Quantification bias in blood alcohol determination by headspace gas chromatography

Elia Mattarucchi, Carlo Peruzzo, Ramona Consuelo Maio, Ursula Andreotta, Marco Mario Ferrario

Ospedale di Circolo e Fondazione Macchi, Laboratory of Toxicology, Varese, Italy – 21100

Marco Mario Ferrario

Università dell’Insubria, Research Centre in Epidemiology and Preventive Medicine (EPIMED), Varese, Italy

All Correspondences to: Elia Mattarucchi Ospedale di Circolo e Fondazione Macchi, Laboratory of Toxicology, Varese, Italy – 21100

ABSTRACT

The quantification of blood alcohol concentration is routinely performed in many forensic laboratories by gas chromatography. In this short report, the influence of the matrix miss-match between samples and calibrators (e.g. the use of aqueous standards to quantify blood samples) and the actual efficiencies of different internal standards used to normalise the ethanol signal were considered. Test samples in blood and water were prepared from a certified reference standard of ethanol. The samples were analysed by gas chromatography using n-propanol and t-butanol as internal standards. The collected data provided evidences that a sub-optimal setting of the considered variables can be responsible for a quantification bias up to 15%. These issues are of practical relevance in the medico-legal sector and should be considered during the process of

method development.

KEY WORDS: blood alcohol concentration, calibrator, internal standard, gas chromatography, bias.

INTRODUCTION:

uman blood samples are the primary type of Hknown^{2, 3}. Method development should start from evidence received for medico-legal testing of tuning of the most important parameters; in this alcohol

concentration by gas chromatography. context we feel that further attention should be paid Whole blood is the sample of choice, since plasma typically entails an overestimation¹. For the purpose of forensic testing, different thresholds of alcohol concentration are set before a person is charged with a fine or crime. The consequent need to produce medico-legal reports underlines the importance of the careful development and validation of the analytical methods applied. The literature on this argument is vast and many possible influencing factors are to the choice of the internal standard and calibrators. The quantification of unknown samples is achieved by interpolation of a calibration curve. For this reason, reference materials at known concentrations of ethanol are widely available on the marketplace⁴. The majority of these materials are aqueous solutions produced according to ISO Guide 34 and satisfy the regulatory needs posed by ISO 17025. However, the opportunity to use aqueous calibrators to quantify blood samples should be carefully evaluated, even if an internal standard is used to correct for the differences among samples and calibrators^{5, 6}. The actual efficacy of the internal standard needs to be experimentally verified. A “good” internal standard is c h e m i c a l l y s i m i l a r t o e t h a n o l w i t h o u t a n y chromatographic overlap with the target substance or other volatiles that could be present in the samples (e.g., acetone, methanol or acetaldehyde). n-propanol is commonly considered the best suited substance for this purpose, even if t-butanol is also used^5,6. The samples can also be diluted

with water up to practically correct for the matrix mismatch (usually a 1:10 dilution is sufficient)⁷. However, this procedure inevitably increases the limit of quantification with evident constraints in dealing with the “zero tolerance” threshold posed for young drivers or commercial vehicle operators.

MATERIALS AND METHODS:

In this report we present some data retrieved from the development of the analytical procedure adopted at our laboratory. The method was arranged starting from the protocol suggested by the producer of the available equipment (i.e. a PerkinElmer Clarus 500 gas chromatographer equipped with a Turbomatrix40 headspace autosampler). The original parameters were: sample equilibration: 30 min 70 °C, vial pressurisation: 15 psi for 3 min, transfer line temperature: 110 °C, injection: 200 °C, oven: 40 °C isothermal, column: Elite-BAC1 (PerkinElmer Cod. N9316579), carrier gas: N₂.n-propanol and t-butanol were both tested as internal standard. Samples were mixed with ammonium sulphate in order to increase the partition coefficient of alcohol in the gas phase and amplify the analytical response⁸. A fixed volume of sample (1 ml) was added to 0.9 ml of 1 M ammonium sulphate and to 0.1 ml of 8 g/L t-butanol or n-propanol (internal standard).

Two type of test samples were prepared: an intermediate series of sample at 0.30, 0.75, 1.50, 3.75, 7.50 g/L of ethanol in water were produced independently from a mother solution 15 g/L. All the samples (including the mother solution) were further diluted 1:5 in water and blood (previously checked for the absence of ethanol). At the end of the process, 2 independent series at 0.06, 0.15, 0.30, 0.75, 1.50 and 3 g/L in blood and water were obtained.

32 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

Quantification bias in blood alcohol…

This dilution scheme ensured that each concentration was independent, and the matrix composition uniform within each series. The second type of samples consists of 4 solutions at the nominal concentrations of 0.5 and 2 g/L in water and blood. These samples were prepared by adding 100 and 400 µl of a 0.5 g/L ethanol standard to a final volume of 1 ml of water or blood.

RESULTS:

The first type of test samples described in the “materials and method” session (i.e. 2 independent series at 0.06, 0.15, 0.30, 0.75, 1.50 and 3 g/L of ethanol in blood and water) were prepared and analysed independently 10 times. For each repetition, blood and water samples were analysed within the same analytical session. For each concentration, it was expected that the average analytical response (i.e. the ethanol/internal standard area ratio) of the blood and water samples would be similar due to normalisation with the internal standard. However, a progressive response difference was observed (Table 1). The disparity becomes clearly significant among samples normalised by t-butanol, while it is not supported by a statistical evidence when n-propanol is used. In order to quantify the possible bias, test samples at the nominal concentrations of 0.5 and 2 g/L in water and blood were used (i.e. the second type of test samples described in the “Materials and methods” section). The 2 couples of samples were produced and analysed independently 10 times using a quantitative method calibrated by commercial certified reference materials based on water solutions. As reported in Table 2, the average error ranges from 2% at 0.5 g/L with n-propanol up to 16% at 2 g/L with t-butanol. The error is indeed negligible, if the analytical samples are matrix matched with the calibrators (i.e. water samples quantified against water calibrators).

DISCUSSION:

The presented data show how a matrix difference between samples and calibrators and/or the actual efficacy of the internal standard can bias the outcome of the alcohol test by gas-chromatography. These factors should be carefully considered during method development. Our data point out the superiority of npropanol for BAC determination. However, a few additional aspects should be considered, especially if t-butanol is selected as internal standard. As the error seems to increase with the amount of ethanol, the use of a fixed correction factor becomes difficult. On the other hand, the practice of making up “in-house” standards by spiking known concentrations of ethanol into “blank” blood samples can lead to regulatory problems associated with the medico-legal sector. The whole process should be carefully controlled and documented, even though it would be unrealistic for a medical laboratory to have enough resources to produce certified reference materials. A possible solution could be the use of commercial blood standards that are industrially manufactured by spiking ethanol into a blood matrix. However, the availability of such materials is still limited and the assigned concentration is usually an average value derived from a number of independent certified laboratories (usually these standards don’t comply with the ISO guide 34 requirements).

CONCLUSIONS:

In conclusion, this report offers some helpful thoughts on the calibration of the analytical methods used to assess blood alcohol concentration for medico-legal purposes. These considerations may seem trivial, but a recent comment by Schug⁹ shows that these analytical issues are still highly relevant. In our practice, the guidelines of the Italian Forensic Toxicologist Group don’t report any specific indication about the internal standard and the calibrators used for alcohol tests¹⁰ and the only way to control the risk of possible bias is to asses experimentally the real efficacy of such measures adopted to minimize the matrix mismatch and to participate periodically to suitable proficiency tests on blood samples.

Table 1. Analytical response of water and blood samples

t-BUTANOL

Blood samples Water samples

0.06	0.0096	0.0009	0.0130	0.0016	-0.0034	3.E-02
0.15	0.0301	0.0031	0.0323	0.0036	-0.0021	5.E-01
0.30	0.0746	0.0020	0.0712	0.0038	0.0034	2.E-01
0.75	0.2281	0.0022	0.1994	0.0034	0.0286	3.E-04
1.50	0.4736	0.0121	0.4073	0.0065	0.0663	1.E-03
3.00	0.9754	0.0407	0.8224	0.0148	0.1530	4.E-03
		t-PROPANOL
	Blood samples		Water samples

0.06	0.0197	0.0012	0.0288	0.0006	-0.0091	1.E-02
0.15	0.0613	0.0034	0.0718	0.0017	-0.0105	6.E-02
0.30	0.1557	0.0136	0.1669	0.0159	-0.0112	5.E-01
0.75	0.4880	0.0287	0.4788	0.0245	0.0093	8.E-01
1.50	0.9824	0.0599	0.9699	0.0424	0.0124	8.E-01
3.00	2.0218	0.0969	1.9658	0.0551	0.0560	6.E-01

Average analytical response (ethanol/internal standard area ratio) of blood and water samples at different concentrations (0.06-3.00 g/L of ethanol) analysed using n-propanol or t-butanol as internal standards (n=10).

Nigerian Biomedical Science Journal Vol. 16 No 3 2019 33

 

Table 2. Ethanol quantification in water and blood samples

Internal Standard t-butanol

Cerilliant® calibrators (water)

Nominal	Av. Quant.	Av. Quant.	Error on blood
Conc. g/L	Water	Blood samples	samples %
	samples
0.50	0.50	0.56	12%
2.00	2.01	2.32	16%

Internal Standard t-butanol

Cerilliant® calibrators (water)

Nominal	Av. Quant.	Av. Quant.	Error on blood
Conc. g/L	Water	Blood samples	samples %
	samples
0.50	0.50	0.51	2%
2.00	2.01	2.11	5%

Average alcohol quantification of 0.5 and 2 g/L ethanol spikes in blood and water analysed using n-propanol or tbutanol as internal standard and Cerilliant® reference materials (water solutions) as calibrators (n = 10).

REFERENCES:

1. Rainey PM. Relation between serum and wholeblood ethanol concentrations. Clin Chem 1993; 39:2288–92.
2. Sklerov JH, Couper FJ. Calculation and verification of blood ethanol measurement uncertainty for headspace gas chromatography. J Anal Toxicol 2011;35:402–10.
3. Scientific Working Group for Forensic Toxicology. SWGTOX standard practices for method validation in forensic toxicology. J Anal Toxicol 2013;37:452–74.
4. Archer M, Vos J, S Visser M. The preparation, assay

Elia Mattarucchi

and certification of aqueous ethanol reference solutions. Accreditation Qual Assur 2007; 12:188–93.

1. Strassnig S, Lankmayr EP. Elimination of matrix effects for static headspace analysis of ethanol. J Chromatogr A 1999;849:629–36.
2. A. Boswell H, Dorman F. Uncertainty of Blood Alcohol Concentration (BAC) Results as Related to Instrumental Conditions: Optimization and Robustness of BAC Analysis Headspace Parameters. Chromatography 2015;2:691–708.
3. Karch S, editor. Forensic Issues in Alcohol Testing. Boca Raton: CRC Press; 2007.30–3.
4. Christmore DS, Kelly RC, Doshier LA. Improved recovery and stability of ethanol in automated headspace analysis. J Forensic Sci 1984;29:1038–44.
5. Schug K. An Indisputable Case of Matrix Effects in Blood Alcohol Determinations. Available at: http://www.chromatographyonline.com/indisputab le-case-matrix-effects-blood-alcoholdeterminations. Accessed: 16 Oct 2018
6. (2017) Guidelines for institutions equipped with laboratories for the detection of substances of abuse for toxicological-forensic and medico-legal purposes on biological samples taken from living subjects – Rev. N° 5. GTFI guidelines. Available at: http://www.gtfi.it/linee-guida/

 

AL Marshad Ibrahim, Hassanein Nagwa, Balakrishnan Bargavi

King Fahad Specialist hospital, Qassim, KSA.

Hassanein Nagwa

Clinical pathology department, Faculty of medicine for girls, Al-Azhar University, Cairo, Egypt.

All Correspondences to: Hassanein Nagwa, King Fahad Specialist hospital, Qassim, KSA.

ABSTRACT

Although the phenotypic features of basophils have been highlighted in literatures, yet some labs generate confusion while discriminating blasts from basophil gate in flow cytometry analysis. This is sometimes due to the overlap of different population on CD45 side scatter plot, and in other situations due to lack of markers which highlight basophils due to financial

jeopardy. Aim of the study: Selection of a proper combination of markers with unique expression on basophils to clearly highlight basophils especially in underresourced labs with limited budget. Methods: Comprehensive basophil phenotyping applied on 40newly diagnosed cases of CML and 20 cases of regenerating marrow post chemotherapy for Precursor B lymphoblastic leukemia with no evidence of abnormal cellsas control. Flow cytometry markers used were CD45, CD38, CD71, CD33, CD22, CD123, HLA-DR, CD34, CD117, CD3, CD4, CD8, CD56, CD11b, HLADR and CD15. Results: Basophils from patients and control samples were clearly discriminated from blasts and lymphocytes by using CD123 and CD22, a unique pattern of expression with perfect back-gating on CD45 side scatter plot which proved the discrimination. Conclusion: CD123 and CD22 will clearly discriminate basophils from different types of population which may occupy the window area as myeloblast, precursor B cells (hematogones), abnormal B cells (lymphoblasts) and mature B cells. This combination should therefore be part of the screening panel used as first line along with CD45in labs with limited budget.

KEY WORDS: Basophil, CML, CD123, CD22.

