- 18 May 2018
Waste products discharged from the digestive tract are composed of up to 75% water, food which is digested but not absorbed, indigestible residue, undigested food, epithelial cells, bile, bacteria, secretion from the digestive tract and inorganic bacteria. Normally an adult human excretes 100-200 grams of feces in a day.
Examination of stool is very helpful in the diagnosis of disease of the gastrointestinal tract as listed below.
Detection of parasites
Stool examination is performed for the detection and identification of worms (adult worms, larvae, segments of worms, ova) and protozoa (cyst or trophozoites). See also: Microscopic Examination of Feces
Stool culture is performed for the evaluation of bacterial infection such as Clostridium difficile, Yersinia, Salmonella, Shigella or Vibrio. Bacterial toxins (such as those released by Clostridium difficile or Clostridium botulinum) can also be identified. See also: Microscopic Examination of Feces
Evaluation of chronic diarrhea
Chronic diarrhea defined as a passage of three or more liquid or loose stools in a day lasting for more than four weeks. Acute diarrhea refers to the passing of three or more liquid or loose stools in a day for less than four weeks. In diarrhea, stool examination is very important part of laboratory investigations. Depending on the nature of the investigation, either a random stool sample or 72- sample or 48-hour sample is collected. A random stool sample is used for the tests of occult blood, pH, fat, white blood cells, microscopy, or culture. A 72- or 48-hour sample is collected and examined for the weight, carbohydrate, fat content, osmolality, or chymotrypsin activity. Causes of chronic and acute diarrhea are listed in Table 988.1 and Figure 988.1 respectively.
1. Watery diarrhea
2. Inflammatory diarrhea
3. Fatty diarrhea
Evaluation of dysentery
Identification of Rotavirus
In infants and young children, Rotavirus is the most common cause of diarrhea. Rotavirus can be identified by the electron microscopic examination of stool. Other techniques, such as latex agglutination, immunofluorescence, or enzyme-linked immunosorbent assay (ELISA) are also used for the detection of Rotavirus in stool.
Chemical tests can be applied on feces to detect excess fat excretion (malabsorption syndrome), occult blood (in ulcerated lesions of the gastrointestinal tract, especially occult carcinoma of the colon) and presence or absence of urobilinogen (obstructive jaundice). See also: Chemical Examination of Feces
Differentiating infection by invasive bacteria (like Salmonella or Shigella) from that due to toxin-producing bacteria (like Vibrio cholerae or Escherichia coli)
Feces is examined for the presence of white blood cells. Increased numbers of polymorphonuclear neutrophils (identified by methylene blue stain from the presence of granules in their cytoplasm) are seen as shown in Figure 988.2. See also: Causes, symptoms, diagnosis, and treatment of Cholera
What is acute leukemia?
It is defined as malignant clonal hematopoietic stem cell disorders characterized by the rapid increase in the number of blast cells in the bone marrow and rapidly progressive fatal course if untreated. Acute leukemia (AL) are primary disorders of the bone marrow, also known as blood cancer.
Classification of acute leukemias
The most widely used classification of acute leukemias is French-American-British (FAB) Co-operative Group classification. The FAB rule for the classification of acute leukemias was originally proposed in 1976. On the basis of the morphology and cytochemistry, they are classified into two major types.
- Acute myeloid leukemia (AML)
- Acute lymphoblastic leukemia (ALL)
Each type is further subclassified. See table 883.1
|Acute myeloid leukemia (AML)|
|M0: Acute myeloid leukemia, minimally differentiated|
|M1: Acute myeloid leukemia without maturation|
|M2: Acute myeloid leukemia, with maturation|
|M3: Acute promyelocytic leukemia M3v: Hypo- or microgranular promyelocytic leukemia|
|M4: Acute myelomonocytic leukemia M4Eo: Acute myelomonocytic leukemia with bone marrow eosinophilia|
M5: Acute monocytic leukemia
|M6: Acute erythroleukemia|
|M7: Acute megakaryocytic leukemia|
|Acute lymphoblastic leukemia (ALL)|
|L1: Lymphoblasts with constant, rounded nuclei, and adequate cytoplasm. Nucleoli are not prominent.|
|L2: More irregular lymphoblast and cytoplasm is available in large quantity. Nucleoli are large and may see one or more nucleoli.|
|L3: Large cells with moderate amount of deeply basophilic cytoplasm; prominent cytoplasmic vacuoles; regular nuclear membrane; 1-2 prominent nucleoli|
In contrast to French-American-British (FAB) classification, World Health Organization (WHO) classification recognizes AML with recurrent cytogenetic abnormalities, AML with multilineage dysplasia, and therapy-related AML as distinct entities.
Patients with clonal, recurrent cytogenetic abnormalities listed in Table 883.2 are considered to have AML irrespective of the percentage of blasts in blood or bone marrow. These patients have characteristic clinical and morphological features and a favorable response to therapy.
1. Acute myeloid leukemia with recurrent genetic abnormalities
2. Acute myeloid leukemia with multilineage dysplasia
3. AML and MDS, therapy related
4. AML, not otherwise categorized
Difference between Acute Myelocytic Leukemia (AML) and Acute Lymphocytic Leukemia (ALL)
|Characteristic Features||Acute Myelocytic Leukemia||Acute Lymphocytic Leukemia|
|Origin of cells||Myeloid series cells||Lymphoid series cells|
|Characteristics of blast cells||Cell is large in size with moderate cytoplasm. Chromatin patterns are fine and lacy. Nucleoli are prominent and are more than two.||Cell is small with scanty cytoplasm. Chromatin patterns are dense. Nucleoli are indistinct and are less than two.|
|Bone marrow||Mixed population of blast and myeloid cells.||Mainly blast cells and very few WBCs or RBCs|
|Sudan black and peroxidase||Positive||Negative|
|Periodic Acid-Schiff (PAS)||Positive in Erythroblast in M6 leukemia||Positive (block patterns) in L1 and L2, and negative in L3|
|Leukocyte Alkaline phosphatase (ALP)||Positive (It is used to differentiate CML from the leukemoid reaction)||Negative|
- Weakness and fatigue
- Low-grade fever
- Bone pain
- Bruising and mild bleeding from gums
- It appears suddenly
Diagnosis of acute leukemia
- This disease is most common in younger age group and more frequent in the children. Mostly found before the age of 20.
- Rapid development of anemia is most common with normocytic and normochromic red cells. You may see some nucleated red cells in the peripheral blood smear.
- Leucocytes (WBCs) count is variable. Mostly leucocytes count is less than 100000/μl.
- Thrombocytopenia (See also Morphology of Platelets)
• Petechiae and purpura may be seen in the skin and mucous membranes.
Laboratory findings in acute myelocytic leukemia (AML)
- Total leucocyte count (TLC) is variable. It is recorded that in 25% of patient's total leucocyte count is < 5000/μl, 25% of the patient has > 50000/μl and 5% to 10% has total leucocyte count between 5000 to 10000/μl.
- Abnormal and immature WBCs are present.
- In 50% of cases, the value of uric acid is raised.
- In bone marrow examination, > 20% blast cells are present. Promyelocytes are numerous especially in M3 (acute promyelocytic leukemia). See table 883.1
- Cytochemical stains, Sudan black, and peroxidase show the positive reaction.
- Auer rods can be seen in the cytoplasm of the cells.
- Nucleated red blood cells may be seen.
- PT, PTT and Thrombin time are elevated.
- Thrombocytopenia may be present and may see the platelets count between 30,000 to 100,000/μl.
Laboratory findings in acute lymphocytic leukemia (ALL)
- Total leucocyte count is variable from low to very high.
- Anemia and thrombocytopenia
- Bone marrow:
It is difficult to find normal RBCs and WBCs.
Very few WBCs are seen.
Characteristically there are blast cells.
Sudan black and peroxidase show the negative reaction.
Auer rods are absent.
Test value for the layman
Examination of peripheral blood smear and bone marrow is advised:
- If the patient has high TLC.
