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Iraqi Journal of Hematology(IJH)

IJH is a Peer Reviewed Scientific biannual Medical Journal published by the

National Center of Hematology Al-Mustansiriyah University, Baghdad-Iraq.

May 2016 Volume 5, Issue 1

Editor director Editor-in-chief

Prof. Dr.Ali Muhammed Jawad FRCP Prof.Dr. Alaa Fadhil Alwan FICMS

Secretary

Dr.Nidhal Kareem Alrahhal MSc, D.CH

Executive editorial Board Advisory board

Prof.Dr.Raad Jaber Musa FICMS Prof.Dr. Naseer Al-Allawi Ph.D (univ.Dohuk)

Prof.Dr.Ban Abass Abdulmajid PhD Prof.Dr. Khalid Nafee CABM (univ.Mosul)

Prof.Dr.Salma Abass Alhadad CABM Prof.Dr. Ali Muslim CABM (USA,Ohaio)

Assist.Prof.Dr.Aladin M.Zubair FICMS Prof.Dr. Ahmed Ibraheem M.D (lebanon)

Assist.Prof.Dr.Alaadin S.Naji FICMS Prof.Dr.Anwar Sheikha FRCP(univ.sulaymani)

Prof.Dr.Mead Kadhim CABM(Univ.Basrah)

Prof.Dr.Subh S. Al-Modalal FICMS(nahrain)

Prof.Dr.Waseem Fadhil CABM.(univ.Nahrain)

Dr.Jaafar Alghaban consultant CABM

Ass.Prof. Adeeb abbas PhD(Uni.mustansiriya)

Ass. Prof. Nabil Salman CABM (Egypt)

First issue published in 2011

1st editor-in-chief Dr.Nabil S. Murad

1st editor director Dr. Adeeb Alshami

Ass. Prof. Raheem Mahdi FICMS(univ.Kufa)

Ass.prof.Alaa Sadik Alawad(Univ.Babylon)

Ass. Prof. Mazin Faisal FICMS(univ.baghdad)

Ass. Prof.Haitham AlRubai FICMS(Baghdad)

Ass. Prof. Ahmed Kudhair FICMS(univ.Erbil)

Dr. Fatin Al-Yassin (Bagdad teach.Hosp)

Dr.Bassam Francis FICMS(Bagh.Teach.Hosp.)

Dr.Asad A. Eledan FICMS(Basrah Teach. Hos)

Dr.Ibrahim K. Ibrahim CABM(Kadhimin Hos)

Dr Abdulmajeed Alwan CABM(Yarmouk Hos)

Dear doctors and colleagues

We would like to congratulate all the colleagues specially those who

work in hematology field, clinical and laboratory for publishing volume 5

issue 1 of the Iraqi Journal of hematology. This issue contains 10 original

articles deal with different topics concerning hematological diseases. We

sincerely hope from the authors to continue their support and cooperation

through sending original articles, case reports, scientific comments and

criticism to the editors in order to keep the journal going on and to keep

raising its standards.

Kind regards

Editor in chief

Editor Director Prof.Dr.Ali Muhammed Jawad FRCP,CABM

Editor in-chief Prof. Dr.Alaa Fadhil Alwan FICMS jnt med, FICMS clin hem

Secretary Dr. Nidhal K. Al-Rahhal M.S.C. (Physiology)-D.CH

Executive Editorial Board Prof. Dr.Raad Jaber Mosa M.Sc,FICMS(hempath)

Prof.Dr. Ban Abbas Abdulmajid Ph.D Molecular path

Prof.Dr.Salma Abass AlHadad CABM ped

Ass.Prof.Dr.Alaadin Mudafar FICMS(hempath)

Ass.Prof.DrAladdin Sahham Naji FICMS jnt med, FICMS clin hem

Instructions to Authors

The Iraqi Journal of Hematology is a periodic peer-reviewed journal published biannually by the National Center of Hematology in collaboration with the Iraqi Society of Hematology. The journal welcomes original articles, case reports and letters to editor in all fields relevant to Hematology. Review articles are also welcomed. However, review articles of high standards will be considered.English language is the standard language of the journal yet Arabic abstracts should be accompanying the original articles.

Papers are accepted on the understanding that the subject matter has not and will not be submitted simultaneously to another journal. The following notes are expected to be considered carefully in writing manuscripts.

1- Manuscripts preparation: the format of the Iraqi Journal of

Hematology complies with the by-standard of the International Committee of Medical Journal Editors (ICMJE) in Vancouver, British Colombia, in 1979 and its last update in February 2006, available on the website www.icmje.org.

2- Three clear and complete copies (including figures and tables) should be submitted. Manuscripts and figures will not be returned to the authors irrespective of the editorial decision to accept, revise or reject them.

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Titles of the paper in Arabic and English.

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Abstracts in English of the review articles and case reports should be unstructured and of not more than 150 words.

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A. Introduction: should state clearly the purpose and rationale of the study.

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F. References: should be in the Vancouver style. They should appear in the text by numbers in the order. List all authors when six or less; when seven or more, list only first six and add et al. Journal titles should be abbreviated in accordance with index Medicus. Examples of correct reference forms are given as follows: Journal: Al-Salihi AR, Hasson EH, Al-Azzawi HT. A short review of snakes in Iraq with special reference to venomous snake bite and their treatment. Iraqi Med J 1987; 36:57-60.

Book chapter: Pen AS. Immunological features of myasthenia gravis. In:Aguayo AJ, Karapti G, editors. Topics in Nerves and Muscle Research. 31st ed. Amsterdam: Experta Medica; 1975; p.123-32.

7- Illustrations: photographs unmounted on glossy paper should be provided with magnification scale if appropriate. Lettering should be in either letraset or stencil of comparable size. Illustrations should be marked on the back with the figure number, title of the paper and name(s) of the author(s) with soft pencil. All photographs, graphs and diagrams should be referred to as figures and should be numbered consecutively in the text in Arabic numerals. The legends to illustrations should be typed on a separate sheet. Tables should be numbered consecutively in the text in Arabic numerals and each typed on a separate sheet. Vertical lines normally will not be printed.

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All submission and correspondence should be sent to:

Editor-in-chief Iraqi Journal of Hematology (IJH)

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Table of Contents

Page Title

1 Inducing of Interleukins -10 and 8 by Epstein Barr Virus in Chronic Lymphocyte

Leukemia………………… Luma Amer Yassir ,Amer Alnajjarr, Dawood S. Dawood

,Alaa Fadhil Alwan

19 Immunohistochemical expression of VEGF in relation to VEGFR and CD34 in

NHL using digital image analysis system……….. Bassam Mohammad Hameed,

Hind Shaker Al-Mamoori, Raad Jaber Musa

32 Evaluation of the changes in iron homeostasis and hepcidin concentration in

preeclamptic patients…………Noora Abd Ali Muhsin, Subuh Salim Al-Mudalal

42 The results analysis of complete blood count during the first half of 2015 at the

Central Child Teaching Hospital in Baghdad………………. Rasha Tariq Jawad ,

Ebtehal Ali Hussien

55 Distribution of red cell antigens according to ABO, Rh and other rare blood

group systems in Kurdish ethnicity…………….Hisham A. Getta, Shaema S.Amin,

Najmaddin Khoshnaw , Belal A. Muhammad

81 Review of Congenital Factor XIII Deficiency in Single Iraqi Teaching Hospital

Lubna Foad Hussain, Obeida Amir Abid

90 CD38 and ZAP-70 as prognostic immunological parameters In patients with

chronic lymphocytic leukemia…………..… Intisar Sh. Ali ,Ghassan A.Al-Anni

,Salman A. Al-Jubury

98 Evaluation the Role of Bone Marrow Examination in Diagnosis of Hematological

Diseases in Hemato-Oncology Centers in Kurdistan Region……….Shorsh Jameel

Ridha , Nawsherwan Sadiq Mohammad , Hoger Ismael Muhammed Sarhang

114 Types of Anaemia in Patients with Rheumatoid Arthritis in Sulaymaniyah-

Kurdistan Region of Iraq……………. Hisham A. Getta, Najmaddin Khoshnaw ,

Alaa Fadhil Alwan ,Sundus F.A, Raouf R. Mirza

129 A Clinical-Hematological Study of Pancytopenia Patients Attending Nanakaly

Hospital in Erbil City………… Alan Isaac Isho , Nawsherwan Sadiq Mohammad,

Saran Abdulqadir Nooruldin

Original article

Iraqi J. Hematology, May 2016, vol.5, Issue 1 1

Inducing of Interleukins-10 and 8 by Epstein Barr Virus in Chronic

Lymphocytic Leukemia

Luma A. Yassir 1, Amer Alnajjar

2, Dawood S. Dawood

3, Alaa Fadhil Alwan

4

1 MSc microbiology/ The national center of hematology/Almustansiriya University/ Baghdad/Iraq

2 Prof. of medical virology/ head Dept. of microbiology / Almustansiriya medical college/Baghdad/Iraq

3 Assistant prof. of medical virology/ Dean of medical technical college/Baghdad / Iraq

4 Prof. of clinical hematology/ director of national center of hematology/Baghdad/ Iraq

Received: 2/12 /2015 Accepted: 14/ 12/ 2015

Abstract:

Background: Many newer studies reported that Epstein- Barr virus (EBV) has

association with chronic lymphocytic leukemia (CLL). The average age of presentation is

of patients with CLL is between 65 and 70 years with male to female ratio is 2:1. Notably,

several studies have reported that expression of Epstein Barr encoding RNA (EBERs) is

associated with progressive or accelerated clinical courses. This type of RNA increases

the level of IL-8 and IL-10 in serum of newly diagnosis of CLL patients.

Objectives: the aims of study were to determine the interleukins 10 and 8 level in newly

diagnosed CLL patients and determine the incidence of EBV infection in patient of CLL.

Materials and Methods: A prospective study conducted at department of clinical

hematology in the national center of hematology in Baghdad, Iraq from January 2013 to

January 2014. It included thirty samples of formalin-fixed, paraffin-embedded tissue of

bone marrow aspirates samples and blood from newly diagnosis B-CLL. They were

diagnosed with CLL according to Binet criteria. The detection of EBV encoded RNAs

(EBER1, EBER2) and also detection for the level of Interleukin (8 and 10) in the serum of

CLL patients were done by in situ hybridization technique.

Results: Histopathological study revealed that all the controls were negative for EBERs

and 46.7% were found to be EBERs positive. There was a correlation between positive

EBERs and tumors stage and also EBERs and IL-10 and with IL-8.

Conclusion: The highest incidence of CLL occurs in the age group 40-80 years old and

males are more liable than females (male/female: 70/30).In situ hybridization technique is

successful method to detect of EBV and positive EBERs. IL-8 is highly significant in

CLL patients and correlates with EBERs and LMP1. There is a correlation between IL-10

EBERs and LMP1 in CLL patients.

Keywords: Epstein-Barr virus, CLL, EBERs, IL-8, IL-10

Inducing of Interleukins -10 and 8 by Epstein Barr Luma A.Y., Amer A., Dawood S. D., Alaa F.A.


