PREPARATION OF BLOOD SMEAR (WEDGE METHOD)

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PREPARATION OF BLOOD SMEAR (WEDGE METHOD)

(1) A small drop of blood (2-3 mm in diameter) is placed in the center line about 1 cm away from one end of a glass slide (typical size of slide is 75 × 25 mm; thickness about 1mm) with a wooden stick or glass capillary. Slide should be clean, dry, and grease-free. Blood sample may be venous (anticoagulated with EDTA) or capillary (finger prick). Better blood cell morphology is obtained if smear is made directly from a skin puncture. If EDTA-anticoagulated venous blood is used, smear should be prepared and stained within 2 hours of blood collection. If venous blood collected in a syringe is used, the last drop of blood in the needle after withdrawing (or first drop while dispensing) should be used.
(2) A 'spreader' slide is placed at an angle of 30° in front of the drop and then drawn back to touch the drop of blood. Blood spreads across the line of contact of two slides.
(3) Smear is made by smooth, forward movement of the 'spreader' along the slide. The whole drop should be used up 1 cm before the end of the slide. The length of the smear should be about 3 cm. The 'spreader' should not be raised above the slide surface till whole drop of blood is spread out.
(4) Smear is rapidly dried by waving it in the air or keeping it under an electric fan. Slow drying causes shrinkage artifact of red cells.
(5) Patient's name or laboratory number and date are written (with a lead pencil, a permanent marker pen, or a diamond pencil) on the thicker end of the smear.
(6) The smear is fixed immediately with absolute methyl alcohol (which should be moisture- and acetone-free) for 2-3 minutes in a covered jar (Absolute ethyl alcohol can also be used, but not methylated spirit as it contains water). Aim of fixation is to prevent washing off of the smear from the slide. Following this, color of the smear becomes light brown. This fixation is desirable even when Leishman stain is used which contains methyl alcohol. This is because Leishman stain may have absorbed moisture leading to poor fixation. If methanol is contaminated with water, sharpness of cell morphology is lost and there is vacuolation of red cells. Methanol should be acetone-free since acetone washes out nuclear stain. (In many laboratories, slide is stained immediately after air-drying without prior fixation, and the results are satisfactory; however, if delay of >4 hours is anticipated between air-drying and staining, the slide should be fixed. If not, a background gray-blue staining of plasma occurs).
 
Notes
(1) Making a 'spreader' slide—a glass slide with absolutely smooth edges should be selected and one or both corners at one end of the slide should be broken off. The 'spreader' slide should be narrower (width of about 15 mm) so that edges of the smear can be examined microscopically. The 'spreader' slide should be discarded after use. If the same is to be reused, its edge should be thoroughly cleaned and dried (otherwise carryover of cells or parasites can occur).
(2) A well-spread blood smear (a) is tongue-shaped with a smooth tail, (b) does not cover the entire area of the slide, (c) has both thick and thin areas with gradual transition, and (d) does not contain any lines or holes.
(3) By changing the angle of the 'spreader' and its speed, thickness of the blood smear can be controlled. In patients with anemia, a thicker smear can be obtained by increasing the angle and the speed of spreading. In patients with polycythemia, a thinner smear is obtained by decreasing the 'spreader' angle and the speed of spreading.
(4) Anticoagulant used may be EDTA (dipotassium salt) or sodium citrate. Heparin should not be used as an anticoagulant for making blood films since it causes platelet clumping and imparts a blue background to the film.
(5) It is recommended to stain blood films in reagent filled Coplin jars (rather than covering them with the staining solution) to avoid formation of stain precipitates due to evaporation.
(6) A drawback of this method is uneven distribution of leukocytes (i.e. monocytes, neutrophils, and abnormal cells are pushed towards the extreme tail end of the smear) and distortion of red cell morphology at the edges.
(7) Blood smear is covered with a coverslip and mounted in a mounting medium (e.g. DPX) for protection against mechanical damage and deterioration of staining with time on exposure to air.
(8) Cleaning of slides: (A) New slides: If new slides are not clean and grease-free, they are left overnight in a detergent solution, washed in running tap water, rinsed in distilled water, and wiped dry with a clean cloth. Before use, they are wiped with 95% methyl alcohol, dried, and then kept covered to protect from dust. (B) Used slides: The used slides are soaked in a detergent solution at 60°C for 20 minutes, washed in running tap water, rinsed in distilled water, and then wiped dry. Before use, they are wiped with 95% methyl alcohol, dried, and then kept covered to protect from dust.

