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Anemia is fatal.RBC count is increased.Hb is severely decreased.MCV is decreased. MCHC is decreased.RDW is increased.Red Blood Cell morphology shows slight hypochromic microcytosis with codocytes, schizocytes, nucleated RBCs.Reticulocytes are increased.Hb electrophoresis demonstrates abnormal pattern on cord blood: Hb A - absentHb Bart's - 80-90%Hb Portland - 0-20%Bone marrow demonstrates marked erythroid hyperplasia.

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.

Clot formation is always abnormal and is often due to increased levels of protein, especially fibrinogen. When the protein level is 1000 mg/dL, clot formation will most likely occur but clots may also form at lower levels of protein. Some clots may be very fine and appear as a thin membrane or "scum" on the surface of the CSF specimen. This type of clot is referred to as a pellicle. Pellicles are composed of fibrinogen and white blood cells. The type of clot formed may give some specific information about the disease state. Some examples are provided in the following table: Example of ConditionType of Clotbacterial meningitispellicle forms in a short time; large clot formation followsTB meningitisweb-like clot (pellicle) after 12-24 hours (enhanced by refrigeration)paresis (type of neurosyphilis)incomplete clotblockage of CSF circulationcompletely clotted due to protein

The reagent strips must be handled and stored properly in order to ensure that results are accurate. The following precautions should be observed: Store strips according to the manufacturer's recommendation. DO NOT expose strips to moisture, direct sunlight or volatile fumes. Remove only enough strips for immediate use and immediately recap the bottle. Avoid contamination of test strips. Do not touch the test areas with fingers or do not lay the test strips directly on the workbench. DO NOT use discolored strips. Compare the color of the unused strip to the negative area on the color chart provided by the company. The color should be similar. Check the expiration date. Re-label the container with a revised expiration date if the manufacturer states a shortened usage period once the container has been opened. Reagent strips must be tested periodically (frequency defined by the laboratory) for clinical reactivity with normal and abnormal urine controls. Urine controls are available commercially or may be prepared and preserved in-house.

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.

Both a normal and an abnormal urine control must be tested with each new lot of reagent strips, and at least every day of patient testing to confirm the accuracy of the reagent strips and the dipstick reader. Some dipstick readers also require periodic calibration. Follow the manufacturer's instructions for calibration procedure and frequency. Quality control results must be recorded, and corrective action must be taken when the results are not in the acceptable range.

The heat and acetic acid test is another semiquantitative test used to confirm the presence of protein in urine. It is more sensitive than the SSA test because the pH of the sample is brought close to the isoelectric point of proteins. However, this test is sometimes considered too sensitive because it can detect trace amounts of protein which are considered normal. The heat and acetic acid test gives false positive results with inorganic iodides, benzoin, tolutamide, and proteoses, similar to the SSA test. Bence-Jones protein consists of dimers of either kappa or lambda light chains from immunoglobulins. This abnormal protein is most often associated with multiple myeloma, but can also be found in cases of lymphoma, macroglobulinemia, leukemia, and other malignancies (Balant and Fabre, 1978). Testing for Bence-Jones protein is not part of the routine urinalysis. However, if Bence-Jones protein is suspected, the heat precipitation test or immunoelectrophoresis can be performed on a urine specimen. The heat precipitation test is based on the protein’s unusual solubility properties. Bence-Jones protein precipitates at temperatures between 40ºC and 60ºC (56ºC optimum), but dissolves again at 100ºC. Upon cooling, the precipitate will reappear around 60ºC and will dissolve again below 40ºC

Under conditions of abnormal carbohydrate metabolism such as occurs in diabetes mellitus, ketones accumulate in the blood (ketonemia) and are excreted in the urine (ketonuria). The accumulation of ketone bodies is often the cause of acidosis and coma in diabetics.

