Clinicians Pocket Reference, 11th Edition

Blood Collection

Venipuncture is discussed in detail in Chapter 13, Venipuncture. The best CBC sample is venous blood drawn with a 22-gauge or larger needle. For a routine CBC, venous blood must be placed in a special hematology lab tube, usually a purple top tube, containing an anticoagulant (EDTA) with which the blood must be mixed gently. Blood for a CBC should be fresh, < 3 h old. Most samples for coagulation studies are submitted in a blue top (citrate) tube. (See Table 13 8 for detailed description of blood collection tubes.) If a capillary fingerstick or heelstick (see Heelstick and Fingerstick) is used, the hematocrit may be falsely low. If the finger has to be "milked," sludging of the RBCs can create a falsely high hematocrit. Wright staining can also be done and viewed as outlined in the next section.

Blood Smears: Wright Stain

Most clinical labs perform automated cell counts. The formal blood smear and Wright stain can provide a manual differential leukocyte count for the evaluation of anemia and other conditions. The slide is usually available for review by students and house staff. The main benefit is to allow identification of abnormal cells and other subtleties that may not be detected with automated systems (Figure 5 1).

Viewing the Film: The Differential WBC

1. Examine the smear in an area where the red cells approximate one another but do not overlap.

2. If the film is too thin or if a rough-edged spreader is used, as many as 50% of the WBCs may accumulate in the edges and tail (see Figure 5 1).

3. WBCs are not randomly dispersed even in a well-made smear. Polys and monos predominate at the margins and tail, and lymphs are prevalent in the middle of the film. To overcome this problem, use the "high dry" or oil immersion objective and count cells in a strip running the entire length of the film. Avoid the lateral edges of the film.

4. If fewer than 200 cells are counted in a strip, count another strip until at least 200 are seen. The special white cell counter found in most labs is ideal for this purpose. In patients receiving chemotherapy, the total count may be so small that only a 25 50 cell differential is possible.

5. In smears of blood from patients with very high white counts, such as those with leukemia, count the cells in any well-spread area where the different cell types are easy to identify. Table 5 1 shows the correlation between the number of cells in a smear and the estimated white cell count. Estimate the platelet count by averaging the number of platelets seen in 10 hpf (under oil immersion) and multiplying by 20,000.

Normal CBC Values

A CBC panel generally includes WBC count, RBC count, Hgb, HCT, MCH, MCHC, MCV, RDW, and usually platelets. The differential is usually ordered separately. Normal CBC, differential, and platelet values are outlined in Tables 5 2 and 5 3.

Normal CBC Variations

Hbg and HCT are highest at birth (20 g/100 mL and 60%, respectively). The values fall steeply to a minimum at 3 mo (9.5 g/100 mL and 32%). Then they slowly rise to near adult levels at puberty; thereafter both values are higher in men. A normal decrease occurs in pregnancy. The number of WBCs is highest at birth (mean of 25,000/mm³) and slowly falls to adult levels by puberty. Lymphs predominate (as much as 60% from the second week of life until age 5 7 y, when polys begin to predominate).

Hematocrit

Because plasma and red cells are lost in equal amounts in acute bleeding, the HCT does not immediately reflect the loss, sometimes not for 2 3 h. In anemia, the red cell indices and reticulocyte count should be checked.

The Left Shift

The degree of nuclear lobulation of PMNs indicates cell age. A predominance of immature cells with only one or two nuclear lobes separated by a thick chromatin band is called a "shift to the left." Conversely, a predominance of cells with four nuclear lobes is called a "shift to the right." (For historical information, left and right designations come from the formerly used manual lab counters, in which the keys for entering stabs were located on the left of the keyboard.) As a rule, 55 80% of PMNs have two to four lobes. More than 20 five-lobed cells/100 WBCs suggests megaloblastic anemia, a six- or seven-lobed poly being diagnostic.

"Bands" or "stabs," the more immature forms of PMNs (the more mature are called "segs"), are identified by the fact that the connections between ends or lobes of a nucleus are greater than one-half the width of the hypothetical round nucleus. In bands or stabs, the connection between the lobes of the nucleus is by a thick band; in segs, by a thin filament. A band is defined as a connecting strip wide enough to reveal two distinct margins with nuclear material in between. A filament is so narrow that no intervening nuclear material is present.

For practical purposes, a left shift is present in the CBC when > 10 12% bands are seen or when the total PMN count (segs plus bands) is > 80.

