1. Select patients with anemia and thrombosis to send for consultation.

2. Specify when to order Coombs test and determine its usefulness.

3. Utilize the classification categories for anemia.

4. Predict which patients are expected to clot and when to perform further testing for etiology of thrombosis.

5. Review the clotting cascade.

Office Evaluation of Anemia

Classification: on normal peripheral blood smear, blood cell (RBC) same size as lymphocyte nucleus; mean corpuscular volume (MCV) equivalent to peripheral smear numbers; hemoglobin (Hb) molecule composed of heme (4 iron moieties) and globin (2 alpha chains and 2 beta chains); globin is scaffolding inside molecule, supporting heme; normal Hb count— in men, >13.5 g/dL (higher than in women [due to no menses, and tes-tosterone receptor on erythroid precursors that increases count); in women, >12 g/dL; Hb <6 g/dL merits concern

Normal RBC production: production factory—bone marrow; foreman—erythropoietin (EPO; drives marrow to produce RBCs); raw materials—nutrients, eg, iron, vitamin B12 , folate; RBC lifespan 100 days; normal reticulocyte count 1%; 1% of RBCs reproduced daily

Screening: Third National Health and Nutrition Examination Survey (NHANES III) demonstrated 10% of patients >65 yr of age anemic (by strict definition, ie, Hb <14 g/dL); of this 10%, 3% have Hb levels <11 g/dL; American Academy of Family Physicians (AAFP) and American College of Physicians (ACP) do not recommend complete blood cell count (CBC) as part of routine health maintenance screening; CBC used to investigate symptoms (not a screening exam); however, bariatric surgery patients need annual screening (have 30% anemia rate, 7% with Hb <10 g/dL)

Anemia: not diagnosis but clinical sign in need of diagnosis

Kinetic classification: 1) not making enough RBCs (hypoproliferation); 2) destroying RBCs (hemolysis); 3) bleeding; clues—MCV elevated secondary to increased reticulocytes (MCV is average of RBC volumes or sizes present); RBC distribution width (RDW) elevated (>15%) secondary to increased number of RBCs; if MCV and RDW elevated, obtain reticulocyte count (1% normal, >3% elevated); reticulocyte count varies in response to disease state; low Hb and normal or low reticulocyte count (eg, Hb of 9 g/dL and reticulocyte count of 1%) may indicate hypoproliferation (anemia should raise reticulocyte count) or problem with marrow response; elevated reticulocyte count indicates RBCs being destroyed, or bleeding occurring, and that marrow has adequate response (eg, sickle cell anemia)

Low RBC production: more common; bone marrow failure—myelodysplastic syndrome (MDS; common starting at 60 yr of age, incidence increases thereafter); aplastic anemia, secondary to pharmaceuticals; erythropoietin (EPO) deficiency—literal (chronic kidney disease) or functional (anemia of chronic disease); missing materials—lack of folate, vitamin B12 , or iron

Hemolytic anemias: increased RBC destruction; helpful tests—lactate dehydrogenase (LDH) elevated; free serum levels of haptoglobin decreased; combination of elevated LDH and low haptoglobin 90% sensitive for hemolytic anemia; indirect bilirubin elevated (overwhelms ability of liver to conjugate it)

Intrinsic hemolytic anemia: hereditary; sickle cell anemia—first disease for which genetic basis discovered (Linus Pauling); single amino acid chain substitution in beta globin is qualitative abnormality; elevated RDW, elevated MCV, elevated reticulocyte count, elevated LDH, low haptoglobin, clear sickle cells on peripheral smear; hemoglobin S on hemoglobin electrophoresis; thalassemia—quantitative defect (decreased rate of synthesis of ≥1 Hb polypeptide chains, or absence of chain); more common in children, can present in asymptomatic adults

Extrinsic hemolytic anemia: acquired autoimmune hemolytic anemia (AIHA); Coombs test—patient has antibodies to own RBCs; activates splenic RBC destruction (hemolysis); Coombs reagent is antibody to antibody; when added to patient’s blood, cross-linking (agglutination) occurs if antibodies present (ie, RBCs clump)

Morphologic classification: 1) macrocytic (RBC larger than lymphocyte nucleus); 2) microcytic (RBC smaller than lymphocyte nucleus); 3) normocytic

Microcytosis: iron deficiency (primary worldwide cause of anemia), thalassemia, and anemia of chronic disease; microcytic RBC has central pallor; iron screening panel must include ferritin level (most important measure of iron storage), iron level, and transferrin saturation (iron divided by total iron binding capacity [TIBC])

Iron deficiency anemia: either bleeding or malabsorbing (eg, bariatric surgery, sprue [less common]); in cases of nonmenstruation and nonbariatric surgery—consider as gastrointestinal bleeding unless proven otherwise, gastroenterology consultation indicated; iron deficiency markers decrease before anemia present (ferritin often undetectable once Hb decreases); ask about pica for ice (common; seen in ≈50% of patients with iron deficiency); ask about restless legs syndrome (RLS; iron deficiency treatable cause of RLS)

