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Sahli’s Method For The Estimation Of Hemoglobin

Dhurba Giri — Fri, 18 Nov 2022 15:33:42 +0000

Hemoglobin (Hb) is a conjugated protein present in Red Blood Cells (RBCs). It serves two important functions in the body- transportation of oxygen and carbon dioxide in between tissues and lungs and acts as a buffer in maintaining blood pH. Normal Hgb is composed of four heme molecules nested in four globin molecules. Among four globin molecules, two chains are alpha chains and two are non-alpha chains. Based on the type of non-alpha chain present, three different types of hemoglobin are found in normal adults.

HbA: 96-98%, Composed of two alpha(α) and two beta(β) globin chains.
HbA2: 1.5-3.5%, Composed of two alpha(α) and two delta(δ) globin chains.
HbF: < 1.0%, Composed of two alpha(α) and two gamma(γ) globin chains.

Hemoglobin can combine with other substances, some normally and some abnormally and can also occur as:

Oxyhemoglobin: Oxygen combined with hemoglobin.
Carboxyhemoglobin: Carbon monoxide (CO) combined with hemoglobin.
Carbaminohemoglobin: Carbon dioxide (CO₂) combined with hemoglobin.
Methemoglobin: Iron oxidized from its ferrous state to ferric state.
Sulfhemoglobin: Sulfur combined with the hemoglobin.
Cyanmethemoglobin: Methemoglobin bonded to cyanide ions.

Methods for Hemoglobin Estimation:

The different methods used for the estimation of hemoglobin can be divided as follows:

Visual methods:
- Sahli’s method
- Dare method
- Haden method
- Wintrobe method
- Tallqvist method
Spectrophotometric methods:
- Oxyhemoglobin method
- Cyanmethemoglobin method
Gasometric method
Automated hemoglobinometry
Other methods:
- Alkaline-hematin method
- Specific gravity method
- Lovibond Comparator method

Sahli’s method for hemoglobin estimation

Sahli’s method, also called as acid hematin method is the visual comparator method for the estimation of hemoglobin. As visual comparison may lead to unacceptable imprecision and accuracy, this method is not recommended nowadays and the use of spectrophotometric methods like Cyanmethemoglobin method is preferred to it.

Principle:

When the blood is added to dilute hydrochloric acid (HCl), hemoglobin present in the RBCs is converted into brown-colored acid hematin. The acid hematin solution is further diluted until it’s color matches exactly with the permanent standard brown glass compared by direct vision.

Requirements:

Specimen:

Capillary or venous blood. Venous blood should be anticoagulated with 1.5-1.8 mg EDTA per mL of blood and mixed immediately.

Instruments:

Sahli’s hemoglobinometer
It is a set of devices that includes a comparator, hemoglobin tube, hemoglobin pipette, and stirrer.
- Comparator: It is a rectangular plastic box with a slot in the middle which accommodates a hemoglobin tube. Brown standard glasses are provided on either side of the slot for color matching. White opaque glass is present at the back to provide uniform illumination.
- Hemoglobin tube: Sahli’s graduated hemoglobin tube is graduated in one side in gram percentage (g%) from 2 to 24, and on the other side in percentage (%) from 20 to 140. The tube is also called Sahli-Adams tube.
- Sahli’s pipette or hemoglobin pipette: It contains only one mark at 20μl or 0.02ml. Unlike WBC and RBC diluting pipettes, it contains no bulb.
- Stirrer: It is a thin glass rod used for stirring the mixture inside the hemoglobin tube.

Reagents:

N/10 Hydrochloric acid (HCl): Mixing 36 grams HCl in distilled water to 1 liter gives 1 N HCl. Diluting it 10 times will give N/10 HCl.
Distilled water

Procedure:

Ensure that the hemoglobinometer tubes and pipette are clean and dry.
Fill the hemoglobinometer tube with N/10 HCl up to its lowest mark i.e. 2 g% or 10% mark with the help of a dropper.
Take blood up to mark in the Sahli’s pipette (20 μl). Wipe the extra blood outside the pipette and deliver it to N/10 HCl in the hemoglobin tube.
Mix and leave it for 10 minutes in order for a complete conversion of hemoglobin to hematin.
Add distilled water drop by drop and stir till color matches with the standard glass of the comparator.
Take the reading at lower meniscus, which directly gives the hemoglobin concentration in 100 ml of blood.

Advantages and Disadvantages of Sahli’s method

Advantages:

Easy to perform and convenient.
Not very time consuming. Can be performed within maximum 15 minutes.
Reagents and apparatus are cheap and easily available. Reagents are less harmful.
Can be used in mass surveys. Doesn’t require electricity.

