CBC, Iron Panel, and Coagulation Notes

Objectives

• Provide an overview of CBC, iron panel, and coagulation laboratory tests with typical normal ranges.
• Describe common pathological conditions related to hemostasis/fibrinolysis and anemia.
• Interpret laboratory values within the context of patient care to provide appropriate recommendations on medication management.

CBC and Iron Panel

• CBC stands for Complete Blood Count, also known as Hemogram
• Measures peripheral blood components:
  • Plasma
  • Red Blood Cells (RBC)
  • White Blood Cells (WBC)
  • Platelets
• Hematopoiesis: process where bone marrow produces blood components

Complete Blood Cell Lab Panel

• Reference Ranges:
  • WBC: 4.0 – 10.5 Thousand/MCL
  • RBC: 4.38 – 5.62 Million/MCL
  • Hgb: 13.5 – 16.9 G/DL
  • Hct: 39.5 – 50 %
  • MCV: 81.5 – 97.0 FL
  • MCH: 27.0 – 33.5 PG
  • MCHC: 32.0 – 35.5 G/DL
  • RDW-CV: 11.6 – 14.4 %
  • PLT: 150 – 400 Thousand/MCL

CBC w/ Differential

• CBCd further differentiates granulocytes.

Descriptive Terminology

• Increased RBC = Polycythemia
• Decreased RBC/Hgb = Anemia
• Increased WBC = Leukocytosis
• Decreased WBC = Leukopenia
• Increased Platelets = Thrombocytosis
• Decreased Platelets = Thrombocytopenia
• Granulocytes = Neutrophils, Basophils, Eosinophils
  • Decrease in granulocytes = Agranulocytosis
  • Increase in granulocytes = Granulocytosis
• Decreased Neutrophils = Neutropenia
• Decrease across cell lineages = Pancytopenia or Cytopenic

Red Blood Cells

• Carries $O<em>2/CO</em>2$ to and from organs
• Reported as # of cells, ~40-45% of blood
• Production stimulated by erythropoietin
• 7 days to maturation in bone marrow, Life span ~120 days
• Mature RBCs lack nuclei

Hematocrit

• Volume of RBCs in a unit volume of whole blood
• $(Hct)(\%)=(RBC / Whole blood volume) \cdot 100$

Hemoglobin

• Iron-rich protein on RBC that carries oxygen from lungs to body and returns $CO_2$ to be exhaled
• Reported as amount of Hb per volume of whole blood
• Higher in men due to increased stimulation from androgen steroids
• Can be lower in females due to blood loss during menstruation

Hemoglobin (cont.)

• Anemia - Not enough RBC/Hgb or dysfunction
  • Hgb < 13.5 gm/dL in men
  • Hgb < 12 gm/dL in women
• Symptoms: SOB, weakness, dizziness, headache, cold hands/feet, pale skin, chest pain, pounding/whooshing in ears. May be asymptomatic
• Examples of common causes: Acute/chronic blood loss, iron deficiency, B12/folate deficiency, kidney dysfunction, hemolytic anemia, sickle cell anemia, anemia of chronic disease

Mean Cell Volume

• MCV = Average size RBCs expressed as femtoliters ($10^{-15}$) or cubic microns ($\mu m^3$
• May help in diagnosing cause of anemia
  • Macrocytic anemia = higher MCV or larger RBC (i.e. Folic acid, B12 deficiency)
  • Microcytic anemia = lower MCV or smaller RBC (i.e. Iron deficiency, thalassemia)
  • Can be falsely elevated with hyperglycemia and agglutination

Other RBC Indices

• MCH (Mean Cell Hemoglobin)
  • Average quantity of Hb in a RBC
  • Can reflect iron supply
• MCHC (Mean Cell Hemoglobin Concentration)
  • Concentration of Hb per volume of cells
  • Independent of cell size

Red Blood Cell Distribution Width

• RDW-CV = The distribution of sizes of RBCs
• Reported as a percentage
• Higher = the more variable sizes of RBCs
• Examples:
  • Normal MCV but patient has a high RDW-CV
  • Patient has a wide range of macro and microcytic RBCs; however seems normal on automated MCV

