Sunday, October 21, 2018

Hematoxylin and Eosin staining : principle, procedure and interpretation

Hematoxylin and Eosin (H & E) staining is the most common staining technique in histopathology. This uses a combination of two dyes, Hematoxylin and Eosin used for demonstration of nucleus and cytoplasmic inclusions in clinical specimens.

Principle

Alum acts as mordant and hematoxylin containing alum stains the nucleus light blue. This turns red in presence of acid, as differentiation is achieved by treating the tissue with acid solution. Bluing step converts the initial soluble red color within the nucleus to an insoluble blue color. The counterstaining is done by using eosin which imparts pink color to the cytoplasm.

Reagents

  1. Harri’s Hematoxylin stain
  2. A = 1 gm hematoxylin in 10 ml ethanol
    B = 20 gm ammonium alum in hot distilled water
    Mix A & B, boil and add 0.5 gm of mercuric oxide and filter.
  3. Eosin solution
  4. Yellow eosin = 1 gm
    Distilled water = 80 ml
    Ethanol = 320 ml
    Glacial Acetic Acid = 2 drops
  5. 0.5% HCl
  6. Dilute ammonia water

Procedure

  1. Deparaffinize the section : flame the slide on burner and place in the xylene. Repeat the treatment.
  2. Hydration : Hydrate the tissue section by passing through decreasing concentration of alcohol baths and water. (100%, 90%, 80%, 70%)
  3. Stain in hematoxylin for 3-5 minutes
  4. Wash in running tap water until sections “blue” for 5 minutes or less.
  5. Differentiate in 1% acid alcohol (1% HCl in 70% alcohol) for 5 minutes.
  6. Wash in running tap water until the sections are again blue by dipping in an alkaline solution (eg. ammonia water) followed by tap water wash.
  7. Stain in 1% Eosin Y for 10 minutes
  8. Wash in tap water for 1-5 minutes
  9. Dehydrate in increasing concentration of alcohols and clear in xylene
  10. Mount in mounting media
  11. Observe under microscope

Result and Interpretation

hematoxylin-and-eosin-staining
  • Nuclei : blue, black
  • Cytoplasm : Pink
  • Muscle fibres : deep red
  • RBCs : orange red
  • Fibrin : deep pink

Friday, October 19, 2018

Differences Between Transudates and Exudates

The closed cavities of the body—namely, the pleuralpericardial, and peritoneal cavities—are each lined by two membranes, visceral membrane and parietal membrane. There is a small amount of fluid between the membranes that is an ultrafiltrate of plasma. When the production and reabsorption of the ultrafiltrate is not balanced, fluid may accumulate, resulting in effusion. Effusions may be classified as transudate or exudate.
differences-between-transudate-and-exudate
Classifying a serous fluid as transudate or exudate can provide a valuable initial diagnostic step and aid diagnosis of underlying cause. Followings are the differences between transudate and exudate :
FeaturesTransudateExudate
CauseUsually develop from imbalances in hydrostatic and oncotic forces in circulation.Usually develop from increased capillary permeability or decreased lymphatic reabsorption.
Associated with-Congestive heart failure
-Fluid overload
-Nephrotic syndrome
-Hepatic cirrhosis
-Malnutrition
-Microbial infections
-Membrane inflammations
-Malignancy
-Connective tissue diseases.
AppearanceClear, thin-colored, pale yellowTurbid, hemorrhagic, straw colored
FibrinogenLow content of fibrinogen (low tendency to clot)High content of fibrinogen (high tendency to clot)
Specific gravity<1.012>1.012
pH>7.3<7.3
Glucose contentSame as plasmaLow (less than 60 mg/dl)
Total ProteinLess than 3 gm/dlMore than 3 gm/dl
Fluid/Serum Protein<0.5>0.5
LDH<0.67 x UNL Serum>0.67 x UNL Serum
WBC count<1000/ul>1000/ul
Differential countMesothelial cells or lymphocytesPolymorphs, lymphocytes or RBCs
CultureSterilePositive

Thursday, October 18, 2018

Coombs Test : Types, Principle, Procedure and Interpretation

The Coomb’s test (also known as Antiglobulin Test or AGT) refers to two clinical blood tests used in immunohematology which are done to find certain antibodies that cause autoimmune haemolysis of red blood cells (erythrocytes). The two types of Coombs tests are:
  • Direct Coombs test
  • Indirect Coombs test

