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ERYTHROPOISES

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ERYTHROCYTE MORPHOLOGY AND PHYSIOLOGY

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RELATED INFORMATION

01. EXPLAIN WHAT IS MEANT BY A BURST FORMING UNIT.

The burst-forming unit - erythrocyte (BFU-E) represents the next stage in differentiation from the myeloid stem cell for the RBC line. At this stage, this cell is committed to eventually become an erythrocyte. The cell has the potential to rapidly proliferate, forming up to 1,000 replicates. This is the most primitive dedicated erythrocyte precursor cell. This cell, if observed on a slide, would be designated as an undifferentiated blast cell.

02. EXPLAIN WHAT IS MEANT BY A COLONY FORMING UNIT - ERYTHROCYTE.

The colony-forming unit - erythrocyte (CFU-E) is the next maturation step toward an erythrocyte. It is an immature cell capable of proliferating up to 64 cells. This cell if observed on a slide would be designated as an undifferentiated blast cell.

Erythrocytes: Morphology and Physiology

This teaching syllabus discusses the morphology and physiology of the erythrocytes appropriate for basic hematology curriculum. All objectives listed are

03. DESCRIBE WHAT HAPPENS TO THE RBC AS IT UNDERGOES ITS VARIOUS MATURATION PROCESSES.

All blood cells originate from the undifferentiated or pluripotential stem cell. There are three general maturation sequences that are common to all cells. [1] All immature cells become progressively smaller as they mature. A. The cytoplasm undergoes changes that affects it staining properties. The cytoplasm in the mature cell demonstrates strong basophilia or blue coloration. This is due to the amount of large amounts of RNA present As the cell matures, the cytoplasmic RNA content decreases and the cytoplasmic hemoglobin content increases.

B. There are changes in the nucleus. In essence, the nucleus undergoes a decrease in mass and becomes smaller. The nuclear material in the immature cell has an affinity for eosinophilic dye to give it a predominately red color. As the nuclear chromatin condenses, the color changes to a predominately dark blue color. The nucleus, as it condenses, becomes more and more coarse and clumped. This causes much variation in the leukocytes. In the erythrocyte, the nuclear mass is ejected, leaving the erythrocyte as a anucleate cell.

The nucleus becomes small and there is variation between the erythroid, myeloid, and lymphoid lines.

C. The nuclear chromatin material in the immature cell changes from its loose, delicate, and spreading strands to form wider strands that are more coarse. The nucleoli that are present in the immature cell as islands of metabolic activity do not have a definite membrane. The nuclear chromatin compresses around the nucleoli to give the appearance of membrane-like structures. The nucleolus consists of RNA that decreases as the cell matures. The function of the nucleolus is to synthesize cytoplasmic RNA. This function ceases in all cell lines, except the lymphocyte,.as the cell matures and the nucleus condenses.

D. Once the maturing erythrocyte can be identified by cytochemical staining, there are eight days in the maturation sequence of the rubriblast(ERYTHROBLAST) to the mature erythrocyte. There is a cell volume decrease in the rubriblast of 500 fL to about 90 fL in the erythrocyte.

0 4. DESCRIBE THE RUBRIBLAST

Synonym: Pronormoblas t. This is the earliest immature cell that can be recognized as an erythrocyte precursor. Its size is 14 to 20 μM. The cytoplasm is deeply basophilic and exists as a small band about the nucleus. The cytoplasmic area adjacent to the nucleus stains lightly and is designated as a perinuclear halo. The cytoplasm is nongranular. The nucleus is large, occupying a large part of the cell. The nuclear to cytoplasm (N/C) ratio is 8/1. The nucleus is round or slightly oval with a fine chromatin pattern characterized by fine clumping. Wright’s stain gives a reddish -purple color. Usually 1-2 nucleoli may be observed. The nucleoli tend to larger (when compared to the nucleoli of the myeloblast) and have a bluish tint. The nucleus may be eccentric. This cell is capable of mitosis, producing two rubriblasts

0 5. DISCUSS THEPRO ERYTHROCYT

Synonym: basophilic erythroblas t) The prorubricyte has an average diameter of 12 to 17 μM. It cytoplasm is very basophilic. Hemoglobin synthesis is occurring but is masked out the large amount of RNA synthesis. The cytoplasm is basophilic and continues to be nongranular. The nucleus is large with a round to slightly oval shape and eccentric. The nuclear to cytoplasm (N/C) ratio is 6/1. The chromatin is condensing, becoming more coarse. Parachromatin areas are appearing. The nucleus takes on a deep purple-red color. Nucleoli (as a rule) are not visible. This cell can undergo mitosis yielding two prorubricytes

Parachromatin is a chromatophilic substance that stains lightly in the nucleus. It is non-gene bearing. Euchromatin is the chromatophilic gene-bearing material (DNA) and globulins. It stains darkly. 06. DISCUSS THE POLYCHROMATIC ERYTHROBLAST .

Synonym: Polychromatic erythroblast/intermediate normoblast. The rubricyte averages 10 to 15 μM in diameter. Its cytoplasm is taking on a pink hue as synthesis in RNA and hemoglobin shifts. The cytoplasm will be variable with colors between blue gray to pink-gray. The cytoplasm continue to be non granular and a perinuclear halo may be present. The nucleus is undergoing changes, becoming more pycnotic, round, and smaller as the chromatin condenses and increases the intensity of its clumping. There are distinct areas of parachromatin. The nucleus will stain darker, a deep blue- purple.. Dependent upon the degree of clumping, the nucleus of some rubricyte’s may take on a clumped appearance. The N/C ratio is around 4/1. This cell represents the last stage in the maturation sequence of erythrocytes in which mitosis can occur. It can divide forming two rubricytes.

07. DISCUSS THELATE NORMOBLAST

Synonym: Orthochromic normoblast. The metarubricyte measures from 7 to 12 μM in diameter. Its cytoplasm is pink to pink-orange. Gray or bluish tones, if observed are due to the presence of residual organelles scattered in the cytoplasm. Hemoglobin production is increased and the cytoplasm has increased in relation to the nucleus. N/C ratio = 1:2. The nucleus is condensed and the chromatin in pycnotic with a homogenous appearance. The nucleus stain blue-black and either centric or eccentric. It is not unusual to find an occasional metarubricyte on the slide with the nucleus protruding from the cell. This cell can squeeze through the capillary walls into systemic circulation by diapedesis.

08. DISCUSS THE RETICULOCYTE.

Synonym: immature erythrocyte, “retic”, juvenile RBC, or neocyte. Its size ranges from 7 to 10 μM, usually slightly larger than a mature RBC. The cytoplasm stains from pink to pink-gray and contains aggregates of RNA reticulum. This reticulum can be seen when the cell is stained with a vital stain. The nucleus is absent. The reticulocyte will quickly eliminate the reticulum, becoming a mature RBC and living an average of 120 days.

The reticulocyte is an index to RBC turnover. Once the nucleus is extruded from the metarubricyte, it takes 4 - 5 days for the reticulocyte to loose it reticulum and become a mature RBC. The “retic” cell spends about three days in the bone marrow. It is slightly larger than the mature RBC and when stained with Wright’s stain, it appears polychromatic (the grayish or bluish tones). The blue tones are referred to as polychromasia or diffuse basophilia. Wright’s stain does not stain or demonstrate the reticulum present in this cell. It is referred to as a polychromatic erythrocyte and represents the “retic” cell. About the third day, the “retic” cell moves into general blood circulation. It will take approximately another 24 hours for the RNA reticulum to disappear from the cell. If there is a demand by the body for more RBC’s in general circulation, then more “retic” cells will be shifted into the blood stream earlier than normal. These larger and more immature forms will be seen on the blood smear as polychromatic “retics”. Because they are the more immature forms, they will circulate in the general circulation longer. The more severe the anemia or blood loss, the greater the number of shifted retics. The normal retic count for the adult is 0.5% to 1.5%. For the newborn, retic counts of 2.5% to 6.5% are considered normal. The newborn’s retic count will fall to the adult level in about two weeks.

The Retic-Production Index (RPI) is a mathematical manipulation to measure erythropoietic activity when “stress” or “prematurely released” reticulocytes are present. It eliminates the error of using the simple reticulocyte calculations that gives it answers in percentages. If the bone marrow production is increased due to erythrocyte stimulation, reticulocytes are being prematurely released into blood circulation (before their usual 2 to 3 day maturation period. These immature retic cells appear as large polychromatophilic erythrocytes. The more reticulum in the cell, the more immature the cell. 09. DISCUSS THE ERYTHROCYTE.

Synonyms: RBC, discocyte, normocyte, akaryocyte, erythroplastid. The mature erythrocyte ranges from 6 to 8 μM. Its cytoplasm stains pink to pink-orange. (NOTE: The color will vary dependent upon the quality of stain and type of stain used. This will be true for all cells seen in a stained blood smear.) It is a non-nucleated, nongranular cell, and contains no inclusions. If inclusions are observed, then something is wrong. It as round and biconcave shape. It is a membrane sack filled with hemoglobin (90%) and water (10%). Enzymes are present so that glycolysis can occur. The membrane is semi-permeable and deformable. RBC’s are subjected to a variety of osmotic forces, undergoing mechanical stressing when passing through the spleen,

liver, and capillaries. Some textbooks reports that the RBC will travel about 300 miles before being “recycled.

10 DISCUSS THE MOVEMENT OF RETICULOCYTES FROM THE BONE MARROW TO PERIPHERAL CIRCULATION AND HOW TO CALCULATE RETICULOCYTE PRODUCTION INDEX.

In normal conditions, the reticulocyte will take about 3.5 days to mature in the bone marrow then they are released into peripheral circulation where they will eliminate the reticulum in about 24 hours to become a mature RBC. If anemia is present in an individual, there is a corresponding increase in erythropoietin production. This hormone decreased the maturation time spent in the bone marrow by the reticulocyte and it precursors. The early released reticulocytes are larger than the normal RBC and take on a bluish hue in Wright’s stain. These cells are called shift cells.

The hematocrit is an index to the degree of anemia. Hematocrit values have been interpolated into maturation time factors for calculating the reticulocyte production index (RPI). A hematocrit range of 40% to 49% has the designation of 1.0. For a hematocrit of 30% to 39%, the designation is 1.5. A hematocrit of 20% to 29% become 2.0 and a hematocrit of 10 to 19 become 2.5. Consider the following table to show the correlation of the hematocrit with bone marrow and peripheral blood.

The RPI become an indicator of the degree of bone marrow response to anemia. This calculation is a shift correction for the corrected reticulocyte count. The formula is as follows:

% retic count (X) hematocrit (L/L)/0.45 (LL) RPI = ------maturation time in peripheral blood

Sample problem: A patient has a reticulocyte count of 6.5% with a hematocrit of 26%. Calculate the Reticulocyte Production Index (RPI)

6.5 (X) 0.26 (L/L)/0.45 (L/L) RPI = ------2.0 6.5 (X) 0.58 3.76 RPI = ------= ------= 1.88 2.0 2.0 This means that the RBC production rate has increased by 1.88 times. This would be deemed to be an inadequate response. By convention, it is agreed that a RPI value less than 2.0 is an inadequate response. A RPI value of 3.0 is considered to be an appropriate bone marrow response to anemia.