Left Shift:

Bacterial infection, toxemia, hemorrhage, myeloproliferative disorders

Right Shift:

Liver disease, megaloblastic anemia, iron deficiency anemia, glucocorticoid use, stress reaction

Reticulocyte Count

Collection: Lavender top tube

The reticulocyte count is not a part of a routine CBC. The reticulocyte count is used in the initial work-up of anemia and in monitoring the effect of hematinic or erythropoietin therapy, monitoring recovery from myelosuppression, or monitoring engraftment after bone marrow transplantation. Reticulocytes are juvenile RBCs with remnants of cytoplasmic basophilic RNA. The presence of these cells is suggested by basophilia of the RBC cytoplasm on Wright stain (polychromasia); however, confirmation requires a special reticulocyte stain. The result is reported as a percentage. Use the following equation to calculate the corrected reticulocyte count for interpretation of the results

This corrected count is an excellent indicator of erythropoietic activity. The normal corrected reticulocyte count = < 1.5%.

Normal bone marrow responds to a decrease in erythrocytes (shown by a decreased HCT) with an increase in the production of reticulocytes. A low reticulocyte count with anemia suggests a chronic disease, a deficiency disease, marrow replacement, or marrow failure.

CBC Diagnostics

See Tables 5 2 and 5 3 for age- and sex-specific normal ranges. Beyond the total WBC count, identification of the specific white cell alteration may aid in the differential diagnosis.

White Cells (Leukocytes)

See Table 5 2.

Increased: Infection, inflammatory process (rheumatoid arthritis, allergy) leukemia, severe stress (physical and emotional), postoperative state (physiologic stress), severe tissue damage (eg, burns), steroids

Decreased: Bone marrow failure (aplastic anemia, infection, tumor, fibrosis, radiation damage), cytotoxic agent or medication (eg, chloramphenicol, linezolid, chemotherapeutic agents), collagen vascular disease such as lupus, liver or spleen disease, vitamin B₁₂ or folate deficiency

Basophils

0 1%

Increased: Chronic myeloid leukemia, aftermath of splenectomy, polycythemia, Hodgkin disease, and, rarely, recovery from infection or hypothyroidism

Decreased: Acute rheumatic fever, pregnancy, aftermath of radiation therapy, steroid therapy, thyrotoxicosis, stress

Eosinophils

1 3%

Increased: Allergy, parasites, skin disease, malignancy, drugs, asthma, Addison disease, collagen vascular disease (mnemonic NAACP: Neoplasm, Allergy/asthma, Addison disease, Collagen vascular disease, Parasites), and pulmonary disease, including L ffler syndrome and PIE

Decreased: Steroids, ACTH, aftermath of stress (infection, trauma, burns), Cushing syndrome

Lymphocytes ("Lymphs")

24 44% See also Lymphocyte Subsets

Increased: Viral infection (AIDS, measles, rubella, mumps, whooping cough, smallpox, chickenpox, influenza, hepatitis, infectious mononucleosis), acute infectious lymphocytosis in children, acute and chronic lymphocytic leukemia

Decreased: (Normal in 22% of population) Stress, burns, trauma, uremia, some viral infections, HIV and AIDS, bone marrow suppression after chemotherapy, steroids, MS

Atypical Lymphocytes

> 20%: Infectious mononucleosis, CMV infection, infectious hepatitis, toxoplasmosis, malignancy < 20%: Viral infections (mumps, rubeola, varicella), rickettsial infections, TB

Monocytes ("Monos")

3 7%

Increased: Bacterial infection (TB, SBE, brucellosis, typhoid, recovery from acute infection), protozoan infection, infectious mononucleosis, leukemia, Hodgkin disease, ulcerative colitis, regional enteritis

Decreased: Lymphocytic leukemia, aplastic anemia, steroid use

PMNs (Polymorphonuclear Neutrophils, Neutrophils, "Polys")

40 76% See also The Left Shift.

Increased

Physiologic (Normal): Severe exercise, last months of pregnancy, labor, surgery, newborn state, steroid therapy

Pathologic: Bacterial infection, noninfective tissue damage (MI, pulmonary infarction, pancreatitis, crush or injury, burn injury), metabolic disorder (eclampsia, DKA, uremia, acute gout), leukemia

Decreased: Pancytopenia, aplastic anemia, PMN depression (a mild decrease is referred to as neutropenia; a severe decrease is called agranulocytosis), marrow damage (x-rays, poisoning with benzene, antitumor drugs), severe overwhelming infection (disseminated TB, septicemia), acute malaria, severe osteomyelitis, infectious mononucleosis, atypical pneumonia, some viral infections, marrow obliteration (osteosclerosis, myelofibrosis, malignant infiltrate), drugs (more than 70, including chloramphenicol, phenylbutazone, chlorpromazine, quinine), vitamin B₁₂ and folate deficiencies, hypoadrenalism, hypopituitarism, dialysis, familial decrease, idiopathic causes

Red Cells

An automated device such as a Coulter Counter is used to measure red cell number, mean corpuscular volume (MCV), and hemoglobin concentration. The hematocrit and other parameters are calculated from those values.

