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Lesson 4-2. Erythrocytes

INTRODUCTION

In the normal development and maturation (erythropoiesis) of the erythrocytic series, the red blood cell undergoes a graduation of morphological changes. This cell development is a gradual transition (as noted in ASCP terminology—they are listed from the most immature to mature cells) and six different stages can be identified. The nomenclature used to describe red blood cells is recommended by the American Society of Clinical Pathologists and the American Medical Association. The terms with some of their synonyms are as given in table 4-1.

ASCP Terminology Synonyms
Rubriblast
Prorubricyte
Rubricyte
Metarubricyte
Diffusely basophilic erythrocyte
Erythrocyte
 Pronormoblast
 Basophilic normoblast
Polychromatophilic normoblast
Orthochromatic normoblast
Polychromatic erythrocyte (Retic)
Normocyte (Mature Red Blood Cell)

Table 4-1. Terminology.

Erythropoiesis is regulated by the intake of substances to build the cells, the storage of these substances, and their proper utilization. When normal erythropoiesis occurs, both the cytoplasm and the nuclei of the cells grow at a synchronized rate. Individual differences in physiology and physical structure of the erythrocyte account for minor morphological changes so often encountered. In certain diseases, these morphological changes may vary to a greater extent. These variations occur in size, shape, staining, and inclusions in the erythrocyte.

ERYTHROCYTIC SERIES

See figures 4-1 through 4-6.

Rubriblast ( Pronormoblast). See figure 4-1.

(1) Size. 12 to 19 microns in diameter. The nuclear to Cytoplasm Ratio (N:C ratio) is 4:1.

Figure 4-1. Erythrocytes series: Rubriblast.

(2) Nucleus. This cell has a large round-to-oval purple nucleus that occupies most of the cell. The nuclear chromatin is arranged in a close mesh network forming a reticular appearance. There are 0-2 light blue nucleoli present within the nucleus.

(3) Cytoplasm. The cytoplasm is dark blue (basophilic), granule-free, and limited to a thin rim (perinuclear halo) around the nucleus. There is no evidence of hemoglobin formation.

Prorubricyte (Basophilic Normoblast). See figure 4-2.

Figure 4-2. Erythrocytes series: Prorubricyte.

(1) Size. 12 to 17microns in diameter. N:C ratio 4:1.

(2) Nucleus. The nucleus is generally round, dark purple, am smaller than the nucleus of the rubriblast. The chromatin is coarse am clumped giving the nucleus a darker stain. Nucleoli are usually not present, but when they are, they appear more prominent than in the rubriblast.

(3) Cytoplasm. The cytoplasm is royal blue and more radiant than in the rubriblast. Cytoplasmic granules are not present.

Rubricyte (Polychromatic Normoblast). See figure 4-3.

Figure 4-3. Erythrocytes series: Rubricyte.

(1) Size. 11 to 15 microns in diameter. N:C ratio 1:1.

(2) Nucleus. The nucleus is dark, round or oval, and smaller than the prorubricyte nucleus. The chromatin material is found in dense, irregular clumps. Nucleoli are not present.

(3) Cytoplasm. The cytoplasm is more abundant than in the precursor cells. It is blue-pink (polychromatic), the pink resulting from the first visible appearance of hemoglobin. Cytoplasmic granules are absent.

Metarubricyte (Orthochromatic Normoblast). See figure 4-4.

Figure 4-4. Erythrocytes series: Metrarubricyte.

(1) Size. 8 to 12 microns in diameter.

(2) Nucleus. This cell has a pyknotic nucleus (a homogeneous blue-black mass with no structure) that is round. The nucleus will be extruded from the cell in the later period of this stage. This is the main difference between the rubricyte and the metarubricyte.

(3) Cytoplasm. The cytoplasm is abundant, reddish to buff pink.

Reticulocyte (Polychromatic Erythrocyte). See figure 4-5.

Figure 4-5. Erythrocytes series: Reticulocyte.

(1) Size. 7 to 10 microns in diameter.

(2) Nucleus. The nucleus is absent.

(3) Cytoplasm. The cytoplasm stains a bluish-buff with Wright’s stain and there is no central light pallor as in the erythrocyte. With supravital staining, this cell will show light blue reticulum strands in the cytoplasm.

Erythrocyte. See figure 4-6.

Figure 4-6. Erythrocytes series: Erythrocyte.

(1) Size. 6 to 8 microns in diameter.

(2) Nucleus. The nucleus is absent.

(3) Cytoplasm. The cytoplasm of the periphery is pinkish red with a central zone of pallor. The central pallor normally does not exceed one-third of the diameter of the cell and the color reflects the amount of hemoglobin present. Erythrocytes are able to change shape in order to transport oxygen. Erythrocytes can become flexible or pliable, and even deformable when traveling through microcirculation.