STAINING OF BLOOD SMEAR

Blood smears are routinely stained by one of the Romanowsky stains. Romanowsky stains consist of a combination of acidic and basic dyes and after staining various intermediate shades are obtained between the two polar (red and blue) stains. Romanowsky stains include May-Grunwald-Giemsa, Jenner, Wright's, Leishman's, and Field's stains. Staining properties of the Romanowsky stains are dependent on two synthetic dyes: methylene blue and eosin. International Committee for Standardization in Haematology has recommended a highly purified standardized stain, which contains azure B and eosin Y; it, however, is very expensive. Romanowsky stains are insoluble in water but soluble in methyl alcohol. Methyl alcohol acts as a solvent as well as a fixative. Staining reaction is pH-dependent. These stains have a tendency towards precipitation and should be filtered before use.
Methylene blue and azure B are basic (cationic) dyes and have affinity for acidic components of the cells (like nucleic acids or basophil granules) and impart purpleviolet color to the nuclear chromatin, dark blue-violet color to the basophil granules, and deep blue color to the cytoplasm of lymphocytes. Eosin is an acidic (anionic) dye and has affinity for basic components like hemoglobin (stained pink-red), and granules in eosinophils (stained orange-red). Neutrophil granules are slightly basic and stain violet-pink or lilac.
Romanowsky stains impart more colours than just blue (from methylene blue or azure B) and red-orange (from eosin Y). Usefulness of the Romanowsky stains lies in their ability to differentially stain leucocyte granules.
A well-stained smear is pink in color in thinner portion and purple-blue in thicker portion. Excess blue coloration can be due to: (i) excessively thick smear, (ii) low concentration of eosin, (iii) impure dyes, (iv) too long staining time, (v) inadequate washing, or (vi) excessive alkaline pH of stain, buffer, or water. Excess red coloration can be due to: (i) impure dyes or incorrect proportion of dyes, (ii) excessive acid pH of stain, buffer, or water (as the red cells take up more acid dye i.e. eosin), (iii) too short staining time, or (iv) excessive washing. If there are granules of stain precipitate (masses of small black dots) on smear, stain needs to be filtered.
Method of Leishman staining is given below:

Reagents
(1) Leishman stain: William Boog Leishman, a British pathologist, modified the original Romanowsky method and devised a stain which is widely known as Leishman's stain. This consists of methylene blue and eosin dissolved in absolute methyl alcohol. Commercially available Leishman stain powder (0.6 gram) is mixed with water-free absolute methyl alcohol (400 ml). Prepared stain should be kept tightly stoppered in a brown bottle and stored in a cool, dark place at room temperature. Exposure to direct sunlight causes deterioration of the stain. After preparation, stain should be kept for 3-5 days before using since it improves the quality of the stain.
(2) Buffered water (pH 6.8).

Method
(1) Air-dry the smear and fix with methanol for 2-3 minutes.
(2) Cover the smear with Leishman stain for 2 minutes.
(3) After 2 minutes, add twice the volume of buffered water and leave for 5-7 minutes. A scum of metallic sheen forms on the surface.
(4) Wash the stain away in a stream of buffered water. Tap water can also be used for washing if it is not highly alkaline or highly acid.
(5) Wipe the back of the slide clean and set it upright in the draining rack to dry.
(6) Mount the slide in a suitable mounting medium (e.g. DPX) with a clean and dry 25 × 25 mm coverslip.
• Red cells: pink-red or deep pink
• Polychromatic cells (Reticulocyt-es): Gray-blue
• Neutrophils: Pale pink cytopla-sm; mauve-purple granules
• Eosinophils: Pale-pink cytoplasm; orange-red granules
• Basophils: Blue cytoplasm; dark blue-violet granules
• Monocytes: Gray-blue cytoplasm; fine reddish (azurophil) granules
• Small lymphocytes: Dark blue cy-toplasm
• Platelets: Purple
• Nuclei of all cells: Purple-violet
Last modified on Tuesday, 25 July 2017 11:34
Dayyal Dg.