11. DESCRIBE HOW TO CALCULATE THE RELATIVE RETICULOCYTE COUNT.

The number of reticulocytes must be determined by counting the retic cells in 1,000 RBC’s. The next step is to divide the number of retic cells by 1,000 and multiply by 100. This will give an answer in percent. Example problem. If you counted 65 retic cells per 1000 RBC’s then divide 65 by 1000. Your answer is 0.065. Multiply this by 100 and obtain 6.5. Your answer is in percent and is the relative count.

12. DISCUSS THE CORRECTED RETICULOCYTE COUNT AND ITS PURPOSE.

The corrected reticulocyte count (CRC) is also called the hematocrit correction and reticulocyte index. A lab report of a relative reticulocyte count may give the appearance of an elevated retic count when it isn’t. the CRC adjusts the actual number of reticulocytes to the hematocrit, giving a more reliable estimate. The formula is as follows: Hematocrit (L/L) CRC = % retic cells (X) ------normal hematocrit (0.45 L/L)

If a patient’s retic count is 6.5% and the hematocrit is 0.28 (L/L), then the formula sets up as

CRC = 6.5% (X) 0.28 / 0.45 = 6.5% (X) 0.62 = 4.04%

A normal CRC is 1% if the hematocrit is between 40 to 48%. If the hematocrit is from 25 to 35% and the CRC falls between 2 and 3%, then “retic” cell production is normal. For a hematocrit that is less than 25%, the CRC should be between 3 and 5 %. The reticulocyte production index (RPI) is the preferred method for determining if there is normal production of reticulocytes.

13. DESCRIBE THE RBC MEMBRANE AND ITS FUNCTION.

The round, biconcave nature of the erythrocyte membrane gives it maximum surface area that is advantageous for gaseous exchange and increased deformability. It composition is approximately 50% protein, 40% lipids, and 10% carbohydrates. Morphologically it is composed of two layers of phospholipids, arranged so that the polar surfaces face the inside and outside of the cell. The non-polar groups are directed to the center of the membrane layer.

The proteins in the RBC membrane account for its shape, structure, and ability to change shape. These proteins are also the channels and pumps to move ions and other molecules in, out, and across the membrane. Some of the proteins function as receptors, many of the proteins function as the RBC antigens (ABO, and Rh), other proteins have enzymatic capability, and all in some degree or another help to stabilize the membrane.

If the molecular composition of the RBC membrane changes, the membrane is affected inducing changes in its shape or ability to transport ions and molecules. If the cholesterol content of the membrane increases, the membrane takes on the appearance of a target cell or spicules develop to form the acanthocyte. If abnormal proteins are incorporated into the membrane, the cell may become an elliptocyte or spherocyte. If proteins are lost, for whatever reason, the integrity of the membrane is compromised and hemolysis will result. It has been found that some of the RBC membrane antigens are essential for membrane integrity.

14 PROVIDE A BRIEF OVERVIEW OF RED BLOOD CELL METABOLISM.

Erythrocytes must be able to metabolize in order to remain viable. The cell has the ability to metabolize glucose through the glycolysis cycle (Embden-Meyerhof anaerobic pathway) for ATP production. ATP is needed to run the membrane pumps (example: Na+ and K+ exchange) which helps to control membrane integrity and cell osmolarity. Energy is required to maintain cell function, membrane shape, and to protect the lipid composition of the cell.

The glycolysis of glucose to ATP provides energy through the Rapoport-Leubering pathway. This is a metabolic strategy to produce 2,3-Diphosphoglycerate (2,3-DPG). 2,3-DPG has an affinity for oxyhemoglobin, which causes the hemoglobin molecule to release it oxygen to the tissues. 2,3-DPG inserts itself between the β-chains of hemoglobin, causing electrostatic interactions, which facilitates displacement of oxygen molecules into the tissues. With increased oxygen pressure in the lungs, the increased number of oxygen molecules displace the 2,3-DPG molecule. Note: 2,3-DPG is also known as 2,3-Bisphosphoglycerate (2,3-BPG).

The Pentose-Phosphate pathway converts oxidized glutathione to it reduced form. Reduced glutathione stabilizes the reduced state of hemoglobin. If reduced hemoglobin changes to the oxidized form, then it will denature and precipitate out as Heinz bodies. Glutathione maintains hemoglobin in its reduced state (Fe++), preventing oxidation of the sulfhydryl groups in the hemoglobin molecules and further reduction to Fe+++ (methemoglobin). Accumulation of methemoglobin will change the structure of the cell membrane, weakening it, and rendering it susceptible to rupture/hemolysis. Increased methemoglobin will eventually precipitate to form Heinz bodies. As a rule, no more than 1% of the cell’s hemoglobin is in the methemoglobin form (some textbooks suggests the 3% may be the upper normal limits for methemoglobin concentration).

15 DISCUSS THE IMPORTANCE OF EVALUATING THE ERYTHROCYTES IN A STAINED BLOOD SMEAR.

Such evaluations enable a conformation of a diagnosis, provides visual criteria for classifying anemias, and describes RBC anomalies in terms of size, shape, and degree of hemoglobinization.

Erythrocyte size varies from microcytic to normocytic to macrocytic. Microcytic is characterized by [1] a MCV = <80 fL and [2] size = <6 μM. Microcytes are observed in iron-deficiency anemias. Normocytic is characterized by [1] MCV = 80 to 100 fL and [2] size = 6 to 9 μM. Macrocytosis, observed in hepatic diseases and vitamin B12 and folic acid deficiency anemias are distinguished by an MCV = >100 fL and size = >9 μM. In macrocytosis, cells tend to maintain a round shape. (Refer to objective 69 for an explanation of the indices.)

Erythrocyte size is described by the term anisocytosis. Anisocytosis is one of the most common forms of abnormal RBC’s and can be associated with a variety of disorders (leukemia, pernicious anemia and other forms of anemia). See Objective #18 to grade the degree of anisocytosis present.

Poikilocytosis indicates a variation in the shape of erythrocytes. A deviation for the normal discoid shape of the erythrocyte is the result of a chemical or physical alteration in the red blood cell membrane or the actual contents of the cell. Because of the variety of shapes seen in erythrocytes, specific names have been assigned to the red blood cell to describe it shape. Examples are: acanthocytes, blister cells, echinocytes, elliptocytes, and target cells. Poikilocytosis may be associated with a variety of anemias.

Erythrocytes hemoglobinization is describes as either normochromic, hypochromic, or hyperchromic. A normochromic RBC describes the presence of a normal amount of hemoglobin in the cell and that it stains uniformly, evenly. Its MCH = 27 to 32 pg (μμgm) and the MCHC = 31 to 37%. (Refer to Objective #69 for an explanation of the indices.) Hypochromasia (also known as hypochromia) indicates that the RBC contains a decreased amount of hemoglobin. Visually, a larger than normal central area of pallor or paleness will be present, with an thin rim of hemoglobin . This is one of the most common forms of abnormal erythrocytes, seen in iron-deficiency anemia and thalassemia. It may also be seen in any hemoglobinopathy. The MCH = <27 pg (μμg) and the MCHC = <31%. The following illustration will assist you in grading the degree of hypochromia.

Hyperchromasia (also called hyperchromia) implies a heavy staining of the red blood cell. Usually it is difficult to correlate over saturation of hemoglobin in the erythrocyte. This type of appearance is seen in extra thick RBC’s or spherocytosis. Indices values may be of little value in this condition. MCH values usually do not differentiate macrocytosis, however MCV values will be increased. MCHC values do not exceed 36 or 37% as a rule. Values of 38%, when obtained, are a maximum value and for all practical purposes are of no value. Polychromasia (also called polychromatophilia) describes the erythrocyte that has taken on a bluish hue. The presence of polychromasia can be correlated to the number of reticulocytes and indicates that the younger and larger RBC’s are being shifted into general circulation.. If polychromasia is observed, a “retic” stain may be needful. Also note the MCV values, these should be slightly increased (slightly greater than 100 fL’s. Refer to the grading scale in Objective #18 for hypochromasia, hyperchromasia, and polychromasia.

16 USING CROSS-SECTIONAL ILLUSTRATIONS AND MEASUREMENTS, IDENTIFY THE TYPES OF ERYTHROCYTES IN DIFFERENT CLINICAL CONDITIONS.

Cell Type Diameter Thickness Volume Example Spherocyte 6.18 μ 3.02 μ 90 fL Chronic hemolytic jaundice Microcyte I 7.07 μ 1.63 μ 63 fL Simple microcytic anemia Macrocyte I 8.89 μ 2.20 μ 135 fL Pernicious anemia Macrocyte II 8.58 μ 1.60 μ 92 fL Obstructive jaundice Normocyte 7.7 μ 2.00 μ 90 fL Normal Microcyte II 6.5 μ 1.50 μ <60 fL Thalassemia

17 DESCRIBE A GRADING SCALE THAT IS APPLICABLE FOR GRADING ANISOCYTOSIS AND POIKILOCYTOSIS.

One method that is used in some laboratories is the following description. Red blood cell anomalies may be graded on a scale of normal, slight, and 1+ to 4+ as follows: Normal = less than 5% of RBC’s differ in shape, size, or hemoglobin intensity from the surrounding pattern of normal round, discoid RBC’s. Slight = approximately 5% to 10% of the RBC’s differ from the normal cells. 1+ = 10% to 25% differ from the normal cells, 2+ = 25% to 50% difference from the normal cells, 3+ = 50% to 75% differ from the normal cells, and 4+ = >75% differences from the normal cells.

The following scale is used by many laboratories in the delta region of the U. S. morphology normal 1+ 2+ 3+ 4+ characteristics limits Macrocytes (>9 μ dia.) 0 - 5 5 - 10 10 - 20 20 - 50 >50 Microcytes (<6 μ dia.) 0 - 5 5 - 10 10 - 20 0 - 50 >50 Hypochromia 0 - 2 3 - 10 10 - 50 50 - 75 >75 Poikilocytosis 0 - 2 3 - 10 10 - 20 20 - 50 >50 Anisocytosis 0 - 2 3 - 10 10 - 20 20 - 50 >50 Acanthocyte none 1 - 5 5 - 10 10 - 20 >20 Burr Cell 0 - 2 3 - 10 10 - 20 20 - 50 >50 Target cell (codocyte) 0 - 2 3 - 10 10 - 20 20 - 50 >50 Tear drop cell (dacryocyte) 0 - 2 2 - 5 5 - 10 10 - 50 >50 Sickle Cell (depranocyte) none (If present in any number, report as positive.) Elliptocyte/Ovalocyte 0 - 2 2 - 10 10 - 20 20 - 50 >50 Helmet cell / Bite cell none 1 - 5 5 - 10 10 - 20 >20 Schistocytes none 1 - 5 5 - 10 10 - 20 >20 Spherocytes 0 - 2 2 - 10 10 - 20 20 - 50 >50 Stomatocytes 0 - 2 2 - 10 10 - 20 20 - 50 >50 Basophilic stippling 0 - 1 1 - 5 5 - 10 10 - 20 >20 Polychromatophilia, adult 0 - 1 2 - 5 5 - 10 10 - 20 >20 Polychromatophilia, infant 1 - 6 7 - 15 15 - 20 20 - 50 >50 Howell-Jolly (HoJo) body none 1 - 2 3 - 5 5 - 10 >10 Pappenheimer body (siderocyte) none 1 - 2 3 - 5 5 - 10 >10

These enumerating values are expresses as the number of occurrences per OIF assuming an average of 200 to 250 RBC’s per the 100X objective.

For grading rouleaux formation, use the following criteria: [1] 1+ = aggregates of 3 to 4 cells [2] 2+ = aggregates of 5 to 10 cells [3] 3+ = numerous aggregates of RBC’s. Only a few free RBC’s are observed.