INTRODUCTION:

low Cytometry has clearly and quickly emerged to Fbe the most helpful technique in hematology lab with predominant role in the diagnose of hematological and non hematological malignancy. Also it can be used for accurate enumeration of bone marrow cells in an objective analytical method¹. Basophil is a unique type of granular myeloid offspring; its granules have high solubility in water, which results in its location in de-granulated cells with low side scatter properties, this criterion make it stand close to lymphocytes and occupy part of blasts window in CD45 side scattered plot. Basophilia is manifested in different condition of benign and malignant diseases. Myeloproliferative malignancies, typically chronic myeloid leukemia (CML) characterize by basophilia, as well as polycythemia rubravera, myelofibrosis and rarely essential thrombocythemia. Basophilia is also seen in benign condition such as hypothyroidism, hypersensitivity reaction, autoimmune disease as

rheumatoid arthritis and ulcerative colitis.²

Many markers have been used to highlight basophil in both normal and diseased condition as CD9, CD13,CD22,CD25,CD33,CD36,CD38,CD45 and CD123,294 and 69 ³

CD22 is one of the Pan B cell markers, however it is not restricted to that lineage as it has been reported as one of basophil marker with a different pattern of expression. In spite of being bright on B cells,it tends to be dimmer on Basophiles,this difference in pattern related to the structure difference of the isoform ligand. ⁴

CD123 is an Interleukin-3 receptor alpha ,it is highly expressed on plasmacytoid dendritic cells and basophils and to lesser degree on monocytes, eosinophil, myeloid and dendritic. It is also expressed on myeloblasts, mature subpopulation of hematogones and mature B cell in certain malignancy such as Hairy cell leukemia.^5,6.

CD25 is an interleukin 2 receptor alpha, its dim expression on basophils had been reported. Its expression on malignancy myeloidprecursor cellsis associated with bad prognostic molecular markers such as FLT3 and DNMT genes.⁷

CD117 is a c- kit marker heavily expressed on Mast cells and also detected on the surface normal myeloblast and promyelocytes, however its expression on normal basophil has not been detected.⁸

Although many markers have been proven to be

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Valuable and Cost Effective Combination…

expressed on basophil, yet a cost effective specific combination has not been discussed for accurate robust identification. This combination could be used in every panel used for identification of WBC, leukemia cells, MRD cells. This combination will discriminate Basophil from myeloblast and also B cell population.

SUBJECT AND METHODS:

40 newly diagnosed cases labeled as CML,18 female and 22 males age range from 22 to 76 years old, 38 cases were in chronic phase of CML and 2 cases were labeled as accelerated phase based on the WHO criteria published 2008. The CML cases were collected over a 4 years period, from January 2014 to December 2018. 20 regenerating phenotypically normal marrows with no residual abnormal cells were assessed for Basophil population as control. 12 pediatric patients (less than 16 years) and 8 adult patient (more than 16 years old). The study was

approved by the research ethical committee of Al Qassimgovernorate.

Flow cytometry analysis:

B o n e m a r r o w a s p i r a t e ( B M A ) s a m p l e s anticoagulated with sodium heparin were used, clotted samples were rejected. Samples were processed within twenty –four hours of extraction. Samples were prepared using gentle processing techniques. Briefly, Antibodies for CML panel were prepared using a four color panel ,which are listed as FITC/PE/PERCP/APC for each tube as follows : 38/33/45/71, 20/10/45/19, 3 / 1 6 + 5 6 / 4 5 / 1 9 , 45/22/123/HLA DR, 15/117/45/34, 25/4/45/11b. The MoAB were used in different combination of flurochrome as fluorescein isothiocyanate (FITC), phycoerythrin (PE), Peridinin Chlorophyll protein complex (per cp), APC (Allophycocyanin). All the antibodies used to analyse basophils were obtained from BD biosciences. Different combination of MoAb used are, CD38(HB7), CD3 (SK7), CD20 (L27), CD15 (MMA), CD25 (2A3), CD45(2D1) were conjugated with FITC, CD33(P67.6), CD10 (MEM-78), CD16 (B73.1), CD56 (NCAM 16.2), CD22 (S-HCL-1), CD117 (104 D2), CD4 (SK3) were conjugated with PE, CD71(L01.1), CD19 (SJ25C1), CD34 (8G12), CD11B (D12), HLA DR (L243) were conjugated with APC , CD123 (7G3) conjugated with PER CP CY5.5 and CD45 (2D1) PER CP was used as a gating marker in all the tubes. The amounts of antibodies added were according to the laboratory titration.

1X10⁶ White Blood cells were added to each tube and incubated at room temperature (RT) in a dark area for 15-20mins. Red cells were lysed using 1x RBC lysing reagent (Becton Dickinson USA)followed by washing cells with cell wash buffer PH 7.0-7.4 (BD) with 1%BSA(bovine serum albumin) as a blocking agent to minimize non-specific binding. In certain

cases when there is non specific binding, cells were incubated for 10 minutes with 50ul of human IgG (immunoglobulin G) for blocking Fc-receptors on the cells. Finally, cells were re-suspended in a cell wash buffer with 1%bovine serum albumin(BSA) for acquisition in flow cytometer.

Acquisition was done after machine calibration by CST beads and optimization of the machine setting. Acquisition of cells was performed on BD FACS Canto2, and analysis was performed using FACS Diva V6 software (Becton Dickinson USA). The cells were gated based onCD45, side scatter allocation of abnormal cells and the expression of markers.

RESULTS:

We examined forty newly diagnosed CML cases 18 female and twenty two males age range from (22-76 years old) 38 diagnosed at the chronic phase and two cases diagnosed in the accelerated phase, also 20 control samples from regenerating normal marrow, originally diagnosed as precursor B lymphoblastic leukemia 12 pediatric patient (less than 16 y) and 8 adult patient (more than 16 years old). We found that certain markers as CD33, Cd38. CD22, C D 1 2 3 , CD11b are invariably expressed for every basophil benign or Malignant (Figure 1, 2). HLA-DR was variably expressed on basophil from CML and control cases in the proportion 60% and 50% respectively. CD117 was not detected on normal basophils as well as CD34 and CD25 on normal basophils while CML in accelerated phase show positivity for CD117 (Figure 3). CD11c were found on normal basophil and abnormal basophils (dim expression).

Seven cases of the control Marrow detected hematogones at different proportion (1-9%) with the characteristic pattern of hematogones. Spectrum of maturation located from the negative CD45 to the brightest location of normal mature B lymphocytes with their side scatter were lower than basophils. CD123 expression was dimmer on hematogones in the seven cases (100% ) than on the basophil, while CD22 versus HLA-Dr was overlapping (Figure 4).

Table.1 show the pattern of expression of different markers on basophils in cases and control.

Marker	CML number (40)	Control samples
		(20) number
CD33	Mod	Mod
CD38	Bright	Bright
CD123	Bright	Bright
CD22	Dim	Dim
CD11b	Dim	Dim
HLA-DR	Dim 24(60%)	Dim 10(50%)
CD117	Positive in
	accelerated phase
	(2/2) 100%	Negative
CD25	Negative	Negative

36 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

Figure. 3 show one CML (accelerated phase) case with positive expression of CD117 on basophil while the blast (blue population) positive for both CD34 and CD117 mean while B cell(red population negative for both.

Hassanein Nagwa

Figure. 4 A control case (regenerating marrow ) show the phenotypic difference between hematogones and Basophil regard CD123, CD22, HLA-DR. Basophils are Magenta in color while hematogones are red.

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Valuable and Cost Effective Combination…

Figure. 5 One CML case show an example of basophil (magenta )negative for HLA-DR while positive for CD123 (brightly) and for CD22 dimly. The B cell appears in red and carry HLA-DR and CD22 brightly, while dim for CD123.

Figure. 6 show basophil (magenta color population

negative for CD25 while positive for CD11b.

DISCUSSION:

Although automatic gating has now been suggested now to be the routine practice, still the manual gate is still the main method for analysis especially in the low budget countries. CD45, side scatter is the standard back gating scatter gram for hematological malignancy’s immunophenotyping. The area of interest is where blast could locate, it is the window area or the whole area, and normally it is occupied by normal myeloblasts and basophils mainly. In the pathological condition, it could be occupied by myeloblasts, lymphoblasts or lymphoma cells as Burkett’s lymphoma.

Accurate determination of occupying population is mandatory for accurate conclusion. Also in MRD, overlap between two populations could lead to confusion and mislabeling of populations detected^9,10. Furthermore, the identification of basophilic

38 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

 

precursors in cases of acute basophilic leukemia and blast crises of CML with basophilic differentiation rely mainly on the knowledge of the robust markers usually used for discriminating this basophils ¹¹.

Basophil is a minor population of mature myeloid cells, sharing myeloid markers as CD33, CD13, CD11b, as well as other markers as CD38, CD123

and CD22. This spectrum of markers expressed, could lead to the improper separation if no unique expression pattern of robust markers is specified. We examined different markers expressed on basophil in different combination and conclude that the combination of CD22 and CD123 will discriminate basophil and separate it accurately from different population located in the blast gate. The extent of CD123 on basophil is unique since it is the brightest marker detected on this cell. This strong expression of CD123 correlates it with the major role in the proliferation and differentiation of basophilis^{12, 13}.

CD22 dimmer expression will isolate it from mature B cells. Although, the argument about CD22 expression on precursor B lymphocytes or lymphoblast may express dim CD22, yet bright CD123 will clearly determine if it is B cell or basophil. In addition, hematogones has very low side scatter comparative to basophils and myeloblasts. Furthermore, lack of CD19 on the basophil which is a fundamental marker used in every lab.since the expression pattern of CD123 at some maturation stages of hematogones and in leukemic condition is far less than that of Basophil. Furthermore, relying on back-gating on CD45, side scatter will clearly discriminate mature B cells with b r i g h t CD45 expression.

Hematogones present with heterogonous spectrum of maturation from negative to moderate CD45 and very low side scatter as well⁵.

Basophil expresses bright CD38 as well as B cell precursor (hematogones) is positive for CD 38 at the same level. However both are less bright than plasma cells which typically locate at the same area on CD45,side scatter and express unique pattern for CD38 ¹². Gating basophil using CD38 versus side scatter was not helpful but the combination of CD38 versus CD33 was able to separate basophils in control group as well as in CML cases¹³.

The expression of CD11b was invariably detected on basophils in both normal and malignant condition which stand to discriminate it from myeloblast which is typically negative forCD11b¹⁴.

Other markers as HLA-DR were recorded in 60% of the cases and around 50% of the gated basophil in the control bone marrow samples. The pattern of expression was dimmer than B cells, however comparable to myeloblasts. Very few cases does not express it, this finding is alarming since scientist use the CD123 positive /HLA-DR negative to gate basophil may lead to losing part of basophil. It is

Hassanein Nagwa

possible that DR expression is caused by cytokine activation or related to the disease itself because basophil is one of the cytokine sensitive cells^{13, 15, 16}.

CONCLUSION:

To conclude, the accurate quantification of different population located in the blast gate is a requirement for proper reporting at diagnosis and follow up of different hematological malignancies. The combination of CD22 and CD123 along with CD45 were definitely and precisely able to highlight and discriminate basophils from other type of cells occupy the blast gate. Identification of a single specific marker with stable expression on basophil is recommended to be investigated in the future to minimize the panel used.

REFERENCES:

1. Jacob MC, Souvignet A, Pont J, Solly 7. F, MondetJ, KesrS, PernolletM, Dumestre PerardC, CamposL, CesbronJY. One tube with eight antibdies for 14-part bone marrow leukocyte differential using flowc ytometry. Cytometry B Clin Cytom 2017 Jul; 92(4): 299-309. doi: 10.1002/cyto.b.21369. Epub 2016 May 6.
2. SProven D,Singel C,Baglin T, Dokal I,2015. White blood cells abnormalities.in Oxford Handbook of clinical hematology. 8. Fourth edition, chapter 13, Oxyford University Press, Great clarend on street, oxford,OX26DP,UK,page 116.
3. Han X, Jorgensen JL, Brahmandam A, Schlette E,

H u h Y O , S h i Y, Aw a g u S , C h e n W. Immunophenotypic study of basophils by multiparameter flow cytometry. Arch Pathol Lab Med. 2008 9. May; 132(5):813-9. doi: 10.1043/1543-10.1186/s13601-016-0100-4. E Collection 2016.

1. Gönen, M., Sun, Z., Figueroa, M. E., Patel, J. P., Abdel-Wahab, O., Racevskis, J., Ketterling, R. P., Fernandez, H., Rowe, J. M., Tallman, M. S., Melnick, A., Levine, R. L. Paietta, E. CD25 expression status improves prognostic risk classification in AML independent of established biomarkers: ECOG phase 3 trial, E1900. Blood,(2012). 120(11), 2297306.
2. Agis, H., Füreder, W., Bankl, H. C., Kundi, M., Sperr, W. R., Willheim, M., Boltz-Nitulescu, G., Butterfield,

J. H., Kishi, K., Lechner, K., …Valent, P. (Comparative immunophenotypic analysis of human mast cells, blood basophils and monocytes. Immunology (1996)., 87(4), 535-43.

1. Saksena A, Gautam P, and Singh TSide scatter versus CD45 flow cytometric plot 2165(2008)132 (813:ISOBBM) 2.0.CO;2. can distinguish acute leukaemia subtypes.

 

, Oto-Obong V. Idah and Bitrus N. Lekshak

Department of Haematology and Blood Transfusion, University of Jos, Nigeria

Ahmed M. Sabo and Oto-Obong V. Idah

Department of Human Physiology, University of Jos, Nigeria.

Thomas P. Yakubu

Department of Pharmacognosy University of Jos, Nigeria

Moses D. Lugos

Department of Medical Laboratory Science, Faculty of Health Sciences & Technology, University of Jos, Nigeria.

