To study hereditary anaemias we need to have the background haematological profile ie "normal values" of a study population; accurate local values for Hb, RBC, PCV are required. Red cell count can be measured accurately only by electronic counters, but these are usually big and need renewable calibrating material. So in the field we need simplified methods: (a) that don’t need intravenous blood but can be done by finger prick, (b) do not need big, heavy equipment, (c) do not need expensive imported equipment and (d) do not
need renewable calibrating material of short life span.
One of the main problems in past surveys, especially where thalassaemia is concerned, is lack of proper red cell indices and accurate Hb A2 estimations.
I contacted Dr. RS Tiwary Director, Regional Medical Research Centre For Tribals for local support to the laboratory work in the field and in RMRC/ICMR laboratories Jabalpur, India: he agreed to provide laboratory back up.
In the first field trip blood samples were collected on filter paper via finger prick. Tribals do not mind and mostly come forward to donate blood through finger prick, because they usually go to local Primary Health Centres for malaria checks. They are scared to see needles and syringes. The response is low for intravenous blood.
In the second field trip blood samples were both finger prick and intravenous, because good amount of blood (5 to 10 ml) was required for DNA studies. The number of samples are shown in table 5.2 In the first field trip samples were tested in London, and in the second field trip the same families were followed up and some fresh families were screened.
Hb electrophoresis and Hb A2 estimation were not performed in the field because they need proper laboratory facilities (electrophoresis tank, spectrophotometer, power pack, buffers) and the field area is far from Jabalpur (where these facilities easily can be obtained). The sickling slide test was not carried out in the field because it needs a microscope and another technician.
Results from field with limited resources need validation: the results were confirmed as far as possible in London.
A field laboratory was assembled with simple portable equipment (table 5.3) to test the feasibility of carrying out the following procedures in the field in primitive conditions. A. Haematological parameters: Hb, RBC, HCT: derived red cell indices MCH, MCV,
Table 5.2 Number of samples in two field trips
Field trip Finger prick Intravenous Mouth wash (DNA studies) Haematological studies DNA studies
First 513 nil nil 400
Second 781 262 63 nil
Total 1294 262 63 400
MCHC needed to detect 6-thalassaemia, a-thalassaemia, iron deficiency, and to provide "normal" haematological values for the tribe.
B. Screening for G6PD deficiency using fluorescence spot test
C. Screening for Iron deficiency using measurement of zinc protoporphyrin level, needed to help interpret Coulter results on red cell indices.
D. Separation of plasma and preparation of lysates for further studies (storage of samples for haematological, DNA analysis).
The following equipment was obtained and tested in London and transported to India together with the necessary chemicals in January 1992.
1. CBC 5 Portable Coulter Counter in carrying case (plus Zapoglobin), (the CBC 5; weighed about 7.7 kg and dimensions; height 355 mm x width 170 mm and depth 380 mm) Accuvettes and Cleaning agent (Coulter clenz)
2. Protofluor Z machine, on loan from Helena laboratories (plus Helena low calibrator, high calibrator and, cyanide reagent).
3. Reagents for G6PD assay (Sigma: G6P, NADP, saponin, oxidised glutathione and Tris buffer).
Equipment obtained in RMRC laboratories, Jabalpur: It included a voltage stabilizer, a portable electricity generator, portable refrigerator, a portable centrifuge, a UV Lamp, and other accessories, distilled water, and Isoton II. This field laboratory was established in the forest rest house at Kharidih village (a study village 257 Kms from Jabalpur). Isoton n prepared in RMRC/ICMR laboratories, Jabalpur as follows:
Reagents:
A. 0.15 molar Sodium dihydrogen ortho-Phosphate NaH2HP0 4 .2 H2 0 (11.7 gm/500 ml distilled water)
B. 0.15 molar di-Sodium Hydrogen ortho-Phosphate (Na2 H PO4 anhydrous 42.6 gm/2000 ml distilled water or
53.4 gm Na 2 HPO 4.2 H2O/2 OOO ml distilled water) C. 0.9% NaCl (18 gm/2000 ml distilled water)
Reagents A and B are mixed in the ratio of 300 ml: 1700 ml- adjusted to pH 7.4. To this 2000 ml of solution C is added. 4.0 gm of Sodium Azide is added as a preservative. The
Table 5.3 Equipment used in the field
Type of measurement Source of blood Equipment weight Reagents made Calibration
Coulter indices Finger prick /venous 17 kg Isoton II in Jabalpur Field G6PD def. Finger prick/venous
UV lamp, 7 kg Field not required
ZPP Finger prick/
venous
8 kg Helena*
reagents
Field
* they could be made in the field also.
above solution is pre filtered through Whatman No. 41 paper and vacuum filtered by 0.2 micron size millepore filter. The filtration step is exceptionally important. The solution must be absolutely particle free, or particles clog the CBC 5 aperture.
Red cell indices CBC 5 portable Coulter Counter
Before leaving London, the red cell values of 20 samples were measured both with the CBC 5 and the Technicon S at Whittington Hospital, London: the results corresponded well. It was concluded that the results obtained in the field should be reliable. I also took the indices of my own blood with the Coulter S in London, so that it could be used in calibrating the machine in the field. In the field and after my return the red cell indices of my own blood (used as a control) measured with CBC 5 and Technicon S corresponded closely. The instructions in the manual were followed closely. The back ground values
of Hb, MCH, MCV, and HCT are checked by following instructions. Once the instrument was calibrated; 40 |xl of whole blood was diluted in a cuvette containing 20 ml of Isoton II; the ratio of whole blood and diluent is 1:500 for Hb estimation; for RBC count 200 |xl of the 1st sample diluted in a 2nd cuvette tube containing 20 ml of Isoton; the ratio of whole blood and diluent is 1:50,000. For each sample RBC, MCH, HCT, and MCV were assessed first and Hb level afterwards. The channel was washed after each Hb estimation to avoid the lysis of the next sample (wave length 525 nm). The CBC 5 was calibrated against my own blood and checked every morning before measuring red cell indices.