Principle of Coombs Test

In certain diseases or conditions, an individual’s blood may contain IgG antibodies that can specifically bind to antigens on the red blood cell (RBC) surface membrane. Red cells coated with complement or IgG antibodies do not agglutinate directly when centrifuged. These cells are said to be sensitized with IgG or complement. In order for agglutination to occur an additional antibody, which reacts with the Fc portion of the IgG antibody, or with the C3b or C3d component of complement, must be added to the system. Because antibodies are gamma globulins, an antibody to gamma globulin can form bridges between red cells sensitized with antibody and cause them to agglutinate.

Direct Coombs Test

The direct Coombs test (also known as the direct antiglobulin test or DAT) is used to detect if antibodies or complement system factors have bound to RBC surface antigens in vivo. A blood sample is taken and the RBCs are washed and then incubated with antihuman globulin. If this produces agglutination of RBCs, the direct Coombs test is positive, a visual indication that antibodies are bound to the surface of red blood cells.
This is the test that is done on the newborn’s blood sample, usually in the setting of a newborn with jaundice. The test is looking for “foreign” antibodies that are already adhered to the infant’s rbcs, a potential cause of hemolysis.
direct-coombs-test

PROCEDURE :

  1. Prepare 5% cell saline suspension of the cells to be tested.
  2. Label 3 tubes as TPC and NC.
  3. In the tube labeled as T (Test), take 2 drops of 5% saline cell suspension to be tested.
  4. In the test tube labeled as PC (Positive control), take 1 drop of anti D sera and 1 drop of Rh +ve pooled cells.
  5. In the test tube labeled as NC (Negative control), take 1 drop of normal saline and one drop of Rh +ve pooled cells.
  6. Add 2 drops of Anti human globulin to each of the tubes.
  7. Mix well and centrifuge for 1 minute at 1500 rpm.
  8. Resuspend the cells by gentle agigation and examine macroscopically and microscopically for agglutination.

Indirect Coombs Test

The indirect Coombs test (also known as the indirect antiglobulin test or IAT) is used to detect in-vitro antibody-antigen reactions. It is used to detect very low concentrations of antibodies present in a patient’s plasma/serum prior to a blood transfusion. In antenatal care, this test is used to screen pregnant women for antibodies that may cause hemolytic disease of the newborn. The IAT can also be used for compatibility testing, antibody identification, RBC phenotyping, and titration studies.
indirect-coombs-test

PROCEDURE :

  1. Label 3 tubes as TPC and NC.
  2. In the tube labeled as T (Test), take 2 drops of test serum.
  3. In the test tube labeled as PC (Positive control), take 1 drop of anti D serum.
  4. In the test tube labeled as NC (Negative control), take 1 drop of normal saline.
  5. Add one drop of 5% cell saline suspension of pooled O Rh +ve cells in each tubes.
  6. Incubate all the tubes at 37°C for 1 hour.
  7. Wash the cells 3 times with normal saline.
  8. Add 2 drops of Anti Human Globulin to each tube.
  9. Keep for 5 minutes and centrifuge at 1500 rpm for 1 minute.
  10. Resuspend the cells and examine macroscopically as well as microscopically for agglutination.

Interpretation

If the blood coagulates, it can be concluded that the patient’s red blood cells have been bound by (his/her own) immunoglobulins. Of course, this isn’t the normal state of affairs, and implies that the patient is experiencing an autoimmune haemolysis of his/her red cells.

Wednesday, October 17, 2018

Cross-Matching : Types, Purpose, Principle, Procedure and Interpretation

Cross Matching is a procedure performed prior to a blood transfusion to determine whether donor blood is compatible (or incompatible) with recipient blood. Compatibility is determined through matching of different blood group systems, the most important of which are the ABO and Rh system, and/or by directly testing for the presence of antibodies against a sample of donor tissues or blood.

