Lecture 1 (pt. 1 up to page 52)

Blood Composition

• Blood constitutes approximately 6-8% of total body weight.

• Plasma comprises 45-60% of blood volume.

• Average blood volume in a 154 lb male is around 5600 ml.

• Normal blood pH is between 7.35 – 7.45.

• Sources for blood collection include:

  • Skin puncture

  • Venous puncture

  • Arterial puncture

Blood Collection Techniques

Skin Puncture

• The specimen obtained is a mixture of arterial and venous blood, often incorrectly referred to as "capillary blood".

• May contain interstitial and intracellular fluid.

• Preferred in pediatrics; useful for glucose monitoring.

• Utilizes a capillary tube for hematocrit measurement.

Skin Puncture Technique

• Site Selection:

  • Infants: Lateral or medial aspect of the heel.

  • Adults: Lateral to the digital pulps.

• Procedure:

  • Warm the area (~42° C - 107° F) to increase blood flow.

  • Prep the site with alcohol.

  • Discard the first drop of blood.

  • Do not "milk" the area to avoid contamination.

Venous Puncture

• Composition of venous specimens depends on the metabolic activity of perfused organs.

• Site Selection:

  • Antecubital fossa:

    • median cubital vein

    • cephalic vein

  • veins in the wrist, hand or ankle, or femoral vein.

Venous Puncture Technique

• Clean the site with alcohol.

• Use a tourniquet proximal to the chosen site.

  • Flexing the hand may assist in venous distention.

  • Anchor vein both distal and proximal to entry point.

  • Needle delivery should be bevel up, entering at a 15° angle and directed along the proximal path of the vein.

• Release the tourniquet once blood flow is established.

Arterial Puncture

• Arterial blood is uniform throughout the body.

• Site Selection:

  • Adult:

    • Radial artery (commonly assessed using the Allen test: blocking of the artery)

    • femoral artery

    • brachial artery

  • Pediatric patients:

    • scalp artery

Arterial Blood Collection

• Deoxygenated blood is pumped from the heart to lungs for oxygenation (right side), then pumped via arteries (left side).

• Commonly collected for arterial blood gas evaluations

  • obtained via radial artery

• Can be drawn directly from the artery or through a vascular access device (VAD) like a femoral arterial line or catheter.

Arterial Puncture Technique (ex. on Radial Artery)

• Local anesthetic infiltration

• Collect blood gases in a heparin tube

• Prep the skin with alcohol

• collect blood distal to the pulse point

• Gently mix the specimen

• apply compression for a minimum of 5 minutes.

• Transport specimens on ice for gas analysis.

Interpretation of Lab Values

• Look for trends in lab values; any abnormal results should be repeated.

• Osler’s Rule:

  • For patients under 60 years of age, aim to attribute all abnormal values to a single pathology (attributed to Dr. William Osler, 1849 - 1919).

Collection Tubes for Specimen Storage

• Serum with coagulants: Red top or SST (tiger top).

• Plasma with anti-coagulants:

  • Citrate (blue top)

  • EDTA (lavender top)

  • Heparin (green top)

  • Fluoride (gray top)

Coagulation Studies

• Collected using a sodium citrate tube (blue top).

• Sodium citrate serves as an anticoagulant (utilized since 1914), acting via calcium chelation to block the coagulation cascade.

• Adenine enhances red cell viability.

• Tests: Prothrombin Time (PT), Partial Thromboplastin Time (PTT), Thrombin Clotting Time.

The Clotting Cascade

Intrinsic Pathway

• Over multiple factors including HK, PK, and various clotting factors leading to thrombin and subsequent fibrin polymer formation.

Extrinsic Pathway

• Involves tissue factor and Factor VII leading to thrombin activation and resulting fibrin creation.

Prothrombin Time (PT)

• Evaluates the extrinsic clotting pathway

  • focusing on vitamin K dependent factors II, VII, IX, and X, as well as protein C and S, synthesized in the liver.

• Conditions that can cause prolonged PT include:

  • vitamin K deficiency

  • impaired fat absorption

  • liver disease

  • Coumadin therapy

    • antibiotics

• Reference values: PT range is 11-13 seconds, varying by laboratory standards

  • International Normalized Ratio (INR) applied for standardization.

• INR calculation:
  $INR={}\frac{observedPT}{controlPT}xISI$

• INR test ensures that PT results are standardized

• PPT used to monitor person’s response to the anticoagulant heparin and can also identify diseases that interfere with blood clotting

• Prophylatic values:

  • Embolism: 2-3

  • Mechanical heart valve: 2.5-3.5

Monitoring and Antibiotics Effect

• Routine PT/INR monitoring is unnecessary for direct oral anticoagulants (DOACs) such as Apixaban (Eliquis) and Rivaroxaban (Xarelto).