All Correspondences to: Changjul L. Singnap

, Department of Haematology and Blood Transfusion, University of Jos, Nigeria

ABSTRACT

Background: It is estimated that over 80% of people still depend mainly on the traditional use of parts of plants and herbs to treat ailments. The medicinal properties of these plants and herbs are linked to the presence of a variety of phytochemicals and their elemental composition. Justification: Every year, 1 in 4 people die of conditions related to thrombosis, with many never knowing their risk for the condition. Prevention of intravascular thrombosis, however, has a narrow therapeutic window, bleeding risk, the incidence of resistance, and unwanted drug interactions, hence the need for anti-thrombotic drugs that deliver more effective prevention of intravascular thrombosis. Aim: The research sought to investigate the effect of Moringaoleifera ethanolic seed extract on Bleeding Time (BT) and Clotting Time (CT), in Wistar albino rats. Materials and Methods: Forty (40)Wistar rats were weighed and randomly divided into two groups, group I=15 rats for BT, group II=15 rats for CT and 10 rats for control tests. After the administration of ethanolic extract of M. oleifera seed at the dose of 100mg/kg, 200mg/kg and 400mg/kg (administered to a group of 5 rats per dose) for 28 days, BT and CT were determined using Tail transection and Lee & White method respectively. The data were analysed using GraphPadPrism (7.03). Result: There was a statistically significant increase at P <0.0001 of BT and CT compared to the control (administered with only distilled water). Conclusion: The prolonged BT and CT indicate that the seed extract of M. oleifera could pose an antagonising effect on both the primary (platelets) and secondary haemostatic activities, a property that can be explored in the management of

thrombotic diseases.

Keywords: Moringaoleifera, Bleeding Time, Clotting Time, Thrombosis.

INTRODUCTION

or thousands of years, plants have been a vital Fsource of medicine. Even today, the World Health Organization (WHO) has reported that up to 80% of people still depend essentially on traditional medications such as the use of herbs to treat ailments.^1,2 Diverse plants phytochemicals and their elemental composition have been

shown to possess medicinal values.^3,4

A growing concern in the development and evaluation of natural antioxidants from medicinal plants mainly in the field of preventive health care and the food industry is reported. Among those herbs, one promising species is Moringa oleifera Lam (Moringa or drumstick tree), which is native to the sub-Himalayan regions of Northwest India.⁴Moringa oleifera (Moringaceae) is a well-known plant in North Eastern,

Nigeria.⁵Lam seed; is a highly valued plant, with an impressive range of medicinal uses and high nutritional value.⁶A good number of traditional medicine references attest to its therapeutic potential, and scientific validation of theseplantsis developing to support at least some of the claims.⁷ Historically, ancient Romans, Greeks and Egyptians, as well as Asian communities, have been using the plant for various medicinal and nutritional purposes.⁸ The plant is used locally for various medicinal purposes by traditionalists and herbalists in Maiduguri, North Eastern Nigeria.⁵ M. oleifera roots and seeds are prescribed for the treatment of snake bites, and scorpion stings and the plant contains a mixture of several hydrolytic enzymes, in which proteases are the key enzymes responsible for the observed pharmacological actions.^9,10 Ajibade et al., 2011 also reported that the seed could reduce the number of platelets, hence the need to look into its effect on haemostatic

 

Investigating the effect of ethanolic extract…

activities in detail.^11,12 Platelets, also called “thrombocytes”, are a component of blood whose function (along with the coagulation factors) is to stop bleeding by clumping and clogging blood vessel injuries. Platelets release a variety of substances that promote blood 1clotting, which causes haemostasis. The normal blood platelets count is between 150 x 10⁹/L and 450 x 10⁹/L (150,000 – 450,000/ mm³). Platelets have no cell nucleus: they are produced by budding-off of the cytoplasm of megakaryocytes.¹³ Formation of this platelets plug (primary haemostasis) is associated with activation of the coagulation cascade; which results in fibrin formation and crosslinking (secondary haemostasis).They also play an important role in the conversion of prothrombin to thrombin because much of the prothrombin first attaches to prothrombin receptors on the platelets already bound to the damaged tissue.14Intravascular thromboses such as deep vein thrombosis (DVT), a major cause of myocardial infarction, stroke and pulmonary embolism, can be caused by abnormal platelet activation, augmented clotting, vascular dysfunction and shear stress due to interrupted blood flow and atherosclerosis.^15,16 This research was therefore designed to investigate the effect ofMoringa oleifera seed extract on bleeding time (BT) and clotting time (CT).

MATERIALS AND METHODS:

Seed Collection and Authentication

Dried seeds of Moringa oleifera were obtained from Lamingo Jos-North local government Area of Plateau State Nigeria.

Preparation of Seed Extract

Good quality seeds were selected for this study. The coat and wings of the seeds were removed and the kernel collected. The kernel was dried for 5 days at room temperature (25 – 38°C) in the Department of Pharmacognosy. Then we produced a fine powder of the seeds by using coffee mill attachment of Moulinex domestic food blender (Moulinex Genuine Blender 1.25 Liter, 400 Watt and White – LM2421EG made in France). The seeds were weighed before and after blending. The quantity after blending was soaked in 4 litres of 70% ethanol at room temperature for 3 days and filtered; the bulked filtrate was evaporated using hot air oven at 41°C for 24 hours. Until needed, the powder produced was stored at room temperature. A fresh solution of the seed extract was prepared when needed. The test solution was prepared by dissolving the fine powder of Moringa oleifera seed in distilled water in the ratio of 10g to 100ml (to give 100mg/ml concentration) and stirred for two minutes before intubation.¹⁷

Experimental Animals

A total of forty (40) male and female Wistar rats weighing between 150g to 250g were used for the research. The rats were kept in a plastic cage at room temperature with twelve hours a night/dark cycle. They had access to their feed and a hygienic environment maintained to prevent infection. The rats were weighed using an electric beam balance at the commencement of the experiments and weekly throughout the duration of the experiment.

Experimental design

A total of 40 Wistar rats were used for this study to determine the BT and CT; 15 rats wereused per test by grouping them into three (5 rats/each dose) 100 mg/kg (low

dose), 200 mg/kg (medium dose) and 400 mg/kg (high dose) of the seed extract in that order. We used 5 rats to control for BT and 5 rats were used to control for the CT. The oro-gastric method of intubation was adapted for the administration of seed extract for experimental rats while distilled water was administered to control groups, these were done in the morning hours before feeding the animals. The rats were intubated daily throughout the period of the experiment (for 28 days).

Sample Collection

Animals were euthanized 24 hours after the last doses administered to them. The rats were weighed and then anaesthetized by placing them in a closed jar containing cotton wool sucked with chloroform and euthanized by cervical dislocation and collected from jugular vein into aodium-citrated sample bottle (at the ratio of 4.5 to 0.5) and the content properly mixed by gentle rolling of the bottle for haematological analysis. The blood parameters analyzed included Bleeding Time (Duke’s method) and Clotting Time (Lee and White method).

Laboratory Methods

Bleeding Time (Tail transection; a modified Duke’s

method)

After 24 hours of the final oral administration of Moringa oleifera seed extract, the bleeding time was measured in rats by modified Duke’s method as described briefly; the tail of the anaesthetized rat was cut 1 cm from the end using a sharp pair of surgical scissors and then the tail lesion blotted with a clean Whatman filter paper every 15 seconds. The interval, from the time the tail incision was made until the time blood was no longer apparently transferred to the filter paper, was recorded as the bleeding time in seconds.¹⁵

Whole blood clotting time (Lee White method)

Three test tubes were placed in a 37°C water bath and 3ml of blood sample was collected from the rats by the jugular method. As soon as blood appeared in the syringe, the stop watch was started. To each of the pre-warmed glass test tubes, 1ml of the blood was added. After the initial 3 minutes of incubation, the three tubes were simultaneously tilted to observe for clot formation at 30 seconds intervals. This procedure was repeated until the blood was clotted. The time taken for the first blood clot to appear was recorded in seconds. Subsequently, the process of tilting was repeated for the remaining 2 tubes until clotting was observed. The average value of clotting time of the three tubes was reported as clotting time of the rat in seconds.

Statistical Analysis

Data collected were expressed as mean ± S.D and analysed using the SPSS software program (Graph PadPrism, 7.03). One Way analysis of variance (ANOVA) was used to determine the level of significance of confidence at 95%, (pvalue< 0.05 was considered significant).¹⁸

RESULTS:

A total of 40 rats were used for the experiment to determine the BT and CT; 5 rats/each dose of: 100 mg/kg (low dose), 200 mg/kg (medium dose) and 400 mg/kg (high dose) of the seed extract in that order. We used 5 rats to control for BT, and5 rats were used to control for the CT. The result showed that the mean value of BT and CT in control groups fall within the normal range of 60-420s and 240-540s respectively. At 100mg/ml of the extract, the mean value of

 

 

BT and CT were 1059.60s and 253.30 seconds respectively, at 200mg/ml BT=

1773.00s, while CT= 346.40s, and at 400mg/ml,

BT= 1917.20s, while CT= 392.00s (Table 1 The effect of ethanolic extract of M. oleifera seed as presented in figures 1 & 2 was seen to be accompanied by significant increase in both BT and CT with increasing concentration of the dosage of extract in the order of 100mg/ml, 200mg/ml, and 400mg/ml (P<0.0001).

Table 1: Mean values of Bleeding Time (BT) & Clotting Time (CT) of experimental ratsfed on daily doses of 100mg/ml, 200mg/ml, and 400mg/ml of ethanolic extract of m. oleiferaseed and a control group on distilled water.

Dose of m.	No. of	Mean BT	Mean CT
oleifera extract	Rats	(Secs)	(Secs)
Control	5	173.20 ± 25.19	240.60	± 12.60
100mg/ml	5	1059.60 ± 52.05	253.30	± 9.32
200mg/ml	5	1773.00 ± 64.43	346.40	± 9.43
400mg/ml	5	1917.20 ± 47.32	392.00	± 9.62

Figure 2: Bar charts showing the dose effect of ethanolic extract of M. oleifera seed onwhole blood Clotting Time of experimental and control rats.

DISCUSSION:

Footnote: The mean and standard deviation of bleeding time and whole blood clotting time ofexperimental and control rats are reported in seconds. While the total is the average values forthe total means of BT and CT.

Figure 1: Bar charts showing the dose effect of ethanolic extract of M. oleifera seed on Bleeding Time of experimental and control rats.

In this work, we investigated the effect of Moringa oleifera on haemostatic profile particularly BT and whole blood CT in vivo using Wistar albino rats.

M. Oleiferawas described as a plant with many medicinal values. The different parts of the plant such as the leaves, seeds, fruits, flowers and back act as cardiac and circulatory stimulants and antihypertensive among others.7 Also, the coagulant property of the seed has been shown to be useful in water purification and treatment processes.¹⁹

We, therefore, sought to investigate the effect of the Moringa oleifera seed extract on blood coagulation in rats. The study showed that Moringa oleifera seed extract might be responsible for the prolonged bleeding and whole blood clotting time in Wistar albino rats as reported in the results section. The average BT of the control (173.20 ± 25 seconds) falls within the normal range of between 120 – 420 seconds (2-7 minutes).

However, at 100mg/kg concentration of the seed extract, a significant increase in the average BT of 1060.0 ± 52.05 seconds (17minutes ± 52.05s) was observed (p ≤0.0001). The result further showed a dosedependent increase with the highest at 1917.20 ± 47.32 seconds (about 33 minutes) at the dose of 400mg/kg. Also, the CT of the control group at 240.60 ± 12.06 seconds (4.10 minute) falls within the normal range of 240 – 540 seconds (4-9 minutes). However, there was no significant difference in the CT at 200mg/ml concentration (346.40 ± 9.43 seconds) of M. oleifera seed extract compared to mean value at 100mg/ml (253.80 ± 9.32 seconds). A statistically significant increase in the mean CT value of 392.00 ± 9.62 seconds was observed compared to CT mean value of 253.80 ± 9.32 seconds at 100mg/ml (P ≤ 0.005).Data revealed that a higher dose of M. oleifera extract seed may prolong both

 

Investigating the effect of ethanolic extract…

bleeding and whole blood clotting time.

The delay in BT and CT may be due to the seed’s ability to cause platelets aggregation hence reducing the number of platelets receptor sites needed for adhesion (the initial stage of bleeding arrest). Our work supported the findings of Luz et al., which reported that a coagulant isolated from M. oleifera seed called Lectin significantly prolongs activated partial thromboplastin time (aPTT) and prothrombin time (PT).²⁰ The research of Luz et al., 2017 was done in vitro using human blood, but we investigated whole blood CT in vivo using rats.

Analysis of variance was performed to compare the mean of extract doses and concentrations which reveals significantly (p≤ 0.05) that all the doses (100mg/ml, 200mg/ml, and 400mg/ml) resulted in increased coagulation (haemostatic) time in rats. The higher the dose the longer it takes for clotting to take place. This means that hypercoagulability (one of the causes of thrombotic events) can be delayed with the right doses of Moringa oleifera seed extract. This findings could be the reason why M. oleifera seeds are used as anti-scorpion, antisnake bites.10

CONCLUSION:

The results of this study showed that M. oleifera seed could delay haemostatic activity for as long as the administered dose lasts, usually within twenty-four hours (24hrs.). Since M. oleifera works similarly to heparin and aspirin; it may be a suitable replacement or another alternative of blood thinners. The seed extract of M. oleifera has the advantage of being a nutritional supplement meaning it has far less side effect if any.

RECOMMENDATIONS:

1. It is necessary to look into this wonderful gift (M. oleifera) to mankind with the aim of developing the seeds as a nutritional supplement or even as a drug which could be recommended in the management of thrombotic events such as deep vein thrombosis (DVT) or atherosclerosis in order to minimize ischaemic heart attack or even stroke since normal haemostasis cannot be possible without the activation of platelets.
2. Further research on the effect of M. oleifera seed tract on other haemostatic profiles such as PTT, PT, clotting factors & co-factors, vWf, and platelets satelitism may be able to reveal more on the efficacy of this plant seed in arresting thrombotic activities.
3. When used as nutritive supplement at the right dose, it can help in the prevention of unnecessary activation of platelets leading to thrombosis; this is because the extract of M. oleifera acts efficiently at both the primary and secondary stages of haemostasis.