Purpose of Cross Matching

The crossmatch is routinely used as the final step of pretransfusion compatibility testing. The purposes of compatibility testing are to detect: irregular antibodies; errors in ABO grouping, and clerical errors in patient identification and result recording. The crossmatch will detect the following:
1. Most recipient antibodies directed against antigens on the donor red blood cells.
2. Major errors in ABO grouping, labeling, and identification of donors and recipients.

Principle

Cross-matching will detect incompatibilities between the donor and recipient that will not be evident on blood typing. There are two types of cross-matches: Major cross-match and Minor cross-match.
The major crossmatch involves testing the patient’s serum with donor cells to determine whether the patient has an antibody which may cause a hemolytic transfusion reaction or decreased cell survival of donor cells. This is the most important cross-match.
The minor crossmatch involves testing the patients cells with donor plasma to determine whether there is an antibody in the donor’s plasma directed against an antigen on the patient’s cells.

Procedure

  1. Prepare donor and recipient blood samples:
    For Major crossmatch : Donor’s red cell and recipient serum or plasma
    For Minor crossmatch : Recipient red cells and donor’s serum or plasma
  2. Prepare 3 – 5% cell suspensions of red cells.
  3. Major Crossmatch:
    Label a test tube. Add two drops of the patient serum and one drop of the appropriate donor cell suspension.
  4. Minor Crossmatch:
    Label a test tube. Add two drops of the appropriate donor serum and one drop of the patient cell suspension.
  5. Mix the tubes and incubate at 37°C for about 45 minutes.
  6. Add two drops of AHG (Antihuman globulin) and mix well.
  7. Centrifuge for 1 minute at 1500 rpm
  8. Read macroscopically and microscopically and record the results

Interpretation

cross-match
The mixture of erythrocytes and serum are observed for hemolysis or microscopically for agglutination. Any evidence of hemolysis/agglutination indicates an incompatible cross-match. Negative results are taken to indicate compatibility.

Zika Virus : Structure, Epidemiology, Pathogenesis, Symptoms, Laboratory Diagnosis and Prevention

The Zika virus (ZIKV) is a flavivirus related to Dengue, Yellow Fever virus, Japanese encephalitis virus and West Nile virus. It is responsible for mosquito-transmitted infection known as Zika fever or Zika disease. Zika Virus is commanding worldwide attention recently because researchers have found evidence that Zika may be linked to birth defects and neurological conditions like microcephaly and Guillain-BarrĂ© syndrome in adults.

SYSTEMATIC CLASSIFICATION OF ZIKA VIRUS

  • Group: Group IV ((+)ssRNA)
  • Family: Flaviviridae
  • Genus: Flavivirus
  • Species: Zika virus

STRUCTURE OF ZIKA VIRUS

zika virus
Zika virions are typically icosahedral-shaped. They are enveloped, 18-45 nanometer in diameter.
The genome is a positive strand RNA enclosed in a capsid and surrounded by a membrane. The RNA contains 10,794 nucleotides encoding 3,419 amino acids.
The virus is inactivated by ether, sodium dexoxycholate and chloroform.

EPIDEMIOLOGY

  • Zika virus was first isolated in 1947 from the blood of a Rhesus monkey (Macaca mulatta) in Zika forest, near Entebbe in Uganda. Subsequently, the virus was recovered from humans and mosquitoes in Uganda, Senegal, Nigeria, Ivory Coast, the Central African Republic and Malaysia.
  • In 2007, a large outbreak of Zika virus infection occured in Yap Island of Micronesia, where nearly 75% of the population was infected.
  • Zika virus did not begin spreading widely in the Western Hemisphere until last May 2015, when the public health authorities of Brazil confirmed an outbreak in northeast Brazil.
  • According to WHO, since Brazil reported the first cases of local transmission, Zika virus has spread to 21 countries and territories of the Americas. The WHO estimates 3 million to 4 million people across the Americas will be infected with the virus in the next year.
zika-virus-epidemiology
Source: Centers for Disease Control and Prevention

PATHOGENESIS OF ZIKA VIRUS

Transmission :

Zika virus spreads to people through mosquito bites. The virus was recovered from mosquitoes of Aedes genus including Aedes africanus, Aedes apicoargenteus, Aedes leuteocephalus, Aedes aegypti, Aedes vitattus and Aedes furcifer. Some evidence suggest that Zika virus can also be transmitted to humans through blood transfusion, perinatal transmission and sexual transmission. However these modes are very rare. The virus was found on one occasion in semen.
The disease cycle continues as Resorvoir host to mosquito to reservoir host; 2-5 days viremia in host, 5-7 days in mosquito, then back to the host.
Information regarding pathogenesis of ZIKV is scarce but mosquito-borne flaviviruses are thought to replicate initially in dendritic cells near the site of inoculation then spread to lymph nodes and the bloodstream. Although flaviviral replication is thought to occur in cellular cytoplasm, One study suggested that ZIKV antigens could be found in infected cell nuclei.

