Study Guide Hematology Test 1

Define and differ whole blood, plasma, and serum

plasma- helps the cells to circulate, fluid fraction, separated from anti-coagulated blood (7% proteins, 91% water, 2% (ions, nutrients, waste products, gases, regulatory substances))

whole blood - anti-coagulant

serum - fluid fraction, separated from clotted blood

define hemoconcentration and hemodilution. What do the lab tests look like? What would be the corrective action?

hemoconcentration - falsely elevates cell counts, increases due to loss of water or plasma from the blood

hemodilution - reduces the concentration of blood components, helps maintain a proper volume of blood without transfusion, IV fluid can dilute the blood

Identify actions of anticoagulants. What color stoppers are used for each?

Ethylene-diamine tetra acetic acid (K²EDTA) - purple top tube, prevents coagulation to occur, used in cell counts and morphologic examination, calcium chelator, blood smears should be made and examined within 2 hrs of collection

sodium citrate - blue top tube, calcium chelator, used in coagulation studies (blood to anticoagulant ration bust be 9:1, low specimen ratio, elevates coag results), used in platelet satellitism (platelet count has to be multiplied by 1.1 to correct for anticoagulant volume)

heparin - green top tube, lithium heparin, inhibits action of thrombin, anti-thrombin activity, used for most chemical testing and not recommended for hematology testing (heparin distorts cellular morphology, interferes with coagulation studies), not recommended for lithium assays - falsey elevates Li values, used in treatment of Bipolar disorders

Identify the main action of the anticoagulants and their actions for EDTA, Na Citrate, and Heparin.

Name the antiseptic used in venipuncture

70% isopropyl alcohol or iodine if they are allergic (povidone-iodine is dried and first drop is wiped away with sterile gauze, subsequent drops are collected of blood.)

name the sites of collection for a skin puncture

fingertip, heel, earlobe

identify the veins commonly used for venipuncture

media cubital vein, median antebrachial, cephalic veins

Explain how to properly identify the patient’s ID

Ask the patient to recite back to you their information so you are not just speaking the info to them and therefore they cannot really confirm or deny it. They could be confused and just say yes.

Name, DOB, medical record number, ask them to identify themselves

Describe how to recognize the EDTA Platelet phenom. How do you correct for this?

platelet satellitism, platelet clumping around neutrophils. EDTA causes this clumping to occur and collecting blood in a citrate tube can be done to repeat the PLT count

List the test performed under the term CBC

Complete blood count

• basic screening test in all patients

• WBC count

• RBC count

• Hematocrit

• Hemoglobin

• Red blood indices: MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration), RDW (red cell distribution width

• platelet count

• white cell differential count (DIff)

define hematopoiesis

production of blood cells and platelets

medullar hematopoiesis - takes place in the bone marrow - primary process

extramedullary hematopoiesis - takes place in other organs such as the spleen or liver, pathological

identify the sites of cell production from fetal to adult development

 Mesoderm, Mesoblastic
 The Yolk Sac
 1st Week - 2-3 Months

 Hepatic
 Liver and Spleen
 Primary site: 2 - 7 Months

 Medullary, Meyloid
 Bone Marrow
 Primary hematopoietic site
 7 Months - Life

Adults - sternum, ribs, vertebra, iliac crest

Pediatric - tibia, femur

list three growth factors responsible for erythropoiesis, red cell differentiation/maturation

 Includes: Interleukins, lymphokines, monokines,
interferons, CSFs
 Colony Stimulating Factors - CSFs
 G-CSF Granulocytes
 GM-CSF Granulocytes and Monocytes, BFU-E
 M-CSF Monocytes
 Erythropoietin
 EPO Erythrocytes development (BFU-E, CFU-E)
 Cytokines affecting RBC maturation:
 SCF (stem cell factor) & Flt3 ligand
 IL1, IL-3, IL6, IL-9, IL-11
 GM-CSF, IL3
 EPO
 Thrombopoietin
 TPO Platelet development

define 4 characteristics of interleukins

1. effective at very low concentrations

2. part of interacting systems with amplification potential

3. have synergistic interactions with other cytokines

4. proteins that exhibit multiple biologic activities such as regulation of autoimmune, inflammatory reactions and hematopoiesis

diagram maturation sequence from pluripotent stem cells to mature erythrocyte. Identify cellular markers of stem cells, myeloid progenitor, lymphoid progenitor

pluripotent stem cell → multipotent stem cell → progenitor cells (monopotent) [Colony forming unit (CFU-GEMM), CFU-E, CFU-G, CFU-Mo, CFU-meg] → precursor blast cell → intermediary cells → mature cells