REFERENCES:

1. Alves, R.R. and I.L. Rosa, Why study the use of animal products in traditional medicines? Journal of ethnobiology and ethnomedicine, 2005. 1(1): p. 5.
2. Maroyi, A., An ethnobotanical survey of medicinal

plants used by the people in Nhema communal area, Zimbabwe. Journal of ethnopharmacology, 2011. 136(2): p. 347-354.

1. Ferreira, P.M.P., et al., Safety and Efficacy of Moringa oleifera Lamarck (1785)— Therapeutic and Toxicological Properties, in Pharmacology and Therapeutics. 2014, InTech.
2. Valdez-Solana, M.A., et al., Nutritional content and elemental and phytochemical analyses of Moringa oleifera grown in Mexico. Journal of Chemistry, 2015. 2015.
3. Zaku, S., et al., Moringa oleifera: An underutilized

tree in Nigeria with amazing versatility: A review.

African Journal of Food Science, 2015. 9(9): p. 456-461.

1. Gopalakrishnan, L., K. Doriya, and D.S. Kumar, Moringa oleifera: A review on nutritive importance and its medicinal application. Food Science and Human Wellness, 2016. 5(2): p. 49-56.
2. Anwar, F., et al., Moringa oleifera: a food plant with

multiple medicinal uses. Phytotherapy Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives, 2007. 21(1): p. 17-25.

1. Petrovska, B.B., Historical review of medicinal plants’ usage. Pharmacognosy reviews, 2012. 6(11): p. 1.
2. Ogbunugafor, H., et al., In vitro and in vivo evaluation of antioxidant properties of Moringa Oleifera ethanolic leaves extract and effect on serum lipid indices in rat. Macedonian Journal of Medical Sciences, 2012. 5(4): p. 397-403.
3. Satish, A., et al., Moringa oleifera Lam.: Protease activity against blood coagulation cascade. Pharmacognosy research, 2012. 4(1): p. 44.
4. Ajayi, I.A., O. Ajibade, and R. Oderinde, Preliminary phytochemical analysis of some plant seeds. Res. J. Chem. Sci, 2011. 1(3): p. 58-62.
5. Day, I.S.C.f.W.T., et al., Thrombosis: a major contributor to the global disease burden. Journal of Thrombosis and Haemostasis, 2014. 12(10): p. 1580-1590.
6. Golebiewska, E.M. and A.W. Poole, Platelet secretion: From haemostasis to wound healing and beyond. Blood reviews, 2015. 29(3): p. 153-162.
7. Eyarefe, O.D., A. Idowu, and J.M. Afolabi, Healing potentials of oral Moringa oleifera leaves extract and tetracycline on methicillin resistant Staphylococcus aureus infected wounds of Wistar rats. Nigerian Journal of Physiological Sciences, 2015. 30(1-2): p. 73-78.
8. Furie, B. and B.C. Furie, Molecular and cellular

30

 

biology of blood coagulation. New England Journal of Medicine, 1992. 326(12): p. 800-806.

1. Bain, B.J., I. Bates, and M.A. Laffan, Dacie and Lewis Practical Haematology EBook. 2016: Elsevier Health

Sciences.

17. J a j a – C h i m e d z a , A . , e t a l . , B i o c h e m i c a l characterization and anti-inflammatory properties of an isothiocyanate-enriched moringa (Moringa oleifera) seed extract. PloS one, 2017. 12(8): p. e0182658.

1. Wongkrajang, P., W. Chinswangwatanakul, and P. Tientadakul, Whole Blood Clotting Time: Variation of

Practice in Coagulation Laboratory, Members of Thailand National External Quality Assessment Scheme. Siriraj Medical Journal, 2017. 63(3): p. 8184.

1. Camacho, F.P., et al., The use of Moringa oleifera as a natural coagulant in surface water treatment. Chemical Engineering Journal, 2017. 313: p. 226-237.
2. de Andrade Luz, L., et al., Structural characterization of coagulant Moringa oleifera Lectin and its effect on hemostatic parameters. International journal of biological macromolecules, 2013. 58: p. 31-36.

 

Maxwell Hubert Antwi

Department of Molecular Medicine, School of Medical Sciences, College of Health Sciences,

Kwame Nkrumah University of Science & Technology, Kumasi, Ghana.

Seth Christopher Yaw Appiah

Department of Sociology and Social Work, Faculty of Humanities and Social Sciences,

Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.

Centre for International Health (CIH), University of Munich Medical School, Ludwig-Maximilians-Universitate of Munchen, Munchen, Germany

All correspondence to: Seth Christopher Yaw Appiah, sychrist2007@gmal.com

ABSTRACT

Background: A major limitation of cervical cytology is the unsuitability of proportion of  smears submitted for analysis and for cytological assessment (unsatisfactory). This study examines the prevalence of unsatisfactory Pap smear, clinical history and diagnosis in the Department of Pathology, Korle-Bu Teaching Hospital (KBTH), Ghana. Materials and Methods: A retrospective review of 15,290 cases spanning 12 years (2005-2016) was carried out at the cytology unit of the Pathology Department of the KBTH. Out of the 15,290 Pap smear records retrieved, 2347 reports were excluded leaving 12,943 for the study. All unsatisfactory smear cases were analyzed and categorized using the Bethesda 2001 System. Results are presented using descriptive statistics. Results: The overall prevalence of unsatisfactory Pap smear was 402 (3.1%). Routine screening smear accounted for 115 (0.9%); reports without clinical history and diagnosis gave 21 (0.2%) and cases with clinical history and diagnosis were 287 (2.2%). The common cause of unsatisfactory Pap smear was scanty cellularity 222 (1.72%). Patient’s history accounted for the least cause of unsatisfactory Pap smear 2 (0.02%). Conclusion: Pap smear results reported as unsatisfactory could

harbor cancer malignancy. Samples should be taken by well-trained persons.

INTRODUCTION

Cancer is a global health concern. Millions of individuals have been diagnosed and several affected people have lost their lives. Cancer of the uterine cervix is a leading public health issue globally and

is the commonest cancer in the female genital tract [1, 2]. Over the past two decades, the incidence of the disease has shown a decline in the developed countries [3, 4], yet in poor resource settings like Ghana, there has not been significant change in both prevalence and incidence. [1, 5]

A report by Ghana health service rated cervical cancer as the topmost cancer affecting women in Ghana, and attributed 50.5% of the condition to human papillomavirus (HPV) types 16 and 18 with 16% as cause of death attributable to cancer [6].

Estimates by the World Health Organization (WHO) posit that cervical cancer case in Ghana will exceed 5000 with mortality of 3300 annually by 2025 if the trend persists [7]. It has become difficult to relegate the condition in the country to the background without attention. Larger percentage of women of reproductive age stand at increased risk of having cancer of the uterine cervix since there is no national screening programme or management readily available [6, 8].

Screening of cervical cancer is uncommon in Ghana as the test is done in few public and private health centers in the country and is lowly patronized by the target group (women) [8, 9]. Nevertheless, risk of dying from cervical

cancer can be reduced with routine Pap test [1, 4]. It has been reported that, the annual global death rates attributable to cervical cancer has declined by 2% ever since Pap smear test was introduced with overall death rate by 74% [10].

The Pap test results indicate various changes relating to clinical observation such as; unsatisfactory, normal, inflammation, benign cellular changes, Atypical Squamous cell of undetermined significance (ASCUS), High Grade Squamous Intraepithelial Lesion (HGSIL) and cervical cancer [2, 11]. Despite its outstanding success, the Pap smear is not 100% perfectly accurate [12-14]. Problems occur at every level from failure of women to get regular Pap smear test in the first place to sampling and interpretation error which is likely to give unsatisfactory results that would need a clinical follow-up [12-14].

The number of false negative reports is of great concern because some are reported as a result of unsatisfactoriness [2, 15]. One of the limitations of cervical cytology is that a proportion of smears submitted for analysis in the light microscope are unsuitable for reliable cytological assessment (3, 4). This category of limitation is considered as unsatisfactory Pap smears [11]. Pap smear according to the Bethseda system can be categorized as follows; Sampling error; this error can be due to slide lacking an adequate number of well-preserved and well-visualized squamous epithelial cells (minimum of 8000 – 12,000 for conventional Pap or 5000 for Liquid-

base pap, thus scanty cellularity) [11, 14].

Processing error includes poor fixation, air-drying artifact or contaminants obscuring over 75% of the epithelial cells and cellular material being too thick or multilayering smear [11, 14]. Obscuring error includes cells being too atrophic, blood and inflammation obscuring over 75% of the epithelial cells, excessive cytolysis [11, 14]. A broken slide which affects smear for cytologic examination is technically unaccepted. Lack of pertinent patient information and clinical history are as cause of unsatisfactory Pap smear. Any of the causes under these errors can be unsatisfactory.

A large prospective study done in Norway found that unsatisfactory Pap smear test results indicated a 1.6 to 4.0 times high risk of harbouring CIN 2/3 or invasive cervical cancer compared with woman with a normal Pap test result [16]. Unsatisfactory category constitutes 1% to 2% of all Pap test [17-19]. There are however little information and knowledge on its prevalence in Ghana and particularly at the nation’s biggest teaching hospital, Korle-Bu Teaching Hospital.

The essence of this study was to provide adequate data and knowledge on unsatisfactory Pap smears’ prevalence and causes in line with their respective clinical history and diagnosis and to help health-care practitioners with measures to minimize any error that could lead to unsatisfactory Pap smear. This study provides baseline data for comparison with similar reviews in future since there is currently no known scientific documented evidence on unsatisfactory Pap smear prevalence in Ghana.

METHODS

Setting

The study was conducted at the Korle-Bu Teaching hospital. The hospital is the largest health facility and also the premier teaching hospital in Ghana. The Korle-Bu Teaching hospital has a bed capacity of over 2000 and offering medical training for medical doctors, nurses and other health professionals. There are 17 clinical and diagnostic departments/units in the hospital. The hospital is host to the National Centre for Radiotherapy and Nuclear Medicine which functions as a referral centre for the management of cancer supporting other specialized services such as renal transplantation, DNA investigations and brachy therapy for the treatment of prostate cancer. The study was carried out using data from the cytology unit of the Pathology Department of the KBTH.

Study Design and Sampling

The study was retrospective review of all Pap smear registry data of women who underwent Pap smear at the cytology unit of the Korle-Bu hospital spanning a period of 12 years (2005-2016). The study used a hospital based registry which has a catchment that covers the entire southern part of Ghana and beyond without any well-defined population. However, under the period the studied, 15,290 conventional Pap smear test reports were retrieved. The cytology unit actively collects data on all Pap smear cases presenting to the hospital for possible diagnosis of cervical cancer. The data sources are often from either women regular visits to the various clinics and wards of admission or by referral to the unit from within

the hospital or outside the hospital. A special case folder is created (folders) within the unit to abstract needed information with succinct case definition. Information on the clinical history of patients was extracted from the medical folders of each client through a careful sifting of the records.

Analysis

The reports taken from the archives had their covers cleaned and the total Pap smear reports were noted as 15,290. Out of the 15,290 reports, 2347 (both missing reports and lack of TZ components reports) were excluded so 12,943 reports were left for the study. The criteria for the unsatisfactory diagnosis were according to the Bethesda System 2001 and their causes categorized as; sampling error due to inadequate number of well preserved and well visualized squamous epithelial cells (minimum of 8000 – 12,000 for conventional Pap smear)—thus scant cellularity (<10% of slide covered by interpretable squamous cells), processing error due to poor fixation, air-drying artifact or contaminants obscuring over 75% of the epithelial cells and cellular material too thick or multilayering smear, presence of obscuring error of the test due to cells being too atrophic with blood and or inflammation obscuring over 75% of the epithelial cells, excessive cytolysis, broken slide, and lack of patient’s history. The 12,943 reports were reviewed for unsatisfactory diagnosis in line with their respective clinical history and diagnosis viz. age, and their causes were noted, counted and grouped into the various categories. The frequencies of the various categories and what constitute them were determined. The prevalence of the unsatisfactory Pap smears in percentages annually were determined and the overall prevalence within that time frame was also known. The prevalence in percentage was calculated as; (total unsatisfactory Pap smears/total Pap smears) multiply by 100. The data was exported into Microsoft Excel and SPSS version 16 for windows and further analysis. Ethics approval was sought from the University of Ghana Ethics approval committee

RESULTS

Population Characteristics

A total number of Pap smears and smears rejected as unsatisfactory reviewed over the twelve year period (Table 1). A total of 15,290 conventional Pap smear test reports were retrieved for review from the archives at the cytology unit of the department of pathology, KBTH over a twelve-year period. Out of the 15,290 Pap smears reports, 529 were missing in the archives, 1818 were limited by lack of transformation zone component and so both (2347 reports) were excluded from this study. The entire Pap smear reports for 2011 could not be found from archives and were excluded. The total Pap smear over the period was 12,943 and unsatisfactory Pap smear was also

1. The average age and age range of women who attended the unit to do Pap smear test were 39.5 and 19 – 80 years respectively. Smears rejected as unsatisfactory were assessed in line with their respective clinical history and diagnosis and their percentages recorded as follows. Unsatisfactory rate with history as routine screening was
2. (0.9%), with clinical history and diagnosis was 287 (2.2%), rate in reports without clinical diagnosis gave 21

