Sputum examination refers to the laboratory examination or test of the material or substance coughed out from the lungs, bronchi, trachea, and larynx. Normally, sputum is mainly composed of mucus and also certain cellular and non-cellular components of host origin. During expectoration, sputum gets contaminated with normal bacterial flora and cells from pharynx and mouth.

Examination of sputum is mainly carried out for:

• Identification of causative agent or organism associated with a particular suspected infection of the lower respiratory tract, e.g.
  – Suspected tuberculosis
  – Pneumonia especially if severe or in an immunocompromised host
  – Pneumocystic carinii pneumonia in HIV-positive patients
  – Suspected fungal infection
  – Infective exacerbation of a chronic disease like bronchiectasis
• Cytological examination for the investigation of viral infections (viral inclusions in cytomegalovirus and herpes simplex infections), fungal infection, asbestosis and malignant cells.

COLLECTION OF SPUTUM

1. Sputum sample is ideally collected in the morning (since secretions accumulate overnight), soon after awakening and before taking any mouthwash or food.
2. Sputum sample is collected in a sterile, clean, dry and wide-mouthed plastic container with a securely fitting screw cap. The container should be of unbreakable or break-resistant plastic and leak-proof to prevent desiccation and aerosol formation, and should have the capacity of about 30 ml.
3. The patient is advised to take a deep breath 2-3 times filling his/her lungs, coughs deeply, and spit into the plastic container. About 2-5 ml of sputum is collected. Sample consisting the only of saliva (watery appearance, clear, and foamy) is not acceptable for laboratory investigations; in such case, another sample should be collected. The container, containing sputum sample, is caped securely and labeled properly.

Induction of Sputum

If the patient is not able to expectorate the sputum spontaneously, inhaling aerosol of 15% sodium chloride (NaCl) and 20% propylene glycol (C₃H₈O₂) for 20 minutes can induce expectoration. Sputum can also be induced by inhaling distilled water in association with chest physiotherapy or by inhaling nebulized hypertonic saline.

For microbiological examination of sputum, sample should be sent to the laboratory immediately. If sputum is allowed to stand, rapid reproduction of contaminating bacterial flora from the throat and oral cavity will occur leading to incorrect results. In inclusion, pathogenic organism, especially Haemophilus influenzae, do not survive for a long time in the collected sample. Sputum sample for bacterial culture should not be refrigerated.

If the sample is to be transported to a remote laboratory for mycobacterial culture, sputum should be collected in 25 ml of the following solution:

• N-acetylpyridinium chloride 5 gm
• Sodium chloride 10 gm
• Distilled water 1000 ml

APPEARANCE OF SPUTUM

Physical appearance of sputum is often indicative and symptomatic of the underlying pathologic process as follows:

• Bloody: Hemoptysis (pulmonary tuberculosis, bronchogenic carcinoma, bronchiectasis, lung abscess, pulmonary infarction, mitral stenosis)
• Bloody and gelatinous (red current jelly): Klebsiella pneumonia
• Rusty: Pneumococcal lobar pneumonia
• Purulent and separating into 3 layers on standing: Lung abscess, bronchiectasis
• Copious amounts of purulent sputum: Bronchopleural fistula, lung abscess, bronchiectasis
• Green: Pseudomonas infection
• Pink, frothy (air bubbles): Pulmonary edema

MICROBIOLOGICAL EXAMINATION OF SPUTUM

Sputum sample is usually adulterated and contaminated with normal flora of the pharynx and oral cavity. Normal flora found in the pharynx and oral cavity are listed below.

• Gram-positive microorganisms: Diptheroids, streptococci (S. pneumoniae, S. viridans), staphylococci (S. epidermidis, S. aureus), lactobacilli, enterococci, Yeasts (Candida spp.), micrococci.
• Gram-negative microorganisms: Coliforms, Haemophilus spp; Neisseria spp; Moraxella catarrhalis, fusobacteria.

Gram staining

Pathogenic organisms found in sputum include—

• Gram-positive: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae.
• Gram-negative:  Klebsiella pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, Yersinia pestis, Pseudomonas aeruginosa.

For bacteriological examination of sputum, sample should be processed in the laboratory within an hour our collection. A small amount of sputum is transferred to a sterile Petri dish and its physical appearance is noted. From the purulent portion of the sputum, a thin smear is made on the grease-free sterile glass slide with a clean stick. The slide is air-dried, fixed and stained with Gram's stain. Entirely watery, mucoid, white, or frothy samples often show squamous epithelial cells covered with the bunches of bacteria; this phenomenon indicates that the sample consists mainly of secretions from the mouth and the throat. Such samples are not acceptable for bacteriological examination (see Figure 989.1). Culture is not carried out if polymorphonuclear neutrophils are less than 10 per epithelial cell.

Because of the presence of various contaminating Gram-positive and Gram-negative microorganism deriving from throat and mouth (normal bacterial flora), Gram-stained smear of sputum should be elucidated carefully.

Morphological appearance of bacterial cells on Gram stained smear is redolent of a particular microorganism as follows:

• Gram-negative diplococci, both intra- and extracellular: Moraxella catarrhalis.
• Gram-positive yeast cells with budding and pseudohyphae: Candida.
• Gram-positive diplococci with surrounding clear space (capsule): S. pneumoniae (see Figure 989.2).
• Gram-negative coccobacilli: H. influenzae.
• Gram-positive cocci in grape-like clusters: S. aureus.
• Large granules with center gram-negative and periphery gram-positive: Actinomyces.

Bacteriological Culture

Culture media is inoculated with a floccule of the purulent portion of sputum for absolute identification of microorganism. Sputum sample is considered as unsuitable for the bacterial culture if it contains >25 squamous epithelial cells/low power field. An ideal sputum sample for bacterial culture contains bronchial epithelial cells, numerous neutrophils (>5/high power field), alveolar macrophages, and few squamous epithelial cells (<10/high power field). Saliva is washed away from sputum with sterile normal saline in order to reduce the amount of contaminating normal bacterial flora in the inoculum. Blood agar plate and chocolate agar (heated blood agar) are inoculated with the washed sputum. The chocolate agar plate is incubated in an atmosphere of extra carbon dioxide (CO₂) and blood agar plate is incubated aerobically. After the incubation for 18 hours, inoculated agar plates are examined for growth; if growth is not sufficient, incubation for further 24 hours is indicated. Antibiotics sensitivity test is carried out only if the amount of bacterial growth is significant.

EXAMINATION OF SPUTUM FOR MYCOBACTERIUM TUBERCULOSIS

Tuberculosis is a crucial public health problem in Pakistan and India. Early diagnosis of pulmonary tuberculosis will lead to early induction of treatment facilitating cure, and also inhibiting the spread of disease to others. In recent times, the number of cases of tuberculosis has increased. World Health Organization called it a global emergency. Multi-drug resistant tuberculosis is also araising on large scale.

Mycobacterium tuberculosis complex comprises of M. tuberculosis, M. africanum, and M. bovis. These tubercle bacilli are the etiologic agents of tuberculosis in the human being. Other mycobacteria are called as non-tuberculosis.

There are two main approaches for the identification tuberculosis.

1. Direct test: This involves detection of M. tuberculosis or its components
2. Indirect test: This consists of detection of cellular or humoral immune response to tuberculosis infection.