Introduction

Chronic lymphocytic leukemia (CLL)

results from neoplastic proliferation of a

mature B by the accumulation of non-

dividing Small lymphocytes. It is

characterized by a persistent

lymphocytosis of more than 10 x 109/

liter and lymphoid intrusion of the bone

marrow of at least 40% (1,2)

With the use of immunological markers,

it is likely to establish the diagnosis of

CLL by B cell clonality, even with

lymphocyte counts of less than 5 x

109/liter. CLL is documented as a disease

entity in the WHO organization and as

the leukemic counterpart of small

lymphocytic lymphoma and CLL the

most common type of adult leukemia in

the United States and Western Europe.(3)

The mutation status of the

immunoglobulin heavy chain variable

region (Ig VH) genes can differentiate

between these two groups: CLL patients

with unmutated immunoglobulin high

variable (Ig VH) genes have a negative

prognosis with rapid progression of the

disease.(3)

Epstein- Barr virus (EBV) infection is

only occasionally detected in CLL by

conventional diagnostic approaches. This

is consistent with in vitro results

suggesting that CLL cells do not

regularly become activated or

immortalized after exposure to EBV,

although this can be achieved after

cytokine activation.(4)

Epstein-Barr virus is the first virus

described to be linked with the human

pathogenesis of tumor. The

topographical distribution of Burkitt

lymphoma were related to areas endemic

with falciparum malaria is supposed to

cause chronic excitement or suppression

of the immune system, making children

more susceptible to the oncogenicity of

EBV.(5,6)

In 1968, EBV was recognized to be

the etiological factor of infectious

mononucleosis. At the same time, EBV

was reported to alter infected B cells to

uncontrolled proliferation.(7)

A large study

in 2009 determined around one in every ten

stomach cancers contained EBV. Studies are

ongoing to decode what role the virus is

playing in this type of cancer and how it

weaves together with other risk factors like

nutrition, genetics, and infection of H.

pylori.(8)

Initiation of EBV lytic program occurs in

memory B cells recirculating during the

lymphoid tissue related with the

oropharyngeal mucosa. Host

http://www.ncbi.nlm.nih.gov/pubmed/19445939

http://scienceblog.cancerresearchuk.org/2014/03/07/a-bugs-life-h-pylori-and-stomach-cancer/

http://scienceblog.cancerresearchuk.org/2014/03/07/a-bugs-life-h-pylori-and-stomach-cancer/



immunosuppression may also generate

viral reactivation in lately infected B

cells, which leads to creative infection.

Nevertheless, the original mechanism of

viral reactivation in vivo is not clearly

understood.(9)

EBV alters B-lymphocyte growth,

causing permanent growth

transformation by regulated expression

of multiple viral genes. These genes

comprise three integral membrane

proteins, latent membrane proteins 1,

2A, and 2B (LMP), 6 EBV nuclear

antigens (EBNA1, 2, 3A, 3B, 3C, and

EBNA-LP), then two small, non-coding

nuclear RNAs (EBERs). The gene linked

products relate with or present homology

to various antiapoptotic molecules,

cytokines, and signal transducers,

endorsing EBV infection,

immortalization, and alteration.(10)

There are two types of EBV viruses ,

EBV-1 and EBV-2, which vary depend

on the EBV nuclear antigen (EBNA)

genes.(11)

.EBV-1 is more common in

most populations and is more competent

in transforming B cells in vitro. EBV-2

is mainly found in parts of Africa and is

related endemic Burkitt lymphoma.(12)

Material and Methods:

This study was designed as a prospective

(case-control) study conducted on the

following main groups during the period

from January 2013 to January 2014.

Thirty formalin-fixed, paraffin

embedded tissue blocks were obtained

from Bone marrow biopsies and blood

samples of B-CLL patients.

The age of the patients ranged between

40-80 years, and the samples were

collected directly from patients in

Baghdad Teaching Hospital, from the

National Center of Hematology and

Twenty Bone marrow biopsies and blood

sera of 20 patients who have

hematological problem other than CLL

as control.

We use In situ hybridization to detected

EBERs in Bone marrow samples In situ

hybridization detection kit from abcam

lot-S01_M61, REF _ WB. 005.50:

Hybridization / detection system for

EBV was purchased from ZytoFast

/Germany Cat. Numbers (T-1070-40)

and ELISA kit for detection Human

IL-10 and Il-8 by (abcam46059

lot:GR162207- 4) and (abcam46032 the

lot: GR151489-6) .



Results:

The distribution of age in the studied

group ranged between 40 - 80 years with

a mean of (60.4) year (table 4.1). Males

constituted 70% of cases and female

30%. The ages of control groups ranged

between 42 - 70 years with a mean of

(55.8) year. Males constituted 76.2% of

control groups and female constituted

23.8%. (Table 1).

As shown in table (2), all controls were

negative for EBERs. Although the

median score and intensity for EBERs

was negative for cases group, the mean

rank for EBERs score, intensity and

composite score (a score resulting from

multiplying the score by intensity) was

significantly higher among cases

compared to controls, figure (1),(2) ,(3)

and Figure( 4 ) show the result of In situ

hybridization. As shown in table (3), the

median IL8 was significantly higher in

CLL cases group (33.1 pg/ml) compared

to control group (22.1 pg/ml),also in

figure (5). A similar pattern was

applicable to IL10. The median IL10 was

also significantly higher in CLL cases

group (29.1 pg/ml) compared to control

group (0 pg/ml), figure (6) . As shown in

table (4), the positive test for EBERs

had no obvious or statistically significant

association with IL8 concentration. IL10

concentration also failed to show any

noticeable linear correlation with

similarly measured IL8 concentration.

Table (1): CLL patients distribution according the age and gender

CLL Control

No % No %

Age (years) <50 5 16.7 4 19.0

50--- 7 23.3 9 42.9

60--- 14 46.7 7 33.3

=>70 4 13.3 1 4.8

Mean±SD(Range

)

60.4±9.2 (40-80) 55.8±8.1 (42-70)

P value 0.392

Gender Male 21 70.0 16 76.2

Female 9 30.0 5 23.8

P value 0.626

.

*Significant difference between proportions using Pearson Chi-square test at 0.05 level



Table 2 : The case-control difference in median score and intensity and composite

score for EBERs viral marker.

Study group

Controls Cases (CLL)

N % N % P

1. EBERs-score

Negative 20 100.0 16 53.3

+ 0 0.0 11 36.7

++ 0 0.0 3 10.0

Total 20 100.0 30 100.0

Range

(Negative to

Negative) (Negative to ++) <0.001

Median Negative Negative

Inter-quartile range

(Negative to

Negative) (Negative to +)

Mean rank 18.5 30.2

2. EBERs-intensity

<0.001

Negative 20 100.0 16 53.3

Weak 0 0.0 8 26.7

Moderate 0 0.0 5 16.7

High 0 0.0 1 3.3

Total 20 100.0 30 100.0

Range

(Negative to

Negative) (Negative to High)



(Negative to

Negative) (Negative to weak)

Mean rank 18.5 30.2

3.

EBERs-composite score (score x

intensity)

<0.001

Range

(Negative to

Negative) (Negative to 6)



(Negative to

Negative) (Negative to 1)

Mean rank 18.5 30.2



Figure 1: Component bar chart showing the case-control difference in EBERs-score.

Figure 2: Component bar chart showing the case-control difference in EBERs-

intensity.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%


100

53.3

0

36.7

0 10

Rel

ativ

e fr

equ

ency

(%

)

++

+

Negative

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%


100

53.3

0

26.7

0

16.7

0 3.3

Rel

ativ

e fr

equ

ency

(%)

High

Moderate

Weak

Negative



Figure 3 : Dot diagram with error bars showing the median (with its inter-quartile

range) EBERSs in cases with CLL compared to controls.

0

1

2

3

4

5

6

Study group

EB

ER

s-co

mpo

site

sco

re (

scor

e x

inte

nsity

)




Figure 4 : Representative expression of EBV-encoded small nuclear early

region (EBERs) in Bone Marrow of CLL patients d . a : positive result

show the Nuclear localization of the EBERs in neoplastic cells by ISH . b :

Negative result.

b

a



Table 3 : The case-control difference in median interleukin concentration.

Study group

Controls Cases (CLL) P

IL8 (pg/ml)

0.03

Range (12.2 - 55.3) (7.8 - 1215)

Median 22.1 33.1

Inter-quartile range (17.9 - 35.4) (21.9 - 70.1)

N 41 30

Mean rank 20.6 29.8

IL10 (pg/ml)

<0.001

Range (0 - 24.9) (0.9 - 43)

Median 0 29.1

Inter-quartile range (0 - 0) (7.7 - 33.9)

N 41 30

Mean rank 12 35.8


range) IL8 (pg/ml) in cases with CLL compared to controls. (Logarithmic scale was

used)

1

10

100

1000

10000

Study group

IL8





range) IL10 (pg/ml) in cases with CLL compared to controls.

Table 4 : The median IL8 concentration (pg/ml) by selected explanatory variables

among cases with CLL.

IL8

Range Median

Interquartile

range N

Mean

Rank P

Age group

(years)

0.6[NS]

<60 (10.6 to 79.4) 31.9 (22.2 to 61.3) 12 14.5

60+ (7.8 to 1215) 36 (21.9 to 130.3) 18 16.2

Gender

0.5[NS]

Female (7.8 to 1215) 55.6 (20 to 401.5) 9 17.2

Male (10.6 to 346.2) 32.8 (24.3 to 46.9) 21 14.8

Positive EBERs

0.1[NS]

Negative (10.6 to 1215) 43.1 (25.3 to 104.9) 16 18

Positive (7.8 to 401.5) 30.4 (17.7 to 44.4) 14 12.7

0

5

10

15

20

25

30

35

40

45

Study group

IL10




Table 5 : The median IL10 concentration (pg/ml) by selected explanatory variables

among cases with CLL.

IL10

Range Median

Interquartile

range N

Mean

Rank P

Age group (years)

0.18[NS]

<60 (0.9 to 38.3) 20.35 (6.25 to 29.85) 12 12.9

60+ (2.5 to 43) 32.3 (9.3 to 34.8) 18 17.3

Positive EBERs

0.63[NS]

Negative (0.9 to 43) 20.75 (8.5 to 33.9) 16 14.8

Positive (2.2 to 43) 32.3 (4 to 33.9) 14 16.3

Discussion :

Epstein- Barr virus has strong

association with variety in B-cell tumors

including Burkitt's lymphoma, Hodgkin

lymphoma, human immunodeficiency

virus, post transplantation lymphoma

disorder and chronic lymphocytic

leukemia. Many studies reported that

CLL patient had evidence of EBV

infection by In situ hybridization for

EBERs and detection of EBV-encoded

EBER transcripts is considered the gold

standard for localizing latent EBV in

tissue samples, as EBER transcripts are

universally expressed in all EBV

associated tumors.(13,14)

The presence of EBERs has been shown

to correlate with progressive or

accelerated clinical course including

transformation to Richter's large cell

lymphoma.(15,16)

However EBERs is also found in

quiescent EBV latency where no protein

is produced and that may be a

suboptimal marker for proliferation or

transformation capability.

