcidin - Acquired Aplastic Anemia  Master regulatory hormone for systemic iron metabolism o approximately 80 to 85% of aplastic anemia cases  Inactivates ferroportin o 2 categories:  Ferroportin: protein in tissues which transports iron from  Idiopathic acquired aplastic anemia – no known cause tissues towards the blood  Secondary acquired aplastic anemia – associated with an  The reason why tissues are able to release iron into the blood identified cause  Prevents the release of iron into the blood  some of the causes include: o chemicals (ex.: insecticides, benzene), viruses (ex.: Epstein-Barr virus), drugs (ex.: chloramphenicol) o Chloramphenicol = drug most frequently implicated in acquired aplastic anemia - Inherited Aplastic Anemia o approximately 15% to 20% of aplastic anemia cases o associated diseases:  Dyskeratosis congenital  Shwachman-Bodian-Diamond syndrome  Fanconi Anemia (FA) IRON DEFICIENCY ANEMIA (IDA)  It is characterized by macrocytosis and reticulocytopenia - Iron deficiency – the most common anemia  most common of the inherited aplastic anemias  a chromosome instability disorder - Possible causes:  characterized by: aplastic anemia, cancer susceptibility, and o Blood loss (especially menstruating women) physical abnormalities  Menstruating women should compensate iron loss by intake of iron supplements or iron rich foods such as legumes, beans,  physical abnormalities may include: dark green leafy vegetables, red meat o skeletal abnormalities (ex.: thumb malformations, o Nutritional deficiency (infants) microcephaly, scoliosis) o ↑iron demand (pregnancy, lactation, adolescence) o skin pigmentation (ex.: hyperpigmentation, o Malignancies of gastrointestinal tract hypopigmentation, café-au-lait lesions) o Hookworm infections o short stature o abnormalities of the eyes, kidneys, and genitals - Clinical features: o Fatigue, weakness, irritability, headache, palpitations, loss of concentration - Fanconi anemia is NOT the same as Fanconi Syndrome. o Shortness of breath o Fanconi syndrome: defect involving the proximal convoluted tubule of o Brittle hair (craving for ice is called pagophagia) kidneys o Pallor PAROXYSMAL NOCTURNAL HEMOGLOBINURIA o Koilonychia (spooning of the fingernails) o Angular cheilosis/chelitis (inflammation of the sides of the mouth) - A.k.a. Marchiafava-Micheli Syndrome o Pica – unusual craving for non-food items such as dirt, clay, laundry - caused by deficiency of complement regulatory proteins, namely: starch, or ice (most common) o DAF (decay-accelerating factor, or CD55) o Glossitis (inflammation of the tongue) o MIRL (membrane inhibitor of reactive lysis, or CD59) - Tests for PNH: STAGES OF IRON DEFICIENCY o Ham’s Acidified Serum Test o Sugar Water Test Hemoglobin Serum TIBC Ferritin  A.k.a. Sucrose hemolysis test; sugar used: sucrose Iron o Flow Cytometry (confirmatory test) Normal Iron Status N N N N Stage 1 N N N ↓ PAROXYSMAL COLD HEMOGLOBINURIA (Storage Iron Depletion) Stage 2 N ↓ ↑ ↓ - A.k.a. Donath-Landsteiner Hemolytic Anemia (Transport Iron Depletion) - Caused by the presence of autoantibodies Stage 3 ↓ ↓ ↑ ↓ (Functional Iron depletion) MICROCYTIC, HYPOCHROMIC ANEMIAS - Thalassemia *characterized by frank iron - Anemia of Chronic inflammation deficiency anemia - Iron deficiency anemia - TIBC: Total Iron Binding Capacity - Lead poisoning (plumbism) - Ferritin: Most sensitive test for IDA - Sideroblastic anemia o Ferritin is the storage form of iron o The only test which becomes abnormal in stage 1 of Iron deficiency - With small cells that have increased central pallor on the smear - MCV and MCHC = both ↓ DIFFERENTIATION OF MICROCYTIC, HYPOCHROMIC ANEMIAS - Microcytic anemia results from an iron level insufficient for maintaining normal erythropoiesis and is characterized