stem cell - CD34

myeloid progenitor - CD33 and CD38

lymphoid progenitor - CD10 and CD38

explain the cause of polychromatic cytoplasm (blueness)

due to increase in Hgb

identify which cells are normally seen in bone marrow and peripheral blood

pronormoblast, basophilic normoblast, polychromatic normoblast, orthochromic normoblast, polychromatic erythrocyte & mature erythrocyte - in bone marrow

in peripheral blood - polychromatic erythrocyte, mature erythrocyte

state the normal M:E ratio

3 to 1

identify the major functions of RBC metabolic pathways

metabolic pathways of RBCs helps them to produce ATP since they lack a mitochondria, almost exclusively through the breakdown of glucose via anaerobic glycolysis

explain the major outcome of the E-M pathway, L-R pathway, HMS pathway in reference to which is anaerobic, aerobic, which accounts for 90% of cell glycoclysis, which protects for oxidation effects of peroxides

EM pathway - anaerobic glycolysis - accounts for 90% of RBC glucose utilization requirements → net result and produces 2ATP molecules

HMS pathway - aerobic glycolysis, 10-15% glucose utilization, protects RBC from accumulation of hydrogen peroxide which denatures hemoglobin, dependent on glucose-6-phosphate dehydrogenase (G6PD), when deficient globin chain denaturation occurs

L-R pathway - production of 2,3-DPG, regulates oxygen carrying capacity, oxygen transport

methemoglobin reductase pathway - maintains iron in the ferrous state (Fe2+), counteracts the oxidized state, Reducing Fe3+ to Fe2+, relies on reducing capacity of nicotinamide adenine dinucleotide (NADH), required for oxygen transport

explain the structure of hemoglobin listing all components and which cell produces it

4 heme molecules

4 Fe2+ atoms

4 globin chains - 2 alpha and 2 beta chains

574 amino acids - 141 aa in alpha chains, 146 aa in beta chains

forming a, B dimers

each peptide coils in 8 helices and 1 Hgb molecule is capable of binding 8 oxygen atoms or 4 molecules of O2

protoporphyrin IX

production: occurs in the mitochondria and cytoplasm of erythroid precursors (pronormoblast through polychromatic erythrocyte)

identify the chromosomes which control hemoglobin production for hemoglobin A, A2, and F

alpha and zeta chains are coded on chromosomes 16

B chains and other globins - coded on chromosome 11

A2 - chromosome 11

A - chromosome 11

F - chromosome 11

list the hemoglobin’s that are considered embryonic hemoglobin

Gower 1

• zeta 2, epsilon 2

Gower 2

• alpha 2, epsilon 2

diagram the biosynthesis of Heme from ALA to heme in detail listing all intermediary porphyrins (enzymes not necessary)

Succinyl CoA + Glycine → Delta-aminolaevulinic acid (ALA) → porphobilinogen → Uroporphyrinogen → Coproporphyrinogen → Protoporphyrinogen → Protoporphyrin → Heme

define oxy-HB. deoxy-Hb. Met-Hb, Carboxy-Hb, Sulf-Hb

oxy-Hb → iron attached to Hb in ferrous Fe2+ state, oxygen attached, imparts a red color to the blood, arterial blood

deoxy-Hb → iron = ferrous Fe2+ state, not carrying oxygen, imparts a dark color to the blood, venous blood

carboxy-Hb → formed when Hb is bound to carbon monoxide, CO has 240 times affinity for Hb to the binding of O2, unable to bind with O2, even when O2 is present, leads to tissue hypoxia and death, caused by cigarette smoking, occupation exposure and intentional exposure

met-Hb → iron = the ferric Fe3+ state, decreased O2 carrying capacity, normally reduce by Met-Hb reductase pathway, ascorbic acid, glutathione reductase, increased in methemoglobin reductase deficiency, hemoglobin M disease (inherited hemoglobin variant), exposure to oxidizing chemicals or drugs

sulf-Hb → sulfur atoms combine irreversibly with heme groups, unable to bind with oxygen, 1/100 the oxygen affinity of Hb A, oxygen delivery is compromised, causes GREEN pigment, erythrocytes carry it until cell is removed from circulation

explain how CO-hemoglobin can bind CO 240 times stronger than O2 and how this leads to asphyxiation

CO forms a more stable bond with the iron atom in the heme group than O2, displacing O2 and preventing it from being transported. has a higher affinity than O2 leading to hypoxia and death

identify the globin chain configs for Hb A, Hb A2, Hb F

Hb A → a2, B2 95-98 % adult Hgb

Hb A2 → a2, delta2 1.5-3.5%

Hb F → 2 alpha, 2 gamma 50-80% newborn fetal Hb adults have: <3% , causes increased oxygen affinity