Disadvantages:

Acid hematin is a suspension, not a true solution. So, some turbidity may result.
This method can’t measure all hemoglobins. It estimates only oxyhemoglobin and reduced hemoglobins. But carboxyhemoglobin, methemoglobin and sulfhemoglobin (which all constitute about 2-12% of total hemoglobin) are not converted to acid hematin.
HbF is not converted to acid hematin. Therefore, Shali’s method is not suitable for measuring hemoglobin in infants upto 3 months.
WBC counts >100,000/cumm will produce turbid solution of acid hematin, which will increase the hemoglobin report by 5-10%.
Chances of visual error are high. Color of acid hematin also fades gradually.
The color of glass standard may fade over time.

References:

Pal, G.K., 2006. Textbook Of Practical Physiology-2Nd Edn. Orient Blackswan.
Ghai, C.L., 2012. A textbook of practical physiology. JP Medical Ltd.
A. V. Naigaonkar. A Manual Of Medical Laboratory Technology.
Cheesebrough, M., 1998. District laboratory practice in tropical countries, part II. Cambridgeshire Tropical Health Technology, Cambridge, UK, 231.
Laboratory Procedures in Clinical Hematology By United States. Department of the Army
Mohan, H. and Mohan, S., 2011. Practical Pathology for Dental Students. JP Medical Ltd.

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Cyanmethemoglobin Method For The Estimation Of Hemoglobin

Dhurba Giri — Thu, 17 Nov 2022 14:53:09 +0000

HbA: 96-98%, Composed of two alpha(α) and two beta(β) globin chains.
HbA2: 1.5-3.5%, Composed of two alpha(α) and two delta(δ) globin chains.
HbF: < 1.0%, Composed of two alpha(α) and two gamma(γ) globin chains.

Hemoglobin can combine with other substances, some normally and some abnormally and can also occur as:

Oxyhemoglobin: Oxygen combined with hemoglobin.
Carboxyhemoglobin: Carbon monoxide (CO) combined with hemoglobin.
Carbaminohemoglobin: Carbon dioxide (CO₂) combined with hemoglobin.
Methemoglobin: Iron oxidized from its ferrous state to ferric state.
Sulfhemoglobin: Sulfur combined with the hemoglobin.
Cyanmethemoglobin: Methemoglobin bonded to cyanide ions.

Methods for Hemoglobin Estimation:

The different methods used for the estimation of hemoglobin can be divided as follows:

Visual methods:
- Sahli’s method
- Dare method
- Haden method
- Wintrobe method
- Tallqvist method
Spectrophotometric methods:
- Oxyhemoglobin method
- Cyanmethemoglobin method
Gasometric method
Automated hemoglobinometry
Other methods:
- Alkaline-hematin method
- Specific gravity method
- Lovibond Comparator method

Cyanmethemoglobin method for hemoglobin estimation

The cyanmethemoglobin method or hemoglobincyanide method is the most accurate method of choice for the measurement of hemoglobin. It is a type of colorimetric/spectrophotometric method which has the following three major advantages over other methods:

Measures all forms of hemoglobin except sulfhemoglobin, which is normally not present in the blood.
Can be easily standardized, and
Cyanmethemoglobin reagent is very stable.

Principle:

Blood is diluted 1:201 in a solution containing potassium ferricyanide (C₆N₆FeK₃) and potassium cyanide (KCN). Potassium ferricyanide oxidizes hemoglobin in the sample to methemoglobin. The methemoglobin further reacts with potassium cyanide to form a stable-colored cyanmethemoglobin (hemiglobincyanide- HiCN) complex. The intensity of the colored complex is measured at 540 nm which is directly proportional to the amount of hemoglobin present in the specimen.

Requirements:

Specimen:

Capillary or venous blood. Venous blood should be anticoagulated with 1.5-1.8 mg EDTA per mL of blood and mixed immediately.

Reagents:

Drabkin’s reagent, pH 7.0–7.4
The original cyanmethemoglobin technique was proposed by Stadie in 1920. This method used separated alkaline ferricyanide and cyanide reagents. A single reagent was introduced by Drabkin and Austin in 1935.

This fluid contains:
- Potassium ferricyanide = 200 mg
- Potassium cyanide = 50 mg
- Potassium dihydrogen phosphate = 140 mg
- Non-ionic detergent = 1 ml
- Distilled or deionized water = to 1 liter
Hemoglobin standard

Procedure:

Label three clean, dry test tubes as Blank (B), Standard (S), and Test (T).
Pipette as follows:

	Blank	Standard	Test
Drabkin’s Reagent	5 ml	5 ml	5 ml
Hemoglobin standard	–	20 µl	–
Sample	–	–	20 µl

Mix well and allow to stand at room temperature (25⁰C) for 5 minutes.
Measure the absorbance of the standard and test sample at 540 nm (green filter) against blank in a colorimeter. The color will be stable for up to several hours.