18 DISCUSS POIKILOCYTOSIS IN ERYTHROCYTES.

Poikilocytosis (poikilocytes) describes the variety of nonspecific shapes that may be observed in RBC’s. Poikilocytosis is an irreversible alteration of the cell membrane and is an indicator of abnormal erythropoiesis due to bone marrow effects and/or abnormal RBC destruction. This is one of the most common forms of abnormal RBC morphology. There is a poikilocytosis expression that occurs as the RBC ages (senescence). The RBC will become pinched, pitted, or notched as the membrane breaks down and sloughs off.

19 DESCRIBE THE SPHEROCYTE.

The spherocyte is an erythrocyte in which the biconcave disc profile is lost. It appears as a smaller and more dense RBC. It is also called a hyperchromic microspherocyte. The spherocyte is formed when there is a defect in the membrane function. The sodium pump causes Na+ retention which increases water retention, increasing the intravascular volume. This cell is observed in immune induced hemolysis, post blood transfusions, and congenital anemia. Comment: Fine needle like projections have been reported as being observed in the membrane surface.

20 DESCRIBE THE ECHINOCYTE.

The echinocyte is a crenated erythrocyte. Laboratory vernacular will refer to this cell as a crenated RBC. Crenation is usually not an indicator of a pathological problem. It is usually an artifact due to [1] loss of intracellular fluid, [2] increased anti-coagulant blood ratios (due to a technique during a phlebotomy or manufacturing error in measuring anticoagulant in the vacutainer tube), [3] slow drying of the blood film, or [4] the patient being dehydrated. Look for rounded, regular, smooth,-tipped projections all around the periphery of the cell. Report as crenated cells present. Sometimes these are graded. Follow lab policy.

21 DESCRIBE THE ACANTHOCYTE.

The acanthocyte is an abnormally crenated RBC. It is the consequences of a defect in the cell membrane. Projections from the cell membrane are irregular and distorted with the apex of the projections being pointed. Synonyms include: thorn cell, spur cell, and spicule. These cell types are observed in abetalipoproteinemia, liver disorders, and lipid metabolism disorders. Their presence has been reported in patients on heparin therapy. Use the grading scale described in Objective #18. Remember that acanthocytes should not be observed in a normal stained blood film. When they are seen, be sure to indicate their presence. Follow lab policy for reporting.

22 DESCRIBE THE BURR CELL.

The burr cells is characterized by abnormal cytoplasmic projections, but not to the same extent as that of the acanthocyte. It is characterized by regular pointed projections with regular shaped curves. There is an overall uniform spacing. These cells are observed in uremia, acute blood loss, stomach cancer, and pyruvate kinase deficiency. Note: Some labs consider the burr cell and the crenated cell as the same cell, therefore do not distinguish between the two.

23 DESCRIBE THE SCHISTOCYTE.

Schistocytes (fragmented cells) are fragments of erythrocytes with wide variation in sizes and shapes, ususally microcytic in size. Schistocytes are seen in vascular lesions, uremia, microangiopathic hemolytic anemias, hemolytic anemias cause by physical agents, and disseminated intravascular coagulation (DIC), whenever there is blood vessel pathology present. Schistocytosis is the result of mechanical trauma in the spleen and interaction with intravascular fibrin strands.

24 DESCRIBE THE ELLIPTOCYTE.

Synonyms: ovalocyte, pencil cell, or cigar cells. Normally about 5% to 10% of the circulating RBC’s are oval. These cells are formed after the erythrocytes matures and leaves the bone marrow. Patients diagnosed with elliptocytosis tend to have normal shaped reticulocytes. The mechanism that causes elliptocytes is not known. There is known to be a hereditary defect present in the RBC cytoskeletal proteins (the spectrin chain). These cells observed in varying percentages in iron deficiency anemia, leukemia associated anemias, thalassemia, and dyserythropoiesis. In most anemias, elliptocytes may make up 10% of the RBC population. Patients with congenital elliptocytosis may demonstrate up to 90% distinctly oval shaped cells.

25 DESCRIBE THE TARGET CELL.

Synonym: Mexican hat cell and codocyte. This cell is characterized by an abnormally thin membrane with an increase incorporation of cholesterol into the cell membrane. It appears because of maldistribution of abnormal hemoglobin or certain materials being deposited into the cell membrane. These cells are more resistant to hypotonic lysis. It is observed in hemoglobinopathy, hepatic diseases, iron deficiency anemia, hemolytic anemia, and splenectomy.

Leptocytes are thin cell that is large in diameter and generally displays a thin rim of hemoglobin at the periphery and a large area of central pallor. This cell is cup-shaped as is the stomatocyte but it has very little depth. Target cells are thought to from from this cell type if the cup depth increases. This cell is seen in liver disease and hypochromic anemias.

26 DESCRIBE THE DEPRANOCYTE.

Synonym: sickle cell and meniscocyte. Lab vernacular refers to these cells as sickle cells. They are associated with the disorder, sickle cell anemia. Like the target cell, sickle cells are resistant to hypotonic lysis. There are two basic types of sickle cells; [1] the oat cell, slightly sickled variation, and/or holly leaf. These RBC collapses into these shapes when there is a reduced oxygen atmosphere. In the presence of a normal oxygen atmosphere, the cells revert to the normal discoid shape. [2] The second type form very distorted filamentous forms. In the presence of a reduced oxygen atmosphere these cells form, but when the oxygen pressure is normalized, they do NOT revert back to the normal discoid shape. Sickle cells are also observed in hemoglobin Sβ-thalassemia anemia and hemoglobin SC anemia. Doe not grade these cells. They are to be reported out as positive, if present.

27 DESCRIBE THE STOMATOCYTE.

The stomatocyte is characterized by a slit-like or narrow rectangular area of pallor in the cell. This cell will be concave on one side and convex on the other. These cells are characterized by a alteration in the permeability of the cell membrane to sodium. It is observed in liver disease, alcoholism, electrolyte imbalance, hereditary stomatocytosis, infectious mononucleosis, lead poisoning, malignancies, and thalassemia minor. Note that these cells may appear as artifacts on a stained blood smear. One textbook states stomatocytes when seen are more apt to be an artifact than a pathological process.

28 DESCRIBE THE SPHEROIDOCYTE.

The spheroidocyte is a thicker than normal erythrocyte. It does have an increased amount of hemoglobin. There is usually a smaller area of pallor in the center of the cell and the area of pallor may be located eccentric.

29 DESCRIBE THE DACRYOCYTE.

Synonym: Tear-drop cell or tennis racket cell. The cell has a definite tear drop shape and the length of the “tail” may vary from cell to cell. Small areas of pallor may be present on the cell. The tear-drop cell is observed in pernicious anemia, thalassemia, myeloid dysplasia, severe anemia, and hemolytic anemia.

30 DESCRIBE THE SIDEROCYTE.

The siderocyte is an erythrocyte that contains deposits of iron in the cytoplasm that stain dark blue with Prussian blue stain. The number of granules vary, often there are more than one granule present in a cell. These granules (which are aggregates of mitochondria, ribosomes and iron particles) may be called Pappenheimer bodies. If more than 10% of the RBC’s contain these granules, then abnormal hemoglobin synthesis is present as seen in hyposplenism and hemolytic anemia. Pappenheimer bodies may aggregate so that they resemble a stack of cannonballs. It is not necessary to enumerate the Pappenheimer bodies, it is sufficient to indicate the presence as positive or negative.

31 DESCRIBE BASOPHILIC STIPPLING.

Basophilic stippling (also called punctate basophilia), is characterized by the presence of numerous granules in the erythrocyte. These blue granules may be fine or coarse and may be intense in color. The granules are aggregates of ribosomes and are evenly distributed in the cell. They are observed in lead poisoning, hemoglobinopathy, alcoholism, and megaloblastic anemias. If the “stippling” is coarse, it may be referred to as punctate stippling. Do not grade basophilic stippling. Report is as “positive” if it is present.

32 DESCRIBE THE HOWELL-JOLLY BODY.

The Howell-Jolly body (or HoJo bodies) are round, purple staining nuclear DNA fragments. They may be 1.0 μM in diameter. They are usually observed in the mature erythrocyte, but may also be seen in the nucleated and immature red blood cell. As a rule, only one Howell-Jolly body is seen per cell and some times two. More than two/cell are not the rule. They are formed during the process of karyorrhexis, usually in the megaloblast. If more than two “HoJo bodies” are present in the red cells, then the patient may have megaloblastic anemia. They are also observed in hemolytic anemias, pernicious anemia, post-operative conditions, splenectomy, or splenic atrophy. If Howell-Jolly bodies are present, report out as positive.

33 DESCRIBE THE CABOT RING.

The Cabot ring is a purple staining ring-like filament or figure-8 and is thought to be formed from the microtubules of the mitotic spindle. This inclusion may be present as a double or triple ring. If observed, it is most likely to be seen in severe anemias (example: pernicious anemia) and lead poisoning. It is generally thought to be due to abnormal erythropoiesis. If present, report it out as positive for Cabot rings.

34 DISCUSS HEMOGLOBIN CRYSTALS.

Hemoglobin crystals are seen as tetragonal shaped crystals, found in Hemoglobin C and Hemoglobin SC disease. If the condition is severe, then up to 10% of the RBC’s may contain these crystals. In the case of Hemoglobin SC disease, the crystals may show greater variation. Hemoglobin C crystals may be demonstrated by washing the red blood cells and suspending them in sodium citrate. Hemoglobin C crystals are precipitated polymers of the beta chains of hemoglobin A. If hemoglobin C crystals are observed, then so are target cells (as a rule). If Hemoglobin C crystals are present, do not enumerate, just report that they are present.

35 DESCRIBE ROULEAUX FORMATION.

Rouleaux formation are RBC’s arranged in rows or stacks. They are sometimes present as a slide artifact due to a delay in the spreading of blood or the settling out phenomenon in the thick portion of the blood smear. Rouleaux appears in chronic inflammatory disorders, multiple myeloma, hyperproteinemia, and Waldenström’s macroglobulinemia. Increased amount of fibrinogen in the blood can cause rouleaux formation. If rouleaux is noted in the thick portion of the stained blood film but not in the thin portion, it is probably an artifact. If rouleaux is noted to extend into the thin monolayer portion of the smear, then it is pathological. If rouleaux is NOT an artifact, but represents some pathologic problem, report as follows: If the cells are arranged in aggregates of 3 to 4 RBC’s, report as 1+; if aggregates of 5 to 10 RBC’s, then report as 2+, and if the aggregates are so numerous that only a few free RBC’s, report as 3+.

36 EXPLAIN HOW TO RECOGNIZE AGGLUTINATION. Agglutination occurs when cold agglutinins or autoimmune hemolytic anemia are present. The RBC’s do not stack as in rouleaux, they will clump randomly. If agglutination is present then the automated RBC counts and cell sizing will not be reliable.

37 DESCRIBE CRESCENT BODIES.

Synonyms: Half-moon cell, semilunar body, crescent cell. These are faintly staining RBC’s that have a quarter-moon shape. They are thought to be ruptured RBC’s. Their size approaches that of a WBC. They are observed in malaria and hemolytic anemias. If an occasional crescent shape is seen, do not report. If a significant number of crescent bodies are noted on the blood spear, include the observation in your report. Do not grade unless required by the laboratory. View the “neighborhood” for poikilocytosis.