Direct tests for the detection of tuberculosis on sputum sample are as follows:

1. Examination of sputum smear
   – Ziehl-Neelsen technique
   – Fluorescence microscopy
2. Molecular Method
3. Culture on standard media
4. Commercial automated culture system

Examination of Sputum Smear

For detection of M. tuberculosis, minimum three sputum samples collected on three different occasions (including at least one early morning sputum sample) need to be examined. A thin sputum smear is prepared on clean, sterile, grease-free glass slide from a yellowish, grayish, opaque, or blood-tinged portion of sputum. Children often ingest sputum and may be unable to cough it up; in such condition sample of fasting gastric juice can be aspirated and examined like sputum.

The smear is stained with Ziehl-Neelsen stain and examined under oil immersion lens in an ordinary light microscope. If the fluorescent microscope is available, smear can be examined after staining it with a fluorochrome (auramine O or auramine-rhodamine).

Ziehl-Neelsen stain of sputum smear: This technique is very simple, rapid and inexpensive. This technique is mainly used for:

• Diagnosis of pulmonary tuberculosis. (A positive sputum smear cases are the major source of spread of infection).
• Determining cure or treatment failure.
• Evaluation of response to anti-tuberculosis treatment.

Ziehl-Neelsen-stained sputum smear is considered as positive if 5000-10000 tubercle bacilli/ml are present in the sputum. Sensitivity of the technique is reported to be 60-80%. Possibilities of detection of tubercle bacilli are increased if multiple sputum samples are examined or if bleach concentration technique is used. In bleach concentration technique, a solution of concentrated sodium hypochlorite (NaOCl) is added to the sputum sample, which causes the liquefaction of mucus and killing of mycobacteria. The sample is kept for overnight sedimentation (or centrifugation), from the sediment of sputum a thin smear is prepared, stained and examined.

With Ziehl-Neelsen staining, mycobacteria apear as bright red straight or slightly curved orb roads (0.2-0.5 μ in width and 2-4 μ in length) against a green or blue background (see Figure 989.3). Mycobacteria, both acid- and alcohol-fast are termed as acid-fast bacilli (AFB). Minimum 100 fields are examined before reporting the smear as negative. If acid-fast bacilli are seen, their number should be reported.

A negative sputum smear does not rule out the diagnosis of tuberculosis since smear may be of poor quality or organisms may be small in number, or sputum sample may not have been collected properly. See also: Acid-Fast Staining: Purpose, Principle, Procedure, and Observation

Fluorescence microscopy: A thin sputum smear is prepared and stained with a fluorochrome (auramine O or auramine-rhodamine). The smear is examined under fluorescence microscope. Mycobacteria appear as bright yellow against a dark background (see Figure 989.4). This technique is very simple and rapid (since the sputum smear is examined under low power lens) and this technique is very useful if the organisms are few in numbers. It is very necessary to confirm a positive sputum smear with Ziehl-Neelsen stain since there is a high rate of false-positive result.

Molecular Method

There are two different methods for the molecular diagnosis of tuberculosis in sputum samples:

• Detection of Mycobacterium tuberculosis in isolates from the culture by nucleic acid probes.
• Direct detection of Mycobacterium tuberculosis in sputum sample.

M. tuberculosis can be rapidly detected directly in sputum samples by identifying DNA sequences specific to it. In M. tuberculosis complex, IS 6110 is the targeted DNA because it is only observed in M. tuberculosis complex. In the genome of M. tuberculosis, multiple copies of this sequence are present. By this method, 10-1000 organisms per ml of sputum can be detected. Other DNA and RNA sequences precise for M. tuberculosis complex can also be targeted.

Laboratory cross-contamination (due to aerosolized PCR products) is also responsible for unreliable and false-positive results. PCR amplifies DNA sequences of both dead and live bacilli, for that reason the test cannot be used to evaluate response to therapy. This test is also expensive. PCR-based assays should be elucidated in the light of clinical features, findings on Ziehl-Neelsen sputum smear and presence of tuberculosis in other family members.

Sputum Culture (Conventional)

For the definitive diagnosis of tuberculosis, pure culture technique is used. M. tuberculosis is isolated from the culture of sputum sample. Sputum culture is usually carried out for:

• Identification of a particular species, if organism other than M. tuberculosis is suspected (for the purpose of incidence, distribution, and control of diseases).
• Drug susceptibility testing.
• Diagnosis in patients who have distinctive radiological and clinical features of tuberculosis but are sputum smear-negative.

Sputum culture is more sensitive as compared to sputum smear examination. It can detect 10 to 100 microorganism in per ml of sputum sample. Its sensitivity for the identification of tuberculosis is 80-85% and specificity is 98%. However, this procedure is very expensive but reliable, around 6 weeks are needed for the result and even longer for the drug susceptibility testing, and earlier decontamination of sputum is required to kill normal bacterial flora.

Contaminating bacteria grows rapidly and digest the culture medium prior tubercle bacilli begin to grow. Therefore, it is necessary to decontaminate the sputum sample by adding 4% sodium hydroxide (used as decontaminating agent).

Standard culture media for the isolation of M. tuberculosis are:

• Solid media: Agar-based (Middlebrook 7H10 or 7H11) or egg-based (Lowenstein-Jensen medium).
• Liquid media: Middlebrook 7H9, Middlebrook 7H12.

The most common solid medium used for the culture is Lowenstein-Jensen medium. Up to 6 weeks are required for the visible mycobacterial growth. For the identification of species, further biochemical tests are performed.

Commercial Automated Culture Systems

Nowadays, rapid automated culture systems are available commercially which can give results within two weeks (instead of six weeks with standard media). However, this procedure is expensive. Examples of such systems are BACTEC™ 460TB system (see Figure 989.5) and BACTEC™ 9050 automatic blood culture analyzer (Becton-Dickinson Diagnostic Instruments Systems, Maryland, USA). These instruments are very sensitive and can detect M. tuberculosis in clinical samples. In this method, broth is used in which radiolabelled ¹⁴C-palmitate has been integrated. Mycobacteria metabolize ¹⁴C-palmitate to radiolabelled ¹⁴CO₂, which is further detected by the instrument.

EXAMINATION OF SPUTUM FOR ORGANISMS OTHER THAN TUBERCLE BACILLI

Further interpretations given below are demonstrated when infection by following organisms is suspected:

• Paragonimus: Saline wet mount of sputum for eggs.
• Histoplasmosis: Giemsa smear.
• Pneumocystis carinii: Bronchoalveolar lavage fluid stained with Giemsa stain and silver stain (see Figure 989.6).
• Yersinia pestis (pneumonic plague): Giemsa smear.
• Aspergillus: Potassium hydroxide wet mount of sputum.
• Yeast-like organisms on Gram’s smear: Sabouraud dextrose agar.

CYTOLOGICAL EXAMINATION OF SPUTUM

Cytological examination of sputum is normally carried out for the diagnosis of bronchogenic carcinoma. Occasionally, it may also be useful in the identification of fungi, protozoa, asbestos bodies and viral inclusions (like those of cytomegalovirus and Herpes simplex virus).

For cytological examination, early morning sputum sample is preferred. For the diagnosis of lung cancer, it is suggested to collect sputum sample daily for first five successive days. This method will increase the chances of detection of malignant cells (see Figure 989.7).