- If the patient has Anemia.
- If the patient has enlarged lymph nodes.
- If the patient has weakness and fever.
- Site: GIT (Gastrointestinal Track)
- Agent: VIBRIO CHOLERA
- They produce smooth, convex, round, colonies which appear opaque and granular in transmitted light.
- They can grow on many kinds of media including enriching media contains bile salt and asparagine.
- They particularly grow on TCB agar (Thiosulfate Citrate Bile Salt agar) and produce yellow colonies.
- They are readily killed by acid and optimum pH for growth is 8.5-9.5.
- Vibrio Cholera contains two types of antigen flagellar (H) and somatic (O).
- Vibrio Cholera contains two types of antigen flagellar (H) and somatic (O).
- All Vibrios shared a single heat labile H antigen.
- The O antigen is composed of heat stable polysaccharides and are classified into 6 serogroups and are further classified into 60 serotypes on the basis of O antigen.
- One serotype of Vibrio Cholera bacilli is responsible for epidemic cholera and is subdivided into two types.
(1) Classical (2) El Tor
- El Tor types Vibrios were different from the classical types in their ability to cause lysis of goa or sheep erythrocyte in a test known as Grieg Test.
- Each of the two biotypes of 01 serotypes of Vibrio is comprised of two or three antigenic factor A, B, and C
- Factor A and B are found in serotype Ogawa, A and C in serotype Inaba and A, B and C in serotype Hikojima.
- Good water supply. Proper treatment of water should be there before supply to the town.
- Proper treatment of sewerage system.
- Personal hygiene and proper sanitation.
- 28 Sep 2017
- Hyperthyroidism: Elevation of both T4 and T3 values along with decrease of TSH are indicative of primary hyperthyroidism.
- 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.
- Factitious hyperthyroidism
- Pituitary TSH-secreting tumor.
- Primary hypothyroidism: The combination of decreased T4 and elevated TSH are indicative of primary hypothyroidism.
- Secondary or pituitary hypothyroidism
- Tertiary or hypothalamic hypothyroidism
- Hypoproteinaemia, e.g. nephrotic syndrome
- Drugs: oestrogen, danazol
- Severe non-thyroidal illness.
- Diagnosis of T3 thyrotoxicosis: Hyperthyroidism with low TSH and elevated T3, and normal T4/FT4 is termed T3 thyrotoxicosis.
- Early diagnosis of hyperthyroidism: In early stage of hyperthyroidism, total T4 and free T4 levels are normal, but T3 is elevated.
- Confirmation of diagnosis of secondary hypothyroidism
- Evaluation of suspected hypothalamic disease
- Suspected hyperthyroidism
- 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.
- Normal response: A rise of TSH > 2 mU/L at 20 minutes, and a small decline at 60 minutes.
- 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.
- Flat response: There is no response; occurs in secondary (pituitary) hypothyroidism.
- Delayed response: TSH is higher at 60 minutes as compared to its level at 20 minutes; seen in tertiary (hypothalamic) hypothyroidism.
Box 864.1 Thyroid autoantibodies
- Hyperthyroidism due to Graves’ disease, toxic multinodular goiter, toxic adenoma, TSH-secreting tumor.
- Hyperthyroidism due to administration of thyroid hormone, factitious hyperthyroidism, subacute thyroiditis.
- Differential diagnosis of high RAIU thyrotoxicosis:
– Graves’ disease: Uniform or diffuse increase in uptake
– Toxic multinodular goiter: Multiple discrete areas of increased uptake
– Adenoma: Single area of increased uptake
- Evaluation of a solitary thyroid nodule:
– ‘Hot’ nodule: Hyperfunctioning
– ‘Cold’ nodule: Non-functioning; about 20% cases are malignant.
|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|
- 28 Sep 2017
|Box 863.1 Terminology in thyroid disorders
|Box 863.2 Thyroid function tests in 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|
|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
- 22 Sep 2017
1. Hypothalamic-pituitary dysfunction:
2. Ovarian dysfunction:
|3. Dysfunction in passages:|
|4. Dysfunction of sexual act: Dyspareunia|
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Measurement of LH, FSH, and estradiol during days 2 to 6: All values are low in hypogonadotropic hypogonadism (hypothalamic or pituitary failure).
- 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).
- Transvaginal ultrasonography: This is done for detection of PCOD.
- Infectious disease: These tests include endometrial biopsy for tuberculosis and test for chlamydial IgG antibodies for tubal factor in infertility.
- 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.
- 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.
- 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.
- 22 Sep 2017
2. Hypothalamic-pituitary dysfunction (hypogonadotropic hypogonadism)
3. Testicular dysfunction:
4. Dysfunction of passages and accessory sex glands:
5. Dysfunction of sexual act:
- History: This includes type of lifestyle (heavy smoking, alcoholism), sexual practice, erectile dysfunction, ejaculation, sexually transmitted diseases, surgery in genital area, drugs, and any systemic illness.
- Physical examination: Examination of reproductive system should includes testicular size, undescended testes, hypospadias, scrotal abnormalities (like varicocele), body hair, and facial hair. Varicocele can occur bilaterally and is the most common surgically removable abnormality causing male infertility.
- Semen analysis: See article Semen Analysis. Evaluation of azoospermia is shown in Figure 861.3. Evaluation of low semen volume is shown in Figure 861.4.
- Chromosomal analysis: This can reveal Klinefelter’s syndrome (e.g. XXY karyotype) (Figure 861.5), deletion in Y chromosome, and autosomal Robertsonian translocation. It is necessary to screen for cystic fibrosis carrier state if bilateral congenital absence of vas deferens is present.
- Hormonal studies: This includes measurement of FSH, LH, and testosterone to detect hormonal abnormalities causing testicular failure (Table 861.2).
- Testicular biopsy: Testicular biopsy is indicated when differentiation between obstructive and non-obstructive azoospermia is not evident (i.e. normal FSH and normal testicular volume).
|Follicle stimulating hormone||Luteinizing hormone||Testosterone||Interpretation|
|Low||Low||Low||Hypogonadotropic hypogonadism (Hypothalamic or pituitary disorder)|
|High||High||Low||Hypergonadotropic hypogonadism (Testicular disorder)|
|Normal||Normal||Normal||Obstruction of passages, dysfunction of accessory glands|
- 08 Sep 2017
- Cephalic or neurogenic phase: This phase is initiated by the sight, smell, taste, or thought of food that causes stimulation of vagal nuclei in the brain. Vagus nerve directly stimulates parietal cells to secrete acid; in addition, it also stimulates antral G cells to secrete gastrin in blood (which is also a potent stimulus for gastric acid secretion) (Figure 859.2). Cephalic phase is abolished by vagotomy.
- Gastric phase: Entry of swallowed food into the stomach causes gastric distension and induces gastric phase. Distension of antrum and increase in pH due to neutralization of acid by food stimulate antral G cells to secrete gastrin into the circulation. Gastrin, in turn, causes release of hydrochloric acid from parietal cells.
- Intestinal phase: Entry of digested proteins into the duodenum causes an increase in acid output from the stomach. It is thought that certain hormones and absorbed amino acids stimulate parietal cells to secrete acid.
- Hydrochloric acid (HCl): This is secreted by the parietal cells of the fundus and the body of the stomach. HCl provides the high acidic pH necessary for activation of pepsinogen to pepsin. Gastric acid secretion is stimulated by histamine, acetylcholine, and gastrin (Figure 859.2). HCl kills most microorganisms entering the stomach and also denatures proteins (breaks hydrogen bonds making polypeptide chains to unfold). Its secretion is inhibited by somatostatin (secreted by D cells in pancreas and by mucosa of intestine), gastric inhibitory peptide (secreted by K cells in duodenum and jejunum), prostaglandin, and secretin (secreted by S cells in duodenum).
- Pepsin: Pepsin is secreted by chief cells in stomach. Pepsin causes partial digestion of proteins leading to the formation of large polypeptide molecules (optimal function at pH 1.0 to 3.0). Its secretion is enhanced by vagal stimulation.