(0.2%) and the overall rate in all diagnosis and routine		unsatisfactory categories were presented according to each
screening was 402 (3.1%).		year. In 2005, total unsatisfactory Pap smear was 63 (4.2%),
Causes of Unsatisfactory Pap Smear and Associated		2006 presented 23 (3.7%), 2007 gave 54 (5.3%), 2008 was
		38 (3.7%), 2009 gave 38 (2.9%), 2010 was 47 (3.7%), 2012
Diagnosis and Clinical History		gave 33 (2.7%), 2013 was 39 (2.8%), 2014 gave 40 (2.5%),
Table 2 shows the various causes of unsatisfactory Pap		2015 was 16 (1.2%), 2016 gave 11 (1.6%) and the overall
smears in line with clinical history and diagnosis. Scanty		annual percentage was 402 (3.1%).
cellularity 77 (0.6%) gave higher number for almost all the		The unsatisfactory categories based on their causes
clinical history and diagnosis with routine screening. This		were reported. Sampling error gave total unsatisfactory
was followed by uterine fibroid 52 (0.4%), infertility 27		rate of 222 (1.72%), processing error gave 46 (0.36%),
(0.2%) (amenorrhea, dysmenorrhea, menorrhagia,		inherent error of the test was 132 (1.02%) and patient
dysuria, leiomyoma), cancer 20 (0.2%) (cervical cancer,		history was 2 (0.02%).
endometrial cancer). Accordingly, the least factors that		Year to Cause Categorization of Unsatisfactory Pap
accounted for the unsatisfactory Pap smear were post-		Smear
menopausal bleeding, bleeding in urine) and cytolysis 2		Table 4 shows the various categorization of
(0.02%)		unsatisfactory Pap smear. Scanty cellularity was the
Cause Specific Categorization of Unsatisfactory Pap		highest recording 222 (1.72%) followed by obscured
Smear		inflammation 69 (0.53%), blood obscurance 56 (0.43%),
The results presented in Table 3 show the frequency and		thick smear 17 (0.13%), with the east being cytolysis 7
annual percentages of the unsatisfactory categories based		(0.05%) and patient history 2 (0.02%).
on their causes. The total and annual percentages of the
	Table 1. Study population characteristics.
	Data		N (%)
	Actual number of conventional Pap smear retrieved		1520 (100%)
	Missing cases in the Archive		529 (3.5%)
	Pap smear data limited by transformation zone component		1818 (10.9%)
	Total number of Pap smears reviewed		12,943
	Total unsatisfactory Pap smears reviewed		401
	Age of women (range) years		9 – 80 yrs
	Mean age (yrs)		39.5
	Unsatisfactory rate in routine screening		115 (0.9%)
	Unsatisfactory rate in clinical history and diagnosis		287 (2.2%)
	Unsatisfactory rate in reports without clinical diagnosis		21 (0.2%)
	Unsatisfactory rate in all diagnosis and routine screening		402 (3.1%)

Table 2. Causes of unsatisfactory Pap smear and associated diagnosis and clinical history.

	Clinical History and Diagnosis
Reason for	RS No. ND No. UF No. Hys/Myo Inf No. Ca PB No. Total
unsatisfactory	%		%	%	%	%	%	%
Scanty cells	77	(0.6)	11 (0.08)	52 (0.4)	20 (0.2)	27 (0.2)	20 (0.2)	15 (0.1)	222 (1.7)
Obscuring
inflammation	18	(0.1)	4 (0.03)	3 (0.02)	17 (0.1)	7 (0.05)	11 (0.08)	9 (0.07)	69 (0.5)
Obscuring blood	10	(0.08)	2 (0.02)	4 (0.03)	4 (0.03)	11 (0.08)	12 (0.09)	13 (0.1)	56 (0.4)
Thick smear	4 (0.03)		1 (0.01)	2 (0.02)	3 (0.02)	3 (0.02)	1 (0.01)	3 (0.02)	17 (0.1)
Drying artifact	3 (0.02)		1 (0.01)	3 (0.02)	2 (0.02)	2 (0.02)	2 (0.02)	2 (0.02)	15 (0.1)
Poor fixation	1 (0.01)		2 (0.02)	3 (0.02)	3 (0.02)	2 (0.02)	1 (0.01)	2 (0.02)	14 (0.1)
Cytolysis	2 (0.02)		0	1 (0.01)	1 (0.01)	1 (0.01)	1 (0.01)	1 (0.01)	7 (0.05)

RS-Routine screening, ND-No diagnosis, UF-Uterine fibroid, Hys/Myo-Hysterectomy/Myomectomy, Inf-Infertility (amenorrhea, dysmenorrhea, menorrhagia, dysuria, leiomyoma), Ca-(cervical cancer, Endometiral cancer), PB-(Post coital bleeding, Post-menopausal bleeding, bleeding in urine).

 

Table 3. Categorization of unsatisfactory Pap smear based on their causes.

Years	Sampling Processing			Patient	Broken	Total Unsat.	Total Pap	%
	Inherent error error			history	slide	Pap smears	smears
	error of the test
2005	34	8	21	0	0	63	1488	4.2
2006	14	1	8	0	0	23	621	3.7
2007	35	9	9	1	0	54	1011	5.3
2008	26	4	8	0	0	38	1023	3.7
2009	25	3	9	1	0	38	1298	2.9
2010	35	3	9	0	0	47	1274	3.7
2012	13	4	16	0	0	33	1215	2.7
2013	17	9	13	0	0	39	1382	2.8
2014	14	5	21	0	0	40	1594	2.5
2015	6	0	10	0	0	16	1336	1.2
2016	3	0	8	0	0	11	701	1.6
	222	46	132	2	0	402	12943	3.1
Total %	1.72	0.36	1.02	0.02	0.00	3.12	–	–

Table 4. Year-to year causes and categorization of unsatisfactory Pap smear.

	Years	cellularity Scant		smear Thick fixationPoor			dryingAir-	obscureBlood Cytolysis inflammation
		Obscured		Patient history Broken slide Unsat papTotal
2005		34	0	5	3	8	2	11	0	0	63
2006		14	0	1	0	1	0	7	0	0	23
2007		35	5	1	3	5	2	2	1	0	54
2008		26	2	1	1	6	0	2	0	0	38
2009		25	2	1	0	5	0	4	1	0	38
2010		35	0	0	3	5	0	4	0	0	47
2012		13	3	0	1	3	1	12	0	0	33
2013		17	1	5	3	5	0	8	0	0	39
2014		14	4	0	1	13	0	8	0	0	40
2015		6	0	0	0	2	2	6	0	0	16
2016		3	0	0	0	3	0	5	0	0	11
	Total	222	17	14	15	56	7	69	2	0	402
	Total Pap
	Smears	12943 %	1.72	0.13	0.11	0.12	0.43	0.05	0.53	0.02	0 3.1

Prevalence to Year Specific Rates of Unsatisfactory Pap Smear

The graph presented in Figure 1 shows that over the years, the highest number of sampling error cases was recorded in 2007 with 35 (5.3%) cases. Highest processing error related cases were recorded in the years 2007 and 2013 (n = 9). No case of broken slide was recorded during the 12 years period. Despite the decline in recorded cases resulting from inherent error of the test from 21 cases in 2005 to a stable 8 and 9 recorded cases over a five year period up to 2010, it increased again in 2012 to 16 recorded cases, fell to 13, increased again to 21 in 2014 and begun to record lower cases in the last two years preceding the end point year for this study (2016).

DISCUSSION

The purpose of this study was to determine the prevalence of unsatisfactory Pap smear in the department of pathology, KBTH and to know the various causes in line with their respective clinical history and diagnosis. The overall prevalence of unsatisfactory Pap smear in this study was 3.1 percent. This estimate is lower when compared to reports from studies done in India, Italy and Taiwan but on the rise when compared to previous studies by Ransdell et al., McGaraghan and Smith-McCune in United States and Netherlands [17, 19-22]. Though the annual prevalence of unsatisfactory Pap smear demonstrates no consistent pattern, year on year prevalence appears to be on the decline. From the annual prevalence in percentages of the unsatisfactory Pap smears, the year 2007 recorded the highest prevalence of 5.3 percent. There was however no

 

suggestive trend or pattern to conclude that the higher the total number of Pap smears or unsatisfactory Pap smear, the higher the prevalence in percentage of unsatisfactory Pap smear to demonstrate any association.

Despite, the study’s inability to determine specific cause attribution to the Pap smear decline, the increasing training and clinical experiences of laboratory scientist at the Cytology Unit of the Korle-Bu Teaching Hospital (KBTH) might play a role.

Though the current study had a lower rate of unsatisfactory Pap smears compared to previously reported findings in India, Italy and Taiwan, the rate (3.1%) remains high because unsatisfactory Pap smear rate of all categories according to reference books should be between 1% to 2% This is against the background that

longitudinal studies of women with unsatisfactory Pap tests have reported an increased risk of epithelial abnormalities [18, 23]. The higher prevalence could be attributed to the conventional approach of Pap smear preparation done at the department but not the liquid-base technology that is able to significantly reduce unsatisfactory rate [14, 24] at the time of the data period. The common reason for unsatisfactory Pap smear in this study was scanty cellularity followed by obscuring inflammation and blood. Scanty cellularity as a cause of unsatisfactory Pap smear has been confirmed in previous studies [17, 20, 23]. It is not surprising to emerge as the most common cause of unsatisfactory Pap smear.

Figure 1. A line plot of graph of unsatisfactory Pap smear prevalence against annual years.

The increasing contribution of scanty cellularity to unsatisfactory Pap smear rates may be explained within the context of its relationship with the technique of sampling. Thus, sample taking by well-trained persons might contribute to reducing the overall rate of unsatisfactory Pap smear. In the current study, though lack of Transformation Zone components in a smear was not considered as unsatisfactory, it is related to sampling technique giving about 1818 excluded reports in this study. The type of sampling device used can also influence specimen adequacy and smear quality (thus present of TZ components) and necessitating the adoption of proper sampling device is encouraged [3, 25]. As reported in previous studies elsewhere [3], scraping the cervix with the extended tip of the spatula followed by using a cytobrush gives adequate samples far better results [3, 25].

Adopting this approach will be key to enhancing the quality of the sampling. In terms of frequency in the current study, obscuring inflammation and blood were the next most frequent causes of unsatisfactory Pap smear and were

related to the obscuring error of the test. Consistent with previously studies by McGaraghan and Smith-McCune in 2000 and by Owens and colleagues in 2013, Obscurance by blood or inflammation as a cause of unsatisfactory Pap smear [14, 20] results from obscuring error of the test. It is the recognition of this error that led to the bringing on board of the liquid-based sampling method that is able to correct the anomaly and generally decreases obscuring problems [24].

This current study had significant rate from this error because, the department of cytology still uses the conventional method of Pap smear preparation. Improved patient preparation or clinician technique (thus not sampling during the patient’s menstrual period) may correct or reduce this cause of the unsatisfactory by obscured Pap [12, 26]. Beyond improving patient preparation, careful attention to transferring of cells onto slide (thus smear preparation), immediate and proper fixation can address thick smear and air-drying problems [3, 25] since these contributed to the causes of unsatisfactory Pap smear under

 

processing error in this study. Better processing of Pap smears therefore yields quality or satisfactory smears for cytological diagnosis [3, 25].

In our present study, patient’s history was labeled as unsatisfactory though the number was not high. Care should be taken to avoid such clerical mistakes. There is little extensive data on how patient history accounts for unsatisfactory Pap smear. However, we found patient history potentially resulting from clerical errors to be responsible for 2 (0.02%) cases of unsatisfactory Pap smear, a development which rarely occurs. None was labeled as unsatisfactory due to technically broken slide and this quality of handling of Pap smear slides should be maintained.

Following a classical definition of unsatisfactory Pap smear as an unreliability in the detection of cervical epithelial abnormalities [18, 27], the definition is relevant in patient management especially within the context of this studies and its findings.

As suggested in previous studies, considering unsatisfactory Pap smears as negative is problematic since negative means absence of disease (SIL or malignancy) and may not prompt adequate follow-up measures [17, 24]. Evidence exists on the possible outcomes when unsatisfactory Pap smears are considered as negative because strong association between false-negatives and unsatisfactory specimens has been amply documented in retrospective studies [17, 18]. Thus, a clear operational definition would need to comply with, for which clinical significance of such smears would need to be determined as well.

The recommended management for unsatisfactory Pap test is to repeat within 2 to 4 months, but this is hardly done in the present study setting due to the patient’s inability to comply usually resulting in missed screening for these women. Similar observations have been reported in low resource settings particularly in different parts of Ghana where infrequent screening opportunities [8, 9] exist. Due to the difficulty with the management of unsatisfactory Pap smear, some studies have posited the need for re-screening though re-screening of previous false-negative Pap smears in patients with current CIN 3 and cancer appears difficult in resource limited settings

1. Notwithstanding, Davey and others study are reassuring in terms of how retrospective re-screening of previously false-negative patients with current CIN 3 and cancer was proven be unsatisfactory after review [13], a finding consistent with earlier studies [16-18]. This calls for the urgent need to have repeated screening whiles minimizing the quick disposition to consider screening results as false-negative.

Unsatisfactory causes were also reviewed in line with their respective clinical history and diagnosis for which unsatisfactory rate from routine screening accounted for 0.86%, whilst and unsatisfactory rate in reports without diagnosis and clinical history was 0.17%.

The clinical history and diagnosis is mostly the primary reason propelling women to do Pap smear in the study setting. The indications as reflected in this current study suggest that patronage by women to do medical check-up for Pap smear is not on the rise because possible predictions could be made from such perspective on the number of women who do Pap smear screening. Mostly, sampling from women with clinical history and diagnosis

of any kind especially malignancy and some benign is a bit challenging and could lead to unsatisfactory smear. This observation carries some veracity particularly when it has been established that additional clinical evaluation, thus inflammation and bleeding associated with CIN 3/carcinoma and some benign cases may cause partially obscured or unsatisfactory Pap tests [16] despite the suggested guidelines [13, 16, 28] on having additional clinical evaluation on women with symptoms and abnormal physical findings [16]. The adoption of continuous quality improvement measures and giving of feedback on quality indicators is critical to decreasing unsatisfactory Pap smears as it has been confirmed in different settings so that CIN 2/3 and invasive carcinoma, are not harboured [15, 24, 28, 29].

