Blood Studies: Hematology and Coagulation



Blood Studies: Hematology and Coagulation






OVERVIEW OF BASIC BLOOD, HEMATOLOGY, AND COAGULATION TESTS


Composition of Blood

The average person circulates about 5 L of blood (1/13 of total body weight), of which 3 L is plasma and 2 L is cells. Plasma fluid derives from the intestines and lymphatic systems and provides a vehicle for cell movement. The cells are produced primarily by the bone marrow and account for blood “solids.” Blood cells are classified as white cells (leukocytes), red cells (erythrocytes), and platelets (thrombocytes). White cells are further categorized as granulocytes (neutrophils, basophils, and eosinophils), lymphocytes, and monocytes.

Before birth, hematopoiesis occurs in the liver. In midfetal life, the spleen and lymph nodes play a minor role in cell production. Shortly after birth, hematopoiesis in the liver ceases and the bone marrow is the only site of production of erythrocytes, granulocytes, and platelets. B lymphocytes are produced in the marrow and in the secondary lymphoid organs; T lymphocytes are produced in the thymus.


Blood Tests

Tests in this chapter are basic screening tests that address disorders of hemoglobin (Hb) and hematopoiesis, synthesis, and function. Blood and bone marrow examinations constitute the major means of determining certain blood disorders (anemia, leukemia and porphyria disorders, abnormal bleeding
and clotting); inflammation; infection; and inherited disorders of red blood cells (RBCs), white blood cells (WBCs), and platelets. Specimens are obtained through capillary skin punctures (finger, toe, heel), dried blood samples, arterial or venous sampling, or bone marrow aspiration. Specimens may be tested by automated or manual hematology instrumentation and evaluation.




BASIC BLOOD TEST


• Complete Blood Count (CBC)/Hemogram

A complete blood count (CBC), also referred to as a hemogram, consists of a WBC count, RBC count, Hb, Hct, RBC indices, and a platelet count. Table 2.2 contains the normal values for a CBC.

The CBC is a basic screening test and is one of the most frequently ordered laboratory procedures. The findings in the CBC give valuable diagnostic information about the hematologic and other body systems, prognosis, response to treatment, and recovery. The CBC consists of
a series of tests that determine number, variety, percentage, concentrations, and quality of blood cells:



  • WBC count: reports the total number of WBCs (leukocytes), which fight infection


  • Differential WBC count (Diff): identifies specific patterns of WBCs by percentage of each cell type (see Differential White Blood Cell Count [Diff; Differential Leukocyte Count] on page 67)


  • RBC count: reports the total number of RBCs, which carry O2 from lungs to blood tissues and CO2 from tissue to lungs


  • Hct: percentage of RBCs’ mass compared to the total volume of blood


  • Hb: main component of RBCs and transports O2 and CO2


  • RBC indices: calculated values of size and Hb content of RBCs; important in anemia evaluations


  • Mean corpuscular volume (MCV)


  • MCHC


  • Mean corpuscular hemoglobin (MCH)


  • Stained red cell examination (film or peripheral blood smear)


  • Platelet count (often included in CBC): Thrombocytes are necessary for clotting and control of bleeding


  • RBC distribution width (RDW): indicates degree variability and abnormal cell size


  • Mean platelet volume (MPV): index of platelet production








TABLE 2.2 Normal Values for Hemogram










































































































































Age


WBC Count (× 103/mm3)


RBC Count (× 106/mm3)


Hb (g/dL)


Hct (%)


MCV (fL)


Birth-2 wk


9.0-30.0


4.1-6.1


14.5-24.5


44-64


98-112


2-8 wk


5.0-21.0


4.0-6.0


12.5-20.5


39-59


98-112


2-6 mo


5.0-19.0


3.8-5.6


10.7-17.3


35-49


83-97


6 mo-1 yr


5.0-19.0


3.8-5.2


9.9-14.5


29-43


73-87


1-6 yr


5.0-19.0


3.9-5.3


9.5-14.1


30-40


70-84


6-16 yr


4.8-10.8


4.0-5.2


10.3-14.9


32-42


73-87


16-18 yr


4.8-10.8


4.2-5.4


11.1-15.7


34-44


75-89


>18 yr (males)


5.0-10.0


4.5-5.5


14.0-17.4


42-52


84-96


>18 yr (females)


5.0-10.0


4.0-5.0


12.0-16.0


36-48


84-96


Age


MCH (pg/cell)


MCHC (g/dL)


Platelets (× 103/mm3)


RDW (%)


MPV (fL)


Birth-2 wk


34-40


33-37


150-450




2-8 wk


30-36


32-36





2-6 mo


27-33


31-35





6 mo-1 yr


24-30


32-36





1-6 yr


23-29


31-35





6-16 yr


24-30


32-36





16-18 yr


25-31


32-36





>18 yr


28-34


32-36


140-400


11.5-14.5


7.4-10.4


Hct, hematocrit; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; MPV, mean platelet volume; RBC, red blood cell; RDW, RBC distribution width; WBC, white blood cell.