In this study all controls showed

negative result EBERs but in patients the

result show that 14 (46%) out of 30 CLL

patients were positive with EBERs.

Results obtained are nearly compatible

to previous study who reported that

(38%) of CLL patients had evidence of

EBV infection proved by EBERs

positively in tumor cells (16)

.

Result demonstrated in this study were in

accordance with 16) Tsimberidou et al

who stated that 12 out of 32 CLL

patients has appositive result.(16)



On the other hand another study found

that 8 of 75 (10.7%) cases showed

EBERs expression restricted to 5–10%

of tumor cells.(17)

This controversy in the above results

may be related to that ISH process

depends on the RNA staining and the

concentration of RNA in the cell .this

method affected by many factors,

including the RNA present in the cell

and concentration of RNA. This

technique is very sensitive. Interleukins-

8 and it's receptor increased in cancer

cells, infiltrating neutrophils, endothelial

cells, and tumor-associated

macrophages (18)

There is no evidence of IL-8 production

by normal B cells but many studies

showed that the natural cellular source

of IL-8 production have been described

to be monocyte/macrophages, T cells,

large granular lymphocytes, fibroblasts,

endothelial cells, mesothelial cells,

keratinocytes, neutrophils ,hepatocytes

and chondrocytes.(19,20,21)

A study which has been done by Celle et

al showed that elevated IL-8 levels may

be founds in the serum of untreated B

cell patient which may be release by B

cells and superannuated of purified B-

CLL cells contain IL8 released

chemotactic activity for neutrophils.(22)

In our study we found that IL-8 was

significantly higher in CLL cases group

the compared with control group. This

result is compatible with other study

published (23,24)

which found that plasma

IL-8 level enhances in CLL patients. The

same result was studied by Yoshizaki et al

who found that IL-8 increased in CLL

patients .(25)

On the other hand another

study found that serum IL-8 level was not

increased in significant level in patient

with CLL when she compared with

healthy control.(26)

The association of EBERs and IL-8 did

not reach the level of statistical

significance. This result is not

compatible with study done by William

et al who found that the stages were

associated with significantly higher

plasma IL-8 levels (P < 0.0001) but

There were no significant difference

between IL-8 production and gender. (27)

While another study found compatible

result with this study where IL8 level in

CLL patients not correlated with CLL

stages. (24)

Notably, serum IL-10 levels are

increased in CLL patients and correlate

with adverse disease features and short

survival.(28,29)

Results obtained in this study revealed

that the IL-10 was significantly higher in

CLL cases group median rang (29.1 pg.



/ml) compared to control group (0 pg/ml)

and the mean rank 35.8 with p<0.001

figure (6)

This result is compatible with other

study(30)

,Which found that IL-10 levels

were higher in CLL patients (median,

5.04 pg/mL; range, undetectable to 74

pg/mL) than in normal volunteers

(median, undetectable; range,

undetectable to 13.68 pg/mL) (P <

.00001) ,and another study found that IL-

10 levels increased in CLL patient of

Iraq and significantly than control group

P<0.05 same study was studied by [31]

who found that Serum levels IL-10 in 20

CLL patient severely dropped in

untreated group (27±11.47 and

0.65±0.23 pg/mL respectively) and

differed significantly healthy in 20

control group (1715.66±1014 pg/mL

respectively)(26)

and David at al who

found that Serum IL-10 levels were also

significantly elevated in CLL

patients. (18)

Other explanations for the increase in

the level of IL-10 in patient with CLL

was demonstrated in other researches

which found that IL-10 is increased in

production by culture of CLL and that

serum IL-10 levels were elevated in five

of the eleven B-CLL patients. These

findings suggest that IL-10 acts as an

autocrine growth factor for B-CLL cells

and cytokine-based therapy might be a

new approach for the treatment of B-

CLL.(33,34)

IL-10 was derived from EBV infected

tumor cells and demonstrated in serum

of CLL and Hodgkin lymphoma patients.

(30,34) . Several studies have reported an

association between EBERs, LMP1and

IL10 stimulation. EBV infection

enhanced production of viral IL-10 and

may also contribute to a local immune

suppression by production of

hydrophobic peptides derived from the

first transmembrane domain of LMP-1

(35) In contract EBERs induce the

transcription of various cytokines

depending on cell type, such as

interleukin-10 (IL-10).

Conclusion: The highest incidence of

CLL occurs in the age group 40-80 years

old and males are more liable than

females (male/female: 70/30).In situ

hybridization technique is successful

method to detect of EBV and positive

EBERs. IL-8 is highly significant in CLL

patients and correlates with EBERs and

LMP1. There is a correlation between

IL-10 EBERs and LMP1 in CLL

patients.



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بواسطة فيروس ابشتاين بار في سرطان الدم الليمفاوي المزمن 8و 01-تحفير االنترلوكين

4، عالء فاضل علوان 3، داود سلمان داود 2، عامر النجار 0لمى عامر ياسر

الجامعة المستنصرية / بغداد / العراقماجستير علم األحياء المجهربة / المركز الوطني ألمراض الدم / 1

أستاذ علم الفيروسات الطبية / رئيس قسم األحياء المجهربة / كلية طب المستنصرية / بغداد / العراق 2

استاذ مساعد. علم الفيروسات الطبية / عميد كلية التقنية الطبية / بغداد / العراق 3

طني لألمراض الدم / بغداد / العراقأستاذ أمراض الدم السريرية / مدير المركز الو 4

:الملخص

سرطان لمرضى المسببات احد ويعتبر عالقة له فايرس بار االبيشتاين ان الدراسات اثبت الخلفية:

وكانت عام( 56-07) بين يتراوح المرضى عمر معدل كان الدراسة هذه في المزمن اللمفاوي الدم

المزمن وكما اثبت اللمفاوي الدم سرطان مرضى في% 2/1 بين تتراوح االناث الى الذكور نسبة

في مصول المرضى . IL-8و IL-10يزيد من افراز EBERsهذه الدراسة ان

: دراسة مستقبلية أجريت في قسم علم أمراض الدم السريري في المركز الوطني المواد والطرق

. وشملت ثالثين 3102يناير إلى 3102لألمراض الدم في بغداد، العراق خالل الفترة من يناير

عينات من واألنسجة الثابتة بالفورمالين جزءا ال يتجزأ من البارافين من عينات نخاع العظام والدم

EBVالمزمن .. وقد أجريت للكشف عن الرنا اللمفاوي الدم سرطان من حديثي التشخيص ب

( في مصل 01و 8(، وكذلك الكشف عن مستوى انترلوكين )EBER1 ،EBER2المشفرة )

المزمن بتقنية التهجين الموقعي. اللمفاوي الدم مرضى سرطان

٪ 4..2ووجد EBERs: كشفت الدراسة النسيجية أن جميع الضوابط كانت سلبية النتائج

اإليجابية EBERsالمزمن . كان هناك ارتباط بين اللمفاوي الدم سرطان في EBERsإيجابية من

.IL-8و مع IL-10و EBERsومرحلة األورام وأيضا

سنة 81-21المزمن في الفئة العمرية اللمفاوي الدم يحدث أعلى معدل النتشار سرطان االستنتاج:

( . تقنية التهجين الموقي هي طريقة 41/21والذكور أكثر عرضة من اإلناث )ذكور / إناث:

ضى الذين يعانون من مهم للغاية في المر IL-8اإليجابية. EBERs و EBVناجحة للكشف عن

IL-10 EBERs. هناك عالقة بين LMP1و EBERsالمزمن ويرتبط اللمفاوي الدم سرطان

المزمن. اللمفاوي الدم سرطان في مرضى LMP1و

IL-8 ،IL-10، المزمن اللمفاوي الدم سرطان : فيروس ابشتاين بار،كلمات البحث

Original article


Immunohistochemical expression of VEGF in relation to VEGFR and

CD34 in NHL using digital image analysis system

Bassam Mohammad Hameed1, Hind Shaker Aboob

2, Raad Jaber Musa

3

1 M.B.Ch.B., Msc. Pathology (haematology), Ph.D. Pathology (haematology) Lecturer at Department of

pathology and forensic medicine /collage of medicine /AL-Nahrain university

2 M.B.Ch.B., FIBMS(path/haemaology) Lecturer at Department of pathology and forensic medicine /collage of

medicine /AL-Nahrain university

3 M.B.Ch.B., M. Sc. Path. (Haem.); F.I.C.M.S.Path.(Haem.) Professor of Haematology at Department of


Received 31/12/2015 accepted 15/2/2016

Abstract

Background: Lymphoma growth and progression appear to be promoted by at least two distinct

angiogenic mechanisms: autocrine stimulation of tumor cells via expression of Vascular Endothelial

Growth Factor(VEGF) and Vascular Endothelial Growth Factor Receptor(VEGFR) by lymphoma

cells, and paracrine influences of the proangiogenic tumor microenvironment on local tumor

vascularity.

Objectives: To assess autocrine effect of VEGF, by studying the correlation of VEGF expression

with its receptor VEGFR expression in NHL. And to assess paracrine effect of VEGF, by studying the

correlation of VEGF expression with CD34 expressed on endothelial cells in Non-Hodgkin

Lymphoma (NHL).

Materials and Methods: A cross sectional study was designed. A total of 66 bone marrow tissue

samples were included in the study, all diagnosed as having NHL according to working formulation.

From each block, 3 sections were taken, and were immunohistochemically stained for CD-34, VEGF

and VEGFR. Scoring of Immunohistochemical staining was performed using specialized automated

cellular image analysis system, Digimizer software, version 3.7.0.

Results: VEGF Immunohistochemical digital parameters named digital labeling index (DLI) was

significantly correlated with the followings; VEGFR (DLI) [P =0.042, r =0.324], CD34 stained area

(A) [P=0.037, r =0.556]. Also VEGFR (DLI) was significantly correlated with CD34 (A).

Conclusion: Autocrine and paracrine effect of VEGF is evident in NHL, as there is positive

correlation between VEGF expression and VEGFR expression, and as tumor vascularity increase with

the increase in VEGF expression.

Keywords: NHL, VEGF, VEGFR

Immunohistochemical expression of VEGF Bassam M.Hameed, Hind Sh. Aboob, Raad J. Musa


Introduction

Tumor angiogenesis mediated by many

mediators in tumor microenviroment, of

these VEGF (vascular endothelial growth

factor) is the most important factor for

angiogenic switch through its interaction

with receptors regulating different aspects

of tumor angiogenesis. Lymphoma growth

and progression as many other tumor

promoted by angiogenesis, this occurs by

two main mechanisms; autocrine

stimulation of tumor cells by expressing

both VEGF and its receptor VEGFR, and

second by paracrine influences of the pro-

angiogenic tumor microenvironment. (1)

Many researchers stated that there is

positive correlation between VEGF

expression with VEFGR expression

in DLBCL lymphoma cells supporting the

concept of autocrine-paracrine role for

VEGF as growth factor for DLBCL

promoting cell survival and

proliferation.(2,3)

In this study we tried to study angiogenesis

via VEGF, VEGFR and microvessel

density in non-Hodgkin lymphoma using

digital analysis system, Advantages of

using digital image system in that scoring

immunohistochemical staining in this

method offer objectivity, reproducibility,

quantification while overcoming manual

scoring method that requires considerable

expertise and is susceptible to

interobserver variability.(4)

Aims of the study

1. To assess autocrine effect of VEGF, by

studying the correlation of VEGF

expression with its receptor VEGFR

expression, in bone marrow biopsy of non-

Hodgkin lymphoma patient using

automated image analysis system.