LIMITATIONS

The major limitation of this study is the possibility for data entry errors from the source data. The study relied on secondary data over the years. However this is the best available data from the cytology unit that has been documented. Our inability to access background characteristic of the women limited the authors from establishing some association with the profiles of the women. Due to the fact that this study was informed by data from a single facility, making generalization of findings difficult. However this study would serve as a baseline for further research to compare all the regional and teaching hospitals to better understand the national prevalence of unsatisfactory Pap smear.

CONCLUSIONS

This study to the best of our knowledge, is the first ever study on unsatisfactory Pap smear in Ghana.

The study identified scanty cellularity as the main cause of unsatisfactorily Pap smear. The 12-year prevalence compared to other studies is higher though incidence appears to be on the decline year on year. It is expected that the cytology unit adopts the liquid base preparation to get a monolayer smear and to lyse any blood obscurance to avoid unsatisfactory Pap smear.

The study has also provided evidence for the need to train staffs who take the Pap smears to increase their confidence in taking the smear due to high number of scanty cellularity’s reports and lack of TZ component whilst encouraging the use of the recommendations that the right sampling tools be used. Computerized based archives should be implemented at the department since there was significant loss of reports and moreover the entire 2011 reports could not be found from archives.

ACKNOWLEDGEMENTS

We acknowledge the Department of Pathology, Cytology Unit, Korle-Bu Teaching Hospital, Accra-Ghana for allowing us to use access data from the unit for the conduct of this study.

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Prevalence of Unsatisfactory Pap Smear and Associated Clinical History and Diagnosis in a Tertiary Teaching Hospital in Ghana

Maxwell Hubert Antwi

Department of Molecular Medicine, School of Medical Sciences, College of Health Sciences,

Kwame Nkrumah University of Science & Technology, Kumasi, Ghana.

Seth Christopher Yaw Appiah

Department of Sociology and Social Work, Faculty of Humanities and Social Sciences,

Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.

Centre for International Health (CIH), University of Munich Medical School, Ludwig-Maximilians-Universitate of Munchen, Munchen, Germany

All correspondence to: Seth Christopher Yaw Appiah, sychrist2007@gmal.com

ABSTRACT

ackground: A major limitation of cervical cytology is the unsuitability of proportion of Bsmears submitted for analysis and for cytological assessment (unsatisfactory). This study examines the prevalence of unsatisfactory Pap smear, clinical history and diagnosis in the Department of Pathology, Korle-Bu Teaching Hospital (KBTH), Ghana. Materials and Methods: A retrospective review of 15,290 cases spanning 12 years (2005-2016) was carried out at the cytology unit of the Pathology Department of the KBTH. Out of the 15,290 Pap smear records retrieved, 2347 reports were excluded leaving 12,943 for the study. All unsatisfactory smear cases were analyzed and categorized using the Bethesda 2001 System. Results are presented using descriptive statistics. Results: The overall prevalence of unsatisfactory Pap smear was 402 (3.1%). Routine screening smear accounted for 115 (0.9%); reports without clinical history and diagnosis gave 21 (0.2%) and cases with clinical history and diagnosis were 287 (2.2%). The common cause of unsatisfactory Pap smear was scanty cellularity 222 (1.72%). Patient’s history accounted for the least cause of unsatisfactory Pap smear 2 (0.02%). Conclusion: Pap smear results reported as unsatisfactory could

harbor cancer malignancy. Samples should be taken by well-trained persons.

INTRODUCTION

ancer is a global health concern. Millions of Cindividuals have been diagnosed and several affected people have lost their lives. Cancer of the uterine cervix is a leading public health issue globally and

is the commonest cancer in the female genital tract [1, 2]. Over the past two decades, the incidence of the disease has shown a decline in the developed countries [3, 4], yet in poor resource settings like Ghana, there has not been significant change in both prevalence and incidence. [1, 5]

A report by Ghana health service rated cervical cancer as the topmost cancer affecting women in Ghana, and attributed 50.5% of the condition to human papillomavirus (HPV) types 16 and 18 with 16% as cause of death attributable to cancer [6].

Estimates by the World Health Organization (WHO) posit that cervical cancer case in Ghana will exceed 5000 with mortality of 3300 annually by 2025 if the trend persists [7]. It has become difficult to relegate the condition in the country to the background without attention. Larger percentage of women of reproductive age stand at increased risk of having cancer of the uterine cervix since there is no national screening programme or management readily available [6, 8].

Screening of cervical cancer is uncommon in Ghana as the test is done in few public and private health centers in the country and is lowly patronized by the target group (women) [8, 9]. Nevertheless, risk of dying from cervical

cancer can be reduced with routine Pap test [1, 4]. It has been reported that, the annual global death rates attributable to cervical cancer has declined by 2% ever since Pap smear test was introduced with overall death rate by 74% [10].

The Pap test results indicate various changes relating to clinical observation such as; unsatisfactory, normal, inflammation, benign cellular changes, Atypical Squamous cell of undetermined significance (ASCUS), High Grade Squamous Intraepithelial Lesion (HGSIL) and cervical cancer [2, 11]. Despite its outstanding success, the Pap smear is not 100% perfectly accurate [12-14]. Problems occur at every level from failure of women to get regular Pap smear test in the first place to sampling and interpretation error which is likely to give unsatisfactory results that would need a clinical follow-up [12-14].

The number of false negative reports is of great concern because some are reported as a result of unsatisfactoriness [2, 15]. One of the limitations of cervical cytology is that a proportion of smears submitted for analysis in the light microscope are unsuitable for reliable cytological assessment (3, 4). This category of limitation is considered as unsatisfactory Pap smears [11]. Pap smear according to the Bethseda system can be categorized as follows; Sampling error; this error can be due to slide lacking an adequate number of well-preserved and well-visualized squamous epithelial cells (minimum of 8000 – 12,000 for conventional Pap or 5000 for Liquid-

base pap, thus scanty cellularity) [11, 14].

Processing error includes poor fixation, air-drying artifact or contaminants obscuring over 75% of the epithelial cells and cellular material being too thick or multilayering smear [11, 14]. Obscuring error includes cells being too atrophic, blood and inflammation obscuring over 75% of the epithelial cells, excessive cytolysis [11, 14]. A broken slide which affects smear for cytologic examination is technically unaccepted. Lack of pertinent patient information and clinical history are as cause of unsatisfactory Pap smear. Any of the causes under these errors can be unsatisfactory.

A large prospective study done in Norway found that unsatisfactory Pap smear test results indicated a 1.6 to 4.0 times high risk of harbouring CIN 2/3 or invasive cervical cancer compared with woman with a normal Pap test result [16]. Unsatisfactory category constitutes 1% to 2% of all Pap test [17-19]. There are however little information and knowledge on its prevalence in Ghana and particularly at the nation’s biggest teaching hospital, Korle-Bu Teaching Hospital.

The essence of this study was to provide adequate data and knowledge on unsatisfactory Pap smears’ prevalence and causes in line with their respective clinical history and diagnosis and to help health-care practitioners with measures to minimize any error that could lead to unsatisfactory Pap smear. This study provides baseline data for comparison with similar reviews in future since there is currently no known scientific documented evidence on unsatisfactory Pap smear prevalence in Ghana.

METHODS

Setting

The study was conducted at the Korle-Bu Teaching hospital. The hospital is the largest health facility and also the premier teaching hospital in Ghana. The Korle-Bu Teaching hospital has a bed capacity of over 2000 and offering medical training for medical doctors, nurses and other health professionals. There are 17 clinical and diagnostic departments/units in the hospital. The hospital is host to the National Centre for Radiotherapy and Nuclear Medicine which functions as a referral centre for the management of cancer supporting other specialized services such as renal transplantation, DNA investigations and brachy therapy for the treatment of prostate cancer. The study was carried out using data from the cytology unit of the Pathology Department of the KBTH.

Study Design and Sampling

The study was retrospective review of all Pap smear registry data of women who underwent Pap smear at the cytology unit of the Korle-Bu hospital spanning a period of 12 years (2005-2016). The study used a hospital based registry which has a catchment that covers the entire southern part of Ghana and beyond without any well-defined population. However, under the period the studied, 15,290 conventional Pap smear test reports were retrieved. The cytology unit actively collects data on all Pap smear cases presenting to the hospital for possible diagnosis of cervical cancer. The data sources are often from either women regular visits to the various clinics and wards of admission or by referral to the unit from within

the hospital or outside the hospital. A special case folder is created (folders) within the unit to abstract needed information with succinct case definition. Information on the clinical history of patients was extracted from the medical folders of each client through a careful sifting of the records.

Analysis

The reports taken from the archives had their covers cleaned and the total Pap smear reports were noted as 15,290. Out of the 15,290 reports, 2347 (both missing reports and lack of TZ components reports) were excluded so 12,943 reports were left for the study. The criteria for the unsatisfactory diagnosis were according to the Bethesda System 2001 and their causes categorized as; sampling error due to inadequate number of well preserved and well visualized squamous epithelial cells (minimum of 8000 – 12,000 for conventional Pap smear)—thus scant cellularity (<10% of slide covered by interpretable squamous cells), processing error due to poor fixation, air-drying artifact or contaminants obscuring over 75% of the epithelial cells and cellular material too thick or multilayering smear, presence of obscuring error of the test due to cells being too atrophic with blood and or inflammation obscuring over 75% of the epithelial cells, excessive cytolysis, broken slide, and lack of patient’s history. The 12,943 reports were reviewed for unsatisfactory diagnosis in line with their respective clinical history and diagnosis viz. age, and their causes were noted, counted and grouped into the various categories. The frequencies of the various categories and what constitute them were determined. The prevalence of the unsatisfactory Pap smears in percentages annually were determined and the overall prevalence within that time frame was also known. The prevalence in percentage was calculated as; (total unsatisfactory Pap smears/total Pap smears) multiply by 100. The data was exported into Microsoft Excel and SPSS version 16 for windows and further analysis. Ethics approval was sought from the University of Ghana Ethics approval committee

RESULTS

Population Characteristics

A total number of Pap smears and smears rejected as unsatisfactory reviewed over the twelve year period (Table 1). A total of 15,290 conventional Pap smear test reports were retrieved for review from the archives at the cytology unit of the department of pathology, KBTH over a twelve-year period. Out of the 15,290 Pap smears reports, 529 were missing in the archives, 1818 were limited by lack of transformation zone component and so both (2347 reports) were excluded from this study. The entire Pap smear reports for 2011 could not be found from archives and were excluded. The total Pap smear over the period was 12,943 and unsatisfactory Pap smear was also

1. The average age and age range of women who attended the unit to do Pap smear test were 39.5 and 19 – 80 years respectively. Smears rejected as unsatisfactory were assessed in line with their respective clinical history and diagnosis and their percentages recorded as follows. Unsatisfactory rate with history as routine screening was
2. (0.9%), with clinical history and diagnosis was 287 (2.2%), rate in reports without clinical diagnosis gave 21

(0.2%) and the overall rate in all diagnosis and routine		unsatisfactory categories were presented according to each
screening was 402 (3.1%).		year. In 2005, total unsatisfactory Pap smear was 63 (4.2%),
Causes of Unsatisfactory Pap Smear and Associated		2006 presented 23 (3.7%), 2007 gave 54 (5.3%), 2008 was
		38 (3.7%), 2009 gave 38 (2.9%), 2010 was 47 (3.7%), 2012
Diagnosis and Clinical History		gave 33 (2.7%), 2013 was 39 (2.8%), 2014 gave 40 (2.5%),
Table 2 shows the various causes of unsatisfactory Pap		2015 was 16 (1.2%), 2016 gave 11 (1.6%) and the overall
smears in line with clinical history and diagnosis. Scanty		annual percentage was 402 (3.1%).
cellularity 77 (0.6%) gave higher number for almost all the		The unsatisfactory categories based on their causes
clinical history and diagnosis with routine screening. This		were reported. Sampling error gave total unsatisfactory
was followed by uterine fibroid 52 (0.4%), infertility 27		rate of 222 (1.72%), processing error gave 46 (0.36%),
(0.2%) (amenorrhea, dysmenorrhea, menorrhagia,		inherent error of the test was 132 (1.02%) and patient
dysuria, leiomyoma), cancer 20 (0.2%) (cervical cancer,		history was 2 (0.02%).
endometrial cancer). Accordingly, the least factors that		Year to Cause Categorization of Unsatisfactory Pap
accounted for the unsatisfactory Pap smear were post-		Smear
menopausal bleeding, bleeding in urine) and cytolysis 2		Table 4 shows the various categorization of
(0.02%)		unsatisfactory Pap smear. Scanty cellularity was the
Cause Specific Categorization of Unsatisfactory Pap		highest recording 222 (1.72%) followed by obscured
Smear		inflammation 69 (0.53%), blood obscurance 56 (0.43%),
The results presented in Table 3 show the frequency and		thick smear 17 (0.13%), with the east being cytolysis 7
annual percentages of the unsatisfactory categories based		(0.05%) and patient history 2 (0.02%).
on their causes. The total and annual percentages of the
	Table 1. Study population characteristics.
	Data		N (%)
	Actual number of conventional Pap smear retrieved		1520 (100%)
	Missing cases in the Archive		529 (3.5%)
	Pap smear data limited by transformation zone component		1818 (10.9%)
	Total number of Pap smears reviewed		12,943
	Total unsatisfactory Pap smears reviewed		401
	Age of women (range) years		9 – 80 yrs
	Mean age (yrs)		39.5
	Unsatisfactory rate in routine screening		115 (0.9%)
	Unsatisfactory rate in clinical history and diagnosis		287 (2.2%)
	Unsatisfactory rate in reports without clinical diagnosis		21 (0.2%)
	Unsatisfactory rate in all diagnosis and routine screening		402 (3.1%)

Table 2. Causes of unsatisfactory Pap smear and associated diagnosis and clinical history.