- Intrinsic factor (IF): IF is necessary for absorption of vitamin B12 in the terminal ileum. It is secreted by parietal cells of stomach.
- 07 Sep 2017
- Gastric intubation for gastric analysis is contraindicated in esophageal stricture or varices, active nasopharyngeal disease, diverticula, malignancy, recent history of severe gastric hemorrhage, hypertension, aortic aneurysm, cardiac arrhythmias, congestive cardiac failure, or non-cooperative patient.
- Pyloric stenosis: Obstruction of gastric outlet can elevate gastric acid output due to raised gastrin (following antral distension).
- Pentagastrin stimulation is contraindicated in cases with allergy to pentagastrin, and recent severe gastric hemorrhge due to peptic ulcer disease.
- It is an invasive and cumbersome technique that is traumatic and unpleasant for the patient.
- Information obtained is not diagnostic in itself.
- Availability of better tests for diagnosis such as endoscopy and radiology (for suspected peptic ulcer or malignancy); serum gastrin estimation (for ZE syndrome); vitamin assays, Schilling test, and antiparietal cell antibodies (for pernicious anemia); and tests for Helicobacter pylori infection (in duodenal or gastric ulcer).
- Availability of better medical line of treatment that obviates need for surgery in many patients.
- 07 Sep 2017
- Hollander’s test (Insulin hypoglycemia test): In the past, this test was used for confirmation of completeness of vagotomy (done for duodenal ulcer).
Hypoglycemia is a potent stimulus for gastric acid secretion and is mediated by vagus nerve. This response is abolished by vagotomy.
In this test, after determining BAO, insulin is administered intravenously (0.15-0.2 units/kg) and acid output is estimated every 15 minutes for 2 hours (8 post-stimulation samples). Vagotomy is considered as complete if, after insulin-induced hypoglycemia (blood glucose < 45 mg/dl), no acid output is observed within 45 minutres.
The test gives reliable results only if blood glucose level falls below 50 mg/dl at some time following insulin injection. It is best carried out after 3-6 months of vagotomy.
The test is no longer recommended because of the risk associated with hypoglycemia. Myocardial infarction, shock, and death have also been reported.
- Fractional test meal: In the past, test meals (e.g. oat meal gruel, alcohol) were administered orally to stimulate gastric secretion and determine MAO or PAO. Currently, parenteral pentagastrin is the gastric stimulant of choice.
- Tubeless gastric analysis: This is an indirect and rapid method for determining output of free hydrochloric acid in gastric juice. In this test, a cationexchange resin tagged to a dye (azure A) is orally administered. In the stomach, the dye is displaced from the resin by the free hydrogen ions of the hydrochloric acid. The displaced azure A is absorbed in the small intestine, enters the bloodstream, and is excreted in urine. Urinary concentration of the dye is measured photometrically or by visual comparison with known color standards. The quantity of the dye excreted is proportional to the gastric acid output. However, if kidney or liver function is impaired, false results may be obtained. The test is no longer in use.
- Spot check of gastric pH: According to some investigators, spot determination of pH of fasting gastric juice (obtained by nasogastric intubation) can detect the presence of hypochlorhydria (if pH>5.0 in men or >7.0 in women).
- Congo red test during esophagogastroduodenoscopy: This test is done to determine the completeness of vagotomy. Congo red dye is sprayed into the stomach during esophagogastroduodenoscopy; if it turns red, it indicates presence of functional parietal cells in stomach with capacity of producing acid.
- Volume of gastric juice: 20-100 ml
- Appearance: Clear
- pH: 1.5 to 3.5
- Basal acid output: Up to 5 mEq/hour
- Peak acid output: 1 to 20 mEq/hour
- Ratio of basal acid output to peak acid output: <0.20 or < 20%
- 07 Sep 2017
- To determine the cause of recurrent peptic ulcer disease:
• To detect Zollinger-Ellison (ZE) syndrome: ZE syndrome is a rare disorder in which multiple mucosal ulcers develop in the stomach, duodenum, and upper jejunum due to gross hypersecretion of acid in the stomach. The cause of excess secretion of acid is a gastrin-producing tumor of pancreas. Gastric analysis is done to detect markedly increased basal and pentagastrinstimulated gastric acid output for diagnosis of ZE syndrome (and also to determine response to acidsuppressant therapy). However, a more sensitive and specific test for diagnosis of ZE syndrome is measurement of serum gastrin (fasting and secretin-stimulated).
• To decide about completeness of vagotomy following surgery for peptic ulcer disease: See Hollander’s test.
- To determine the cause of raised fasting serum gastrin level: Hypergastrinemia can occur in achlorhydria, Zollinger-Ellison syndrome, and antral G cell hyperplasia.
- To support the diagnosis of pernicious anemia (PA): Pernicious anemia is caused by defective absorption of vitamin B12 due to failure of synthesis of intrinsic factor secondary to gastric mucosal atrophy. There is also absence of hydrochloric acid in the gastric juice (achlorhydria). Gastric analysis is done for demonstration of achlorhydria if facilities for vitamin assays and Schilling’s test are not available (Achlorhydria by itself is insufficient for diagnosis of PA).
- To distinguish between benign and malignant ulcer: Hypersecretion of acid is a feature of duodenal peptic ulcer, while failure of acid secretion (achlorhydria) occurs in gastric carcinoma. However, anacidity occurs only in a small proportion of cases with advanced gastric cancer. Also, not all patients with duodenal ulcer show increased acid output.
- To measure the amount of acid secreted in a patient with symptoms of peptic ulcer dyspepsia but normal X-ray findings: Excess acid secretion in such cases is indicative of duodenal ulcer. However, hypersecretion of acid does not always occur in duodenal ulcer.
- To decide the type of surgery to be performed in a patient with peptic ulcer: Raised basal as well as peak acid outputs indicate increased parietal cell mass and need for gastrectomy. Raised basal acid output with normal peak output is an indication for vagotomy.
- 05 Sep 2017
Box 855.1 Determination of basal acid output, maximum acid output, and peak acid output
- Volume: Normal total volume is 20-100 ml (usually < 50 ml). Causes of increased volume of gastric juice are—
• Delayed emptying of stomach: pyloric stenosis
• Increased gastric secretion: duodenal ulcer, Zollinger-Ellison syndrome.
- Color: Normal gastric secretion is colorless, with a faintly pungent odor. Fresh blood (due to trauma, or recent bleeding from ulcer or cancer) is red in color. Old hemorrhage produces a brown, coffee-ground like appearance (due to formation of acid hematin). Bile regurgitation produces a yellow or green color.
- pH: Normal pH is 1.5 to 3.5. In pernicious anemia, pH is greater than 7.0 due to absence of HCl.
- Basal acid output:
• Normal: Up to 5 mEq/hour.
• Duodenal ulcer: 5-15 mEq/hour.
• Zollinger-Ellison syndrome: >20 mEq/hour.
Normal BAO is seen in gastric ulcer and in some patients with duodenal ulcer.
- Peak acid output:
• Normal: 1-20 mEq/hour.
• Duodenal ulcer: 20-60 mEq/hour.
• Zollinger-Ellison syndrome: > 60 mEq/hour.
• Achlorhydria: 0 mEq/hour.
Normal PAO is seen in gastric ulcer and gastric carcinoma. Values up to 60 mEq/hour can occur in some normal individuals and in some patients with Zollinger-Ellison syndrome.
In pernicious anemia, there is no acid output due to gastric mucosal atrophy. Achlorhydria should be diagnosed only if there is no free HCl even after maximum stimulation.
- Ratio of basal acid output to peak acid output (BAO/PAO):
• Normal: < 0.20 (or < 20%).
• Gastric or duodenal ulcer: 0.20-0.40 (20-40%).
• Duodenal ulcer: 0.40-0.60 (40-60%).
• Zollinger-Ellison syndrome: > 0.60 (> 60%).
