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Published in Clinical Pathology
Thursday, 17 August 2017 21:35
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.
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)
Fasting plasma glucose ≥ 126 mg/dl (≥ 7.0 mmol/L)
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.
Figure 837.1 Blood glucose values in normal individuals
Figure 837.1 Blood glucose values in normal individuals, prediabetes, and diabetes mellitus
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).
Figure 837.2 Oral glucose tolerance curve
Figure 837.2 Oral glucose tolerance curve
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.
  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.
Table 837.1 Interpretation of oral glucose tolerance test
Parameter Normal Impaired fasting glucose Impaired glucose tolerance Diabetes mellitus
(1) Fasting (8 hr) < 100 100-125 ≥ 126
(2) 2 hr OGTT < 140 < 140 140-199 ≥ 200
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
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.
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, HbA1C)
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 HbA1c 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 HbA1c.
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%.
Box 837.1 Glycated hemoglobin 
  • Hemoglobin A1C of 6% corresponds to mean serum glucose level of 135 mg/dl. With every rise of 1%, serum glucose increases by 35 mg/dl. Approximations are as follows:
    – Hb A1C 7%: 170 mg/dl
    – Hb A1C 8%: 205 mg/dl
    – Hb A1C 9%: 240 mg/dl
    – Hb A1C 10%: 275 mg/dl
    – Hb A1C 11%: 310 mg/dl
    – Hb A1C 12%: 345 mg/dl
  • Assesses long-term control of DM (thus indirectly confirming plasma glucose results or self-testing results).
  • Assesses whether treatment plan is working
  • Measurement of glycated hemoglobin does not replace measurement of day-to-day control by glucometer devices.
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.
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.
Urinary Albumin Excretion
Diabetes mellitus is one of the leading causes of renal failure. Diabetic nephropathy develops in around 20-30% of patients with type 1 or type 2 DM. Diabetic nephropathy progresses through different stages as shown in Figure 837.3. Hypertension also develops along the course of nephropathy with increasing albumin excretion. Evidence indicates that if diabetic nephropathy is detected early and specific treatment is instituted, further progression of nephropathy can be significantly ameliorated. Early detection of diabetic nephropathy is based on estimation of urinary albumin excretion. In all adult patients with DM, usual reagent strip test for proteinuria should be carried out periodically. Positive test means presence of overt proteinuria or clinical proteinuria and may be indicative of overt nephropathy. In all such patients quantitation of albuminuria should be carried out to plan appropriate therapy. If the routine dipstick test for proteinuria is negative, test for microalbuminuria should be carried out.
Figure 837.3 Evolution of diabetic nephropathy
Figure 837.3 Evolution of diabetic nephropathy. In 80% of patients with type 1 DM, microalbuminuria progresses in 10-15 years to overt nephropathy that is then followed in majority of cases by progressive fall in GFR and ultimately end-stage renal disease. Amongst patients with type 2 DM and microalbuminuria, 20-40% of patients progress to overt nephropathy, and about 20% of patients with overt nephropathy develop end-stage renal disease. Abbreviation: GFR: Glomerular filtration rate
The term ‘microalbuminuria’ refers to the urinary excretion of albumin below the level of detection by routine dipstick testing but above normal (30-300 mg/ 24 hrs, 20-200 μg/min, or 30-300 μg/mg of creatinine). Albumin excretion rate is intermediate between normal (normal albumin excretion in urine is < 30 mg/24 hours) and overt albuminuria (> 300 mg/24 hours). Significance of microalbuminuria in DM is as follows:
  • 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.
Specific therapeutic interventions such as tight glycemic control, administration of ACE (angiotensinconverting enzyme) inhibitors, and aggressive treatment of hypertension significantly slow down the progression of diabetic nephropathy.
In type 2 DM, screening for microalbuminuria should begin at the time of diagnosis, whereas in type 1 DM, it should begin 5 years after diagnosis. At this time, a routine reagent strip test for proteinuria is carried out; if negative, testing for microalbuminuria is done. Thereafter, in all patients who test negative, screening for microalbuminuria should be repeated every year.
Screening tests for microalbuminuria include:
Reagent strip tests to detect microalbuminuria are available. Positive results should be confirmed by more specific quantitative tests like radioimmunoassay and enzyme immunoassay. For diagnosis of microalbuminuria, tests should be positive in at least two out of three different samples over a 3 to 6 month period.
Lipid Profile
Abnormalities of lipids are associated with increased risk of coronary artery disease (CAD) in patients with DM. This risk can be reduced by intensive treatment of lipid abnormalities. Lipid parameters which should be measured include:
  • Total cholesterol
  • Triglycerides
  • Low-density lipoprotein (LDL) cholesterol
  • High-density lipoprotein (HDL) cholesterol
The usual pattern of lipid abnormalities in type 2 DM is elevated triglycerides, decreased HDL cholesterol and higher proportion of small, dense LDL particles. Patients with DM are categorized into high, intermediate and low-risk categories depending on lipid levels in blood (Table 837.2).
Table 837.2 Categorization of cardiovascular risk in diabetes mellitus according to lipid levels (American Diabetes Association)
Category Low density lipoproteins High density lipoproteins Triglycerides
High-risk ≥130 < 35 (men) ≥ 400
    < 45 (women)  
Intermediate risk 100-129 35-45 200-399
Low-risk < 100 > 45 (men) < 200
    > 55 (women)  
Annual lipid profile is indicated in all adult patients with DM.
The three most serious acute metabolic complications of DM are:
  • Diabetic ketoacidosis (DKA)
  • Hyperosmolar hyperglycemic state (HHS)
  • Hypoglycemia
The typical features of DKA are hyperglycemia, ketosis, and acidosis. The common causes of DKA are infection, noncompliance with insulin therapy, alcohol abuse and myocardial infarction. Patients with DKA present with rapid onset of polyuria, polydipsia, polyphagia, weakness, vomiting, and sometimes abdominal pain. Signs include Kussmaul’s respiration, odour of acetone on breath (fruity), mental clouding, and dehydration. Classically, DKA occurs in type 1, while HHS is more typical of type 2 DM. However, both complications can occur in either types. If untreated, both events can lead to coma and death.
Hyperosmolar hyperglycemic state is characterized by very high blood glucose level (> 600 mg/dl), hyperosmolality (>320 mOsmol/kg of water), dehydration, lack of ketoacidosis, and altered mental status. It usually occurs in elderly type 2 diabetics. Insulin secretion is adequate to prevent ketosis but not hyperglycemia. Causes of HHS are illness, dehydration, surgery, and glucocorticoid therapy.
Differences between DKA and HHS are presented in Table 837.3.
Table 837.3 Comparison of diabetic ketoacidosis and hyperosmolar hyperglycemic state
Parameter Diabetic ketoacidosis Hyperosmolar hyperglycemic state
1. Type of DM in which more common  Type 1 Type 2 
2. Age  Younger age  Older age
3. Prodromal clinical features  < 24 hrs  Several days
4. Abdominal pain, Kussmaul’s respiration  Yes  No
5. Acidosis  Moderate/Severe  Absent
6. Plasma glucose  > 250 mg/dl  Very high (>600 mg/dl)
7. Serum bicarbonate  <15 mEq/L  >15 mEq/L
8. Blood/urine ketones  ++++  ±
9. β-hydroxybutyrate  High  Normal or raised
10. Arterial blood pH  Low (<7.30)  Normal (>7.30)
11. Effective serum osmolality*  Variable  Increased (>320)
12. Anion gap**  >12  Variable
*Osmolality: Number of dissolved (solute) particles in solution; normal: 275-295 mOsmol/kg*Osmolality: Number of dissolved (solute) particles in solution; normal: 275-295 mOsmol/kg
** Anion gap: Difference between sodium and sum of chloride and bicarbonate in plasma; normal average value is 12
Laboratory evaluation consists of following investigations:
  • 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.
Testing for ketone bodies: Ketone bodies are formed from metabolism of free fatty acids and include acetoacetic acid, acetone and β-hydroxybutyric acid.
Indications for testing for ketone bodies in DM include:
  • 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).
An increased amount of ketone bodies in patients with DM indicate impending or established diabetic ketoacidosis and is a medical emergency. Method based on colorimetric reaction between ketone bodies and nitroprusside (by dipstick or tablet) is used for detection of both blood and urine ketones.
Test for urine ketones alone should not be used for diagnosis and monitoring of diabetic ketoacidosis. It is recommended to measure β-hydroxybutyric acid (which accounts for 75% of all ketones in ketoacidosis) for diagnosis and monitoring DKA.
  • Venous plasma glucose:
    Fasting: 60-100 mg/dl
    At 2 hours in OGTT (75 gm glucose): <140 mg/dl
  • Glycated hemoglobin: 4-6% of total hemoglobin
  • Lipid profile:
    – Serum cholesterol: Desirable level: <200 mg/dl
    – Serum triglycerides: Desirable level: <150 mg/dl
    – HDL cholesterol: ≥60 mg/dl
    – LDL cholesterol: <130 mg/dl
    – LDL/HDL ratio: 0.5-3.0
  • C-peptide: 0.78-1.89 ng/ml
  • Arterial pH: 7.35-7.45
  • Serum or plasma osmolality: 275-295 mOsm/kg of water.

