Lab Tests For Anemias

There are three cardinal Red Cell Measurements that determine anemia: RBCs (red blood cell count), hemoglobin levels, and hematocrit levels.

At first glance, it may seem that these three are redundant since low levels of all three indicate anemia when levels fall and a correction when they rise. While all three tend to run parallel, they are not always straight.

For example, in one type of anemia, RBCs may be lower than would be expected for observed levels of hemoglobin. This happens in macroctic anemias. To further distinguish problems, three calculated Red Cell Indices have been devised.

These are MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), and MCHC (mean corpuscular hemoglobin concentration).

Iron Deficiency Anemia Testing
The most common tests measure the number and size of the red blood cells and the cells’ hemoglobin content. A sensitive test that will detect a developing iron deficiency before it is full-blown, it measures the amount of transferrin in the blood and the amount of iron it is carrying.

At the very beginning of an iron deficiency, before these levels fall, the transferrin concentration rises for a short time. Other tests measure iron stores. Iron stores are measured directly (by bone marrow biopsy) or indirectly (by blood tests that measure serum ferritin, transferrin saturation, or total iron-binding capacity).

A sensitive indicator of heme synthesis is the amount of free erythrocyte protoporphyrin (FEP) within erythrocytes. However, the results of these tests can be misleading because of such complicating factors as infection, pneumonia, a blood transfusion, or iron supplements. Bone marrow studies may reveal depleted or absent iron stores (done by staining) and normoblastic hyperplasia.

Diagnosis must rule out such other forms of anemia as that from thalassemia minor, malignancy, and chronic inflammatory, hepatic and renal disease.

Characterisitic blood study results that could indicate anemia include the following:

• low hemoglobin levels (males, low hematocrit levels (males, low serum iron levels, with high binding capacity
• low serum ferritin levels
• low RBC count, with microcytic and hypochromic cells (in early stages, RBC count may be normal except in infants and children)
• decreased MCV

RBC count: Normally, there are 4.7 million to 6.1 million red cells per microliter of adult males. For females, the normal range is 4.2-5.4 million/µl. The problem with using only this test for anemia is that all red cells are not equal. In the various types of anemia and other hematologic conditions, the size and weight of each red cell can vary markedly. Sometimes, the number of cells is normal; but the mass of each cell may be abnormally low, indicating anemia despite a normal RBC count.

Hemoglobin concentration: It is very difficult to measure the concentration in individual red cells, so the first step is to ‘pop’ all the red cells and let the hemoglobin flow out into the blood sample where it becomes evenly distributed in the plasma. This process is called hemolysis (hemo meaning blood and lysis meaning destruction).

Since hemoglobin is a colored substance, its concentration can easily be measured by shining a light through the thickness of the specimen to determine how much light is blocked. This process is called ‘colorimetry’. The lower the concentration of the colored substance, the more light shines through. The only problem is that, when hemoglobin is released, it goes berserk chemically and starts to break down, losing some of its light-absorbing qualities. To offset this problem, the sample is chemically transformed into a substance called cyanmethemoglobin. It is this solution that is analyzed by colorimetry; and, after a few mathematical calculations, the original concentration of hemoglobin can be determined.

• For males, the normal level is 14-18 grams per deciliter of blood (a deciliter is 1/10 of a liter)
• For females, the normal level is 12-16 g/dL

However, most practitioners use the lower figure of 12 for both men and women when giving a diagnosis of anemia. But not every doctor does this. Some doctors allow elderly people a lower level without initiating further diagnostic workups. Since anemia is common in the elderly, this practice needs to be revised.

Hematocrit (PCV): The hematocrit is defined as the fraction of the volume of whole blood occupied by the red blood cells and is a measurement of volume, not mass. Another name is Packed Cell Volume (PCV), expressed in a percentage of the total blood volume. To determine this, a clinician spins a volume of blood in a centrifuge to separate the red blood cells from the plasma. Low values indicate a reduced number of red blood cells, and thus anemia. Normal levels are as follows:

• Adult men: 40%-54%
• Adult women: 37%-47%
• Children ages 2-5: 34%
• Children ages 6-12: 37%

MCV: Mean corpuscular volume is a direct or calculated measure to determine the average size of a red blood cell. This is calculated by dividing the hematocrit by the RBC count and multiplying by a unit conversion factor. Such a measure helps to classify the type of anemia. The reference range for MCV is 80-94 femtoliters (a femtoliter is one quadrillionth of a liter), commonly abbreviated 80-94 fL. In iron deficiency anemia, the red blood cells are smaller than average (microcytic). In folate and B12 deficiencies, the red blood cells are larger than average (macrocytic). One in which the MCV falls within the reference range is a normocytic anemia.