Thalassemia: alpha (4 globin genes) —≥2 globin chain deletions present before tests abnormal; microcytic with MCV <80 fL and Hb ≈11 g/dL; typically asymptomatic; more severe type, alpha thalassemia major (more common in black and Asian patients; usually presents in childhood); on Hb electrophoresis, alpha thalassemia silent; common in adult medicine (more common than beta); beta (2 globin genes)—variable penetrance for missing second gene; heterogeneous presentation; classically, Mediterranean ethnicity; elevated Hb A2 and Hb F on electrophoresis; classic thalassemia findings—normal automated RBC count, low Hb level (measure of surface area of RBC sphere), and low MCV (eg, Hb 9 g/dL, MCV 65 fL, RBC 4 million/mm3 ); check iron panel

Anemia of chronic disease: usually normocytic (but can be microcytic); if ferritin (acute phase reactant) level >100 ng/ mL, can exclude iron deficiency; case presentation—38-yr-old man presents with fatigue, cough, and 20-lb weight loss; borderline microcytic, elevated platelet count (either as acute phase reactant or from iron deficiency), and normal RDW; chest x-ray showed nonmalignant fibrothorax; underwent thoracotomy with decortication; at 1-yr recovery, anemia resolved; low iron, low TIBC, and elevated ferritin classic for anemia of chronic disease

Macrocytic anemia: associated with vitamin (B12 and folate) deficiency, pharmaceutical agents (those affecting DNA synthesis), and MDS (especially in elderly); vitamin deficiency—folate and vitamin B12 (recycles folate); thymidylate synthesis rate-limiting in DNA synthesis (folate derivatives essential cofactors); vitamin B12 necessary to maintain nerve function (eg, neurotransmitters, membrane phospholipids) via S-adenosylmethionine; folate deficiency (no DNA synthesis in nucleus, RNA in cytoplasm adequate); folate effective for blood issues, vitamin B12 effective for neurologic problems; macro-ovalocytes on peripheral smear classic finding for vitamin B12 and folate deficiencies; megaloblastic anemia (not equal to macrocytosis) describes bone marrow finding of mature cytoplasm and immature nucleus; folate not stored in body (alcoholics can become folate-deficient in ≤2 wk); B12 stored for 3 yr in liver (eg, anorexic individual can have sustained malnutrition before B12 deficiency manifests); drugs affecting DNA synthesis—chemotherapeutic agents; hydroxyurea; zidovudine (AZT); dilantin; MDS—median age at diagnosis, mid 60s; usually macrocytic anemia; diagnosis requires bone marrow sample; typically asymptomatic, can progress to leukemia; prognosis extremely variable; macrocytosis without anemia also associated with alcoholism or liver disease

Normocytic anemia: difficult diagnosis; examples include anemia of chronic disease (usually normocytic), chronic kidney disease (CKD), and EPO deficiency (both normocytic by definition); speaker utilizes creatinine clearance calculation (eg, Modification of Diet in Renal Disease [MDRD], Cockcroft-Gault) for clarification; eg, if patient elderly and serum creatinine >1.5 mg/dL, creatinine clearance calculation may reveal low creatinine clearance (eg, <35 mL/min), which is sufficient for anemia

Testing: CBC—provides RDW, MCV, and white blood cell and platelet counts; use CBC to direct further testing; comprehensive metabolic profile (CMP)—allows evaluation of liver function, renal function, calcium, and total protein; additional tests (based on type of anemia)—if microcytic (iron studies, C-reactive protein [CRP], and erythrocyte sedimentation rate [ESR]); if macrocytic (vitamin B12 , folate, LDH, and haptoglobin [Coombs test if LDH and haptoglobin suggest hemolytic anemia]); if normocytic (serum protein electrophoresis, EPO [debatable]); all types of anemia— check reticulocyte count and peripheral smear

When to consider consultation: abnormal peripheral smear; any unexplained anemia; hemolytic anemia; and transfusion-dependent anemia

Thrombophilia: Is the Patient a Clotter?

Case presentations: patient 1—female college student; athlete; 22 yr of age; taking oral contraceptives (OCs) for 1 yr; presents with dyspnea and chest pain; no family history of clots, and no comorbidities; computed tomography (CT) shows loss of continuity between right main branch and main trunk of pulmonary artery (pulmonary embolus [PE]), large left pleural effusion, and acute right heart failure; patient anticoagulated with unfractionated heparin in intensive care unit; patient extremely labile (difficult to anticoagulate); ICU team questioned whether patient antithrombin-deficient; speaker’s reaction (based on rarity of scenario) that “by definition, she is a clotter”; whether there is a defined thrombophilic state undetermined; patient 2—man; 49 yr of age; factor V Leiden (FVL) homozygote; first clot when patient 40 yr old; on lifetime enoxaparin (Lovenox) after warfarin (Coumadin) failure; presents with bruising; patient 3—man; 45 yr of age; presents with headaches; family history positive for clotting disorder (actually, brother of patient 2); magnetic resonance imaging (MRI) of brain shows small-vessel ischemic changes; FVL does not explain arterial thromboses; patient has never had clot; however, due to positive family history, he has 75% chance of having defined thrombophilic state