	Clinical History and Diagnosis
Reason for	RS No. ND No. UF No. Hys/Myo Inf No. Ca PB No. Total
unsatisfactory	%		%	%	%	%	%	%
Scanty cells	77	(0.6)	11 (0.08)	52 (0.4)	20 (0.2)	27 (0.2)	20 (0.2)	15 (0.1)	222 (1.7)
Obscuring
inflammation	18	(0.1)	4 (0.03)	3 (0.02)	17 (0.1)	7 (0.05)	11 (0.08)	9 (0.07)	69 (0.5)
Obscuring blood	10	(0.08)	2 (0.02)	4 (0.03)	4 (0.03)	11 (0.08)	12 (0.09)	13 (0.1)	56 (0.4)
Thick smear	4 (0.03)		1 (0.01)	2 (0.02)	3 (0.02)	3 (0.02)	1 (0.01)	3 (0.02)	17 (0.1)
Drying artifact	3 (0.02)		1 (0.01)	3 (0.02)	2 (0.02)	2 (0.02)	2 (0.02)	2 (0.02)	15 (0.1)
Poor fixation	1 (0.01)		2 (0.02)	3 (0.02)	3 (0.02)	2 (0.02)	1 (0.01)	2 (0.02)	14 (0.1)
Cytolysis	2 (0.02)		0	1 (0.01)	1 (0.01)	1 (0.01)	1 (0.01)	1 (0.01)	7 (0.05)

RS-Routine screening, ND-No diagnosis, UF-Uterine fibroid, Hys/Myo-Hysterectomy/Myomectomy, Inf-Infertility (amenorrhea, dysmenorrhea, menorrhagia, dysuria, leiomyoma), Ca-(cervical cancer, Endometiral cancer), PB-(Post coital bleeding, Post-menopausal bleeding, bleeding in urine).

 

Table 3. Categorization of unsatisfactory Pap smear based on their causes.

Years	Sampling Processing			Patient	Broken	Total Unsat.	Total Pap	%
	Inherent error error			history	slide	Pap smears	smears
	error of the test
2005	34	8	21	0	0	63	1488	4.2
2006	14	1	8	0	0	23	621	3.7
2007	35	9	9	1	0	54	1011	5.3
2008	26	4	8	0	0	38	1023	3.7
2009	25	3	9	1	0	38	1298	2.9
2010	35	3	9	0	0	47	1274	3.7
2012	13	4	16	0	0	33	1215	2.7
2013	17	9	13	0	0	39	1382	2.8
2014	14	5	21	0	0	40	1594	2.5
2015	6	0	10	0	0	16	1336	1.2
2016	3	0	8	0	0	11	701	1.6
	222	46	132	2	0	402	12943	3.1
Total %	1.72	0.36	1.02	0.02	0.00	3.12	–	–

Table 4. Year-to year causes and categorization of unsatisfactory Pap smear.

	Years	cellularity Scant		smear Thick fixationPoor			dryingAir-	obscureBlood Cytolysis inflammation
		Obscured		Patient history Broken slide Unsat papTotal
2005		34	0	5	3	8	2	11	0	0	63
2006		14	0	1	0	1	0	7	0	0	23
2007		35	5	1	3	5	2	2	1	0	54
2008		26	2	1	1	6	0	2	0	0	38
2009		25	2	1	0	5	0	4	1	0	38
2010		35	0	0	3	5	0	4	0	0	47
2012		13	3	0	1	3	1	12	0	0	33
2013		17	1	5	3	5	0	8	0	0	39
2014		14	4	0	1	13	0	8	0	0	40
2015		6	0	0	0	2	2	6	0	0	16
2016		3	0	0	0	3	0	5	0	0	11
	Total	222	17	14	15	56	7	69	2	0	402
	Total Pap
	Smears	12943 %	1.72	0.13	0.11	0.12	0.43	0.05	0.53	0.02	0 3.1

Prevalence to Year Specific Rates of Unsatisfactory Pap Smear

The graph presented in Figure 1 shows that over the years, the highest number of sampling error cases was recorded in 2007 with 35 (5.3%) cases. Highest processing error related cases were recorded in the years 2007 and 2013 (n = 9). No case of broken slide was recorded during the 12 years period. Despite the decline in recorded cases resulting from inherent error of the test from 21 cases in 2005 to a stable 8 and 9 recorded cases over a five year period up to 2010, it increased again in 2012 to 16 recorded cases, fell to 13, increased again to 21 in 2014 and begun to record lower cases in the last two years preceding the end point year for this study (2016).

DISCUSSION

The purpose of this study was to determine the prevalence of unsatisfactory Pap smear in the department of pathology, KBTH and to know the various causes in line with their respective clinical history and diagnosis. The overall prevalence of unsatisfactory Pap smear in this study was 3.1 percent. This estimate is lower when compared to reports from studies done in India, Italy and Taiwan but on the rise when compared to previous studies by Ransdell et al., McGaraghan and Smith-McCune in United States and Netherlands [17, 19-22]. Though the annual prevalence of unsatisfactory Pap smear demonstrates no consistent pattern, year on year prevalence appears to be on the decline. From the annual prevalence in percentages of the unsatisfactory Pap smears, the year 2007 recorded the highest prevalence of 5.3 percent. There was however no

 

suggestive trend or pattern to conclude that the higher the total number of Pap smears or unsatisfactory Pap smear, the higher the prevalence in percentage of unsatisfactory Pap smear to demonstrate any association.

Despite, the study’s inability to determine specific cause attribution to the Pap smear decline, the increasing training and clinical experiences of laboratory scientist at the Cytology Unit of the Korle-Bu Teaching Hospital (KBTH) might play a role.

Though the current study had a lower rate of unsatisfactory Pap smears compared to previously reported findings in India, Italy and Taiwan, the rate (3.1%) remains high because unsatisfactory Pap smear rate of all categories according to reference books should be between 1% to 2% This is against the background that

longitudinal studies of women with unsatisfactory Pap tests have reported an increased risk of epithelial abnormalities [18, 23]. The higher prevalence could be attributed to the conventional approach of Pap smear preparation done at the department but not the liquid-base technology that is able to significantly reduce unsatisfactory rate [14, 24] at the time of the data period. The common reason for unsatisfactory Pap smear in this study was scanty cellularity followed by obscuring inflammation and blood. Scanty cellularity as a cause of unsatisfactory Pap smear has been confirmed in previous studies [17, 20, 23]. It is not surprising to emerge as the most common cause of unsatisfactory Pap smear.

Figure 1. A line plot of graph of unsatisfactory Pap smear prevalence against annual years.

The increasing contribution of scanty cellularity to unsatisfactory Pap smear rates may be explained within the context of its relationship with the technique of sampling. Thus, sample taking by well-trained persons might contribute to reducing the overall rate of unsatisfactory Pap smear. In the current study, though lack of Transformation Zone components in a smear was not considered as unsatisfactory, it is related to sampling technique giving about 1818 excluded reports in this study. The type of sampling device used can also influence specimen adequacy and smear quality (thus present of TZ components) and necessitating the adoption of proper sampling device is encouraged [3, 25]. As reported in previous studies elsewhere [3], scraping the cervix with the extended tip of the spatula followed by using a cytobrush gives adequate samples far better results [3, 25].

Adopting this approach will be key to enhancing the quality of the sampling. In terms of frequency in the current study, obscuring inflammation and blood were the next most frequent causes of unsatisfactory Pap smear and were

related to the obscuring error of the test. Consistent with previously studies by McGaraghan and Smith-McCune in 2000 and by Owens and colleagues in 2013, Obscurance by blood or inflammation as a cause of unsatisfactory Pap smear [14, 20] results from obscuring error of the test. It is the recognition of this error that led to the bringing on board of the liquid-based sampling method that is able to correct the anomaly and generally decreases obscuring problems [24].

This current study had significant rate from this error because, the department of cytology still uses the conventional method of Pap smear preparation. Improved patient preparation or clinician technique (thus not sampling during the patient’s menstrual period) may correct or reduce this cause of the unsatisfactory by obscured Pap [12, 26]. Beyond improving patient preparation, careful attention to transferring of cells onto slide (thus smear preparation), immediate and proper fixation can address thick smear and air-drying problems [3, 25] since these contributed to the causes of unsatisfactory Pap smear under

 

processing error in this study. Better processing of Pap smears therefore yields quality or satisfactory smears for cytological diagnosis [3, 25].

In our present study, patient’s history was labeled as unsatisfactory though the number was not high. Care should be taken to avoid such clerical mistakes. There is little extensive data on how patient history accounts for unsatisfactory Pap smear. However, we found patient history potentially resulting from clerical errors to be responsible for 2 (0.02%) cases of unsatisfactory Pap smear, a development which rarely occurs. None was labeled as unsatisfactory due to technically broken slide and this quality of handling of Pap smear slides should be maintained.

Following a classical definition of unsatisfactory Pap smear as an unreliability in the detection of cervical epithelial abnormalities [18, 27], the definition is relevant in patient management especially within the context of this studies and its findings.

As suggested in previous studies, considering unsatisfactory Pap smears as negative is problematic since negative means absence of disease (SIL or malignancy) and may not prompt adequate follow-up measures [17, 24]. Evidence exists on the possible outcomes when unsatisfactory Pap smears are considered as negative because strong association between false-negatives and unsatisfactory specimens has been amply documented in retrospective studies [17, 18]. Thus, a clear operational definition would need to comply with, for which clinical significance of such smears would need to be determined as well.

The recommended management for unsatisfactory Pap test is to repeat within 2 to 4 months, but this is hardly done in the present study setting due to the patient’s inability to comply usually resulting in missed screening for these women. Similar observations have been reported in low resource settings particularly in different parts of Ghana where infrequent screening opportunities [8, 9] exist. Due to the difficulty with the management of unsatisfactory Pap smear, some studies have posited the need for re-screening though re-screening of previous false-negative Pap smears in patients with current CIN 3 and cancer appears difficult in resource limited settings

1. Notwithstanding, Davey and others study are reassuring in terms of how retrospective re-screening of previously false-negative patients with current CIN 3 and cancer was proven be unsatisfactory after review [13], a finding consistent with earlier studies [16-18]. This calls for the urgent need to have repeated screening whiles minimizing the quick disposition to consider screening results as false-negative.

Unsatisfactory causes were also reviewed in line with their respective clinical history and diagnosis for which unsatisfactory rate from routine screening accounted for 0.86%, whilst and unsatisfactory rate in reports without diagnosis and clinical history was 0.17%.

The clinical history and diagnosis is mostly the primary reason propelling women to do Pap smear in the study setting. The indications as reflected in this current study suggest that patronage by women to do medical check-up for Pap smear is not on the rise because possible predictions could be made from such perspective on the number of women who do Pap smear screening. Mostly, sampling from women with clinical history and diagnosis

of any kind especially malignancy and some benign is a bit challenging and could lead to unsatisfactory smear. This observation carries some veracity particularly when it has been established that additional clinical evaluation, thus inflammation and bleeding associated with CIN 3/carcinoma and some benign cases may cause partially obscured or unsatisfactory Pap tests [16] despite the suggested guidelines [13, 16, 28] on having additional clinical evaluation on women with symptoms and abnormal physical findings [16]. The adoption of continuous quality improvement measures and giving of feedback on quality indicators is critical to decreasing unsatisfactory Pap smears as it has been confirmed in different settings so that CIN 2/3 and invasive carcinoma, are not harboured [15, 24, 28, 29].

LIMITATIONS

The major limitation of this study is the possibility for data entry errors from the source data. The study relied on secondary data over the years. However this is the best available data from the cytology unit that has been documented. Our inability to access background characteristic of the women limited the authors from establishing some association with the profiles of the women. Due to the fact that this study was informed by data from a single facility, making generalization of findings difficult. However this study would serve as a baseline for further research to compare all the regional and teaching hospitals to better understand the national prevalence of unsatisfactory Pap smear.

CONCLUSIONS

This study to the best of our knowledge, is the first ever study on unsatisfactory Pap smear in Ghana.

The study identified scanty cellularity as the main cause of unsatisfactorily Pap smear. The 12-year prevalence compared to other studies is higher though incidence appears to be on the decline year on year. It is expected that the cytology unit adopts the liquid base preparation to get a monolayer smear and to lyse any blood obscurance to avoid unsatisfactory Pap smear.

The study has also provided evidence for the need to train staffs who take the Pap smears to increase their confidence in taking the smear due to high number of scanty cellularity’s reports and lack of TZ component whilst encouraging the use of the recommendations that the right sampling tools be used. Computerized based archives should be implemented at the department since there was significant loss of reports and moreover the entire 2011 reports could not be found from archives.

ACKNOWLEDGEMENTS

We acknowledge the Department of Pathology, Cytology Unit, Korle-Bu Teaching Hospital, Accra-Ghana for allowing us to use access data from the unit for the conduct of this study.

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An assessment of the availability of Medical Laboratory Services in primary health care centres in Ogbia LGA, Bayelsa State, Nigeria.

Atiegha C and Suama P

Department of Medical Laboratory Sciences, College of Health Technology Otuogidi-Ogbia, Bayelsa State.

Stella U. K.

Department of Medical Laboratory Science, Rivers State University, Port-Harcout.

Gbarebe B

Department of Health Information Management, College of Health Technology, Otuogidi-Ogbia, Bayelsa State.

All correspondence to: E-mail: achristo40@gmail.com

ABSTRACT

Services at the Primary Health Care (PHC) are the first set of health services that are provided at the grass-root or rural level. A medical laboratory is a place for examination of materials derived from human body for the purposes of providing information on diagnosis of diseases.