Clinical laboratory professional specialized to external quality assessment (proficiency testing) schemes for Laboratory medicine and clinical pathology. | Author/Writer/Blogger

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  • TOTAL THYROXINE (T4)
    Total serum thyroxine includes both free and protein-bound thyroxine and is usually measured by competitive immunoassay. Normal level in adults is 5.0-12.0 μg/dl.
     
    Test for total thyroxine or free thyroxine is usually combined with TSH measurement and together they give the best assessment of thyroid function.
     
    Causes of Increased Total T4
     
    1. Hyperthyroidism: Elevation of both T4 and T3 values along with decrease of TSH are indicative of primary hyperthyroidism.
    2. Increased thyroxine-binding globulin: If concentration of TBG increases, free hormone level falls, release of TSH from pituitary is stimulated, and free hormone concentration is restored to normal. Reverse occurs if concentration of binding proteins falls. In either case, level of free hormones remains normal, while concentration of total hormone is altered. Therefore, estimation of only total T4 concentration can cause misinterpretation of results in situations that alter concentration of TBG.
    3. Factitious hyperthyroidism
    4. Pituitary TSH-secreting tumor.
     
    Causes of Decreased Total T4
     
    1. Primary hypothyroidism: The combination of decreased T4 and elevated TSH are indicative of primary hypothyroidism.
    2. Secondary or pituitary hypothyroidism
    3. Tertiary or hypothalamic hypothyroidism
    4. Hypoproteinaemia, e.g. nephrotic syndrome
    5. Drugs: oestrogen, danazol
    6. Severe non-thyroidal illness.
     
    Free Thyroxine (FT4)
     
    FT4 comprises of only a small fraction of total T4, is unbound to proteins, and is the metabolically active form of the hormone. It constitutes about 0.05% of total T4. Normal range is 0.7 to 1.9 ng/dl. Free hormone concentrations (FT4 and FT3) correlate better with metabolic state than total hormone levels (since they are not affected by changes in TBG concentrations).
     
    Measurement of FT4 is helpful in those situations in which total T4 level is likely to be altered due to alteration in TBG level (e.g. pregnancy, oral contraceptives, nephrotic syndrome).
     
    Total and Free Triiodothyronine (T3)
     
    Uses
     
    1. Diagnosis of T3 thyrotoxicosis: Hyperthyroidism with low TSH and elevated T3, and normal T4/FT4 is termed T3 thyrotoxicosis.
    2. Early diagnosis of hyperthyroidism: In early stage of hyperthyroidism, total T4 and free T4 levels are normal, but T3 is elevated.
     
    A low T3 level is not useful for diagnosis of hypothyroidism since it is observed in about 25% of normal individuals.
     
    For routine assessment of thyroid function, TSH and T4 are measured. T3 is not routinely estimated because normal plasma levels are very low.
     
    Normal T3 level is 80-180 ng/dl.
     
    Free T3: Measurement of free T3 gives true values in patients with altered serum protein levels (like pregnancy, intake of estrogens or oral contraceptives, and nephrotic syndrome). It represents 0.5% of total T3.
     
    Thyrotropin Releasing Hormone (TRH) Stimulation Test
     
    Uses
     
    1. Confirmation of diagnosis of secondary hypothyroidism
    2. Evaluation of suspected hypothalamic disease
    3. Suspected hyperthyroidism
     
    This test is not much used nowadays due to the availability of sensitive TSH assays.
     
    Procedure
     
    • A baseline blood sample is collected for estimation of basal serum TSH level.
    • TRH is injected intravenously (200 or 500 μg) followed by measurement of serum TSH at 20 and 60 minutes.
     
    Interpretation
     
    1. Normal response: A rise of TSH > 2 mU/L at 20 minutes, and a small decline at 60 minutes.
    2. Exaggerated response: A further significant rise in already elevated TSH level at 20 minutes followed by a slight decrease at 60 minutes; occurs in primary hypothyroidism.
    3. Flat response: There is no response; occurs in secondary (pituitary) hypothyroidism.
    4. Delayed response: TSH is higher at 60 minutes as compared to its level at 20 minutes; seen in tertiary (hypothalamic) hypothyroidism.
     