Calculation:

Calculate the concentration of hemoglobin in the specimen using the following formula:

Alternatively, a calibration graph can be prepared using multiple standards of varying concentration and the result can be obtained quickly by checking hemoglobin concentration which corresponds to obtained absorbance. This is markedly acceptable when a large number of specimens are daily processed on the same instrument.

References:

Pal, G.K., 2006. Textbook Of Practical Physiology-2Nd Edn. Orient Blackswan.
DR .Sara Fadhil Bunea, Medical laboratory techniqes, Human physiology practical.
Haemoglobin Reagent Product Insert – Tulip Diagnostics

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May Grunwald-Giemsa Stain: Principle, Preparation and Procedure

Dhurba Giri — Sat, 19 Feb 2022 15:40:22 +0000

May Grunwald-Giemsa stain(MGG) is a type of Romanowsky stain, which is used routinely for staining of air-dried cytological smears, blood, and bone marrow smears. Cytological preparations made from FNAC and serous fluids are normally processed by May Grunwald-Giemsa stain. It is useful for studying cellular morphology and is superior to PAP stain to study cytoplasm, granules, vacuoles, and basement membrane.

Principle

May Grunwald-Giemsa stain is a combination of two stains: May Grunwald stain and Giemsa stain.

May Grunwald stain is alcohol-based stain composed of methylene blue and eosin.
Giemsa stain is alcohol-based stain composed of methylene blue, eosin and azure B.

The working principle of MGG stain is the same as that of other Romanowksy stains. Polychromatic Romanowksy dyes contain different ratios of methylene blue (and the reagent-related thiazine dyes, such as azure B), as the cation (+vely charged, basic) component, and eosin Y as the anion (-vely charged, acidic) component. Cation and anion components in combination produce the well-known Romanowsky effect or metachromasia.

The solvent methanol initially fixes the cells. The basic dyes carry net positive charges; consequently, they stain nuclei (because of the negative charges of phosphate groups of DNA and RNA molecules), granules of basophil granulocytes, and RNA molecules of the cytoplasm. The eosin carries a net negative charge and stains red blood cells and granules of eosinophil granulocytes. Buffer solution of pH 6.5-6.8 is used to enable the dye to precipitate and bind well with the cellular material.

Reagents

May Grunwald Stain

Stock solution:
May Grunwald dye= 0.3 gm
Methanol = 100 ml
Working Solution:
Stock solution= 20 part
Phosphate Buffer (pH 6.8)= 30 part

Giemsa Stain

Stock Solution:
Giemsa Powder= 1 gm
Glycerine= 66ml
Absolute ethanol= 66ml
Mix giemsa and glycerine, place in 60C oven for 30 minutes 2 hr. Add 66ml methanol
Working Solution:
Stock Giemsa= 50 drops
Distilled Water=50ml

Procedure

Prepare a thin smear and air dry.
Fix smears for 5-10 minutes with methanol.
Stain the smear in May Grunwald working solution for 10 minutes.
Rinse in pH 6.8 buffer.
Stain the slides with diluted Giemsa stain for 30 minutes.
Wash the smears with distilled water and let them dry.
Mount the slide with DPX and examine under microscope.

Results

Erythrocytes: Light pink to light purple
Platelets: Granules – Reddish purple
Lymphocytes/monocytes: Nuclei – Dark purple, Cytoplasm – Sky blue
Neutrophils: Nuclei – Dark blue, Granules – Reddish purple, Cytoplasm – Pale pink
Eosinophils: Nuclei – Blue, Granules – Red/orange red, Cytoplasm – Blue
Basophils: Nuclei – Dark blue, Granules – Purple

References

Dey, P. (2018). Basic and advanced laboratory techniques in histopathology and cytology. Springer Singapore.
Matutes, E., Pickl, W. F., Van’t Veer, M., Morilla, R., Swansbury, J., Strobl, H., … & Ludwig, W. D. (2011). Mixed-phenotype acute leukemia: clinical and laboratory features and outcome in 100 patients defined according to the WHO 2008 classification. Blood, The Journal of the American Society of Hematology, 117(11), 3163-3171.
Product Information May-Grünwald Giemsa, Avantor Performance Materials.

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Arneth Count for Neutrophils: Principle, Procedure and Clinical Significance

Dhurba Giri — Sat, 02 Jan 2021 06:02:28 +0000

The Arneth count is the determination of the percentage distribution of different types of neutrophils on the basis of their nuclear lobes. It is not commonly used in modern medicine. Joseph Arneth classified neutrophils into following five types.