Sputum sample may be either spontaneously produced or artificially induced. If the patient is not able to expectorate the sputum spontaneously, inhaling aerosol of 15% sodium chloride (NaCl) and 20% propylene glycol (C₃H₈O₂) for 20 minutes can induce expectoration. This normally results in the induction of sufficient sputum sample.

The sputum sample should be sent to the laboratory just after the collection without the addition of any fixative. If the sample is to be transported to a remote laboratory, prefixation of sputum with Saccomano’s fixative is recommended. This involves collection of sputum in a mixture of 2% carbowax and 50% ethyl alcohol.

In the laboratory, a thin sputum smear is prepared on clean, sterile, grease-free glass slide from a yellowish, grayish, opaque, or blood-tinged portion, or from tissue fragments in sputum and stained with Papanicolaou technique. The sputum sample is considered as adequate for cytological examination bronchial epithelial cells or alveolar macrophages are seen in the smear.

The average sensitivity is about 65% in sputum examination for detection of malignant cells. Sensitivity increases as per following conditions:

• Size of tumor is large.
• Lesion is centrally located rather than at the periphery of the lung.
• Histologic type of carcinoma is of squamous nature rather than adenocarcinoma or small cell carcinoma.
• Increased numbers of sputum samples are examined.

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

Bacteriologic examination

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.

Figure 988.1 Classification and causes of acute diarrhea

Evaluation of dysentery

Differentiate between bacillary dysentery and amebic dysentery is done by the identification of the causative organism in the stool. See also: Microscopic Examination of Feces

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 examination

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.

1. Acute myeloid leukemia (AML)
2. Acute lymphoblastic leukemia (ALL)

Each type is further subclassified. See table 883.1

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.

Difference between Acute Myelocytic Leukemia (AML) and Acute Lymphocytic Leukemia (ALL)

Sign and symptoms of acute leukemia

1. Weakness and fatigue
2. Low-grade fever
3. Bone pain
4. Bruising and mild bleeding from gums
5. It appears suddenly

Diagnosis of acute leukemia

1. This disease is most common in younger age group and more frequent in the children. Mostly found before the age of 20.
2. 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.
3. Leucocytes (WBCs) count is variable. Mostly leucocytes count is less than 100000/μl.
4. 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)

1. 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.
2. Abnormal and immature WBCs are present.
3. In 50% of cases, the value of uric acid is raised.
4. In bone marrow examination, > 20% blast cells are present. Promyelocytes are numerous especially in M3 (acute promyelocytic leukemia). See table 883.1
5. Cytochemical stains, Sudan black, and peroxidase show the positive reaction.
6. Auer rods can be seen in the cytoplasm of the cells.
7. Nucleated red blood cells may be seen.
8. PT, PTT and Thrombin time are elevated.
9. Thrombocytopenia may be present and may see the platelets count between 30,000 to 100,000/μl.

Laboratory findings in acute lymphocytic leukemia (ALL)

1. Total leucocyte count is variable from low to very high.
2. Anemia and thrombocytopenia
3. 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:

1. If the patient has high TLC.
2. If the patient has Anemia.
3. If the patient has enlarged lymph nodes.
4. If the patient has weakness and fever.

Why are these tests performed?

1. These tests are performed for the diagnosis of bleeding disorders.
2. APTT is performed to distinguished the functionality of the clotting factors I, II, V, VII, IX, X, XI, and XII.
3. APTT is used to check the treatment of the patient taking heparin or other medicine for blood thinning.

See: Role of laboratory tests is bleeding disorders

Collection of Sample

Venous blood sample is collected from antecubital fossa in a test tube containing trisodium citrate (3.2%), with the anticoagulant to blood proportion being 1:9. See also: Prothrombin time (PT): Collection of Specimen.

Precautions

1. Sample handling is very sensitive, false and raised values are obtained if the ratio of blood and anticoagulant is not correct.
2. Plasma is stable for one hour if kept at 4º C.
3. Plasma can be preserved for 28 days if frozen.

Principles

Activated Partial Thromboplastin Time (APTT)

Plasma is incubated with an activator (which initiates intrinsic pathway of coagulation by contact activation). Phospholipid (also called as partial thromboplastin) and calcium are then added and clotting time is measured.

Partial Thromboplastin Time (PTT)

It is one stage test. It distinguishes the functionality of the clotting factors I, II, V, VIII, X, XI, and XII. Both Activated Partial Thromboplastin Time (APTT) and Partial Thromboplastin Time (PTT) have the same clinical significance but Activated Partial Thromboplastin Time (APTT) is more reliable as compare to Partial Thromboplastin Time (PTT) due to its sensitivity.

Prothrombin Time (PT)

Tissue thromboplastin and calcium are added to plasma and clotting time is determined. The test determines the overall efficiency of extrinsic and common pathways.

International Sensitivity Index (ISI) and International Normalized Ratio (INR)

International Sensitivity Index (ISI) of a particular tissue thromboplastin is derived (by its manufacturer) by comparing it with a reference thromboplastin of known ISI. For standardization and to obtain comparable results, it is recommended to report PT (in persons on oral anticoagulants) in the form of an International Normalized Ratio (INR).

International Normalized Ratio (INR) is calculated by the following formula.

Purpose of INR: The INR is calculated to evaluate the following conditions.

1. Atrial fibrillation
2. Thrombophilia
3. Cardiomyopathy
4. Prosthesis (Replacement of heart valve)
5. Venous thromboembolism
6. Antiphospholipid syndrome

Normal Values

Technique of APTT, PTT, and PT is different in different laboratories therefore normal values varies with the lab to lab. A normal control is always run with the patient's sample. In general, normal values are below.

• APTT: 30-40 seconds
• PTT: 60-70 seconds
• PT: 11-16 seconds
• INR: 1-1.5

Critical Values

• APTT: > 70 seconds (Usually it is considered as panic value. If APTT is greater then 100 seconds, spontaneous bleeding may occur.)
• INR: > 5.0 (In Deep Vein Thrombosis (DVT) patient on warfarin treatment, an expected value of INR is between 2.0 to 3.0.)

Reasons for the high results

1. Disseminated intravascular coagulopathy (DIC )
2. Factor XII deficiency
3. Cirrhosis
4. Hemophilia A and B
5. Von Willebrand’s disease
6. Hypofibrinogenemia
7. Vitamin K deficiency
8. Malabsorption
9. Leukemia
10. Fibrin breakdown products
11. All congenital deficiencies of Intrinsic system coagulation factors
12. Drugs

The significance of APTT, PTT, PT, and INR test for the layman

• Patients, taking medication for blood thinning or on heparin treatment, are advised for these laboratory investigations.

Why is this test performed?

1. This hormone test is evaluated in different conditions, such as Adrenal insufficiency, in Acromegaly and Cushing Syndrome.
2. In Addison's disease, the level of Adrenocorticotropic hormone (ACTH) is noted more than 1000 pg/ml.
3. In Adrenal carcinoma, Adenoma, and Adrenocortical insufficiency, the level of Adrenocorticotropic hormone (ACTH) decreases.

Collection of Sample

For the estimation of Adrenocorticotropic hormone (ACTH), patient’s plasma is needed. Blood is collected in a chilled plastic test tube containing EDTA or heparin and blood is placed in cold ice-water.

The sample is centrifuged at 4º C, plasma is separated and stored at -20º C immediately within 15 minutes of the blood collection.

See: Uses of anticoagulants for hematological investigations

Note: For the diagnosis of Cushing Syndrome, the blood sample is collected in between 6 PM to 11 PM.