Globin chain configs Hb H, Hb I, Hb barts, Hb S, Hb C

Hb S → (alpha 2, beta 2 6gluc → val) beta chain hemoglobinopathy, substitution of glutamic acid with valine, sickle cell disease (homo or hetero), insoluble in dithionite solns, will be positive for dithionite solubility test

Hb C → (alpha 2, beta 2 6 glu → lysine) substitution of glutamic acid with lysine in 6th position, see Hb-C crystals in RBCs and results in hemolysis of RBCs, hemolytic anemia, increased frequency in individuals from western africa, italy, greece, turkey, and middle east

Hb H → Beta 4 (B4), inconsistent with life, associated with alpha thalassemia, still born, rapid electrophoretic migration (anode), unstable and when stained with crystal violet, precipitate as Heinz bodies

Hb Bart’s → Gamma 4, inconsistent with life, associated with Hydrops fetalis - alpha thalassemia, still born, rapid electrophoretic migration (anode)

Hb I → (alpha 2 16 glu → lysine, beta 2) first described in 1955 in afro-american family, characterized by faster electrophoretic mobility in alkaline pH similar to Hb H

describe principle of Hb electrophoresis. explain mobility of major hemoglobin types

Alkaline electrophoresis, cellulose acetate agar, pH 8.4-8.6, Hb is ionized in alkaline buffer, Hbs have net negative charge, Negative charged Hb migrate to + Pole
 + pole = Anode
 Migrates to neutrality or pI (Isoelectric
point)

 Order or Migration:
 Origin to + pole (Anode)
 C , S, F , A , H , BARTS , I (anode)

identify two Hb bands that migrate in the same regions with Hb C and Hb S

Hb S → D and G

Hb C → E, A2, O

what is the chemical principle of hemoglobin determination using automated methods?

Cyan-methemoglobin methods

spectral-absorbance

KCN

K3Fe(CN)6

oxidation of Hb to Met-Hb

KCN converts Met-Hb to Cyan - Met-Hb

absorbance spectrum at 540 nm

absorbance is compared to a standard

Specific Gravity method → compared to CuSO4 solution, used in red cross, CuSo4 solution SG + 1.053 = Hg of 12.5 g/dl

Hg >= 12.5 g/dl will sink

Hg <= 12.5 g/dl will float

indicating donor is anemic

list and describe the tests for Hb F or characteristics of Hb F

Hb F → Alkali denaturation, Hb F is resistant to denaturation with strong alkali solutions (clear solution)

• Adult Hb is not → turbid solution

Hb F potential plasma marker of FRBCs in colorectal tumors

Increased HbF synthesis in adults is suggested to relate to different pathways in the activation of the y-globin gene

growth factors are shown to induce Hb F production

Hb F is resistant to acid elution - cells will look red pink with Kleihauer Acid elution

describe the Dithionite solubility test. Identify which hemoglobin it is used to screen for? How is the turbidity interpreted?

sickle cell disease/ trait

Hb S is screened for and is insoluble in dithionite solutions, resulting in a positive test, turbidity is positive result.

identify the abnormal hemoglobin on a patient giver their test results and using their national origin as a clue

diagram a hemoglobin electrophoresis plate, showing the migration of hemoglobin bands for a normal patient, cord blood, and ASC control

Trouble-shoot and explain what should be performed next if you suspect that two hemoglobin’s may be migrating in the same electrophoretic region

explain how to differentiated hemoglobin C from E and hemoglobin S from D using alkaline and acid electrophoretic techniques

differentiate by their difference in charger

Hb C moves fastest at pH of 5.25 while Hb A is slowest. HbC has greater net positivity than Hb S

Hb S separates from Hb D and G on acid electrophoresis while on alkaline they migrate together. Hb D and G are further differentiated from S in that they produce a negative result on the hemoglobin solubility test.

Given the race of the patient, explain how this may be used to aid in the identification of abnormal hemoglobins, e.g if a patient is from southeast Asia, how can this be used to differ hemoglobin C from Hb E?