by abnormal results of iron Serum Iron TIBC Serum FEP studies. ferritin - Early development of a microcytic anemia may reveal reduced iron stores, Thalassemias ↑ N ↑ N but an obvious anemia has not developed. Anemia of Chronic ↓ ↓ ↑ ↑ inflammation ANEMIA OF CHRONIC INFLAMMATION (ACI) Iron deficiency ↓ ↑ ↓ ↑ anemia - Was originally called ACD (anemia of chronic disease) Lead poisoning N to ↑ (Adults) N N ↑ - Most common anemia among hospitalized patients - Normal person: Low iron levels cause the release of iron from storage N to ↓ (Children) - Patients with ACI: body stores have abundant iron; but iron from storage is Sideroblastic ↑ N ↑ Mixed anemias (↑ and ↓) not released and red cells are deficient in iron  SIDEROBLASTIC ANEMIAS NOTES - develop when the production of protoporphyrin or the incorporation of iron - Impaired DNA synthesis - considered as root cause of megaloblastic into protoporphyrin is prevented anemia - SIDEROBLAST – nucleated RBC precursor with cytoplasmic iron granules - DNA synthesis is dependent on an important structure: thymidine - SIDEROCYTE – anucleate RBC w/ iron granules triphosphate (TTP). This structure cannot be made unless it receives a - Iron is trapped in the mitochondria (cannot be fully utilized in Hb synthesis) methyl group from methyl tetrahydrofolate or folic acid. Vitamin B12 is the cofactor responsible for transferring the methyl group to methyl - Ring sideroblasts – hallmark of the sideroblastic anemias tetrahydrofolate. Adequate amounts of vitamin B12 and folic acid are - The “rings” in ring sideroblasts = mitochondria loaded with iron important to the formation of TTP. If TTP cannot be made, then it is - Dimorphic peripheral blood picture (normochromic and hypochromic replaced by deoxyuridine triphosphate. The synthesis of this structure RBCs) results in nuclear fragmentation and destruction of cells and impaired cell division. MACROCYTIC, NORMOCHROMIC ANEMIAS - Vitamin B12 is also known as Cobalamin. - Megaloblastic anemia - Folate is the general term used for any form of the folic acid. - Non-megaloblastic anemia - Cyanocobalamin - synthetic form of vitamin B12 (found only in *MCV = High, MCHC = Normal supplements) - Methylcobalamin - naturally occurring form that may be obtained from MEGALOBLASTIC ANEMIA NON-MEGALOBLASTIC ANEMIA either food sources or supplements - Vitamin B12 deficiency - Aplastic anemia - In either folate or vitamin B12 deficiency, patients may experience: - Folate deficiency - Chronic liver disease o fatigue, weakness, and shortness of breath - Myelodysplasia - Alcoholism o glossitis (loss of epithelium on the tongue results in a smooth surface - Erythroleukemia - Bone marrow failure and soreness) - Some drugs o gastritis, nausea, or constipation - Blood pictures seen in folate deficiency and vitamin B12 deficiency are DIFFERENTIATING MEGALOBLASTIC NON- indistinguishable. However, their clinical presentations differ. FACTORS ANEMIA MEGALOBLASTIC - In vitamin B12 deficiency, neurologic symptoms may be noticeable and ANEMIA neuropsychiatric symptoms may also be present. 1. Some of the Vitamin B12 deficiency – may be o Liver disease causes caused by: o Alcoholism Neurologic symptoms: Neuropsychiatric o memory loss symptoms: - inadequate intake (dietary (chronic) o Bone marrow o numbness o personality deficiency is infrequent) failure o tingling in toes and fingers changes - increased need (occurs during o loss of balance o psychosis pregnancy, lactation and o further impairment of walking by loss of growth) vibratory sense, especially in the lower - competition (ex. limbs Diphyllobothrium latum) - lack of intrinsic factor (IF) - Dietary deficiency of vitamin B12 is infrequent. However, this condition is o ex. Gastrectomy [surgical possible for