Serum Osmolality can also be calculated by the following formula recommended by American Diabetes Association:
Effective serum osmolality (mOsm/kg) = (2 × sodium mEq/L) + Plasma glucose (mg/dl)
  • 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


Published in Clinical Pathology
Wednesday, 16 August 2017 12:56
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.
Box 836.1 Indications for measurement of β human chorionic gonadotropin

• Early diagnosis of pregnancy
• Diagnosis and management of gestational trophoblastic disease
• As a part of maternal triple test screen
• Follow-up of malignant tumors that produce β human chorionic gonadotropin.
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.
  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).
Figure 836.1 Level of human chorionic gonadotropin during pregnancy
Figure 836.1 Level of human chorionic gonadotropin during pregnancy
Box 836.2 Diagnosis of early pregnancy

• Positive serum hCG test: 8 days after conception or 3 weeks after last menstrual period (LMP)
• Positive urine hCG test: 21 days after conception or 5 weeks after LMP
• Ultrasonography for visualization of gestational sac:
– Transvaginal: 21 days after conception or 5 weeks after LMP
– Transabdominal: 28 days after conception or 6 weeks after LMP
Human chorionic gonadotropin is synthesized by syncytiotrophoblasts (of placenta) and detectable amounts (~5 mIU/ml) appear in maternal serum about 8 days after conception (3 weeks after LMP). In the first trimester (first 12 weeks, calculated from day 1 of LMP) of pregnancy, hCG levels rapidly rise with a doubling time of about 2 days. Highest or peak level is reached at 8-10 weeks (about 100,000 mIU/ml). This is followed by a gradual fall, and from 15-16 weeks onwards, a steady level of 10,000-20,000 mIU/ml is maintained for the rest of the pregnancy (Figure 836.1). After delivery, hCG becomes non-detectable by about 2 weeks.
Box 836.2 shows minimum time required for the earliest diagnosis of pregnancy by hCG test and ultrasonography (USG).
Two types of pregnancy tests are available:
  • Qualitative tests: These are positive/negative result types that are done on urine sample.
  • Quantitative tests: These give numerical result and are done on serum or urine. They are also used for evaluation of ectopic pregnancy, failing pregnancy, and for follow-up of gestational trophoblastic disease.
Ectopic Pregnancy
Ectopic pregnancy refers to the implantation of blastocyst at a site other than the cavity of uterus. The most common of such sites (>95% cases) is fallopian tube. Early diagnosis and treatment of tubal ectopic pregnancy is essential since it can lead to maternal mortality (from rupture and hemorrhage) and future infertility. Ectopic pregnancy is a leading cause of maternal death during first trimester. Diagnosis of ectopic pregnancy can be readily made in most cases by ultrasonography and estimation of β-subunit of human chorionic gonadotropin.
Early diagnosis of unruptured tubal pregnancy can be made by quantitative estimation of serum hCG and ultrasonography. In normal intrauterine pregnancy, hCG titer doubles every 2 days until first 40 days of gestation. If hCG rise is abnormally slow, then an unviable pregnancy (either ectopic or abnormal intrauterine pregnancy) should be suspected.
Transabdominal USG can detect gestational sac in intrauterine pregnancy 6 weeks after LMP. The level of hCG in serum at this stage is >6500 mIU/ml. If gestational sac is not visualized at this level of hCG, then there is a possibility of ectopic pregnancy. Transvaginal ultrasonography can detect ectopic pregnancy average 1 week earlier than abdominal ultrasonography; it can detect gestational sac if β-hCG level is 1000-1500 mIU/ml. Therefore, if gestational sac is not visualized in the presence of >1500 mIU/ml of β-hCG level, an ectopic pregnancy can be suspected.
Early diagnosis of ectopic pregnancy provides the option of administration of intramuscular methotrexate (rather than surgery), which causes dissolution of conceptus. This improves the chances of patient’s future fertility. Serial measurements of hCG after surgical removal of ectopic pregnancy can help in detecting persistence of trophoblastic tissue.
Termination of pregnancy before fetus becomes viable (i.e. before 20 weeks) is called as abortion.
In threatened abortion, vaginal bleeding is present but internal os is closed and process of abortion, though started, is still reversible. It is possible that pregnancy will continue.
Serial quantitative titers of hCG showing lack of expected doubling of hCG level and USG are helpful in diagnosis and management of abortion.
Gestational Trophoblastic Disease (GTD)
It is characterized by proliferation of pregnancyassociated trophoblastic tissue. The two main forms of GTD are hydatidiform (vesicular) mole (benign) and choriocarcinoma (malignant). Clinical features of GTD are as follows:
  • Short history of amenorrhea followed by vaginal bleeding.
  • Size of uterus larger than gestational age; uterus is soft and doughy on palpation with no fetal parts and no fetal heart sounds.
  • Excessive nausea and vomiting due to high hCG.
  • Characteristic snowstorm appearance on pelvic USG.
Quantitative estimation of hCG is helpful in diagnosis and management of GTD.
Trophoblastic cells of GTD produce more hCG as compared to the trophoblasts of normal pregnancy for the same gestational age. Concentration of hCG parallels tumor load. Also, hCG continues to rise beyond 10 weeks of gestation without reaching plateau (as expected at the end of first trimester).
After evacuation of uterus, weekly estimation of hCG is advised till subsequent three (weekly) results are negative; following evacuation of vesicular mole, hCG becomes undetectable (after 2-3 months) on follow-up in 80% of cases. Plateau or rising hCG indicates persistent GTD. In such cases, chemotherapy is indicated.
Negative results for hCG after therapy should be regularly followed up every 3 months for 1-2 years.
These are classified into two main groups:
  • Biological assays or bioassays
  • Immunological assays
In bioassay, effect of hCG is tested on laboratory animals under standardized conditions. There are several limitations of bioassays like need for animal facilities, need for standardization of animals, long time required for the test results, low sensitivity, and high cost. Therefore, bioassays have been replaced by immunological assays.
In Ascheim-Zondek test, urine from pregnant woman is injected into immature female mice. Formation of hemorrhagic corpora lutea in ovaries (after 4 days) is a positive test. Friedman test is similar except that urine is injected into female rabbit. In rapid rat test, injection of urine containing hCG into female rats is followed by hyperaemia and hemorrhage in ovaries. Yet another test measures release of spermatozoa from male frog after injection of urine containing hCG.
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.
Figure 836.2 Principle of agglutination inhibition test for diagnosis of pregnancy
Figure 836.2 Principle of agglutination inhibition test for diagnosis of pregnancy
In latex particle agglutination inhibition test (Figure 836.2), anti-hCG antibodies are incubated with patient’s urine. This is followed by addition of hCGcoated latex particles. If hCG is present in urine, anti-hCG serum is neutralized, and no agglutination of latex particles occurs (positive test). If there is no hCG in urine, there is agglutination of latex particles (negative test). This is commonly used as a slide test and requires only a few minutes.
Sensitivity of agglutination inhibition test is >200 units/liter of hCG.
Radioimmunoassay, enzyme immunoassay, and radioimmunometric assay are more sensitive and reliable than agglutination inhibition assay.
Quantitative tests are employed for detection of very early pregnancy, estimation of gestational age, diagnosis of ectopic pregnancy, evaluation of threatened abortion, and management of GTD.
  • Serum human chorionic gonadotropin:
    – Non-pregnant females: <5.0 mIU/ml
    – Pregnancy: 4 weeks after LMP: 5-100 mIU/ml
    – 5 weeks after LMP: 200-3000 mIU/ml
    – 6 weeks after LMP: 10,000-80,000 mIU/ml
    – 7-14 weeks: 90,000-500,000 mIU/ml
    – 15-26 weeks: 5000-80000 mIU/ml
    – 27-40 weks: 3000-15000 mIU/ml
 Further Reading:


Published in Clinical Pathology
Tuesday, 15 August 2017 23:54
 Box 835.1 Contributions to semen volume
• Testes and epididymis: 10%
• Seminal vesicles: 50%
• Prostate: 40%
• Cowper’s glands: Small volume
Semen (or seminal fluid) is a fluid that is emitted from the male genital tract and contains sperms that are capable of fertilizing female ova. Structures involved in production of semen are (Box 835.1):
  • 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.
Normal values for semen analysis are shown in Tables 835.1 and 835.2.
Table 835.1 Normal values of semen analysis (World Health Organization, 1999)
Test Result
1. Volume ≥2 ml
2. pH 7.2 to 8.0
3. Sperm concentration ≥20 million/ml
4. Total sperm count per ejaculate ≥40 million
5. Morphology ≥30% sperms with normal morphology
6. Vitality ≥75% live
7. White blood cells <1 million/ml
8. Motility within 1 hour of ejaculation  
    • Class A ≥25% rapidly progressive
    • Class A and B ≥50% progressive
9. Mixed antiglobuiln reaction (MAR) test <50% motile sperms with adherent particles
10. Immunobead test <50% motile sperms with adherent particles
Table 835.2 Biochemical variables of semen analysis (World Helath Organization, 1992)
 1. Total fructose (seminal vesicle marker) ≥13 μmol/ejaculate 
 2. Total zinc (Prostate marker)  ≥2.4 μmol/ejaculate
 3. Total acid phosphatase (Prostate marker)  ≥200U/ejaculate
 4. Total citric acid (Prostate marker)  ≥52 μmol/ejaculate
 5. α-glucosidase (Epididymis marker)  ≥20 mU/ejaculate
 6. Carnitine (Epididymis marker)  0.8-2.9 μmol/ejaculate
Box 835.2 Tests done on seminal fluid
• Physical examination: Time to liquefaction, viscosity, volume, pH, color
• Microscopic examination: Sperm count, vitality, motility, morphology, and proportion of white cells
• Immunologic analysis: Antisperm antibodies (SpermMAR test, Immunobead test)
• Bacteriologic analysis: Detection of infection
• Biochemical analysis: Fructose, zinc, acid phosphatase, carnitine.
• Sperm function tests: Postcoital test, cervical mucus penetration test, Hamster egg penetration assay, hypoosmotic swelling of flagella, and computer-assisted semen analysis
Availability of semen for examination allows direct examination of male germ cells that is not possible with female germ cells. Semen analysis requires skill and should preferably be done in a specialized andrology laboratory.
  1. Investigation of infertility: Semen analysis is the first step in the investigation of infertility. About 30% cases of infertility are due to problem with males.
  2. To check the effectiveness of vasectomy by confirming absence of sperm.
  3. To support or disprove a denial of paternity on the grounds of sterility.
  4. To examine vaginal secretions or clothing stains for the presence of semen in medicolegal cases.
  5. For selection of donors for artificial insemination.
  6. For selection of assisted reproductive technology, e.g. in vitro fertilization, gamete intrafallopian transfer technique.
Semen specimen is collected after about 3 days of sexual abstinence. Longer period of abstinence reduces motility of sperms. If the period of abstinence is shorter than 3 days, sperm count is lower. The sample is obtained by masturbation, collected in a clean, dry, sterile, and leakproof wide-mouthed plastic container, and brought to the laboratory within 1 hour of collection. The entire ejaculate is collected, as the first portion is the most concentrated and contains the highest number of sperms. During transport to the laboratory, the specimen should be kept as close to body temperature as possible (i.e. by carrying it in an inside pocket). Ideally, the specimen should be obtained near the testing site in an adjoining room. Condom collection is not recommended as it contains spermicidal agent. Ejaculation after coitus interruptus leads to the loss of the first portion of the ejaculate that is most concentrated; therefore this method should not be used for collection. Two semen specimens should be examined that are collected 2-3 weeks apart; if results are significantly different additional samples are required.
Box 835.3 Semen analysis for initial investigation of infertility
• Volume
• pH
• Microscopic examination for (i) percentage of motile spermatozoa, (ii) sperm count, and (iii) sperm morphology
The tests that can be done on seminal fluid are shown in Box 835.2. Tests commonly done in infertility are shown in Box 835.3. The usual analysis consists of measurement of semen volume, sperm count, sperm motility, and sperm morphology.
Terminology in semen analysis is shown in Box 835.4.
 Box 835.4 Terminology in semen analysis

