Hemoglobin Information and Courses from MediaLab, Inc.

Thalassemia is best thought of as a group of disorders rather than a single disease. They demonstrate a hemoglobin synthesis disorder in which there exists a defect in the rate of production of one or more of the globin chains. This defect results from either a heterozygous or homozygous deletion or inactivation of a globin chain gene.

In thalassemia there is often an excess production or accumulation of globin chains produced by genes that are not effected by the thalassemia deletion. In alpha thalassemia this may be seen as gamma chain tetramers (hemoglobin Bart's) in the unborn child and as beta chain tetramers (hemoglobin H) in adults. Tetramer accumulation often leads to red blood cell damage and hemolytic anemia.

Alpha thalassemia intermedia (Hemoglobin H Disease) results from a deletion of three out of four alpha chain loci. Infants born with alpha thalassemia intermedia appear normal at birth but often develop anemia and splenomegaly by the end of their first year. Hepatomegaly is not a common finding and there may be some association with mental retardation. Due to the hemolytic nature of this anemia, there may be an increase in respiratory infections, leg ulcers and gallstones. Skeletal changes are not commonly seen in hemoglobin H disease. Every ethnic group can have occurrences of hemoglobin H disease; but it is most often seen in Southeast Asian, the Middle East and the Mediterranean islands. Development and life expectancy are usually normal, but some affected individuals may require splenectomy and transfusion therapy.

Chromosome 16 contains the genetic codes for the zeta and alpha hemoglobin chains.Each chromosome has two loci alpha chains 1 and 2. This equals a total of four loci of material coding for the alpha hemoglobin chain. See the image for a visual representation of these loci.In the genotypic notation of alpha thalassemia an "" represents the presence of an alpha locus. A "-" represents a deletion of a locus.The notation for the normal number of alpha loci is /. The amount of Hb A produced by this normal gene is 97-98 %.(drawing modified from Harmening, 1999)

When three loci of alpha chains are deleted (--/-) or inactive, only 70-90% of Hemoglobin A is made. The excess beta chains that remain unpaired form the tetramers of Hemoglobin H.(drawing modified from Harmening, 1999)

After considering the results of the brilliant cresyl blue stain, the clinical laboratory scientist decided to repeat the hemoglobin electrophoresis on this patient. This time, she shortened the electrophoresis time by fifteen minutes.The results of the electrophoresis, represented in the image below, show a band in the area of Hb H. Hemoglobin H travels quickly during alkaline electrophoresis, and a shorter electrophoresis time was needed to ensure that HbH remained on the acetate paper. HbF is still present as it was on the original electrophoresis, but it is blended into the Hb A band.

Anemia is mild to absent.RBC count is increased.Hb is slightly decreased.MCV is decreased. MCHC is slightly decreased.RDW is normal to slightly increased.Red Blood Cell morphology shows slight hypochromic microcytosis.Reticulocytes are normal to slightly increased.Hb electrophoresis demonstrates a normal pattern in adults:Hb A - 97-98% Hb A2 - 1-2.5% Hb F - <1%. Neonates have 5-15% Bart's Hemoglobin (gamma chain tetramers).Hb H inclusions are rarely seen.Bone marrow demonstrates erythroid hyperplasia.

Bilirubin is formed as a result of hemoglobin degradation. Normally, senescent red blood cells are removed from circulation and the bilirubin that is formed is processed by the liver. The normal level of bilirubin in the serum of adults is 0.2-1mg/dl. Bilirubin levels increase with liver disorders and also in anemia that is a result of a hemolytic process. Patients may display jaundice when serum bilirubin levels exceed 2mg/dl.Persons with alpha thalassemia intermedia usually have an increased bilirubin level, because of ongoing hemolysis. This bilirubin is typically the unconjugated fraction of bilirubin.

Hemoglobin electrophoresis is the movement of hemoglobin proteins in an electric field at a fixed pH.Because the various hemoglobins are comprised of different combinations of globin chains (normal or abnormal), they will demonstrate different degrees of mobility. Typically, when a thalassemia or hemoglobinopathy is suspected, an alkaline electrophoresis is performed which may be confirmed with acid electrophoresis.For an alkaline hemoglobin electrophoresis, a hemolysate is applied to cellulose acetate which is electrophoresed in a buffer at pH 8.4-8.6. At this pH hemoglobin proteins move from cathode to anode. The proteins are visualized by the application of a dye which also makes them measurable by densitometry.