Coefficient of variation is commonly used as a means of measuring the variability of an instrument. The data are gathered by recording the values for the normal and abnormal controls for each test run. At the end of the month, the standard deviation, mean, and coefficient of variation are calculated. The testing data for a particular instrument might look like this: January February March Normal Control s CV 100.9 2.43 2.41 103.1 2.99 2.90 102.0 2.21 2.17 Abnormal Control s CV 209.5 4.41 2.11 211.6 4.00 1.89 206.8 3.95 1.91 The coefficient of variation stays fairly constant from month to month. If there is a sudden increase, there might be a problem with the method or the equipment.In the clinical laboratory, the use of CV as a measure of relative variability should not be confused with the use of the standard deviation as a measure of absolute variability. For example, support physicians agreed that for accurate patient treatment, the inherent variability in a glucose method should be less than 5 mg/dL. In this case, neither the hexokinase nor the orthotoluidine method is acceptable. It does not matter which is more precise if neither is precise enough to result in adequate patient care.

Under normal conditions, Howell-Jolly bodies are thought to be remnants of nuclear fragments due to incomplete expulsion of the nucleus. In pathological conditions, they are aggregates of chromosomes which have separated from the mitotic spindle during abnormal mitosis. Single or multiple Howell-Jolly bodies may be found in a red cell. A single HJ body in a red cell may be seen in megaloblastic anemia, hemolytic anemia such as sickle cell anemia and after splenectomy. Megaloblastic anemia or abnormal erythropoiesis is usually present when multiple Howell-Jolly bodies are observed in a single cell.

Bernard-Soulier Syndrome is a genetic platelet disorder characterized by abnormal platelet function tests, unusually large platelets, and a moderate decrease in platelet count. Clinically, patients present with mucotaneous bleeding of varying severity, as well as having gingival bleeds, epistaxis, purpura, and gastrointestinal hemorrhaging. Treatment can range from the administration of iron supplements up to red cell replacement therapy if the episodic bleeding is severe enough to warrant it.

In some laboratories the anticoagulated sample is used to prepare concentrated smears. Placing the fluid in a Wintrobe tube and centrifuging it separates the sample into four layers:fat and perivascular cellsplasmabuffy layer - myeloid and nucleated erythroid cellserythrocytesThe volume of each layer is measured using the scale on the Wintrobe tube and then the percentage of each layer is calculated. Next the plasma is removed and a smear is made from the buffy coat and top of the red cell layer. Either the manual push method or cytospin technique may be used to make the smears. They may be stained with a variety of cytochemical stains. Concentrated smears are used to examine cell morphology and demonstrate the presence of abnormal cells when the marrow is hypocellular. The smears cannot be used for differential counts or evaluation of cellularity.

A rare abnormal variant of a plasma cell called a flame cell is shown here. Not the red tinged area at the periphery. These cells contain more immunoglobulin than normal plasma cells.

Quality control charts are examined to see if there are problems in the procedure being tested. The Westgard rules are one tool that can help to determine whether there is a problem, and whether that problem is due to random or systematic error.The six Westgard multi-rules are: 12S rule: this rule applies when at least one result falls more than two standard deviations above or below the mean. This is a signal that the run must be examined in further detail, and does not in itself warrant discarding the run. However, if all of the results are with in 2s, the run should be accepted. 13s rule: this rule applies when a result falls outside of the 3s limit. The run is rejected, and a random error has probably occurred. 22S rule: this rule applies when two consecutive results exceed the +2 or the -2 standard deviation limit. The controls could be normal, abnormal, or one of each. A violation of this rule usually indicates a systematic error. The run is rejected. R4S rule: this rule applies when the difference between the highest and lowest result of a run exceeds 4 standard deviations. This rule detects random errors. The run is rejected. 41S rule: this rule applies when four consecutive control samples all exceed the +1 or the -1 limit. The controls could be normal, abnormal, or a combination of the two. This rule detects systematic errors. The run is rejected. 10x rule: this rule applies when 10 consecutive controls all fall on the same side of the mean, either above or below. This rule detects a systematic error. The run is rejected.Some labs choose not to use all of the Westgard rules; however, it is recommended that all labs use at least two rules, one that can detect systematic error and one that can detect random error.