vegans (strict vegetarians) who do NOT eat meat, eggs, or removal of the stomach], dairy products. Helicobacter pylori - Sources of Vitamin B12: infection, Pernicious anemia o liver o meat o oysters and clams - The only natural source of o fish o eggs o cheese and other dairy products vitamin B12 are animals, not plants - Vitamin B12 is NOT destroyed by cooking. - Strict vegetarians are prone to - Pregnancy needs a significant increase in folate to fulfill the requirements Vitamin B12 deficiency related to rapid fetal growth, uterine expansion, placental maturation, and expanded blood volume. Folate deficiency during pregnancy can lead to PA (Pernicious Anemia) impaired formation of the fetal nervous system, resulting in neural tube - characterized by the presence defects (like spina bifida). of antibodies against parietal - Sources of Folate: cells and antibodies against IF. o leafy green o liver o fortified breakfast - Parietal cells are cells in the vegetables o whole grains o oranges stomach which produce o broccoli o beef o cereals intrinsic factor and o dried beans o some fruits, hydrochloric acid o dairy products especially - Intrinsic factor is important for vitamin B12 absorption - Folates - heat labile (overcooking of foods can ↓ their nutritional value) - Pernicious means dangerous LABORATORY TESTS USED TO DIAGNOSE VITAMIN B12 DEFICIENCY Folate deficiency – may be caused AND FOLATE DEFICIENCY by: - inadequate intake SCREENING TESTS Folate deficiency Vitamin B12 - increased need (No difference) deficiency - renal dialysis Complete blood count ↓Hb, Hct, RBCs, ↓Hb, Hct, RBCs, WBCs, PLTS WBCs, PLTS Acute erythroleukemia MA NMA ↑MCV, MCH ↑MCV, MCH 2. Presence of hypersegmented neutrophils Present Absent Manual differential count Hypersegmented Hypersegmented (with >6 nuclear lobes) neutrophils, neutrophils, REMEMBER: Oval macrocytes, Oval macrocytes, o Normal neutrophils have 3 to 5 nuclear lobes. Anisocytosis, Anisocytosis, o Hypersegmented neutrophils - essentially pathognomonic for megaloblastic Poikilocytosis Poikilocytosis anemia RBC inclusions RBC inclusions 3. Shape of the macrocytes Oval Round Absolute reticulocyte ↓ ↓ 4. Presence of megaloblasts in the bone marrow Present Absent count Serum total and indirect ↑ ↑ - Megaloblasts: giant RBC precursors in the bone bilirubin marrow Serum lactate ↑ ↑ dehydrogenase  Folate Vitamin B12  Some of the findings include: SPECIFIC DIAGNOSTIC TESTS o Hypochromic RBCs with basophilic stippling deficiency deficiency Serum Vitamin B12 N ↓ o Toxic granulation in neutrophils Serum folate ↓ N or ↑ o Elevated Zinc Erythrocyte Protoporphyrin test RBC folate ↓ N or ↓ Serum methylmalonic acid N ↑ 2.) HEREDITARY PORPHYRIAS Serum/plasma homocysteine ↑ ↑ o examples include the: holoTC assay (Holotranscobalamin) N ↓ THREE HEMATOLOGICALLY SIGNIFICANT PORPHYRIAS - Mean cell hemoglobin (MCH) - elevated by the ↑volume of the cells (ERYTHROPOIETIC PORPHYRIAS): - Mean cell hemoglobin concentration (MCHC) - usually within the reference Congenital Erythropoietic X-linked Differentiating interval (Hb production is unaffected.) Erythropoietic Protoporphyria Erythropoietic Porphyria Protoporphyria factors - Other possible findings in megaloblastic anemia: teardrop cells, nucleated RBCs, Howell-Jolly bodies, basophilic stippling, and Cabot rings (a.k.a. Gunther’s - Once in the enterocyte, the vitamin B12 is then liberated from IF and bound disease) to transcobalamin (previously called transcobalamin II) and released into the blood. In the plasma, 10 to 30% of the vitamin B12 is bound to Uroporphyrinogen III Ferrochelatase ALA-synthase 2 transcobalamin (75% is bound to transcobalamin I and III, referred to as affected Enzyme synthase deficiency deficiency (gain of function) the haptocorrins). - The