See: Procedures for the collection of blood for hematological investigations

Precautions

1. Collect the blood sample in a chilled plastic test tube containing EDTA or heparin.
2. Avoid high carbohydrates diet, take the low-carb diet.
3. Avoid physical activity for 12 hours before the collection of the blood sample.
4. Stop medication such as corticosteroids, 48 hours before the collection of blood sample.
5. An anxious collection of the blood sample may increase the level of Adrenocorticotropic hormone (ACTH).

Normal Values

• 6 to 8 AM: < 80 pg/ml or < 18 pmol/L (SI units)
  6 to 11 MP: < 50 pg/ml or < 11 pmol/L (SI units)
  or less than 120 pg/ml
• According to another references:
  8 AM:  < 120 pg/mL
  4 to 8 PM: < 85 pg/mL
  Cord blood: 50 to 570 pg/mL
  Newborn: 10 to 185 pg/mL

Reasons for the increased level of ACTH

1. Cushing syndrome
2. Addison's disease
3. Stress
4. Ectopic ACTH syndrome

Reasons for the decreased level of ACTH

1. Secondary adrenal insufficiency
2. Exogenous steroid administration
3. Hypopituitarism
4. Adrenal adenoma or carcinoma

The significance of Adrenocorticotropic hormone (ACTH) test for the layman

1. This test is advised in abnormal metabolism of lipids.
2. This test is advised to the patients of Diabetes Mellitus (DM).
3. This test is performed for the diagnosis of Cushing syndrome.
4. This test is advised if there are truncal obesity and thin extremity.

Why is this test performed?

1. The test is performed for the diagnosis of prostatic carcinoma by the estimation of total acid phosphatase (TAP) and the prostatic component.
2. The test is also performed for the examination for the presence of semen in medicolegal cases by a vaginal swab. Due to the high level of acid phosphatase in semen, its presence indicates recent sexual intercourse. Level of ≥50 U/sample is considered as positive evidence of semen.

Collection of Sample

For the test of total acid phosphatase (TAP) and the prostatic acid phosphatase (PAP), morning sample is preferred. About 3 to 5 ml blood is collected in a vacutainer and blood is allowed to clot. Then blood is centrifuged for 10 minutes at 4500 rpm in order to get the clear serum. The test is performed immediately. However, the sample can be preserved for 24 hours if kept at 2-8º C.

See: Procedures for the collection of blood for hematological investigations

Precaution

1. The sample has very poor stability in the whole blood, therefore serum is separated immediately and the test is performed within an hour.
2. The sample is unstable for the test of ACP if the pH is more then 7.0.
3. The sample is unstable for the test of ACP at room temperature (> 37º C).
4. Hemolyzed serum causes the false-positive result. Increased values can be observed in hemolyzed serum.

Normal Values

• Total acid phosphatase (TAP)
  2.5 to 3.7 ng /mL
  or < 3.0 mg /L
• Prostatic acid phosphatase (PAP)
  < 2.5 ng/mL (0 to 0.6 U/L)
• Other references
  Adult: 0.13 to 0.63 units/L at 37° C or 2.2 to 10.5 units/L (SI units)
  Child: 8.6 to 12.0 units/mL at 30° C
  Newborn:10.4 to 16.4 units /mL at 30°C

Reasons for the increased level

1. Prostatic carcinoma
2. Prostatitis
3. Benign prostatic hyperplasia
4. Metastatic carcinoma of the prostate
5. Metastases to the bones

Moderately raised level seen in, other than prostatic carcinoma

1. Hyperparathyroidism
2. Niemann-Pick disease
3. Sickle cell anemia
4. Prostatitis and Benign prostatic hyperplasia ( BPH )
5. Any cancer that has given metastasis to the bones.
6. Urinary retention
7. Multiple myelomas
8. Myeloid Leukemia
9. Paget disease
10. Liver diseases like cirrhosis
11. Renal diseases
12. Thrombocytosis
13. Gaucher’s disease

The significance of acid phosphatase test in medicolegal cases

Due to the high level of acid phosphatase in semen, its measurement is very important in rape cases. For the collection of a sample from the victim's body, following methods are applied.

1. Direct aspiration or saline lavage
2. Vaginal swab is obtained from the victim's vagina, and the sample is placed in 2.5% broth and can preserve at 4° C.

The significance of acid phosphatase test for the layman

1. The test is performed for the diagnosis of prostatic carcinoma.
2. This is carried out in cases of alleged rape or sexual assault.

Purpose of the test

The test is performed to assess the adrenal cortex function, and evaluate and monitor the adrenal tumors and hyperplasia.

How to collect the urine sample for the 17-ketosteroids test?

1. For the test of 17-ketosteroids in urine, 24 hours urine sample is required. Urine is collected in a container, containing one gram boric acid or 6 ml hydrochloric acid (HCl).
2. The first urine sample is discarded and then collected the all urine sample for 24 hours.
3. The 24 hours urine sample is transferred to the pathology laboratory for laboratory findings.

Why is boric acid used for the collection of the urine sample?

Boric acid converts the urine into a bacteriostatic medium, which inhibits the growth of bacteria in urine and preserves the urine for its bacteriological examination.

Precautions

Some medicines like aspirin, diuretics, antibiotics, birth control medication and hormones therapy (e.g. Estrogen) may interfere with laboratory findings, so avoid such types of medicine before preparing yourself for the laboratory test.

Normal Values

• Males: 8-20 mg/24-hour
• Females: 6-15 mg/24-hour
• Elderly males > 70 years: 3-12 mg/24-hour
• Elderly females > 70 years: 3-13 mg/24-hour
• Infants: < 1 mg/24-hour
• 1 to 5 years: < 5 mg/24-hour

Reasons for the increased level of 17-ketosteroids in the 24-hour urine sample

• Pregnancy
• Stein-Leventhal syndrome
• Hyperpituitarism
• Ectopic ACTH-secreting tumors
• Administration of ACTH
• Cushing’s syndrome
• Congenital adrenal hyperplasia 
• Testosterone secreting or androgen-secreting tumors of:
  (1) Ectopic ACTH-secreting tumors
  (2) Ovaries
  (3) Testes

Reasons for the decreased level of 17-ketosteroids in the 24-hour urine sample

• Hypopituitarism
• Severe infections 
• Klinefelter's syndrome
• Addison’s disease
• Severe stress
• Nephrosis
• Debilitating diseases
• Chronic diseases
• Castration
• Myxedema
• Drugs that can decrease the level of 17-ketosteroids include:
  (a) Probenecid
  (b) Estrogens
  (c) Reserpine
  (d) Thiazide diuretics
  (e) Salicylates (prolonged use)
  (f) Birth control pills

The significance of 17-ketosteroids for the layman

1. The test is advised if the females has hairs on her face.
2. The test is performed to elaborate any abnormality or disorders of the adrenal gland.

CHOLERA is a specific infectious disease that affects the lower portion of the intestine and is characterized by violent purging, vomiting, muscular cramp, suppression of urine and rapid collapse. It can a terrifying disease with massive diarrhea. The patient’s fluid losses are enormous every day with severe rapid dehydration, death comes within hours.

ETIOLOGY

• Site: GIT (Gastrointestinal Track)
• Agent: VIBRIO CHOLERA

MORPHOLOGICAL CHARACTER

Gram-negative, curved rods, non-capsulated, non-spore, motile by means of flagella (polar) they occur singly.