MCH: Mean corpuscular hemoglobin is defined as the average mass of hemoglobin in each red cell. This is calculated by dividing the hemoglobin concentration of whole blood by the RBC count and multiplying by a unit conversion factor. The reference range for MCH is 27-31 picograms (a picogram is one trillionth of a gram), commonly abbreviated 27-31 pg. This is technically not a very important red cell index because it tends to parallel the MCV.

MCHC: Mean corpuscular hemoglobin concentration is the average concentration of hemoglobin in the red cells. It is calculated by dividing the whole blood hemoglobin concentration by the hematocrit and applying a unit conversion factor. The reference range is 32 to 36 grams per deciliter, abbreviated 32-36 g/dL. When the level falls below the normal range, it is classified as hypochromic anemia (very rare). If the level is higher than the normal range, it is hyperchromic anemia and if the MCHC is within the reference range, it is called normochromic anemia.

Sickle Cell Testing
Infant screening is done with blood samples collected at birth from the umbelical cord or from a heel prick. However, testing is not usually done before the child is eighteen weeks old unless there is a high risk for sickle cell disease or anemia. Blood samples are analyzed to see what kinds of hemoglobin the infant is producing. If abnormal hemoglobin is found, the physician will want to have the DNA of the sample analyzed for a genetic diagnosis. Often, the testing procedure is repeated – starting with a new blood sample – to make sure that the results are accurate. If commercial laboratories do the testing, the procedures they generally use are as follows:

• high performance liquid chromatography (often this is the testing method used first to identify which type of hemoglobin is present. These results will be confirmed with the hemoglobin electrophoresis method)
• isoelectric focusing (identifies hemoglobin bands by their migration in an electric field and abnormal specimens are presumed positive for sickle cell disease and will be suject to further testing)
• hemoglobin electrophoresis

If the sickle cell trait is evident, a CBC (complete blood count) will show the following:

• RBCs (red blood cells) will be low and WBC (white blood cells) will be elevated.
• Platelet counts and hemoglobin may be low or normal.
• Erythrocyte sedimentation rate is decreased.
• Serum iron is increased.
• RBC survival time is decreased.
• Reticulocyte count is increased.

Hemoglobin electrophoresis: Electrophoresis tests were the first technique used in the discovery of abnormal hemoglobin in those with sickle cell disease. Testing variations of this type was developed by Dr. Linus Pauling and still widely used today to diagnose sickle cell disease. This test is not only able to determine if a patient has the disease or the trait, but it can also indicate if the person has such related blood disorders as thalassemia, hemoglobin C, or hemoglobin D. Electrophoresis testing used to be very time-consuming and expensive, although it can usually be done now in under an hour. However, improvements have made it quicker, simpler, less costly, and more reliable.

The electrophoresis equipment consists of two tanks with a strip of a solid material stretched between them. The solid material may be made from cellulose acetate and looks like a strip of paper; or it can consist of a thin layer of agarose, a seaweed extract, placed on a plastic sheet. Electrodes are placed into each tank and the tanks filled with a buffered solution. An electrical circuit is formed that conducts current from one electrode, through a buffer, across the solid material, into the other tank of buffer, and finally into the second electrode.

If a solution of hemoglobin is placed on the solid strip and a voltage applied to the electrode, the hemoglobin molecules will move toward the negative electrode. When the blood of a person with sickle-cell anemia is analyzed next to that of a normal individual, the hemoglobins are seen to migrate to different locations. The sickle cell hemoglobin has a greater positive charge than normal hemoglobin and will move toward the negative electrode at a faster rate. The blood of a person with sickle cell trait has two different hemoglobins: one moves like the normal molecule while the other migrates like that of a person with sickle cell anemia.

TechniCon system: It is an automated test. A drop of blood is put into a little cup and mixed with a special chemical formula. The machine reads the solution by making a tracing on graph paper. If the person has sickle cell disease or sickle cell trait, the line makes a tall peak. If the test comes out positive, a second test can be done to indicate whether the person has the disease or the trait. In the second test, the height of the peak depends on how much sickle hemoglobin there is. The peak observed for a person with sickle cell disease will be twice as high as the peak for someone with sickle cell trait.

Transcranial Doppler imager: It is an ultrasound device which allows doctors to try to prevent strokes in children by using this test to identify which children have the greatest risk. It has been found that changes occur in the brain blood vessels of many children with sickle cell disease. The arteries gradually narrow, making them more likely to become clogged, causing the blood cells to sickle. Such plugs of blood cells could cut off blood flow to parts of the brain, resulting in death of brain tissue (a stroke). The Doppler imager shows the narrowed areas that are potential trouble spots. This allows the doctor to be better able to identify patients who are at a greater risk for strokes and can treat them accordingly.