Normal values occur in gastric ulcer or gastric carcinoma.
|Increased gastric acid output||Decreased gastric acid output|
|• Duodenal ulcer||• Chronic atrophic gastritis|
|• Zollinger-Ellison syndrome||1. Pernicious anemia|
|• Hyperplasia of antral G cells||2. Rheumatoid arthritis|
|• Systemic mastocytosis||3. Thyrotoxicosis|
|• Basophilic leukemia||• Gastric ulcer|
|• Gastric carcinoma|
|• Chronic renal failure|
- 30 Aug 2017
- Brown: Normal
- Black: Bleeding in upper gastrointestinal tract (proximal to cecum), Drugs (iron salts, bismuth salts, charcoal)
- Red: Bleeeding in large intestine, undigested tomatoes or beets
- Clay-colored (gray-white): Biliary obstruction
- Silvery: Carcinoma of ampulla of Vater
- Watery: Certain strains of Escherichia coli, Rotavirus enteritis, cryptosporidiosis
- Rice water: Cholera
- Unformed with blood and mucus: Amebiasis, inflammatory bowel disease
- Unformed with blood, mucus, and pus: Bacillary dysentery
- Unformed, frothy, foul smelling, which float on water: Steatorrhea.
- Sedimentation techniques: Ova and cysts settle at the bottom. However, excessive fecal debris may make the detection of parasites difficult. Example: Formolethyl acetate sedimentation procedure.
- Floatation techniques: Ova and cysts float on surface. However, some ova and cysts do not float at the top in this procedure. Examples: Saturated salt floatation technique and zinc sulphate concentration technique.
- 30 Aug 2017
- Occult blood
- Excess fat excretion (malabsorption)
- Reducing sugars
- Fecal osmotic gap
- Fecal pH
- Intestinal diseases: hookworms, amebiasis, typhoid fever, ulcerative colitis, intussusception, adenoma, cancer of colon or rectum.
- Gastric and esophageal diseases: peptic ulcer, gastritis, esophageal varices, hiatus hernia.
- Systemic disorders: bleeding diathesis, uremia.
- Long distance runners.
- Ingestion of peroxidase-containing foods like red meat, fish, poultry, turnips, horseradish, cauliflower, spinach, or cucumber. Diet should be free from peroxidase-containing foods for at least 3 days prior to testing.
- Drugs like aspirin and other anti-inflammatory drugs, which increase blood loss from gastrointestinal tract in normal persons.
- Foods containing large amounts of vitamin C.
- Conversion of all hemoglobin to acid hematin (which has no peroxidase-like activity) during passage through the gastrointestinal tract.
- Deficiency of pancreatic lipase (insufficient lipolysis): chronic pancreatitis, cystic fibrosis.
- Deficiency of bile salts (insufficient emulsification of fat): biliary obstruction, severe liver disease, bile salt deconjugation due to bacterial overgrowth in the small intestine.
- Diseases of small intestine: tropical sprue, celiac disease, Whipple’s disease.
- Microscopic stool examination after staining for fat: A random specimen of stool is collected after putting the patient on a diet of >80 gm fat per day. Stool sample is stained with a fat stain (oil red O, Sudan III, or Sudan IV) and observed under the microscope for fat globules (Figure 845.2). Presence of ≥60 fat droplets/HPF indicates steatorrhea. Ingestion of mineral or castor oil and use of rectal suppositories can cause problems in interpretation.
- Quantitative estimation of fecal fat: The definitive test for diagnosis of fat malabsorption is quantitation of fecal fat. Patient should be on a diet of 70-100 gm of fat per day for 6 days before the test. Feces are collected over 72 hours and stored in a refrigerator during the collection period. Specimen should not be contaminated with urine. Fat quantitation can be done by gravimetric or titrimetric method. In gravimetric method, an accurately weighed sample of feces is emulsified, acidified, and fat is extracted in a solvent; after evaporation of solvent, fat is weighed as a pure compound. Titrimetric analysis is the most widely used method. An accurately weighed stool sample is treated with alcoholic potassium hydroxide to convert fat into soaps. Soaps are then converted to fatty acids by the addition of hydrochloric acid. Fatty acids are extracted in a solvent and the solvent is evaporated. The solution of fat made in neutral alcohol is then titrated against sodium hydroxide. Fatty acids comprise about 80% of fecal fat. Values >7 grams/day are usually abnormal. Values >14 grams/day are specific for diseases causing fat malabsorption.
- 28 Aug 2017
(Plasma sodium × Urine creatinine)
- Causes of increased specific gravity:
a. Reduced renal perfusion (with preservation of concentrating ability of tubules),
e. Urinary tract obstruction.
- Causes of reduced specific gravity:
a. Diabetes insipidus
b. Chronic renal failure
c. Impaired concentrating ability due to diseases of tubules.
- 27 Aug 2017
- 27 Aug 2017
- Pre-renal azotemia: shock, congestive heart failure, salt and water depletion
- Renal azotemia: impairment of renal function
- Post-renal azotemia: obstruction of urinary tract
- Increased rate of production of urea:
• High protein diet
• Increased protein catabolism (trauma, burns, fever)
• Absorption of amino acids and peptides from a large gastrointestinal hemorrhage or tissue hematoma
- Diacetyl monoxime urea method: This is a direct method. Urea reacts with diacetyl monoxime at high temperature in the presence of a strong acid and an oxidizing agent. Reaction of urea and diacetyl monoxime produces a yellow diazine derivative. The intensity of color is measured in a colorimeter or spectrophotometer.
- Urease- Berthelot reaction: This is an indirect method. Enzyme urease splits off ammonia from the urea molecule at 37°C. Ammonia generated is then reacted with alkaline hypochlorite and phenol with a catalyst to produce a stable color (indophenol). Intensity of color produced is then measured in a spectrophotometer at 570 nm.
- It is produced from muscles at a constant rate and its level in blood is not affected by diet, protein catabolism, or other exogenous factors;
- It is not reabsorbed, and very little is secreted by tubules.
Causes of Increased Serum Creatinine Level
- Pre-renal, renal, and post-renal azotemia
- Large amount of dietary meat
- Active acromegaly and gigantism
- Increasing age (reduction in muscle mass)
- Jaffe’s reaction (Alkaline picrate reaction): This is the most widely used method. Creatinine reacts with picrate in an alkaline solution to produce spectrophotometer at 485 nm. Certain substances in plasma (such as glucose, protein, fructose, ascorbic acid, acetoacetate, acetone, and cephalosporins) react with picrate in a similar manner; these are called as non-creatinine chromogens (and can cause false elevation of serum creatinine level). Thus ‘true’ creatinine is less by 0.2 to 0.4 mg/dl when estimated by Jaffe’s reaction.
- Enzymatic methods: These methods use enzymes that cleave creatinine; hydrogen peroxide produced then reacts with phenol and a dye to produce a colored product, which is measured in a spectrophotometer.
- Increased BUN with normal serum creatinine:
• Pre-renal azotemia (reduced renal perfusion)
• High protein diet
• Increased protein catabolism
• Gastrointestinal hemorrhage
- Increase of both BUN and serum creatinine with disproportionately greater increase of BUN:
• Post-renal azotemia (Obstruction to the outflow of urine)
Obstruction to the urine outflow causes diffusion of urinary urea back into the blood from tubules because of backpressure.
Causes of Decreased BUN/Creatinine Ratio (<10:1)
- Acute tubular necrosis
- Low protein diet, starvation
- Severe liver disease
(72 × Serum creatinine in mg/dl)
The agents used for measurement of GFR are:
- Exogenous: Inulin, Radiolabelled ethylenediamine tetraacetic acid (51Cr- EDTA), 125I-iothalamate
- Endogenous: Creatinine, Urea, Cystatin C
- A small amount of creatinine is secreted by renal tubules that increase even further in advanced renal failure.
- Collection of urine is often incomplete.
- Creatinine level is affected by intake of meat and muscle mass.
- Creatinine level is affected by certain drugs like cimetidine, probenecid, and trimethoprim (which block tubular secretion of creatinine).