CULTURAL CHARACTER

1. They produce smooth, convex, round, colonies which appear opaque and granular in transmitted light.
2. They can grow on many kinds of media including enriching media contains bile salt and asparagine.
3. They particularly grow on TCB agar (Thiosulfate Citrate Bile Salt agar) and produce yellow colonies.
4. They are readily killed by acid and optimum pH for growth is 8.5-9.5.

BIOCHEMICAL CHARACTER

They ferment sucrose and maltose but not arabinose. They are oxidase positive which make them different from enteric Gram-negative rods. Some are halotolerant while others are halophilic require presence of NaCl for their growth.

ANTIGENIC CHARACTER

• 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.

CHOLERA TOXIN

V. Cholera elaborated an enterotoxin that is responsible for the loss of fluid is Cholera, called CHOLERAGEN. It is a polymeric protein with a molecular weight 84,000 daltons containing two major domains. The domain "A" with molecular weight 28,000 daltons, play the key role in the biological activity of the Choleragen. The domain "B" is also known as CHOLERAGENOID with a molecular weight 56,000 daltons bind the toxin to its receptors on host cell surface. it is also the immunologically active region of the toxin.

Vibrio Cholera has been shown to produce a second toxin called ZONULA OCCULUDENS TOXIN (ZOT). This toxin disintegrates the tight junction between enterocytes, allowing escape of water and electrolytes.

MODE OF ACTION OF TOXIN

The toxin subunit "A" and "B" promote the entry of subunit "A" into the cell, "B" subunit is responsible for attachment of toxin to the epithelial cell of the small intestine. This subunit alters the activity of the regulatory protein, that controls the activity of enzyme, Adenylate Cyclase. This enzyme converts the ATP (Adenosine Tri-Phosphate) into CAMP (Cyclic Adenosine 5 Mono Phosphate). This increase in cyclic AMP level causes loss of water, electrolytes and result in diarrhea. This may lead to death because of dehydration and acidosis.

PATHOGENESIS

Cholera occurs in epidemic form under the condition of overcrowding, floods, wars, and famine. Humans are the only known natural hosts. A person may have to ingest 10⁸ – 10¹⁰ organism to become infected. Vibrio Cholera is transferred from one person to another by ingestion of contaminated water or foodstuff. The contact with the carrier can also contribute to epidemics.

The Cholera bacilli find their way into the small intestine where they proliferate and elaborate the Choleragen. The toxin elevates the produces a massive secretion of isotonic fluid into the lumen of the intestine.

CLINICAL FINDING

The incubation period is few hours to 4 days. After incubation, there is sudden onset of nausea, vomiting, diarrhea with abdominal cramps, rapid dehydration and loss of fluid electrolytes. Mortality rate without treatment is 25% to 50%.

LABORATORY DIAGNOSIS

Diagnosis of Cholera patient by physical examination of stool, direct microscopic examination. Culture technique and also by agglutination method.

SMEAR

Smear made from stool sample is not distinctive however darkfield microscopy or phase contrast microscopy can show motile Vibrios.

CULTURE

There is rapid growth on peptone agar, TCB’s near pH 9 colony can be picked after 18-24 hours of incubation.

AGGLUTINATION

Agglutination test using anti O group on serum and also by the biochemical reaction.

EPIDEMIOLOGY

Man is the only host of Cholera disease and spread of infection is from person to person with contaminated water, food or flies. In many intensive 1% to 5% of exposed susceptible person developed the disease. The carrier state seldom exceeds 3 to 4 weeks.

PREVENTION

1. Good water supply. Proper treatment of water should be there before supply to the town.
2. Proper treatment of sewerage system.
3. Personal hygiene and proper sanitation.

TREATMENT

Individual infected with Cholera require rehydration adequately by giving a solution of Oral Rehydration Salts (ORS) containing sodium chloride, sodium bicarbonate, potassium chloride and glucose. During the epidemic, 80-90% of diarrhea patient can be treated by oral rehydration alone but the patient who becomes severely dehydrate must be given intravenous fluid.

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.

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.

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.

1. Confirmation of diagnosis of secondary hypothyroidism
2. Evaluation of suspected hypothalamic disease
3. 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.

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.

• 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. 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.

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.

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.

1. 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.
2. 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.
3. 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.
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.
5. Hormonal studies: This includes measurement of FSH, LH, and testosterone to detect hormonal abnormalities causing testicular failure (Table 861.2).
6. 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).

• 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.
• Mucus
• Intrinsic factor (IF): IF is necessary for absorption of vitamin B₁₂ in the terminal ileum. It is secreted by parietal cells of stomach.

• 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.

1. 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.
   
   
2. 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.
   
   
3. 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.
   
   
4. 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).
   
   
5. 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%

1. 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.
2. To determine the cause of raised fasting serum gastrin level: Hypergastrinemia can occur in achlorhydria, Zollinger-Ellison syndrome, and antral G cell hyperplasia.
3. 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).
4. 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.
5. 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.
6. 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.

1. 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.
2. 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.
3. pH: Normal pH is 1.5 to 3.5. In pernicious anemia, pH is greater than 7.0 due to absence of HCl.
4. 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.
5. 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.
6. 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.

Chemical examination of feces is usually carried out for the following tests (Figure 845.1):

• Occult blood
• Excess fat excretion (malabsorption)
• Urobilinogen
• Reducing sugars
• Fecal osmotic gap
• Fecal pH

Test for Occult Blood in Stools

Presence of blood in feces which is not apparent on gross inspection and which can be detected only by chemical tests is called as occult blood. Causes of occult blood in stools are:

1. Intestinal diseases: hookworms, amebiasis, typhoid fever, ulcerative colitis, intussusception, adenoma, cancer of colon or rectum.
2. Gastric and esophageal diseases: peptic ulcer, gastritis, esophageal varices, hiatus hernia.
3. Systemic disorders: bleeding diathesis, uremia.
4. Long distance runners.

Occult blood test is recommended as a screening procedure for detection of asymptomatic colorectal cancer. Yearly examinations should be carried out after the age of 50 years. If the test is positive, endoscopy and barium enema are indicated.

Tests for detection of occult blood in feces: Many tests are available which differ in their specificity and sensitivity. These tests include tests based on peroxidase-like activity of hemoglobin (benzidine, orthotolidine, aminophenazone, guaiac), immunochemical tests, and radioisotope tests.

Tests Based on Peroxidase-like Activity of Hemoglobin

Principle: Hemoglobin has peroxidase-like activity and releases oxygen from hydrogen peroxide. Oxygen molecule then oxidizes the chemical reagent (benzidine, orthotolidine, aminophenazone, or guaiac) to produce a colored reaction product.

Benzidine and orthotolidine are carcinogenic and are no longer used. Benzidine test is also highly sensitive and false-positive reactions are common. Since bleeding from the lesion may be intermittent, repeated testing may be required.

Causes of False-positive Tests

1. 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.
2. Drugs like aspirin and other anti-inflammatory drugs, which increase blood loss from gastrointestinal tract in normal persons.

Causes of False-negative Tests

1. Foods containing large amounts of vitamin C.
2. Conversion of all hemoglobin to acid hematin (which has no peroxidase-like activity) during passage through the gastrointestinal tract.