    Antithyroid Antibodies
     
    Box 864.1 Thyroid autoantibodies
     
    • Useful for diagnosis and monitoring of autoimmune thyroid diseases.
    • Antimicrosomal or antithyroid peroxidase antibodies: Hashimoto’s thyroiditis
    • Anti-TSH receptor antibodies: Graves’ disease
    Various autoantibodies (TSH receptor, antimicrosomal, and antithyroglobulin) are detected in thyroid disorders like Hashimoto’s thyroiditis and Graves’ disease. Antimicrosomal (also called as thyroid peroxidase) and anti-thyroglobulin antibodies are observed in almost all patients with Hashimoto’s disease. TSH receptor antibodies (TRAb) are mainly tested in Graves’ disease to predict the outcome after treatment (Box 864.1).
     
    Radioactive Iodine Uptake (RAIU) Test
     
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    Causes of Increased Uptake
     
    • Hyperthyroidism due to Graves’ disease, toxic multinodular goiter, toxic adenoma, TSH-secreting tumor.
     
    Causes of Decreased Uptake
     
    • Hyperthyroidism due to administration of thyroid hormone, factitious hyperthyroidism, subacute thyroiditis.
     
    Uses
     
    RAIU is most helpful in differential diagnosis of hyperthyroidism by separating causes into those due to increased uptake and due to decreased uptake.
     
    Thyroid Scintiscanning
     
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    Interpretation
     
    • Differential diagnosis of high RAIU thyrotoxicosis:
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      – Toxic multinodular goiter: Multiple discrete areas of increased uptake
      – Adenoma: Single area of increased uptake
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    Interpretation of thyroid function tests is shown in Table 164.1.
     
    Table 864.1 Interpretation of thyroid function tests
    Test results Interpretations
    1. TSH Normal, FT4 Normal Euthyroid
    2. Low TSH, Low FT4 Secondary hypothyroidism
    3. High TSH, Normal FT4 Subclinical hypothyroidism
    4. High TSH, Low FT4 Primary hypothyroidism
    5. Low TSH, Normal FT4, Normal FT3 Subclinical hyperthyroidism
    6. Low TSH, Normal FT4, High FT3 T3 toxicosis
    7. Low TSH, High FT4 Primary hyperthyroidism
     
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  • DISORDERS OF THYROID
    Box 863.1 Terminology in thyroid disorders
    • Primary hyper-/hypothyroidism: Increased or decreased function of thyroid gland due to disease of thyroid itself and not due to increased or decreased levels of TRH or TSH.
    • Secondary hyper-/hypothyroidism: Increased or decreased function of thyroid gland due to increased or decreased levels of TSH.
    • Tertiary hypothyroidism: Decreased function of thyroid gland due to decreased function of hypothalamus.
    • Subclinical thyroid disease: A condition with abnormality of thyroid hormone levels in blood but without specific clinical manifestations of thyroid disease and without any history of thyroid dysfunction or therapy.
    • Subclinical hyperthyroidism: A condition with normal thyroid hormone levels but with low or undetectable TSH level.
    • Subclinical hypothyroidism: A condition with normal thyroxine and triiodothyronine level along with mildly elevated TSH level.
    Among the endocrine disorders, disorders of thyroid are common and are only next in frequency to diabetes mellitus. They are more common in women than in men. Functional thyroid disorders can be divided into two types depending on activity of the thyroid gland: hypothyroidism (low thyroid hormones), and hyperthyroidism (excess thyroid hormones). Any enlargement of thyroid gland is called as a goiter. Terminology related to thyroid disorders is shown in Box 863.1.
     
    Hyperthyroidism
     
    Hyperthyroidism is a condition caused by excessive secretion of thyroid hormone. Causes of hyperthyroidism are listed in Table 863.1.
     
    Table 863.1 Causes of hyperthyroidism
    1. Graves’ disease (Diffuse toxic goiter)
    2. Toxicity in multinodular goiter
    3. Toxicity in adenoma
    4. Subacute thyroiditis
    5. TSH-secreting pituitary adenoma (secondary hyperthyroidism)
    6. Trophoblastic tumours that secrete TSH-like hormone: choriocarcinoma, hydatidiform mole
    7. Factitious hyperthyroidism
     
    Clinical Characteristics
     
    Clinical characteristics of hyperthyroidism are nervousness, anxiety, irritability, insomnia, fine tremors; weight loss despite normal or increased appetite; heat intolerance; increased sweating; dyspnea on exertion; amenorrhea and infertility; palpitations, tachycardia, cardiac arrhythmias, heart failure (especially in elderly); and muscle weakness, proximal myopathy, and osteoporosis (especially in elderly).
     