These tests are described in detail in the following pages.



Normal Findings



Interfering Factors


RBC Count



  • Many physiologic variants affect outcomes: posture, exercise, age, altitude, pregnancy, and many drugs.


Hematocrit



  • Physiologic variants affect Hct outcomes: age, gender, and physiologic hydremia of pregnancy.


Hemoglobin



  • Physiologic variations affect test outcomes: high altitude, excessive fluid intake, age, pregnancy, and many drugs.


Mean Corpuscular Hemoglobin Concentration



  • High values may occur in newborns and infants.


  • Presence of leukemia or cold agglutinins may increase levels. Mean corpuscular hemoglobin concentration (MCHC) is falsely elevated with a high blood concentration of heparin.



MCH



  • Hyperlipidemia and high heparin concentrations falsely elevate MCH values.


  • WBC counts >50,000/mm3 falsely elevate Hb values and falsely elevate the MCH.


WBC Count



  • Hourly variation, age, exercise, pain, temperature, and anesthesia affect test results.


Neutrophils and Eosinophils



  • Physiologic conditions such as stress, excitement, exercise, and obstetric labor increase neutrophil levels. Steroid administration affects levels for up to 24 hours.


  • The eosinophil count is lowest in the morning and then rises from noon until after midnight. Do repeat tests at the same time every day. Stressful states such as burns, postoperative states, and obstetric labor decrease the count. Drugs such as steroids, epinephrine, and thyroxine affect eosinophil levels.


Platelets



  • Physiologic factors include high altitudes, strenuous exercise, excitement, and premenstrual and postpartum effects.


  • A partially clotted blood specimen affects the test outcome.



TESTS OF WHITE BLOOD CELLS


• White Blood Cell (WBC) Count (Leukocyte Count)

WBCs (leukocytes) are divided into two main groups: granulocytes and agranulocytes. The granulocytes receive their name from the distinctive granules that are present in the cytoplasm of neutrophils, basophils, and eosinophils. However, each of these cells also contains a multilobed nucleus, which accounts for their also being called polymorphonuclear (PMN) leukocytes. In laboratory terminology, they are often called “polys” or PMNs. The agranulocytes, which consist of the lymphocytes and monocytes, do not contain distinctive granules and have nonlobular nuclei that are not necessarily spherical. The term mononuclear leukocytes is applied to these cells.

The endocrine system is an important regulator of the number of leukocytes in the blood. Hormones affect the production of leukocytes in the blood-forming organs, their storage and release from the tissue, and their disintegration. A local inflammatory process exerts a definite chemical effect on the mobilization of leukocytes. The life span of leukocytes varies from 13 to 20 days, after which the cells are destroyed in the lymphatic system; many are excreted from the body in fecal matter.

Leukocytes fight infection and defend the body by a process called phagocytosis, in which the leukocytes actually encapsulate foreign organisms and destroy them. Leukocytes also produce, transport, and distribute antibodies as part of the immune response to a foreign substance (antigen).

The WBC count serves as a useful guide to the severity of a disease process. Specific patterns of leukocyte response can be expected in various types of diseases as determined by the differential count (showing percentages of each of the different types of leukocytes). Leukocyte and differential counts, by themselves, are of little value as aids to diagnosis unless the results are related to the clinical condition of the patient—only then is a correct and useful interpretation possible. Signs and symptoms of increased WBCs include fever, bruising, petechiae, fatigue, anemia, bleeding of mucous membranes, weight loss, and frequent infections.