N:C Ratio - Nuclear: cytoplasmic Ratio - The ratio of nuclear volume to cytoplasmic volume within any one cell.Neoplasm - Any new and abnormal growth, such as a tumor.Neutrophilic Granules - Specific granules present in the cytoplasm of neutrophils. These granules resemble pencil stippling and stain a lilac color due to their affinity for both basic and acid dyes.Phagocyte - Any cell that ingests microorganisms or other cells and foreign particles.Phagocytosis - The ingestion and destruction of microorganisms or other foreign particles.Plasma - The fluid portion of blood in which the various blood cells are suspended.PF3 (platelet Factor 3) - A lipoprotein component of the platelet membrane; functions as a surface catalyst during blood coagulation.Pseudopod - A temporary protrusion of the cytoplasm of a cell.Refractile - Capable of refracting or changing the direction of light.Senescence - The process or condition of growing old.Serotonin - A constituent of blood platelets and other cells and organs; induces constriction of the blood vessels.Specific Granules - Granules found in cells of the more mature stages of the granulocytic series. They have distinct staining reactions which differ with each type of granulocyte.T-cell - Thymus derived lymphocyte which mediates cellular immunity.Thrombocyte (Platelet) - A circular or oval disk found in the blood; concerned with hemostasis.Thymus - A ductless gland-like body situated in the anterior mediastinal cavity; reaches its maximum development during the early years of childhood.Vacuole - Any small space or cavity formed in the cytotoplasm of a cell.

Prolonged bleeding time may indicate:Reduced numbers of platelets.Poorly functioning platelets, or:Medications such as aspirin, which inhibit platelet function, have been recently taken. Abnormal blood vessels may also prolong bleeding time.

Filling a light blue-topped tube to its recommended volume is especially critical; if it is filled incompletely, coagulation results will be incorrectly reported as abnormal.If a short draw is anticipated, a “partial collection” tube which contains less anticoagulant and requires less blood may be used.The light blue topped collection tube shown on the left requires reduced blood volume, and is filled only to the line.

In the late 1950’s, Dr. William Youden (1900-1971) developed what has now become known as the Youden Plots. This statistical technique involves both normal and abnormal controls and graphically helps to differentiate between systematic and random errors. The inner square of the plot (yellow) represents one standard deviation (1SD). The next larger square (green) represents 2SD, and the outer square (blue) represents 3SD. A horizontal median line is drawn parallel to the X-axis and a second median line is drawn parallel to the Y-axis. The intersection of the two median lines is called the Manhattan Median. One or two 45-degree lines are drawn through the Manhattan Median. The results of at least two different levels of controls (e.g. Level 1/Level 2 or Normal/Abnormal) are then plotted on the chart as X-axis versus Y-axis.

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.

In a study on the reporting of red blood cell morphology abnormalities conducted in Ontario, Canada (Hookey L, Dexter D, Lee DH, Laboratory Hematology 7:83-88, 2001), fewer than 50% of 33 participants used the same term to describe the quantitative frequency of peripheral blood abnormalities. Seven blood smears, each containing one of several abnormal erythrocytes-- schistocytes, teardrop cells, acanthocytes, and Howell-Jolly bodies--were evaluated by 32 participants. The participants were asked to document their evaluations from a list of quantitative terms. There was a heterogeneity in the use of terms "rare," "slight," "occasional," "few," "mild", "present," "moderate," "many," and "marked." Choices of terms were subjective without points of reference. Guidelines for establishing standardized qualitative estimations of abnormal erythrocytes in the peripheral smear are presented as follows: 1+ = 2 - 4/Oil Immersion Field (OIF) 2+ = 5 - 7/OIF 3+ = 8 - 10/OIF 4+ = >10/OIF. The terms "few," "moderate," "many," and "marked" may be substituted for the 1+ - 4+ grading system, but only when their specific points of reference are universally understood in tandem with the above guidelines. A comment should be triggered if any erythrocyte abnormalities are seen in numbers >3/OIF including, but not limited to, polychromasia, basophilic stippling, nucleated RBC's, and Howell-Jolly bodies. Rouleaux or RBC agglutination are important findings and must be documented.