Stage	Nuclear lobes	Normal Range
Stage 1 (N1)	Nucleus is unilobed. The nucleus is C or U shaped.	2-5%
Stage 2 (N2)	Nucleus is bilobed. Two lobes are separated by thin strands.	20-30%
Stage 3 (N3)	Nucleus is trilobed. Three lobes are separated by thin strands.	40-50%
Stage 4 (N4)	Nucleus is tetralobed. Four lobes are separated by thin strands.	10-15%
Stage 5 (N5)	Nucleus is pentalobed. Five lobes are separated by thin strands.	2-5%

Principle of Arneth Count

The staging of neutrophils is based on the degree of maturity. The younger neutrophils contain fewer nuclear lobes than the older ones. The neutrophils enter the bloodstream mostly as bilobed cells, but the number of lobes increase to 5 or more by the end of their short life span of 8-10 hours.

Neutrophils are grouped into different types (N1, N2, N3, N4 and N5) on the basis of nucelar lobes by examining a stained smear under oil-immersion objective. In case of difficulty in determing nuclear lobes, two other parameters can be considered to stage the neutrophils.

The number of granules: the younger cells contain more granules.
The size oth the cell: cell size decreases as the cell advances on age.

Procedure

Prepare a thin blood smear as in DLC, stain with Leishman/Wright’s stain.
Examine the smear under low power objective to assess the distribution of cells.
Count neutrophils under oil immersion objective as N1, N2, N3, N4 and N5 for neutrophils having 1, 2, 3, 4 and 5 nuclear lobes respectively. Count the cells in the same zigzag pattern as in DLC.
Count at least 100 neutrophils and and enter your observations in a tabular format. Note the percentage distribution of various stages of neutrophils and plot a graph.

Clinical Significance of Arneth Count

The Arneth count is used to determine the number of younger or older neutrophils in the cirulation. As it reveals the production of neutrophils, it indirectly reflects the activity of the bone marrow. When there are more younger cells in circulation, the change is called shift to left and when there are more older cells in the circulation, the change is called shift to right.

Shift to Left

The total cells counted in N1, N2 and N3 are more than 80%. It indicates that bone marrow is hyperactive. Because left shift occurs due to increased production of cells, this is also called regenerative shift. Conditions related to this are:

Acute pyogenic infections
Tuberculosis
Hemorrhage
Irradiation

Shift to Right

The total cells counted in N4 and N5 are more than 20%. It indicates that bone marrow is hypoactive. Because right shift occurs due to hypofunction of bone marrow, this is also called degenerative shift. Conditions related to this are:

Megaloblastic anemia
Aplastic anemia
Septicemia
Uremia

References

Pal, Gopal Krushna. Textbook of practical physiology. Orient Blackswan, 2001.
Indu, Khurana. Textbook of medical physiology. Elsevier 1 (2009): 777.

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Wright’s Stain : Preparation, Principle, Procedure and Results

Dhurba Giri — Thu, 25 Jul 2019 14:58:04 +0000

Wright’s stain is a type of Romanowsky stain, which is commonly used in hematology laboratory for the routine staining of peripheral blood smears. It is also used for staining bone marrow aspirates, urine samples and to demonstrate malarial parasites in blood smears.
Wright’s stain is named for James Homer Wright, who devised the stain in 1902 based on a modification of Romanowsky stain. The stain distinguishes easily between the blood cells and hence became widely used for performing differential WBC counts and evaluate the morphology of blood cells.

Principle

Wright’s stain is a polychromatic stain consisting of a mixture of Eosin and Methylene blue. As the Wright stain is methanol based, it doesn’t require a fixation step prior to staining. However, fixation helps to reduce water artefact that can occur on humid days or with aged stain.

Methanol fixes the cells to the slide. Eosin Y is an acidic anionic dye and methylene blue is basic cationic dye. When diluted in buffered water, ionization occurs. Eosin stains the basic components such as hemoglobin and eosinophilic granules an orange to pink color. Methylene blue stains acidic cellular components such as nucleic acid and basophilic granules in varying shades of blue. The neutral components of the cells are stained by both components of the dye, producing variable colors.

Reagents

The dye may be purchased as a powder which is then mixed to methanol or a ready-made solution may be obtained.