2. To assess paracrine effect of VEGF, by

studying the correlation of VEGF

expression with CD34 expressed on

endothelial cells, in bone marrow biopsy

of non-Hodgkin lymphoma patient using

automated image analysis system.

Materials and methods

A cross sectional study was designed, a

total of 66 tissue samples (paraffin block

of bone marrow biopsy) were included in

the study. All the samples were taken from

the (Medical city/ teaching laboratories),

cases presented during the period 2008-

2010 as all diagnosed having NHL

according to working formulation by

histopathological examination for primary

lymph node biopsy. From each block, 3

sections of 5µm thickness were taken,

each section were immunohistochemically

stained for CD-34, VEGF and VEGFR.



The procedure was carried out according

to manufacturer's instructions. Taking

sections and mounted on Fisher brand

positively charged slides. Than slides

deparaffinized, and placed in DAKO

antigen retrieval (PH 6 for VEGF, PH 9

for VEGFR, 1700 for CD34) after heating

slides with antigen retrieval solution in

microwave for 20 min, slides let to cool

down. Later on LSAP (DAKO staining kit)

used for staining, after blocking

endogenous peroxidase, and incubation of

primary antibody for 30 min. Scoring of

immunohistochemical staining was

performed using specialized automated

cellular image analysis system, Digimizer

software, version 3.7.0.

Image capture

Using a light microscope, each

immunohistochemically stained slide was

scanned with 10 × objectives for the

positive brown immunostaining, and with

40× objective three fields that reflect the

best of the overall immunostaining of the

entire slide were chosen and captured

using a Sony digital camera (cyber-shot

DSC-W510). Captured images of

4000×3000 pixels were saved on PC in an

uncompressed JPG format.

Image analysis

Each image was analyzed by Digimizer

software (Version 3.7.0). Determination of

immunostaining intensity was done by

using the Magic Wand tool in the toolbar

menu in digimizer program (see figure 1).

The tolerance level of the Magic Wand

tools was adjusted so that the entire

positive cells were selected. The

measurements comprised:

1. Color Intensity (I): which measures the

average intensity of the brown color for

the selected objects depending on the

expression of antigens in the cells.

2. Fractional area stained (A) = [(mean

area× Number of objects) / area of a single

image field] 100%

3. Digital Labeling Index (DLI): first used

by Al-Sinjery, G. M. [5], this tool is

calculated according to the following

formula:

DLI= [Fractional area(A) × reverse

Intensity(I)].

This digital parameter, is the best

representative for the expression because it

combines both the Fractional area and the

Intensity of immunohistochemical

staining.

Statistical analysis:

Statistical analysis was performed with

SPSS (statistical package for social

sciences) version 16 and Excel 2007

programs.



Figure 1. showing snap shot for digimizer softwear window

Results

Correlation between digital parameters of

digimizer softwear of VEGF and other

angiogenic markers in NHL: Digital

parameter for VEGF digital labeling index

(DLI) which combines staining intensity

and area staining was significantly

correlated with the followings; VEGFR

digital labelling index (DLI), CD34

staining area (A), as well as VEGFR (DLI)

was significantly correlated with the CD34

staining area (A). figure (II)



Figure II. Correlation chart shows positive correlation between VEGF (DLI) and VEGFR-

1(DLI),(P =0.042, r =0.324).

Figure III. Correlation chart shows positive correlation between VEGF (DLI) and CD34

(A),(P=0.037, r =0.556).

y = 0.553x + 21.406

0

20

40

60

80

100

120

0 20 40 60 80 100 120

VEG

F D

LI

VEGFR DLI

y = 0.9586x + 13.552

0

20

40

60

80

100

120

140

0 10 20 30 40 50 60

VEG

F

DLI

CD34 area



Figure IV. Correlation chart shows positive correlation between VEGFR-1 (DLI) and

CD34(A),(P=0.041, r = 0.303).

Figure V. Trephine biopsy section from a patient with Folliculer lymphoma showing

paratrabecular infiltration& random focal . H&E, x 4 objective.

y = 1.1056x - 1.5145

-20

0

20

40

60

80

100

120

0 10 20 30 40 50 60

VG

EFR

DLI

CD34 area



Figure VI. Trephine biopsy section from a patient with B-SLL showing nodular

infiltration . with D20 + immunohistochemistry (X 4 objective.).

Figure VII. Trephine biopsy section from a patient with B-DLL showing diffuse

infiltration. With CD20 + immunohistochemistry (X 10 objective.)



Figure VIII. Trephine biopsy section from a patient with B-DLL stained with

immunohstochemistry showing positive VEGF-A lymphoma cells (arrows). With

cytoplasmic brown staining (40X).



Figure IX. Trephine biopsy section from a patient with B-DLL showing endothelial cells

positive for CD34 stained with immunohstochemistry (40X).

Discussion

In Iraq many researchers studied

angiogenesis in different malignancies like

prostate adenocarcinoma, multiple

myeloma , renal cell carcinoma, gastric

adenocarcinoma.(6-9)

Kareem and Jaafer

studied angiogenesis in haematolymphoid

tumors, Kareem studied MVD in NHL,

while Jaafer studied VEGF in CLL.(10,11)

But most of these studies used

microvessel density as a marker of

angiogenesis, with manual count,

restricting their result to blood vessel

count. Another study done by Qasim B. et

al pushed the work step forward by using

digimizer software for CD34+ blood

vessels , this make assessment more

subjective and computer based.(12)

This work tried to assess angiogenic

markers in NHL, by studying VEGF

expression and its autocrine effect via its

receptor VEGFR expression and the

paracrine effect via CD34+ vessel

expression, using Digimizer software to

analyze their immunohistochemical

expression, instead of manual count for

blood vessels and manual score for

VEGF/VEGFR expression.

Many researcher suggest that VEGF may

play dual roles in tumor angiogenesis,

first through signaling to endothelial cells



promoting them to form new blood vessels

resulting in angiogenesis and second via

signaling to tumor cells as an

autocrine/paracrine growth factor.(2)

This

research assessed the interaction among

vascularity (CD34+ area) and local

expression of VEGF and VEGFR in NHL

cases.

VEGF expression had positive correlation

with VEGFR expression in different

digimizer softwear parameters (intensity

"I", area "A", digital labeling index

"DLI"). This may be due to autocrine

effect of VEGF. Both VEGF and VEGFR

had positive correlation with lymphoma

vascularity expressed by CD34 area "A",

also reinforcing the concept of paracrine

effect of VEGF. These findings are similar

to findings of previous work.(10,13-18)

The VEGF immunohistochemistry is

reflective of effective local VEGF

signaling. It is expected that MVD

increases with VEGF expression. Average

lymphoma vascularity labeled by CD34+

endothelial cells did increase with

strength of VEGF staining with statistical

significant in the present work, and this

was also demonstrated in the study by

Berthold et al . (19)

Dita et al have shown

that higher MVD is present in DLCL

specimens expressing higher levels of

VEGF.(16)

This finding is consistent with a

paracrine role of VEGF elaborated by

lymphoma cells in tumor angiogenesis.(3)

Conclusion

Autocrine and paracrine effect of VEGF is

evident in NHL, as there is positive

correlation between VEGF expression and

VEGFR expression, furthermore as tumor

vascularity increases expressed by CD34

stained area with the increase in VEGF

expression.



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8. Mazin, J.I., NabeelA.Rasheed, and A.H.

Manal, Microvessel density in Renal Cell

Carcinoma. Journal of the Faculty of

Medicine 2010. 52(4): p. 424-425.

9. Al-kaptan A.H,I., Uses of CD31

monoclonal Antibody for the Assessment

Of Angiogenesis as a prognostic Factor in

Gastric Adenocarcinoma. Journal of the

Faculty of Medicine 2005. 47(1): p. 42-49.

10. Kareem, G.M., A Study of

Angiogenesis Measured by Expression of

CD34 antigen in Non-Hodgkin's

Lymphoma, in A thesis submitted to the

council of the College of Medicine at Al-

Nahrain University. 2006, AL-Nahrain:

baghdad.

11. Ja’afar A.M., In Situ Hybridization

Analysis of p-53 and Bcl-2 Oncogenes and

Angiogenesis Factors VEGF and MMP-9



in Chronic Lymphocytic Leukemia, in A

Thesis Submitted to College of Medicine

and Committee of Graduate Studies

University of Baghdad. 2008, Baghdad.

12. Qasim, B.J., Assessment of

Immunohistochemical Expression of

Molecular Markers: Matrix

Metalloproteinase -7 (MMP-7), CD34,

p53, bcl2, Proliferating Cell Nuclear

Antigen (PCNA), Estrogen and

Progesterone Receptors in Human

Colorectal Carcinogenesis Using

Specialized Automated Cellular Image

Analysis System. , in A thesis submitted to

the Council of college of Medicine/ Al-

Nahrain University in partial fulfillment of

the requirements for the degree of Doctor

of Philosophy in pathology. 2011, Al-

Nahrain: Baghdad.

13. Wang, E.S., et al., Targeting autocrine

and paracrine VEGF receptor pathways

inhibits human lymphoma xenografts in

vivo. Blood, 2004. 104(9): p. 2893-2902.

14. Lee, Y.K., et al., VEGF receptor

phosphorylation status and apoptosis is

modulated by a green tea component,

epigallocatechin-3-gallate (EGCG), in B-

cell chronic lymphocytic leukemia. Blood,

2004. 104(3): p. 788-94.

15. Gratzinger, D., et al., Prognostic

significance of VEGF, VEGF receptors,

and microvessel density in diffuse large B

cell lymphoma treated with anthracycline-

based chemotherapy. Lab Invest, 2007.

88(1): p. 38-47.

16. Dita G, et al., Microvessel density and

expression of vascular endothelial growth

factor and its receptors in diffuse large B-

cell lymphoma subtypes. Am J Pathol,

2007. 170: p. 1362-9.

17. Tzankov, A., et al., Angiogenesis in

nodal B cell lymphomas: a high

throughput study. Journal of Clinical

Pathology, 2006. 60(5): p. 476-482.

18. Zhang, W., et al., Expression of tumor-

associated macrophages and vascular

endothelial growth factor correlates with

poor prognosis of peripheral T-cell

lymphoma, not otherwise specified.

Leukemia & Lymphoma, 2011. 52(1): p.

46-52.

19. Streubel, B., et al., Lymphoma-specific

genetic aberrations in microvascular

endothelial cells in B-cell lymphomas. N

Engl J Med, 2004. 351(3): p. 250-9.

Correspondence to

Dr. Bassam Mohammad Hameed

Department of pathology and forensic

medicine /collage of medicine /AL-

Nahrain university

Email: [email protected]

Mobile: 07700664914

Immunohistochemical expression of VEGF Bassam M.Hameed, Hind Sh. Aboob, Raad J.