Normal Findings

Black adults: 3.2 to 10.0 × 103 cells/mm3 or × 109/L or 3200 to 10,000 cells/mm3

Adults: 4.5 to 10.5 × 103 cells/mm3 or × 109/L or 4500 to 10,500 cells/mm3

Children:

0 to 2 weeks: 9.0 to 30.0 × 103 cells/mm3 or × 109/L or 9000 to 30,000 cells/mm3

2 to 8 weeks: 5.0 to 21.0 × 103 cells/mm3 or × 109/L or 5000 to 21,000 cells/mm3

2 months to 6 years: 5.0 to 19.0 × 103 cells/mm3 or × 109/L or 5000 to 19,000 cells/mm3

6 to 18 years: 4.8 to 10.8 × 103 cells/mm3 or × 109/L or 4800 to 10,800 cells/mm3







Interfering Factors



  • Hourly rhythm: There is an early-morning low level and late-afternoon peak. Age, gender, exercise, medications, pregnancy, pain, temperature, altitude, and anesthesia affect test results.


  • Age: In newborns and infants, the WBC count is high (10,000/mm3 to 20,000/mm3 or 10 × 109/L to 20 × 109/L); the count gradually decreases in children until the adult values are reached between 18 and 21 years of age.


  • Any stressful situation that leads to an increase in endogenous epinephrine production and a rapid rise in the leukocyte count



• Differential White Blood Cell Count (Diff; Differential Leukocyte Count)

The total count of circulating WBCs is differentiated according to the five types of leukocytes, each of which performs a specific function.

The differential count is expressed as a percentage of the total number of leukocytes (WBCs). The distribution (number and type) of cells and the degree of increase or decrease are diagnostically significant. The percentages indicate the relative number of each type of leukocyte in the blood. The absolute count of each type of leukocyte is obtained mathematically by multiplying its relative percentage by the total leukocyte count. The formula is:

Relative value (%) × WBC (cells/mm3) = Absolute value (cells/mm3)










Function of Circulating White Blood Cells According to Leukocyte Type





















Cell


These Cells Function to Combat


Neutrophils


Pyogenic infections (bacterial)


Eosinophils


Allergic disorders and parasitic infestations


Basophils


Parasitic infections, some allergic disorders


Lymphocytes


Viral infections (measles, rubella, chickenpox, infectious mononucleosis)


Monocytes


Severe infections, by phagocytosis









Differential For Leukocyte Count








































































































Age


Bands/Stab (%)


Segs/Polys (%)


Eos (%)


Basos (%)


Lymphs (%)


Monos (%)


Metas (%)


Birth-1 wk


10-18


32-62


0-2


0-1


26-36


0-6



1-2 wk


8-16


19-49


0-4


0-0


38-46


0-9



2-4 wk


7-15


14-34


0-3


0-0


43-53


0-9



4-8 wk


7-13


15-35


0-3


0-1


41-71


0-7



2-6 mo


5-11


15-35


0-3


0-1


42-72


0-6



6 mo-1 yr


6-12


13-33


0-3


0-0


46-76


0-5



1-6 yr


5-11


13-33


0-3


0-0


46-76


0-5



6-16 yr


5-11


32-54


0-3


0-1


27-57


0-5



16-18 yr


5-11


34-64


0-3


0-1


25-45


0-5



>18 yr


3-6


50-62


0-3


0-1


25-40


3-7


0-1


Bands or stab cells, immature forms of neutrophils; Segs, segmented neutrophils; Polys, polymorphonuclear neutrophils; Eos, eosinophils; Basos, basophils; Lymphs, lymphocytes; Monos, monocytes; Metas, metamyelocytes.


The differential count alone has limited value; it must always be interpreted in relation to the WBC count. If the percentage of one type of cell is increased, it can be inferred that cells of that type are relatively more numerous than normal, but it is not known whether this reflects an actual increase in the (absolute) number of cells that are relatively increased or an absolute decrease in cells of another type. On the other hand, if the relative (percentage) values of the differential count and the total WBC count are both known, it is possible to calculate absolute values that are not subject to misinterpretation.

Historically, a differential count was done manually, but current hematology instruments do an automated differential count. The count is based on a combination of hydrodynamic focusing and fluorescent dyes in newer instrumentation. However, not all samples can be evaluated by automated methods. When a leukocyte count is extremely low or high, a manual count may be necessary. Extremely abnormal leukocytes, such as those in leukemia, also have to be counted by hand. The automated instrument has built-in quality control that senses abnormal cells and flags the differential. A microscopic count must then be done.


• Segmented Neutrophils (Polymorphonuclear Neutrophils, PMNs, Segs, Polys)

Neutrophils, the most numerous and important type of leukocytes in the body’s reaction to inflammation, constitute a primary defense against microbial invasion through the process of phagocytosis. These cells can also cause some body tissue damage by their release of enzymes and endogenous
pyogens. In their immature stage of development, neutrophils are referred to as “stab” or “band” cells. The term band stems from the appearance of the nucleus, which has not yet assumed the lobed shape of the mature cell.