identify and list the clinical significance of increased and decreased RBC values

decreased - anemia (aplastic, iron deficiency, hemolytic, b12 deficiency, folic acid deficiency anemia, anemia of blood loss), anemia of chronic diseases such as RA and SLE, leukemia, hypothyroidism, liver disease, kidney disease

increased - polycythemia (1, 2), heart failure, lung disease (emphysema, COPD, pulmonary fibrosis), hypoxia, CO exposure, erythrocytosis (increased RBC prod), congenital heart disease, severe dehydration, smoking, living in high altitude, performance enhancing drugs 9 anabolic steroids, testosterone, erythropoietin)

identify or state the reference normal values for RBC, Hb, Hct, MCV, MCH, MCHC

RBC → 4.0 - 6.0 × 10^6 / uL

Hct → 35-50%

Hb → 12-18 g/dL

MCV → 80-100 fl

MCH → 26-34 pg/cell

MCHC → 32-36 g/dl

define MCV, MCH, MCHC

MCV - measures the average volume of RBC and classified anemia, categorizes RBC by size, mean corpuscular volume

MCH - measures the avg concentration of hemoglobin in RBC

MCHC - mean corpuscular hemoglobin concentration, avg concentration of hemoglobin per volume of RBC (100 ml), categorizes RBC according to their concentration of hemoglobin, hypochromic or normochromic

Identify and list the equations for MCV, MCH, MCHC and be able to calculate the indices given the RBC count, Hb and Hct Values

MCHC - Hgb (g/dL) x 100 / Hct (%)

MCH = Hb (g/dL) x 10 / RBC

MCV = Hct x 10 / RBC

name or list at least five diseases or conditions for the clinical significance for decreased or elevated hemoglobin and hematocrit values

decreased hemoglobin -

 Anemia
 Aplastic anemia
 Iron deficiency
anemia
 Thalassemia
 Hemolytic anemia
 B12 deficiency and
folate acid anemia
 Anemia of blood loss
 Anemia of Chronic
Diseases
 RA & SLE
 Leukemia
 Hypothyroidism
 Liver Disease
 Renal Disease
 Hemolytic reactions
 Transfusion reactions of
incompatible blood
 Reaction to chemicals or
drugs
 Reaction to infectious agents
 Clostridium sp.
 Reaction to physical
agents,
 severe burn
 prosthetic heart valves

increased:  Polycythemia
 Primary and Secondary
 Hemoconcentration of the blood
 Chronic obstructive pulmonary disease
 Congestive heart failure

Explain the physical affects of altitude and age on RBC, Hb, and Hct values

age : normal RBC of newborn is higher than adult, drop to lowest point in life at 2 to 4 months, will increase in adulthood and lead to decreases in RBC count over time

altitude: higher the altitude the greater the increase in RBC, decreased oxygen content of the air stimulates the RBC to rise (erythrocytosis)

RBC, Hb and Hct all correlate so all will increase with RBC and decrease with RBC

Name who always wins the NY or Boston Marathon and explain why with reference to high altitudes and EPO cycles

Identify which RBC tests are actually measured and which are calculated

RBC count, MCV, and Hgb are measured directly and Hct, MCH, MCHC, and RDW are calculated

Explain the affect on RBC, Hb, and Hct values of leaving a tourniquet on too long during blood collection

Explain the affect on RBC, Hb, and Hct values if a blood sample was collected above an IV site

GIven the MCV, MCH, and MCHC results from a patient, identify if one should expect to see microcytic hypochromic cells, normocytic or macrocytic cells under the microscope

Given the MCV, MCH, and MCHC results for a patient that may have iron deficiency anemia, B12 deficiency or Folic acid deficiency anemia

define the terms anisocytosis, poikilocytosis, and anisochromia

anisocytosis - variations in cell size

poikilocytosis - variations in cell shape

anisochromia - variation in cell color

name at least four disease that are associated with decreased MCV and MCHC values

1. iron deficiency

2. thalassemia

3. lead poisoning

4. porphyria

5. sideroblastic anemia

name or identify at least two diseases that are associated with increased MVC

1. megaloblastic anemia - vitamin b12 deficiency or folic acid deficiency

2. pernicious anemia - impaired b12 absorption, intrinsic factor deficiency, gastrectomy

3. anemia of liver disease

4. increased reticulocyte prod.

identify what disease is associated with a decreased MCH and MCHC

1. hypochromic anemia - fe deficiency anemia, thalassemia, lead poisoning, porphyria, sideroblastic anemia

identify the physiologic cause for the following cells

schistocytes, acanthocytes, stomatocytes, spherocytes, elliptocytes, ovalocytes, target cells, bite cells, dacrocytes, tear drop cells

echinocytes - depletion of ATP, osmotic imbalances, prolonged exposure to anticoagulant

acanthocytes - alteration in membrane lipid content, loss of membrane integrity, high cholesterol to phospholipid ratio

elliptocytes - membrane defect in spectrin and protein 4.1

ovalocytes - reduced ATP and 2,3-DPG

target cells/codocytes - membrane defect - excess membrane cholesterol and phospholipid, deceased Hb

dacrocytes, tear drop cells - squeezing during splenic passage

stomatocytes - abnormally increased cation influx results in increased permeability to sodium, net influx of water, overhydration, and swelling

schistocytes - due to membrane damage

bite cells - missing membrane, G6PD deficiency, hemolytic anemia, hemoglobinopathies

helmet cells/ keratocytes - DIC, hemolytic anemia

spherocytes - genetic membrane defect - spectrin assembly, poor protein 4.1 to sepctrin binding, actin-spectrin band 3 complex defect, defective cytoskeleton

drepanocytes - presence of Hb S - crystallized under low oxygen tension conditions

match or identify the composition of RBC inclusions with what inclusion is made of e.g (HJ bodies with DNA)