Musa


في االورام CD34 )و VEGFRوارتباطه ب ) VEGF)التقين الوناعي النسيجي للوعاهل )

هوجكن باستخذام نظام رقوي لتحليل الصوراللوفاوية عذا

3, ا.د.رعذ جابر هوسى 2, م.د.هنذ شاكر عبعوب 1م.د.بسام دمحم حويذ

جايؼح انهش \كهح انطة\يذسط ف فشع ػهى االيشاع وانطة انؼذن 1

جايؼح انهش \كهح انطة\يذسط ف فشع ػهى االيشاع وانطة انؼذن 2

جايؼح انهش \كهح انطة\اسرار ف فشع ػهى االيشاع وانطة انؼذن 3

ولخصال

: ا ى األوساو انهفاوح وانرقذو تركىها ك ا رى ي قثم اث ػهى األقم ي اناخ الخلفية

انخالا انسشطاح ػ طشق انرؼثش األوػح انذيىح غشائ ػايم autocrineانشضح: انرحفض

ا ( ي قثم خالا سشطVEGFR( واألوػح انذيىح غشائ ػايم ى يسرقثالخ )VEGFانى )

ػهى األوػح proangiogenicانغذد انهفاوح، وانرأثشاخ ظش انظاوي ي انكشوح انىسو

انذيىح انسشطاح انحه.

يغ VEGF، ي خالل دساسح ػالقح انرؼثش VEGFي autocrineذقى ذأثش األهذاف:

ى تطاح االوػح، . ونرقى ذأثش ظش انظاوي ي ػايمNHLف VEGFRيسرقثالخ انرؼثش

أػشب ػ انخالا انثطاح ف سشطا انغذد CD34يغ VEGFي خالل دساسح ػالقح انرؼثش

(.NHLغش هىدجك ) انهفاوح

ي ػاخ األسجح نخاع انؼظى 66: ذى ذظى دساسح يقطؼح. أدسجد يا يجىػه الوواد والطرق

3. ي كم ػح أخزخ working formulationوفقا NHLف انذساسح، ى ال ذشخض ل

. VEGFR، ػايم ى تطاح االوػح وCD-34ل immunohistochemicallyأقساو، وطثغد

انخهىح طىسج وذحهم ان يرخظض ظاو تاسرخذاو انرشقى ذفز ذى

.3.7.3، اإلطذاس Digimizerتثشايج

( إنى حذ DLIاناػى انؼهاخ انشقح اسه يؤشش انؼالياخ انشقح ) VEGFاسذثظ تائج :الن

(Aيطقح انهى ) CD34[، 3.324، ص = P = 0.042] VEGFR (DLI)كثش يغ يا ه،:

[P = 0.037 = أضا 3.556، ص .]VEGFR (DLI اسذثظ تشكم يهحىظ يغ )CD34 (A.)

، NHLوظش انظاوي ي ػايم ى تطاح االوػح هى واضح ف Autocrineذأثش الخالصة:

، وكا صادج األوػح انذيىح VEGFRوانرؼثش VEGFكا أ هاك ػالقح إجاتح ت انرؼثش

.VEGFنهىسو راسة يغ صادج ف انرؼثش

NHL ،VEGF ،VEGFR: ألكلوات الوفتاحية

Original article


Evaluation of the changes in iron homeostasis and hepcidin concentration in

preeclamptic patients

Noora Abd Ali Muhsin1, Subuh Salim Al-Mudalal

2, Bassam Mohammad Hameed

3

1 M.B.CH.B /AL-Imamin AL-Kadhmin medical city

2 Professor of Haematopathology/ M.B.Ch.B /M.Sc /F.I.C.M.S. (Pathology/Haematology)/ Department of


3 Msc. Pathology (haematology), Ph.D. Pathology (haematology) Lecturer Department of pathology and forensic

medicine /collage of medicine /AL-Nahrain university


Abstract

Background: Plasma iron is increased in preeclampsia (PE) in comparison to normal pregnant women.

The relation between iron homeostasis and inflammation is hepcidin. Hepcidin is an acute phase reactant

protein which has major role in iron hemostasis.

Objectives: To evaluate serum iron, total iron binding capacity (TIBC), serum ferritin and serum

hepcidin levels in preeclamptic pregnant women in relation to non preeclamptic pregnant women, who

were not on any iron supplement regimen.

Materials and Methods: This case control study was conducted on twenty pregnant women in the third

trimester of pregnancy suffering from preeclampsia that had not received iron supplement or had blood

transfusion within last three months, those patients attended the obstetrics and gynecology department at

(AL-Imamin AL-Kadhmin medical city) between May to August 2013. Along with those patient twenty

non preeclamptic pregnant women who were age and gestational age matched were included as control

group. Moreover any subject presented with active infection, chronic diseases, chronic blood loss or twin

pregnancy was excluded. A total of 5 ml of venous blood sample was obtained from each patient and

control and tested for measurement of Hb, PCV, and MCHC by automated device whereas, serum of iron

and TIBC, ferritin and hepcidin were measured by ELISA technique.

Results: The mean level of Hb, PCV, MCHC, serum iron, serum ferritin and serum hepcidin in

preeclamptic patients were higher than those of control group (P value of < 0.05).There was a non-

significant correlation between serum iron and hepcidin in preeclamptic patients (r=0.234, P= 0.32)

whereas there was a significant strongly positive correlation between serum iron and hepcidin in the

control group (r=0.839, P = 0.003).

Conclusions: In preeclamptic patients serum iron concentration is increased in spite of high hepcidin

concentration which might indicate a resistance to the iron-decreasing action of hepcidin.

Keywords: preeclampsia, hepcidin; pregnancy; iron regulation

Evaluation of the changes in iron homeostasis Noora A.A.M, Subuh S. Al-Mudalal, Bassam M.H.


Introduction

Preeclampsia, a systemic syndrome

manifested primarily by hypertension and

proteinuria, presents mainly in the second

half of pregnancy, and affects approximately

3% to 5% of pregnancies worldwide.(1)

Normal women has a decrease in serum iron

and ferritin during the third trimester of

pregnancy as their stores of iron are depleted

because of fetoplacental demand and

required expansion of red cell mass.(2,3)

Plasma iron concentrations and ferritin are

increased, whereas total iron binding

capacity (TIBC) is decreased in

preeclampsia. The increase in plasma iron is

in contrast to inflammation characteristic for

preeclampsia. The link between iron

homeostasis and inflammation is hepcidin.(4)

Hepcidin, a negative regulator of iron

absorption and Recycling (5)

, is a small

peptide produced by the hepatocytes in

response to increased body iron and

inflammation.(6)

The increase in plasma iron

concentrations despite high hepcidin

concentrations in preeclampsia might

indicate a resistance to the iron-decreasing

action of hepcidin.(4,7)

The aims of the study were to evaluate

serum iron, total iron binding capacity

(TIBC), serum ferritin and serum hepcidin

levels in pregnant women suffering from

preeclampsia and to compare them with non

preeclamptic pregnant women.


This study was conducted on twenty

preeclamptic pregnant women in the third

trimester of pregnancy who were attending

the obstetrics and gynecology department at

(AL-Imamin AL-Kadhmin medical city)

between May to August 2013. Along with

twenty non preeclamptic pregnant women

served as control and who were age and

gestational age matched and both groups had

a parity of three or less and twin pregnancy

were excluded. All of the preeclamptic

patients were diagnosed previously by

obstetrical specialist and the mean arterial

pressure (MAP) was measured for all

pregnant women.

MAP = [(2×diastolic blood

pressure)+systolic blood pressure] / 3.

A written consent for participation in the

study was obtained from each subject

included in the study.



Moreover pregnant women included in the

study were not receiving iron supplement

and had not received blood transfusion

within the last three month. Also both

groups should not suffer from active

infection, any chronic disease, and chronic

blood loss.

Blood sampling:

A total of 5 ml of venous blood sample was

obtained from each patient and control by

venipuncture from the cubital fossa under

aseptic technique. The blood sample was

divided into two smaller samples as follows:

1-First sample comprised of two milliliters

of blood in Ethylene diamine tetra acetic

acid (EDTA) tube for measurement of Hb,

PCV, MCHC by automated haematology

analyzer (Sysmex KX-21N, Japan).

2-Second sample comprises of three

milliliters of blood in plain tube to obtain

serum by centrifugation of clotted blood for

measurement of iron and UIBC, ferritin and

hepcidin. Serum Iron and UIBC was

estimated by the direct colorimetric assay

using automated analyzer (ARCHITECT

c4000, Abbott, USA) and by commercially

available kit using for (IRON, 6K95-30 and

6K95-41, ARCHITECT, Abbott, USA), and

for UIBC (UIBC LIQUID, 4P79-30,

ARCHITECT, Abbott,USA). The TIBC was

calculated from serum iron concentration

plus unsaturated iron binding capacity

(UIBC).Ferritin was calculated by enzyme

immunoassay based on standard method

using automated immunoanalyzer

(Minividas, Biomerieux, France) and

commercially available kit (VIDAS Ferritin,

30 411, Biomerieux, France). Hepcidin was

calculated in patient and control serum by

Enzyme linked immunosorbent assay

(ELISA) based on biotin double antibody

sandwich technology using standard enzyme

reader (ELISA Reader, Diagnostic

automation inc, USA) and a commercially

available kit (Human Hepcidin ELISA kit,

MBS164980, MyBioSource, USA).

Computerized statistical analysis were

performed using SPSS (statistical package

of social sciences), version 16 with

Microsoft office excel 2007.The numeric

data were expressed as mean±SD. Student t-

test was used to estimate the difference

between two means. Correlation test was

used to find the association between two

numerical variables.



Results

This study included twenty preeclamptic

pregnant women in third trimester of

pregnancy with mean age was 28.5±4.9

years. Along with twenty non preeclamptic

pregnant women who were age and

gestational age matched and who served as

control group, whose mean age was

28.3±6.3years. The mean arterial pressure was

significantly higher in preeclamptic patient

(114.9±8.9 mmHg ) than the mean arterial

pressure control group (87.8±6.2 mmHg)

Haematological and biochemical parameters

of preeclamptic patients and control group

were presented in Table 1, showing that the

mean level of Hb,PCV,MCHC as well as

mean level of serum iron, serum ferritin,

serum hepcidin of preeclamptic patients

were significantly higher than those of

control group. Only the mean level of TIBC

was lower in preeclamptic patients than

control group.

