Hematology Tests

Blood consists of red cells (erythrocytes), white cells (leukocytes), and platelets (thrombocytes), suspended in a liquid called plasma. A CBC usually includes white blood cell count (WBC), red blood cell count (RBC), hemoglobin, hematocrit, red cell indices (MCV, MCH, MCHC), and platelet count. Some others tests listed under the CBC include red cell distribution width (RDW), mean platelet volume (MPV) and a differential examination of the quality and quantity of various white cells reported either in percent or absolute terms.

Reference values for the different parts of the CBC are difficult to list as some vary by age, sex, and altitude. Also different instruments will have different principles of measurement that can impact on the final values.

Hemoglobin (Hb)

Hemoglobin is a large complex protein made up of globin chains and heme found inside the red cells. Heme contains iron which is the portion of the protein that actually binds to oxygen in the lungs and releases it into the tissues. This is one of those test that will vary in reference ranges although it is fair to say that values around or under 10 are usually seen in patients with some signs or symptoms of anemia such as shortness of breath or fatigue or pallor.

Hematocrit (Hct)

If you think of the hemoglobin test as measuring just the weight of the protein in the red cells, the hematocrit is the percent of volume that is taken up by red cells. It is also called the packed RBC volume.

RBC indices (Mean Cell Volume/MCV, Mean Cell Hemoglobin/ MCH, Mean Cell Hemoglobin Concentration/MCHC)

Counting cells does not tell you much about their quality. These values try to describe the red cell population. Think of the 4th grade math question: "If 12 apples cost $1.20, how much does 1 apple cost?" If you know the red cell count and you know how much volume these cells take up (Hct), then by dividing the hematocrit by the red cell count will give you the average (or mean) cell volume. Similarly, if you know the total weight of hemoglobin and the red count, then you can find out the average or mean weight of hemoglobin in each cell. And, if you know the weight of hemoglobin and the volume of blood that is red cells, you can determine what percentage of an average red cell is taken up by hemoglobin. The benefit of these indices is that you can quickly narrow down the potential causes of anemia; the disadvantage is that these number assume a similar population of cells. For example, if the MCV is 90, does that mean all your cells are 90 femtoliters in volume or could some be 85 and 95 (within reference limits), or 75 and 105 (decidedly out of reference limits)?

Relative or Red cell Distributive Width (RDW)

This is a new test that can be performed only with modern instrumentation. The instrument measures every red cell it counts, finds the average, and compares each red cell size to that average. This indicates the variability in the size of the RBCs. Using this test with the indices allows you to answer questions about cell size and quality quickly.

While every laboratory will have developed their own specific reference ranges, you could assume that an MCV of 80 - 100 femtoliters, an MCH of 29 - 31 picograms, an MCHC of 30 - 35% would generally reflect a population of cells described as normochromic normocytic. An MCV less than 80 describes microcytes or small cells. An MCV greater than 100 reflects macrocytes. An MCHC less than 30 usually is taken to mean that the cells are not adequately filled with hemoglobin. When looking at red cells, it is the hemoglobin that gives the cell its red color so less than adequate hemoglobin is known as hypochromia.

White Blood Cell Count (WBC)

White cells are easily separated from red cells in that a mature red cell does not contain a nucleus. Cells are broken and only nuclei are counted. This works quite well unless immature red cells that still contain nuclei are present. These red cell nuclei are counted and the result is a falsely elevated number. Corrected white cell counts have had the red cell eliminated from the report.

Differential

White blood cells are evaluated by a differential count, which reports percentages of the types of WBCs present. These are neutrophils which fight infection (also known as polys and bands, polymorphonuclear leukocytes, PMN’s, grans, segs and nonsegs), lymphocytes which produce antibodies and other immune system activities (lymphs, ly), monocytes which also fight infection (mono’s), eosinophils (eos) and basophils (basos) which are involved with allergies. The red cells are also evaluated for size, shape, color and the presence of any abnormalities

Manual differentials are performed by taking a drop of blood, spreading it on a slide, staining it, and evaluating 100 cells individually for quality and changes in morphology. For patients with elevated white cell counts, differentials of 200 cells or greater might be done. Automated differentials are performed by either testing for specific compounds within the cells or comparing their size, shape, and content. Most instruments will count thousands of cells. For both types of differentials, the numbers are reported in percentages.

