Chart 8.6 Morphology of normal and abnormal blood cells in Romanowsky stained blood film
8.9 Reticulocyte count Methaemoglobin reduction test
Reticulocyte count
Value of test:Reticulocytes are immature red cells normally present in small numbers in the blood (up to 2%). Reticulocyte numbers increase when there is an increase in erythropoietic activity. A reticulocyte count assesses bone marrow activity, e.g. whether there is an effective erythropoietic response when there is a reduction in the number of red cells due to haemolysis or haemorrhage. A reticulocyte count is also of value in monitoring the erythropoietic response of an anaemic patient following treatment.
Principle of test
An isotonic solution of a supravital stain (i.e. one that stains living material) such as New methylene blue or brilliant cresyl blue is incubated with a few drops of blood. To detect riboso- mal RNA in reticulocytes, the red cells must be stained while they are still living (not fixed). A thin preparation is made and the reticulocytes counted microscopically. Reticulocytes are recognized by the violet-blue stained granules of ribosomal RNA (reticulin) they contain. The reticulocyte count is expressed as a percentage, or preferably in absolute numbers when an electronic analyzer RBC count is available.
Reagent
10 g/l (1% W/v) New methylene Reagent No. 61 blue or brilliant cresyl blue (No. 17)*
*New methylene blue gives better staining of reticulin in retic- ulocytes than brilliant cresyl blue. It is chemically different from other forms of methylene blue.
Keep the stain refrigerated and always filter it before use.
Specimen: Use well-mixed EDTA anticoagulated blood or if the patient is a young child, use free- flowing capillary blood.
Test method
1 Filter 2–3 drops of the stain into a small tube or vial.
7 Report the reticulocyte count as the average of the two counts performed (from the two separate preparations).
Quality control
Quality control of reticulocyte counts should include filtering the stain before use and checking the best staining time to use when a new batch of stain is made. Always perform duplicate counts (preferably using a different person for each count). The two counts should agree within 20% of each other.
Sources of error
● Not mixing adequately the stained blood prior to making the blood films.
● Not counting the cells accurately or counting too few cells.
● Using stain which has become contaminated and has not been filtered before use.
● Confusing Heinz bodies (see following text) or precipitated stain for the reticulin of reticulocytes.
Interpretation of reticulocyte counts Reference range*
*Figures should be checked locally
Infants at birth . . . 2–5%
Children and adults . . . 0.5–2.5%
Note: Whenever an RBC count is available, express the reticulocyte in absolute numbers.
Raised reticulocyte counts:Found when there is an increase in red blood cell production as occurs in:
– Haemolytic anaemias (with effective erythro- poiesis).
– Following acute blood loss
– After iron therapy for iron deficiency anaemia or specific therapy for megaloblastic anaemia Reticulocyte responses are higher with haemolysis than haemorrhage.
Decreased reticulocyte count: Associated with ineffective erythropoiesis or decreased production of red cells.
Heinz bodies in reticulocyte preparation Heinz bodies consist of altered (precipitated) haemo- globin caused by oxidative damage. They can be found in the red cells of patients with G6PD deficiency during a haemolytic crisis (see subunit 8.2), in some forms of thalassaemia, in conditions 2 Add about 4 drops* of EDTA anticoagulated
blood or capillary blood and mix well.
*The amount of blood used is not critical. Use at least twice the volume of blood to stain if the patient is severely anaemic.
3 Incubate at room temperature for 20 minutes or 10–15 minutes at 35–37C.
4 Mix gently to resuspend the red cells and using a capillary or plastic bulb pipette, transfer a drop of the stained blood to each of two slides. Spread to make two evenly spread thinfilms. Wave the slides back and forth to air-dry the films. Protect the films from dust and insects until the count can be performed.
5 Count the reticulocytes microscopically. Use the 10 objective (with reduced condenser iris diaphragm) to check the distribution of the red cells. Select an area where the red cells can be seen individually, add a drop of immersion oil, and examine using the oil immersion objective (open more the condenser iris diaphragm).