Strokes are the most serious side effect among children with sickle cell disease. Studies have found up to 17% of sickle cell patients have strokes which occur most often in childhood and adolescence. The result may be learning disabilities, paralysis, or even death.

Tests not reliable: Two unreliable tests are the metabisulfite test and the solubility test. They are not sensitive enough to distinguish between sickle cell disease and sickle cell trait.

Metabisulfite test: Although the sodium metabisulfite test is fairly simple and rather inexpensive to perform, it is not reliable. A drop of blood is put onto a glass slide with a drop of the chemical sodium metabisulfite. The slide is covered with a coverslip and sealed with Vaseline to keep out oxygen. After a short time, the technician checks for sickling. In addition to being unable to distinguish between sickle cell disease and sickle cell trait, this test may also produce false negatives and false positives, as well as being unreliable in testing newborns.

Solubility test: The sickledex is another unreliable method used in large-scale testing programs. A drop of blood is put in a test tube along with several chemicals that cause the outer membrane of the red blood cells to burst, allowing their contents to leak out (a process known as hemolysis). Normal hemoglobin dissolves readily in the solution in the test tube, but hemoglobin S is less soluble. Therefore, if the fluid turns cloudy after a short time, it means sickle cells are present. If the fluid is clear, the blood is normal. Like the sodium metabisulfite test, major disadvantages of the Sickledex test are: it cannot distinguish between the sickle cell disease and the sickle cell trait; and, it is expensive and unreliable for newborns.

The dithionite test: It is a variation of the sickledex test. It uses the same chemicals as the Sickledex test, but everything is automated. Since the test tube readings are done by machine, the process is faster and less costly; but, like the other two tests, it cannot be used to screen newborns. Even babies with two sickle cell genes have too little HbS in their blood to give a positive reading. During fetal development and for a short time after birth, the infant’s blood contains mainly fetal hemoglobin (HbF), in which two alpha chains are combined with two gamma globin chains instead of two beta chains.

Hemolytic Anemia Testing
One or more lab tests are generated in order to distinguish between anemias caused by lowered cell production (hyporegenerative anemias) or by shortened red cell life span (hemolytic anemias). This distinction is made through one or more of the following means.

Reticulocyte count: This is the most important test in the initial classification of a normocytic anemia. Hemolytic anemias are usually normocytic normochromic, meaning that the average red cell is of normal size and concentration of hemoglobin, which is not surprising since production is completely normal, too. The problem is that something is causing these normal red cells to be destroyed much faster than they can be replaced. The result is that the number of immature reticulocytes in the blood rises severalfold above the normal level of less than 1.5%. In fact, in a severe case of hemolytic anemia, the retic count may be as high as 20%. This test is highly reliable, inexpensive, and easily done in even the most primitive labs. Despite this, many physicians fail to make use of it.

Blood smear examination: In some types of hemolytic anemia, the shape of the red cells is abnormal. Their appearance can easily be assessed through a peripheral smear examination. A drop of blood is placed on a glass microscope slide, and the edge of another slide is drawn into the drop. Just before the drop has spread over the interface between the edge of the second slide and the surface of the first slide, the second slide is smartly scraped over the length of the first slide. The result is a flame-shaped smear on the first slide. The end of the smear at which the blood was first dropped is too thick to examine; but, out toward the feathered edge at the opposite end, the smear is just thin enough to allow the cells to be seen. The technique of creating smears is actually quite difficult, and it takes quite a bit of practice by technicians before they are considered proficient. Since nothing can be seen on an unstained smear, one of the Romanowsky stains (usually Wright) is employed to make the cells visible. Reticuloyces cannot be distinguished from other red cells on the Wright stain, however; so the next step is to examine them microscopically at two levels of magnification (100X and 1000X). The lower power is for screening the smear for rare abnormal cells and the higher one is for discerning the details of the cells. With rare exception, the only people truly qualified to evaluate a blood smear thoroughly are hematologists and laboratorians.

Serum bilirubin level: Because of the accelerated destruction of red cells in hemolytic anemia, heme is broken down more rapidly. Unconjugated (indirect) bilirubin accumulates in the plasma because the liver, that normally collects, conjugates, and excretes it, is overwhelmed. The level of bilirubin is measured in serum with a very simple, routine test. If the level is high enough (about 2 mg/dL), clinical jaundice is usually noticed. In hemolytic anemias where the liver function is normal, the serum bilirubin rarely exceeds 7 mg/dL. Much higher levels (up to 50 mg/dL) may be observed in patients with diseases of the liver or bile ducts. Because jaundice is seen in these diseases, as well as in hemolytic anemias, the doctor is interested in knowing the relative proportions of conjugated (direct) and unconjugated bilirubin in the serum. In liver and biliary tract disease, there is more conjugated than unconjugated bilirubin in the serum. In hemolytic anemia, the reverse is true. Thus, the serum bilirubin test typically yields three results: total, indirect, and direct bilirubin.