Immunochemical Tests

These tests specifically detect human hemoglobin. Therefore there is no interference from animal hemoglobin or myoglobin (e.g. meat) or peroxidase-containing vegetables in the diet.

The test consists of mixing the sample with latex particles coated with anti-human haemoglobin antibody, and if agglutination occurs, test is positive. This test can detect 0.6 ml of blood per 100 grams of feces.

Radioisotope Test Using ⁵¹Cr

In this test, 10 ml of patient’s blood is withdrawn, labeled with ⁵¹Cr, and re-infused intravenously. Radioactivity is measured in fecal sample and in simultaneously collected blood specimen. Radioactivity in feces indicates gastrointestinal bleeding. Amount of blood loss can be calculated. Although the test is sensitive, it is not suitable for routine screening.

Apt test: This test is done to decide whether blood in the vomitus or in the feces of a neonate represents swallowed maternal blood or is the result of bleeding in the gastrointestinal tract. The test was devised by Dr. Apt and hence the name. The baby swallows blood during delivery or during breastfeeding if nipples are cracked. Apt test is based on the principle that if blood is of neonatal origin it will contain high proportion of hemoglobin F (Hb F) that is resistant to alkali denaturation. On the other hand, maternal blood mostly contains adult hemoglobin or Hb A that is less resistant.

Test for Malabsorption of Fat

Dietary fat is absorbed in the small intestine with the help of bile salts and pancreatic lipase. Fecal fat mainly consists of neutral fats (unsplit fats), fatty acids, and soaps (fatty acid salts). Normally very little fat is excreted in feces (<7 grams/day in adults). Excess excretion of fecal fat indicates malabsorption and is known as steatorrhea. It manifests as bulky, frothy, and foul-smelling stools, which float on the surface of water.

Causes of Malabsorption of Fat

1. Deficiency of pancreatic lipase (insufficient lipolysis): chronic pancreatitis, cystic fibrosis.
2. Deficiency of bile salts (insufficient emulsification of fat): biliary obstruction, severe liver disease, bile salt deconjugation due to bacterial overgrowth in the small intestine.
3. Diseases of small intestine: tropical sprue, celiac disease, Whipple’s disease.

Tests for fecal fat are qualitative (i.e. direct microscopic examination after fat staining), and quantitative (i.e. estimation of fat by gravimetric or titrimetric analysis).

1. 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.
2. 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.

Test for Urobilinogen in Feces

Fecal urobilinogen is determined by Ehrlich’s aldehyde test (see  Article “Test for Detection of Urobilinogen in Urine”). Specimen should be fresh and kept protected from light. Normal amount of urobilinogen excreted in feces is 50-300 mg per day. Increased fecal excretion of urobilinogen is seen in hemolytic anemia. Urobilinogen is deceased in biliary tract obstruction, severe liver disease, oral antibiotic therapy (disturbance of intestinal bacterial flora), and aplastic anemia (low hemoglobin turnover). Stools become pale or clay-colored if urobilinogen is reduced or absent.

Test for Reducing Sugars

Deficiency of intestinal enzyme lactase is a common cause of malabsorption. Lactase converts lactose (in milk) to glucose and galactose. If lactase is deficient, lactose is converted to lactic acid with production of gas. In infants this leads to diarrhea, vomiting, and failure to thrive. Benedict’s test or Clinitest™ tablet test for reducing sugars is used to test freshly collected stool sample for lactose. In addition, oral lactose tolerance test is abnormal (after oral lactose, blood glucose fails to rise above 20 mg/dl of basal value) in lactase deficiency. Rise in blood glucose indicates that lactose has been hydrolysed and absorbed by the mucosa. Lactose tolerance test is now replaced by lactose breath hydrogen testing. In lactase deficiency, accumulated lactose in the colon is rapidly fermented to organic acids and gases like hydrogen. Hydrogen is absorbed and then excreted through the lungs into the breath. Amount of hydrogen is then measured in breath; breath hydrogen more than 20 ppm above baseline within 4 hours indicates positive test.

Fecal Osmotic Gap

Fecal osmotic gap is calculated from concentration of electrolytes in stool water by formula 290-2([Na⁺] + [K⁺]). (290 is the assumed plasma osmolality). In osmotic diarrheas, osmotic gap is >150 mOsm/kg, while in secretory diarrhea, it is typically below 50 mOsm/kg. Evaluation of chronic diarrhea is shown in Figure 845.3.

Fecal pH

Stool pH below 5.6 is characteristic of carbohydrate malabsorption.

1. Pre-renal azotemia: shock, congestive heart failure, salt and water depletion
2. Renal azotemia: impairment of renal function
3. Post-renal azotemia: obstruction of urinary tract
4. 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

1. 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.
2. 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.

1. Pre-renal, renal, and post-renal azotemia
2. Large amount of dietary meat
3. Active acromegaly and gigantism

1. Pregnancy
2. Increasing age (reduction in muscle mass)

1. 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.
2. 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.

1. Increased BUN with normal serum creatinine:
   • Pre-renal azotemia (reduced renal perfusion)
   • High protein diet
   • Increased protein catabolism
   • Gastrointestinal hemorrhage
2. 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.

• Acute tubular necrosis
• Low protein diet, starvation
• Severe liver disease

• Exogenous: Inulin, Radiolabelled ethylenediamine tetraacetic acid (⁵¹Cr- EDTA), ¹²⁵I-iothalamate
• Endogenous: Creatinine, Urea, Cystatin C

1. A small amount of creatinine is secreted by renal tubules that increase even further in advanced renal failure.
2. Collection of urine is often incomplete.
3. Creatinine level is affected by intake of meat and muscle mass.
4. Creatinine level is affected by certain drugs like cimetidine, probenecid, and trimethoprim (which block tubular secretion of creatinine).

• Estimation of Creatinine Clearance from Serum Creatinine by Prediction Equations

• 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

1. Nephrotic syndrome in adults (most common indication)
2. Nephrotic syndrome not responding to corticosteroids in children.
3. Acute nephritic syndrome for differential diagnosis
4. Unexplained renal insufficiency with near-normal kidney dimensions on ultrasonography
5. Asymptomatic hematuria, when other diagnostic tests fail to identify the source of bleeding
6. Isolated non-nephrotic range proteinuria (1-3 gm/24 hours) with renal impairment
7. Impaired function of renal graft
8. Involvement of kidney in systemic disease like systemic lupus erythematosus or amyloidosis

1. Uncontrolled severe hypertension
2. Hemorrhagic diathesis
3. Solitary kidney
4. Renal neoplasm (to avoid spread of malignant cells along the needle track)
5. Large and multiple renal cysts
6. Small, shrunken kidneys
7. Acute urinary tract infection like pyelonephritis
8. Urinary tract obstruction

1. 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.
2. Arteriovenous fistula
3. Infection
4. Accidental biopsy of another organ or perforation of viscus (liver, spleen, pancreas, adrenals, intestine, or gallbladder)
5. Death (rare).

1. Patient’s informed consent is obtained.
2. Ultrasound/CT scan is done to document the location and size of kidneys.
3. 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.
4. Patient is sedated before the procedure.
5. Patient lies in prone position and kidney is identified with ultrasound.
6. The skin over the selected site is disinfected and a local anesthetic is infiltrated.
7. 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.
8. 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.
9. The biopsy should be placed in a drop of saline and examined under a dissecting microscope for adequacy.
10. 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.