6 Count systematically (i.e. consecutive fields), 500 red cells (1 000 if there are very few reticulo- cytes), noting the number that are reticulocytes.
Calculate the percentage of reticulocytes (see following text).
Appearance of reticulocytes
Reticulocytes appear as pale green-blue stained cells containing dark blue-violet inclusions in the form of small granules, distributed irregularly as shown in colour Plate 110. Mature red cells stain pale green-blue.
Counting reticulocytes: A convenient method of counting reticulocytes is to reduce the size of the microscope field by inserting in each eyepiece a circular piece of black (opaque) paper which has a punched out hole of about 5 mm.
To calculate % of reticulocytes:
– Using a hand tally counter, count a total of 500 red cells, noting on paper the number of cells that are reticulocytes (alternatively use two hand tally counters or a white cell differ- ential counter).
– Multiply the number of reticulocytes counted by 2.
– Divide the figure by 10 to obtain the per- centage figure.
associated with unstable haemoglobin, and follow- ing splenectomy.
Appearance of Heinz bodies
In reticulocyte preparations, Heinz bodies stain a lighter blue violet than the reticulin of reticulocytes and are usually seen as small granules lying close to the periphery of the cell or protruding through the cell membrane (see colour Plate 108). They vary in size from 1–3 m in diameter. Several Heinz bodies may be present in a single cell. If seen, report as
‘Heinz bodies present’.
Note: Heinz bodies are best stained using isotonic crystal violet or methyl violet stain (0.5 g dissolved in 100 ml physiological saline). The staining method is the same as that described for staining reticulocytes.
Haemoglobin H in reticulocyte preparation Haemoglobin H ( 4) is an unstable haemoglobin which can form in the red cells of patients with thalassaemia trait (small numbers of cells affected, i.e. up to 1%), and in patients with HbH disease (more than 10% of cells affected). HbH disease is described in subunit 8.2. Haemoglobin H inclusions are best stained using brilliant cresyl blue and the reticulocyte staining technique previously described but allowing 1 hour for staining.
Appearance of Haemoglobin H inclusions
Cells containing HbH are easily recognized by the many small pale blue granules they contain. The cells have been likened to golf balls. The appearance of HbH in a red cell is shown in colour Plate 106.
Note: Heinz bodies and the reticulin of reticulocytes, stain much darker than Haemoglobin H inclusions.
Methaemoglobin reduction test Screening for G6PD deficiency
Value of test:The methaemoglobin reduction test is one of the simpler and less expensive tests to screen for G6PD deficiency. As explained in subunit 8.2, reduced G6PD activity in red cells can cause acute intravascular haemolysis following exposure to oxidant agents or fava beans (favism), neonatal jaundice and less commonly, chronic haemolytic anaemia. The severity of clinical symptoms is mainly dependent on the variant of defective G6PD gene inherited. For the main laboratory findings associ- ated with a haemolytic crisis, see subunit 8.2.
8.9
Principle of test
Haemoglobin is oxidized to methaemoglobin (Hi) by sodium nitrite. The redox dye, methylene blue activates the pentose phosphate pathway, resulting in the enzymatic conversion of Hi back to haemoglobin in those red cells with normal G6PD activity. In G6PD deficient cells there is no enzymatic recon- version to haemoglobin.
Reagents
● Methylene blue, 0.4 mmol/l Reagent No. 57
● Sodium nitrite-glucose reagent*
*The reagent must be prepared fresh on the day of use. To make 40 ml:
Sodium nitrite . . . 0.5 g Glucose . . . 2.0 g Dissolve the chemicals in 40 ml of distilled (deionized) water.
Preparation of reagents in tubes for long term storage To store the reagents in dried ready to use form:
– Mix equal volumes of methylene blue reagent with sodium nitrite-glucose reagent.
– Dispense in 0.2 ml amounts into small glass tubes.