Incubation Period :

The incubation period (the time from exposure to appearance of symptoms) of Zika virus disease is not clear, but is likely to be 3-12 days.

Signs and Symptoms :

About only 20-25% of people infected with Zika virus develop symptoms. The most common symptoms of Zika virus infection are:
  • Fever
  • Maculopapular rashes
  • Joint pain (arthritis, arthralgia)
  • Conjunctivitis(red eyes)
  • Muscle pain
The zika infection is more serious as it is associated with two neurological conditions:
  • Microcephaly : A serious birth defect in which babies have small heads and incomplete brain development. This may occur when mother gets infected during the first trimester of pregnancy.
  • Guillain-BarrĂ© syndrome : Guillain-Barre syndrome (GBS) is a rare disorder where a person’s own immune system damages the nerve cells, causing muscle weakness and sometimes, paralysis.

LABORATORY DIAGNOSIS OF ZIKA VIRUS

There is no widely available test for Zika infection. In most people, diagnosis is based on clinical symptoms and epidemiological circumstances (such as Zika outbreak in the patient’s area or trips to areas where the virus is circulating). Diagnostic tests for ZIKV infection include :
  1. Polymersase Chain Reaction :
    Nucleic acid detection by reverse transcriptase-polymerase chain reaction (RT-PCR) targeting the non-structural protein 5 genomic region is the primary means of diagnosis. It is useful in the first 3-5 days after the onset of symptoms.
  2. Serological Tests :
    An ELISA has been developed to detect IgM to ZIKV only after five days. Because it is closely related to dengue and yellow fever, it may cross-react with antibody tests for those viruses.
  3. Nucleic Acid Amplification Test :
    Nucleic acid amplification test (NAT) for detection of viral RNA can also be performed.
  4. Plaque Reduction Neutralization Assay :
    The Plaque reduction neutralization assay generally has improved specificity over immunoassays, but may still yield cross-reactive results in secondary flavivirus infections.

TREATMENT OF ZIKA VIRUS

No specific vaccine or medications are available to prevent or treat ZIKA virus infections. The symptoms are mild – when they appear at all – and usually require only rest, nourishment and other supportive care.

PREVENTION AND CONTROL OF ZIKA VIRUS

  1. Elimination and control of mosquito : Avoid allowing standing water in outdoor containers so that they do not become mosquito breeding sites, avoid accumulating garbage, use mosquito nets in windows and doors.
  2. Prevention of mosquito bites : Personal protection measures to avoid mosquito bites should be applied when staying in risk areas, sleep under mosquito nets, do not travel in affected areas.
  3. Public awareness about Zika and mosquitoes : Peoples should be made aware about the disease and it’s preventive measures. They should take the basic precautions to protect themselves from the disease.

Erythrocyte Sedimentation Rate (ESR) : Principle, Methods of Determination and Clinical Significance

The erythrocyte sedimentation rate (ESR) is a common hematological test for nonspecific detection of inflammation that may be caused by infection, some cancers and certain autoimmune diseases. It can be defined as the rate at which Red Blood Cells (RBCs)sediment in a period of one hour.

PRINCIPLE OF ESR

When anticoagulated blood is allowed to stand in a narrow vertical glass tube, undisturbed for a period of time, the RBCs – under the influence of gravity- settle out from the plasma. The rate at which they settle is measured as the number of millimeters of clear plasma present at the top of the column after one hour(mm/hr). This mechanism involves three stages:
  • Stage of aggregation : It is the initial stage in which piling up of RBCs takes place. The phenomenon is known as Rouleaux formation. It occurs in the first 10-15 minutes.
  • Stage of sedimentation : It is the stage of actual falling of RBCs in which sedimentation occurs at constant rate. This occurs in 30-40 minutes out of 1 hour, depending upon the length of the tube used.
  • Stage of packing : This is the final stage and is also known as stationary phase. In this, there is a slower rate of falling during which packing of sedimented RBCs in column occurs due to overcrowding. It occurs in final 10 minutes in 1 hour.