Furthermore , there was a non significant

correlation between serum iron and hepcidin

in preeclamptic patients (r=0.234, P= 0.32)

whereas this correlation was strongly

positive in normal pregnant control group

(r=0.839, P = 0.003). (Figure 1)



Table 1: Haematological and biochemical parameters of preeclamptic patients and control group

*Significant P value(<0.05)



Figure 1. Correlation between serum iron and hepcidin in control group (P = 0.003)



Discussion

The present study had revealed that iron

profile (includes S.iron, TIBC, S.ferritin was

within normal range in preeclamptic patient

although they were not receiving any iron

supplement. Whereas the iron profile in

normal pregnancy women was that of iron

deficiency anemia, which was expected

since they were not receiving iron

supplements. Those results were in

agreement with studies done by Tasneem

zafar et al (3)

, Gergely Toldi et al (4)

and

Margaret P. Rayman et al(8)

. These results

may be attributed to that, in preeclampsia

there is impairment of trophoblast invasion

to the maternal spiral arteries resulting in

necrotic and hemorrhagic areas in the

placental tissue. The injured red blood cells

in those area will be a source of free iron

radical. (8,9)

Additionally, Margaret P. Rayman et al in

their study had found that, iron released

from red-cell destruction of damaged

placenta are clearly capable of initiating and

propagating lipid peroxidation, results in

endothelial-cell damage in preeclamptic

patients.(8)

So we may propose that

disturbance of iron status is a cause and

result for preeclamptic changes.

Consequently, preeclamptic patients had

normal Hb, PCV and MCHC levels, whereas

control group had low Hb, PCV and MCHC

levels since those patients had not received

iron supplement. Those results were in

agreement with studies done by Tasneem

zafar et al.(3,10)

This study showed that, the mean level of

serum hepcidin in preeclamptic patients was

within normal range and significantly higher

than that control group and there was no

correlation of its level with S. iron. This

result was in agreement with study done by

Gergely Toldi et al, who found that

increased in the pro-inflammatory cytokine;

interleukin-6 (IL-6) in preeclamptic patients

as a result of inflammatory reaction, results

in high hepcidin concentration in those

patients.(4)

despite of high S. iron level

which might indicate a resistance to the

iron-decreasing action of hepcidin. This

resistance to hepcidin may be due to

functional or structural abnormalities of

ferroportin. It is well known that normally

the chronic inflammation decreases iron

availability through decreasing the release of

iron from the macrophages to the plasma as

a result of raised serum hepcidin (11)

, but this

drive is absent in preeclamptic patients.(4,7)

Thus we may propose that in preeclamptic

patients the ongoing inflammation had no



effect on iron status through hepcidin.(7,12)

Where as in normal pregnant women

hepcidin level was significantly decrease in

relation to low S. iron level .

Conclusion

In preeclamptic patients serum iron

concentration is increased despite of high

hepcidin concentration thus we may suggest

that pregnant women not receiving iron

therapy and even more had normal Hb , high

S.iron ,and high hepcidin may suggest an

impeding preeclampsia , close follow up is

recommended , thus for these pregnant

women iron status should be assessed before

giving iron supplement.



Reference:

1. Maynard, S.E. and S.A. Karumanchi,

Angiogenic factors and preeclampsia. Semin

Nephrol, 2011. 31(1): p. 33-46.

2. Malek-mellouli, M., et al., [Iron status in

pregnant women and its changes during

preeclampsia]. Tunis Med, 2013. 91(10): p.

577-82.

3. Zafar, T. and Z. Iqbal., Iron status in

preeclampsia. Professional Medical Journal,

2008. 15(1): p. 74-80.

4. Toldi, G., et al., Hepcidin concentrations

and iron homeostasis in preeclampsia. Clin

Chem Lab Med, 2010. 48(10): p. 1423-6.

5. Collins, J.F., M. Wessling-Resnick, and

M.D. Knutson, Hepcidin regulation of iron

transport. J Nutr, 2008. 138(11): p. 2284-8.

6. Camaschella, C. and L. Silvestri,

Molecular mechanisms regulating hepcidin

revealed by hepcidin disorders.

ScientificWorldJournal, 2011. 11: p. 1357-

66.

7. Kandi, S., et al., Pre Eclampsia and Iron

Status: A Review. American Journal of

Medical and Biological Research, 2014.

2(6): p. 121-123.

8. Rayman, M.P., et al., Abnormal iron

parameters in the pregnancy syndrome

preeclampsia. Am J Obstet Gynecol, 2002.

187(2): p. 412-8.

9. Valenzuela, F.J., et al., Pathogenesis of

preeclampsia: the genetic component. J

Pregnancy, 2012. 2012: p. 632732.

10. Koenig, M.D., et al., Hepcidin and iron

homeostasis during pregnancy. Nutrients,

2014. 6(8): p. 3062-83.

11. Choudhry, V.P., Hepicidin and its role in

iron metabolism. Indian J Pediatr, 2010.

77(7): p. 787-8.

12. Basher, K. and K. Deb, Alteration in

iron status in pre eclampsia. Mymensingh

Med J, 2006. 15(1): p. 22-4.

Correspondence to

Dr. Bassam Mohammad Hameed

Department of pathology and forensic

medicine /collage of medicine /AL-Nahrain

university


Mobile: 07700664914

Evaluation of the changes in iron homeostasis Noora A.A.M, Subuh S. Al-Mudalal,

Bassam M.H.


عند الحوامل المصابات بتسمم الحمل تركيز الهبسيدين وتوازن الحديد التغيرات فيتقييم

M.B.Ch.B محسن د.نورا عبد علي

M.B.Ch.B/M.Sc/F.I.C.M.S.(Haematology) ا.د.صبح سالم عبد اللطيف المدلل

Msc. Pathology (haematology), Ph.D. Pathology (haematology) م.د.بسام محمد حميد

ملخصال

يتم زيادة بالزما الحديد في تسمم الحمل بالمقارنة مع النساء الحوامل الطبيعيين و العالقة بين توازن الحديد الخلفية.:

وااللتهاب هي هيبسيدين. هيبسيدين هو بروتين تفاعلي حاد المرحلة الذي له دور كبير في توازن الحديد.

ة ، مصل الفيريتين وهيبسيدين المصل في النساء مستويات حديد المصل، ومجموع الحديد ملزم قدر تقييم األهداف:

الحوامل المصابات بنسمم الحمل فيما يتعلق بالنساء الحوامل االصحاء، الذين لم يكونوا على أي نظام عالجي للحديد.

يعانين من اللواتيأجريت هذه الدراسة على عشرين من النساء الحوامل في الثلث الثالث من الحمل المواد والطرق:

تسمم الحمل و لم يعطين الحديد و لم يتم نقل الدم لهم في األشهر الثالثة األخيرة، هوالء الحوامل حضرن الى قسم

. وإلى جانب هؤالء المرضى 3102أغسطس عام -أمراض النساء والتوليد في مستشفى االمامين الكاظمين بين مايو

ي نفس العمر والعمر الحملي وأدرجت كمجموعة مراقبة عشرين امرأة حامل غيرمصابات بتسمم الحمل الذين كانوا ف

مطابقة. وعالوة على ذلك تم استبعاد أي حامل مع عدوى نشطة، او لديها أمراض مزمنة، اوفقدان الدم المزمن أو

مل من عينة من الدم الوريدي من كل مريض ومراقبة واختبار لقياس 5الحمل التوأم. تم الحصول على ما مجموعه

، الفيريتين وهيبسيدين TIBCبواسطة جهاز آلي في حين تم قياس مصل الحديد MCHC، وPCVغلوبين، الهيمو

.ELISAبواسطة تقنية

، حديد المصل، الفيريتين في المصل وهيبسيدين PCV ،MCHCمستوى الهيموجلوبين، كان متوسط النتائج:

( كان. 1.15من > Pالمجموعة الضابطة )قيمة أعلى من تلك التي تسمم الحملالمصل في المرضى الذين يعانون

= rهناك عالقة غير ذات داللة إحصائية بين الحديد في الدم وهيبسيدين في المرضى الذين يعانون تسمم الحمل )

1.320 ،P = 0.32 في حين كان هناك ارتباط إيجابي قوي كبير بين الحديد في الدم وهيبسيدين في السيطرة على )

(.r =1.820 ،P = 0.003المجموعة )

لديهم زيادة تركيز الحديد في الدم على الرغم من تركيز هيبسيدين العالية كان الحوامل مع تسمم الحمل االستنتاجات:

التي قد تشير إلى وجود مقاومة للعمل على خفض الحديد من هيبسيدين.

: تسمم الحمل، هيبسيدين. حمل؛ تنظيم الحديدألكلمات المفتاحية

Original article


The results analysis of complete blood count during the first half of

2015 at the Central Child Teaching Hospital in Baghdad.

Rasha Tariq Jawad1 , Ebtehal Ali Hussien

2

1MD. M.B.Ch.B. F.I.C.M.S/Hematopathology ,Senior Specialized Hematopathologist, Manager of the

Hematology Unit, Laboratory Department / Central Child Teaching Hospital ,Baghdad ,Iraq.

2 BMLT, Laboratory technician / Hematology Unit, Laboratory Department / Central Child Teaching

Hospital ,Baghdad ,Iraq.


Abstract:

Background: Complete blood count (CBC) is the most commonly requested test by

physicians. The central child teaching hospital is the second largest pediatric hospital in

IRAQ. We review the CBC for the first half of 2015 at that hospital.

Objectives: this study was arranged to explore the prevalence of anemia, leucocytosis,

leucopenia, thrombocytosis, thrombocytopenia, using automated CBC counter Emerald &

Ruby Abbott for outpatient and inpatients respectively.

Patients and methods: A retrospective study included a total of 19341 patient attended

central child teaching hospital (from the 1st of January -1

st of July 2015). All of them did

complete blood picture test using ruby Abbott and emerald Abbott hematology autoanalyzer,

14418 of them were inpatients and 4723 patients attended to the outpatient clinic.

Results: the results were analyzed as anemia, leucocytosis, leucopenia, thrombocytosis,

thrombocytopenia for the whole patients and for each month to the inpatients so that to

analyze the result according to season. Anemia was seen more commonly in the inpatients

(30.79%) than the outpatient (20.8%). Leucopenia was seen more in outpatients (17.15%)

than in inpatients (8.1%), while leukocytosis shown to be (10.8%) of inpatients and 2% of

outpatients. Thrombocytopenia was (8.3%) of inpatient and (3.1%) of outpatients. Regarding

the monthly incidence we found that the highest percentage of anemia in outpatients seen at

May (42%), while the lowest was seen at January (19.76%).Leucocytosis and thrombocytosis

both showed the highest percentage at January (19.76%), (15.29%) respectively.

Conclusion: anemia, leucocytosis, and thrombocytosis seen in inpatients more than

outpatients ,leucopenia seen in outpatients more than inpatients ,thrombocytosis showed near

results for in and out patients but a little higher for inpatients.

Keywords: analysis, CBC, children

The results analysis of complete blood count .Rasha Tariq Jawad , Ebtehal Ali Hussien


Introduction:

Complete blood picture is the most

commonly requested test by physician

as it is easy and highly informative for

patient’s health. It requires minimum

amount of anti-coagulated whole blood

and done on automated blood count

machine lasting few minutes to get the

results.

The primary field of interest is

whether the patient have anemia,

whether the WBC count have evidence

of infection and whether the platelets

count affect hemostasis.(1)

Pediatric

anemia refers to a hemoglobin or

hematocrit level lower than the age-

adjusted reference range for healthy

children. Physiologically, anemia is a

condition in which reduced hematocrit

or hemoglobin levels lead to

diminished oxygen-carrying capacity

that does not optimally meet the

metabolic demands of the body.