38 DESCRIBE THE MICROSPHEROCYTE.

Microspherocytes appear in the blood as small round cells and are the result of intravascular hemolysis. This cell type is seen in patient who receive burns over a minimum of 15% of their bodies. It is thought that the heat, that the burned part of the body experiences exerts a direct effect upon the RBC’s to produce fragmentation, budding, and microspherocyte formation. Experiments conducted by heating RBC’s to 49 oC demonstrated this fragmentation phenomenon of erythrocytes. These cells are mechanically and osmotically fragile and are rapidly removed from circulation. This phenomenon is characterized by tiny cell diameters of 2 to 4 μM and a MCV that is <60 fL.

N O T E There is a hemolytic anemia disorder in which the cell membrane protein (spectrin) is abnormal. The RBC will fragment, producing similar fragments as seen in burn patients. These RBC fragments are called “pyropoikilocytes. 39 DESCRIBE HELMET CELLS.

Helmet cells are fragmented cells or schistocytes. They are also called “bite” cells. They are cells that are defective and when traveling through the spleen, the macrophages failed to remove the total cell. They are observed in pulmonary emboli, myeloid metaplasia, and disseminated intravascular coagulation (DIC).

40 DESCRIBE THE KNIZOCYTE.

Knizocytes are RBC’s with more than two concavities. They tend to appear on the blood smear as having a dark staining bar in the center of the cell with two areas of pallor on either side. They have been describes as resembling a pinched bottle How these are formed is not understood. They are observed in hemolytic anemia and hereditary spherocytosis.

41 DESCRIBE THE BLISTER CELL.

The blister cell is formed when the cell is injured and a portion of the hemoglobin leaks out. If the blister breaks, a keratocyte is formed. Once the keratocyte is formed, they are a fragile cell and will disappear from circulation in a few hours. It is seen in end-stage renal disease, as an indicator of pulmonary emboli, also seen in sickle cell anemia, and microangiopathic hemolytic anemia. Also, the vacuole in blister cells is known to rupture and form keratocytes and/or schistocytes.

42 DESCRIBE THE KERATOCYTE.

Synonym: horn cell. These cells form when an erythrocyte is “snagged” by a fibrin strand and the cell is partially cut into. Part of the cell fuses back leaving two or three horn-like projections. The keratocyte is a fragile cell and remains in circulation for only a few hours. These are observed in diffuse intravascular coagulation (DIC). There is a recommendation that keratocytes be reported as schistocytes. Follow lab protocol in reporting these cell types.

43 DESCRIBE NORMAL ADULT HEMOGLOBIN.

Hemoglobin (hgb) is a red colored, conjugated, large molecular weight protein (mw = 64,458) that makes up about 28% of the RBC mass. Most of the RBC mass is water. Each adult hemoglobin molecule (designated as hgb A) consists of a quaternary protein molecule that consists of four globulin (polypeptide) sub-units. The four globulin chains constitute a tetramer. Two of the sub-units are designated as α-chains and the other two subunits are the β-chains. Each subunit contains one heme structure which binds the oxygen molecule to form oxyhemoglobin. See the following illustration:

The hemoglobin chain is manufactured in the cytoplasm of the cell by the ribosomes. Hemoglobin synthesis begin in the prorubricyte (basophilic normoblast). By the time the developing erythrocyte has matured to the metarubricyte stage, about 66% of the hemoglobin formation has been completed. The completion of hemoglobin synthesis occurs in the reticulocyte. The heme structure is manufactured in the mitochondria and cytoplasm in five basic steps. [Step 1] The Kreb’s cycle provide a porphyrin precursor in the mitochondrion, [Step 2] The formation of the porphyrin ring occurs in the cytoplasm, [Step 3] The porphyrin rings are assembled into the coproporphyrinogen III (CPG). [Step 4] The CPG molecule is transferred into the mitochondrion for transforming to protoporphyrinogen IX (PPG). [Step 5] The final step is inserting a single ferrous (Fe++) molecule to form heme. Heme is expelled from the mitochondrion to the cytoplasm where it combines with an α- or β-globulin subunit to form a hemoglobin monomer. Two α-hgb monomers and two β-hgb monomers combine to form the hemoglobin tetramer.

The function of hemoglobin is to transport oxygen to the tissues and return carbon dioxide to the lungs. Each erythrocyte contains about 300 million hemoglobin molecules and there are about 30 trillion RBC in the average adult body. One gram of hemoglobin can combine with 1.34 mL of oxygen. In one liter of blood, about 195 mLs of oxygen is bound to hemoglobin and 3 mLs. of oxygen is carried in the free form.

The globulin chain determines the classification of the type of hemoglobin.

Hemoglobin Structure Stage of Life % in Adult % in Newborn

Gower I ζ2ε2 0-5 weeks Embryo None up to 40 Gower II α2ε2 4-13 weeks Embryo None up to 35 Portland ζ2γ2 4-13 weeks Embryo None up to 35 Fetal (F) α2γ2 Newborn and Adult <1.0 80 A1 α2β2 Newborn and Adult 97 20 A2 α2δ2 Newborn and Adult 2.5 <0.5

44 DISCUSS IRON IN HEMOGLOBIN SYNTHESIS.

Iron is necessary for the production of hemoglobin. There is about 4.0 grams of iron in the body. An estimated 65% of the iron is bound up as hemoglobin and up to 30% is stored in the liver, spleen, and bone marrow. The remainder of the iron is bound to myoglobin, transferrin, and ferritin. Iron is not synthesized in the body and must be incorporated through the diet on a regular basis. Dietary iron is in both the ferrous (Fe++) and ferric (Fe+++) forms in the GI tract. Only the ferrous form is absorbed and this occurs primarily in the jejunum and duodenum. The body does an excellent job of recycling iron so that only about 10% of dietary iron is being absorbed in the healthy individual.. Some of the ferric iron is reduced to its ferrous form by the acidity of the stomach. Once the ferrous iron is absorbed by the intestinal mucosal cells, it is reduced to the ferric state as it combines with the protein apoferritin (a β1-globulin) to form the storage molecule ferritin. It has been estimated that one apoferritin molecule can bind up to 4,000 iron molecules. Once the mucosal cells are saturated with iron, absorption ceases and any unabsorbed iron is excreted in the feces. The mucosal cells release ferric (Fe+++) iron on body demand and release from ferritin allows Fe+++ to be reduced to its ferrous (Fe++) form as it is taken up by the plasma transport molecule, transferrin (TRF) to form a ferric-transferrin complex. This complex is taken to the bone marrow or other cells needing iron. The complex attaches to the cell’s receptor, then the membrane invaginates, and the iron molecules are encased within a vacuole within the cytoplasm. The iron dissociates from the complex and goes to the mitochondria where it is incorporated into heme or bound with apoferritin to form ferritin. The transferrin is ejected from the cell to repeat its transport function. The iron incorporated as ferritin forms aggregates as it is stored in nucleated RBC’s. Note: Fe++ and Fe+++ does not exist in the free form. With special stains, these iron stores can be seen in the erythrocytes designated as siderocytes.

Transferrin is a glycoprotein (a β1-globulin) and is synthesized in the liver. Transferrin (TRF) is known to exist in more than 30 variant forms, yet each variant can bind and transport iron. TRF transports iron from the intestinal mucosal cells and the mononuclear phagocyte system (originally called the reticuloendothelial system [RES]) to other cellular sites for metabolic activity. Most of the iron is delivered to the developing rubriblast and the remainder for the synthesis of myoglobin, cytochromes, catalases, peroxidases, and flavoproteins. TRF (type C) is the predominant from in American Caucasians and Afro-Americans. 45 BRIEFLY DESCRIBE THE VALUE OF THE TOTAL IRON BINDING CAPACITY TEST IN HEMATOLOGY.

The total iron-binding capacity test (TIBC) is a test procedure that totally saturates the protein transferrin (TRF) [but it will include other proteins with iron binding capability] with iron. The TIBC test measures the resulting iron concentration to arrive at the available amount of transferrin (hence it is functional measurement of transferrin concentration). The normal value for the adult male and female is 250 to 460 μg/dL. Actually this test tends to over-measure the actual amount of transferrin because iron can also be bound by albumin and certain other plasma proteins. In normal conditions, when iron values are about 70 to 180 μg/dL, an estimated 30% to 35% of the transferrin molecules are bound with iron. In the TIBC test, from 15% to 50% transferrin saturation can be demonstrated in the normal individual with values for female being somewhat lower than for the male. This test can be useful to help diagnose disorders involving iron metabolism or anemias. The TIBC (ug/dL) can be calculated indirectly by multiplying the serum transferrin value times 1.25. 46 DESCRIBE THE VALUE OF SERUM IRON TESTING IN HEMATOLOGY. The adult body contains about 3 to 5 grams of iron, with 2 to 2.5 grams being located in the hemoglobin. A small amount (less than 150 mg) is found in myoglobin (an oxygen binding protein of muscle). Iron is also a ligand or cofactor in enzymes, enabling the enzyme (such as peroxidases, catalases, and cytochromes) to be functionally active. Iron can be stored as ferritin and hemosiderin, which serves as an important storage pool of iron. If an iron deficiency condition arises, these two pools will become diminished. Iron deficiency is one of the most prevalent disorders, affecting 15% of the world population. Iron is decreased in iron deficiency anemia, malnutrition, malignancy, chronic infection, and anemia of chronic disease. Normal values are shows as follows: Male (adult): 50 to 160 μL/dL Female (16 to 40 y/o): 45 to 150 μL/dL Child: 50 to 120 μL/dL Newborn: 100 to 250 μL/dL Iron is increased in iron poisoning, hematochromatosis, viral hepatitis, and sideroblastic anemia. 47 EXPLAIN HOW THE TRANSFERRIN SATURATION TEST CAN BE USED IN HEMATOLOGY. This test is also known as the percent saturation test and is simply a mathematical comparision of the serum iron to the TIBC. Calculate it as follows: Divide the Total Iron (μg/dL) by TIBC (μg/dL) and multiply by 100% to obtain the % saturation. Example problem: if the total iron is 100 μg/dL and divided by TIBC of 350 μg/dL, a result of 0.2857 is obtained. Multiple 0.2857 by 100% and the % saturation (or transferrin saturation) becomes 28.57 %. The μg/dL for both total iron and TIBC cancel out and vanish to leave a percent value. Normal values are as follows: Male (adult): 20 to 55% Female (16 to 40 y/o) 15 to 50% Child, newborn, and infant: 12 to 50% 48 EXPLAIN HOW THE FERRITIN TEST CAN BE USED IN HEMATOLOGY. Ferritin is a multi-unit (24-subunits) protein that takes on the form of a shell and contains approximately 4,500 iron atoms. The iron is deposited in the core of this large molecle as a ferric hydroxyphosphate complex. In this way, the toxic action of extra iron is prevented. If the ferritin shell is void of iron, it is then known as apoferritin. It is found in most cells and can be quickly mobilized to store iron. Little ferritin is found in human plasma, but will be elevated if there is an excess of iron. The amount of ferritin in plasma is used as an index of body iron stores. The synthesis of ferritin is connected to the level of iron in the cells. When iron levels are high, mRNA (ferritin type) is activated (or stabilized) to produce more ferritin. When iron stores are decrease, the ferritin type mRNA is destablilized (deactivated) and production of ferritin ceases. Normal levels of ferritin are as follows: Adult (male): 20 to 250 μg/L Female (16 to 40 y/o): 10 to 120 μg/L Child: 7 to 140 μg/L Newborn: 25 to 200 μg/L Ferritin is elevated in sideroblastic anemia and hemochromatosis, but it is decreased in iron deficiency anemia. 49 EXPLAIN HOW THE TRANSFERRIN TEST CAN BE USED IN HEMATOLOGY. Transferrin is a beta-1-globulin, a glycoprotein that is synthesized in the liver. It transports iron through the circulatory system to cells that require iron. There are transferrin receptors on the cell allowing the binding the molecule to attach. The transferrin enters the cell via endocytosis and the iron detaches from the transferrin molecule and is passed into the cytoplasm where it is bound to ferritin to be held until ready for use. The transferrin molecule does not detach from the cytoplasmic membrane receptor and it will be returned back to outside the cell where it dissasociates from the receptor and reenters the plasma to pick up more iron. Normal values for plasma transferrin are: Adult (male): 200 to 380 mg/dL Female (16 to 40 y/o) 200 to 380 mg/dL Child: 200 to 360 mg/dL Newborn: 130 to 275 mg/dL Transferrin is increased in iron deficiency anemia and decreased in iron overload and hemochromatosis. If transferrin is tested for, it may be to access the nutritional health of the patient. If there is an inflammatory process going on in the body, its concentration will decrease. 50 DISCUSS THE ANOMALY HEMOCHROMATOSIS. Hemochromatosis can express itself an autosomal recessive disorder with a progressive increase in iron stores. This creates a toxic condition and can lead to organ impairment and damage. Males are affected more than females and the disorder usually expresses itself around the age of 40 (or older). When the disorder expresses itself, up to 4 mg of iron (possibly more) can be absorbed daily and the iron is deposited directly to parenchymal cells (liver, pancreas, heart, and other organs). Consequently, patients are at high risk for cardiac damage, liver cancer, and diabetes (type I). Hemochromatosis may be of the acquired type and in this case the iron is deposited into the reticuoendothelial cells of the liver, spleen, and bone marrow. If the condition persists, then iron is deposited in the parenchymal cells as in the hereditary type. The acquired form of hemochromatosis may be a secondary response to anemias or multiple blood transfusions. Chronic liver disease with alcoholism may present with increased iron levels in the tissues. In hemochromatosis the serum iron test, transferrin saturation, and serum ferritin, are increased. The TIBC is decreased. 51 DISCUSS FREE ERYTHOCYTE PROTOPORPHYRIN (FEP) AND HOW ITS MEASUREMENT IS HELPFUL IN HEMATOLOGY.