- Establish the diagnosis
- Assess severity and activity of disease
- Assess prognosis by noting the amount of scarring
- To plan treatment and monitor response to therapy
- Nephrotic syndrome in adults (most common indication)
- Nephrotic syndrome not responding to corticosteroids in children.
- Acute nephritic syndrome for differential diagnosis
- Unexplained renal insufficiency with near-normal kidney dimensions on ultrasonography
- Asymptomatic hematuria, when other diagnostic tests fail to identify the source of bleeding
- Isolated non-nephrotic range proteinuria (1-3 gm/24 hours) with renal impairment
- Impaired function of renal graft
- Involvement of kidney in systemic disease like systemic lupus erythematosus or amyloidosis
- Uncontrolled severe hypertension
- Hemorrhagic diathesis
- Solitary kidney
- Renal neoplasm (to avoid spread of malignant cells along the needle track)
- Large and multiple renal cysts
- Small, shrunken kidneys
- Acute urinary tract infection like pyelonephritis
- Urinary tract obstruction
- Hemorrhage: As renal cortex is highly vascular, major risk is bleeding in the form of hematuria or perinephric hematoma. Severe bleeding may occasionally necessitate blood transfusion and rarely removal of kidney.
- Arteriovenous fistula
- Accidental biopsy of another organ or perforation of viscus (liver, spleen, pancreas, adrenals, intestine, or gallbladder)
- Death (rare).
- Patient’s informed consent is obtained.
- Ultrasound/CT scan is done to document the location and size of kidneys.
- Blood pressure should be less than 160/90 mm of Hg. Bleeding time, platelet count, prothrombin time, and activated partial thromboplastin time should be normal. Blood sample should be drawn for blood grouping and cross matching, as blood transfusion may be needed.
- Patient is sedated before the procedure.
- Patient lies in prone position and kidney is identified with ultrasound.
- The skin over the selected site is disinfected and a local anesthetic is infiltrated.
- A small skin incision is given with a scalpel (to insert the biopsy needle). Localization of kidney is done with a fine bore 21 G lumbar puncture needle. A local anesthetic is infiltrated down to the renal capsule.
- A tru-cut biopsy needle or spring loaded biopsy gun is inserted under ultrasound guidance and advanced down to the lower pole. Biopsy is usually obtained from lateral border of lower pole. Patient should hold his/her breath in full inspiration during biopsy. After obtaining the biopsy and removal of needle, patient is allowed to breath normally.
- The biopsy should be placed in a drop of saline and examined under a dissecting microscope for adequacy.
- Patient is turned to supine position. Vital signs and appearance of urine should be monitored at regular intervals. Patient is usually kept in the hospital for 24 hours.
- Hematoxylin and eosin (for general architecture of kidney and cellularity)
- Periodic acid Schiff: To highlight basement membrane and connective tissue matrix.
- Congo red: For amyloid.
- 17 Aug 2017
- Diagnosis of DM
- Screening of DM
- Assessment of glycemic control
- Assessment of associated long-term risks
- Management of acute metabolic complications.
- Chemical methods:
– Orthotoluidine method
– Blood glucose reduction methods using neocuproine, ferricyanide, or copper.
- Enzymatic methods: These are specific for glucose.
– Glucose oxidase-peroxidase
– Glucose dehydrogenase
- Fasting blood glucose: Sample for blood glucose is withdrawn after an overnight fast (no caloric intake for at least 8 hours).
- Post meal or postprandial blood glucose: Blood sample for glucose estimation is collected 2 hours after the subject has taken a normal meal.
- Random blood glucose: Blood sample is collected at any time of the day, without attention to the time of last food intake.
- Patient should be put on a carbohydrate-rich, unrestricted diet for 3 days. This is because carbohydrate-restricted diet reduces glucose tolerance.
- Patient should be ambulatory with normal physical activity. Absolute bed rest for a few days impairs glucose tolerance.
- Medications should be discontinued on the day of testing.
- Exercise, smoking, and tea or coffee are not allowed during the test period. Patient should remain seated.
- OGTT is carried out in the morning after patient has fasted overnight for 8-14 hours.
- A fasting venous blood sample is collected in the morning.
- Patient ingests 75 g of anhydrous glucose in 250-300 ml of water over 5 minutes. (For children, the dose is 1.75 g of glucose per kg of body weight up to maximum 75 g of glucose). Time of starting glucose drink is taken as 0 hour.
- A single venous blood sample is collected 2 hours after the glucose load. (Previously, blood samples were collected at ½, 1, 1½, and 2 hours, which is no longer recommended).
- Plasma glucose is estimated in fasting and 2-hour venous blood samples.
|Parameter||Normal||Impaired fasting glucose||Impaired glucose tolerance||Diabetes mellitus|
|(1) Fasting (8 hr)||< 100||100-125||—||≥ 126|
|(2) 2 hr OGTT||< 140||< 140||140-199||≥ 200|
- Low-risk pregnant women need not be tested if all of the following criteria are met, i.e. age below 25 years, normal body weight (before pregnancy), absence of diabetes in first-degree relatives, member of an ethnic group with low prevalence of DM, no history of poor obstetric outcome, and no history of abnormal glucose tolerance.
- Average-risk pregnant women (i.e. who are in between low and high risk) should be tested at 24-28 weeks of gestation.
- High-risk pregnant women i.e. those who meet any one of the following criteria should be tested immediately: marked obesity, strong family history of DM, glycosuria, or personal history of GDM.
- One step approach
- Two step approach
- Fasting: 95 mg/dl
- 1 hour: 180 mg/dl
- 2 hour: 155 mg/dl
- 3 hour: 140 mg/dl
- Periodic measurement of glycated hemoglobin (to assess long-term control).
- Daily self-assessment of blood glucose (to assess day-to- day or immediate control).
Numerous prospective studies have demonstrated that a good control of blood glucose reduces the development and progression of microvascular complications (retinopathy, nephropathy, and peripheral neuropathy) of diabetes mellitus. Mean glycated hemoglobin level correlates with the risk of these complications.
|Box 837.1 Glycated hemoglobin
Goal of tight glycemic control in type 1 DM patients on insulin can be achieved through self-monitoring of blood glucose by portable blood glucose meters.
- It is the earliest marker of diabetic nephropathy. Early diabetic nephropathy is reversible.
- It is a risk factor for cardiovascular disease in both type 1 and type 2 patients.
- It is associated with higher blood pressure and poor glycemic control.
- Albumin to creatinine ratio in a random urine sample
- Urinary albumin excretion in a 24-hour urine sample.
- Total cholesterol
- Low-density lipoprotein (LDL) cholesterol
- High-density lipoprotein (HDL) cholesterol
|Category||Low density lipoproteins||High density lipoproteins||Triglycerides|
|High-risk||≥130||< 35 (men)||≥ 400|
|< 45 (women)|
|Low-risk||< 100||> 45 (men)||< 200|
|> 55 (women)|
- Diabetic ketoacidosis (DKA)
- Hyperosmolar hyperglycemic state (HHS)
|Parameter||Diabetic ketoacidosis||Hyperosmolar hyperglycemic state|
|1. Type of DM in which more common||Type 1||Type 2|
|2. Age||Younger age||Older age|
|3. Prodromal clinical features||< 24 hrs||Several days|
|4. Abdominal pain, Kussmaul’s respiration||Yes||No|
|6. Plasma glucose||> 250 mg/dl||Very high (>600 mg/dl)|
|7. Serum bicarbonate||<15 mEq/L||>15 mEq/L|
|8. Blood/urine ketones||++++||±|
|9. β-hydroxybutyrate||High||Normal or raised|
|10. Arterial blood pH||Low (<7.30)||Normal (>7.30)|
|11. Effective serum osmolality*||Variable||Increased (>320)|
|12. Anion gap**||>12||Variable|
|Osmolality: Number of dissolved (solute) particles in solution; normal: 275-295 mOsmol/kg
** Anion gap: Difference between sodium and sum of chloride and bicarbonate in plasma; normal average value is 12
- Blood and urine glucose
- Blood and urine ketone
- Arterial pH, Blood gases
- Serum electrolytes (sodium, potassium, chloride, bicarbonate)
- Blood osmolality
- Serum creatinine and blood urea.