In DM, applications of laboratory tests are as follows:

• Diagnosis of DM
• Screening of DM
• Assessment of glycemic control
• Assessment of associated long-term risks
• Management of acute metabolic complications.

LABORATORY TESTS FOR DIAGNOSIS OF DIABETES MELLITUS

Diagnosis of DM is based exclusively on demonstration of raised blood glucose level (hyperglycemia).

The current criteria (American Diabetes Association, 2004) for diagnosis of DM are as follows:

Typical symptoms of DM (polyuria, polydipsia, weight loss) and random plasma glucose ≥ 200 mg/dl (≥ 11.1 mmol/L)

Or

Fasting plasma glucose ≥ 126 mg/dl (≥ 7.0 mmol/L)

Or

2-hour post glucose load (75 g) value during oral glucose tolerance test ≥ 200 mg/dl (≥ 11.1 mmol/L).

If any one of the above three criteria is present, confirmation by repeat testing on a subsequent day is necessary for establishing the diagnosis of DM. However, such confirmation by repeat testing is not required if patient presents with (a) hyperglycemia and ketoacidosis, or (b) hyperosmolar hyperglycemia.

The tests used for laboratory diagnosis of DM are (1) estimation of blood glucose and (2) oral glucose tolerance test.

Estimation of Blood Glucose

Measurement of blood glucose level is a simple test to assess carbohydrate metabolism in DM (Figure 837.1). Since glucose is rapidly metabolized in the body, measurement of blood glucose is indicative of current state of carbohydrate metabolism.

Glucose concentration can be estimated in whole blood (capillary or venous blood), plasma or serum. However, concentration of blood glucose differs according to nature of the blood specimen. Plasma glucose is about 15% higher than whole blood glucose (the figure is variable with hematocrit). During fasting state, glucose levels in both capillary and venous blood are about the same. However, postprandial or post glucose load values are higher by 20-70 mg/dl in capillary blood than venous blood. This is because venous blood is on a return trip after delivering blood to the tissues.

When whole blood is left at room temperature after collection, glycolysis reduces glucose level at the rate of about 7 mg/dl/hour. Glycolysis is further increased in the presence of bacterial contamination or leucocytosis. Addition of sodium fluoride (2.5 mg/ml of blood) maintains stable glucose level by inhibiting glycolysis. Sodium fluoride is commonly used along with an anticoagulant such as potassium oxalate or EDTA. Addition of sodium fluoride is not necessary if plasma is separated from whole blood within 1 hour of blood collection.

Plasma is preferred for estimation of glucose since whole blood glucose is affected also by concentration of proteins (especially hemoglobin).

There are various methods for estimation of blood glucose:

• Chemical methods:
  – Orthotoluidine method
  – Blood glucose reduction methods using neocuproine, ferricyanide, or copper.

Chemical methods are less specific but are cheaper as compared to enzymatic methods.

• Enzymatic methods: These are specific for glucose.
  – Glucose oxidase-peroxidase
  – Hexokinase
  – Glucose dehydrogenase

Chemical methods have now been replaced by enzymatic methods.

Terms used for blood glucose specimens: Depending on the time of collection, different terms are used for blood glucose specimens.

• 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.

Oral Glucose Tolerance Test (OGTT)

Glucose tolerance refers to the ability of the body to metabolize glucose. In DM, this ability is impaired or lost and glucose intolerance represents the fundamental pathophysiological defect in DM. OGTT is a provocative test to assess response to glucose challenge in an individual (Figure 837.2).

American Diabetes Association does not recommend OGTT for routine diagnosis of type 1 or type 2 DM. This is because fasting plasma glucose cutoff value of 126 mg/dl identifies the same prevalence of abnormal glucose metabolism in the population as OGTT. World Health Organization (WHO) recommends OGTT in those cases in which fasting plasma glucose is in the range of impaired fasting glucose (i.e. 100-125 mg/dl). Both ADA and WHO recommend OGTT for diagnosis of gestational diabetes mellitus.

Preparation of the Patient

• 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.

Test

1. A fasting venous blood sample is collected in the morning.
2. 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.
3. 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).
4. Plasma glucose is estimated in fasting and 2-hour venous blood samples.

Interpretation of blood glucose levels is given in Table 837.1.

OGTT in gestational diabetes mellitus: Impairment of glucose tolerance develops normally during pregnancy, particularly in 2nd and 3rd trimesters. Following are the recent guidelines of ADA for laboratory diagnosis of GDM:

• 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.

Initially, fasting plasma glucose or random plasma glucose should be obtained. If fasting plasma glucose is ≥ 126 mg/dl or random plasma glucose is ≥ 200 mg/dl, repeat testing should be carried out on a subsequent day for confirmation of DM. If both the tests are normal, then OGTT is indicated in average-risk and high-risk pregnant women.

There are two approaches for laboratory diagnosis of GDM

• One step approach
• Two step approach

In one step approach, 100 gm of glucose is administered to the patient and a 3-hour OGTT is performed. This test may be cost-effective in high-risk pregnant women.

In two-step approach, an initial screening test is done in which patient drinks a 50 g glucose drink irrespective of time of last meal and a venous blood sample is collected 1 hour later (O’Sullivan’s test). GDM is excluded if glucose level in venous plasma sample is below 140 mg/dl. If level exceeds 140 mg/dl, then the complete 100 g, 3-hour OGTT is carried out.

In the 3-hour OGTT, blood samples are collected in the morning (after 8-10 hours of overnight fasting), and after drinking 100 g glucose, at 1, 2, and 3 hours. For diagnosis of GDM, glucose concentration should be above the following cut-off values in 2 or more of the venous plasma samples:

• Fasting: 95 mg/dl
• 1 hour: 180 mg/dl
• 2 hour: 155 mg/dl
• 3 hour: 140 mg/dl

LABORATORY TESTS FOR SCREENING OF DIABETES MELLITUS

Aim of screening is to identify asymptomatic individuals who are likely to have DM. Since early detection and prompt institution of treatment can reduce subsequent complications of DM, screening may be an appropriate step in some situations.

Screening for type 2 DM: Type 2 DM is the most common type of DM and is usually asymptomatic in its initial stages. Its onset occurs about 5-7 years before clinical diagnosis. Evidence indicates that complications of type 2 DM begin many years before clinical diagnosis. American Diabetes Association recommends screening for type 2 DM in all asymptomatic individuals ≥ 45 years of age using fasting plasma glucose. If fasting plasma glucose is normal (i.e. < 100 mg/dl), screening test should be repeated every three years.

Another approach is selective screening i.e. screening individuals at high risk of developing type 2 DM i.e. if one or more of the following risk factors are presentobesity (body mass index ≥ 25.0 kg/m2), family history of DM (first degree relative with DM), high-risk ethnic group, hypertension, dyslipidemia, impaired fasting glucose, impaired glucose tolerance, or history of GDM. In such cases, screening is performed at an earlier age (30 years) and repeated more frequently.

Recommended screening test for type 2 DM is fasting plasma glucose. If ≥126 mg/dl, it should be repeated on a subsequent day for confirmation of diagnosis. If <126 mg/dl, OGTT is indicated if clinical suspicion is strong. A 2-hour post-glucose load value in OGTT ≥200 mg/dl is indicative of DM and should be repeated on a different day for confirmation.