Free erythrocyte protoporphyrin is s product of the heme synthesis pathway. This molecule escapes from the synthesis pathway and exists as a nonheme protoporphyrin in the erythrocyte. It can be measured and the information it provides is clinically useful. Normal reference levels range from 170 to 770 μg/L. If a condition arises in which there is a iron deficiency or an impairment of the utilization of iron, FEP will increase. Also lead poisoning will signficantly increase this product. This testing procedure is a helpful screening test for iron deficiency. It was used to screen for lead posioning, but more sensitive tests have replace it as a screening test. It is also increased in anemia of chronic disease and may be in siderblastic anemia (but not always). It is usually normal in hemochromatosis and thalassemia trait. 52 DISCUSS HEMOSIDERIN AND HOW IT MEASUREMENT IS HELPFUL IN HEMATOLOGY.

Hemosiderin is a poorly defined molecule that is formed by the degradation of ferritin, yet still contains iron. It can be detected by Prussian Blue stain and is seen histologically with there is excessive storage of iron. The iron stored in the cell is an insoluble cellular inslusion of Fe3+ complexed with ferritin. These complexes form granules (from 1 to 2 μm in diameter) that serve as as storage form of iron when there is insufficient levels of apoferritin. The iron to ferritin ratio is much greater as hemosiderin than in ferritin. Hemosiderin can release it iron, but does so at a very slow rate. Hemosiderin, when seen on unstained smears, appears as yellow granules. When present in Wright's or Wright's-Giemsa stain, the granules are bownish-blue coloration. The presence or absence of hemosiderin is determined by cytochemical staining of bone marrow using Prussian blue stain which is the preferred and precise technique, which yields bluish granules. It is used to assess body iron stores. If hemosiderin granules is absent, then the body iron stores are depleted. The presence of granules may be reported as a numerical value from 0 to 4+, where 2+ represents a normal or adequate iron store. Normal marrow will have 30 to 50% of the erythroblasts containing specks of hemosiderin. These cells are called sideroblasts. In the normal metabolism of hemoglobin, iron can be captured by the renal tubules and complexed with storage proteins and ferritin to form tubular hemosiderin. When these cells are sloughed off, hemosiderin can be demonstrated in the urine (hemosiderinuria). Normally hemosiderin is not found in the urine. If present, it indicates an unexplained anemia or chronic intravascular hemolysis. 53 DISCUSS THE FATE/DESTRUCTION OF THE ERYTHROCYTE.

The life span of an erythrocytes averages about 120 days for an adult male but only 109 days for an adult female. As the RBC circulates in vessels with a larger diameter, the cell is a typical biconcave cell. When it circulates through capillaries, the diameter reduces to 2 to 3 μM and the cell must “squeeze” through. In this process, the cell experiences physical and osmotic stresses that causes the loss of much of it internal plasma. This ability to migrate through a channel smaller than the cell is called deformability. Repeated passages through the capillaries is traumatic and results in the significant changes in the cell membrane which leads to its removal from circulation. These changes includes membrane damage and decreased efficiency of the metabolic pathways. RBC destruction occurs primarily in the spleen, liver, and bone marrow. The rupture of the membrane allow the escape of hemoglobin. Hemoglobin is degraded to heme and globulin. The “globin” polypeptide chain is degraded to smaller peptide units and joins the amino acid pool. The amino acid pool is not an actual storage place for amino acids. Amino acids cannot be stored in special sites as is fat in adipocytes. This is a conceptual concept in which it is understood that there are small amounts of amino acids present in cells and in circulating blood. Remember that proteins are present in all cells and tissues. These proteins can be mobilized during times of stress (starvation or times of fasting). Also, amino acids can be metabolized to glycogen to be used as a source of energy or to triacylglycerol to be stored as fat. The heme of hemoglobin is degraded to iron and the porphyrin ring in the macrophage. The ring is opened by the enzyme heme oxidase to produce carbon monoxide and biliverdin. Carbon monoxide is formed when the α-carbon in the methane bridge of the ring structure is broken to form biliverdin. CO escapes and some will bind with hemoglobin, but most is eventually exhaled in the lungs. Biliverdin is quickly reduced to bilirubin. Most of the released bilirubin is immediately bound to albumin to form an unconjugated bilirubin. When the unconjugated bilirubin enters the liver, the bilirubin-albumin complex is broken and bilirubin recombined with glucuronic acid to form conjugated bilirubin. In the conjugated state, bilirubin is now polar and lipid insoluble. It is in this form that bilirubin is excreted into the bile and passes into the small intestine. In the small intestine, the conjugated bilirubin is called mesobilinogen and it is acted upon by bacteria to form stercobilinogen, then stercobilin. These are pigmented compounds. Up to 80% of the conjugated bilirubin will be excreted as stercobilinogen and stercobilin. The remainder is reabsorbed into general circulation and recycles through the liver or is excreted through the kidneys. In the urinary system, stercobilinogen and stercobilin are secreted as urobilinogen and urobilin, even though they are the same compounds.

Some bilirubin will circulate in the plasma as a free form (unbound). The albumin- bilirubin complex and the free bilirubin are referred to as unconjugated bilirubin. These are water insoluble and alcohol soluble. These give the indirect van den Bergh reaction. Bilirubin complexed with glucuronic acid is designated as conjugated bilirubin and is water soluble and alcohol insoluble. The conjugated form gives the direct van den Bergh reaction.

54 LIST THIRTEEN CHANGES THAT OCCURS IN THE AGING PROCESS OF THE RBC.

[1] increased membrane bound IgG [8] cell becomes more spherical [2] increased cell density [9] increased intracellular viscosity [3] increased intracellular sodium 10] increased methemoglobin [4] decrease enzyme activity [11] decrease intracellular potassium [5] decrease hgb affinity for oxygen [12] decrease phospholipids [6] decreased cell cholesterol [ [13] decrease in sialic acid [7] changes in MCHC and MCV

55 LIST THE NORMAL HEMOGLOBIN VALUES EXPECTED FOR NINE AGE GROUPS.

Birth ...... 17 to 23 g/dL 6 to 10 years . . . . . 10.5 to 14.6 g/dL 2 months ...... 9 to 14 g/dL 11 to 15 years . . . . 11.4 to 15.4 g/dL 12 months ...... 11.8 g/dL >16 to 50 (male) ...... 14. to 18 g/dL five years ...... 12.6 g/dL >16 to 50 (female) . . . . 12 to 16 g/dL > 50 years . . . . . values may increase slightly

56 LIST FIVE FACTORS THAT AFFECT THE ERYTHROCYTE VALUES.

[1] Physiological. Taller and heavier individuals tend to have increased RBC counts. [2] Psychic. Fear and excitement tend to increase the RBC count. [3] Sexual differences. Males tend to have higher RBC counts. Testosterone promotes erythropoiesis. Estrogen tend to decrease erythropoiesis. [4] Barometric pressure. Increasing altitudes has an augmenting effect that increases the RBC count. [5] Muscular activity. Physical conditioning and training leads to elevated RBC counts. RBC values drop to normal ranges as the person settles back into a routine that maintains his physical conditioning.

NOTE: Anything that will increase the RBC count will also have the same effect the hemoglobin and hematocrit determinations.

57 DESCRIBE HOW THE GLOBIN CHAINS DIFFER ON THE HEMOGLOBIN MOLECULE.

The globin chains that are part of the hemoglobin molecule are sequences of amino acids which may be designated as peptide chains. The different globulin chains are designated by an Greek letter. Examine the following listing for the types of globin chains found in hemoglobin

Greek letter # of amino acids comments designation Alpha (α) 141 Beta (β) 46 Delta (δ) 146 differs from β-form by 10 amino acids Gamma (γ) 146 differs from β-form by 39 amino acids Epsilon (ε) 146 embryonic stage only Zeta (ζ) 146 embryonic stage only

The embryonic forms of globin (ζ and ε) appear only in the first three months of embryonic development and then disappears. The globin portion of the hemoglobin molecule will consist of two sets of globin pairs. For example, two α-globin and two β- globin molecules is characteristic of hemoglobin A. Two ζ-globin and two γ-globin molecules is characteristic of the Portland type of hemoglobin.

58 BRIEFLY DESCRIBE SIX TYPE OF NORMAL HEMOGLOBIN MOLECULES.

[1] See Objective #43 for the description of Hemoglobin A1, the normal adult hemoglobin. This hemoglobin is the major oxygen carrier in the human from about three months to death. It appears in the fifth month of gestation. [2] Hemoglobin F. This is a hemoglobin normal to the fetus. Appearing about the fifth week of gestation, it will increase and peak about the seventh month, and can make up to 95% of the total hemoglobin. About the time of birth the amount of Hgb F will be reduced to about 80% of the total hemoglobin, as the developing body increased the rate of Hgb A1 production. About six months after birth, Hgb A1 has almost totally replaced Hgb F. By the child’s third birthday, <1.0% of the total hemoglobin is the F- type. Hemoglobin F has a higher affinity for oxygen and can “pick-up” the low levels of placental or uterine oxygen. It consists of two α-globins and two γ-globins. Hgb F is easier to oxidize to methemoglobin and it is also more resistant to alkaline denaturation than other hemoglobins. [3] Hemoglobin Gower I. This is an embryonic hemoglobin, found in trace amounts. It consists of two ζ-globins and two ε-globins. It can be detected on in the first three months of embryonic life. [4] Hemoglobin Gower II. This is the most important of the embryonic hemoglobins and will make up as much as 60% of the total embryonic hemoglobin. It persist only during the first three months of life. It consists of two ε-globins and two α-globins. [5] Hemoglobin Portland. This is an embryonic hemoglobin, found in trace amounts. It consists of two γ-globins and two ζ-globins.