vitamin B12-transcobalamin complex, called holotranscobalamin (holoTC), is the metabolically active form of Vitamin B12. Autosomal recessive Autosomal X-linked dominant Inheritance PATHOPHYSIOLOGIC CLASSIFICATION dominant 1.) ANEMIAS CAUSED BY DECREASED PRODUCTION OF RBCs - Aplastic anemia - Megaloblastic anemia - Photosensitivity, - Photosensitivity; - Photosensitivity; - Iron deficiency anemia hemolytic anemia, anemia is mild if mild microcytic, - Thalassemia red-pigmented present hypochromic - Anemia of chronic renal failure urine, erythrodontia anemia with - Anemia of endocrine disorders - Prognosis for this reticulocyte - Anemia of chronic inflammation (ACI) disease is response is - Anemia associated w/ marrow infiltration significantly possible - Sideroblastic anemia WORSE than for the other Clinical features 2.) ANEMIAS CAUSED BY INCREASED DESTRUCTION porphyrias, with - Intracorpuscular abnormality death occurring at a o Membrane defect: -hereditary spherocytosis young age in many  hereditary elliptocytosis cases.  pyropoikilocytosis - Some of the other o Enzyme deficiency clinical features:  G-6-PD o Excessive hair  pyruvate kinase growth  porphyria o Deformed  globin abnormality – hemoglobinopathies (e.g. Hb SS, CC, SC)  paroxysmal nocturnal hemoglobinuria fingers and fingernails - Extracorpuscular abnormality o mechanical: microangiopathic hemolytic anemia, etc. o Scars o infection: malaria, Bartonella, Ehrlichia, Babesia o Port wine red o chemical and physical agents: drugs, toxins, burns urine o antibody-mediated: acquired hemolytic anemia due to warm-reacting - Associated with the - - Additional antibodies legend of the Notes werewolves 3.) BLOOD LOSS (HEMORRHAGE) PORPHYRIAS - Diseases characterized by impaired production of HEME - Heme biosynthesis: o occurs in all metabolically active cells containing mitochondria o most prominent in bone marrow and liver. o Erythroid marrow is the MAJOR heme-forming tissue, generating 85% of the daily heme requirement - “Porphyria” - derived from the Greek word “porphyra”, which means purple - Primary cause: specific enzyme deficiencies in the heme biosynthetic pathway - Products from earlier stages of the pathway accumulate in cells that actively produce heme (like the RBCs and hepatocytes). - Excess porphyrins leak from cells as they age or die (may be excreted in urine or feces). - Accumulated products deposit in tissues as well. - Some of the accumulated products are fluorescent (deposition in skin can lead to photosensitivity with severe burns upon exposure to sunlight). - Only 3 porphyrias have hematologic manifestations (others have a greater effect on the liver). Even in those with hematologic effects, the hematologic impact is relatively minimal, and photosensitivity is a greater clinical problem. - May be ACQUIRED or HEREDITARY 1.) ACQUIRED PORPHYRIAS o an example is:  Lead poisoning  Lead is able to inhibit some enzymes like pyrimidine 5'-nucleotidase and ferrochelatase  Pyrimidine-5-nucleotidase: enzyme needed to remove the RNA remnants from the reticulocytes  Ferrochelatase: a.k.a. heme synthetase; enzyme needed to insert the Fe+2 at the center of the protoporphyrin ring  LABORATORY FEATURES OF THE 3 HEMATOLOGICALLY SIGNIFICANT PORPHYRIAS Laboratory Features Congenital Erythropoietic Porphyria Erythropoietic Protoporphyria X-linked Erythropoietic Protoporphyria (CEP) (EPP) (XLEPP) Red blood Protoporphyrin Normal ↑↑↑ ↑ cells Uroporphyrin ↑↑↑ Normal Normal Coproporphyrin ↑↑ Normal Normal Urine Porphobilinogen Normal Normal Normal Uroporphyrin ↑↑↑ Normal Normal Coproporphyrin ↑↑ Normal Normal Feces Protoporphyrin Normal ↑↑ Normal or ↑ Coproporphyrin ↑ Normal Normal Confirmatory ↓↓ Uroporphyrinogen III synthase ↓↓ Ferrochelatase activity ↑↑ ALA-synthase