This test determines the presence of neutrophilia or neutropenia. Neutrophilia is an increase in the absolute number of neutrophils in response to invading organisms and tumor cells. Neutropenia occurs when too few neutrophils are produced in the marrow, too many are stored in the blood vessel margin, or too many have been called to action and used up.


Normal Findings

Absolute count: 3000 to 7000/mm3 or 3 to 7 × 109/L

African American adults: 1.2 to 6.6 × 109/L

Differential: 50% of total WBC count

0% to 3% of total PMNs are stab or band cells





Interfering Factors



  • Physiologic conditions such as stress, excitement, fear, vomiting, electric shock, anger, joy, and exercise temporarily cause increased neutrophils. Crying babies have neutrophilia.


  • Obstetric labor and delivery cause neutrophilia. Menstruation causes neutrophilia.


  • Steroid administration: Neutrophilia peaks in 4 to 6 hours and returns to normal by 24 hours (in severe infection, expected neutrophilia does not occur).


  • Exposure to extreme heat or cold


  • Age



    • Children respond to infection with a greater degree of neutrophilic leukocytosis than adults do.


    • Some elderly patients respond weakly or not at all, even when infection is severe.


  • Resistance



    • People of any age who are weak and debilitated may fail to respond with a significant neutrophilia.


    • When an infection becomes overwhelming, the patient’s resistance is exhausted and, as death approaches, the number of neutrophils decreases greatly.


  • Myelosuppressive chemotherapy


  • Many drugs cause increases or decreases in neutrophils.









TABLE 2.3 Leukocyte Abnormalities and Diseases



























































Abnormality


Description


Associated Diseases


Toxic granulation


Coarse, black or purple, cytoplasmic granules


Infections or inflammatory diseases; acute reactive state


Döhle bodies


Small (1-2 μm), blue, cytoplasmic inclusions in neutrophils


Infections or inflammatory diseases, burns


Pelger-Huët anomalies


Neutrophil with bilobed nucleus or no segmentation of nucleus; chromatin is coarse, and cytoplasm is pink with normal granulation


Hereditary (congenital), myelogenous leukemia


May-Hegglin anomaly


Basophilic, cytoplasmic inclusions of leukocytes; similar to Döhle bodies


May-Hegglin syndrome (hereditary), includes thrombocytopenia and giant platelets


Alder-Reilly anomaly


Prominent azurophilic granulation in leukocytes; similar to toxic granulation; granulation is seen better with Giemsa stain


Hereditary, mucopolysaccharidosis


Chédiak-Higashi anomaly


Gray-green, large cytoplasmic inclusions that are fused giant lysosomes (phospholipids)


Chédiak-Higashi syndrome; few cases of acute myeloid leukemia


Lupus erythematosus (LE) cells


Neutrophilic leukocyte with a homogeneous red-purple inclusion that distends the cell’s cytoplasm


LE and other collagen diseases, chronic hepatitis, drug reactions, serum sickness (not naturally occurring in the body—must be induced to form by mechanical trauma in vitro)


Tart cell


Neutrophilic leukocyte with a phagocytized nucleus of a granulocyte that retains some nuclear structure


Drug reactions (e.g., penicillin, procainamide) or actual phagocytosis


Myeloid “shift to left”


Presence of bands, myelocytes, metamyelocytes, or promyelocytes


Infections, intoxications, tissue necrosis, myeloproliferative syndrome, leukemia (chronic myelocytic), leukemoid reaction, pernicious anemia, hyposplenism


Hypersegmented neutrophil


Mature neutrophil with more than five distinct lobes


Megaloblastic anemia, hereditary constitutional hypersegmentation of neutrophils; long-term chronic infection


Leukemic cells (e.g., lymphoblasts, myeloblasts)


Presence of lymphoblasts, myeloblasts, monoblasts, myelomonoblasts, promyelocytes (none normally present in peripheral blood)


Leukemia (acute or chronic), leukemoid reaction, severe infectious or inflammatory diseases, myeloproliferative syndrome, intoxications, malignancies, recovery from bone marrow suppression


Auer bodies


Rodlike, 1-6 μm long, red-purple, refractile inclusions in neutrophils


Acute myelocytic leukemia or myelomonocytic leukemia


Smudge cell


Disintegrating nucleus of a ruptured leukocyte


Increased numbers in leukemic blood, particularly in acute lymphocytic leukemia or chronic lymphocytic leukemia when WBC count is >10,000/mm3 or >10 × 109/L





• Eosinophils

Eosinophils, capable of phagocytosis, ingest antigen-antibody complexes and become active in the later stages of inflammation. Eosinophils respond to allergic and parasitic diseases. Eosinophilic granules contain histamine (one third of all the histamine in the body).