• For patients on warfarin, regular INR monitoring is critical. Early evaluation (within 3-7 days after prescribing antibiotics) significantly reduces bleeding risks.

Data from Studies on Overanticoagulation

• The need for careful monitoring during antibiotic administration in patients on stable warfarin regimens was observed in data from Glasheen et al.

• Key findings included varying effects of different antibiotics on INR levels, indicating the extent of anticoagulation risk with certain medications:

  • Terazosin (n=333): Decrease of -0.15, with no significant incidence

  • Azithromycin (n=158): Increase of 0.51, affecting 31% and 16% of patients

  • Levofloxacin (n=258): Increase of 0.85, affecting 33% and 19% of patients

    • WORST CULPRIT

  • TMP/SMX (n=120): Increase of 1.76, affecting 69% and 44% of patients

Partial Thromboplastin Time (PTT)

• PTT is the best singular test for coagulation

  • screening disorders related to fibrin formation and monitoring heparin therapy.

• Reference Values:

  • PTT is typically 60-85 seconds

  • aPTT is typically 30-40 seconds.

Thrombin Clotting Time (TCT)

• Assesses the terminal steps of the coagulation pathway by evaluating fibrin-fibrinogen interaction.

• Thrombin induced clotting is very rapid

  • TCT is elevated/expedited when:

    • heparin is present interfering with thrombin action

      • when fibrin degradation products exist

        • fibrinogen levels < 100mg/dl

• Reference Value: Between 10-15 seconds.

Antifactor Xa Assay

• Utilized for monitoring anticoagulant therapies, especially unfractionated heparin (UFH) and low molecular weight heparins (LMWH).

• Used in specific situations of factor deficiencies or lupus anticoagulants.

• Reference values (CHECK PPT)

Other Clotting Tests and Their Applications

• Bleeding Time: Evaluates platelet disorders

  • Ivy method - forearm with cuff @ 40mm/Hg <5 min

  • Duke method - ear lobe <3 min

• Lee-White Clotting Time:

  • older method assessing clot formation time

  • ranging between 4-8 minutes as normal

D-Dimer Assay

• Blood test checking for clotting problems, specifically measuring D-dimer, a breakdown product of cross-linked fibrin clots.

  • detected with ELISA assay

• Positive results indicate potential clotting issues such as deep venous thrombosis (Homan’s sign positive, red, swelling and pain), pulmonary embolism, and disseminated intravascular coagulation.

  • increased amounts of D dimer

Complete Blood Count (CBC)

• Key data includes red and white blood cell counts, crucial for assessing cell morphology.

• EDTA purple/ lavender top tubes are commonly used as EDTA prevents platelet aggregation.

Red Blood Cell (RBC) Metrics

Red Blood Cell Count

• Expressed in cells per unit volume, normally biconcave, ranging 6-8 µm in diameter.

• Life Span: Approximately 120 days, with reference values as follows:

  • Males: 4.6 - 6.2 x 10^6/ml

  • Females: 4.2 - 5.4 x 10^6/ml

Reticulocytes

• Immature RBCs circulating for around 24 hours. Increases in reticulocyte counts are common during anemic states when bone marrow response is increased to make more RBCs than normal (hemolytic anemia).

  • may be referred to polychromatophilia

• Reference Values: About 1% (range 0.5 to 1.8%).

Hemoglobin

• Acts as the primary component for oxygen and carbon dioxide transport; concentration determined by specific gravity.

  • depth of staining may be a guide

• Reference Values:

  • Males: 13.5 - 18 g/dl

  • Females: 12 - 16 g/dl

Hematocrit

• Measure of packed red cell volume expressed as a percentage or decimal fraction.

• Reference Values:

  • Males: 40 - 54%

  • Females: 38 - 47%

Red Blood Cell Indices

• Key calculations include Mean Corpuscular Volume (MCV), Mean Cell Hemoglobin (MCH), and Mean Cell Hemoglobin Concentration (MCHC) which classify and characterize anemias.

Mean Corpuscular Volume (MCV)

• Reflects the average volume of a red blood cell.

• MCV = Hct x 10 /RBC

• Reference Values:

  • Males: 80 - 98 fl

  • Females: 81 - 99 fl

Mean Cell Hemoglobin (MCH)

• Denotes the average weight of hemoglobin per RBC.

• Reference Values:

  • MCH = Hb/RBC

    • Males: 26 - 32 pg

    • Females: 26 - 32 pg

Mean Cell Hemoglobin Concentration (MCHC)

• The ratio of hemoglobin amount to hematocrit level.

  • MCHC = Hb/Hct

• Reference Values: 32 - 36%.

Red Blood Cell Distribution Width (RDW)

• Used to estimate anisocytosis, which is the variation in red blood cell size.