Candidates for clotting: those >45 yr of age; pregnant patients and those in post-partum period; surgical and trauma patients (due to inflammation and immobility); patients on OCs with another cofactor; malignancy (active cancer)

Risk for clot: FVL most common inherited thrombophilia; relative risk (RR) of heterozygote FVL woman taking OCs 30 times that of general population (at 20 yr of age, patient’s absolute risk 10/100,000 woman-years; RR, 300/100,000 woman-years; individual risk 0.3%/yr); patients on hormone replacement therapy (HRT; lower estrogen than OCs) have 10 times RR (absolute risk for 50-yr-old woman, 1200/100,000 woman-years; individual risk 1.2%/yr)

Risk for recurrence: previous deep venous thrombosis (DVT); previous PE (considered sentinel event); most recurrences early (10% of patients with DVT or PE reclot within 6 mo); 30% of patients with PE have recurrent PE within 10 yr; studies show resumption of increased recurrence risk when anticoagulation stopped

Who to test: patients who clot without provocation; young patients on OCs (especially with PE); individuals with second clots; those with clots in unusual places (eg, Budd-Chiari syndrome, renal or dural vein thrombosis, mesenteric veins [such patients may have treatable disease]); patients with family history of primary relative with clot

What to test: clotting cascade—tissue factor (in endothelium of blood vessel) converts factor VII to VIIa; VIIa reacts with X, and changes to Xa; Xa with factor Va converts prothrombin (also known as factor II) to thrombin; generation of thrombin key to thrombosis; thrombin both powerful coagulant and anticoagulant; procoagulation—thrombin changes fibrinogen to fibrin (cross-linked fibrin clot); thrombin activates platelets, factor XIII, factor V, and factor VIII; anticoagulation—antithrombin (formerly, antithrombin III) binds to thrombin to turn it off (slow process); heparin— any type (eg, unfractionated, low molecular weight) acts as catalyst and speeds conversion; patient with deficiency of antithrombin heparin-resistant (not able to bind and remove thrombin; possible scenario for patient 1); protein C and its cofactor protein S—natural anticoagulants; become activated protein C (APC), which inhibits factor Va; factor Va critical step in thrombosis (Va with Xa converts prothrombin to thrombin); FVL patients resistant to normal APC and unable to turn off anticoagulation; prothrombin 20210 (procoagulant) gene mutation leads to higher levels of prothrombin, which increases thrombin; incidence of risk factors in thromboembolic disease (in unscreened population)—antithrombin deficiency, 2%; protein C, 2%; protein S, 2%; FVL, ≤20%; prothrombin 20210, ≈5%; testing during acute thrombosis— natural anticoagulants decrease (used up by clot); antithrombin, protein C, and protein S decrease (caution, these decreases expected; do not label patient as “in middle of big clotting storm”); testing patients on coumadin—vitamin K-dependent anticoagulant factors (factors II, VII, IX, and X, and proteins C and S) decrease; not affected by clot status—FVL and prothrombin (genetic mutations) tested with polymerase chain reaction (PCR) primers

Treatment: acute treatment of DVT and PE identical; etiology of clot matters only with secondary prophylaxis; PE requires prophylaxis for ≥6 mo; speaker’s preference—in PE with right heart failure, treat for 1 yr to avoid, eg, pulmonary hypertension (treat one to prevent other)

Case presentations (continued): patient 4—woman; 75 yr of age; active; FVL heterozygote; past history significant for 3 pregnancies and surgeries; no previous clotting issues; presents with recent history of DVT after extended bus/airplane trip; had been on warfarin (Coumadin) for 6 mo; patient advised to discontinue anticoagulation, with strict precaution to get up every hour when travelling; if PE present, consider longer prophylaxis

D-dimer study: premise—whenever anticoagulation stopped, risk for clots increases; D-dimer product of clot breakdown; positive if patient repeatedly clotting and lysing; study design—enrolled patients with first unprovoked thromboembolic event, who received ≥3 mo of warfarin therapy; D-dimer assessed 1 mo after stopping warfarin; patients positive for D-dimer randomized to more warfarin or nothing; patients negative for D-dimer advised to stay off warfarin; results—of 385 normal D-dimer patients, not anticoagulated, 24 had recurrent clot; of 120 abnormal D-dimer patients, not anticoagulated, 18 (10%) had recurrences; of 103 abnormal D-dimer patients, on anticoagulation, 3 had recurrences; conclusion—stopping anticoagulation has significant incidence of recurrent VTE, reduced by resuming treatment

Long-term anticoagulation: consider stopping anticoagulation for noncompliant patients, patients at high risk for falls, and patients with gastritis; however, every major trial on thrombosis shows that stopping anticoagulation resumes clot risk; treating past 6 mo becomes secondary prophylaxis to prevent next clot (treatment for first 6 mo evidence-based for thrombophilia)

How to test: hypercoagulable screen (antithrombin III, protein C, and protein S); PCR testing for FVL (increased incidence for those of northern European descent) and prothrombin; consultation to discuss risks and benefits of anticoagulation

Consultation referrals: unprovoked thrombus; recurrent thrombus; unusually located thrombus