activity tests activity Genetic testing ↑↑ free erythrocyte protoporphyrin and zinc Genetic testing protoporphyrin - Single arrow – minimal increase or decrease - Double arrows – moderate increase or decrease - Triple arrows – marked increase or decrease HEMOGLOBINOPATHIES Associated with decreased oxygen affinity: - Qualitative globin synthesis defect 10. Hb Agenogi 11. Hb Beth Israel - Due to differences in the arrangement of amino acids in the polypeptide 12. Hb Yoshizuka chain - Major Groups of Hemoglobinopathies: HEMOGLOBIN S o Alpha-hemoglobinopathies o Beta-hemoglobinopathies (most frequently encountered) - Glutamic acid on the 6th position of the B chain is replaced by valine o Gamma-hemoglobinopathies - Defined by the structural formula: o Delta-hemoglobinopathies 𝛼2 𝛽26𝐺𝑙𝑢→𝑉𝑎𝑙 - The name of the hemoglobinopathy depends on the affected globin chain - When oxygenated, Hb S is fully soluble (sickling happens when oxygen - Of the three adult hemoglobins (A1, A2, F), only hemoglobin A2 is affected drops at the issue level). When oxygen is released from the molecule, a by beta hemoglobinopathies since Hb A2 is made up of α2β2 (2 alpha + 2 conformational change happens which results in polymerization of beta) hemoglobin molecules leading to the formation of tactoids or crystals (in turn, this causes the cells to become rigid). β-hemoglobinopathies - Degree of sickling depends on the concentration of hemoglobin S in the Homozygous β- Heterozygous β- RBC: hemoglobinopathies hemoglobinopathies o If the concentration of Hemoglobin S is 80 to 100% (as in sickle cell - Both β genes: mutated - One of the β genes: normal anemia), sickling of the red blood cells occur readily at only slightly - Hb A1 is absent - Other β gene: mutated decreased oxygen concentrations - Abnormal hemoglobin: - Hemoglobin A1 is greater than or o If the concentration of Hemoglobin S is only 20 to 40% (as in the sickle becomes the predominant equal to the amount of abnormal cell trait), oxygen concentrations must be much lower before sickling hemoglobin type hemoglobin occurs. - Examples: - Examples: - Sickle cells obstruct blood low to tissues and organs (resulting in tissue o Sickle cell disease o Sickle cell trait death organ infarction, and pain).  Genotype: Hb SS  Genotype: Hb AS  S gene is the  A is the normal beta mutated beta gene gene  S is the mutated beta  S gene is gene inherited  Resistant to P. o Hb C disease falciparum  Genotype: Hb CC o Hb C trait  C gene is the  Genotype: Hb AC mutated beta  A is the normal beta gene gene  C gene is  C is the mutated beta inherited gene Examples of abnormal hemoglobins present in certain β-hemoglobinopathies: 1. Hb S - Most common - Most severe TESTS FOR HEMOGLOBIN S - Structural formula: 𝛼2 𝛽26𝐺𝑙𝑢→𝑉𝑎𝑙 SCREENING - At the 6th position of the beta chain, the glutamic acid is replaced by TESTS valine PRINCIPLE POSSIBLE RESULTS 2. Hb C - 2nd most common - Whole blood is POSITIVE (+) - 𝛼2 𝛽2 6𝐺𝑙𝑢→𝐿𝑦𝑠 mixed with sodium - presence of either sickle cells or "holly- - At the 6th position of the beta chain, the glutamic acid is replaced by metabisulfite (a leaf” form of the RBCs lysine reducing agent - Note: "Holly-leaf” form of RBCs is 3. Hb E which frequently found in the sickle cell trait. Sodium metabisulfite method - 3rd most common deoxygenates Hb) Bear in mind, however, that using this - 𝛼2 𝛽2 26𝐺𝑙𝑢→𝐿𝑦𝑠 o Reducing method, it is NOT possible to - At the 26th position of the beta chain, the glutamic acid is replaced by agent removes differentiate sickle cell trait from sickle lysine oxygen from cell anemia. Although in sickle cell M hemoglobins (associated with methemoglobinemia and cyanosis): the test anemia, the sickling reaction happens 4. Hb M-Saskatoon environment more rapidly than in sickle