• First indicator changes in conditions like:

  • iron-deficiency anemia or related to chronic blood loss.

• Reference Values: 11.6 - 14.6% with average being 13.1%.

Morphological Examination of Erythrocytes

Color

• reflection of hemoglobin concentration

• Normochromic: Proper concentration of hemoglobin.

  • center of biconcave disc is 1/3 diameter of RBC

• Hypochromic: Central area is enlarged and paler, as seen in MCH and MCHC-related anemia.

  • ex. iron deficient anemia

• Hyperchromic: Smaller, more intensely stained central area

  • indicates megaloblastic anemia.

• Anisochromia

  • variation in the color of RBCs, unequal hemoglobin content

    • ex. dimporphic anemia

• Polychromatophilia

  • RBCs stained with different dyes and their appearances

  • If more staining than normal:

    • indicates prescence of residual RNA within RBC

    • cells are larger or lack central pallor

    • represents a reticulocyte or immature RBC

    • may be referred to as a shift cell

Size

• Microcytes: Abnormally small RBCs.

  • ex. MCV < 80

• Macrocytes: Abnormally large RBCs.

  • ex. MCV > 100

• Anisocytosis: Variation in size, characteristic in most anemia.

Shape

• Poikilocytosis: Variation in shape.

  • ex. tear drop, helmet shaped, oval, pear

• Elliptocytosis: RBCs are elliptical in shape

  • ex. iron deficient anemia, myelofibrosis, megaloblastic anemia, and sickle cell anemia

• Spherocytes:

  • hereditary, hemolytic disease

    • HALLMARK SIGN: splenic condition

• Target cells: Excess membrane relative to volume

  • observed in liver disease and related hypochromia.

• Shistocytes:

  • RBC fragments, megaloblastic anemia, microangiopathic hemolytic anemia

• Acanthocytes:

  • coarse, irregularly spaced, variably sized crenation, resembling many pointed stars

  • IRREGULARLY SPICULATED

• Echinocytes: REGULARLY SPICULATED

Anemia Classifications

Normocytic Anemia

• Due to increased blood loss or decreased RBC production

  • identified by reticulocyte counts

    • elevated with increased blood loss

  • anemia of chronic disease

• MCV 80-100

Increased Red Cell Loss

• Causes include

  • Acute blood loss (e.g., trauma)

    • ex. third spacing (retroperitoneal pooling)

  • Hemolytic disorders

    • e.g., hereditary spherocytosis or elliptocytosis

    • sickle cell anemia

    • G6PD deficiencies

      • moth ball cells

      • burr cells

      • vulnerable to oxidation

Decreased Red Cell Production

• Reticulocyte count is not elevated

• Primary marrow disease

• Resulting from conditions such as hypoproliferative states due to virus

  • e.g., Human parvovirus B19 (FIFTH DISEASE)

    • decreased erythropoietin production, hypothyroidism, liver disease

Specific Types of Anemia

Anemia of Chronic Disease

• Related to chronic inflammation; often normocytic, normochromic.

  • defective RBC production

  • faulty incorporation of iron

• Hemoglobin levels may range from 9-11 g/dl.

  • MCHC ~32 (may border on hypochromic)

Macrocytic Anemia

• Results from DNA synthesis disorders in erythrocyte precursors due to folate or B12 deficiencies.

  • Folate is absorbed in upper intestines and lasts a few months

    • deficiency due to drug therapy:

      • ex. methotrexate, phenytoin, bactrim

  • B12 is absorbed in the ILEUM and binds with INTRINSIC FACTOR

    • deficiency = PERNICIOUS ANEMIA

    • MEGALOBLASTIC ANEMIA

      • Typical signs include neurological, psychological, and cardiac symptoms.

        • test using SCHILLING TEST, an ANTIBODY ASSAY for:

          • ABs against parietal/IF

            • due to gastric mucosal atrophy, autoimmunue reaction to gastric parietal cells or intrinsic factor

Microcytic Anemia

• Defined by Hb levels < 7.5 g/dl; associated with iron-deficiency, thalassemias, and sideroblastic anemia

  • decrease synthesis/availability of IRON, PORPHYRIN, and GLOBIN

Iron Deficiency Anemia

• 80% iron is recycled

• erythropoiesis will not increase if transferrin saturation is maintained between 20-60%

• iron absorption occurs in the proximal small intestine

• transferrin has 2 iron binding sites

  • clearance of Fe+2 is 60-90 min

• Present with pallor, tachycardia, wide pulse pressure, vertigo and headache

• Lab findings show low serum iron and ferritin levels with elevated Total Iron Binding Capacity (TIBC).

  • Daily iron needs:

    • male: 1 mg/day

    • female: 1.4 mg/day

ENDS PAGE 52