• Normozoospermia: All semen parameters normal
• Oligozoospermia: Sperm concentration <20 million/ml (mild to moderate: 5-20 million/ml; severe: <5 million/ml)
• Azoospermia: Absence of sperms in seminal fluid
• Aspermia: Absence of ejaculate
• Asthenozoospermia: Reduced sperm motility; <50% of sperms showing class (a) and class (b) type of motility OR <25% sperms showing class (a) type of motility.
• Teratozoospermia: Spermatozoa with reduced proportion of normal morphology (or increased proportion of abnormal forms)
• Leukocytospermia: >1 million white blood cells/ml of semen
• Oligoasthenoteratozoospermia: All sperm variables are abnormal
• Necrozoospermia: All sperms are non-motile or non-viable
The aim of post-vasectomy semen analysis is to detect the presence or absence of spermatozoa. The routine follow-up consists of semen analysis starting 12 weeks (or 15 ejaculations) after surgery. If two successive semen samples are negative for sperms, the semen is considered as free of sperm. A follow-up semen examination at 6 months is advocated by some to rule out spontaneous reconnection.
Further Reading:


Published in Clinical Pathology
Tuesday, 15 August 2017 01:44
These tests are available only in specialized andrology laboratories. The tests are not standardized thus making interpretation difficult. If used singly, a sperm function test may not be helpful in fertility assessment. They are more predictive if used in combination.
Postcoital (Sims-Huhner) Test
This is the examination of the cervical mucus after coitus and assesses the ability of the sperm to penetrate the cervical mucus. The quality of the cervical mucus varies during the menstrual cycle, becoming more abundant and fluid at the time of ovulation (due to effect of estrogen); this facilitates penetration of the mucus by the spermatozoa. Progesterone in the secretory phase increases viscosity of the mucus. Therefore cervical mucus testing is scheduled just before ovulation (determined by basal body temperature records or follicular sizing by ultrasonography). Postcoital test is the traditional method to detect the cervical factor in infertility. Cervical mucus is aspirated with a syringe shortly before the expected time of ovulation and 2-12 hours after intercourse. Gross and microscopic examinations are carried out to assess the quality of cervical mucus (elasticity and drying pattern) and to evaluate the number and motility of sperms (Box 834.1). If ≥ 10 motile sperms are observed the test is considered as normal. An abnormal test may result from: (a) poor quality of cervical mucus due to wrong judgment of ovulation, cervicitis or treatment with antioestrogens (e.g. Clomid), and (b) absence of motile sperms due to ineffective technique of coitus, lack of ejaculation, poor semen quality, use of coital lubricants that damage the sperm, or presence of antisperm antibodies. Antisperm antibodies cause immotile sperms, or agglutination or clumping of sperms; they may be present in either partner. If cervical factor is present, intrauterine insemination is the popular treatment. The value of the postcoital test is disputed in the medical literature.
Box 834.1 Interpretation of postcoital test
  • Normal: Sperms are normal in amount and moving forward in the mucus; mucus stretches atleast 2 inches (5 cm) and dries in a fern-like manner.
  • Abnormal: Absence of sperms or large number of sperms are dead or sperms are clumped; cervical mucus cannot stretch 2 inches (5 cm) or does not dry in a fern-like manner.
This test can be carried out if semen analysis is normal, and the female partner is ovulating and fallopian tubes are not blocked. It is also done if antisperm antibodies are suspected and male partner refuses semen analysis.
Cervical Mucus Penetration Test
In this test, greatest distance traveled by the sperm in seminal fluid placed and incubated in a capillary tube containing bovine mucus is measured. Majority of fertile men show score >30 mm, while most infertile men show scores <20 mm.
Hamster Egg Penetration Assay
Hamster oocytes are enzymatically treated to remove the outer layers (that inhibit cross-species fertilization). They are then incubated with sperms and observed for penetration rate. It can be reported as (a) Number of eggs penetrated (penetration rate <15% indicates low fertility), or as (b) Number of sperm penetrations per egg (Normal >5). This test detects sperm motility, binding to oocyte, and penetration of oocyte. There is a high incidence of false-negative results.
Hypo-osmotic Swelling of Flagella
This test assesses the functional integrity of the plasma membrane of the sperm by observing curling of flagella in hypo-osmotic conditions.
Computer-assisted Semen Analysis
Computer software measures various characteristics of the spermatozoa; however, its role in predicting fertility potential is not confirmed.