Screening for type 1 DM: Type 1 DM is detected early after its onset since it has an acute presentation with characteristic clinical features. Therefore, it is not necessary to screen for type 1 DM by estimation of blood glucose. Detection of immunologic markers (mentioned earlier) has not been recommended to identify persons at risk.

Screening for GDM: Given earlier under OGTT in gestational diabetes mellitus.

LABORATORY TESTS TO ASSESS GLYCEMIC CONTROL

There is a direct correlation between the degree of blood glucose control in DM (both type 1 and type 2) and the development of microangiopathic complications i.e. nephropathy, retinopathy, and neuropathy. Maintenance of blood glucose level as close to normal as possible (referred to as tight glycemic control) reduces the risk of microvascular complications. There is also association between persistently high blood glucose values in DM with increased cardiovascular mortality.

Following methods can monitor degree of glycemic control:

• Periodic measurement of glycated hemoglobin (to assess long-term control).
• Daily self-assessment of blood glucose (to assess day-to- day or immediate control).

Glycated Hemoglobin (Glycosylated Hemoglobin, HbA_1C)

Glycated hemoglobin refers to hemoglobin to which glucose is attached nonenzymatically and irreversibly; its amount depends upon blood glucose level and lifespan of red cells.

Hemoglobin + Glucose ↔ Aldimine → Glycated hemoglobin

Plasma glucose readily moves across the red cell membranes and is being continuously combined with hemoglobin during the lifespan of the red cells (120 days). Therefore, some hemoglobin in red cells is present normally in glycated form. Amount of glycated hemoglobin in blood depends on blood glucose concentration and lifespan of red cells. If blood glucose concentration is high, more hemoglobin is glycated. Once formed, glycated hemoglobin is irreversible. Level of glycated hemoglobin is proportional to the average glucose level over preceding 6-8 weeks (about 2 months). Glycated hemoglobin is expressed as a percentage of total hemoglobin. Normally, less than 5% of hemoglobin is glycated.

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.

The terms glycated hemoglobin, glycosylated hemoglobin, glycohemoglobin, HbA₁, and HbA_1c are often used interchangeably in practice. Although these terms refer to hemoglobins that contain nonenzymatically added glucose residues, hemoglobins thus modified differ. Most of the studies have been carried out with HbA_1c.

Glycated hemoglobin should be routinely measured in all diabetic patients (both type 1 and type 2) at regular intervals to assess degree of long-term glycemic control. Apart from mean glycemia (over preceding 120 days), glycated hemoglobin level also correlates with the risk of the development of chronic complications of DM. In DM, it is recommended to maintain glycated hemoglobin level to less than 7%.

Spurious results of glycated hemoglobin are seen in reduced red cell survival (hemolysis), blood loss, and hemoglobinopathies.

In DM, if glycated hemoglobin is less than 7%, it should be measured every 6 months. If >8%, then more frequent measurements (every 3 months) along with change in treatment are advocated.

There are various methods for measurement of glycated hemoglobin such as chromatography, immunoassay, and agar gel electrophoresis.

Role of glycated hemoglobin in management of DM is highlighted in Box 837.1.

Self-Monitoring of Blood Glucose (SMBG)

Diabetic patients are taught how to regularly monitor their own blood glucose levels. Regular use of SMBG devices (portable glucose meters) by diabetic patients has improved the management of DM. With SMBG devices, blood glucose level can be monitored on day-to-day basis and kept as close to normal as possible by adjusting insulin dosage. SMBG devices measure capillary whole blood glucose obtained by fingerprick and use test strips that incorporate glucose oxidase or hexokinase. In some strips, a layer is incorporated to exclude blood cells so that glucose in plasma is measured. Aim of achieving tight glycemic control introduces the risk of severe hypoglycemia. Daily use of SMBG devices can avoid major hypoglycemic episodes.

SMBG devices yield unreliable results at very high and very low glucose levels. It is necessary to periodically check the performance of the glucometer by measuring parallel venous plasma glucose in the laboratory.

Portable glucose meters are used by patients for day-to-day self-monitoring, by physicians in their OPD clinics, and by health care workers for monitoring admitted patients at the bedside. These devices should not be used for diagnosis and population screening of DM as they lack precision and there is variability of results between different meters.

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.

Glycosuria

Semiquantitative urine glucose testing for monitoring of diabetes mellitus in home setting is not recommended. This is because (1) even if glucose is absent in urine, no information about blood glucose concentration below the renal threshold (which itself is variable) is obtained (Normally, renal threshold is around 180 mg/dl; it tends to be lower in pregnancy (140 mg/dl) and higher in old age and in long-standing diabetics; in some normal persons it is low), (2) urinary glucose testing cannot detect hypoglycemia, and (3) concentration of glucose in urine is affected by urinary concentration. Semiquantitative urine glucose testing for monitoring has now been replaced by self-testing by portable glucose meters.

LABORATORY TESTS TO ASSESS LONG-TERM RISKS

• 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
• Triglycerides
• Low-density lipoprotein (LDL) cholesterol
• High-density lipoprotein (HDL) cholesterol

• Diabetic ketoacidosis (DKA)
• Hyperosmolar hyperglycemic state (HHS)
• Hypoglycemia

• 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.

• 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

• Venous plasma glucose: > 450 mg/dl
• Strongly positive test for glucose and ketones in urine
• Arterial pH: < 7.2 or > 7.6
• Serum sodium: < 120 mEq/L or > 160 mEq/L
• Serum potassium: < 2.8 mEq/L or > 6.2 mEq/L
• Serum bicarbonate: < 10 mEq/L or > 40 mEq/L
• Serum chloride: < 80 mEq/L or > 115 mEq/L

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.

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

1. 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.
2. 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.
3. Early diagnosis of ectopic pregnancy: Trans-vaginal ultrasonography (USG) and quantitative estimation of hCG are helpful in early diagnosis of ectopic pregnancy (before rupture).
4. Evaluation of threatened abortion: Serial quantitative estimation of hCG is helpful in following the course of threatened abortion.
5. Diagnosis and follow-up of gestational trophoblastic disease (GTD).
6. 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.
7. Follow-up of ovarian or testicular germ cell tumors, which produce hCG.

Normal Pregnancy

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).

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

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.

Abortion

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

Bioassays

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.

Immunological Assays

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.

REFERENCE RANGES

• 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

• 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.

• 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

1. Mix one drop of semen with 1 drop of eosin-nigrosin solution and incubate for 30 seconds.
2. A smear is made from a drop placed on a glass slide.
3. 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.
4. The result is expressed as a proportion of viable sperms against non-viable as an integer percentage.

1. 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).
2. 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.
3. 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.
4. 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
   
   
5. Normal sperm count is ≥ 20 million/ml (i.e. ≥ 20 × 10⁶/ml). Sperm count < 20 million/ml may be associated with infertility in males.

1. Normal sperm
2. 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
3. Defects in neck:
   • Bent neck and tail forming an angle >90° to the long axis of head
4. Defects in middle piece:
   • Asymmetric insertion of midpiece into head
   • Thick or irregular midpiece
   • Abnormally thin midpiece
5. Defects in tail:
   • Bent tails
   • Short tails
   • Coiled tails
   • Irregular tails
   • Multiple tails
   • Tails with irregular width
6. Pin heads: Not to be counted
7. Cytoplasmic droplets
   • > 1/3rd the size of the sperm head
8. Precursor cells: Considered abnormal