Reading from cathode to anode (left to right): #1 slight amount of Hb A2, mostly Hb A #2 near equal amounts of Hb C and Hb A #3 Hb A and Hb H #4 Hb A2, Hb S and Hb A #5 control specimen Hb F and Hb A #6 control specimen Hb C, Hb S and Hb A

A densitometer tracing of the hemoglobin electrophoresis gel is made in order to quantitate the bands present. Below is a normal gel and its corresponding tracing. Hemoglobin A is 98% and A2 is 1.5%. Notice that Hemoglobin F is usually present in small enough amounts that its tracing may blend into that of Hemoglobin A. A separate procedure may need to be performed if quantitation of HbF is important.

Hemoglobin H, consisting of beta chain tetramers, is an unstable hemoglobin which forms precipitates just below the cell's membrane and can be observed when red blood cells are stained with Brilliant Cresyl Blue (BCB).

Cells in which unstable hemoglobin is not present or has not yet precipitated will appear to have a smooth surface.

Thalassemias are part of a group of hemoglobin synthesis disorders in which a defect exists in the rate of production of one or more of the globin chains. This defect results from either a heterozygous or homozygous deletion or inactivation of a globin chain gene.Thalassemias are named according to the affected gene or the globin chain that is showing reduced or absent synthesis.Globin chain loci are found on: chromosome 11 (beta, delta, epsilon, and gamma) chromosome 16 (alpha, and zeta)

Beta thalassemia major, B0/B0 (two gene mutations, deletions or combination) results in very few to no beta chains being produced.Hemoglobin A levels are at or near 0%.(drawing modified from Harmening, 1999)

In Beta thalassemia minor, B0/B, one beta gene locus is completely deleted or inactive.Hemoglobin A production is down to 70% - 85% in this state of beta thalassemia.(drawing modified from Harmening, 1999)

Delta-beta thalassemia intermedia exists when both gene loci for beta and delta chains are deleted or inactive on one chromosome, while the other chromosome contains a beta chain gene that is partially deleted or inactive. Delta-Beta 0/ Beta+sIn this state the majority of hemoglobin will be Hb F, with very little Hb A and A2 present.(drawing modified from Harmening, 1999)

Bilirubin is formed as a result of hemoglobin degradation. Normally, senescent red blood cells are removed from circulation and the bilirubin that is formed is processed by the liver. The normal level of serum bilirubin for adults is 0.2-1mg/dL.Bilirubin levels increase with some liver disorders and also in anemia that is a result of a hemolytic process. Patients may display jaundice when serum bilirubin levels exceed 2mg/dL.Persons with beta thalassemia major usually have an increased bilirubin level. This bilirubin is typically the unconjugated fraction of bilirubin.

Of the hemoglobins normally present in an adult, Hb A migrates the fastest, followed by Hb F. Hb A2 moves only slightly from the point of origin near the cathode.Abnormal hemoglobins show the following migration patterns:Hb C migrates with Hb A2 near the cathode.Hb S lies between Hb A2 and Hb F.Hb H and Bart's hemoglobin are unstable and very fast moving, with Hb H being the faster of the two. They are located nearer the anode past Hb A .Relative migrations of hemoglobin variants on alkaline electrophoresis can be seen below.

This densitometer tracing corresponds to the pattern for normal distribution of hemoglobin.

The laboratory findings in this case represent classic findings seen in beta thalassemia minor including: erythrocytosis, decreased hemoglobin, normal hematocrit, normal RDW, and the presence of codocytes (target cells). This patient does have a mild anemia, but some patients with beta thalassemia minor have no anemia. Hemoglobin electrophoresis confirms this diagnosis, showing an increased Hb A2 level and decreased Hb A.In addition, the slightly increased iron and slightly decreased TIBC contradict a suspicion of iron deficiency. These chemistry results are typical for beta thalassemia, even though the red blood cells are microcytic and hypochromic.

Bilirubin, a product of hemoglobin breakdown, is characterized by its yellow pigment. The presence of bilirubin in urine is always abnormal. It is important to note that unconjugated bilirubin cannot be excreted by the kidneys because it is bound to albumin and is not soluble in water. In the liver, bilirubin combines with glucuronic acid through the action of a glucuronyl transferase to form water soluble bilirubin diglucuronide. Under normal circumstances, conjugated bilirubin passes from the bile duct and then to the intestinal tract. Intestinal bacteria reduce conjugated bilirubin to urobilinogen. Approximately half of the urobilinogen is excreted in the feces; most of the other half is recirculated through the liver. A small amount of urobilinogen bypasses the liver and is excreted in the urine.