This test is used to diagnose allergic infections, assess severity of infestations with worms and other large parasites, and monitor response to treatment.


Normal Findings

Absolute count: 0 to 0.7 × 109/L

Differential: 0% to 3% of total WBC count




Interfering Factors



  • Daily rhythm: Normal eosinophil count is lowest in the morning and then rises from noon until after midnight. For this reason, serial eosinophil counts should be repeated at the same time each day.


  • Stressful situations, such as burns, postoperative states, electroshock, and labor, cause a decreased count.


  • After administration of corticosteroids, eosinophils disappear.


  • See Appendix E for drugs that affect test outcomes.




• Basophils

Basophils, which constitute a small percentage of the total leukocyte count, are considered phagocytic. The basophilic granules contain heparin, histamines, and serotonin. Tissue basophils are called mast cells and are similar to blood basophils. Normally, mast cells are not found in peripheral blood and are rarely seen in healthy bone marrow.

Basophil counts are used to study chronic inflammation. There is a positive correlation between high basophil counts and high concentrations of blood histamines, although this correlation does not imply cause and effect. It is extremely difficult to diagnose basopenia because a 1000 to 10,000 count differential would have to be done to get an absolute count.


Normal Findings

Absolute count: 15 to 50/mm3 or 0.02 to 0.05 × 109/L

Differential: 0% to 1.0% of total WBC count




Interfering Factors

See Appendix E for drugs that affect test outcomes.



• Monocytes (Monomorphonuclear Monocytes)

These agranulocytes, the largest cells of normal blood, are the body’s second line of defense against infection. Histiocytes, which are large macrophagic phagocytes, are classified as monocytes in a differential leukocyte count. Histiocytes and monocytes are capable of reversible transformation from one to the other.

These phagocytic cells of varying size and mobility remove injured and dead cells, microorganisms, and insoluble particles from the circulating blood. Monocytes escaping from the upper and lower respiratory tracts and the gastrointestinal and genitourinary organs perform a scavenger function, clearing the body of debris. These phagocytic cells produce the antiviral agent called interferon.

This test counts monocytes, which circulate in certain specific conditions such as tuberculosis, subacute bacterial endocarditis, and the recovery phase of acute infections.


Normal Findings

Absolute count: 100 to 500/mm3 or 0.1 to 0.5 × 109/L

Differential: 3% to 7% of total WBC count, or 0.03 to 0.07 of total WBC count





Interfering Factors

See Appendix E for drugs that affect test outcomes.



• Lymphocytes (Monomorphonuclear Lymphocytes); CD4, CD8 Count; Plasma Cells

Lymphocytes are small mononuclear cells without specific granules. These agranulocytes are motile cells that migrate to areas of inflammation in both early and late stages of the process. These cells are the source of serum immunoglobulins and of cellular immune response and play an important role in immunologic reactions. All lymphocytes are manufactured in the bone marrow. B lymphocytes mature in the bone marrow, and T lymphocytes mature in the thymus gland. B cells control the
antigen-antibody response that is specific to the offending antigen and is said to have “memory.” The T cells, the master immune cells, include CD4+ helper T cells, killer cells, cytotoxic cells, and CD8+ suppressor T cells.

Plasma cells (fully differentiated B cells) are similar in appearance to lymphocytes. They have abundant blue cytoplasm and an eccentric, round nucleus. Plasma cells are not normally present in blood.

This test measures the number of lymphocytes in the peripheral blood. Lymphocytosis is present in various diseases and is especially prominent in viral disorders. Lymphocytes and their derivatives, the plasma cells, operate in the immune defenses of the body.


Normal Findings

Lymphocytes: 25% to 40% of total leukocyte count (relative value) or 1500 to 4000 cells/mm3 or 1.5 to 4.0 × 109/L

Plasma cells: 0% or none

CD4 count: total WBC count × lymphocytes (%) × lymphocytes (%) stained with CD4

CD4/CD8 ratio: >1.0




Interfering Factors



  • Physiologic pediatric lymphocytosis is a condition in newborns that includes an elevated WBC count and abnormal-appearing lymphocytes that can be mistaken for malignant cells.