In some patients percentages might be misleading so absolute values of the types of WBC , i.e., the number of white blood cells multiplied by the percentage seen are valuable in diagnosing illness or following therapy. Persons receiving chemotherapy often have decreased WBC. If a patient’s absolute granulocyte count (ANC or AGC) goes below 2,000 cells, then physicians become concerned about the possibility of infection. A number below 1,000 is cause for greater concern and less than 500 usually lands the patient in the hospital.

Platelets (PLT)

Platelets are essential for the clotting of blood. One could think of them as tying together the clotting mechanism and the damaged area. When platelets are low, it may take longer for the blood to clot. When platelet counts are too high, unnecessary blood clots may occur. Strangely enough, bleeding can also happen if the platelets interfere with each other! Platelet function is affected by many drugs; one of the most well known is aspirin. Easy bruising may be a sign of a decreased platelet count. Bruising like Goldilocks’ companions comes in three "sizes". The largest is called hematoma; it’s multicolored, raised, has very well defined edges, and painful. The middle one is flat, usually red/blue and not well defined edges. Many normal women get these on their thighs from bumping into furniture, etc. The smallest is called petechiae. These looks something like freckles. They occur in multiples, may be itchy, and are caused by very small blood vessel damage. Petechiae can be seen in nosebleeds and gum bleeding.

Mean Platelet Volume (MPV)

This is a test similar to the MCV and we don’t know a whole lot about what it tells us.

White Blood Cells found in the normal differential.

Granulocytes Cells that contain large visible granules are sometimes called granulocytes. They can be separated into 3 distinct cell lines, based on the reaction of the granules to the most commonly used stain in Hematology, the Wright stain. The stain is a pH based stain. Structures that favor the basic stain stain dark blue or basophilic; while those that favor the acid stain, eosin, stain bright red-orange. Some structures seem indifferent to the stain and are called neutral. The most numerous cell line of the granulocytes contain both light blue and light pink granules. As a result they are called neutrophils. This cell line is considered the first line of defense against most bacteria. It takes 6 steps for this cell to mature from a myeloblast to a fully mature cell. There have been several different ways to identify these cells so the following names are more or less synonymous: The most mature cells is called polymorphonuclear leukocytes (polys or PMN’s) or segmented neutrophils (segs). One step from fully mature is the band or nonsegmented cell. Both these cells types are functional; the older one seems just a little bit faster. These cells are usually between 50 - 70% of all of the cells seen in a normal differential performed on an adult. These numbers do not work for infants and young children.

Cells whose granules stain bright red orange are called eosinophils and are part of the allergic response. Those granules contain histamine among other proteins. They should constitute between 0 - 4% of a normal differential for an adult.

Cells whose granules stain dark blue are called basophils and are also involved in allergic reactions.

Monocytes are a type of cross over cell. Many people call them granulocytes because they do contain granules but the granules are not large or easily seen. At any rate, monocytes are your basic "junk food" eaters. They are primarily responsible for removing dead or damaged cells and constitute less than 10% of the adult differential.

Lymphocytes are cells that do not contain large numbers of any granules. They are responsible for producing antibodies against foreign material such as antigens found in viruses and some bacteria. They also are active against malignancy. In the adult, they range between 20 and 45% of the cells seen in the differential.

When a lymphocyte is actively defending against an antigen, there will be changes seen in the cell and the cell is called a "reactive" lymphocyte.

Red blood cells in the differential

Normal red cells should be monotonous. They should all be about the same size, biconcave in shape, well filled with hemoglobin, and be about the same color. Changes in these descriptions are clues to many disease processes. Changes are usually graded as slight/moderate/ marked or 1+, 2+, 3+, and 4+ with 4+ being the most unusual. Terms that are used to describe these changes include: anisocytosis (changes in size), poikilocytosis (changes in shape), hypochromia (less than adequate hemoglobin) and polychromasia/polychromatophilia (changes in color).