[6] Hemoglobin A2. This hemoglobin type makes up to 3% of normal adult hemoglobin and consists of two α-globins and two δ-globins. It appears late in the fetal life and is not produced in any significant quantities. It is designated as a minor hemoglobin.

59 DESCRIBE METHEMOGLOBIN.

Synonyms: hemiglobin (Hi). Methemoglobin (non-functional hemoglobin) is the result of the oxidation of Fe++ (ferrous iron) in the hemoglobin to the ferric state (Fe+++). This oxidized form of hemoglobin cannot bind oxygen. Up to 3% of the total hemoglobin in the body may be converted to methemoglobin daily. The body has a means of counteracting this oxidation process through the enzyme, NADH dependent methemoglobin reductase (also called diaphorase). This is an efficient reduction system, reducing methemoglobin 250 times faster than a hemoglobin molecule can be oxidized. As a rule, the methemoglobin levels never exceed 1.0%. If methemoglobin levels reach 10%, cyanosis can develop. Blood plasma will begin to take on a brownish appearance. If levels approach 60%, then hypoxia occurs. Heinz bodies may be seen in this anomaly.

In Hemoglobin M disease, the affected individual will demonstrate greater than 2% methemoglobin levels. This disorder can be acquired (caused by toxic substances), presence of a Hemoglobin M variant, the presence of a defective enzyme (NADH- diaphorase), or a NADH-diaphorase deficiency. The normal mechanism for converting methemoglobin to normal functional hemoglobin is as shown.

Oxidation is the loss of electrons. A molecule can give up an electron or hydrogen ion to another molecule. Reduction is to gain an electron. It can mean for something to undergo a reaction. In the laboratory, a popular testing method for determine the amount of hemoglobin is the cyanmethemoglobin method. This method requires reducing hemoglobin to the methemoglobin form before it can be reduced to cyanmethemoglobin. Hemoglobin electrophoresis is diagnostic for this disorder. Nitrates and chlorates can cause oxidation of hemoglobin to methemoglobin.

60 EXPLAIN HOW THE EMBDEN-MEYERHOF PATHWAY OPPOSES METHEMOGLOBIN ACCUMULATION.

In the oxidation of hemoglobin to methemoglobin, if an electron is lost, the ferrous iron ( Fe++) is oxidized to ferric iron (Fe+++). Conversely, if an electron is added to ferric iron, ferrous iron is formed. The Embden-Meyerhof pathway (Glycolysis cycle) produces NADH, a nucleotide molecule that can function as an electron donor. RBC’s contain the enzyme, NADH-methemoglobin reductase (also knows as diaphorase). NADH is produced as glyceraldehyde (which gives up an electron and NAD captures it) is converted to 1,3-diphosphoglycerate. The resulting NADH molecule can then donate its electron to methemoglobin reducing it to functional hemoglobin.

61 DISCUSS SULFHEMOGLOBIN.

Sulfhemoglobin is formed with sulfur levels build up in the body. This is a stable hemoglobin that cannot combine with or carry oxygen. Sulfhemoglobin is usually seen when methemoglobin is present in higher than normal amounts. It is formed with hemoglobin is exposed to drugs, such as trinitrotoluene, acetanilid, aniline dyes, sulfur containing cathartics, inorganic sulfides, phenacetin, or sulfonamides. Sulfhemoglobin is found in normal blood, but in concentrations <1.0%. This type of hemoglobin once formed, remains with the cell until the cell is removed from circulation, at which time the sulfhemoglobin is removed. Sulfhemoglobin cannot be reduced by ascorbic acid or methylene blue as can methemoglobin, but can combine with carbon monoxide to form carboxysulfhemoglobin. Sulfhemoglobin cannot be converted to cyanmethemoglobin and measured by this standard procedure. Sulfhemoglobin can be measured examining a prepared hemolysate at 610 nm wavelength. 62 DISCUSS CARBOXYHEMOGLOBIN.

Carboxyhemoglobin (Hgb∙CO)is a carbon monoxide derivative of hemoglobin and is usually found in normal blood at concentrations <1.0% of the total hemoglobin. Carbon monoxide has an affinity for hemoglobin that is 200 time greater than that of oxygen. People who smoke have concentrations of carboxyhemoglobin of approximately 5.0% of the total hemoglobin. People who live in cities have a higher level of Hgb∙CO than those who live in rural areas. When Hgb∙CO concentrations reach 10%, judgment is impaired and at 50% concentration, unconsciousness is a real risk. Also other symptoms include respiratory failure, followed by the risk of death. A simple procedure for testing for carbon monoxide poisoning is to hemolyze 0.5 mLs of blood with 20 mLs of distilled water and adding 1.0 mL of 1N NaOH. If a light cherry red color appears, then the blood contains more than 20% carboxyhemoglobin. Carboxyhemoglobin can be measured spectrometrically by comparing the δ-absorption bands with that of oxyhemoglobin.

63 DESCRIBE THE DIFFERENCE BETWEEN DACIE’S AND DRABKIN’S CYANMETHEMOGLOBIN REAGENTS.

Dacie’s reagent is made with distilled water, potassium ferricyanide, and potassium cyanide. Drabkin’s reagent is similar except it contains sodium bicarbonate. Drabkin’s reagent is preferred over the Dacie reagent. The sodium bicarbonate facilitates the lysis of the RBC’s.

64 DESCRIBE THE PRINCIPLE OF THE CYANMETHEMOGLOBIN METHOD.

When whole blood is added to the reagent, potassium ferricyanide converts hemoglobin to methemoglobin. In the next reaction step, methemoglobin reacts with potassium cyanide to form cyanmethemoglobin (a stable colored compound). The amount of cyanmethemoglobin formed is measured spectrophotometrically at 540 nm wavelength. Cyanmethemoglobin is the standard for hemoglobin determinations because it measures all hemoglobins except sulfhemoglobin. It is very easy to standardize, forms a stable solution, and is a rapid and easily reproducible procedure. There are some sources of concern, these being, [1] cyanide is toxic, but it takes from 4 to 6 liters of Drabkin’s solution to form a lethal dose. [2] Commercial solutions tend to be unstable and have a short shelf life. To avoid this problem, refrigerate and/or keep in the dark. [3] Testing blood from heavy smokers tend to introduce significant error into the procedure. As much as 10% of their blood can be in the form of carboxyhemoglobin. It takes about one hour for cyanmethemoglobin reagent to convert Hgb∙CO to cyanmethemoglobin. [4] If lipemic blood is used is used to determine hemoglobin concentrations (may that never happen), there will be increased turbidity in the reagent-blood mixture and the reading will be increased. [5] Globulin diseases (examples: multiple myeloma and Waldenstrom’s macroglobinemia) cause high readings. In Drabkin’s reagent the abnormal proteins will precipitate, causing increased turbidity. (Note: K2CO3 , if added to the Drabkin’s solution, it will prevent these proteins from precipitating out.)

NOTE: Objectives 65-68 are abbreviated descriptions of obsolete technologies that are included for their historical interest. These four objectives will not be included in any tests.

65 DESCRIBE THE ACID HEMATIN METHOD FOR HEMOGLOBIN DETERMINATIONS.

This is a method in which dilute HCl is added to blood to form acid hematin. This is a slightly inaccurate method and requires a Sahli-Hellige hemoglobinometer that contains a colored glass standard for comparison.

66 DESCRIBE THE ALKALI HEMATIN METHOD FOR HEMOGLOBIN DETERMINATIONS.

This is a method in which dilute NaOH is added to blood and heated. It’s major disadvantage was that it did not measure hemoglobin F. The color of the alkali hematin solution is compared to known standards or measured in a colorimeter at a wavelength of 610.

67 DESCRIBE THE OXYHEMOGLOBIN METHOD FOR HEMOGLOBIN DETERMINATIONS.

This is a method where whole blood is mixed with 0.007 NH4OH (N/150) and hemoglobin is converted to the oxyhemoglobin form. It is an accurate and stable method. It’s disadvantage lies in its sensitivity to copper. Any copper present will convert oxyhemoglobin to methemoglobin. Sodium carbonate (0.1%) may be substitute for ammonium hydroxide. The oxyhemoglobin solution is measured photometrically at 550 wavelength.

68 DESCRIBE THE COLOR INDEX CONCEPT OF EVALUATION RED BLOOD CELLS.

This is an interesting, but outmoded method of evaluating the amount of hemoglobin in the cell. It is a mathematical method that requires the RBC count and hemoglobin value. It was calculated by dividing the Hgb reading by the first two numbers in the RBC count. It is based upon the principle that a 100% reading is equal to a hemoglobin concentration of 14.5 g/dL..

69 EXPLAIN THE HEMATOCRIT .

Synonyms: packed cell volume, “crit”. The hematocrit (HCT) is the percentage of the total volume of blood that is occupied by packed red blood cells. It is the simplest and most accurate of the laboratory procedures. The hematocrit results are preferred (as a rule) over that of the RBC count and allows for calculation of the indices. Regarding the manual hematocrit, the following is applicable: [1] The buffy coat can provide a “rough” estimate of the WBC count. A rule-of -thumb estimate is a buffy coat of ≤1.0% is generally represents a normal WBC count. [2] The buffy coat is not to be included as part of hematocrit reading. [3] A distinctly colored plasma layer can indicate the presence an icteric condition. A. Normal values are as follows: • Adult male = 42% to 53% • Adult female = 36% to 46% • Newborn = 50% to 62% • One year = 31% to 39%

70 DESCRIBE THE BUFFY COAT.

The buffy coat is a distinct white layer or “button” between the plasma and RBC’s. Platelets (a very thin yellow-white layer) will layer over WBC cells (the thicker grayish-white layer). The buffy coat is a convenient way to collect a large number of leukocytes for evaluating a blood film for leukemia, leukopenia, megaloblasts, LE cells, etc. The buffy coat from a normal healthy individual will be 1% or less. If the buffy coat is greater than 1%, then this indicates an increased WBC count and a possible pathology.

71 LIST NINE SOURCES OF ERROR POSSIBLE IN HEMATOCRIT TESTING.

[1] Improper sealing of tube. [2] Increased anticoagulant ratio causing increased cell shrinkage which causes a decreased hematocrit. [3] Time and speed of the centrifuge. [4] Improper mix of blood. [5] Including the buffy coat into the hematocrit determination., [6] Incorrectly using the hematocrit reader. [7] If the patient has an anemic condition (See comment below). [8] Hemorrhage (decreases hct). [9] Not removing first drop of blood of a fingerstick (Tissue fluid is in the first drop and will dilute the specimen).

Comment: Anemias can affect the hematocrit by increasing or decreasing the reading. For example, if the patient has microcytosis with anemia, the HCT will be decreased. Other examples include: [1] macrocytosis will cause an increased HCT and [2] poikilocytosis may facilitate trapping of plasma in the spaces about the cells, causing an increase in the hematocrit.