cell trait, this 5. Hb M-Milwaukee-1 - In such conditions, observation must NOT be relied upon 6. Hb M-Milwaukee-2 (Hyde-Park) Hb S existing in the to distinguish between the two RBC causes the conditions. Associated with increased oxygen affinity: formation of sickle 7. Hb Hiroshima shaped RBCs NEGATIVE (-) 8. Hb Rainier - Normal looking or slightly crenated 9. Hb Bethesda RBCS  - When RBCs are added POSITIVE (+) HEMOGLOBIN C to the working solution - Turbid solution (black lines on the - Glutamic acid on the 6th position of the beta chain is replaced by lysine. Sodium dithionite tube test (Solubility test) containing sodium reader scale are NOT visible through - Defined by the structural formula: dithionite (reducing the test solution) agent) and saponin 6𝐺𝑙𝑢→𝐿𝑦𝑠 𝛼2 𝛽2 (hemolytic agent), the NEGATIVE (-) red cells immediately - Clear solution (black lines on the lyse. reader scale are visible through the - Two crystals related to Hb C: - Hb S (and sickling test solution) 1. Hb SC crystals o Characteristic appearance: hemoglobins), in the - Note: If this test is positive, a Hb reduced state (in a “Washington monument” electrophoresis should be performed concentrated buffer o Found protruding the RBC membrane on the specimen solution), forms liquid - On the other hand, Hb crystals and produces a 2. Hb CC crystals: electrophoresis results showing the o Characteristic appearance: turbid appearance presence of Hb S should be “Bar of gold” confirmed by a positive sodium o Found within the RBC membrane dithionite tube test (solubility test) THALASSEMIAS - Quantitative globin synthesis defect - Initially called “Thalassic (Greek for "great sea") Anemia” - Other names: o Hereditary Leptocytosis o Mediterranean Anemia - Reduction or total absence of synthesis of one or more of the globin chains - Thalassemias – named according to the chain with reduced or absent synthesis - Mutations affecting the α- or β-globin gene – most clinically significant (the reason: Hb A [a2β2] is the major adult hemoglobin) - Thalassemia occurs in all parts of the world. However, its distribution is concentrated in the "thalassemia belt". - Thalassemia Belt o extends from the Mediterranean east through the Middle East and India to Southeast Asia and South to Northern Africa o Its geographic location coincides with areas in which malaria is prevalent o Thalassemia minor (heterozygous thalassemia) seems to impart resistance to malaria. - Individual and family histories are important in thalassemia diagnosis. The Hemoglobin - If performed alone, it serves as a screening test ethnic background of the person should be investigated because of the Electrophoresis for hemoglobin S increased prevalence of particular gene mutations in specific populations. (Cellulose - If performed along with sodium dithionite tube test, - Clinical findings that suggest thalassemia include (these are particularly Acetate) it becomes a confirmatory test for hemoglobin S prominent in untreated or partially treated β-thalassemia major): - In an alkaline buffer (8.4 to 8.6) hemoglobin is a o pallor (due to the anemia) Note: Considered negatively charged molecule o jaundice (due to hemolysis) as the primary o splenomegaly (due to sequestration of abnormal red cells, too much - During electrophoresis, the Hb molecules travel screening extravascular hemolysis, and some extramedullary erythropoiesis) toward the anode (+) because of their net negative procedure to o skeletal deformities (due to the massive expansion of the bone charge detect variant marrow cavities) (abnormal) - The difference in the net charge of the Hb molecule defines its mobility and reveals itself by - Clinical manifestations of thalassemia arise from: hemoglobins o Decreased or absent production of a specific