Staining Solution
Wright’s stain powder = 1.0 gm
Water free methanol = 400 ml
Phosphate buffer (0.15M, ph 6.5/6.8)
Potassium dihydrogen phosphate, anhydrous = 0.663 gm
Disodium hydrogen phosphate, anhydrous = 0.256 gm
Distilled water = 100 ml

Procedure

Prepare a film of blood or bone marrow on a microscopic slide and allow to air dry.
Place the air-dried smear on the slide staining rack, smear side facing upwards.
Cover the blood film with undiluted staining solution. The undiluted stain fixes and partially stains the smear.
Let stand for 2-3 minutes.
Add approximately equal amount of buffered water (pH 6.5). The diluted stain shouldn’t overflow. Mix by gentle blowing. A metallic sheen (or green ‘scum’) should appear on the slide if mixing is appropriate.
Leave for 5 minutes.
Without disturbing the slide, flood the distilled water and wash until the thinner parts of the film are pinkish red.
Allow the slide to dry at room temperature and examine under microscope.

Results

Cells	Result
Erythrocytes	Yellowish-red
Neutrophils	Nucleus: Dark purple Granules: Reddish ileac granules Cytoplasm: Pale pink
Eosinophils	Nucleus: Blue Granules: Red to orange red Cytoplasm: Blue
Basophils	Nucleus: Purple to dark blue Granules: Dark purple
Lymphocytes	Nucleus: Dark purple Cytoplasm: Sky blue
Monocytes	Nucleus: Dark purple Cytoplasm: Mosaic pink and blue
Platelets	violet to purple granules

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Laboratory Diagnosis of Megaloblastic Anemia (MBA)

Dhurba Giri — Sun, 21 Jul 2019 03:34:11 +0000

Anemia is a condition of decrease in number of circulating red blood cells (and hence hemoglobin) below a normal range for age and sex of the individual, resulting in decreased oxygen supply to tissues. Megaloblastic anemia is a type of macrocytic anemia, which is characterized by defective DNA synthesis and presence of distinct megaloblasts in the bone marrow. This anemia is caused due to the deficiency of Vitamin B12 and/or Folic acid. Less commonly, also due to acquired abnormalities of their metabolism.

Megaloblastic anemia (MBA) may cause a problem in differential diagnosis from other conditions which may cause macrocytosis. Macrocytosis is a normal finding in newborns and during pregnancy but also occurs in alcoholism, hepatic disease, hemolytic states and hypothyroidism. This topic will discuss about laboratory investigations for the differential diagnosis of MBA from those conditions, along with some preliminary investigations.

Hematological Tests

1. Hemoglobin and Hematocrit

According to WHO, the criteria for anemia is when adult males have Hemoglobin levels <13 g/dL and adult females have <12 g/dL. As the iron deficiency worsens, both Hb and PCV decline together.

Hb >12 g/dl : Not anemic
Hb 10–11 g/dl : Mild anemia
Hb 8–9 g/dl : Moderate anemia
Hb 6–7 g/dl : Marked anemia
Hb 4–5 g/dl : Severe anemia
Hb < 4 g/dl : Critical

2. Red Cell Indices

MCV and MCH are increased. MCHC remains normal and RDW is elevated.

Mean Corpuscular Volume (MCV): It is the average volume of the RBC expressed in femtoliters. It becomes >97 fL in MBA (normal 82–98 fL). MCV values above 125 fl are almost always associated with MBA.
Mean Corpuscular Hemoglobin (MCH): MCH indicates the amount of Hemoglobin (weight) per RBC and is expressed as picograms. MCH will be >32 pg in MBA (normal 27–32 pg).
Mean Corpuscular Hemoglobin Concentratration (MCHC): The MCHC measures the average concentration of hemoglobin in a red blood cell. MCHC remains normal in between 31–36 g/dL.
Red Cell Distribution Width (RDW): RDW is a quantitative measure of anisocytosis. In MBA, RDW is elevated in roughly two-thirds of all cases (normal 11.5–14.5%).

CBC & Peripheral Blood Smear

There may be a pancytopenia (decreased RBC, WBC and platelets count). Oval macrocytes, hypersegmanted neutrophils and Howell-jolly bodies are the characteristic triad of abnormalities in megaloblastic anemia.

1. Red Blood Cells (RBCs)

Macrocytosis: RBCs are usually larger than normal. Macrocytic and oval (egg shaped macro-ovalocyte) is diagnostic feature.
Most macrocytes lack the central pallor.
Anisopoikilocytosis: Marked variation in shape and size of the red cells. Ovalocytes, tear-drop cells and some fragmaneted cells are usually found.
Inclusions: Howell-jolly bodies, Cabot rings and basophilic stippling may be seen.

2. White Blood Cells (WBCs)

Leukopenia: decreased WBC count.
Hypersegmented neutrophils (Right shift): When there are at least 5 five-lobed neutrophils per 100 WBCs or at least 1 six-lobed neutrophil is noted.

3. Platelets

Platelet count is usually reduced. Giant platelets may also be observed.

4. Reticulocyte Count

The reticulocyte count is normal or low.