72 DESCRIBE HOW TO CALIBRATE THE PACKING TIME OF A HEMATOCRIT CENTRIFUGE.

Being by preparing several hematocrits for centrifugation using the same blood specimen. On the first centrifugation, spin the specimens for two minutes. Repeat centrifugation steps by increasing centrifuge time at 30 second intervals. Look for consistent readings and a translucent appearance in the packed RBC’s. Normal centrifugation time should fall between three and five minutes.

73 LIST FOUR REASONS WHY A PATIENT MAY HAVE AN ELEVATED HEMATOCRIT.

[1] Pathological polycythemia, [2] physiological polycythemia, [3] shock associated with surgery, burns, or traumas, and [4] dehydration.

74 LIST FIVE REASONS WHY A PATIENT MAY HAVE A DECREASED HEMATOCRIT.

[1] anemias, [2] hydration, [3] pregnancy, [4] receiving IV fluids, and [5] cardiac decompensation (a failure to maintain a good blood circulation).

75 DISCUSS THE CLINICAL SIGNIFICANCE OF THE RED BLOOD CELL INDICES.

The indices are a set of mathematical calculations that define the size and hemoglobin content of erythrocytes. The requires measurement of hemoglobin (g/dL), hematocrit (%), and the RBC count (μL). Synonyms for the indices are {1] red cell indices, [2] mean cell values, [3] average cells values, [4] mean corpuscular values, [5] blood indices, and [6] erythrocyte indices. All indices should be confirmed by visual examination of a stained peripheral blood smear. The indices calculations are a valuable means of helping to differentiate anemias. The three indices are as follows:

Mean Corpuscular Volume (MCV) . This calculates the average volume of the RBC in a given sample of blood. Calculate as follows: HCT / RBC/μL (X) 10 = MCV. The MCV is expresses in fL. fL = femtoliter (10-15/L). (Note: fL = μM3). Sample problem: HCT = 45% and RBC count = 4.85 X 106/μL (use only the first three digits, 4.85)? Answer = 92.78 fL Solution: MCV = 45 divided by 4.85 times 10 = 92.7835 OR 92.78 fL.

MCV values are significant in determining if the RBC is microcytic, normocytic, or macrocytic. If <80 fL, then microcytic; if between 80 and 100 fL, then normocytic; and if >100 fL, then macrocytic. Examples: [1] pernicious anemia = 99.7 to 146.7 fL, [2] iron deficiency anemia = 56.7 to 88.8 fL, and β-thalassemia minor = 53.7 to 73.5 fL There may be a rare occasion in which femtoliters must be calculated in femtomoles. If this is the case, simply multiply fL times 0.0155.

Mean Corpuscular Hemoglobin (MCH) represents the amount of hemoglobin (by weight) in the average erythrocyte in a sample of blood. Calculate as follows: MCH (pg) = Hgb (g/dL) / RBC (μL). pg = picogram (10-12). (NOTE: pg = μμg) Sample problem: Hgb = 15.0 g/dL, RBC = 4.85 (x) 106. Answer = 30.93 pG. Solution: MCH = 15 divided by 4.85 times 10 = 30.9278 OR 30.93 pG

Both the MCV and MCH have the same denominators (RBC count/μL). A relationship between the Hgb and Hct is maintained. Because the Hct is usually 3X that of Hgb, the MCV will usually be 3X the MCH in normal individuals. The MCH is considered to be of lesser importance in describing anemias then the MCV and MCHC. If the MCH is <27 pg, then it tends to be characteristic of microcytic anemias and normocytic, hypochromic anemias. If the MCH falls between 27 to 32 pg, then this indices is normal. A MCH >32 pg has been perceived as being indicative of macrocytic anemias. Because the MCH does NOT take into account the size of the cell, it must be interpreted by considering the MCV. The reason why the MCH has been deemed to less clinically significant in evaluating anemias is that in some anemias, there is a somewhat proportional change in the hemoglobin content (based on the MCH) with a change in the size of the cell (based on the MCV), BUT the MCHC remains normal, therefore the anemia is designated as normochromic. In other anemias, with a decrease in cell size (MCV) and a corresponding decrease in the MCH, If the MCHC is also decreased the anemia is hypochromic. It is found that a decrease in the MCH (<26 pG) or increase in MCH (>32 pG) does not mean that the anemia should be respectively classified as hypochromic or hyperchromic. Remember that under certain conditions, the MCH can be falsely elevated. This is also true for the MCV and MCHC. Mean Corpuscular Hemoglobin Concentration (MCHC) is considered to be an absolute value and expresses the concentration of hemoglobin in terms of average weight in the average RBC in a sample of blood and expressed as a percent value. The MCHC is now being expressed as g/dL. It is the ratio of the hemoglobin weight to RBC volume. Calculate the MCHC (%) as follows: Hgb (g/dL) / Hct (%) (X) 100. Sample problem: Hgb = 15 g/dL and Hct = 45%. Answer = 33.3 g/dL (%). Solution: MCHC = 15 divided by 45 times 100 = 33.3333 or 33.3 g/dL

If the MCHC values <31%, then it is suggestive of iron deficiency anemia. A value of 31 to 37% is normal. Values >37 are suggestive of hyperchromia. (NOTE: MCHC values >38 should not occur. Normal RBC’s contain maximum amounts of hemoglobin.) If a MCHC value >38% occurs, then revisit the indices, an error in calculation is very likely. If a blood sample contains rouleaux or RBC agglutination, then an electronically counted and determined Hct may be falsely low, which will affect the MCH. A blood specimen with lipemic plasma may result in abnormally low MCHC values. Hypochromia characteristically occurs in iron deficiency anemias, thalassemia, and defective iron metabolism. Hyperchromic values usually indicates shape changes. It is not the usual rule to find the term hyperchromia included in many anemia classification schemes. Note: In the past the MCHC has normally been reported as a percent value. Lab’s continue to report the MCHC value this way. There is a trend to report this indices as g/dL because it represents the average concentration of hemoglobin per cell size and hemoglobin is classically reported as g/dL. N O T E Accurate calculations of hemoglobin, hematocrit, and RBC count determinations are required for accurate indices calculations. If an error of 5% or greater occurs in any of the three parameters, the indices results are invalid. It is important for the laboratory to use electronic counters, quality reagents, reliable controls, and precision volumes in measurement and glassware. 76 LIST EXAMPLES ILLUSTRATING HOW HEMOGLOBIN, HEMATOCRIT AND RBC COUNTS CAN BE USED FROM THE ERYTHROCYTE INDICES TO PRODUCE A PICTURE OF ERYTHROCYTE MORPHOLOGY.

[1] Normochromic, normocytic erythrocytes. Hgb = 14.0 g/dL MCV = 91.9 fL (80 - 100 fL) Hct = 41% MCH = 31.3 pg (27 - 32 pg) RBC = 4.5 X 106/μL MCHC = 34.1% (31 - 36 %) Erythrocytes are normal in size and contain a normal concentration of hemoglobin.

[2] Hypochromic, normocytic erythrocytes. Hgb = 9.6 g/dL MCV = 85.4 fL (80 - 100 fL) Hct = 38% MCH = 21.6 pg (27 - 32 pg) RBC = 4.45 X 106/μL MCHC = 25.3% (31 - 36 %) Erythrocytes are normal in size but contain less hemoglobin than normal

[3] Hypochromic, microcytic erythrocytes. Hgb = 8.9 g/dL MCV = 65.9 fL (80 - 100 fL) Hct = 29% MCH = 20.2 pg (27 - 32 pg) RBC = 4.40 X 106/μL MCHC = 30.7% (31 - 36 %) Erythrocytes are smaller than normal and contain less hemoglobin than normal.

[4] Normochromic, macrocytic erythrocytes. Hgb = 8.0 g/dL MCV = 112.6 fL (80 - 100 fL) Hct = 26% MCH = 34.6 pg (27 - 32 pg) RBC = 2.31 X 106/μL MCHC = 30.8% (31 - 36 %) Erythrocytes are larger than normal (MCV) and contain a larger amount of hemoglobin (MCH). Notice that the MCHC is normal, therefore the RBC’s are normochromic.

[5] Hypochromic, macrocytic erythrocytes. Hgb = 5.6 g/dL MCV = 113.7 fL (80 - 100 fL) Hct = 24% MCH = 26.5 pg (27 - 32 pg) RBC = 2.11 X 106/μL MCHC = 23.3% (31 - 36 %) Erythrocytes are large (MCV). Both MCHC and MCV are low, therefore hypochromia.

77 EXPLAIN THE PRINCIPLE OF THE “RULE OF THREE”.

A general application of mathematics can be used to verify the validity of the RBC count, along with the hemoglobin and hematocrit values. This concept is valid as long as the visual inspection of the stained blood film demonstrates normochromic, normocytic red blood cells. Look at the following examples for how the “rule of three” is suppose to work. Assume that the lab reported the following normal values for a pre-surgery patient: RBC count = 4.85 X 106/μL Hgb = 14.5 g/dL Hct = 44%

[1] If the RBC count is 4.85 X 106/μL, then 3 X 4.85 = 14.55 (g/dL) for hemoglobin. This is a confirming value because it falls within ± 3 units. [2] If the hemoglobin is 14.5%, then 3 X 14.5 = 43.5. This is a confirming value because it falls within ± 3 units [3] Divide the hematocrit (44%) by 3 to obtain an answer of 14.7 (g/dL). This is a confirming value for hemoglobin because it falls within ± 3 units. [4] Divide the hemoglobin (14.5) by 3 to obtain an answer of 4.83. This is a confirming value of the RBC count because it falls within ± 3 units.

The rule of three will not function for a patient with a normocytic, hypochromic anemia. If the lab reported the following values: RBC count = 4.20 X 106/μL Hct = 39% Hgb = 8.9 g/dL

[1] 4.20 X 3 = 12.6 (g/dL for hemoglobin) Does not follow the rule of three [2] 8.9 X 3 = 26.7 (% for the hematocrit). Does not follow the rule of three [3] 39 ÷ 3 = 13 (g/dL for hemoglobin). Does not follow the rule of three. [4] 8.9 ÷ 3 = 2.96 (X 106/μL for the RBC count). Does not follow the rule of three.

If the rule of three indicates a discrepancy, then an investigation should be undertaken to determine this represents the results of a pathology, in which case no action is required. If not a pathology, then corrective action must be implemented, whether the problem is due to the instrument or the specimen.

78 DISCUSS THE RED CELL DISTRIBUTION WIDTH IN RBC EVALUATIONS.

The red cell distribution width (RDW) is a mathematical calculation (built into the automated instrumentation) that uses the MCV and RBC count to measure the variation in the RBC volume distribution. The following formula is used: SD [RBC volume distribution] (x) 100/MCV = RDW (%) The normal range is 11.5% to 14.5%. If the RDW value is low, the RBC population is even, all cells are in the same size range. Low readings suggest that there is little clinical significance because there is little or no deviation in the size of the erythrocytes. If the RDW is increased, then it is clinically significant, indicating increased sizes with variation. Examination of a blood smear with an increased RDW should reveal a picture of anisocytosis. RDW values should be interpreted with caution and only after evaluating the blood smear and histogram. If there is a true increase in the variation of cell sizes, the base of the RBC histogram should be broader. Remember, when you are examining a blood smear, to see up to 6% variation in RBC sizes is not abnormal.