- At diagnosis of diabetes mellitus
- At regular intervals in all known cases of diabetes, during pregnancy with pre-existing diabetes, and in gestational diabetes
- In known diabetic patients: during acute illness, persistent hyperglycemia (> 300 mgs/dl), pregnancy, and clinical evidence of diabetic acidosis (nausea, vomiting, abdominal pain).
- Venous plasma glucose:
Fasting: 60-100 mg/dl
At 2 hours in OGTT (75 gm glucose): <140 mg/dl
- Glycated hemoglobin: 4-6% of total hemoglobin
- Lipid profile:
– Serum cholesterol: Desirable level: <200 mg/dl
– Serum triglycerides: Desirable level: <150 mg/dl
– HDL cholesterol: ≥60 mg/dl
– LDL cholesterol: <130 mg/dl
– LDL/HDL ratio: 0.5-3.0
- C-peptide: 0.78-1.89 ng/ml
- Arterial pH: 7.35-7.45
- Serum or plasma osmolality: 275-295 mOsm/kg of water.
Serum Osmolality can also be calculated by the following formula recommended by American Diabetes Association:
- Anion gap:
– Na+ – (Cl– + HCO3–): 8-16 mmol/L (Average 12)
– (Na+ + K+) – (Cl– + HCO3–): 10-20 mmol/L (Average 16)
- Serum sodium: 135-145 mEq/L
- Serum potassium: 3.5-5.0 mEq/L
- Serum chloride: 100-108 mEq/L
- Serum bicarbonate: 24-30 mEq/L
- 16 Aug 2017
Pregnancy tests detect human chorionic gonadotropin (hCG) in serum or urine. Although pregnancy is the most common reason for ordering the test for hCG, measurement of hCG is also indicated in other conditions as shown in Box 836.1.
Human chorionic gonadotropin is a glycoprotein hormone produced by placenta that circulates in maternal blood and excreted intact by the kidneys. It consists of two polypeptide subunits: α (92 amino acids) and β (145 amino acids) which are non-covalently bound to each other. Structurally, hCG is closely related to three other glycoprotein hormones, namely, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). The α subunits of hCG, LH, FSH, and TSH are similar, while β subunits differ and confer specific biologic and immunologic properties. Immunological tests use antibodies directed against β-subunit of hCG to avoid cross-reactivity against LH, FSH, and TSH.
Box 836.1 Indications for measurement of β human chorionic gonadotropin
• Early diagnosis of pregnancy
• Diagnosis and management of gestational trophoblastic disease
• As a part of maternal triple test screen
Syncytiotrophoblastic cells of conceptus and later of placenta synthesize hCG. Human chorionic gonadotropin supports the corpus luteum of ovary during early pregnancy. Progesterone, produced by corpus luteum, prevents ovulation and thus maintains pregnancy. After 7-10 weeks of gestation, sufficient amounts of progesterone are synthesized by placenta, and hCG is no longer needed and its level declines.
CLINICAL APPLICATIONS OF TESTS FOR HUMAN CHORIONIC GONADOTROPIN
- Early diagnosis of pregnancy: Qualitative serum hCG test becomes positive 3 weeks after last menstrual period (LMP), while urine hCG test becomes positive 5 weeks after LMP.
- Exclusion of pregnancy before prescribing certain medications (like oral contraceptives, steroids, some antibiotics), and before ordering radiological studies, radiotherapy, or chemotherapy. This is necessary to prevent any teratogenic effect on the fetus.
- Early diagnosis of ectopic pregnancy: Trans-vaginal ultrasonography (USG) and quantitative estimation of hCG are helpful in early diagnosis of ectopic pregnancy (before rupture).
- Evaluation of threatened abortion: Serial quantitative estimation of hCG is helpful in following the course of threatened abortion.
- Diagnosis and follow-up of gestational trophoblastic disease (GTD).
- Maternal triple test screen: This consists of measurement of hCG, α-fetoprotein, and unconjugated estriol in maternal serum at 14-19 weeks of gestation. The maternal triple screen identifies pregnant women with increased risk of Down syndrome and major congenital anomalies like neural tube defects.
- Follow-up of ovarian or testicular germ cell tumors, which produce hCG.
In women with normal menstrual cycle, conception (fertilization of ovum to form a zygote) occurs on day 14 in the fallopian tube. Zygote travels down the fallopian tube into the uterus. Division of zygote produces a morula. At 50-60-cell stage, morula develops a primitive yolk sac and is then called as a blastocyst. About 5 days after fertilization, implantation of blastocyst occurs in the uterine wall. Trophoblastic cells (on the outer surface of the blastocyst) penetrate the endometrium and develop into chorionic villi. There are two main forms of trophoblasts—syncytiotrophoblast and cytotrophoblast. Placental development occurs from chorionic villi. After formation of placenta, the conceptus is called as an embryo. When embryo develops most major organs, it is called as fetus (after 10 weeks of gestation).
Box 836.2 Diagnosis of early pregnancy
• Positive serum hCG test: 8 days after conception or 3 weeks after last menstrual period (LMP)
• Positive urine hCG test: 21 days after conception or 5 weeks after LMP
• Ultrasonography for visualization of gestational sac:
– Transvaginal: 21 days after conception or 5 weeks after LMP
– Transabdominal: 28 days after conception or 6 weeks after LMP
Human chorionic gonadotropin is synthesized by syncytiotrophoblasts (of placenta) and detectable amounts (~5 mIU/ml) appear in maternal serum about 8 days after conception (3 weeks after LMP). In the first trimester (first 12 weeks, calculated from day 1 of LMP) of pregnancy, hCG levels rapidly rise with a doubling time of about 2 days. Highest or peak level is reached at 8-10 weeks (about 100,000 mIU/ml). This is followed by a gradual fall, and from 15-16 weeks onwards, a steady level of 10,000-20,000 mIU/ml is maintained for the rest of the pregnancy (Figure 836.1). After delivery, hCG becomes non-detectable by about 2 weeks.
Box 836.2 shows minimum time required for the earliest diagnosis of pregnancy by hCG test and ultrasonography (USG).
Two types of pregnancy tests are available:
- Qualitative tests: These are positive/negative result types that are done on urine sample.
- Quantitative tests: These give numerical result and are done on serum or urine. They are also used for evaluation of ectopic pregnancy, failing pregnancy, and for follow-up of gestational trophoblastic disease.
Ectopic pregnancy refers to the implantation of blastocyst at a site other than the cavity of uterus. The most common of such sites (>95% cases) is fallopian tube. Early diagnosis and treatment of tubal ectopic pregnancy is essential since it can lead to maternal mortality (from rupture and hemorrhage) and future infertility. Ectopic pregnancy is a leading cause of maternal death during first trimester. Diagnosis of ectopic pregnancy can be readily made in most cases by ultrasonography and estimation of β-subunit of human chorionic gonadotropin.
Early diagnosis of unruptured tubal pregnancy can be made by quantitative estimation of serum hCG and ultrasonography. In normal intrauterine pregnancy, hCG titer doubles every 2 days until first 40 days of gestation. If hCG rise is abnormally slow, then an unviable pregnancy (either ectopic or abnormal intrauterine pregnancy) should be suspected.
Transabdominal USG can detect gestational sac in intrauterine pregnancy 6 weeks after LMP. The level of hCG in serum at this stage is >6500 mIU/ml. If gestational sac is not visualized at this level of hCG, then there is a possibility of ectopic pregnancy. Transvaginal ultrasonography can detect ectopic pregnancy average 1 week earlier than abdominal ultrasonography; it can detect gestational sac if β-hCG level is 1000-1500 mIU/ml. Therefore, if gestational sac is not visualized in the presence of >1500 mIU/ml of β-hCG level, an ectopic pregnancy can be suspected.