Bone Marrow Examination

Cellularity: Moderately to markedly hypercellular.
M:E ratio: Due to marked erythroid hyperplasia, M:E ratio is reversed ranging from 1:1 to 1:6 (normal 2:1 to 4:1).
Erythropoiesis: Megaloblastic type. Megaloblasts are large, abnormal counterparts of normoblasts showing asynchrony of nuclear and cytoplasmic maturation. As developing megaloblasts die in marrow (intramedullary hemolysis), there is ineffective erythropoiesis.
Myelopoiesis: Adequate in smaer. May display giant metamyelocytes and band forms.
Megakaryopoiesis: Normal or increased in number.
Bone marrow iron: Markedly increased, demonstrated by negative Prussian blue reaction.

Biochemical Tests

Common Biochemical Tests for both vitamin B12 and Folic acid deficiency
Serum homocysteine	↑
Serum bilirubin	↑
Serum iron and ferritin	↑
Plasma lactate dehydrogenase (LDH)	↑
Serum vitamin B12/folate	↓

Specific tests for vitamin B12 deficiency
Serum vitamin B12 levels	↓
Vitamin B12 metabolites: Serum methylmalonic acid Urinary excretion of methylmalonic acid	↑ ↑
Schilling test	for vitamin B12 absorption

Specific tests for vitamin B12 deficiency

Serum vitamin B12 levels

↓

Vitamin B12 metabolites:

Serum methylmalonic acid
Urinary excretion of methylmalonic acid

↑
↑

Schilling test

for vitamin B12 absorption

Specific tests for Folic acid deficiency
Serum folic acid levels:	↓
FIGLU in urine	excessively excreted

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Cross Matching : Types, Principle, Procedure and Interpretation

Dhurba Giri — Tue, 02 Jul 2019 18:43:12 +0000

Cross matching is a procedure performed prior to transfusion of blood or blood products to detect any serological incompatibilities in the blood of donor and recipient. Before a donor’s blood is transfused into a recipient, there should be no antigens or antibodies in both, that would react with each other resulting in transfusion reaction. Cross matching is designed to prevent such transfusion reactions which may occur after transfusion.

Cross matching plays important role to detect:

Most recipient antibodies directed against donor’s red blood cell antigens.
Most donor antibodies directed against recipient’s red blood cell antigens.
Major errors in ABO grouping, labeling and identification of donor and recipients.

Principle of Cross Matching

Cross matching is based on the principle of serological detection of any clinically significant irregular/unexpected antibodies in either donor or recipient’s blood. There are two types of cross matches:

Major Cross Match: It involves testing the donor’s red cells with recipient’s serum to determine the presence of any antibody which may cause hemolysis or agglutination of donor red cells.This is more important than minor cross match.
Minor Cross Match: It involves testing of donor’s plasma with recipient’s red cells to determine the presence of any antibody which may cause hemolysis or agglutination of recipient’s red cells.

Type	Donor’s	Recipient’s
Major Cross Match	Red Cells	Serum/Plasma
Minor Cross Match	Serum/Plasma	Red Cells

Procedure of Cross Matching

There are different methods for cross matching, as shown in table. Among them most commonly used technique is Anti-human globulin (AHG) cross match.

Method of Cross Match	Detects Antibody of Type :
Saline Cross Match	IgM
Albumin Cross Match	IgG
Anti-Human Globulin (AHG) Cross Match	IgG

Major Cross Match

Prepare donor and recipient’s blood sample: Donor’s red cells and recipient’s serum/plasma.
Prepare 3-5% saline cell suspension of red cells.
Label a test tube.
Add two drops of recipient’s serum and one drop of donor cell suspension.
Mix and incubate the tubes at 37 degree Celsius for about 60 minutes.
Decant the serum completely and wash the cells three times in saline.
Add two drops of Anti-human Globulin (AHG) and mix. Allow to stand at room temperature for 5 minutes.
Centrifuge at 1500 rpm for 1 minute.
Observe macroscopically and microscopically for agglutination.
If macroscopic agglutination is not observed, transfer a small amount onto a glass slide and examine for microscopic agglutination. Rouleaux is not an indication of incompatibility.

Minor Cross Match

Prepare donor and recipient’s blood sample: Recipient’s red cells and donor’s serum/plasma.
Label a test tube.
Add two drops of donor’s serum and one drop of recipient’s cell suspension.
Mix and incubate the tubes at 37 degree Celsius for about 60 minutes.
Decant the serum completely and wash the cells three times in saline.
Add two drops of Anti-human Globulin (AHG) and mix. Allow to stand at room temperature for 5 minutes.
Centrifuge at 1500 rpm for 1 minute.
Observe macroscopically and microscopically for agglutination.
If macroscopic agglutination is not observed, transfer a small amount onto a glass slide and examine for microscopic agglutination. Rouleaux is not an indication of incompatibility.