    The triad of Graves’ disease consists of hyperthyroidism, ophthalmopathy (exophthalmos, lid retraction, lid lag, corneal ulceration, impaired eye muscle function), and dermopathy (pretibial myxoedema).
     
    Box 863.2 Thyroid function tests in hyperthyroidism
    • Thyrotoxicosis:
      Serum TSH low or undetectable
      – Raised total T4 and free T4.
    • T3 toxicosis:
      – Serum TSH undetectable
      – Normal total T4 and free T4
      – Raised T3
    Laboratory Features
     
    In most patients, free serum T3 and T4 are elevated. In T3 thyrotoxicosis (5% cases of thyrotoxicosis), serum T4 levels are normal while T3 is elevated. Serum TSH is low or undetectable (< 0.1 mU/L) (Box 863.2).
     
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    Features of primary and secondary hyperthyroidism are compared in Table 863.2.
     
    Table 863.2 Differences between primary and secondary hyperthyroidism
    Parameter Primary hyperthyroidism Secondary hyperthyroidism
    1. Serum TSH Low Normal or high
    2. Serum free thyroxine High High
    3. TSH receptor antibodies May be positive Negative
    4. Causes Graves’ disease, toxic multinodular goiter, toxic adenoma Pituitary adenoma
     
    Evaluation of hyperthyroidism is presented in Figure 863.1.
     
    Figure 863.1 Evaluation of hyperthyroidism
    Figure 863.1 Evaluation of hyperthyroidism. TSH: thyroid stimulating hormone; FT4: free T4; FT3: free T3; TRAb: TSH receptor antibody; TRH: Thyrotropin releasing hormone
     
    Hypothyroidism
     
    Hypothyroidism is a condition caused by deficiency of thyroid hormones. Causes of hypothyroidism are listed in Table 863.3. Primary hypothyroidism results from deficient thyroid hormone biosynthesis that is not due to disorders of hypothalamus or pituitary. Secondary hypothyroidism results from deficient secretion of TSH from pituitary. Deficient or loss of secretion of thyro-tropin releasing hormone from hypothalamus results in tertiary hypothyroidism. Secondary and tertiary hypothyroidism are much less common than primary. Plasma TSH is high in primary and low in secondary and tertiary hypothyroidism. Differences between primary and secondary hypothyroidism are shown in Table 863.4.
     
    Table 863.3 Causes of hypothyroidism 
    1. Primary hypothyroidism (Increased TSH)
      • Iodine deficiency
      • Hashimoto’s thyroiditis
      Exogenous goitrogens
      • Iatrogenic: surgery, drugs, radiation
    2. Secondary hypothyroidism (Low TSH): Diseases of pituitary
    3. Tertiary hypothyroidism (Low TSH, Low TRH): Diseases of hypothalamus
     
    Table 863.4 Differences between primary and secondary hypothyroidism
    Parameter Primary hypothyroidism Secondary hypothyroidism
    1. Cause Hashimoto’s thyroiditis Pituitary disease
    2. Serum TSH High Low
    3. Thyrotropin releasing hormone stimulation test Exaggerated response No response
    4. Antimicrosomal antibodies Present Absent
     
    Box 863.3 Thyroid function tests in hypothyroidism
    • Primary hypothyroidism
      – Serum TSH: Increased (proportional to degree of hypofunction)
      – Free T4: Decreased
      – TRH stimulation test: Exaggerated response
    • Secondary hypothyroidism
      – Serum TSH: Decreased
      – Free T4: Decreased
      – TRH stimulation test: Absent response
    • Tertiary hypothyroidism
      – Serum TSH: Decreased
      – FT4: Decreased
      – TRH stimulation test: Delayed response
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    In severe cases, myxoedema coma (an advanced stage with stupor, hypoventilation, and hypothermia) can occur.
     
    Laboratory Features
     
    Laboratory features in hypothyroidism are shown in Box 863.3.
     
    Normal serum thyroxine (T4 and FT4) coupled with a moderately raised TSH (>10 mU/L) is referred to as subclinical hypothyroidism. It is associated with bad obstetrical outcome, poor cognitive development in children, and high risk of hypercholesterolemia and progression to overt hypothyroidism.
     