Hematocrit

Men 40 54%; women 37 47%

Calculated from MCV and red cell number; the percentage volume of red cells in a given volume of blood

Increased: Primary polycythemia (polycythemia vera), secondary polycythemia (reduced fluid intake or excess fluid loss), congenital or acquired heart and lung disease, high altitude, heavy smoking, tumors (renal cell carcinoma, hepatoma)

Decreased: Megaloblastic anemia (folate or B₁₂ deficiency); iron deficiency anemia; sickle cell anemia or other hemoglobinopathy; acute or chronic blood loss; sideroblastic anemia, hemolysis; anemia due to chronic disease, dilution, alcohol, or drugs

MCH (Mean Cellular [Corpuscular] Hemoglobin)

27 31 pg (SI: pg)

The amount of hemoglobin in the average red cell. Calculated as

Increased: Macrocytosis (megaloblastic anemia, high reticulocyte count)

Decreased: Microcytosis (iron deficiency, sideroblastic anemia, thalassemia)

MCHC (Mean Cellular [Corpuscular] Hemoglobin Concentration)

33 37 g/dL (SI: 330 370 g/L)

The average concentration of Hbg in a given volume of red cells. Calculated as

Increased: Very severe, prolonged dehydration; spherocytosis

Decreased: Iron deficiency anemia, overhydration, thalassemia, sideroblastic anemia

MCV (Mean Cell [Corpuscular] Volume)

78 98 m³ (SI: fL)

The average volume of red blood cells; measured directly with the automated cell counter

Increased/Macrocytosis: Megaloblastic anemia (B₁₂, folate deficiency), macrocytic (normoblastic) anemia, reticulocytosis, myelodysplasia, Down syndrome, chronic liver disease, treatment of AIDS with AZT, chronic alcoholism, cytotoxic chemotherapy, radiation therapy, phenytoin (Dilantin) use, hypothyroidism, newborn state

Decreased/Microcytosis: Iron deficiency, thalassemia, some cases of lead poisoning or polycythemia

Normal: Anemia of chronic disease, acute blood loss, primary bone marrow failure

RDW (Red Cell Distribution Width)

11.5 14.5%

RDW is a measure of the degree of anisocytosis (variation in RBC size) and is determined with an automated counter.

Increased: Many types of anemia (iron deficiency, pernicious, folate deficiency, thalassemia), liver disease

Platelets

150,000 450,000 L

Platelet counts may be normal in number but abnormal in function, as occurs in aspirin therapy. Abnormalities of platelet function are assessed by bleeding time and platelet aggregation studies.

Increased: Sudden exercise, trauma, fracture, aftermath of asphyxia, aftermath of surgery (especially splenectomy), acute hemorrhage, myeloproliferative disorders, leukemia, aftermath of childbirth, carcinoma, cirrhosis, iron deficiency

Decreased: DIC, ITP, TTP, HUS, congenital disease, marrow suppressants (chemotherapy, alcohol, radiation), burns, snake and insect bites, leukemia, aplastic anemia, hypersplenism, infectious mononucleosis, viral infection, cirrhosis, massive transfusion, HELLP syndrome (a severe form of preeclampsia with microangiopathic hemolysis, elevated liver function test results, and low platelet count), preeclampsia and eclampsia, prosthetic heart valve, more than 30 drugs (NSAIDs, anticonvulsants, aspirin, thiazides, others)

Lymphocyte Subsets

Specific monoclonal antibodies are used to identify specific T and B cells. Lymphocyte subsets are useful in the diagnosis of AIDS and various types of leukemia and lymphoma. The designation CD (clusters of differentiation) has replaced the older antibody designations. Results are most reliably reported as an absolute number of cells/L rather than as a percentage. A CD4/CD8 ratio < 1 is seen in AIDS. Absolute CD4 count is used to determine when to initiate therapy with antiretroviral agents or to administer prophylaxis of certain infections, eg, PCP. The CDC considers an HIV-positive patient to have AIDS if the CD4 count < 200.