Anemia is not a specific disease entity

but is a condition caused by various

underlying pathologic processes. (2)

In

addition, conditions are emphasized in

which anemia is the only hematologic

abnormality. The combination of

anemia with leucopenia, neutropenia,

or thrombocytopenia may suggest a

more global failure of hematopoiesis,

caused by conditions such as aplastic

anemia, Fanconi anemia,

myelofibrosis, or leukemia, or may

suggest a rapid destruction or trapping

of all blood elements, such as

hypersplenism, localized coagulopathy

in a large hemangioma or

hemophagocytic lymphohistiocytosis

(HLH) or macrophage activation

syndrome (MAS) (2)

. The WHO show

that the prevalence of anemia is

(47.4%) among preschool aged

children (3)

prevalence of anemia in

Iraq in children under 5 from 1995-

2011 is 36% (4)

. Inpatients prevalence

of anemia seen more than general

population ,a study in India show that

anemia in inpatient in one tertiary

pediatric hospital was( 72.79%)(5)

,

another study in Brazil showed the

prevalence of anemia in pediatric

hospital to be (56.6% ) 10(6)

.while it is

59%,and 24% respectively in the

general population of these countries(4)

.

Leukocytosis is a common laboratory

finding encountered in hospital

practice, in the emergency department

or a medical or surgical setting.

Generally, the vast majority of cases of



leukocytosis are reactive and benign—

resulting from liberation of various

cytokines that stimulate the

development of leukocyte precursors

and release of mature cells from the

marrow, endothelium, and spleen.

Most reactive episodes of leukocytosis

are neutrophilic in nature

(“neutrophilia”) and result from a

myriad of stressors. (7)

Occasionally,

the total leukocyte count will exceed

25,000 cells/mm3, with the majority of

cells being polymorphonuclear

leukocytes. This scenario is referred to

as a leukemoid reaction, and may be

difficult to differentiate from a

leukemic picture, hence the name

“leukemoid.” As such, in patients with

leukemoid reactions, the main

differential diagnostic concerns include

chronic myelogenous leukemia, acute

myelogenous leukemia, or other

myeloproliferative disorders. A typical

leukemoid reaction, however, occurs in

the presence of an acutely stressful or

inflammatory/infectious event, such as

hemorrhage, hemolysis, febrile

episodes, sepsis, trauma, pancreatitis,

or Clostridium difficile

infection. Clostridium difficile has the

unique propensity of causing

leukemoid reactions in cases of severe

colitis, and likely results from

liberation of cytotoxins (7)

. Leukopenia

is a less commonly encountered

problem in routine practice than is

leukocytosis. n general, leukopenia

may result from decreased marrow

production of leukocytes precursors,

by peripheral destruction or

sequestration of circulating leukocytes,

or by autoimmune cellular damage or

destruction. The most common

etiology of leucopenia is decreased

marrow production due to a variety of

disorders that damage the developing

leukocyte mass in the bone marrow (7).

A low platelet count

(thrombocytopenia) may also be

detected in the complete blood count.

Thrombocytopenia which is defined as

platelet less than 150× 103

per µL. It is

often discovered incidentally on

complete blood picture test patient

with platelet count greater than 50×103

per µL rarely have symptoms. Platelet

count from 30 to 50 × 103 per µL

rarely manifest as purpura. A count

from 10 to 30× 103 per µL may cause

bleeding with minimal trauma .a

platelets count less than 5×103 per µL

may cause spontaneous bleeding This

may be due to bone marrow problems,

some medications or , immunologic or

genetic problems, advanced liver

disease, or cancers such as leukemia(8)

thrombocytosis is often associated with

http://www.antimicrobe.org/new/eSepsis.htm

http://www.antimicrobe.org/new/b113.asp

http://www.emedicinehealth.com/thrombocytopenia_low_platelet_count/article_em.htm

http://www.emedicinehealth.com/thrombocytopenia_low_platelet_count/article_em.htm

http://www.emedicinehealth.com/liver/article_em.htm

http://www.emedicinehealth.com/liver/article_em.htm

http://www.emedicinehealth.com/leukemia_quiz_iq/quiz.htm



infection or an inflammation such as

osteomyelitis or rheumatoid arthritis

,hematological causes include chronic

blood loss red cell destruction

,splenectomy and rebound following

recovery from bone marrow

suppression. Moderately increased

platelet count does not usually have

pathological implication (9)

.

The complete blood count values are

usually reported based on the number

of cells in a specific volume of blood.

Fully automated multichannel

instrument usually measure from 8 to

20 component .automated instruments

have high level of precision ,if

instrument are carefully calibrated and

their correct operation is ensured by

quality control procedure they produce

test that are generally accurate (9)

.A

complete blood count may be done as

part of a regular physical examination.

Normal values for the complete blood

count (CBC) tests depend on age, sex,

how high above sea level you live, and

the type of blood sample .each ethnic

group has its normal range and many

literatures establish their own normal

range for specific hospitals in these

towns (10,11,)

. Table (1) shows the

normal value of infant and children

which clarify the difference with age.

What Affects the Test (9)

Factitiously low WBC occasionally

occurs as consequence of WBC

agglutination ,prolonged sample

storage ,abnormally fragile

cells(leukemia)

A very high WBC count is more

common and usually result from

failure of RBC lysis .this occurs

with RBC of neonates or uremia or

the presence of abnormal

hemoglobin such as S or C Hb.

Low platelets count may be due to

giant platelets. Identified as red cells

or of EDTA-induced platelets.

Clumping

High platelet count may be due to

marked microcytosis or fragmented

red cells or fragmented WBC or due

to bacteria or fungi.

The automated MCV& Hct are

prone to certain errors. those

resulting from microclots or partial

clotting ,extreme microcytosis and

the presence of cryoglobulin or cold

agglutinins the latter cause factitious

elevation of MCV as the

agglutinated cells counted as single

cell.

Patients and Methods:

A total of 19141 patients attended and

did complete blood count (CBC) at the



Central Child Teaching Hospital in

Baghdad Iraq for the period from the

1st of January to the 1

st of July 2015.

We retrospectively analyzed CBC

results of those patients. 14418 was

hospitalized (inpatients), 4723 was

outpatients. 2 ml of venous blood was

collected in k3 EDTA tubes from the

patients, and the CBC for inpatients

made on Rupy Abbott autoanalyzer

working on principle of electrical

impedance. the outpatient CBC done

on Emerald Abbott Autoanalyzer. The

test done at the hematological units of

the outpatients laboratory and the

central laboratory. the result was

compared with the schedule of

hematological normal values for

normal infants and normal children

that is published on Dacie and Lewis

practical hematology eleventh edition

(9) The results of the first half of 2015

is analyzed according to the incidence

of anemia ,leucocytosis , leucopenia,

thrombocytosis , thrombocytopenia.

The automated counters measure the

hemoglobin concentration by

modification of the manual HiCN

method with cyanide reagent ,RBC and

other blood cells counted by aperture

impedance , blood is highly diluted in

a buffered electrolyte solution under

controlled condition the diluted blood

passed through the aperture scattering

light or change the potential between

tow electrodes (depending on the

technology used) which last as long as

the cell take to pass ,the number of

pulses indicate the count ,the height of

the pulse indicate the volume of the

cell passing through (MCV) .WBC is

determined in whole blood in which

red cells have been lysed .Platelets are

counted in whole blood using the same

technique with an upper threshold is

needed to separate platelets from red

cells and a lower threshold to separate

plate lets from debris and electronic

noise. Three part differential count

assigns cells to categories usually

designated: granulocytes (large cells),

lymphocytes (small cells), and

monocytes (mononuclear or middle

cells .three part differential counts

from a single channel instrument (1,9,12)

The normal values may differ slightly

based on the reference range and the

machine used in the laboratory and,

therefore, the results may be slightly

different from one laboratory to the

next (10,11)

. The normal reference range

is typically provided and printed with

the results of the complete blood count

for accurate interpretation. Different

laboratories may report slightly

different reference ranges.(13)



Table (1); hematological values for normal infants and normal children (Dacie

and Lewis 11th

edition) (9)

Parameter birth Day

3

Day

7

Day

14

1

month

2

month

3-6

months

1 year 2-6

years

6-12

years

RBC×1012

/L 5-7 4-6.6 4.9-6.3 3.6-6.2 3-5.4 3.1-

4.3

4.1-5.3 3.9-

5.1

4-5.2

Hb g/dl 14-22 15-21 13.5−21.5 12.5−

20.5

11.5-

16.5

9.4-14 11.1-14.1 11-

14

11.5-

15.5

Pcv(range) 45-75 45-67 42-66 31-71 33-53 28-42 30-40 30-38 34-40 35-45

MCV 100-120 92-118 88-126 86-124 92-

116

87-

103

68-84 72-84 75-78 75-95

MCH 31-37 30-36 24-30 25-29 24-30 25-33

retics×109/L 120-400 50-350 50-100 20-60 30-50 40-100 30-100

WBC×109/L 10-26 7-23 6-22 5-21 5-15 6-18 6-16 5-15 5-13

N×109 4-14 3-5 3-6 3-7 3-9 1-5 1-6 1-7 1.5-8 2-8

L×109/L 3-8 2-8 3-9 3-16 4-10 4-12 3.5−11 6-9 1-5

M×109/L 0.5-2 0.5-1 0.1-1.7 0.3-1 0.4−

1.2

0.2-1.2 0.2 -1.0

E×109/L 0.1-1 0.1-2 0.1−0.8 0.1−0.9 0.2-1 0.2 - 1.0

Platelets 100−450 210−500 160−500 170−500 200-

500

210-

650

200-550 200-

490

170-

410

The reported values are either directly

measured or calculated by the machine.

The measured values are:

WBC number

RBC number

Mean Corpuscular Volume

(MCV)

Hemoglobin (Hg)

Platelet count

Mean platelet volume(MPV)

The calculated values are:

Hematocrit (HCT)

Mean Corpuscular Hemoglobin

(MCV)


Concentration (MCHC)


(MCH)

Red Cell Distribution Width

(RDW)



Results

A total of 19141 patients included in

this study with age range from (1day-

14 years old). About third of the

inpatients (30.79 %) and only 20.8% of

the outpatients were with low

hemoglobin concentration (table

2).leucopenia seen in 8.1% of

inpatient and 17.15% of the outpatients

while leucocytosis seen in 10.8%. of

the inpatients and 2% of the

outpatients . Thrombocytopenia seen in

8.3% of the inpatients and 3.11% of

the outpatients while thrombocytosis

seen in 6.8% of the inpatients and

5.14% of the out patients (Table 2).

Table (3) showed that the highest

percentage of anemia was at May

(42,9%) and the lowest was seen at

January (19.76%). The highest

percentage of leucocytosis was seen at

January (19.76%) for inpatients, the

lowest percentage seen at June (4.1%).

Leucopenia was seen comparable for

the whole five months and a lower

percentage in June (5.1%) (Table 4).