Published in Clinical Pathology
Tuesday, 15 August 2017 01:04
The role of antisperm antibodies in causation of male infertility is controversial. The immunological tests done on seminal fluid include mixed antiglobulin reaction (MAR test) and immunobead test.
The antibodies against sperms immobilize or kill them, thus preventing their passage through the cervix to the ovum. The antibodies can be tested in the serum, seminal fluid, or cervical mucus. If the antibodies are present bound to the head of the sperm, they will prevent the penetration of the egg by the sperm. If antibodies are bound to the tail of the sperm, they will retard motility.
a. SpermMAR™ test: This test can detect IgG and IgA antibodies against sperm surface in semen sample. In direct SpermMAR™ IgG test, a drop each of semen (fresh and unwashed), IgG-coated latex particles, and anti-human immunoglobulin are mixed together on a glass slide. At least 200 motile spermatozoa are examined. If the spermatozoa have antibodies on their surface, antihuman immunoglobulin will bind IgG-coated latex particles to IgG on the surface of the spermatozoa; this will cause attachment of latex particles to spermatozoa, and motile, swimming sperms with attached particles will be seen. If the spermatozoa do not have antibodies on their surface, they will be seen swimming without attached particles; the latex particles will show clumping due to binding of their IgG to antihuman immunoglobulin.
In direct SpermMAR™ IgA test, a drop each of fresh unwashed semen and of IgA-coated latex particles, are mixed on a glass slide. The latex particles will bind to spermatozoa if spermatozoa are coated with IgA antibodies.
In indirect SpermMAR™ tests, fluid without spermatozoa (e.g. serum) is tested for the presence of antisperm antibodies. First, antibodies are bound to donor spermatozoa which are then mixed with the fluid to be analyzed. These antibodies are then detected as described above for direct tests.

Atleast 200 motile spermatozoa should be counted. If >50% of spermatozoa show attached latex particles, immunological problem is likely.
b. Immunobead test: Antibodies bound to the surface of the spermatozoa can be detected by antibodies attached to immunobeads (plastic particles with attached anti-human immunoglobulin that may be either IgG, IgA, or IgM). Percentage of motile spermatozoa with attached two or more immunobeads are counted amongst 200 motile spermatozoa. Finding of >50% spermatozoa with attached beads is abnormal.