Accuracy of results obtained with CBC 5 Coulter Counter
To check the accuracy of results obtained with CBC 5 in the field, in comparison to electronic Coulter Counter in London, after my return 206 blood samples were assessed by both CBC 5 and Technicon S from mixed patients who were attending the hospital at Whittington Hospital, London. For CBC 5, pippeting and measuring with required amount of Isoton II and mixing the red cells are done manually, whereas all aspects are automatic for Technicon S. Results were recorded and analyzed to see the difference between both the instruments.
Hb Level: The haemoglobin levels are same with Coulter CBC 5 and Technicon S (Figure 5.3).
MCH: The mean cell haemoglobin was slightly higher with the CBC 5 in comparison to Technicon S in most of the samples. The mean difference of MCH was 0.5 pg (higher for CBC 5): this may be because the CBC 5 gives slightly lower RBC count than Technicon S. There may be a slight problem in screening in borderline cases ie, where the MCH is between 25-27 pg, but the observed values were clearly acceptable (figure 5.4).
MCV: The mean cell volume is slightly lower with CBC 5 than Technicon S. The observed difference may be due to instrument variation (figure 5.5).
MCHC values are high for CBC 5 compared to Technicon S, as is to be expected from the smaller MCV. The modal value is 32 for Technicon, 34 for CBC 5 (figure 5.6). In
this study Hb and the MCH values were the most important parameters, and it is concluded that these results obtained in the field are acceptably reliable.
Zinc protofluor machine fo r screening fo r iron deficiency
The Helena haematofluorometer is a simple machine which measures fluorescence in whole red cells on a slide, at a fixed wave length (424 nm) characteristic of zinc protoporphyrin (ZPP). Increased fluorescence gives a semiquantitative indication of iron deficiency or lead poisoning. Haem is formed in the developing red cell by insertion of iron into a formed protoporphyrin ring. In iron deficiency or impaired iron utilization (eg lead poisoning) zinc is inserted into protoporphyrin instead of iron, to make zinc protoporphyrin (ZPP). The ZPP is stable and remains in the red cells and can be measured by its fluorescence as above. Fluorescence at 424 nm is therefore an indicator of the available iron supply at the time the cell was formed. A raised ZPP/haem ratio (>80) is characteristic of iron deficiency, and lead poisoning, and may also occur in anaemia associated with (eg) chronic infection.
Principle
The ranges provided by Helena Laboratories are: <80 pmol ZPP/haem ratio = Normal
>80 jxmol ZPP/haem ratio = Iron deficient
The haemoglobin must be fully oxygenated to avoid spectral shifts. "Protoflour reagents" added to test blood convert the haemoglobin to cyanmethaemoglobin, which is stable and has the same spectral characteristics as oxyhaemoglobin. Reagents and procedure are followed according to kit method of Helena laboratories.
Fluorescence spot method fo r G6PD deficiency screening
The fluorescent spot test (Beutler 1966; Beutler and Mitchell 1968) is reliable, sensitive and usable on stored blood. It is based on measuring the fluorescence of NADPH produced when G6P and NADP are added to haemolysate. The mixture is spotted on to filter paper after incubation and observed in Ultra-Violet light.
Principle
In presence of glucose-6-phosphate dehydrogenase (G6PD) and NADP, G6P is oxidised to 6-phosphogluconate in the reaction,
G6P + N A D P --- 6PG +NADPH
When a G6PD deficient haemolysate is incubated with G6P and NADP a smaller amount of NADPH is formed. NADPH fluoresces at long wavelength.
To make the test more sensitive, oxidized glutathione (GSSG) is included in the reaction mixture. In the presence of GSSG, NADPH is re-oxidised by glutathione reductase present in the lysate.
GSSG + NADPH + H --- 2GSH + NADP
The screening test therefore measures the difference between G6PD activity and glutathione reductase activity. When the G6PD level is normal NADPH continues to accumulate, but when levels are deficient, little or no NADPH accumulates.
Because of some modification made for this study I give here the full method modified from Beutler and Mitchell 1968 as used in Hammersmith Hospital.
Reagents:
i. D-G6P 10 mM/1 or 305 m g/100 ml
ii. NADP 7.5 mM/1 or 60 mg/10 ml
iii. Saponin 750 mM/1 or 10 gm/1
iv. Tris Hcl Buffer pH 7.8
V. Oxidized Glutathione (GSSG) 8 mM/1 or 49 gm/1
The above reagents are mixed in following proportion:
2 parts of G6P
1 part of NADP
2 parts of Saponin
3 parts of Tris HCl Buffer
1 part of GSSG
2 parts of Distilled water
The combined reagent is stable at 20°C for two years and at 4°C for 2 months.
Fresh blood samples (Second field trip)
No. of individuals 60 ---
Hb in gm /dl
§ ■ CBC 5 H Tech. S
Figure 5.3 Frequency distribution of Hb level with CBC 5 and Technicon
No. of individuals
17 18 19 20 21 22 23 24 26 26 27 28 29 30 31 32 3 3 34
MCH in pg
CBC 5 Tech. S
100
6 6 - 6 9 6 0 - 6 4 6 6 - 6 9 7 0 - 7 4 7 6 - 7 9 6 0 - 8 4 8 6 - 8 9 9 0 - 9 4 9 6 - 9 9 1 0 0 - 1 0 4 1 0 6 - 1 0 9