Results and Interpretation

Compatible donor and recipient blood should show no agglutination in both major and minor cross match. Blood which shows incompatibility in major cross match should never be transfused, because the large plasma volume of the recipient blood containing antibodies can destroy the donor’s red cells easily. The minor incompatibility is less important because the donor’s serum which contains the antibodies is diluted in the recipient’s own plasma, making the antibodies very dilute and ineffective.

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Laboratory Diagnosis of Iron Deficiency Anemia (IDA)

Dhurba Giri — Thu, 06 Dec 2018 06:34:30 +0000

Anemia is the condition of decrease in number of circulating red blood cells (and hence hemoglobin) below a normal range for age and sex of the individual, resulting in decreased oxygen supply to tissues. Iron deficiency anemia is a type of microcytic hypochromic anemia, which is the most common nutritional disorder. Iron is an essential element in the synthesis of hemoglobin.

Iron deficiency anemia (IDA) may cause a problem in differential diagnosis from other hypochromic anemias like beta-thalassemia trait, alpha-thalassemia trait, HbE disease, sideroblastic anemia or anemia due to chronic diseases. This topic will discuss about laboratory investigations for the differential diagnosis of IDA from those conditions, along with some preliminary investigations.

Hematological Tests

1. Hemoglobin and Hematocrit

Hb >12 g/dl : Not anemic
Hb 10–11 g/dl : Mild anemia
Hb 8–9 g/dl : Moderate anemia
Hb 6–7 g/dl : Marked anemia
Hb 4–5 g/dl : Severe anemia
Hb < 4 g/dl : Critical

2. Red Cell Indices

MCV, MCH and MCHC are reduced. RDW is raised.

Mean Corpuscular Volume (MCV): It is the average volume of the RBC expressed in femtoliters. It becomes <80 fL in IDA (normal 82–98 fL).
Mean Corpuscular Hemoglobin (MCH): MCH indicates the amount of Hemoglobin (weight) per RBC and is expressed as picograms. MCH will be <25 pg in IDA (normal 27–32 pg).
Mean Corpuscular Hemoglobin Concentratration (MCHC): The MCHC measures the average concentration of hemoglobin in a red blood cell. MCHC goes below 27 g/dL(normal 31–36 g/dL).
Red Cell Distribution Width (RDW): RDW is a quantitative measure of anisocytosis. In IDA, RDW is increased and >15%. It is earliest sign of iron deficiency (normal 11.5–14.5%).

Peripheral Blood Smear

1. Red Blood Cells (RBCs)

Microcytosis: RBCs are usually smaller than normal. Dimorphic blood picture is seen with a dual population of red cells of which one is macrocytic and the other microcytic and hypochromic when iron deficiency is associated with severe folate or vitamin B12 deficiency.
Hypochromasia: Central pallor in RBCs is more than 1/3.
Poikilocytosis: Elliptical forms are common, and elongated pencil (cigar) shaped cells may be seen. Target cells and Teardrop cells may also be present in small numbers. Severe anemia shows ring/pessary cells.

2. White Blood Cells (WBCs)

WBCs are usually normal in number, but can increase due to chronic marrow stimulation in long-standing cases. There may be associated eosinophilia if iron deficiency is secondary to hookworm infestation.

3. Platelets

Platelet count is usually normal, but may be slightly to moderately increased, especially in patients who are bleeding.

4. Reticulocyte Count

The reticulocyte count is low in relation to the degree of anemia.

Bone Marrow Examination

Cellularity: moderately hypercellular.
M:E ratio: varies from 2:1 to 1:2 (normal 2:1 to 4:1).
Erythropoiesis: hyperplasia and micronormoblastic maturation.
Myelopoiesis: normal.
Megakaryopoiesis: normal.
Bone marrow iron: Absent. “Gold standard” test, demonstrated by negative Prussian blue reaction.

Biochemical Tests

Serum iron profile studies are used to establish a differential diagnosis of microcytic, hypochromic anemia.

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Sudan Black B Stain : Purpose, Principle, Procedure and Interpretation

Dhurba Giri — Sun, 18 Nov 2018 06:31:18 +0000

Sudan Black B (SBB) is a fat soluble dye which has very high affinity for neutral fats and lipids. SBB staining is useful for for the differentiation of Acute myeloid leukemia (AML) from Acute lymphoid leukemia (ALL). It is similar to that of Myeloperoxidase (MPO) staining pattern of leukocytes and monocytes. It has few advantages over MPO:

SBB can be used for staining smears more than 2 weeks old.
SBB stains both azurophilic and specific granules in neutrophils, whereas MPO stains azurophilic granules only.
There is only a little fading of the SBB stain over time.