    Evaluation of hypothyroidism is presented in Figure 863.2
     
    Figure 863.2 Evaluation of hypothyroidism
    Figure 863.2 Evaluation of hypothyroidism. TSH: thyroid stimulating hormone; FT4: free T4; TRH: Thyrotropin releasing hormone
  • FEMALE INFERTILITY: CAUSES AND INVESTIGATIONS
    The ovaries are the sites of production of female gametes or ova by the process of oogenesis. The ova are released by the process of ovulation in a cyclical manner at regular intervals. Ovary contains numerous follicles that contain ova in various stages of development. During each menstrual cycle, up to 20 primordial follicles are activated for maturation; however, only one follicle becomes fully mature; this dominant follicle ruptures to release the secondary oocyte from the ovary. Maturation of the follicle is stimulated by follicle stimulating hormone (FSH) secreted by anterior pituitary (Figure 862.1). Maturing follicle secretes estrogen that causes proliferation of endometrium of the uterus (proliferative phase). Follicular cells also secrete inhibin which regulates release of FSH by the anterior pituitary. Fall in FSH level is followed by secretion of luteinizing hormone (LH) by the anterior pituitary (LH surge). This causes follicle to rupture and the ovum is expelled into the peritoneal cavity near the fimbrial end of the fallopian tube. The fallopian tubes conduct ova from the ovaries to the uterus. Fertilization of ovum by the sperm occurs in the fallopian tube.
     
    Figure 862.1 The hypothalamus pituitary ovarian axis
    Figure 862.1 The hypothalamus-pituitary-ovarian axis 
     
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    Figure 862.2 Normal menstrual cycle
    Figure 862.2 Normal menstrual cycle
     
    Causes of Female Infertility
     
    Causes of female infertility are shown in Table 862.1.
     
    Table 862.1 Causes of female infertility
    1. Hypothalamic-pituitary dysfunction:
    • Hypothalamic causes
      – Excessive exercise
      – Excess stress
      – Low weight
      – Kallman’s syndrome
      Idiopathic
    • Pituitary causes
      – Hyperprolactinemia
      Hypopituitarism (Sheehan’s syndrome, Simmond’s disease)
      – Craniopharyngioma
      – Cerebral irradiation
     2. Ovarian dysfunction:
    • Polycystic ovarian disease (Stein-Leventhal syndrome)
    • Luteinized unruptured follicle
    • Turner’s syndrome
    • Radiation or chemotherapy
    • Surgical removal of ovaries
    • Idiopathic
     3. Dysfunction in passages:
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      Infections: Tuberculosis, gonorrhea, Chlamydia
      – Previous surgery (e.g. laparotomy)
      – Tubectomy
      Congenital hypoplasia, non-canalization
      Endometriosis
    • Uterus
      – Uterine malformations
      – Asherman’s syndrome
      – Tuberculous endometritis
      Fibroid
    • Cervix: Sperm antibodies
    • Vagina: Septum
     4. Dysfunction of sexual act: Dyspareunia
     
    Investigations
     
    Evaluation of female infertility is shown in Figure 862.3.
     
    Figure 862.3 Evaluation of female infertility
    Figure 862.3 Evaluation of female infertility. FSH: Follicle stimulating hormone; LH: Luteinizing hormone; DHEA-S: Dihydroepiandrosterone; TSH: Thyroid stimulating hormone; ↑ : Increased; ↓ : Decreased
     
    Tests for Ovulation
     
    Most common cause of female infertility is anovulation.
     