The test for blood on the urine reagent strip is based on the peroxidase-like activity of hemoglobin which catalyzes the reaction of cumene hydroperoxide and 3, 3', 5, 5' tetramethylbenzidine. The test is sensitive to free hemoglobin, myoglobin and a minimum of 5 intact red cells per microliter of urine.

No blood is found in the urine of healthy individuals although samples from menstruating females, frequently, but not always, test positive for blood. Hematuria is associated with renal or genital urinary disorders in which the bleeding is the result of irritation to the involved organs or trauma. Examples include renal calculi, pyelonephritis, glomerulonephritis, tumors, trauma or exposure to toxic chemicals or drugs and/or strenuous exercise. Hemoglobinuria may be due to the lysis of red cells within the urinary tract. If it is caused by intravascular hemolysis, the hemoglobin is then filtered through the glomeruli. In the normal individual, the hemoglobin molecule attaches to haptoglobin and in this way bypasses the kidney filtration system. When the hemoglobin/haptoglobin system is overwhelmed, as in cases of hemolytic anemia, severe burns, transfusion reaction, infection or strenuous exercise, hemoglobin passes into the urine.

A false positive urobilinogen reaction may occur with the dipstick method when substances known to react with Ehrlich's reagent such as sulfonamides and p-aminosalicylic acid are present in the urine. Drugs that contain Azo dyes, such as Azo Gantrisin®, have a gold color that masks the reaction, causing a false positive reaction. Atypical color reactions may be obtained in the presence of high concentrations of p-aminobenzoic acid. The dipstick urobilinogen test cannot detect porphobilinogen in a urine specimen. Porphobilinogen is a molecule formed during the synthesis of the heme portion of hemoglobin.

Bilirubin is formed as a result of the breakdown of hemoglobin from erythrocytes in the reticuloendothelial system. It becomes bound to albumin and transported through the blood to the liver. This free or unconjugated bilirubin is insoluble in water and cannot be filtered through the glomerulus of the kidney. In the liver, bilirubin becomes conjugated with glucuronic acid to form bilirubin diglucuronide. This conjugated bilirubin, which is also called direct bilirubin, is water soluble and is excreted by the liver through the bile duct and into the duodenum.

A 63 year old man was seen in the emergency room with the complaints of sudden onset of fever, chills, and abdominal pain, accompanied by mild diarrhea. The blood pressure was 140/84, the pulse rate 82/minute, and the body temperature 39.8C. A blood sample was drawn for a complete blood count, and a blood culture.A second blood culture was drawn from the opposite arm, with 10 ml of blood being placed into each an aerobic and an anaerobic bottle, following customary practice.The complete blood count revealed a hemoglobin of 15.8 mg/dl, a hematocrit of 45%, and a white blood count of 4.2/L. The neutrophils were 39%, lymphocytes 45%, monocytes 10%, eosinophils 4% and basophils 2%. The platelet count was 255/L. The patient was admitted to the hospital for further work-up and empiric antibiotic therapy.Within 24 hours after admission, the body temperature had decreased to 38.2C, although the mild diarrhea persisted.A stool toxin test for Clostridium difficile was negative and neither enteric pathogens nor Campylobacter species were recovered in stool culture after 24 hours incubation. Fecal neutrophils were not seen on direct examination. The anaerobic blood culture became positive 36 hours after inoculation.

In statistics, a variable is any quantity that is a part of a data point. Variables can either be dependent or independent. An independent variable is a quantity that is directly controlled by the observer or experimenter. The dependent variable, as its name suggests, depends on the independent variable. The dependent variable is often the quantity you want to measure, and it the result of the experiment or test.For example, you may want to determine the relationship between hemoglobin concentration and age. You select people of various ages, and then test their hemoglobin concentrations. Age is the independent variable, and is controlled by the experimenter (you can select which ages are in the experiment). The dependent variable is the resulting hemoglobin concentration.In some cases, these criteria may not be useful in determining which variable should be the independent variable, such as determining the correlation between the readings given by two different instruments for the same samples. In that case, there might be other criteria for selecting the independent variable.