  • Exercise, emotional stress, and menstruation can cause an increase in lymphocytes.


  • African Americans normally have a relative (not absolute) increase in lymphocytes.


  • See Appendix E for drugs that affect outcomes.




• Lymphocyte Immunophenotyping (T and B Cells)

Lymphocytes are divided into two categories, T and B cells, according to their primary function within the immune system. In the body, T and B cells work together to help provide protection against infections, oncogenic agents, and foreign tissue, and they play a vital role in regulating self-destruction or autoimmunity.

Most circulating lymphocytes are T cells with a life span of months to years. The life span of B cells is measured in days. B cells (antibody) are considered bursa or bone marrow dependent and are responsible for humoral immunity (in which antibodies are present in the serum). T cells (cellular) are thymus derived and are responsible for cellular immunity. T cells are further divided into helper T (CD3+, CD4+) cells and suppressor T (CD3+, CD8+) cells.

Evaluation of lymphocytes in the clinical laboratory is performed by quantitation of the lymphocytes and their subpopulations and by assessment of their functional activity. These laboratory analyses have become an essential component of the clinical assessment of two major disease states: lymphoproliferative states (e.g., leukemia, lymphoma), in which characterization of the malignant cell in terms of lineage and stage of differentiation provides valuable information to the oncologist to guide prognosis and appropriate therapy; and immunodeficient states (e.g., HIV infection, organ transplantation), in which the alterations in the immune system that occur secondary to infection are evaluated.

The method of lymphocyte quantitation and characterization is based on the detection of cell surface markers by very specific monoclonal antibodies. For cell surface immunophenotyping, flow cytometry has become the method of choice. Cell surface phenotyping is accomplished by reacting cells from an appropriate specimen with one or more labeled monoclonal antibodies and passing them through a flow cytometer, which counts the proportion of labeled cells.


Normal Findings for Adult Peripheral Blood by Flow Cytometry

T and B surface markers:

Total T cells (CD3+): 53% to 88%

Helper T cells (CD3+, CD4+): 32% to 61%


Suppressor T cells (CD3+, CD8+): 18% to 42%

B cells (CD19+): 5% to 20%

Natural killer cells (CD16+): 4% to 32%

Absolute counts (based on pathologist’s interpretation):

Total lymphocytes: 660 to 4600/mm3 (0.6 to 4.6 × 109/L)

Total T cells (CD3+): 812 to 2318/mm3

Helper T cells (CD3+, CD4+): 589 to 1505/mm3

Suppressor T cells (CD3+, CD8+): 325 to 997/mm3

B cells (CD19+): 92 to 426/mm3

Natural killer cells (CD16+): 78 to 602/mm3

Lymphocyte ratio:

Helper-to-suppressor T-cell ratio >1.0






STAINS FOR LEUKEMIAS

Several special WBC staining methods are used to diagnose leukemia, amyloid disease, lymphoma, and erythroleukemia; to differentiate erythema myelosis from sideroblastic anemia; to monitor progress and response to therapy; and to detect early relapse. Amyloid refers to starchlike substances deposited in certain diseases (e.g., tuberculosis, osteomyelitis, leprosy, Hodgkin’s disease, and carcinoma).


• Sudan Black B (SBB) Stain

The Sudan Black B (SBB) stain aids in differentiation of the immature cells of acute leukemias, especially acute myeloblastic leukemia. The SBB stains a variety of fats and lipids that are present in myeloid leukemias but are not present in the lymphoid leukemias.


Reference Values


Positive Reactions

Granulocytic cells (neutrophils and eosinophils)

Myeloblasts

Promyelocytes

Neutrophilic myelocytes

Metamyelocytes, bands, and segmented neutrophils

Eosinophils at all stages

Monocytes and precursors


Variable Reactions

Basophils


Negative Reactions (Sudanophobia)

Lymphocytes and lymphocytic precursors

Megakaryocytes and thrombocytes (platelets)

Erythrocytes

Erythroblasts may display a few granules that represent mitochondrial phospholipid components.




Interfering Factors

There are cases of acute leukemia in which the cytochemical stains are not useful and fail to reveal the differentiating features of any specific cell line.