– Dry the contents of the tubes at room temperature.
– Stopper the tubes and store in the dark at room tempera- ture.
Note: In dried form the reagents are stable for up to 6 months.
Blood sample:EDTA anticoagulated venous blood is suitable. It should not be collected during a haemolytic crisis but when the patient has recovered and reticulocyte numbers have fallen back to normal levels. This is because reticulocytes contain higher levels of G6PD and may mask low G6PD activity in mature red cells. The blood must be tested within 8 hours of being collected. When the patient is anaemic, use a plasma reduced blood sample (remove suffi- cient plasma until the PCV is about 0.40).
Test method
1 Take 3 small glasstubes and label Test, Normal, Deficient.
2 Pipette into each tube as follows:
Tube Test Normal Deficient
control control Sodium
nitrite-glucose
reagent (fresh) 0.1 ml – 0.1 ml Methylene
blue reagent 0.1 ml – –
Patient’s blood 2 ml 2 ml 2 ml
● Homozygous sickle cell anaemia(HbSS) in which a person inherits a haemoglobin S (HbS) gene from both parents. It is common in Africa, parts of India, the Middle East, and the Caribbean.
● Sickle cell haemoglobin C (HbSC) disease in which a person inherits HbS gene from one parent and HbC gene from the other. It is found in West Africa.
● Sickle cell thalassaemia in which a person inherits HbS gene and one of the thalassaemia genes. Clinically, HbS/ O thalassaemia is the more severe form and is found in North Africa (and elsewhere). HbS/ thalassaemia is found mainly in West Africa and those of West African descent (e.g. Caribbean).
● Sickle cell haemoglobin DPunjab (HbSDPunjab) diseasein which HbS is inherited with HbDPunjab. It is found amongst Sikh people.
Sickle cell trait
In sickle cell trait (HbAS) there is a heterozygous inheritance of HbS with HbA. The condition is asymptomatic with 30–40% of haemoglobin in the red cells being HbS and the remainder, normal haemoglobin. Under conditions of hypoxia, (low oxygen level) sickling may occur, e.g. when mountain climbing at high altitude, during pro- longed anaesthesia, in severe pneumonia, or during prolonged intensive exercise (e.g. military training).
Haematological investigation of sickle cell disease
In district laboratories the following are the tests used to investigate suspected sickle cell disease and to identify sickle cell trait carriers (to assist in family counselling):
Measurement of haemoglobin (see subunit 8.4).
Sickle cell slide test to detect HbS in red cells.
HbS solubility filtration test to differentiate sickle cell anaemia from other sickle cell disorders.
Examination of a Romanowsky stained thin blood film for features associated with sickle cell disease.
Haemoglobin electrophoresis: When the tests per- formed at district level indicate that a person has a sickle cell disorder, this should be confirmed by haemoglobin electrophoresis in the nearest Haematology Reference Laboratory or Sickle Cell 3 Stopper the tubes and mix well (gentle mixing).
Incubate all three samples at 35–37C for 90 minutes.
4 Take 3 large tubes (15 ml capacity) and label as described in step 1. Pipette 10 ml of distilled (deionized) water into each tube.
5 Transfer 0.1 ml of well mixed sample from the Test, Normal, and Deficient tubes to the large tubes. Mix the contents of each tube.
6 Examine the colour of the solution in each tube.
Interpretation of test results
Colour of test solution . . . Normal G6PD activity is similar to the
red colour of the Normaltube
Colour of test solution . . . Reduced G6PD activity is similar to the (G6PD deficiency in brown colour of the homozygote) Deficienttube
Note: Results from a heterozygote are midway between normal G6PD activity and G6PD deficiency in the homozygote.
Quality control
Follow the technique exactly.
The main sources of error when performing the methaemoglobin reduction test are:
● Testing blood which has a high reticulocyte count or too low a haemoglobin concentration (see previous text).
● Not using freshly made sodium nitrite-glucose reagent.