Early diagnosis of ectopic pregnancy provides the option of administration of intramuscular methotrexate (rather than surgery), which causes dissolution of conceptus. This improves the chances of patient’s future fertility. Serial measurements of hCG after surgical removal of ectopic pregnancy can help in detecting persistence of trophoblastic tissue.
Termination of pregnancy before fetus becomes viable (i.e. before 20 weeks) is called as abortion.
In threatened abortion, vaginal bleeding is present but internal os is closed and process of abortion, though started, is still reversible. It is possible that pregnancy will continue.
Serial quantitative titers of hCG showing lack of expected doubling of hCG level and USG are helpful in diagnosis and management of abortion.
Gestational Trophoblastic Disease (GTD)
It is characterized by proliferation of pregnancyassociated trophoblastic tissue. The two main forms of GTD are hydatidiform (vesicular) mole (benign) and choriocarcinoma (malignant). Clinical features of GTD are as follows:
- Short history of amenorrhea followed by vaginal bleeding.
- Size of uterus larger than gestational age; uterus is soft and doughy on palpation with no fetal parts and no fetal heart sounds.
- Excessive nausea and vomiting due to high hCG.
- Characteristic snowstorm appearance on pelvic USG.
Quantitative estimation of hCG is helpful in diagnosis and management of GTD.
Trophoblastic cells of GTD produce more hCG as compared to the trophoblasts of normal pregnancy for the same gestational age. Concentration of hCG parallels tumor load. Also, hCG continues to rise beyond 10 weeks of gestation without reaching plateau (as expected at the end of first trimester).
After evacuation of uterus, weekly estimation of hCG is advised till subsequent three (weekly) results are negative; following evacuation of vesicular mole, hCG becomes undetectable (after 2-3 months) on follow-up in 80% of cases. Plateau or rising hCG indicates persistent GTD. In such cases, chemotherapy is indicated.
Negative results for hCG after therapy should be regularly followed up every 3 months for 1-2 years.
LABORATORY TESTS FOR HUMAN CHORIONIC GONADOTROPIN
These are classified into two main groups:
- Biological assays or bioassays
- Immunological assays
In bioassay, effect of hCG is tested on laboratory animals under standardized conditions. There are several limitations of bioassays like need for animal facilities, need for standardization of animals, long time required for the test results, low sensitivity, and high cost. Therefore, bioassays have been replaced by immunological assays.
In Ascheim-Zondek test, urine from pregnant woman is injected into immature female mice. Formation of hemorrhagic corpora lutea in ovaries (after 4 days) is a positive test. Friedman test is similar except that urine is injected into female rabbit. In rapid rat test, injection of urine containing hCG into female rats is followed by hyperaemia and hemorrhage in ovaries. Yet another test measures release of spermatozoa from male frog after injection of urine containing hCG.
These are rapid and sensitive tests for detection and quantitation of hCG. Variable results are obtained by different immunological tests with the same serum sample; this is due to differences in specificity of different immunoassays to complete hCG, β-subunit, and β-core fragment. A number of immunological tests are commercially available based on different principles like agglutination inhibition assay, enzyme immunoassay including enzyme linked immunosorbent assay or ELISA, radioimmunoassay (RIA), and immunoradiometric assay.
A commonly used qualitative urine test is agglutination inhibition assay. Early morning urine specimen is preferred because it contains the highest concentration of hCG. Causes of false-positive test include red cells, leukocytes, bacteria, some drugs, proteins, and excess luteinizing hormone (menopause, midcycle LH surge) in urine. Some patients have anti-mouse antibodies (that are used in the test), while others have hCG-like material in circulation, producing false-positive test. Anti-mouse antibodies also interfere with other antibody-based tests and are known as ‘heterophil’ antibodies. Fetal death, abortion, dilute urine, and low sensitivity of a particular test are causes of false-negative test. Renal failure leads to accumulation of interfering substances causing incorrect results.
In latex particle agglutination inhibition test (Figure 836.2), anti-hCG antibodies are incubated with patient’s urine. This is followed by addition of hCGcoated latex particles. If hCG is present in urine, anti-hCG serum is neutralized, and no agglutination of latex particles occurs (positive test). If there is no hCG in urine, there is agglutination of latex particles (negative test). This is commonly used as a slide test and requires only a few minutes.
Sensitivity of agglutination inhibition test is >200 units/liter of hCG.
Radioimmunoassay, enzyme immunoassay, and radioimmunometric assay are more sensitive and reliable than agglutination inhibition assay.
Quantitative tests are employed for detection of very early pregnancy, estimation of gestational age, diagnosis of ectopic pregnancy, evaluation of threatened abortion, and management of GTD.
- Serum human chorionic gonadotropin:
– Non-pregnant females: <5.0 mIU/ml
– Pregnancy: 4 weeks after LMP: 5-100 mIU/ml
– 5 weeks after LMP: 200-3000 mIU/ml
– 6 weeks after LMP: 10,000-80,000 mIU/ml
– 7-14 weeks: 90,000-500,000 mIU/ml
– 15-26 weeks: 5000-80000 mIU/ml
– 27-40 weks: 3000-15000 mIU/ml
Further Reading: SEMEN ANALYSIS FOR INVESTIGATION OF INFERTILITY
- 15 Aug 2017
Box 835.1 Contributions to semen volume
• Testes and epididymis: 10%
• Seminal vesicles: 50%
• Prostate: 40%
• Cowper’s glands: Small volume
- Testes: Male gametes or spermatozoa (sperms) are produced by testes; constitute 2-5% of semen volume.
- Epididymis: After emerging from the testes, sperms are stored in the epididymis where they mature; potassium, sodium, and glycerylphosphorylcholine (an energy source for sperms) are secreted by epididymis.
- Vas deferens: Sperms travel through the vas deferens to the ampulla which is another storage area. Ampulla secretes ergothioneine (a yellowish fluid that reduces chemicals) and fructose (source of nutrition for sperms).
- Seminal vesicles: During ejaculation, nutritive and lubricating fluids secreted by seminal vesicles and prostate are added. Fluid secreted by seminal vesicles consists of fructose (energy source for sperms), amino acids, citric acid, phosphorous, potassium, and prostaglandins. Seminal vesicles contribute 50% to semen volume.
- Prostate: Prostatic secretions comprise about 40% of semen volume and consist of citric acid, acid phosphatase, calcium, sodium, zinc, potassium, proteolytic enzymes, and fibrolysin.
- Bulbourethral glands of Cowper secrete mucus.
|1. Volume||≥2 ml|
|2. pH||7.2 to 8.0|
|3. Sperm concentration||≥20 million/ml|
|4. Total sperm count per ejaculate||≥40 million|
|5. Morphology||≥30% sperms with normal morphology|
|6. Vitality||≥75% live|
|7. White blood cells||<1 million/ml|
|8. Motility within 1 hour of ejaculation|
|• Class A||≥25% rapidly progressive|
|• Class A and B||≥50% progressive|
|9. Mixed antiglobuiln reaction (MAR) test||<50% motile sperms with adherent particles|
|10. Immunobead test||<50% motile sperms with adherent particles|
|1. Total fructose (seminal vesicle marker)||≥13 μmol/ejaculate|
|2. Total zinc (Prostate marker)||≥2.4 μmol/ejaculate|
|3. Total acid phosphatase (Prostate marker)||≥200U/ejaculate|
|4. Total citric acid (Prostate marker)||≥52 μmol/ejaculate|
|5. α-glucosidase (Epididymis marker)||≥20 mU/ejaculate|
|6. Carnitine (Epididymis marker)||0.8-2.9 μmol/ejaculate|
|Box 835.2 Tests done on seminal fluid
• Physical examination: Time to liquefaction, viscosity, volume, pH, color
• Microscopic examination: Sperm count, vitality, motility, morphology, and proportion of white cells
• Immunologic analysis: Antisperm antibodies (SpermMAR test, Immunobead test)
• Bacteriologic analysis: Detection of infection
• Biochemical analysis: Fructose, zinc, acid phosphatase, carnitine.