globin chain, which the speed with which it migrates to the positive pole. reduces hemoglobin synthesis and generates microcytic, hypochromic RBCs; and o Unequal production of the α- or β-globin chains causing an imbalance in the α/β chain ratio (leads to a markedly decreased survival of erythrocytes and their precursors) - REMEMBER: The α/β chain imbalance is more significant and determines the clinical severity of the thalassemia. The mechanism and the degree of shortened red cell survival are dissimilar for the β-thalassemias and α- thalassemias. SCREENING TESTS TESTS EXPECTED FINDINGS CONFIRMATORY PRINCIPLE Complete Blood ↓ Hb, ↓ Hct, ↓ MCV, ↓ MCH, ↓ MCHC, Slight to moderate TESTS Count (CBC) ↑ Reticulocyte count Hemoglobin - Migration distances of the different hemoglobins Peripheral Varying degrees of microcytosis, hypochromia, target Electrophoresis are based on the electrophoretic charge of the Blood Smear cells, anisocytosis, NRBCs (nucleated red blood cells), (Citrate Agar) molecules and their adsorption to the agar poikilocytosis and RBC inclusions (examples include: compound basophilic stipplings, Howell-Jolly bodies, Pappenheimer Note: Used to bodies, Hb H inclusion bodies) confirm variant Iron studies Serum ferritin and serum iron: Normal or ↑ TIBC: Normal hemoglobins and (to rule out IDA) further differentiates - Confirmatory test: Molecular Genetic Tests hemoglobin S from D and G, and hemoglobin C from hemoglobins E, OArab, CHarlem ALPHA THALASSEMIA β-thalassemia βsilent/βsilent - Moderate clinical symptoms, intermedia β+/βsilent transfusion-independent - typically caused by a deletion of one, two, three or all four of the α-globin β0/βsilent - Mild to moderate hemolytic genes (results in reduced or absent production of a chains anemia, microcytic, hypochromic - Genotype in normal persons: αα/αα (4 α-genes) RBCs Clinical No. of Genotype Remarks syndromes Deleted NOTE Gene Note: The following are examples of β-thalassemic genes and their Silent carrier One of (-α/αα) - Asymptomatic definitions: state four - β0 = β-globin gene mutation in which no β chains are produced (α-thalassemia) genes - β+ = β-globin gene mutation that results in 5% to 30% reduction in β α-thalassemia Two of (--/αα) - Mild microcytic chain production trait four or hypochromic anemia - βsilent = β-globin gene mutation that results in mildly decreased β chain (aka: α- genes (-α/-α) production thalassemia minor) Hb H disease Three of (--/-α) - Mild microcytic WHITE BLOOD CELLS four hypochromic anemia genes - Nucleated cells that function in body defense. - Hb H found in adults - Hb Bart found in neonates - RBC vs WBC: RBCs are anucleate while WBCs are nucleated - Reference ranges - Hb H may coexist with Hb Constant Spring (a more o Adult (males and females): 4.5 to 11.0 x 109/L (SI)  4,500 to 11,000 /mm3 (conventional) severe disease than Hb H) o Newborn: 13.5 to 38.0 x 109/L (SI) Hb Bart Four of (--/--) - Hb Bart’s Disease  13,500 to 38,000/mm3 (conventional) hydrops fetalis four - MOST SEVERE FORM OF syndrome genes - How to convert WBC count from conventional units to SI? ALPHA THALASSEMIA o Ex. 4,200/mm3 (conventional) to SI (a.k.a. Hb Bart - Most severe form may Syndrome, α- o Move three decimal points to the left cause stillbirth/hydrops o Answer: 4.2 x 109/L Thalassemia fetalis Major) o Move three decimal points to the right if converting from SI to - NOTE: Fetus commonly conventional cannot survive gestation, - NOTE: The difference between mm3 and µL is INSIGNIFICANT (either of causing stillbirth with the two may be utilized in reporting the WBC count using the conventional hydrops fetalis. units) - However, presently, with the help of intrauterine NORMAL WBC COUNTS AND DIFFERENTIAL COUNTS transfusion and the (In different ages) neonatal intensive care AGE TOTAL WBC (x109/L) LYMPHOCYTES (%) unit, survival may be NEUTROPHILS (%) (in range) EOSINOPHILS (%) MONOCYTES (%) possible (survivors will have severe transfusion- dependent anemia like patents suffering from β- thalassemia major). - Bone marrow transplant or cord blood transplant may 6 months 6.0 to 17.5 32 61 5 3 be helpful. 