Examine the information in Table II and Figure 2, and see if you can answer the following questions: Which hemoglobin category contains the most observations? How many women had a hemoglobin between 12 and 13 gm/dL? How many women had a hemoglobin greater than 12 gm/dL? How many had a hemoglobin of greater than 13, but less than 15 gm/dL? Answers: the category 12 - 14 had the most observations, and 35 women (25+9+1) had a hemoglobin count greater than 12. However, questions 2 and 4 cannot be answered with the information presented.You can see that the process of grouping the information into categories can result in the loss of some information, but for most purposes the benefits of readability outweigh the information loss.

Agarose gels are chemically purified forms of agar, a polysaccharide extracted from seaweed. The gel pores allow for separation of proteins based on their individual charge and mass. Agarose gel will naturally clear after drying the separated proteins.Common clinical uses of agarose gel electrophoresis (AGE) are separations of plasma proteins, hemoglobin variants, lipoproteins, and isoenzymes. The gels come prepackaged with a plastic template to lay over gel for sample application or slots etched in the gel for these samples.

IEF's greatest advantage is its high resolution, resulting in greater separation of solutes. IEF of serum proteins results in many more bands; these bands are sharper because each pH region is very narrow. Performing IEF is easier because the placement of sample application is not important. The sample and ampholytes can be mixed before application; the ampholytes will migrate, create the gradient, and then the proteins separate and migrate.Some isoenzymes and variant hemoglobins in prenatal screening are separated with IEF. Detection of oligoclonal bands in gamma-globulin is a newer use of IEF. IEF is commonly used as one of the separations in two-dimensional electrophoresis.

Although the nucleus has been extruded, the reticulocyte is still considered immature because it retains numerous organelles needed for hemoglobin production, such as ribosomes, mitochondria, and fragments of the Golgi apparatus. The reticulocyte is slightly larger (10 microns) than the mature erythrocyte. A reticulocyte normally remains in the bone marrow for one or two days before entering the circulation and its final 24 hours of maturation. The red cell is mature when hemoglobin production is complete and the organelles have disintegrated. Reticulocytes normally make up 0.5 - 1.5% of the peripheral blood red cells. They appear blue/gray on the Wright's stained smear. The residual RNA in the cytoplasm causes the blue/gray color. The terms, polychromasia or polychromatophilic, are used to describe these cells on a Wright's stained preparation. A supravital stain such as new methylene blue N or brilliant cresyl blue is used to stain reticulocytes for an actual count.

1. Beutler E. Iron storage disease: Facts, fiction and progress. Blood Cells Mol Dis. 2007;39:140-7.2. Higgins T, Beutler E, Doumas BT. Hemoglobin, iron, and bilirubin. In: Burtis CA, editor. Teitz Fundamentals of Clinical Chemistry. 6th ed. Saunders Elsevier, 2008.3. Ganz T. Hepcidin, a key regulator of iron metabolism and mediator of anemia and inflammation. Blood 2003;102(3):78-8.4. Andrews NC, Schmidt PJ. Iron homeostasis. Annu Rev Physiolo. 2007;69:69-85.5. Murtagh LJ, Whiley M, Wilson S, et al. Unsaturated iron binding capacity and transferrin saturation are equally reliable in detection of HFE hemochromatosis. Am J Gastroenterol. 2002;97(8):2093-9.6. Haddy TB, Castro OL, Rana SR. Hereditary hemochromatosis in children, adolescents, and young adults. Am J Pediatr Hematol Oncol 1988;10:23-4.7. Edwards CQ, Ajoika RS, Kushner JP. Hemochromatosis: A genetic definition. In Barton JC, Edwards CQ, eds. Hemochromatosis: Genetics, Pathophysiology, Diagnosis and Treatment. Cambridge, UK:Cambridge Univ Pr 2000:8-11.8. Whitlock EP, Garlitz BA, Harris EL , et al. Screening for Hereditary Hemochromatosis: A Systematic Review for the U.S. Preventive Services Task Force. Ann Intern Med. 2006; 145: 209-23.9. Wallace DF, Subramaniam VN. Non-HFE haemaochromatosis. World J Gastroenterol. 2007;13(35):4690-8.10. Tavill AS. Diagnosis and management of hemochromatosis. Hepatology. 2001;33:1321-811. Qaseem A, Aronson M, Fitterman N, Snow V, Weiss KB, Owens DK, et al. Screening for hereditary hemochromatosis: a clinical practice guideline from the American College of Physicians. Ann Intern Med. 2005;143:517-21.12. Phatak PD, Bonkovsky HL, and Kowdley KV. Hereditary Hemochromatosis: time for targeted screening. Ann Intern Med. 2008; 149(4): 270 – 2.13. Brissot P, deBels F. Current approaches to the management of hemochromatosis. Hematology Am Soc Hematol Educ Program. 2006:36-41. 14. Guidance for industry: Variances for blood collection from individuals with hereditary hemochromatosis. http://www.fda.gov/cber/gdlns/hemchrom.htm Accessed 12/17/08.