Platelets in the differential

Platelets cannot be counted with any accuracy when viewing the blood smear. They are only be estimated and no comments about they ability to act in clotting can be made. So you are left with comments such as adequate or appears decreased/increased. Comments can be made about their size but since platelets swell when they come into contact with the anticoagulants used in the collection of blood for a CBC, this may sometimes not be very important,

Reticulocyte count (Retic count) Red cells are formed in the bone marrow. As the cells matures, it goes through several stages. The last one prior to leaving the marrow as a mature red cells is called the reticulocyte. Counting reticulocytes can correlate with the ability of the marrow to produce red cells. Elevated retic’s may mean that he marrow is capable of producing increased amounts of red cells. Decreased retic counts may mean that there is some damage to the marrow’s red cell making apparatus. This test aids in the diagnosis of anemia and is an indicator of response to therapy for anemia. Either blood from a lavender topped tube or fingerstick can be used. Reticulocyte counts may be done manually at the microscope or by automated methods. The normal range is approximately 0.5 - 2.0 %, but varies with age and population.

Sedimentation Rate (ESR, Sed rate)

The erythrocyte sedimentation rate is a non-specific indicator of inflammation. It is measured by the degree of settling of red blood cells in a specific time period, usually one hour. There are several methods for determination of the ESR; the most common are the Wintrobe and the Westergren, named for the developers of the procedure. Blood for these procedures is drawn into either a lavender or blue top tube, depending on the procedure. There is also an automated method for determining the sedimentation rate. There is no special patient preparation.

ESR are best used when comparing changes over time. A single test does not give much usable information. Some things that can cause an elevated ESR include exercise, arthritis, rheumatic fever, myocardial infarct (MI), infections, some malignancies, menstruation, and normal pregnancy after the third month. The normal range varies by the method used.

Thrombosis, Warfarin, and Protimes

Thrombosis is the unexpected development of a blood clot in a vein or an artery that plugs the vessel. Blood in the blocked vessel cannot reach critical tissue, and the tissue dies. Thrombosis can occur in arteries, causing heart attacks and strokes, or in veins, causing thrombophlebitis, deep vein thrombosis, and pulmonary emboli (see table 1).

Table 1: Types of Thrombosis

Common Name	Clinical Name	Site of Clot	Type
Heart attack	Coronary thrombosis causing myocardial infarction	Coronary arteries that nourish the heart	Arterial
Stroke	Cerebrovascular accident	Cerebral arteries that nourish the brain	Arterial
Thrombophlebitis	Superficial venous thrombosis	Superficial leg veins	Venous
	Deep venous thrombosis	Deep leg veins that return blood to the heart	Venous
Pulmonary embolism	Pulmonary thrombotic embolus	Clots from deep leg veins travel to the lung and block arteries	Venous

Thrombosis is a major affliction of humankind. In the USA, at least one in a thousand people suffers a venous thrombotic event every year. The real number could be even higher since the symptoms of pulmonary emboli resemble other disorders and often go undiagnosed. Further, each year there are 500,000 deaths from heart attacks and 500,000 strokes resulting in 100,000 deaths. Those who survive strokes are often faced with severe disabilities.

People with certain conditions have increased thrombosis risk requiring preventive treatment. Atrial fibrillation, diabetes, cancer, autoimmune diseases like systemic lupus erythematosis (SLE), knee and hip surgery, and chronic inflammatory conditions all may lead to thrombosis if no anticoagulant therapy is given.

Clot-dissolving Drugs are Used First to Stop Thrombosis

When a thrombosis victim arrives at the hospital, the doctor stops the clotting process and prevents additional damage by directing a clot-dissolving drug to the clot via cardiac catheterization. The three "clotbusters" used in the USA are tissue plasminogen activator (TPA), streptokinase, and urokinase. All three are effective at rapidly reestablishing blood flow, but, without long-term treatment, the injured spot in the vessel may clot again. Repeat clotting, called rethrombosis, is life-threatening, so the doctor must start anticoagulant therapy soon after the clot-dissolving therapy is completed.

Anticoagulants Prevent Rethrombosis

Table 2 Oral Anticoagulant; USA Trademarks

• Warfarin
• Warfarin sodium (generic)
• Coumarin
• Panwarfin
• Sofarin
• Coumadin
• Dicumarol

The Protime Test

Coumadin’s dosage must be carefully controlled because the safe therapeutic target range is narrow. Each patient needs a different amount depending on their weight, diet, general health, and activity level. People taking Coumadin must adhere very closely to their dosages and schedule, and must have blood collected at regular intervals for "protime" tests. The word protime is a contraction of prothrombin time, or "PT," a test of blood clotting that measures the effects of Coumadin in the blood. The name comes from prothrombin, which is another name for coagulation factor II.