2 years 6.0 to 17.0 33 59 5 3 4 years 5.5 to 15.5 42 50 5 3 BETA THALASSEMIA 6 years 5.0 to 14.5 51 42 5 3 8 years 4.5 to 13.5 53 39 4 2 - caused by mutations that affect the B-globin gene complex 16 years 4.5 to 13.0 57 35 5 3 - no deletion in the beta gene 21 years 4.5 to 11.0 59 34 4 3 - Normal persons: 2 beta genes only (1 from the mother, 1 from the father) - Predominant WBC in an adult: neutrophils Clinical Examples Remarks - Predominant WBC in children 6 lobes - Megaloblastic anemias, myelodysplastic syndromes, Neutrophils hereditary neutrophil hypersegmentation, myelokathexis - NOTE: Myelokathexis pertains to a rare hereditary condition characterized by normal granulocyte production; nevertheless, there is impaired release into the blood (leads to neutropenia). Here, neutrophils appear hypermature. There may be hypersegmentation, hypercondensed chromatin, and pyknotic changes in this condition. Pelger-Huet Anomaly - Failure of the neutrophil nucleus to segment - Most common genetic disorder of WBCs (a.k.a. True/Congenital PHA) - Pelger-Huet Cell - Autosomal dominant disorder o “pince nez" or "spectacle" form of neutrophil nucleus - Decreased nuclear segmentation (bilobed, unilobed), coarse o Hyposegmented neutrophil (with 1 to 2 nuclear lobes) chromatin clumping pattern potentially affecting all o Unilobed = round, ovoid, or peanut shaped leukocytes, although morphologic changes are MOST o Bilobed = spectacle-like ("pince nez") morphology obvious in mature neutrophils. with nuclei attached by a thin filament - A result of a mutation in the lamin β-receptor gene - Lamin β-receptor = an inner nuclear membrane protein; Acquired PHA plays a major role in leukocyte nuclear shape changes that aka: Pseudo-PHA occur during normal maturation - Psuedo-Pelger Huet Cell - Homozygous PHA: all neutrophils are affected and o neutrophils that resemble Pelger-Huet cells demonstrate round nuclei (hyposegmented and hypogranular) - Heterozygous PHA: 55% to 93% of the neutrophil population o may be seen in acute myeloid leukemia, chronic are affected (there is generally a mixture of all of the myeloproliferative neoplasms, and myelodysplastic aforementioned nuclear shapes) syndromes (MDS), HIV infection, tuberculosis, Mycoplasma pneumoniae, and severe bacterial - Neutrophils show normal granulation in true PHA. infections - Neutrophils in PHA appear to function normally. Lupus Erythematosus (LE) - Usually a neutrophil that has ingested the antibody- - Usually an in vitro phenomenon Cell coated nucleus of another neutrophil or has engulfed the - Found in systemic lupus erythematosus (SLE) but may homogenous, globular nuclear mass of a destroyed cell also be found in comparable connective tissue disorders Rieder Cell - Similar to normal lymphocytes BUT the nucleus is - Found in chronic lymphocytic leukemia or can then be notched, lobulated, and cloverleaf-like artificially formed through blood film preparation Flame Cell - An abnormal plasma cell with intensely eosinophilic or - Found in IgA Myeloma A.k.a. Thesaurocyte "flamingo cytoplasm” Grape Cell - An abnormal plasma cell with a cytoplasm that is - Found in Multiple Myeloma A.k.a. Morula cell, Mott cell completely filled with Russell Bodies (round, discrete globules containing antibodies) Alder-Reilly Anomaly - Characterized by dense azurophilic granulation in all - Granulation results from an abnormal deposition and st