Published in Clinical Pathology
Monday, 14 August 2017 22:02
The most important test in semen analysis for infertility is microscopic examination of the semen.
The first laboratory assessment of sperm function in a wet preparation is sperm motility (ability of the sperms to move). Sperm motility is essential for penetration of cervical mucus, traveling through the fallopian tube, and penetrating the ovum. Only those sperms having rapidly progressive motility are capable of penetrating ovum and fertilizing it.
Principle: All motile and non-motile sperms are counted in randomly chosen fields in a wet preparation under 40× objective. Result is expressed as a percentage of motile spermatozoa observed.
Method: A drop of semen is placed on a glass slide, covered with a coverslip that is then ringed with petroleum jelly to prevent dehydration, and examined under 40× objective. Atleast 200 spermatozoa are counted in several different microscopic fields. Result is expressed as a percentage of (a) rapidly progressive spermatozoa (moving fast forward in a straight line), (b) slowly progressive spermatozoa (slow linear or non-linear, i.e. crooked or curved movement), (c) non-progressive spermatozoa (movement of tails, but with no forward progress), and (d) immotile spermatozoa (no movement at all) (WHO critera). Sperms of grades (c) and (d) are considered to be poorly motile (asthenospermia). Normally, ≥ 25% of sperms show rapid progressive motility, or ≥ 50% of sperms show rapid progressive and slow progressive motility.
If the proportion of motile spermatozoa is < 50%, then proportion of viable sperms should be determined by examining an eosin preparation.
Principle: A cell with intact cell membrane (a vital or viable cell) will not take up the eosin Y and will not be stained, while a non-viable or dead cell will have damaged cell membrane, will take up the dye, and will be stained pink-red (Figure 832.1). Another stain (e.g. nigrosin) may  be used to stain the background material. The test is performed if motility is abnormal.
Figure 832.1 Eosin nigrosin stain
Figure 832.1 Eosin-nigrosin stain. Dead sperms are stained pink-red, while live sperms are stained white
  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.
Seventy-five percent or more of sperms are normally live or viable.
Principle: The sperm count is done after liquefaction in a counting chamber following dilution and the total number of spermatozoa is reported in millions/ml (106/ml).
  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.
A smear is prepared by spreading a drop of seminal fluid on a glass slide, stained, and percentages of normal and abnormal forms of spermatozoa are counted. The staining techniques used are Papanicolaou, eosinnigrosin, hematoxylin-eosin, and Rose Bengal-toluidine blue stain. Atleast 200 spermatozoa should be counted under oil immersion. Percentages of normal and abnormal spermatozoa should be recorded.
Normal morphology: A spermatozoon consists of three main components: head, neck, and tail. Tail is further subdivided into midpiece, main (principle) piece, and end piece (Figure 832.2 and Box 832.1).
Figure 832.2 Morphology of spermatozoa
Figure 832.2 Morphology of spermatozoa
Head is pear-shaped. Most of the head is occupied by the nucleus which has condensed chromatin and few areas of dispersed chromatin (called nuclear vacuoles). The anterior 2/3rds of the nucleus is surrounded by acrosomal cap. Acrosomal cap is a flattened membranebound vesicle containing glycoproteins and enzymes. These enzymes are required for separation of cells of corona radiata and dissolution of zona pellucida of ovum during fertilization.
Neck is a very short segment that connects the head and the tail. Centriole in the neck gives rise to axoneme of the flagellum. Axoneme consists of 20 microtubules (arranged as a central pair surrounded by 9 peripheral doublets) and is surrounded by condensed fibrous rings.
Middle piece is the first part of the tail and consists of central axoneme surrounded by coarse longitudinal fibers. These are surrounded by elongated mitochondria that provide energy for movement of tail.
Principle or main piece constitutes most of the tail and is composed of axoneme that is surrounded by 9 coarse fibers. This central core is surrounded by many circularly arranged fibrous ribs.
Endpiece is the short tapering part composed of only axoneme.
Normally, > 30% of spermatozoa should show normal morphology (WHO, 1999). The defects in morphology that are associated with infertility in males include defective mid-piece (causes reduced motility), an incomplete or absent acrosome (causes inability to penetrate the ovum), and giant head (defective DNA condensation).
Box 832.1 Normal sperm morphology
• Total length of sperm: About 60 μ
• Total length of sperm: About 60 μ
• Head:
   – Length: 3-5 μ
   – Width: 2-3 μ
   – Thickness: 1.5 μ
• Neck: Length: 0.3 μ
• Middle piece:
   – Length: 3-5 μ
   – Width: 1.0 μ
• Principal piece:
   – Length: 40-50 μ
   – Width: 0.5 μ
• End piece: 4-6 μ
Abnormal morphology (Figure 832.3): WHO morphological classification of human spermatozoa (1999) is given below:
  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
Figure 832.3 Abnormal morphological sperm forms
Figure 832.3 Abnormal morphological sperm forms: (1) Normal sperm, (2) Large head, (3) Small head, (4) Tapered head, (5) Pyriform head, (6) Round head, (7) Amorphous head, (8) Vacuoles in head, (9) Round head without acrosome, (10) Double head, (11) Pin head, (12) Round head without acrosome and thick midpiece, (13) Coiled tail, and (14) Double tail

Round cells on microscopic examination may be white blood cells or immature sperm cells. Special stain (peroxidase or Papanicolaou) is required to differentiate between them. White blood cells >1 million/ml indicate presence of infection. Presence of large number of immature sperm cells indicates spermatogenesis dysfunction at the testicular level.


Published in Clinical Pathology
Monday, 14 August 2017 13:42
Biochemical markers (Table 831.1) can be measured in semen to test the secretions of accessory structures. These include fructose (seminal vesicles), zinc, citric acid or acid phosphatase (prostate), and α-glucosidase or carnitine (epididymis).
Table 831.1 Biochemical variables of semen analysis (World Helath Organization, 1992)
1. Total fructose (seminal vesicle marker) ≥13 μmol/ejaculate
2. Total zinc (Prostate marker) ≥2.4 μmol/ejaculate
3. Total acid phosphatase (Prostate marker) ≥200U/ejaculate
4. Total citric acid (Prostate marker) ≥52 μmol/ejaculate
5. α-glucosidase (Epididymis marker) ≥20 mU/ejaculate
6. Carnitine (Epididymis marker) 0.8-2.9 μmol/ejaculate
Resorcinol method is used for detection of fructose. In this test, 5 ml of resorcinol reagent (50 mg resorcinol dissolved in 33 ml concentrated hydrochloric acid; dilute up to 100 ml with distilled water) is added to 0.5 ml of seminal fluid. The mixture is heated and brought to boil. If fructose is present, a red-colored precipitate is formed within 30 seconds.
Absence of fructose indicates obstruction proximal to seminal vesicles (obstructed or absent vas deferens) or a lack of seminal vesicles. In a case of azoospermia, if fructose is absent, it is due to the obstruction of ejaculatory ducts or absence of vas deferens, and if present, azoospermia is due to failure of testes to produce sperm.