Normal Lymphocyte Subsets

• Total lymphocytes 660 4600/L
• T cells 644 2201 L (60 88%)
• B cells 82 392 L (3 20%)
• Helper/inducer T cells (CD4) 493 1191 L (34 67%)
• Suppressor/cytotoxic T cells (CD8) 182 785 L (10 42%)
• CD4/CD8 ratio > 1

RBC Morphology Differential Diagnosis

The following are erythrocyte abnormalities and the associated conditions. General terms include poikilocytosis (irregular RBC shape such as sickle or burr) and anisocytosis (irregular RBC size such as microcytes and macrocytes).

Basophilic Stippling: Lead or heavy-metal poisoning, thalassemia, severe anemia

Burr Cells (Acanthocytes): Severe liver disease; high levels of bile, fatty acids, or toxins

Heinz Bodies: Drug-induced hemolysis

Helmet Cells: Microangiopathic hemolysis (TTP, HUS, HELLP syndrome), hemolytic transfusion reaction, transplant rejection

Howell Jolly Bodies: Asplenia

Nucleated RBCs: Severe bone marrow stress (eg, hemorrhage, hemolysis, hypoxia), marrow replacement by tumor, extramedullary hematopoiesis

Polychromasia: A bluish red cell on routine Wright stain suggests reticulocytes

Sickling: Sickle cell anemia

Schistocytes: DIC, microangiopathic anemia, severe burns, drug effect (CSA, tacrolimus, ticlopidine, others)

Spherocytes: Hereditary spherocytosis, immune hemolysis, severe burns, ABO transfusion reaction

Target Cells (Leptocytes): Thalassemia, hemoglobinopathies, liver disease, any hypochromic anemia, aftermath of splenectomy

WBC Morphology Differential Diagnosis

The following are conditions associated with changes in the normal morphology of WBCs.

Auer Rods: AML

D hle Inclusion Bodies: Severe infection, burns, malignancy, pregnancy

Hypersegmentation: Megaloblastic anemia

Toxic Granulation: Severe illness (sepsis, burn, high fever)

Coagulation and Other Hematologic Tests

The coagulation cascade is shown in Figure 5 2. A variety of coagulation-related and other blood tests follow.

Anti-Xa Test (Anti Factor Xa, Anti Activated Factor X)

Anti-Xa heparin units per milliliter of plasma Collection: Blue top tube

Used to monitor heparin therapy when the PTT cannot be used (ie, patient with lupus anticoagulant). Only test for monitoring low-molecular-weight (LMW) heparin (eg, Lovenox)

Therapeutic:

LMW heparin: 0.5 1.0 anti-Xa units/mL

Prophylaxis:

LMW heparin: 0.2 0.4 anti-Xa units/mL

Activated Clotting Time (ACT)

114 186 s Collection: Black top tube from instrument manufacturer

A bedside test used in the operating room, dialysis unit, or other facility to document neutralization of heparin (ie, after CABG, heparin is reversed) or monitoring of antithrombin inhibitors (bivalirudin)

Increased:

Heparin, some platelet disorders, severe clotting factor deficiency

Antithrombin III (AT-III)

17 30 mg/dL or 80 120% of control value Collection: Blue top tube, patient must be off heparin for 6 h

Used in evaluation of thrombosis

Decreased:

Autosomal-dominant familial AT-III deficiency, PE, severe liver disease, late pregnancy, oral contraceptives, nephrotic syndrome (lost in urine with resulting increase in factor II and X activity), DIC, heparin therapy (> 3 d)

Increased:

Warfarin (Coumadin), after MI

Bleeding Time

Duke, Ivy < 6 min; Template < 10 min Collection: A bedside test performed by technicians. After a small incision is made, the wound is wicked with filter paper every 30 s until the fluid is clear.

In vivo test of hemostasis, platelet function, local tissue factors, and clotting factors. Nonsteroidal medications should be stopped 5 7 d before the test, because these agents can affect platelet function.

Increased:

Thrombocytopenia, von Willebrand disease, defective platelet function, drugs such as NSAIDs, uremia

Coombs Test, Direct (Direct Antiglobulin Test)

Normal = negative Collection: Purple top tube

Patient's erythrocytes; test for the presence of antibody on the patient's cells and for the screening for autoimmune hemolytic anemia

Positive:

Autoimmune hemolytic anemia, hemolytic transfusion reaction, some drug sensitizations (methyldopa, levodopa, cephalosporins, penicillin, quinidine), hemolytic disease of the newborn (erythroblastosis fetalis)

Coombs Test, Indirect (Indirect Antiglobulin Test/Autoantibody Test)

Normal = negative Collection: Purple top tube

Patient's serum; check for cross-match before blood transfusion. Test for antibodies against red cell antigens in the patient's serum.