VARIATIONS IN ERYTHROCYTES

Size.

(1) Anisocytosis. Anisocytosis (see figure 4-7) is a variation in the size of erythrocytes beyond the normal limits. Cells of varying size are seen in the same fields.

(2) Macrocytes. Macrocytes are erythrocytes larger than 9 microns in diameter. These cells may be found in liver disease.

(3) Microcytes. These erythrocytes are smaller than 6 microns in diameter. These cells are found in thalassemia and other anemias.

Shape.

(1) Poikilocytosis. This term describes a marked variation in the shape of erythrocytes. Poikilocytes can be pear-shaped, comma-shaped, oval- shaped, or various other bizarre forms (see figure 4-7). These cells are encountered in pernicious anemia and many other types of anemia.

Figure 4-7. Variations in erythrocytes: Marked Poikilocytosis.
Anisocytosis and target cells.

(2) Sickle cell (Drepanocytes). Sickle cells (figure 4-8) are abnormal erythrocytes that assume a crescent or sickle-shaped appearance under conditions of reduced oxygen tension. The presence of sickle cells is an inherited abnormality due to the presence of hemoglobin S. Sickle cell anemia is encountered primarily in Blacks.

Figure 4-8. Variations in erythrocytes: Poikilocytosis: Sickle cells.

(3) Spherocytes. These are abnormal erythrocytes that are spherical in shape, having a diameter smaller than normal, and a darker stain (without central pallor) than normal erythrocytes. These cells are found in instances of hemolytic anemias and are particularly characteristic of congenital hemolytic jaundice, a hereditary disorder.

(4) Ovalocvtes (elliptocyte). These cells are abnormal erythrocytes that have an oval or “sausage” shape (see figure 4-9). They can be found in hereditary elliptocytosis.

Figure 4-9. Variations in erythrocytes: Elliptocytes (oval erythrocytes).

(5) Target cells (codocyte). Target cells (figure 4-10) are erythrocytes that have deeply stained (pink) centers and borders, separated by a pale ring, giving them a target-like appearance. They are associated with liver disease and certain hemoglobinopathies.

Figure 4-10. Variations in erythrocytes:
a. Metarubricyte, b. Target Cell, c. Crenated RBC

(6) Burr cells (echinocyte). Burr cells (figure 4-11) are triangular or crescent-shaped erythrocytes with one or more spiny projections on the periphery. These cells are seen in uremia, acute blood loss, cancer of the stomach and pyruvate kinase deficiency.

Figure 4-11. Variations in erythrocytes: Crenated RBC burr cells. Acanthocytes 2 leukocytes.

(7) Acanthocytes (spur cells). Acanthocytes are irregularity-shaped erythrocytes with long spiny projections. They are seen in a congenital abnormality characterized by serum concentration of low density (beta) lipoproteins.

(8) Crenated erythrocytes. This condition occurs when blood films dry too slowly and the surrounding plasma becomes hypertonic. There is no pathological significance when they are found in blood smears.

(9) Schistocytes. These are red blood cell fragments. Frequently these cells have a hemispherical shape (helmet cells).

(10) Rouleaux formation. This phenomenon is adherence of erythrocytes to one another presenting a stack-of-coins appearance. It occurs in conditions characterized by increased amounts of fibrinogen and globulin.

Staining.

(1) Hypochramia. Hypochramia (figure 4-12) is a condition in which the normal central pallor is increased due to decreased hemoglobin content. This condition is characteristic of many anemias.

Figure 4-12. Variations in erythrocytes: Hypochromic macrocytic erythrocytes.

(2) Polychromatophilia. This term describes non-nucleated erythrocytes that show bluish coloration instead of light pink. Polychromatophilia is due to the fact that the cytoplasm of these cells does not mature, resulting in the abnormal persistence of the basophilic cytoplasm of the earlier nucleated stages.

Inclusions.

(1) HoweII-Jolly bodies. These are nuclear remnants found in the erythrocytes of the blood in various anemias. They are round, dark violet granules about one micron in diameter (see figure 4-13). Generally, only one Howell-Jolly body will be found in any one red cell. However, two or more may sometimes be present. HoweII-Jolly bodies generally indicate absent or non-functioning spleen. They occur in megalobastic anemia and in other forms of nuclear maturation defects.

Figure 4-13. Variations in erythrocytes:
a. Metarubricyte. b. Howell-Jolly bodies.

(2) Cabot's rings (ring bodies). These are bluish threadlike rings found in the red cells in the blood of patients with severe anemias (figure 4-14). They are interpreted as remnants of the nuclear membrane and appear as ring or “figure-eight' structures. Usually only one such structure will be found in any one red cell.