Phlebotomy is considered the treatment of choice for patients with iron overload due to hereditary hemochromatosis (HH). Each unit of blood contains approximately 200 to 250 mg of iron. As erythrocytes are removed by phlebotomy, iron stores are mobilized and utilized in the production of new, circulating erythrocytes. Through periodic phlebotomies, stored iron is removed until iron-deficient erythropoiesis is induced. The initial, or iron reduction, phase of treatment typically consists of removing one unit (450 mL) of whole blood once or twice weekly. Prior to beginning phlebotomy, the patient’s hemoglobin and hematocrit must be checked to ensure that the patient is not anemic. A sample for serum ferritin is also collected at this time.Initial treatment goals include inducing iron deficient hematopoiesis without the development of debilitating symptoms of anemia. A hemoglobin concentration of 10.0 to 12.0 g/dL is often used as a target range. The initial treatment phase continues until excess stored iron is removed and ferritin levels decrease to approximately 50 ng/mL. (13) Ferritin and hemoglobin levels are periodically monitored during this phase. The number of phlebotomies needed to reduce iron levels and induce anemia is related to the degree of initial iron overload. Patients may be referred to a hematologist or gastroenterologist during the initial treatment phase. Many patients receive therapeutic phlebotomy services in a hospital or doctor’s office, but patients may also undergo phlebotomy at a blood center. Blood collected from persons with HH may be used for transfusion or as blood products if it has been collected from a facility with an approved variance from the US Food and Drug Administration. Not all blood centers have applied for or been granted this variance.(14)The initial treatment phase continues until excess stored iron is removed and ferritin levels decrease to approximately 50 ng/mL. Removal of excess stored iron may take from one month to three years.

Within the hematopoietic cords each cell line has a specific location for development. Erythroid precursors are located near a venous sinusoid and cluster around a macrophage. This is referred to as an erythroblastic island. Developing red cells obtain iron needed for hemoglobin production from macrophages. Megakaryocytes are also located close to a venous sinus. They extend their cytoplasm in fingerlike projections through the sinus wall in order to release their platelets directly into the blood in the sinus. Immature granulocytes lie within the hematopoietic cords. The metamyelocyte stage is the first stage of the granulocyte series that is motile and able to move toward the sinus area. Mature neutrophils, eosinophils and basophils enter the sinusoidal blood through the basement membrane. As maturing erythrocytes also move toward the sinus wall any remaining nuclei are lost as the red cells move through small openings in the cells lining the sinus wall.

Specialists in radioassay use radionuclides to determine the chemical makeup of body fluids such as blood and urine. Specialists in blood gas analysis evaluate lung and breathing function by levels of oxygen, carbon dioxide, pH, and hemoglobin with automated tests. Specialists in histology examine cellular and tissue samples using fixation, dehydration, embedding, microtomy, frozen sectioning, staining, and other similar techniques. Histology specialists licensed as technicians can perform specimen processing, embedding, cutting, staining, and frozen sectioning only under the general supervision of a director, supervisor, or technologist. Specialists in cytology process and interpret samples relating cytopathological disease. Non-gynecological cytology preparations can be screen by a specialist in cytology but final review and interpretation must be done by a physician.