Positive:

Isoimmunization from previous transfusion, autoimmune hemolytic anemia, incompatible blood or medications (eg, methyldopa)

Factor V (Leiden) Mutation

Normal = negative Collection: Lavender or blue top tube

Factor V Leiden (activated protein C [APC] resistance) is the most common hereditary blood coagulation disorder in the United States (5 7%). Heterozygotes have thrombosis risk (thrombophilia) three to eight times that of the general population. The risk among homozygotes is 140 times that of the general population. A PCR and reverse dot blot genetic test.

Positive:

Factor V mutation

Fibrin D-Dimers

(See also Chapter 4, D-Dimer.)

Negative or < 0.25 mcg/mL Collection: Blue, green, or purple top tube

Evidence of fibrin formation, fibrin cross-linking, and fibrinolysis

Increased:

DIC, thromboembolic disease (PE, arterial or venous thrombosis)

Fibrin Degradation Products (FDP), Fibrin Split Products (FSP)

< 10 mcg/mL Collection: Blue top tube

Generally replaced by the fibrin D-dimer as a screen for DIC

Increased:

DIC (usually > 40 mcg/mL), any thromboembolic condition (DVT, MI, PE), hepatic dysfunction

Fibrinogen

123 370 mg/dL (SI: 1.23 3.7 g/L) (Panic levels < 100 or > 500) Collection: Blue top tube

Most useful in management of DIC and congenital hypofibrinogenemia

Increased:

Inflammatory reaction, oral contraceptives, pregnancy, cancer (kidney, stomach, breast)

Decreased:

DIC (sepsis, amniotic fluid embolism, abruptio placentae), surgery (prostate, open heart), neoplastic and hematologic conditions, acute severe bleeding, burns, venomous snake bite, congenital disorder

Mixing Studies (Circulating Anticoagulant Screen)

Collection: Blue top tube

Used to evaluate prolonged PT or PTT. Normal plasma is mixed with patient plasma, and the abnormal clotting time is measured again in the mix. If the clotting time corrects, a factor deficiency exists. Assay for factors VIII, IX, XI, and XII to identify the specific factor (note: warfarin may also give this result). If the clotting time does not correct, an inhibitor is present (ie, lupus anticoagulant [associated with thrombosis and habitual abortion], heparin, argatroban, high-dose danaparoid, specific factor inhibitor). Prolonged RVVT (Russell viper venom time) is used to diagnose lupus anticoagulant. (RVV activates factor X.)

Partial Thromboplastin Time (Activated Partial Thromboplastin Time, PTT, aPTT)

27 38 s Collection: Blue top tube

Used to evaluate the intrinsic coagulation system (see Figure 5 2). Most often used to monitor heparin therapy

Increased:

Heparin, defect in the intrinsic coagulation system (except factors VII and XIII), prolonged application of tourniquet before drawing of sample, hemophilia A and B, von Willebrand disease (sometimes normal), lupus anticoagulant (antiphospholipid antibody), DIC

Prothrombin Time (PT)

11.5 13.5 s (INR, normal = 0.8 1.4) Collection: Blue top tube

Used to evaluate the extrinsic coagulation system (see Figure 5 2), which includes factors I, II, V, VII, and X. The use of INR instead of patient/control ratio to guide warfarin therapy is now the standard. INR provides a more standardized result; measures the control against a WHO standard reagent. Therapeutic INR is 2 3 for DVT, PE, TIAs, and atrial fibrillation. Mechanical heart valves require an INR of 2.5 3.5 (see also Chapter 22, Table 22 10). Not affected by heparin

Increased:

Drugs (warfarin), vitamin K deficiency, fat malabsorption, liver disease, prolonged application of tourniquet before drawing of sample, DIC, massive transfusion

Sedimentation Rate (Erythrocyte Sedimentation Rate, ESR)

Collection: Lavender top tube

A nonspecific test; high sensitivity and low specificity. Most useful in serial measurement to follow the course of disease (eg, polymyalgia rheumatica or temporal arteritis)

Wintrobe Scale:

Men, 0 9 mm/h; women, 0 20 mm/h

Increased:

Any type of infection, inflammation, rheumatic fever, endocarditis, neoplasm, AMI, multiple myeloma

Thrombin Time

10 14 s Collection: Blue top tube

Measure of conversion of fibrinogen to fibrin and fibrin polymerization. Used to detect the presence of heparin and hypofibrinogenemia

Increased:

Systemic heparin, DIC, fibrinogen deficiency, congenitally abnormal fibrinogen molecules