RBC Related Labs

• Reticulocyte Count
  • Indirect measurement of new RBC productions
  • Reticulocytes circulate in blood ~2 days before maturing
  • Normally ~1% (rate of production/release); > 2.5% may indicate hemolysis
• Peripheral Smear
  • Additional information on functional status of bone marrow/RBC production
  • Provides details on cell morphologies
  • Typically reserved for diagnosing hematologic disorders
• Coombs Test
  • Differentiates between hemolytic anemia or immune response
    • Direct Coombs = tests antibodies bound to erythrocytes
    • Indirect Coombs = measures antibodies in serum
  • Positive Direct Coombs indicated antibody-mediated hemolysis

Iron Panel

• Recommend drawing before iron infusions and blood transfusion (> 4 days) for accurate baseline levels
• Reference Range Units:
  • Transferrin 203 – 362 mg/dL
  • Iron 49 – 181 mcg/dL
  • TIBC 284 – 507 mcg/dL
  • % Saturation 20 – 55 %
  • Ferritin 23 – 233 ng/mL

Iron Panel Components

• Transferrin: protein in plasma that transports iron
• Iron: concentration of iron bound to transferrin (Can be decreased during infection)
• Total Iron Binding Capacity (TIBC)
  • Each transferrin molecule can carry two iron atoms
  • All sites are filled and iron concentration measured
• % Transferrin Saturation (%Sat) = $(\frac{serum\ iron}{TIBC}) \cdot 100$
  • Percent of transferrin sites with iron ready for erythropoiesis
• Ferritin: protein that stores/releases iron in the body (Best indicator of iron deficiency or overload. Acute phase reactant / inflammatory marker)

Iron Deficiency Anemia

• Caused by bleeding or inadequate iron uptake
• Typical labs: ↓ Hg, Hct, RBC, may also have ↓MCV, +/- Symptoms, ↓ Serum Iron, ↓ Ferritin, High ↑ TIBC, TSat may be low
• Treatment options: IV/PO iron supplement, RBC transfusion
• Iron panel can be used to monitor for efficacy and/or iron overload

Drugs associated with Hemolytic Anemia

• Acquired: Beta-lactamase, cephalosporins, Isoniazid, Levodopa, Methyldopa, Penicillins, Chemotherapy, Quinidine, Ribavirin, Rifampin
• High risk in G6PD Deficiency: Chloroquine, Dapsone, Methylene blue, Nitrofurantoin, Primaquine, Probenacid, Rasburicase, Sulfonamides

White Blood Cells (aka leukocytes)

• WBC = Granulocytes, Monocytes, & Lymphocytes (Cells that protect body from infection)
  • High WBC can suggest infection/inflammation vs. neoplastic vs medication
  • Low WBC can suggest failure/disruption of hematopoiesis in marrow vs immune destruction vs sequestration
• Medications that can affect WBC
  • Increase: steroids, granulocyte colony stimulating factors, epinephrine
  • Decrease: clozapine, myelosuppressive chemotherapy, carbamazepine

Granulocyte - Neutrophils

• Segmented Neutrophils
  • ~55-70% of total WBC
  • “Immediate Response” cells  Increases with bacterial infections
  • Other common causes include neoplastic disorders, ischemia, autoimmune, and medications
  • Lifetime < 1 day
• Neutrophil Bands
  • Immature neutrophils
  • “Left Shift”

Absolute Neutrophil Count (ANC)

• Classifications:
  • Mild Neutropenia = ANC < 1500 cells/microL or < 1.5 K/microL
  • Moderate Neutropenia = ANC < 1000 cells/microL or < 1.0 K/microL
  • Severe Neutropenia = ANC < 500 cells/microL or < 0.5 K/microL
• $ANC = WBC \frac{cells}{\mu L} \cdot \frac{segs + bands}{100}$
• Sample Calculation:
  • The WBC count is 5.2 thousands/microL and reported segmented neutrophils is 48.3% and bands are 4%. What is the ANC?
  • $ANC = 5.2 \frac{K}{\mu L} \cdot \frac{48.3\% + 4\%}{100} = 2.7 \frac{K}{\mu L} = 2700 \frac{cells}{\mu L}$

Granulocytes – Eosinophils & Basophils

• Eosinophils
  • ~0-5% of WBC
  • Increases with drug allergy, asthma, inflammation, parasite infections, and some dermatologic disorders
• Basophils
  • ~0-1% of WBC
  • Increases with inflammation or hypersensitivity reactions

Lymphocytes

• ~20-40% for WBC
• Increases with viral infections and lymphomas
• Decreases with immune suppressive therapies/states (i.e. myelosuppressive chemotherapy, HIV, or systemic steroids)
• Two Types:
  • T lymphocytes: directly attacks infected cells/tumors and regulates immune cell function
  • B lymphocytes: creates antibodies against foreign material