• Periodic Acid-Schiff (PAS) Stain

The periodic acid-Schiff (PAS) stain aids in the diagnosis of acute lymphoblastic leukemia (ALL). Early myeloid precursors and erythrocyte precursors are negative. As granulocytes mature, they increase in PAS positivity, whereas mature RBCs stay negative. The PAS stain cannot be used to distinguish between ALL and AML or between benign and malignant lymphocytic disorders.


Normal Findings

Lymphoblasts: stain (positive)

Myeloblasts: do not stain (negative)






• Terminal Deoxynucleotidyl Transferase (TDT) Stain

The thymus is the primary site of terminal deoxynucleotidyl transferase (TDT)-positive cells, and TDT is found in the nucleus of the more primitive T cells. A thymus-related population of TDT-positive cells resides in the bone marrow (normally a minor population, 0% to 2%). TDT is increased in >90% of cases of ALL of childhood. A minor (5% to 10%) population of patients with acute nonlymphoblastic leukemia has TDT-positive blasts. TDT-positive blasts are prominent in some cases of chronic myelogenous leukemia (CML), relating to the development of an acute blast phase. TDT has been reported to assist in establishing the diagnosis of ALL. TDT-positive cases of blast-phase CML correlate with a positive response to chemotherapy (vincristine and prednisone).


Normal Findings

Negative in nonlymphoblastic leukemia

Negative in peripheral blood

0% to 2% positive in bone marrow






• Leukocyte Alkaline Phosphatase (LAP) Stain

Neutrophils are the only leukocytes to contain various amounts of alkaline phosphatase.

The leukocyte alkaline phosphatase (LAP) stain is used as an aid to distinguish chronic granulocytic leukemia from a leukemoid reaction. A leukemoid reaction is a high WBC count that may look like leukemia but is not. In remission of CML, the LAP may return to normal. In the blast phase of CML, the LAP may be elevated.


Normal Findings

40 to 100 LAP units






Interfering Factors



  • Any physiologic stress, such as third-trimester pregnancy, labor, or severe exercise, causes an increased LAP score.


  • Steroid therapy increases LAP score.


  • CML with infection increases LAP score.



• Tartrate-Resistant Acid Phosphatase (TRAP) Stain

The malignant mononuclear cells of leukemic reticuloendotheliosis (hairy cell leukemia) are resistant to inhibition by tartaric acid. There is evidence that the reaction is not entirely specific because tartrate-resistant acid phosphatase (TRAP) reactions have been reported in prolymphocytic leukemia and malignant lymphoma and in some cases of infectious mononucleosis.


Normal Findings

No TRAP activity






TESTS FOR RED BLOOD CELLS

Many tests look at the RBCs: their number and size, amount of Hb, rate of production, and percent composition of the blood. The RBC count, Hct, and Hb are closely related but different ways to look at the adequacy of erythrocyte production. The same conditions cause an increase (or decrease) in each of these indicators.


• Red Blood Cell (RBC) Count (Erythrocyte Count)

The main function of the RBC (erythrocyte) is to carry oxygen from the lungs to the body tissues and to transfer carbon dioxide from the tissues to the lungs. This process is achieved by means of the Hb in the RBCs, which combines easily with oxygen and carbon dioxide and gives arterial blood a bright red appearance. To enable use of the maximal amount of Hb, the RBC is shaped like a biconcave disk—this affords more surface area for the Hb to combine with oxygen. The cell also is able to change its shape when necessary to allow for passage through the smaller capillaries.

The RBC count, an important measurement in the evaluation of anemia or polycythemia, determines the total number of erythrocytes in a microliter (cubic millimeter) of blood.


Normal Findings





Interfering Factors



  • Posture: When a blood sample is obtained from a healthy person in a recumbent position, the RBC count is 5% lower. (If the patient is anemic, the count will be lower still.)


  • Dehydration: Hemoconcentration in dehydrated adults (caused by severe burns, untreated intestinal obstruction, severe persistent vomiting, or diuretic abuse) may obscure significant anemia.


  • Age: The normal RBC count of a newborn is higher than that of an adult, with a rapid drop to the lowest point in life at 2 to 4 months. The normal adult level is reached at age 14 years and is maintained until old age, when there is a gradual drop (see Table 2.4).


  • Falsely high counts may occur because of prolonged venous stasis during venipuncture.


  • Stress can cause a higher RBC count.


  • Altitude: The higher the altitude, the greater the increase in RBC count. Decreased oxygen content of the air stimulates the RBC count to rise (erythrocytosis).