Erythrocytes are produced in the bone marrow and released into the peripheral blood where they may remain for approximately 120 days before senescence.Their main function is the transport of the respiratory gases (oxygen and carbon dioxide) between the lungs and body tissues.Each erythrocyte can be thought of as an "envelope" containing hemoglobin.Each hemoglobin molecule contains iron which has a high affinity for oxygen.As a result, when an erythrocyte passes through one of the capillaries of the lungs, it picks up oxygen.The oxygen is transported through the blood to the tissues where it is released.Carbon dioxide from the tissues then diffuses into the RBC where it undergoes chemical changes.About 70% of the altered carbon dioxide diffuses into the plasma, 25% binds to the hemoglobin molecule, and 5% goes into simple solution within the red cell.In each of these three ways carbon dioxide is transported from the body tissues back to the lungs, where it is released.

When the results on Mr. John Ready were called to the nurse, she was very surprised that the result of his CBC was normal. The nurse explained to the lab tech that Mr. John Ready had a known diagnosis of lower GI bleeding. His hemoglobin had been very low for the past 24 hours because of the internal bleeding, and she thought it was very surprising that his hemoglobin had normalized so quickly without having received a blood transfusion. Mr. Ready’s doctor decided the patient should be redrawn to ensure a correct result. The nurse further questioned if the phlebotomist could possibly have drawn the wrong patient because earlier that day Mr. Ready had been moved to room 831, and room 825 was presently occupied by a patient named Walter Redding. If Julie had checked the patient’s armband, she would have realized that the patient in 825 was the wrong patient.Relevant topics:Importance of patient ID, Patient identification continued, Specimen labeling, Specimen labeling Continued, Blood bank specimens

Also known as Complete Blood Count (CBC) and is run on whole blood.Blood is tested for quantity and quality of different blood cell types, including: White Blood Cells (WBC Count) Red Blood Cells (RBC Count) Platelets (Platelet Count) Blood is also tested for hemoglobin & hematocrit (H&H).

Red blood cells contain hemoglobin, which carries oxygen from the lungs to the tissues of the body. Hemoglobin gives blood its red color. Red blood cells are shown in the photomicrograph of a stained blood smear to the right.

A ten-year-old boy came to a physician's attention because of recent jaundice and icteric sclerae. The immediate laboratory work revealed: Hct 24%(normal 36%-47%), MCV 79.5 fl (normal 78-95fl),RDW 13%(normal 11.5-15.0%). His blood smear findings are reflected in these photomicrographs. Note particularly the spherocytes in the upper picture. Some resemble a half-blister with the other half of the cell containing solidly-staining hemoglobin. These are called eccentrocytes. When present, they should trigger a search for red cell hereditary G-6PD deficiency and the oxidant that triggered hemolysis. These morphological findings are only clues; specific testing for G-6PD deficiency should be performed. The blue arrows in the upper photomicrograph are directed toward solid-staining spherocytes in which the cell membrane is beaded by inclusions wrapped within the cell membrane, suggesting the remains of denatured hemoglobin. Included on the smear is a target cell, several acanthocytes, a smudge cell, and a few schistocytes. The lower photomicrograph is supravital staining of affected red blood cells, verifying the presence of Heinz bodies. This disorder was first recognized during the Korean war in 10% of black American soldiers given the antimalarial drug primiquine.

G6PD deficiency occurs in the same geographic distribution as malaria. It has been theorized that enzyme deficient cells are more resistant to malarial parasites than normal cells.When hemolysis is triggered, the appearance of the red blood cells is modulated by activity of the spleen.Spherocytes, schistocytes, and nucleated red blood cells may appear in the peripheral blood.Denatured hemoglobin removed by an active spleen may leave bite cells, identified by the arrows in this photomicrograph, suggesting the presence of G6PD deficiency.

Schistocyte is a general term for a fragmented red blood cell that may assume various shapes, some with horn-like projections (keratocytes), triangle-forms (triangulocytes), and helmet shapes, as illustrated in the upper photograph. Schistocytes are formed when erythrocytes are forced through a vessel blocked with interlacing fibrin strands and the red cells are sliced into fragments. True schistocytes are devoid of central pallor. These damaged cells continue to circulate while healing their torn edges. Finally, they are removed by the spleen. Bite cells (lower photograph) appear when an abnormal hemoglobin aggregate (Heinz body) is nibbled out of a red cell's cytoplasm by the spleen leaving a bitten apple appearance. Glucose 6-PD deficiency secondary to chemical poisoning or injury by oxidant drugs are settings for Heinz body formation, and the telltale bite cells remain as evidence. Hemolytic anemia associated with severe liver disease is another setting where bite cells are formed.