Platelets (aka thrombocytes)

• Small fragment of cells involved in primary hemostasis during coagulation. Life span ~7-10 days
• Primary hemostasis
  • First response to blood vessel injury
  • Platelet aggregation to create initial fibrin clot
• High number of platelets = theoretically higher clotting risk (i.e. embolic strokes, DVT/PE)
  • Examples: inflammation, hematologic disorder
• Low number of platelets = Increased bleeding risk
  • Typically keep > 30-50k especially if other bleeding risk factors
  • Spontaneous bleeding can occur with platelets < 20k
  • Examples: Medication, infection, neoplasm, autoimmune, DIC

Coagulation Labs

Coagulation Tests

• Extensive tests available measuring hemostasis, fibrinolysis, and thrombosis
• Performed on plasma portion of blood
• Useful in diagnosis, monitoring, and guiding treatment decisions in setting of coagulopathies

Background: Hemostasis & Fibrinolysis

• Hemostasis = Stopping or Preventing Bleeding
  1. Primary hemostasis:
     • platelet aggregation to create initial platelet plug
  2. Secondary Hemostasis or Coagulation Cascade:
     • Enzymatic cascade
     • Goal is to create fibrin that will strengthen initial clot framework
• Fibrinolysis = Clot degradation

Background: Coagulation Cascade

• Two Pathways:
  1. Intrinsic
     • Contact system initiated
  2. Extrinsic
     • Activated by tissue factor (Factor III) that presents when blood vessel injured
• Pathways converge to the common pathway with factor X activation to Xa

Common Anticoagulation and Fibrinolytics

• Direct FaXa Inhibitors: RivaroXAban, ApiXAban, EdoXAban, BetriXAban
• Indirect FXa Inhibitor: Fondaprinux
• Direct Thrombin Inhibitors: Argatroban, Bivalirudin, Dabigatran
• Unfractionated Heparin (UFH) (more thrombin)
• Low molecular weight heparin (LMWH) i.e. enoxaparin, dalteparin (more FXa)
• Warfarin affects Factor II, VII, IX, X. Inhibits Protein C and S initially (procoagulable state)
• TPA agents: Alteplase, Tenecteplase, Reteplase

Background: Thrombosis

• Thrombosis = Pathologic formation of a clot
• Virchow’s Triad

Common Coagulation Tests

• PT
• INR
• aPTT
• Anti-Xa
• Thrombin time
• Fibrinogen
• D-dimers
• Example Reference Range Units:
  • PT: 12.0 – 14.2 Seconds
  • INR: < 1.2 -
  • aPTT: 24.2 – 36.7 Seconds
  • Thrombin time: 12-19 Seconds
  • D-Dimers: < 500 ng/mL
  • Fibrinogen: 155-439 mg/dL
  • Anti-Xa: 0.3 – 0.7 IU/mL

Prothrombin Time

• PT = Measures time required for clot formation during extrinsic and common coagulation
• Potential causes of prolonged PT:
  • Warfarin, UFH, LMWH, DOACs
  • Vitamin K deficiency
  • Factor deficiencies
  • Liver dysfunction
  • Fibrinogenemia
  • DIC

International Normalized Ratio (INR)

• INR corrects for differences in thromboplastin reagents used in PT
• Normal range < 1.2
• Higher = Decreased clotting or “thinner” blood (Highr bleeding risk)
• Primarily used for Warfarin management
  • Goal INR 2-3 for history Afib/DVT/PE
  • Goal INR 2.5-3.5 for history of mitral valve replacement
• Used as surrogate measure of synthetic liver function (Ex. MELD score, Child-Pugh)
• Causes of PT will also prolong INR

Vitamin K Antagonist Warfarin Mechanism Overview

• Vitamin K: Cofactor involved in amino acid γ- carboxyglutamate formation that is required for synthesis of blood clotting proteins (Factors II, VII, IX, X and Protein C/S)
• Warfarin: Inhibits vitamin K epoxide reductase complex 1 (VKORC1) leading to decreased synthesis of clotting factors
• INR increases with prolonged time to clot