Red cell morphology can be defined as the appearance of the erythrocytes on a Wright's stained smear.Careful examination of the red cells for the purpose of identifying abnormalities is part of the differential procedure. This examination is important because it may provide valuable diagnostic information to the physician, as well as provide a quality control mechanism to verify red cell indices values as determined by automated or manual methods.Evaluating red cell morphology involves differentiating normal morphology from abnormal and artificial morphology. The abnormal morphology covered in this unit may be seen in a variety of disorders.

Red blood cells can vary in size (diameter/volume) from smaller than normal, microcytes, to larger than normal, macrocytes. When red cells of normal size, microcytes and macrocytes are present in the same field, the term anisocytosis is used.Since the purpose of this unit is to acquaint you with the appearance (identification) of abnormal red cell morphology, percentages of abnormalities present will not be considered. It is important to be aware that rating red cell morphology for the purpose of reporting it is a skill which must be learned before you are able to complete this aspect of a differential count.

Semen analysis can provide important information related to the function of the male reproductive system but, even when results are within normal limits, it does not ensure that a male is fertile. A normal semen analysis result does not mean that all causes of male infertility have been ruled out. One reason for this is that there can be considerable differences between one semen analysis result and another in a single individual. On the other hand, an abnormal result does not always mean that a couple cannot conceive a pregnancy. Men with suboptimal sperm counts have been known to father children. Also, infection, trauma, stress, febrile illness and medications can cause temporary subfertility. For all of these reasons multiple specimens are recommended for a complete analysis of the semen.

There are a number of abnormalities of sperm morphology. Abnormal heads can include enlarged head, double head, round head, constricted head, amorphous head, pinhead, and acute tapering forms. There are also heads with abnormal numbers of vacuoles. Midpiece abnormalities include distended and thin midpiece regions. Abnormal tails include short tails, double, triple or multiple tails, coiled tails, broken tails, or absent tail. Cytoplasmic droplets are also seen in some specimens. These are large regions of cytoplasm just below the head assumed to represent failure of complete sperm maturation or a sign of either toxicity or oxidation. There have also been reports that cytoplasmic droplets may be artifacts from the fixation and staining for morphology analysis.

Appearance of sperm with abnormal tails: a: short tail b: double tail c: multiple tail d: coiled tail wound around the head

A sperm count is considered normal if it is over 20 million sperm/ml. Although lower counts are considered abnormal by World Health Organization (WHO) standards (see the WHO manual, 1999), it is sometimes possible for men with significantly reduced counts to father a pregnancy.

A third type of epithelial cell are transitional epithelial cells. They are often pear-shaped with a thin tail on one end. Transitional epithelial cells are not clinically significant unless they are seen in large numbers or have abnormal morphology which may indicate transitional cell carcinoma. Catherization may cause increased numbers of transitional epithelial cells.

There are a number of crystals which are seen less frequently but are of considerable significance when they appear. Their presence should be verified by further testing and confirmed by a supervisor or pathologist before reporting the results. Polarized light can aid in crystal identification.

Once the microscopic examination is completed, it is important to decide whether the results are normal or abnormal. Correlation involves comparing the microscopic findings with the macroscopic findings. If the results are consistent with each other, the urinalysis may be reported. If a discrepancy exists, the microscopic results cannot be reported. The findings that do not correlate must be repeated. The following table illustrates results which may be found together in a urinalysis. Microscopic Macroscopic Casts (may be accompanied by mucous) Possible positive protein reaction White Blood Cells (bacteria may accompany WBCs in microscopic) Possible positive protein reaction Possible alkaline pH (fresh) Possible cloudy urine Red Blood Cells Possible positive blood reaction Possible positive protein reaction Possible negative blood reaction (if only a few RBCs are seen) Possible cloudy urine Possible red or brown urine Bacteria (may be accompanied by WBCs) Possible alkaline pH (fresh) Possible positive protein reaction Possible cloudy urine Possible positive nitrite reaction Yeast (may be accompanied by WBCs) Possible glucose Possible cloudy urine Crystals Should suggest approximate pH Possible cloudy urine Possible high sp. gravity Trichomonas Possible cloudy urine due to increased WBCs and mucous Report the microscopic findings if they correlate with the macroscopic. Report common crystals if requested or when an unusual number of one type is present. Do not report abnormal crystals unless confirmed by further tests and pathologist. Do not report sperm.