Urine urobilinogen: This is part of the routine urinalysis dipstick test. In the hemolytic state, the liver churns out conjugated bilirubin at full tilt. Some of this is eventually converted to urobilinogen in bowel bacteria, reabsorbed, and excreted in the urine, where increased amounts are detected in the dipstick test.

Serum haptoglobin level: Because of the sloppy spilling of hemoglobin by RES (reticuloendothelial system) while it disposes of red cells, in times of brisk hemolysis, more hemoglobin is spilled. This is quickly turned into haptoglobin and cleared from the plasma by the RES. If hemolysis is rapid enough, all of the haptoglobin is used up and undetectable in the serum by a lab test. Therefore, the serum haptoglobin can be used as a measure of hemolysis – the lower the serum haptoglobin, the worse the hemolysis.

Urine hemoglobin: Normally, the kidneys do such a good job in keeping red cells from falling out of the blood vessels that no hemoglobin is detectable in the urine. The routine dipstick urinalysis can detect any hemoglobin that escapes into the urine in the course of brisk hemolysis. A positive hemoglobin test by dipstick is not an indication of hemoglobinuria however, since any of a number of conditions can cause blood in the urine, including simple urinary tract infections.

Pernicious Anemia Testing
Blood results that suggest pernicious anemia include the following:

• decreased hemoglobin (4 to 5 g/100ml) and decreased RBCs;
• increased mean corpuscular volume (MCV>120), mainly because larger-than-normal RBCs each contain increased amounts of hemoglobin, meaning corpuscular hemoglobin concentration is also increased;
• possible low WBC, low platelet counts, and large, malformed platelets;
• serum vitamin B12 assay levels elevated serum LDH.

Bone marrow aspiration reveals erythroid hyperplasia (crowded red bone marrow) with an increased number of megaloblasts and only a few normally developing RBCs. Gastric analysis shows the absence of free hydrochloric acid after histamine or pentagastrin injection.

Schilling test is the definitive test for pernicious anemia. In this test, the patient fasts for 12 hours and then receives a small oral dose (0.5 to 2 mcg) of radioactive vitamin B12. Two hours later, a larger dose (1 mg) of nonradioactive vitamin B12 is given IM, as a parenteral flush; and the radioactivity of a 24-hour urine specimen is measured. About 7% of the radioactive B12 dose is excreted in the first 24 hours; but those with pernicious anemia will excrete less than 3%, leaving the remainder unabsorbed and it passes into the stool. When the Schilling test is repeated with IF added, the test shows normal excretion of vitamin B12. Important serologic findings may include IF antibodies and antiparietal cell antibodies.

Thalassemia Testing
To differentiate between iron deficiency and thalassemia, RBC indices can be helpful. The hemoglobin concentration will generally be lower in iron deficiency, but the distinguishing factors will be in elevated HbA and HbF. These will be elevated in beta thalassemia but normal or decreased in alpha thalassemia or iron deficiency anemia.

The Mentzer Index was developed to help distinguish these two anemias. It is calculated by dividing the RBC count into the MCV. If the quotient is less than 13, thalassemia is more likely; but if the quotient is greater than 13, it is more likely to indicate iron deficiency.

• Thalassemia major: RBCs and hemoglobin are decreased; reticulocytes, bilirubin, and urinary and fecal urobilinogen are elevated; low serum folate level, indicating increased folate utilization by the hypertrophied bone marrow. Peripheral blood smear reveals target cells (extremely thin and fragile RBCs), microcytes, pale nucleated RBCs, and marked anisocytosis. Skull and skeletal x-rays show a thinning and widening of the marrow space in the skull and long bones, possible granular appearance in the bones of the skull and vertebrae, possible areas of osteoporosis in the long bones, and deformities (rectangular or biconvex) of the phalanges. Quantitative hemoglobin studies show a significant rise in Hb F and a slight increase in Hb A2. Diagnosis must rule out iron deficiency anemia, which also produces slightly lowered hemoglobin (hypochromia) and notably small (microcytic) RBCs.
• Thalassemia intermedia: RBCs are hypochromic and microcytic, but the anemia is less severe than that in thalassemia major.
• Thalassemia minor: RBCs are slightly hypochromic and microcytic. Quantitative hemoglobin studies show a significant increase in HbA levels and a moderate rise in HbF levels.