The objective of this study is to assess the level of provision of medical laboratory services in primary health care centers in Ogbia local government area of Bayelsa-State, Nigeria. This study was carried out in 20 functional primary health facilities. A descriptive survey design involving questionnaire and a check list was used to assess information on the issue. The results reveal that only 20 primary health facilities operate in the LGA of over 55 communities. Among them only 9 health facilities are offering skeletal medical laboratory services. It further revealed a grossly under staffing of medical laboratory personnel. Medical laboratory services in the health centers in Ogbia Local Government of Bayelsa State, Nigeria are yet to be fully developed. We recommend therefore that policy makers should focus their attention in providing the needed manpower and equipments to boost health care services in these health care facilities.

Key words: Laboratory, PHC, Diagnosis, Scientist, Technician, Grass root.

INTRODUCTION

n India the National Rural Health Mission (NRHM) Itaught wise to look at the ways in which the missions and goals of Primary Health Care (PHC) had been strengthened in the rural settings. This process is carried out in order to explore the avenues for more upgrading. As part of the commitment of the NRHM to improve public health care services, one of its core strategies had been to strengthen PHCs to meet the level of the countries Public

Health Standards, IPHS (1).

However, the World Health Organization (WHO) had defined health as a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity. Health can also be seen as the ability to adapt and manage physical, mental and social challenges throughout life. ‘Health for All’ is a programming goal of the WHO, which envisions securing the health and well- being of people around the world that has been popularized since the 1970s. It is the basis for the WHO’s primary health care strategy to promote health, human dignity and enhanced quality of life (2).

Furthermore, health service is seen as the essential part of a Nation’s social security in which the state is compulsorily obligated by its constitutional provisions to direct its policies toward ensuring that there are adequate medical services for all persons, including management, prevention and treatment of diseases. Health care services

in Nigeria are rendered as an integrated services. However, it was further reorganized into three tiers by the Federal Civil Service in the year 1988 under the auspices of the federal ministry of health. The tiers are the primary, secondary and tertiary health care’s (3).

Services at the PHC are the first set of health services that are provided at the grass-root or rural level. Health care (services) here requires contact with the community health facilities. This level of health care service identifies the health chalenges of the inhabitants and resolve them using appropriate technologies. Referring much serious and complicated cases to the secondary or tertiary health care service delivery. It is the cornerstone of the rural health services (4).

These days quality health care of patients has become more dependent on findings of medical laboratory investigations (5).

Good Laboratory policies are the decisions which are taken in consultation with other medical staff to enable the laboratory to operate reliably and efficiently in accord with other departments (6).

Medical laboratory can be defined in several ways by different authors: a specialized laboratory for carrying out investigations (routine or research) on human specimen sin order to generate accurate and reliable result; where tests are carried out on clinical specimens in order to get information about the health of a patient pertaining to the diagnosis, treatment and prevention of diseases; a place for

14 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

An assessment of the availability of Medical…

measurement and examination of materials derived from human body (fluids, tissues, excreta, cells, etc.) for the purposes of providing information on diagnosis, prognosis, prevention or treatment of diseases; a branch of medical science that plays a vital role in public health and disease control; is where clinical pathology tests are carried out on clinical specimens to obtain information about the health of a patient and to aid in diagnosis, treatment and prevention of diseases( 7,8).

Medical laboratory science as a multidisciplinary field encompassed medical microbiology, haematology, clinical chemistry and histopathology. The discovery of microscope by Antony Van L., a German Scientist revolutionized the field of medical sciences (9). The place of medical laboratory science in medicine and public health is very fundamental. Medical laboratory science is key in disease identification, disease control and surveillance and in treatment of patients. Yet this very important services is still under provided or utilized (10).

Medical Laboratory personnel includes multiple categories of Medical laboratory science practitioners who have different levels of education and training ranging from medical laboratory assistants, medical laboratory technicians and medical laboratory scientists with their particular functions accordingly (11).

There are mainly two types of medical laboratories that process the majority of medical specimens. They are the hospital based and private based laboratories. Hospital based laboratories are medical laboratories that are located within the hospital that perform tests on patient whereas private laboratories are located outside the hospital that also perform tests on patients (12,13). Although specific routine tests are not limited to few, it is expedient that medical laboratory facility at the primary health care level perform at least these routine tests such as urinalysis, pregnancy test, blood grouping/Rhesus typing, Haemoglobin genotyping, haemoglobinestimation, tuberculosis test and serological assays (4).

Number of medical laboratory analysis had greatly impacted on the operations of PHC. Medical Laboratory diagnosis as the branch of diagnostic activities of PHC is saddled with the duty of producing laboratory tests results needed for the effective treatment of diseases (8,10).

It is clear that the role of medical laboratory in medicine and the society at large can and will never be over-emphasized. Though, there had been series of complains concerning the services in the laboratory at PHC level; they provide the physicians and other health care givers the information to detect disease, predisposition to disease, establish prognosis, guide patients management, monitor efficacy of treatment, confirm or refute a diagnosis. Reports obtained revealed that patients got inadequate satisfaction in regard to primary health care service delivery. The low turn up could be attributed to the non-equipment in the laboratory and bad information management system. It is apparent that patients do not effectively assess the services of medical laboratory at the primary health care level, possibly due to its non-establishment at the level of primary health care provider or simply due to lack of political will to make it functional (14, 15, 16). Despite these great functions and benefits of medical laboratory services, it had been reported that this service had been under assessed in general health practices at the PHC level (17).

However in the practice of modern Medicine, medical services had proven to be comprehensive and of global health standard only with the support of basic laboratory facilities to serve as the important determinant influencing the utilization of health services (18).

It is therefore clear that primary goal of PHC entail the involvement of ‘appropriate technology’ which is designed to equip the health facilities to render effective and quality medical care (19).

The ‘Bedrock” of modern medicine they say is the Medical laboratory. It plays a role that cannot be quantified in the three tiers of health services and the society at large. Therefore, this study is aimed at assessing the medical laboratory services in primary health care centres in Ogbia local government area of Bayelsa-State, Nigeria.

MATERIALS AND METHODS

This study was carried out in 20 functional primary health care facilities in Ogbia Local Government Area of Bayelsa State. A descriptive survey designed was used to assess the Medical Laboratory Services in the region. Subjects

The study subjects were the head of the Health facilities. This instrument was further divided into two sections: section ‘A’ includes names of community, health facilities and staff cadre in the laboratory, while section ‘B’ includes type of tests conducted. Ethical approval was obtained from the officers in charge from the respective health facilities and the committee of ethics and man power development, college of Health Technology, Otuogidi, Ogbia-Town, Bayelsa state.

PROCEDURE

An interview guide/checklist was used as method of data collection. The data were analyzed and results were presented in tables.

RESULT

From the data generated, only 20 communities had health centres, of which 9(45.0%) operate medical laboratory services while 11(55.0%) had no medical laboratory services (figure 1).

Out of the 9 available medical laboratories, the staff manning them are 5(55.6%) medical laboratory technicians, 1(11.1%) are medical laboratory assistants and 3(33.3%) are community health extension workers (Table 1).

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Table 1: Personnel in charge of the laboratory

Personnel	Frequency	Percentage (%)
Medical Laboratory Technician	5	55.6
Medical Laboratory Assistant	1	11.1
Community Health Extension Worker	3	33.3

Of the 9 functional medical laboratories, the quality of services are 4(44.4%) good, 5(55.6%) are fair and 0(0.0%) and poor (Table 2).

Table 2: Quality of MLS

Quality	Frequency	Percentage (%)
Good	4	44.4
Fair	5	55.6
Poor	0	00.0

Of these 9 functional medical laboratories, 3(33.3%) offer regular services while 6(66.7%) have irregular services (figure 2).

Figure 2 Regularity of laboratory services provided

Table 3 gives the laboratory tests carried out in each of the health centres. In these 9 functional health facilities, 8(88.9%) parameters:Blood grouping, Hb-genotype, Hb-estimation, Hepatitis B, urinalysis, malaria parasites test, pregnancy test and HIV screening are carried out Otuogidi health facility, 6(66.7%) parameters: Blood grouping, Hb-estimation, urinalysis, malaria parasites test, pregnancy test and HIV screening are done in Otuoke health facility, 5(55.6%) parameters: HIV screening, Hb-estimation, urinalysis, malaria parasites test and pregnancy test are done in Otuakeme health facility, 4(44.4%) parameters: HIV screening, urinalysis, malaria parasites test and pregnancy test are carried out in Emeyal health facility, 3(33.3%) parameters: HIV screening, urinalysis, malaria parasites test and pregnancy test are done in Otuobagi health facility, 2(22.2%) parameters: HIV screening and malaria parasites test are done in Kolo health facility, while 3(33.3%) parameters: HIV screening, malaria parasites test and pregnancy test are carried out in Okodi, Oloibiri and Ewoi health facilities respectively (Table 3).

16 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

An assessment of the availability of Medical…

Table 3: Panel of Tests done in MLS

Laboratory’s Location	Test Done	Frequenc
		y
PHC Otuogidi	Blood grouping, Hb-Genotype, Hb-estimation,
	Hepatitis B, Urinalysis, MP Test, PT Test & HIV	8
	Screening
PHC Otuoke	ABO Blood grouping, Hb-estimation, HIV
	screening, Urinalysis, MP Test &PT Test	6
PHC Otuakeme	HIV screening, Hb-estimation, Urinalysis, MP
	Test & PT Test	5
PHC Emeyal	HIV Screening, Urinalysis, MP Test & PT Test
PHC Otuobagi	HIV screening, Urinalysis, MP Test & PT Test	4
PHC Okodi	HIV screening, MP Test & PT Test	3
PHC Oloibiri	HIV screening, MP Test & PT Test	3
PHC Ewoi	HIV screening, MP Test & PT Test	3
PHC Kolo	HIV screening & MP Test	3

DISCUSSION

Our study revealed that there are only 20 established primary health care centres in Ogbia Local Government Area of Bayelsa-State (which has over 55 communities) that are providing health services to the people in the communities. This finding is not in tandem with NRHM, 2005 which has the commitment of improving public health care service deliveries as one of its core strategies to strengthen PHCs to meet the level of Public Health Standards (1).

With this limited number of health facility, it is obvious that most of the persons residing in most communities within the rural areas in the local government had no access to quality health services. This issue also negates what Dibia, (2002) pointed out. He said health service is an essential part of a Nation’s social security which mandated the state to direct its policies toward ensuring adequate medical services for all persons (3).

The fact that of the twenty existing Health facilities only 9 are offering medical laboratory services which indicates that the quality of health service delivery rendered to the people is grossly inefficient and as such, undermined the role of medical laboratory services. This finding are also in contrast with the agreement of the World Health Assembly, that said WHO advocates for basic laboratory services and using appropriate technologies to support and strengthen clinical and public health activities at the PHC level (19,20).

The study further revealed an inappropriate staff strength and cadre working in the medical laboratory department that may not be able to face more rigorous analysis. There are also high tendencies of releasing wrong or falsified tests results because there are no qualified and trained medical laboratory scientists to verify and ascertain the validity of the generated tests results. We identified the presence of untrained, incompetent, non-qualified and non-licensed laboratory workers in all the laboratories. Invariably, the facilities are operated by quacks. It is therefore obvious that the tests results generated in these facilities may not be accurate.

This study also revealed a massive substandard, poorly functional and a dilapidated medical laboratory system with series of complains of dissatisfaction from patients. This had really hampered the progress to patients’ treatment. This findings are in agreement with that of Coulter & Ellim, 2006, attributing a poor functional medical laboratory and monitoring scheme majorly to the lack of political will, thereby causing health set back during treatment (15).

The quality of medical laboratory services depend greatly on the personnel involve in the diagnostic processes. Poor staffing posed an impediment to patients’ satisfaction to service deliveries. Our findings are in consonant with Sambo et al., 2010, whose record revealed that patients returns home with insufficient satisfaction owing to poor service delivery at the primary health care level (16).

The type of laboratory tests done is so skeletal and inconsequential. These array of tests varies significantly from one facility to the other that are managing to provide laboratory services. This findings negates that of Azad, 2012, who highlighted some of the essential laboratory services that should be provide at the primary health care level (4).

CONCLUSION

World Health Organization (WHO) believed in ‘Health for all’. However, this declaration had not been achieved because so many persons or patients are still deprived from accessing quality health services. These services are carried by trained, certified and license medical laboratory scientists only and as such, the services of medical laboratory scientists are often demanded by the requesting officers (physicians) or the patients concern. Medical laboratory science is the bed rock of modern medicine hence, when medical scientists are not integrated in patients’ management, the patients had been denied to accessing quality health service delivery. The role of medical laboratory scientists is so pivotal in health services and the society at large. Ironically, there are only 20 primary health care facilities that offered health services in Ogbia Local Government Area of Bayelsa State, of which

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only 9 are offering a skeletal medical laboratory services in other to attend to the health need of the people which is grossly inadequate. The medical laboratories are not furnished, no equipment or reagents. The staff strength is totally inadequate characterized with unqualified and non-licensed personnel. Common routine clinical tests that cut across all the units in the laboratory are not comprehensively done. Results generated are not reliable and reproducible consequent upon, detrimental to patients’ treatment. Lack of political will by the ruling class is another impediment that had resulted in the dilapidated nature of the health industry hence, hampered the WHO programme of ‘Health for All’, since patients cannot and are not accessing quality medical laboratory services at the third tier of health services. We therefore recommend that the authorities ( state and local government as well as federal agencies) should commence the equipmenting, staffing and training of all cadre of laboratory personnel as well as the provision of new laboratries for the health facilities that do not have them in order to enhance quality and efficient health care for the inhabitants.

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