Published in Clinical Pathology
Monday, 14 August 2017 13:04
Examination is carried out after liquefaction of semen that occurs usually within 20-30 minutes of ejaculation.
Normal semen is viscous and opaque gray-white in appearance. After prolonged abstinence, it appears slightly yellow.
Immediately following ejaculation, normal semen is thick and viscous. It becomes liquefied within 30 minutes by the action of proteolytic enzymes secreted by prostate. If liquefaction does not occur within 60 minutes, it is abnormal. The viscosity of the sample is assessed by filling a pipette with semen and allowing it to flow back into the container. Normal semen will fall drop by drop. If droplets form ‘threads’ more than 2 cm long, then viscosity is increased. Increased semen viscosity affects sperm motility and leads to poor invasion of cervical mucus; it results from infection of seminal vesicles or prostate.
Volume of ejaculated semen sample should normally be > 2 ml. It is measured after the sample has liquefied. Volume < 2.0 ml is abnormal, and is associated with low sperm count.
4. pH
A drop of liquefied semen is spread on pH paper (of pH range 6.4-8.0) and pH is recorded after 30 seconds. Normal pH is 7.2 to 8.0 after 1 hour of ejaculation. The portion of semen contributed by seminal vesicles is basic, while portion from prostate is acidic. Low pH (< 7.0) with absence of sperms (azoospermia) suggests obstruction of ejaculatory ducts or absence of vas deferens. Low pH is usually associated with low semen volume (as most of the volume is supplied by seminal vesicles).


Published in Forensic Pathology
Sunday, 13 August 2017 17:08
This includes examination of material obtained from vagina, stains from clothing, skin, hair, or other body parts for semen. This is carried out in cases of alleged rape or sexual assault.
Collection of Sample
  • Vagina: Direct aspiration or saline lavage
  • Clothing: When scanned with ultraviolet light, semen produces green white fluorescence. A small piece (1 m2) of clothing from stained portion is soaked in 1-2 ml of physiologic saline for 1 hour. A similar piece of clothing distant from the stain is also soaked in saline as a control.

Presence of motile sperms in vaginal fluid indicates interval of < 8 hours. Smears prepared from collected samples are stained and examined for the presence of sperms.
Acid phosphatase is determined on vaginal or clothing samples. 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.
When semen is positively identified in vaginal fluid or other sample, test can be carried out for the presence of blood group substances in the same sample. The ‘secretor’ individuals (80% individuals are secretors) will secrete the blood group substances in body fluids, including semen.
This test detects the presence of choline found in high concentration in semen. To several drops of sample, add equal volume of reagent (iodine 2.54 g, potassium iodide 1.65 g, distilled water 30 ml); in positive test rhombic or needle-like crystals of periodide of choline form. False-positive tests can occur due to high choline content of some other body fluids.


Published in Clinical Pathology
Sunday, 13 August 2017 02:34
Definition of microscopic hematuria is presence of 3 or more number of red blood cells per high power field on microscopic examination of urinary sediment in two out of three properly collected samples. A small number of red blood cells in urine of low specific gravity may undergo lysis, and therefore hematuria may be missed if only microscopic examination is done. Therefore, microscopic examination of urine should be combined with a chemical test.
These detect both intracellular and extracellular hemoglobin (i.e. intact and lysed red cells) as well as myoglobin. Heme proteins in hemoglobin act as peroxidase, which reduces hydrogen peroxide to water. This process needs a hydrogen donor (benzidine, orthotoluidine, or guaiac). Oxidation of hydrogen donor leads to development of a color (Figure 828.1). Intensity of color produced is proportional to the amount of hemoglobin present.
Chemical tests are positive in hematuria, hemoglobinuria, and myoglobinuria.
Figure 828.1 Principle of chemical test for red cells
Figure 828.1 Principle of chemical test for red cells, hemoglobin, or myoglobin in urine
Benzidine Test
Make saturated solution of benzidine in glacial acetic acid. Mix 1 ml of this solution with 1 ml of hydrogen peroxide in a test tube. Add 2 ml of urine. If green or blue color develops within 5 minutes, the test is positive.
Orthotoluidine Test
In this test, instead of benzidine, orthotoluidine is used. It is more sensitive than benzidine test.
Reagent Strip Test
Various reagent strips are commercially available which use different chromogens (o-toluidine, tetramethylbenzidine).
Causes of false-positive tests:
  • Contamination of urine by menstrual blood in females
  • Contamination of urine by oxidizing agent (e.g. hypochlorite or bleach used to clean urine containers), or microbial peroxidase in urinary tract infection.
Causes of false-negative tests:
  • Presence of a reducing agent like ascorbic acid in high concentration: Microscopic examination for red cells is positive but chemical test is negative.
  • Use of formalin as a preservative for urine
Evaluation of positive chemical test for blood is shown in Figure 828.2.
Figure 828.2 Evaluation of positive chemical test for blood in urine
Figure 828.2 Evaluation of positive chemical test for blood in urine
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