Here’s how the protime works. The laboratory scientist collects a small blood specimen in a tube with an anticoagulant that keeps the blood from clotting. In the laboratory she separates the blood cells from the liquid portion by centrifuging the specimen. She then precisely measures a small amount of plasma (that’s the liquid portion of blood), adds a chemical called "thromboplastin" and measures how long it takes for the plasma to clot. The time interval from addition of thromboplastin to clotting is called the prothrombin time. For normal individuals the protime result is about 12 seconds, but in people taking Coumadin it is longer, up to 20 to 25 seconds.

Protime Variations

In a busy laboratory, scientists may perform 200 or 300 protimes a day using automated instruments. Protime results vary from laboratory to laboratory depending on the type of instrument, the brand of thromboplastin used, and the operator’s technique. A patient taking Coumadin could, on one day, have a protime of 19 seconds at one laboratory, 21.5 at another, and 23 seconds at a third. If nothing were done about this variation, a person would have to always have their protimes done at the same laboratory each time to avoid repeated, and possibly erroneous, dosage adjustments.

In the 1980s, laboratory scientists learned to minimize the protime inter-lab variation problem by developing the international normalized ratio (INR). Every laboratory compares their protime results to an international standard and, using a mathematical formula, reports the product as an INR number. For example, blood from a person taking no Coumadin would have an INR near 1.0, whereas a person taking Coumadin should have an INR in the therapeutic target range of 2-3. The therapeutic range is extremely important. If a person who needs Coumadin has an INR result below 2, the dosage is too low, and there is a risk of rethrombosis. INRs between 3 and 3.5 are relatively safe, but above that, hemorrhage is likely. The doctor reviews the INR each time a protime is done, and adjusts the Coumadin dosage to keep the INR in the therapeutic target range throughout the period of therapy.

Unexpected Protime Variations

Laboratory scientists have shown that all INR results vary slightly, so doctors do not usually adjust the dosage if successive results are within 15% of each other. Once in a while, however, a more extreme variation may be seen. Why? The most likely cause is a skipped or mistimed pill. It is important for the patient to take their Coumadin pills at the times and dosages prescribed, usually around 5 milligrams per day. If a pill is accidentally missed, the patient is instructed to take it as soon as he realizes the error and get a new protime done within the next few days. Diet may also cause variation. Lettuce, cabbage, broccoli, greens, and liver contain high vitamin K concentrations and can reduce Coumadin’s effectiveness. Most people eat more fresh vegetables in the summer when they are readily available. Their INR drops, reflecting their dietary change (table 4).

Table 4: Causes of Protime (INR) Variability

• Missed dosage
• Dietary changes: foods high in vitamin K
• Blood collection variations: short draw, clots, high temperature, delay cause high INRs
• Blood collection variations: long draw, prolonged chilling, shaking cause low INRs

Blood collection errors also cause protime changes. The laboratory scientist must ensure that the blood reaches the collection tube "fill line" and must gently mix it within seconds of the time it is collected to make sure it does not clot. "Short draws" or clotted blood both give erroneously high INRs. Blood that is transported at temperatures above 80° Fahrenheit or stored at room temperature for more than 24 hours also causes high INRs. On the other hand, if the scientist overfills the blood collection tube, or the blood is stored in the refrigerator for more than 24 hours, the INR will be falsely low. And if the blood is shaken too vigorously in mixing, its cells may rupture, also causing false low values. If there is any suspicion that a collection error has occurred, the test should be repeated before the dosage is adjusted.

Summary

Coumadin therapy has saved many lives and is essential for the prevention of rethrombosis after a thrombotic event. It is also used to prevent thrombosis in high risk individuals. Coumadin is safe and effective, but it has a narrow therapeutic dosage range that requires regular laboratory monitoring. The protime test is the standard test of therapeutic effectiveness, and Coumadin dosages are adjusted to keep the protime within the therapeutic range, an INR of 2 to 3. Although protime results are normalized around the world through the application of the INR, patients, laboratory scientists, and doctors must watch carefully for variations caused by changes in dosage, diet, or specimen collection errors.

Back to Lab Testing Home Page