Figure 4-14. Variations in erythrocytes: (a) Cabot's ring.

(3) Basophilic stippling. Round, small, blue-purple granules of varying size in the cytoplasm of the red cell represent a condensation of the immature basophilic substance (see poly-chromatophilia) that normally disappears with maturity. This is known as basophilic stippling (figure 4-15). It can be demonstrated by standard staining techniques in contrast to reticulocyte filaments that require a special stain. Stippling occurs in anemias and heavy metal poisoning (lead, zinc, silver, mercury, bismuth) and denotes immaturity of the cell.

Figure 4-15. Variations in erythrocytes: (a) Basophilic stippled erythrocyte.

(4) Heinz-Ehrlich bodies. These are small inclusions found primarily in those hemolytic anemias induced by toxins. They are round, refractile bodies inside the erythrocyte and are visible only in unfixed smears. It is thought that they are proteins that have been dematured and that they are an indication of erythrocyte injury.

(5) Siderocytes. These are erythrocytes containing iron deposits. These deposits indicate an incomplete reduction of the iron from ferric to the ferrous state that is normally found in hemoglobin. Prussian blue stain must be used to readily demonstrate these cells.

e. Megaloblastic Erythrocytes. The development of megaloblastic cells is caused by a deficiency of vitamin B12 or folic acid. Pernicious anemia is a disease considered to be due to a deficiency in vitamin B12 and/or certain related growth factors. With this deficiency, the erythrocytes do not mature normally and are generally larger than normal. The most notable characteristic of this abnormal maturation is a difference in the rates of maturation of the cytoplasm and the nucleus. The development of the nucleus is slower than that of the cytoplasm, so that in the more mature of the nucleated forms a spongy nucleus as well as an exceptionally large size may be observed. Nuclear chromatin in the megaloblast is much finer and is without the clumps observed in the rubriblast. Such development is termed asynchronism. The mature cell is large (about 10 microns) and is termed a megalocyte. The younger cells of this series are named by adding the suffix “pernicious anemia type," that is rubricyte, pernicious anemia type, and so forth. See figures 4-16 and 4-17.

Figure 4-16. Variations in erythrocytes:
(a) Rubricytes (pernicious anemia).

Figure 4-17. Variations in erythrocytes:
(a) Metarubricyte (pernicious anemia).

 

Lesson 5-2. Manual Counts, Other Body Fluids

CEREBROSPINAL FLUID CELL COUNTS

Cerebrospinal fluid is delivered to a counting chamber and examined microscopically for blood cells. Normally, spinal fluid is clear. If it is cloudy, dilute before charging the counting chamber.

Reagent. Normal saline.

Procedure.

NOTE: Set up cell counts on spinal fluids within 30 minutes after withdrawal of the specimen.
  1. Clear spinal fluid is set up as follows:

(a) With a transfer pipet introduce a drop of well-mixed spinal fluid into both counting chamber of a Hemacytometer.

CAUTION: Avoid contamination by careful handling of spinal fluid.

(b) Examine the entire ruled area for the presence of cellular elements. If both leukocytes and erythrocytes are observed, note the condition of the red cells (fresh or crenated).

(c) Count all cells in the entire ruled area (0.9 cu mm).

  1. Turbid sample – Perform dilution using normal saline.
  • Slightly hazy – 1:10 dilution.
  • Hazy – 1:20 dilution.
  • Mix the specimen well.
  • Discard the fluid in the capillary portion of the pipet.
  • Charge the counting chamber and allow the cells to settle for five minutes.
  • Under low-power magnification count all cells in the entire ruled area (0.9 cu mm).
  • Switch to high-power and perform a rough differential count on prepared smear.

Calculations.

Clear spinal fluid:

Number of cells counted x dilution factor x area factor x depth factor = total cells / µl

Turbid spinal fluid:

Number of cells counted X dilution (10) x area factor x depth factor = total cells / µl

Very clouded spinal fluid:

Number of cells counted X dilution (20) x area factor x depth factor = total cells / µl

Sources of Error.

  • Improper collection of blood specimens causes variable results.
  • Wet or dirty pipets.
  • Not allowing cells to settle for an adequate amount of time.
  • Poor pipetting technique causes high or low counts. Poor pipetting technique includes:
  • Undershooting Unopette with blood.
  • Overfilling Unopette with blood.
  • Air bubbles in the shaft.
  • Not mixing the blood specimen thoroughly.
  • Failure to expel 3 or 4 drops in the pipet tips before charging the Hemacytometer.
  • Overfilling the chamber of the hemacytometer, which causes erroneously high counts.
  • Not mixing the diluted specimen prior to filling the Hemacytometer.
  • Uneven distribution of cells in the counting chamber causes erroneous results.
  • Counting artifacts.
  • Dirty or scratched Hemacytometer.
  • Failure to mix anticoagulated blood thoroughly before use.