    1. Regular cycles, mastalgia, and laparoscopic direct visualization of corpus luteum indicate ovulatory cycles. Anovulatory cycles are clinically characterized by amenorrhea, oligomenorrhea, or irregular menstruation. However, apparently regular cycles may be associated with anovulation.
    2. Endometrial biopsy: Endometrial biopsy is done during premenstrual period (21st-23rd day of the cycle). The secretory endometrium during the later half of the cycle is an evidence of ovulation.
    3. Ultrasonography (USG): Serial ultrasonography is done from 10th day of the cycle and the size of the dominant follicle is measured. Size >18 mm is indicative of imminent ovulation. Collapse of the follicle with presence of few ml of fluid in the pouch of Douglas is suggestive of ovulation. USG also is helpful for treatment (i.e. timing of coitus or of intrauterine insemination) and diagnosis of luteinized unruptured follicle (absence of collapse of dominant follicle). Transvaginal USG is more sensitive than abdominal USG.
    4. Basal body temperature (BBT): Patient takes her oral temperature at the same time every morning before arising. BBT falls by about 0.5°F at the time of ovulation. During the second (progestational) half of the cycle, temperature is slightly raised above the preovulatory level (rise of 0.5° to 1°F). This is due to the slight pyrogenic action of progesterone and is therefore presumptive evidence of functional corpus luteum.
    5. Cervical mucus study:
      Fern test: During estrogenic phase, a characteristic pattern of fern formation is seen when cervical mucus is spread on a glass slide (Figure 862.4). This ferning disappears after the 21st day of the cycle. If previously observed, its disappearance is presumptive evidence of corpus luteum activity.
      Spinnbarkeit test: Cervical mucus is elastic and withstands stretching upto a distance of over 10 cm. This phenomenon is called Spinnbarkeit or the thread test for the estrogen activity. During the secretory phase, viscosity of the cervical mucus increases and it gets fractured when stretched. This change in cervical mucus is evidence of ovulation.
    6. Vaginal cytology: Karyopyknotic index (KI) is high during estrogenic phase, while it becomes low in secretory phase. This refers to percentage of super-ficial squamous cells with pyknotic nuclei to all mature squamous cells in a lateral vaginal wall smear. Usually minimum of 300 cells are evaluated. The peak KI usually corresponds with time of ovulation and may reach upto 50 to 85.
    7. Estimation of progesterone in mid-luteal phase (day 21 or 7 days before expected menstruation): Progesterone level > 10 nmol/L is a reliable evidence of ovulation if cycles are regular (Figure 862.5). A mistimed sample is a common cause of abnormal result.
     
    Figure 862.4 Ferning of cervical mucosa
    Figure 862.4 Ferning of cervical mucosa
     
    Figure 862.5 Serum progesterone during normal menstrual cycle
    Figure 862.5 Serum progesterone during normal menstrual cycle
     
    Tests to Determine the Cause of Anovulation
     
    1. Measurement of LH, FSH, and estradiol during days 2 to 6: All values are low in hypogonadotropic hypogonadism (hypothalamic or pituitary failure).
    2. Measurement of TSH, prolactin, and testosterone if cycles are irregular or absent:
      Increased TSH: Hypothyroidism
      Increased prolactin: Pituitary adenoma
      Increased testosterone: Polycystic ovarian disease (PCOD), congenital adrenal hyperplasia (To differentiate PCOD from congenital adrenal hyperplasia, ultrasound and estimation of dihydroepiandrosterone or DHEA are done).
    3. Transvaginal ultrasonography: This is done for detection of PCOD.
     
    Investigations to Assess Tubal and Uterine Status
     
    1. Infectious disease: These tests include endometrial biopsy for tuberculosis and test for chlamydial IgG antibodies for tubal factor in infertility.
    2. Hysterosalpingography (HSG): HSG is a radiological contrast study for investigation of the shape of the uterine cavity and for blockage of fallopian tubes (Figure 862.6). A catheter is introduced into the cervical canal and a radiocontrast dye is injected into the uterine cavity. A real time X-ray imaging is carried out to observe the flow of the dye into the uterine cavity, tubes, and spillage into the uterine cavity.
    3. Hysterosalpingo-contrast sonography: A catheter is introduced into the cervical canal and an echocontrast fluid is introduced into the uterine cavity. Shape of the uterine cavity, filling of fallopian tubes, and spillage of contrast fluid are noted. In addition, ultrasound scan of the pelvis provides information about any fibroids or polycystic ovarian disease.
    4. Laparoscopy and dye hydrotubation test with hysteroscopy: In this test, a cannula is inserted into the cervix and methylene blue dye is introduced into the uterine cavity. If tubes are patent, spillage of the dye is observed from the ends of both tubes. This technique also allows visualization of pelvic organs, endometriosis, and pelvic adhesions. If required, endometriosis and tubal blockage can be treated during the procedure.
     
    Possible pregnancy and active pelvic or vaginal infection are contraindications to tubal patency tests.
     
    Figure 862.6 Hysterosalpingography
    Figure 862.6 Hysterosalpingography
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