The results are abnormal. The presence of glucose is not a normal finding. However, the two glucose methods correlate well with each other.The specific gravity does not correlate well with the glucose. A large amount of glucose should elevate the urine specific gravity. The specific gravity result should therefore be rechecked before reporting. The presence of 3+ bacteria, does not correlate well with scant white cells and lack of turbidity. The technologist should question whether the specimen was held at room temperature for a protracted period prior to examination.

This slide is also from a patient who has Alder-Reilly anomaly. Notice that neutrophil seen in this slide has granulation which is much heavier than in the previous slide. The amount of granulation may vary from cell to cell with some cells being unaffected. A lymphocyte showing abnormal granules is also present in this slide.

Auer rods are red staining, needle-like bodies seen in the cytoplasm of myeloblasts, and/or progranulocytes in leukemia. Auer rods are cytoplasmic inclusions which result from an abnormal fusion of the primary (azurophilic) granules. Single or multiple Auer rods may be seen in the cytoplasm of a cell. If more than one is present, they are frequently close together and may even be overlapping. Their identification is very important because, if found, they can confirm the presence of myeloblasts indicating the presence of a myeloid (non-lymphoblastic) leukemia. They can also be seen in myeloid blast crisis in chronic granulocytic leukemia. Auer rods are never seen in lymphoblasts. This differentiation is important because the treatment of lymphoblastic and myeloblastic leukemia are different. Auer Rods always classified as pathological.

Several additional familial and congenital disorders associated with atypical inclusions in WBCs are now recorded. These individual syndromes carry the following names: Fechtner, Alport, Epstein, Sebastian, and Paris-Trousseau.Fechtner syndrome( Peterson etal,Blood 65:397-406,1985)was described with 8 family members spanning 4 generations presenting with varying degrees of nephritis, deafness,and congenital cataracts. The syndrome is likely a variant of Alport syndrome with the addition of leukocyte inclusions and macrocytothemia. Several more cases involving other families have been reported. The inclusions resemble toxic Doehle bodies or those of the May-Hegglin anomaly by light microscopy, but are ultrastructurally unique.Alport syndrome in itself is autosomal dominant, X-linked , hereditary and characterized by sensorineural deafness and hereditary nephritis. It is believed to result from abnormal glycopeptide synthesis in renal basement membranes. Recurrent hematuria and slowly progressive renal insufficiency are clinical findings. Cataracts and platelet abnormalities may be added features.Epstein syndrome is essentially Alport syndrome with the addition of macrothrombocytopenia (Seri, et al. Hum Genet 110:182-186, 2002). Neutrophil inclusions are absent in this disorder; neutrophilic inclusions are considered part of the Fechtner syndrome. The Sebastian platelet syndrome is a variant of hereditary macrothrombocytopenia combined with neutrophil inclusions that differ from Doehle bodies, but are similar to those inclusions in Fechtner syndrome. (Greinacher, et al, Blut 61:282-288, 1990).Paris-Trousseau syndrome includes large platelets containing giant alpha granules identifiable in the peripheral blood.(Breton-Gorius, Blood 85:1805,1995)

In 1952 Chediak (a Cuban physician) reported a childhood disorder in which abnormal cytoplasmic inclusions appeared in the neutrophils of four family members. In 1954 Higashi reported a similar abnormality in an 11-month old Japanese infant. These inclusions were identified as lysosomal in origin and found in this rare autosomal recessive disorder Death was usually related to recurrent infections or hemmorhage though now some of the affected patients live to reproduce. Ocular and cutaneous albinism, increased susceptibility to pyogenic infections, abnormal granules in neutrophils, and a bleeding tendency are prominent findings. The striking neutrophilic inclusions appear as coarse intra-cytoplasmic azurophilic granules (see photograph).These granules arise from dilated portions of the Golgi-endoplasmic reticulum lysosomal apparatus. Aleutian mink and other animals are known to have Chediak-Higashi syndrome. Azurine pelts from infected mink were once prized by coat makers.