Hemoblobin H disease follows deletions of 3 of the 4 alpha globulin chains. Beta chains, unable to bind with insufficient numbers of alpha chains, form beta chain tetramers, or HbH.These beta chain tetramers appear as numerous dot size inclusions in erythrocyte cytoplasm, best seen in supravital brilliant cresyl blue stains (lower photograph).The most common molecular defect in alpha thalassemia is DELETION, not MUTATION; whereas, in beta thalassemia, the molecular defect is MUTATION.Leptocytes, as illustrated in the upper photograph,(lepto, derived from a Greek word meaning thin, fine, or slight), are characteristic of HbH disease. They have thinner cell membranes than the cells we recognize as target cells. They stain more lightly than normal erythrocytes and their centers are almost colorless.Subtle changes perhaps, but worth keeping in mind

The patient whose blood smear is shown in the photograph was a 32-year-old female from Virginia who came to the high country of Colorado to ski. The day after arrival, she experienced shortness of breath, fatigue, and upper abdominal pain. She was seen in a medical center in the mountains where a working diagnosis of altitude sickness was made. A CBC revealed RBCs 5.1 x 1012/L, hemoglobin 12.8g/dL, MCV 60fL, hematocrit 40.9%, and normal total WBC, differential, and platelet count. The RDW was normal. Further questioning revealed a previous diagnosis of heterozygous beta-chain thalassemia. No other abnormal hemoglobins were found on hemoglobin electrophoresis, but HbA-2 was elevated to 5%, supporting the diagnosis of beta thalassemia. The patient's poikylocytosis and anisocytosis may be a clue to an underlying erythrocyte abnormality. Persons with iron deficiency anemia may experience various degrees of hypoxia upon arriving at high altitudes. Those with sickle cell disease and thalassemia minor (as in this case) may experience bone pain or other symptoms of "crisis" and/or alteration in the appearance of their erythrocytes upon sudden high altitude exposure. The classic teaching is that in differentiating iron deficiency anemia from thalassemia, increased RDW would favor iron deficiency; normal RDW favors thalassemia.

The family (cited in the previous case history) was from a region of Thailand where the physician knew HbE carriers are prevalent. Homozygous hemoglobin E is common in Southeast Asia and presents with very mild anemia and seldom requires transfusion. Over 30 million people in the world are HbE carriers, making this abnormal hemoglobin almost as common as HbS. Hemoglobin E is uncommon in North America and in Europe, but with changing immigration patterns, hemoglobinopathy E cannot be ignored. Peripheral blood smear findings of target cells, microspherocytes, red cell hypochromia, a few red blood cell fragments, and nucleated red blood cells require evidence from hemoglobin electrophoresis to establish a diagnosis. Clinically, a very important and severe syndrome is hemoglobin E/beta thalassemia in which there is hemolysis requiring repeated transfusions. The patient has a severe anemia, low MCV (50's), and high RBC. This is characteristic of Hgb E/beta thalassemia.

The peripheral blood smear of HbH disease presented before is reviewed in the upper photograph.As mentioned, these leptocytes are pale-staining with hemoglobin confined to a thin, flat, cell membrane.Illustrated in the lower photograph are target cells or codocytes (a term derived from a Greek word for hat)Membrane accumulations of phospholipids and cholesterol (particularly in obstructive jaundice) promote target cell formation.When these cells are spread out on a glass slide, a central bump of hemoglobin appears to produce the target, a manifestation of excess cellular membrane compared to the amount of hemoglobin inside.The early descriptions of thalassemias, then called hereditary leptocytosis (Mediterranean anemia, Cooley's anemia), include description of leptocyes, which may have represented HbH disease.

Ovalocytes are rod shaped erythrocytes with nearly parallel lateral walls. If the long axis of an erythrocyte is no more than twice as long as the short axis, the cell is an ovalocyte. If the long axis is more than twice as long as the short axis, the cell is an elliptocyte. Hemoglobin tends to collect at each end of these cells. The ends of the cells are rounded and never pointed, to be differentated from sickle cells. Ovalocytes present in greater than 25% of red cells on the blood smear are characteristic of hereditary ovalocytosis. The oval shape is attributed to a defect in horizontal red cell membrane protein interactions. Lesser numbers of circulating ovalocytes may be present in various anemias including megaloblastic, sideroblastic, iron deficiency, and in thalassemias. A rare ovalocyte (less than 1%) may be found on almost any peripheral blood smear. Resistance to malarial infection may be a beneficial attribute of hereditary ovalocytosis.