METHOS OF ESR DETERMINATION

There are two main methods to determine ESR :
  • Wintrobe’s method
  • Westergren’s method
Each method produces slightly different results. Mosely and Bull (1991) concluded that Wintrobe’s method is more sensitive when the ESR is low, whereas, when the ESR is high, the Westergren’s method is preferably an indication of patient’s clinical state.

WINTROBE’S METHOD

wintrobe-tube-esr
This method uses Wintrobe’s tube, a narrow glass tube closed at the lower end only. The Wintrobe’s tube has a length of 11 cm and internal diameter of 2.5 mm. It contains 0.7-1 ml of blood. The lower 10 cm are in cm and mm. The marking is 0 at the top and 10 at the bottom for ESR. This tube can also be used for PCV. The marking is 10 at the top and 0 at the bottom for PCV.

REQUIREMENTS :

  • Anticoagulated blood (EDTA, double oxalate)
  • Pasteur pipette
  • Timer
  • Wintrobe’s tube
  • Wintrobe’s stand

PROCEDURE :

  1. Mix the anticoagulated blood thoroughly.
  2. By using Pasteur pipette, fill the Wintrobe’s tube upto ‘0’ mark. There should be no bubbles in the blood.
  3. Place the tube vertically in ESR stand and leave undisturbed for 1 hour.
  4. At the end of 1 hour, read the result.

NORMAL VALUE :

For males : 0-9 mm/hr
For females 0-20 mm/hr

WESTERGREN’S METHOD

It is better method than Wintrobe’s method. The reading obtain is magnified as the column is lengtheir. The Westregren tube is open at both ends. It is 30 cm in length and 2.5 mm in diameter. The lower 20 cm are marked with 0 at the top and 200 at the buttom. It contains about 2 ml of blood.
westergren-method-esr

REQUIREMENTS :

  • Anticoagulated blood (0.4 ml of 3.13% trisodium citrate solution + 1.6 ml blood)
  • Westergren tube
  • Westergren stand
  • Rubber bulb (sucker)

PROCEDURE :

  1. Mix the anticoagulated blood thoroughly.
  2. Draw the blood into the tube upto 0 mark with the help of rubber bulb.
  3. Wipe out blood from bottom of the tube with cotton.
  4. Set the tube upright in stand. Make sure the pipette fits snugly to eliminate possible leakage and that the pipette is in vertical position.
  5. Leave the tube undisturbed for 1 hour.
  6. At the end of 1 hour, read the result.

NORMAL VALUE :

For males : 0-10 mm/hr
For females : 0-15 mm/hr

Clinical Significance of ESR

The erythrocyte sedimentation rate (ESR) is a non-specific test. It is raised in a wide range of infectious, inflammatory, degenerative, and malignant conditions associated with changes in plasma proteins, particularly increases in fibrinogen, immunoglobulins, and C-reactive protein. The ESR is also affected by many other factors including anaemia, pregnancy, haemoglobinopathies, haemoconcentration and treatment with anti-inflammatory drugs.

Causes of a significantly raised ESR :

  • All types of anemias except sickle cell anemia
  • Acute and chronic inflammatory conditions and infections including:
    – HIV disease
    – Tuberculosis
    – Acute viral hepatitis
    – Arthritis
    – Bacterial endocarditis
    – Pelvic inflammatory disease
    – Ruptured ectopic pregnancy
    – Systemic lupus erythematosus
  • African trypanosomiasis (rises rapidly)
  • Visceral leishmaniasis
  • Myelomatosis, lymphoma, Hodgkins disease, some tumours
  • Drugs, including oral contraceptives

Causes of Reduced ESR :

  • Polycythaemia
  • Poikilocytosis
  • Newborn infants
  • Dehydration
  • Dengue haemorrhagic fever
  • and other conditions associated with haemoconcentration