Discussion.

If more than 100 leukocytes per cu mm are present, centrifuge the undiluted specimen, make a smear, and stain with modified Wright's stain. Perform a routine differential count and also estimate the ratio of erythrocytes to leukocytes.

NOTE: It may be necessary to use egg albumin or cell-free serum to make the sediment adhere to the slide.

Normally the spinal fluid is water clear. It can be turbid if cell count is 500 or more cells per cu mm. If there is fresh blood with spontaneous clotting, the indications are those of a bloody tap. Xanthochromia develops after subarachnoid hemorrhage has been present for a few hours and is due to disintegration of blood pigments. Xanthochromia may also develop from tumors, abscesses, and inflammation.

Cell counts above ten are considered to be evidence of intracranial disease. The predominant cell in most viral infections, syphilis, and tuberculous meningitis is the lymphocyte. Bacterial infections due to meningococcus, pneumococcus, and so forth, usually result in a predominance of the neutrophil. Cerebral and extradural abscesses as well as subdural hemorrhages produce a neutrophilic response although bacteria are not demonstrated.

Biochemical, bacteriological, virological, serological, and hematological ands are all necessary to reflect the true condition of the cerebrospinal fluid. The current laboratory standing operating procedures should give guidance for the most efficient method to accomplish all the necessary ands.

Normal Value: Zero to five cells per cu mm (chiefly lymphocytes).

SEMEN ANALYSIS

Semen analysis involves gross examination (volume, color, turbidity, viscosity, and pH) and microscopic examination (motility and spermatozoa count).

Reagent. Tap water.

Collection Instructions. A physician will usually give the instructions; however, the patient should be reminded of several critical points.

  • The patient may be required to abstain from intercourse for 48 to 72 hours.
  • The specimen is collected in a clean container that has been pre- warmed to body temperature.
  • The specimen should be delivered to the laboratory within 1 hour.
  • The specimen must be kept at body temperature (37oC) and not subjected to extremes of heat or cold.

Gross Examination.

  • Record. the time of collection and receipt of the specimen.
  • Measure and record the volume.
  • Observe and record the color (white, gray, yellow, and so forth), turbidity (clear, opalescent, opaque, and so forth), and viscosity (viscid, gelatin, liquid).
  • Determine the pH with a pH reagent strip and record this.
Motility Examination.
  1. When the specimen becomes fluid (within 15 to 30 minutes after collection, the semen liquifies by the action of fibrinolysin), place one drop on a slide (pre-warmed to 37şC) and place a cover slip on it.
  2. Under high dry power, count motile and nonmotile spermatozoa in two or more areas until a total of at least 200 spermatozoa have been observed. It is necessary to focus through the entire depth of a given field so as to include nonmotile spermatozoa that may have settled to the bottom of the slide. Only those that move forward actively are considered motile. Record the percent of motile spermatozoa seen.
  3. Repeat this procedure in three hours and six hours, using a new drop from the original specimen each time.

Spermatozoa Count.

  1. Make a 1:20 dilution of seminal fluid with diluent (tap water).
  2. Mix sample thoroughly and charge a Hemacytometer.
  3. Count the spermatozoa in the same manner as you would count white blood cells.
  4. After counting the sperm, examine the morphology and report the percent of abnormal forms. Morphologically normal sperm are quite uniform in appearance. Any sperm with rounded, enlarged, small, or bilobed heads are abnormal. Abnormal tails are enlarged, small, irregular in length, absent, or multiple. See figure 5-3 for morphology of spermatozoa.

Figure 5-3. Morphology of spermatozoa.

Calculations.

Sources of Error.

  • Delay in analysis results in a lower percentage of motile forms and a lower count.
  • Temperature extremes cause spermatozoa to die.

 Discussion.

Semen analyses are usually performed as part of infertility studies or following a vasectomy.

Semen analysis can be performed for medico-legal cases involving rape or to support or disprove a denial of paternity on the grounds of sterility.

Semen is derived from the following: testes, seminal vesicles, prostate, epididymides, vasa deferentia, bulourethral glands, and urethral glands.

Normal Values.

  • Volume: 2.0-5.0 ml.
  • pH: 7.3-7.8.
  • Motility: > 50-60 percent.
  • Spermatozoa Count: 20-160 x 106 sperm/ ml.
 

Exercises for Lesson 5

 

David L. Heiserman, Editor

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Revised: June 06, 2015