Poikylocytosis that includes tear-drop shaped erythrocytes, schistocytes, and target cells is present in both the upper and lower photographs. In addition, macrocytes are present, two of which (one in each field) have coarse basophilic stippling. The stippling may represent abnormal hemoglobin synthesis. These stippled erythrocytes remain in circulation in the absence of pitting by a spleen.

This peripheral blood smear is from a patient with pernicious anemia, which results from an inability to absorb the vitamin B12 needed for DNA synthesis. Since many cells are destroyed in the bone marrow, decreased numbers of red cells are present in the circulating blood, resulting in anemia. However, the red cells that are present are generally macrocytes and are filled with hemoglobin.

Sickle cells, also referred to as drepanocytes, are formed as a result of the presence of hemoglobin S in the red cell. As the red cell ages, it becomes rigid as it passes through the low oxygen tension atmosphere of the small capillaries in the body. In the absence of oxygen, hemoglobin S polymerizes into rods, causing the sickle cell shape. The shape of sickle cells can vary from cigar-shaped, as shown in the upper image, to the more severe sickled-form, shown in the bottom image.

Codocytes are thin-walled cells showing a darkly-stained center area of hemoglobin which has been separated from the peripheral ring of hemoglobin. When viewed under the electron microscope. these cells have a cup-shaped appearance. A codocyte is seen in the center of this slide.

The prekeratocyte becomes a keratocyte when the vacuole ruptures, leaving a damaged cell that resembles a 'helmet' as seen in this image. Keratocytes are also referred to as 'horn' cells because they resemble a red cell with two horns.

In addition to the amount of hemoglobin present, the color of the cell must also be considered. Completely mature red cells appear buff-colored, while slightly immature non-nucleated red cells (reticulocyte stage) appear blue/gray on Wright's stained smears due to the presence of residual ribonucleic acid (RNA).The terms used to describe these cells are polychromasia or polychromatophilia. Polychromatophilic cells are frequently larger in size than mature red cells and can be distinguished from both types of macrocytes by this distinctive color.

Erythrocytes, when spread on a glass slide, show varying degrees of central pallor as noted in the previous exercise. This central pallor is related to the hemoglobin concentration present in the red cells.When viewing normal mature red cells, the central area (one-third of the cell) is white, while buff-colored hemoglobin is visible in the outer two-thirds of the cell. The mean corpuscular hemoglobin concentration (MCHC, 32-36 gm/dl of red blood cells), is the indice value which is used to verify the presence of adequate hemoglobin concentration in the cells visible on the peripheral smear.

When the results on Mr. John Ready were called to the nurse, she was very surprised that the result of his CBC was normal. The nurse explained to the laboratory technologist that Mr. John Ready had a known diagnosis of lower GI bleeding. His hemoglobin had been very low for the past 24 hours because of the internal bleeding, and she thought it was very surprising that his hemoglobin had normalized so quickly without having received a blood transfusion. Mr. Ready’s doctor decided the patient should be redrawn to ensure a correct result. The nurse further questioned if the phlebotomist could possibly have drawn the wrong patient because earlier that day Mr. Ready had been moved to room 831, and room 825 was presently occupied by a patient named Walter Redding. If Julie had properly identified the patient by asking him to state his name and then checking the name and identification number on the wristband, she would have realized that the patient in 825 was the wrong patient.

Mr. R.M., a 55-year old male, was admitted to a hospital emergency department with severe lower gastrointestinal bleeding. His history revealed multiple prior transfusions, the last of which he received five years earlier.Physical examination revealed hemodynamic instability (systolic BP 60 mmHg). Blood tests revealed a hemoglobin (Hb) of 8 g/dL (80 g/L) and a hematocrit (HCT) of 28% (0.28). The patient received aggressive fluid resuscitation with Ringer's lactate and was sent to the operating room (OR) for an emergency laparotomy.The physician ordered four units of Red Blood Cells to be crossmatched.Two units of uncrossmatched group O Rh-negative Red Blood Cells were also ordered and authorized for immediate emergency transfusion.

Sometimes the red cells become so swollen that the cell membrane ruptures, causing the cell to lose hemoglobin. These empty membranes are known as "ghost" cells.