79 DISCUSS THE PRINCIPLE OF THE ERYTHROCYTE SEDIMENTATION RATE (ESR).

The erythrocyte sedimentation rate (ESR) is a simple and commonly performed laboratory test that is useful in monitoring inflammatory disorders or differentiating between diseases. This procedure is nonspecific. German physician (Christian Friedrich Nasse) in 1836, discovered that changes in the plasma proteins caused the variable rates at which RBC’s wold fall in whole blood. It would be a century before the significance of this finding would be recognized. The ESR procedure was introduced to the medical profession about 1915 by a Swedish physician. Alf Westergren (Swedish physician) developed the basic sedimentation rate procedure that is still in current use and bears his name. A popular synonym for ESR is “sed rate”.

In inflammatory changes or other disorders, the plasma will under go changes in fibrinogen, α-globulins, and/or β-globulins. These plasma changes will enhance rouleaux formation and speed the fall of the erythrocytes. Plasma changes causes changes in the RBC surface permitting aggregation of the RBC (rouleaux formation) which in turn increases RBC mass. The rate at which RBC’s fall is directly proportional to the erythrocyte mass and also inversely proportional to the plasma viscosity. In the normal healthy individual, the red blood cell does not form rouleaux. Absence of rouleaux leaves the RBC as a small mass and settling phenomenon of whole blood will proceed slowly.

There are two procedures employed by the laboratory: [1] Westergren method. This is the preferred procedure and is in more common use than the other method. A blood specimen drawn in EDTA or Sodium Citrate is required. The original method required diluting four parts of whole blood with one part of sodium citrate. The method has been modified to combining 2.0 mLs of whole blood (drawn in EDTA) with 0.5 mLs of 0.85% NaCl. Air bubbles invalidate the test. The diluted blood is placed in a special Westergren pipet and allowed to “settle out” during a one hour interval. Normal ESR Westergren values are: Males: <50 years = 0 to 15 mm/hr >50 years = 0 to 20 mm/hr Females: <50 years = 0 to 20 mm/hr >50 years = 0 to 30 mm/hr Children: 0 to 10 mm/hr N O T E NCCLS specifications for the Westergren method are strict. The tube must be a thick wall plastic or glass tube at least 300 mm in length. The tube must support a column of blood 200 mm in length with a bore of 2.65 mm (± 0.15 mm). Markings must be from 0 to 180. The supporting rack must hold the tube exactly vertical with no more than a 2o variation. The rack must also be able to support the tube vibration free. [2] The Wintrobe method uses a heavy walled tube that is 115 mm in length and a bore of 3.0 mm. This tube was originally used to perform a macrohematocrit which has become an obsolete test. The tube was also designed to perform an ESR. The tube is marked with two series of graduations from 0 to 100 graduations that run in opposite directions. The rack used to hold the Wintrobe tube must be vertical and vibration free. This test procedure uses EDTA anticoagulant whole blood without dilution and no air bubbles.

Normal reference ranges are: men: 0 to 9 mm/hr women: 0 to 20 mm/hr children: 0 to 13 mm/hr

Cautions to be applied in performing the ESR test are: [1] If clots, any size, are in the tube, it invalidate the test. [2] The concentration of anticoagulant must be correct or the results will be skewed. Too much anticoagulant will increase the ESR. To low a concentration will results in clots. [3] Invert the tube of blood after collection several times to assure a good mixing of anticoagulant. [4] Patients diagnosed with severe anemia should not be tested by this technique. Results are invalid. [5] If sodium or potassium oxalate and heparin are used as anticoagulants, the RBC’s will undergo some degree of shrinkage, causing a falsely elevated ESR. [6] Tilting of the ESR tube will increase the ESR. [7] The blood (at the beginning of the test) must be set at the zero mark for accuracy. [8] Bubbles in the tub invalidate the test. [9] If the blood sets at room temperature for more than two hours, the cells will become more spherical, skewing the test. Such cells cannot under rouleaux. [10] Do not test patients with such diagnosis as sickle cell anemia, thalassemia, or spherocytosis. These conditions causes decreased ESR results.

The ESR is increased in the following conditions: acute infections pregnancy acute appendicitis (after 24 hours) pyogenic arthritis chronic infections rheumatic fever hepatitis rheumatoidal arthritis inflammation ruptured ectopic pregnancy menses subacute bacterial endocarditis myocardial infarction tuberculosis multiple myeloma nephrosis Waldenstrom’s macroglobulinemia

80 DISCUSS THE CONCEPT OF THE ZETA SEDIMENTATION RATIO (ZSR).

Also referred to as the ZSR test, this is a procedure that requires the uses of special 75 mm long capillary tubes (with an inner diameter of 2.0 mm) and a “Zetafuge”. This is a technique of using series of controlled centrifuge speeds to compact and disperse the erythrocytes. The first spin is at low speed for 45 seconds which forces the cells against the vertical wall of the capillary tube to increase the rate of rouleaux formation. The “zetafuge” will stop and the tubes are rotated 180O and spun a second time for 45 seconds to move the rouleaux formed cells back across the inner diameter of the tube. This step is repeated two more times. The result is that the inter-face of the red blood cell column and plasma forms a unique curve dependent upon the negative charge on the RBC membrane that normally keeps the RBC’s apart. The degree of the net charge on the membrane affects the degree of packing and spacing between the RBC’s that results in the formation of a unique curve that contains a meniscus design

called the “knee of the curve”. It is at this curve that the measurement is taken. An ordinary hematocrit reader may be used. See the above illustration. A standard hematocrit is also performed and the results used in the calculation of the ZSR. The formula is ZSR% = Hct% ÷ Zetacrit X 100. This procedure is dependent upon the behavior of RBC’s as they approach each other under a specific, standardized gravitational force. The reference range for males and females are as follows: Normal = 40% to 51% Borderline = 51% to 54% Slightly elevated = 55% to 59% Moderately elevated = 60% to 64% Markedly elevated = > 64%

The procedure takes about four minutes to run and is not affected by anemias. It is affected by fibrinogen and gamma globulin concentrations which will reduce the normal negative charges on the surface of the RBC. It is an alternate technique for obtaining the erythrocyte sedimentation rate.

81 EXPLAIN THE CONCEPT (PRINCIPLE) OF HEMOGLOBIN ELECTROPHORESIS.

Electrophoresis is a testing principle that take advantage of charged particles migrating in an electric field. The hemoglobin molecule carries an electrical charge because it contains both carboxyl (COO−) and protonated nitrogen (NH3+) groups. The number of these two groups determines the strength of the charge on the molecule. If the hemoglobin molecule contains more carboxyl groups, the molecule will carry a stronger negative charge that cancels out the weaker positive charge from the protonated groups. In an electric field this molecule will migrate toward the positive electrode (the anode). If the protonated groups predominate, then the molecule will migrate toward the cathode (negative charged electrode). There are several factors that affect the rate at which the molecule will migrate toward an electrode. These are the net charge on the molecule, the size (mass) of the molecule, the shape of the molecule, the strength of the electrical field, the chemical and physical properties of the supporting medium, and the temperature of the electrophoresis system.

The hemoglobin molecule is made up of repeating units of amino acids and the number and type of amino acids determine its charge. There are more than 450 known variants of hemoglobin that result form amino acid substitutions made in the hemoglobin molecule. For example, sickle cell hemoglobin differs from normal (A1) hemoglobin by a single amino acid. Sickle cell hemoglobin contains valine instead of glutamic acid at the sixth position on the β-globulin chain. Substitutions can alter the solubility, stability, and function of the hemoglobin molecule. As a result of this one amino acid molecule difference, sickle cell hemoglobin will migrate slower than normal adult hemoglobin.

If a hemoglobin migrates in an electric field faster than normal adult hemoglobin, it is designated as a fast hemoglobin. If it migrates slower, then it is a slow hemoglobin. The rate at which a hemoglobin molecule migrates in an electric field is known as its Rf value.

Routine hemoglobin electrophoresis tests are routinely performed first on cellulose acetate strips at a pH of 8.6. When two or more hemoglobin species (such as Hgb D, Hgb S, and Hgb G) are found to migrate at the same speed, then the electrophoresis may be repeated on a different or secondary system such as citrate agar at a pH of

6.0. Note that hemoglobins A2, C, E, and O Arab will migrate at the same Rf value on cellulose acetate strips at a pH of 8.6 and have to be differentiated using citrate agar at a pH of 6.0.

To set up a hemoglobin electrophoresis procedure, a blood sample (venous or capillary blood) is obtained from the patient and a hemolysate prepared using red blood cells. Using EDTA or heparin as anticoagulants does not interfere with the electrophoresis procedure. The hemolysate can be stored frozen for future use. The major source of error in performing an electrophoresis procedure is poor technique. Other sources of error include deteriorated reagents, incorrect pH, equipment malfunction, contamination of agar or acetate strips, improperly stored or prepared hemolysates, and bacterial contamination.

The interpretation of results is based upon known migration patterns of the different types of hemoglobins and comparing these patterns with known controls. Refer to the following illustration of hemoglobin electrophoresis patterns. Remember that normal blood contains A1, A2, and F hemoglobins.

Table 1. Hemoglobin Types and Percentages

82 DESCRIBE IN GENERAL TERMS, ANEMIA AND WHAT INFORMATION IN THIS UNIT OF STUDY IS HELPFUL IN THE DIAGNOSIS OF ANEMIA. Anemia is a disorder in which there is a decrease in the ability of blood to carry and deliver oxygen to the tissues/cells. In the clinical laboratory, when anemia is present, there will be a decrease in the number of erythrocytes/μL (RBC count) or in grams of hemoglobin or a decrease in the red blood cells mass (hematocrit). Usually anemia is an expression of a primary disorder or disease. The physician's role (with clinical laboratory testing) then become that of determining the primary cause and treating the anemia if necessary. As a "rule of thumb", the body will lose about 0.85% of its red blood cells mass daily. This represents about 20 mls of blood that is lost. The body in turn must replenish that loss. If this loss cannot be replenished, then anemia will develop. Anemia can develop with the loss of RBC's exceeds the ability of the body to maintain an appropriate level of production or there is some event occurring in the body that prevents RBC production. The physician will evaluate the patient's clinical symptoms and medical history to help him/her to diagnose the presence and the type of anemia. The physician's review of the patient's medical history will include any or all of the following: A. family history, if any members experienced anemic conditions B. the patient's diet and consumption of snack foods C. any medications that are currently prescribed D. if their home/work environment exposes them to chemicals

The physician will consider the symptoms that are being described: A. fatigue and weakness B. appetite loss C. discomfort in breathing D. changes in the rate of the heart beat or unusual ryhthms E. headaches or other body discomfort F. fainting and/or dizziness

The evaluation will include physical examination to include: A. enlargement of the spleen and/or liver B. pallor in the skin, cheeks, and/or fingernails C. blood pressure D. changes in the nails of the fingers and toes E. neural changes and irritability

The physician will turn to the laboratory to collect blood and perform all or any combination of the following tests: A. CBC (hemoglobin, RBC count, WBC count, hemoglobin, hematocrit, indices. | platelet count, RDW, PMV, and a stained peripherial blood smear evaluation. B. Reticulocyte count and a retic stained blood smear evaluation. C. Serum iron, TIBC, transferrin test, percent saturation, ferritin test, and free erythrocyte protoprophyrin (FEP) test. D. Hemosiderin in the urine. The information obtained in the medical history and physical evaluation along with that provided by the laboratory will help the physician classify the type of anemia. Anemia's may be classified as either functional (pathophysiological), morphological, maturation defect, proliferation defect, or a possible combination of two or more of these classifications.

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