  • Pregnancy: There is a relative decrease in RBC count when the body fluid increases in pregnancy, with the normal number of erythrocytes becoming more diluted.


  • There are many drugs that may cause decreased or increased RBC count. See Appendix E for drugs that affect test outcomes.


  • The EDTA blood sample tube must be at least three fourths filled or values will be invalid because of cell shrinkage caused by the anticoagulant.


  • The blood sample must not be clotted (even slightly) or the values will be invalid.



• Hematocrit (Hct); Packed Cell Volume (PCV)

The word hematocrit means “to separate blood,” which underscores the mechanism of the test because the plasma and blood cells are separated by centrifugation.

The Hct test is part of the CBC. This test indirectly measures the RBC mass. The results are expressed as the percentage by volume of packed RBCs in whole blood (packed cell volume [PCV]). It is an important measurement in the determination of anemia or polycythemia.


Normal Findings

Women: 36% to 48% or 0.36 to 0.48

Men: 42% to 52% or 0.42 to 0.52

Children:

0 to 2 weeks: 44% to 64% or 0.44 to 0.64

2 to 8 weeks: 39% to 59% or 0.39 to 0.59

2 to 6 months: 35% to 49% or 0.35 to 0.49

6 months to 1 year: 29% to 43% or 0.29 to 0.43

1 to 6 years: 30% to 40% or 0.30 to 0.40

6 to 16 years: 32% to 42% or 0.32 to 0.42

16 to 18 years: 34% to 44% or 0.34 to 0.44







Interfering Factors



  • People living at high altitudes have high Hct values as well as high Hb and RBC values.


  • Normally, the Hct slightly decreases in the physiologic hydremia of pregnancy.


  • The normal values for Hct vary with age and gender. The normal value for infants is higher because the newborn has many macrocytic red cells. Hct values in females are usually slightly lower than in males.


  • There is also a tendency toward lower Hct values in men and women older than 60 years of age, corresponding to lower RBC count values in this age group.


  • Severe dehydration from any cause falsely raises the Hct.



• Hemoglobin (Hb)

Hb, the main component of erythrocytes, serves as the vehicle for the transportation of oxygen and carbon dioxide. It is composed of amino acids that form a single protein called globin, and a compound called heme, which contains iron atoms and the red pigment porphyrin. It is the iron pigment that combines readily with oxygen and gives blood its characteristic red color. Each gram of Hb can carry 1.34 mL of oxygen per 100 mL of blood. The oxygen-combining capacity of the blood is directly proportional to the Hb concentration rather than to the RBC count because some RBCs contain more Hb than others. This is why Hb determinations are important in the evaluation of anemia.

The Hb determination is part of a CBC. It is used to screen for disease associated with anemia, to determine the severity of anemia, to monitor the response to treatment for anemia, and to evaluate polycythemia.

Hb also serves as an important buffer in the extracellular fluid. In tissue, the oxygen concentration is lower, and the carbon dioxide level and hydrogen ion concentration are higher. At a lower pH, more oxygen dissociates from Hb. The unoxygenated Hb binds to hydrogen ion, thereby raising the pH.
As carbon dioxide diffuses into the RBC, carbonic anhydrase converts carbon dioxide to bicarbonate and protons. As the protons are bound to Hb, the bicarbonate ions leave the cell. For every bicarbonate ion leaving the cell, a chloride ion enters. The efficiency of this buffer system depends on the ability of the lungs and kidneys to eliminate, respectively, carbon dioxide and bicarbonate. Refer to the discussion of ABGs in Chapter 14.


Normal Findings

Women: 12.0 to 16.0 g/dL or 120 to 160 g/L

Men: 14.0 to 17.4 g/dL or 140 to 174 g/L

Children:

0 to 2 weeks: 14.5 to 24.5 g/dL or 145 to 245 g/L

2 to 8 weeks: 12.5 to 20.5 g/dL or 125 to 205 g/L

2 to 6 months: 10.7 to 17.3 g/dL or 107 to 173 g/L

6 months to 1 year: 9.9 to 14.5 g/dL or 99 to 145 g/L

1 to 6 years: 9.5 to 14.1 g/dL or 95 to 141 g/L

6 to 16 years: 10.3 to 14.9 g/dL or 103 to 149 g/L

16 to 18 years: 11.1 to 15.7 g/dL or 111 to 157 g/L



Sep 25, 2018 | Posted by in PATHOLOGY & LABORATORY MEDICINE | Comments Off on Blood Studies: Hematology and Coagulation
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