Basophilic Stippling - RNA and ribosome/mitochondrial aggregates (wright’s tain and supravital stains)

Cabot Rings - residual nuclear membrane or mitotic spindle (wright’s stain)

Howell Jolly Bodies - Nuclear DNA remnants (wright’s stain)

Pappenheimer bodies - clusters of free iron. non-heme iron (wright’s stain and prussian blue stain)

siderodic granules - non-nucleated - siderocyte and granules in NRBC - ringed sideroblast (prussian blue stain)

Heinz bodies - precipitated, denatured, unstable hemoglobin (crystal violet stain/supra vital stains)

malarial pigments - wright’s stains

what is the possible clinical significance of basophilic stippling

lead intoxication, heavy metal poisoning, thalassemia, severe anemia, accelerated erythropoiesis

identify what prussian blue stains with

identify the indices associated with macrocytes, microcytes, and normocytes

describe the relationship between polychromatic erythrocytes and reticulocytes

name tests that would be positive in Favism and G6PD deficiency

appearance of bite cells, G6PD deficiency, oxidation of hemoglobin

Fluorescent spot test, Quantitative Spectrophotometric Assay, Enzyme chronographic test

identify the cause of rouleaux and agglutination

agglutination - antibody induced

rouleaux - increased plasma proteins and decreased zeta potential - natural repulsion mechanism

identify what cells indices is measured by the RDW

Reticulocytes

explain when Shuffner’s granules would be expected to be found and in what species

in causes of parasites such as P. vivax and P. ovale

name at least three causes for a positive Heinze body test

• G6PD deficiency

• Post Splenectomy

• Methemoglobinemia Hb M

• Alpha Thalassemia - Hb H, Barts

• Drugs related to hemolytic anemia, drug poisoning

diagram an algorithm for using the reticulocytes count, beginning with 9Hb and Hct

identify the clinical significance for ESR, Reticulocyte count, Sickledex and positive Heinz body prep

used in the diagnosis of occult disease - inflammation, infections, autoimmune disorders, multiple myeloma, malignancy

reticulocyte count - differentiate anemia, increased BM activity, indicator erythrocyte production, check effectiveness of treatment, check recovery of bone marrow failure, determine effects of radioactive substances on exposed workers

heinz bodies - G6PD deficiency, post splenectomy, methemoglobinemia Hb M, alpha thalassemia (Hb H, bart’s), drugs related to hemolytic anemia (anemia caused by drug poisoning)

identify which factors increase ESR values

inflammation, acute infections, autoimmune disease (RA & SLE), multiple myeloma, malignancy, polymyalgia rheumatica, temporal arteritis, tissue necrosis, anemia, pregnancy

Explain what needs to be performed when suspected sickle cells are identified

name the stain is used in Retic prep

new methylene blue - supra vital stain

calculate a correct reticulocyte count and then calculate an absolute retic count

#retic / 1000 × 100 OR # retics / 10 = % retics

absolute retic count = % retics x total RBC (10^6, 10^12) =

corrected retic count = RPI + retic % x patient’s Hct/Normal Hct

describe what anemia an increased reticulocyte count can differentiate

hemolytic anemia - immune hemolytic, RBC membrane, hemoglobinopathy, RBC enzyme deficits, toxin exposure

diagram an algorithm to differentiate hemolytic anemia from other anemia using the reticulocyte procedure

identify which erythrocytes have increased and decreased osmotic fragility

decreased osmotic fragility - target cells

increased osmotic fragility - spherocytes

given the results for an osmotic fragility test, identify if the patient has HS or thalassemia with target cells

turbidity is positive for Hb S or thalassemia but if clear and see through then negative

sickle cell anemia or sickle cell trait

explain what the G6PD spot fluorescent test is used to detect

detects G6PD and looks for NADP → NADPH with uv light

interpret what it means when the spot fluorescent test is positive for

when positive - means you do not have deficiency for G6PD and it is present, but if absent and no fluorescence then you have the deficiency