• Sperm function tests: Postcoital test, cervical mucus penetration test, Hamster egg penetration assay, hypoosmotic swelling of flagella, and computer-assisted semen analysis
- Investigation of infertility: Semen analysis is the first step in the investigation of infertility. About 30% cases of infertility are due to problem with males.
- To check the effectiveness of vasectomy by confirming absence of sperm.
- To support or disprove a denial of paternity on the grounds of sterility.
- To examine vaginal secretions or clothing stains for the presence of semen in medicolegal cases.
- For selection of donors for artificial insemination.
- For selection of assisted reproductive technology, e.g. in vitro fertilization, gamete intrafallopian transfer technique.
|Box 835.3 Semen analysis for initial investigation of infertility
• Microscopic examination for (i) percentage of motile spermatozoa, (ii) sperm count, and (iii) sperm morphology
| Box 835.4 Terminology in semen analysis
• Normozoospermia: All semen parameters normal
• Oligozoospermia: Sperm concentration <20 million/ml (mild to moderate: 5-20 million/ml; severe: <5 million/ml)
• Azoospermia: Absence of sperms in seminal fluid
• Aspermia: Absence of ejaculate
• Asthenozoospermia: Reduced sperm motility; <50% of sperms showing class (a) and class (b) type of motility OR <25% sperms showing class (a) type of motility.
• Teratozoospermia: Spermatozoa with reduced proportion of normal morphology (or increased proportion of abnormal forms)
• Leukocytospermia: >1 million white blood cells/ml of semen
• Oligoasthenoteratozoospermia: All sperm variables are abnormal
• Necrozoospermia: All sperms are non-motile or non-viable
- PHYSICAL EXAMINATION OF SEMEN FOR INVESTIGATION OF INFERTILITY
- BIOCHEMICAL ANALYSIS OF SEMEN FOR INVESTIGATION OF INFERTILITY
- MICROSCOPIC EXAMINATION OF SEMEN FOR INVESTIGATION OF INFERTILITY
- IMMUNOLOGIC ANALYSIS OF SEMEN FOR INVESTIGATION OF INFERTILITY
- SPERM FUNCTION TESTS OR FUNCTIONAL ASSAYS
- EXAMINATION FOR THE PRESENCE OF SEMEN IN MEDICOLEGAL CASES
- 15 Aug 2017
Atleast 200 motile spermatozoa should be counted. If >50% of spermatozoa show attached latex particles, immunological problem is likely.
- Mix one drop of semen with 1 drop of eosin-nigrosin solution and incubate for 30 seconds.
- A smear is made from a drop placed on a glass slide.
- The smear is air-dried and examined under oilimmersion objective. White sperms are classified as live or viable, and red sperms are classified as dead or non-viable. At least 200 spermatozoa are examined.
- The result is expressed as a proportion of viable sperms against non-viable as an integer percentage.
- Semen is diluted 1:20 with sodium bicarbonateformalin diluting fluid (Take 1 ml liquefied semen in a graduated tube and fill with diluting fluid to 20 ml mark. Mix well).
- A coverslip is placed over the improved Neubauer counting chamber and the counting chamber is filled with the well-mixed diluted semen sample using a Pasteur pipette. The chamber is then placed in a humid box for 10-15 minutes for spermatozoa to settle.
- The chamber is placed on the microscope stage. Using the 20× or 40× objective and iris diaphragm lowered sufficiently to give sufficient contrast, number of spermatozoa is counted in 4 large corner squares. Spermatozoa whose heads are touching left and upper lines of the square should be considered as ‘belonging’ to that square.
- Sperm count per ml is calculated as follows:
Sperm count = Sperms counted × correction factor × 1000
Number of squares counted × Volume of 1 square
= Sperms counted × 20 1000
4 × 0.1
= Sperms counted × 50, 000
- Normal sperm count is ≥ 20 million/ml (i.e. ≥ 20 × 106/ml). Sperm count < 20 million/ml may be associated with infertility in males.
• Total length of sperm: About 60 μ
• Total length of sperm: About 60 μ
– Length: 3-5 μ
– Width: 2-3 μ
– Thickness: 1.5 μ
• Neck: Length: 0.3 μ
• Middle piece:
– Length: 3-5 μ
– Width: 1.0 μ
• Principal piece:
– Length: 40-50 μ
– Width: 0.5 μ
• End piece: 4-6 μ
- Normal sperm
- Defects in head:
• Large heads
• Small heads
• Tapered heads
• Pyriform heads
• Round heads
• Amorphous heads
• Vacuolated heads (> 20% of the head area occupied by vacuoles)
• Small acrosomes (occupying < 40% of head area)
• Double heads
- Defects in neck:
• Bent neck and tail forming an angle >90° to the long axis of head
- Defects in middle piece:
• Asymmetric insertion of midpiece into head
• Thick or irregular midpiece
• Abnormally thin midpiece
- Defects in tail:
• Bent tails
• Short tails
• Coiled tails
• Irregular tails
• Multiple tails
• Tails with irregular width
- Pin heads: Not to be counted
- Cytoplasmic droplets
• > 1/3rd the size of the sperm head
- Precursor cells: Considered abnormal
|1. Total fructose (seminal vesicle marker)||≥13 μmol/ejaculate|
|2. Total zinc (Prostate marker)||≥2.4 μmol/ejaculate|
|3. Total acid phosphatase (Prostate marker)||≥200U/ejaculate|
|4. Total citric acid (Prostate marker)||≥52 μmol/ejaculate|
|5. α-glucosidase (Epididymis marker)||≥20 mU/ejaculate|
|6. Carnitine (Epididymis marker)||0.8-2.9 μmol/ejaculate|
- Vagina: Direct aspiration or saline lavage
- Clothing: When scanned with ultraviolet light, semen produces green white fluorescence. A small piece (1 m2) of clothing from stained portion is soaked in 1-2 ml of physiologic saline for 1 hour. A similar piece of clothing distant from the stain is also soaked in saline as a control.
- 13 Aug 2017
1. MICROSCOPIC EXAMINATION OF URINARY SEDIMENT
Definition of microscopic hematuria is presence of 3 or more number of red blood cells per high power field on microscopic examination of urinary sediment in two out of three properly collected samples. A small number of red blood cells in urine of low specific gravity may undergo lysis, and therefore hematuria may be missed if only microscopic examination is done. Therefore, microscopic examination of urine should be combined with a chemical test.
2. CHEMICAL TESTS
These detect both intracellular and extracellular hemoglobin (i.e. intact and lysed red cells) as well as myoglobin. Heme proteins in hemoglobin act as peroxidase, which reduces hydrogen peroxide to water. This process needs a hydrogen donor (benzidine, orthotoluidine, or guaiac). Oxidation of hydrogen donor leads to development of a color (Figure 828.1). Intensity of color produced is proportional to the amount of hemoglobin present.
Chemical tests are positive in hematuria, hemoglobinuria, and myoglobinuria.
Make saturated solution of benzidine in glacial acetic acid. Mix 1 ml of this solution with 1 ml of hydrogen peroxide in a test tube. Add 2 ml of urine. If green or blue color develops within 5 minutes, the test is positive.
In this test, instead of benzidine, orthotoluidine is used. It is more sensitive than benzidine test.
Reagent Strip Test
Various reagent strips are commercially available which use different chromogens (o-toluidine, tetramethylbenzidine).
Causes of false-positive tests:
- Contamination of urine by menstrual blood in females
- Contamination of urine by oxidizing agent (e.g. hypochlorite or bleach used to clean urine containers), or microbial peroxidase in urinary tract infection.
Causes of false-negative tests:
- Presence of a reducing agent like ascorbic acid in high concentration: Microscopic examination for red cells is positive but chemical test is negative.
- Use of formalin as a preservative for urine
Evaluation of positive chemical test for blood is shown in Figure 828.2.