Principle of Sudan Black B Stain

As SBB is a fat soluble dye, it stains lipids such as sterols, neutral fats and phospholipids. These are present in azurophilic and secondary granules of myelocytic and lysosomal granules of monocytic cells. During staining, the dye leaves the solvent because of its high solubility in lipids than solvent. On microscopic examination, varying degree of black colored pigments are seen in the positive reaction.

Requirements

Sample: Fresh anticoagulated whole blood or bone marrow smear may be used. The slides must be fixed as soon as possible.
Fixative: 40% formaldehyde solution vapor
Stain: SBB 0.3 g in 100 ml absolute ethanol
Phenol buffer: Dissolve 16 g crystalline phenol in 30 ml absolute ethanol. Add to 100 ml distilled water in which 0.3 g Na2HPO4.12H2O has been dissolved
Working SBB stain solution: Add 40 ml buffer to 60 ml SBB solution (The composition of working stain may slightly vary upon different products.)
Counterstain: May–Grunwald–Giemsa or Leishman stain.

Procedure of Sudan Black B Stain

Fix air dried smears in formalin vapour, formaldehyde or formalin-ethanol fixative for 10 minutes.
Wash gently in water for 5-10 minutes.
Place the slides in the working stain solution for 1 hour in a Coplin jar with a lid on.
Remove and flood the slides with 70% alcohol for 30 seconds. Tip the 70% alcohol off and flood again. Repeat this three times.
Rinse in running tap water and air dry.
Counterstain without further fixation with Leishman stain or May–Grunwald–Giemsa stain.
Air dry and examine microscopically.

Results and Interpretation

Production of black and granular pigment indicates positive reaction.

Lipids are present in azurophilic and secondary granules of myelocytic cells. Hence, these are SBB positive. The staining becomes more intense as cells mature from myeloblast to mature forms. Basophils are generally not positive but may show bright red/purple metachromatic staining of the granules.
Lipids are present in lysosomal granules of monocytic cells. Monocytic cells show variable reactions, from negative to weakly positive.
Lymphoid cells are SBB negative. However, in ALL, less than 3% of blast cells show positive reaction.

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Myeloperoxidase (MPO) Stain : Purpose, Principle, Procedure and Interpretation

Dhurba Giri — Tue, 06 Nov 2018 14:33:07 +0000

Myeloperoxidase is a lysosomal enzyme present in the primary azurophilic granules of neutrophils, eosinophils and to a certain extent, monocytes. Lymphocytes do not contain myeloperoxidase.

Purpose of Myeloperoxidase stain

Myeloperoxidase (MPO) stain is useful for differentiating the blasts of acute myeloid leukemia (AML) from those of acute lymphoblastic leukemia (ALL). It is also used to diagnose congenital deficiency of neutrophil myeloperoxidase.

Principle of MPO stain

In presence of hydrogen peroxide, myeloperoxidase present in the leukocyte granules oxidize substrates from colorless form to an insoluble blue/brown derivative at the site of the activity. Benzidine or 3,3′-diaminobenzidine or p-phenylenediamine dihydrochloride are used as substrates.

Requirements

Fixative: 10% formal ethanol or buffered formal acetone.
Substrate: Benzidine or 3,3′-diaminobenzidine or p-phenylenediamine dihydrochloride
Buffer: Sorensen’s phosphate buffer, pH 7.3
3% Hydrogen Peroxide
Working substrate: Add 30 mg DAB in 60 ml buffer, add 120 ul hydrogen peroxide and mix.
Counterstain: Hematoxylin

Procedure of MPO stain

Fix air dried smears in formal ethanol or buffered formal acetone for 60 seconds.
Rinse thoroughly in running tap water for 30 seconds.
Cover the smear with working substrate and incubate for 10 minutes.
Wash gently with running tap water for 30 seconds.
Counterstain with hematoxylin for 3-5 minutes.
Rinse in running tap water and air dry.
Examine under microscope.

Interpretation

Myeloperoxidase is present in the primary granules of myeloid cells. Early myeloblasts are negative, with granular positivity appearing progressively as they mature.
In many cases of AML (without maturation-M1, with maturation-M2 and promyelocytic leukemia-M3), Myeloperoxidase activity has been found in more than 80% blasts. Auer rods are strongly MPO positive.
Cells of monocytic series display a less intense positive reaxtion that is characterized by fine granular deposits scattered throughout the cell.
Lymphoblasts and lymphoid cells are MPO negative.

Positive MPO Reaction	Negative MPO Reaction
Neutrophilic granulocytes except blast forms	Basophils (weakly positive)
Eosinophils	Lymphocytic cell series
Monocytes except blast forms	Erythrocyte cell series

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