Examples of Medications that Affect INR

• Medications that increase INR
  • Antibiotics: azithromycin, metronidazole, bactrim, fluoroquinolones, macrolides
  • Antifungals: azoles
  • Chemotherapies: imatinib, fluorouracil (5-FU)
  • Amiodarone
  • Allopurinol
  • SSRIs (i.e. fluoxetine, sertraline)
• Medications that decrease INR
  • Antibiotics: Dicloxacillin, nafcillin, oxacillin, rifampin
  • Antiepileptics (Carbamazepine, phenobarbital, phenytoin)
  • Saint John’s Wort
  • Vitamin K (including diet)
• False elevations can occur with daptomycin, oritavancin, telavancin (Drug-lab interaction)

Activated Partial Thromboplastin Time (aPTT)

• Measures time to clot formation during intrinsic and common clotting pathway
• Not well standardized hence range will differ based on reagent lot and instruments used
• Poor specificity/sensitivity – isolated prolonged aPTT common (Work-up further based on clinical picture)
• Possible causes for prolongation
  • Factor deficiency
  • Heparin therapy
  • Liver disease
  • Fibrinogenemia
  • False elevations with oritavancin, telavancin

Heparin Mechanism

• Antithrombin: most important thrombin (FIIa) inhibitor
• Heparin products bind to antithrombin (AT)  promotes AT binding to thrombin (FIIa) and FXa  Inhibits coagulation

Heparin/DTI and aPTT Monitoring

• aPTT can be used to monitor unfractionated heparin (UFH) and direct thrombin inhibitors (e.g. argatroban, bivalrudin) that has unpredictable PK/PD
• Target typically 1.5 to 2.5x increase in aPTT from patient’s baseline
• Heparin resistance
  • Can occur in patients with high levels of FVIII or low AT levels
  • Recommended to use anti-Xa instead

Anti-factor Xa Activity Assay (Anti-Xa)

• Preferred method of monitoring UFH at most institutions (Typical goal range 0.3 to 0.7 U/mL)
• Heparin binds to AT in assay and neutralizes FXa present in reagent
  • Higher heparin in plasma  Lower free FXa  Increased anticoagulation effect/Decreased clotting risk
• Can also be used for low molecular weight heparin (i.e. enoxaparin, fondaparinux) when variable PK expected
  • Ex. Pregnancy, obesity, low body weight, pediatrics, renal dysfunction
  • BID dosing: 0.6 – 1 unit/mL (kU/L) for enoxaparin
  • QD dosing: > 1 unit/mL (kU/L) for enoxaparin

Thrombin Time (TT)

• Measures fibrinogen formation and/or thrombin inhibition
• Typical range 14.7-19.5 seconds
• Not commonly ordered
• Common causes:
  • Heparin
  • Hyperfibrinogenemia
  • Dysfibrinogenemia
  • Direct thrombin inhibitors

D-dimer & Fibrinogen

• D-dimer
  • Created when cross-linked fibrin clot is cleaved by plasmin
  • Increased D-dimer = clot lysis in circulation
  • Useful in diagnosis of disseminated intravascular coagulation (DIC)
• Fibrinogen assay
  • Protein involved in forming clots
  • Recommended in bleeding, cirrhosis, DIC patients
  • Guides in treatment recommendation (i.e. transfusion)

Disseminated Intravascular Coagulation (DIC)

• Phenomenon where activation of coagulation and fibrinolysis causes production of thrombin and plasmin as well as consumption of coagulation factors and inhibitors
• Typical lab findings: Prolonged PT/aPTT, decreased fibrinogen, elevated D-dimers, and thrombocytopenia
• Clinical scenarios warranting DIC monitoring: sepsis, malignancy, obstetric complications, massive trauma/injury, acute hemorrhage
• Can result in both hyperfibrinolytic state (increased bleeding) (more common) or prothrombotic state (normal PT, normal/shortened aPTT, & normal/elevated fibrinogen)

Other coagulation labs

• Thromboelastography (TEG) & Rotational thromboelastography (ROTEM): Measure clotting or reaction time. Nearly point of care although more common CC/OR settings to differentiate coagulopathy vs bleeding. Helps in guiding transfusions giving peri-surgery
• Individual factor levels can be measured
  • Examples: Factor VIII Deficiency Hemophilia A, Factor IX Deficiency  Hemophilia B, VWF  Von Willebrand Disease
  • Treatment with factor replacement therapies

Trust but Verify

• Interferences from IV solutions (i.e. heparin locks, gtts)
• Hemolysis
  • Can falsely decrease RBCs, HCT, aPTT
• Hemodilution or Hemoconcentration
• Conditions that affect blood cells
• Automated CBCd errors
• Human error