Patients with thrombocytosis were

higher in January (15.9%) and the

lowest percentage was seen in June

(2.35%), while thrombocytopenia

showed the highest percentage in April

(12.72%) and the lowest in January

(2.68%) (Table 5).

Table (2): percentage of high Hb, WBC, platelet, and low Hb, WBC, platelet for

inpatient and outpatient.

Test IP(N=14419) % OP(N=4723) %

Low Hb. 4438 30.79% 984 20.8

Low WBC 1166 8.1% 810 17.15%

High WBC 1562 10.8% 96 2%

Low plt. 1194 8.3% 147 3.11%

High plt. 981 6.8% 243 5.14%



Table (3): the percentage of anemic patients in the inpatients for each month and

the total of the 1st half of 2015.

Month No. of patient Patients with low

Hb.

Percentage

January 2419 478 19.76%

February 2560 873 34.1%

March 2227 767 34.49%

April 2367 569 24.03%

May 2383 1023 42,93%

June 2462 728 29.56%

Total 14418 4438 30,79%

Table (4) the percentage of leucopenia& leucocytosis for each month and the

total of the 1st half of 2015.

Month No. of

patients

Patients

with low

WBC

Percentage

Patients with

high

WBC

Percentage

January 2419 212 8.76% 478 19.76%

February 2560 203 7.92% 218 8.51%

March 2227 183 8.21% 306 13,74%

April 2367 231 9.75% 269 11.36%

May 2383 212 8.9% 191 8.01%

June 2462 125 5.1% 100 4.1%

Total 14418 1166 8.1% 1562 10.8%



Table (5): the percentage of thrombocytopenia and thrombocytosis for each

month and the total of the 1st half of 2015.

Month Pat. No. Low plt. Percentage High plt. Percentage

January 2419 65 2.68% 370 15.29%

February 2560 137 5.35% 87 3.39%

March 2227 192 8.62% 261 11.71%

April 2367 301 12.72% 78 3.29%

May 2383 249 10.45% 127 5.32%

June 2462 250 10.15% 58 2.35%

Total 14418 1194 8.3% 981 6.8%

Discussion

The result of our study showed that

anemia seen in 30% of the hospitalized

patient and 20% of the outpatient was

anemic. prevalence of anemia seen

differ from one country to another 10%

of hospitalized adult patients seen to be

anemic in one study(14)

while another

study in Brazil show that 56% of

hospitalized children under the age of

5 are anemic.(6)

.in a study in India

which chose the same age as ours they

show that anemia seen in 72% of the

hospitalized patients (5)

. The studies

show that anemia worldwide seen in

43%undre 5 years(14)

, even in the

developed world 21-26%

become anemic at some point during

their childhood and adolescence(5)

.

Leucopenia in this study seen in

(8.1% ) of the inpatient and (17.15%)

of the outpatient . as we depend on the

table of Dacie and Lewis for CBC

count ,a WBC below 5×109

/L

considered leucopenia that why we as

middle eastern people should establish

our own normal range (10,12)

,even in

the west black people have a lower

normal leukocytes than white and the

books ask the physician not to do

unneeded test for those patients and to

put this in mind (7)



Leucocytosis seen in 10.8% of

hospitalized patient and 2% of

outpatients. That is seems logical as

usually hospitalized patients are more

ill than outpatients.

Thrombocytosis seen in 6.8 % of the

inpatients and 5.14% of the outpatients

.thrombocytosis in children is common

but usually without symptoms the

cause mostly infection ,trauma, surgery

,renal disease and chronic

inflammation .in a study in Taiwan

thrombocytosis seen in 7.6% of

hospitalized patient in (KMCH)

hospital which is very near to our

result (8)

.

Thrombocytopenia which is defined

as platelet less than 150× 103

per µL .

It is often discovered incidentally on

complete blood picture test patient

with platelet count greater than 50×103

per µL rarely have symptoms. Platelets

count from 30 to 50 × 103 per µL

rarely manifest as purpura. A

count from 10 to 30 may cause

bleeding with minimal trauma .A

platelets count less than 5 may cause

spontaneous bleeding in the current

study 8% of hospitalized patients seen

to be thrombocytopenic .this include

all thrombocytopenia below 150× 103

which have so many reasons. all the

literatures consider ITP as the main

cause in children the incidence 100

cases per million person annually

50% occur in children .

Psuedothrombocytopenia from EDTA

dependent agglutinin should be put in

mined (15)

.

Conclusion :

anemia, leucocytosis, and

thrombocytosis seen in inpatients

more than outpatients ,leucopenia seen

in outpatients more than inpatients

,thrombocytosis show near results for

in and out patients but a little higher

for inpatients.



References

1. Fischbach FT, Dunning MB.

Manual of Laboratory and Diagnostic

Tests, 8th ed. Philadelphia: Lippincott

Williams and Wilkins.2009

2. Susumu I , Robert J Arceci.

Pediatric Acute Anemia.

http://emedicine.medscape.com/article/954

506-overview

3. WHO. Worldwide Prevalence of

Anemia 1993-2005: Global Database

ON Anemia. Geneva :WHO;2008.

4. Stevens GA, Finucane MM, De-

Regil LM, et al. Global, regional, and

national trends in hemoglobin

concentration and prevalence of total

and severe anemia in children and

pregnant and non-pregnant women for

1995-2011: a systematic analysis of

population-representative data. The

Lancet Global Health 2013; 1(1):

e16-e25.

5. Firdose S, Siddaraju P. Anemia

among hospitalized children at a

multispecialty hospital, Bangalore.

India. Journal of family medicine and

primary care .2014 Jan-Mar;3(1):48-53

6. Eliane S. , Campos G, Emídio C.

de Albuquerque, Ilma K, Grande de

Arruda, Prevalence of anemia in under

five-year-old children in a children's

hospital in Recife, Brazil Rosemary

Ferreira dos Santos,. Rev Bras

Hematol.2011; 33(2): 100–104.

7. Mark A, Marinella MD. Infectious

disease. Leukocytosis and leucopenia.

http://www. antimicrobe.org /new

/e19.asp

8. Chen HL, Chioou SS, Sheen JM.

Thrombocytosis in children at one

medical center of southern Taiwan.

Acta Paediatrca Tiawinaca1999 ,

40(5):309-313.

9. Carol Briggs ,Barbara J.Bain. Daicie

and Lewis .Practical Hematology

.Basic Hematological Techniques

Eleventh Edition .2012

10. Buseri FI, Siaminabo IJ, Jeremiah

ZA, Reference values of hematological

indices of infants, children, and

adolescents in Port Harcourt, Nigeria,

July 2010 Volume 2010:2 Pages

65—70.

11. Alexander H, Judith K, Benjamin

M, Peter GK, Bertrand L,

Haematological and biochemical

reference intervals for infants and

children in Gabon. Tropical Medicine

and International Health, 2011. March

;volume 16 (3) pp 343–348

http://www.ncbi.nlm.nih.gov/pubmed/?term=dos%20Santos%20RF%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=dos%20Santos%20RF%5Bauth%5D

https://www.dovepress.com/pathology-and-laboratory-medicine-international-archive80-v236



12. Chris Higgins. Understanding

laboratory investigations: a guide for

nurses, midwives and healthcare

professionals’ .third edition. Part three

Hematology tests.2013.Wiley-

Blackwell publication

13. Rachoin JS, Cerceo E, Milcarek B,

Hunter K, Gerber DR Prevalence and

impact of anemia in hospitalized

patients. South Med J. 2013

Mar;106(3):202-6.

14. Rehemah. Simbauranga, Erasmus

kamugisha. Prvalence and factors

associated with severe anemia amongst

under 5 children at bugando medical

center ,mwanza, tanzania. BMC

Hematology.2015.

15. Ropert guaer. Omack WA..

Thrompocytopenia. American Family

Physicion. Medical Center Family

Medicine Residency ,Fort Bragg, north

carolina. 2012 march15; 85(6): 612-

622

Correspondence to:

Dr.Rasha Tariq Jawad

Manager of the Hematology Unit,

Laboratory Department / Central Child

Teaching Hospital, Baghdad, Iraq.



مستشفى الطفل المركزي في 5102تحليل نتائج تعداد الدم الكامل خالل النصف األول من عام

التعليمي في بغداد

2ابتهال علي حسين ، 1 رشا طارق جواد

0MD /M.B.Ch.B/ F.I.C.M.S اختصاص امراض الدم المختبري ، مدير وحدة أمراض الدم، قسم /

المختبرات / مستشفى الطفل المركزي التعليمي في بغداد، العراق.

5 BMLTوحدة أمراض الدم، قسم المختبر / المستشفى الطفل المركزي التعليمي في بغداد، ي، فني مختبر /

العراق.

الملخص:

( هو االكثر طلبا عادة كتحليل دم من قبل األطباء. المستشفى التعليمي الطفل CBCتعداد الدم الكامل )الخلفية:

في 5102للنصف األول من عام CBCالمركزي هو ثاني أكبر مستشفى لألطفال في العراق. نستعرض نتائج

هذه المستشفى.

تم ترتيب هذه الدراسة الستكشاف مدى انتشار فقر الدم، فرط الكريات البيض، نقص الكريات البيض، األهداف:

لمرضى العيادة االستشارية CBCكثرة الصفيحات، نقص الصفيحات الدموية، وذلك باستخدام المحلل اآللي

مستشفى على التوالي.والمرضى الداخلين لل

المستشفى التعليمي المركزي ورا مريض حض 04390دراسة استعادية، كان ما مجموعه المرضى والطرق:

يوليو( لكل واحد منهم تم اجراء اختبار صورة الدم باستخدام روبي ابوت المحلل التلقائي. 0يناير من 0)من

المرضى إلى العيادة الخارجية. 9753من المرضى كانوا داخلون للردهات وحضر 09901

تم تحليل النتائج على النحو التالي: فقر الدم، فرط الكريات البيض، نقص الكريات البيض، كثرة النتائج:

الصفيحات، نقص الصفيحات الدموية للمرضى كلهم وعن كل شهر للمرضى الراقدين بحيث تكون تحليل النتيجة

٪(. 1، 51٪( من العيادات الخارجية )74، 31عا في المرضى المنومين )وفقا للموسم. فقر الدم كان أكثر شيو

٪(، في 1،0٪( من المرضى المنومين في )02، 07واعتبر نقص الكريات البيض أكثر في العيادات الخارجية )

٪ من العيادات الخارجية. 5٪( من المرضى المنومين و01.1حين أن زيادة عدد الكريات البيضاء أظهرت أن )

٪( من مرضى العيادات الخارجية. وفيما يتعلق 3.0٪( من المرضى الداخليين و )1.3نقص الصفيحات )كان

٪(، في حين 95اإلصابة الشهرية وجدنا أن أعلى نسبة فقر الدم لدى مرضى العيادات الخارجية شهدت في مايو )

لصفيحات سواء أظهر أعلى ٪(. فرط الكريات البيض وا04.71كان ينظر إلى أدنى مستوى في كانون الثاني )

٪( على التوالي.02.54٪(، ) 04.71نسبة في كانون الثاني )

الخالصة: فقر الدم، فرط الكريات البيض، وكثرة الصفيحات شوهدت اكثر في المرضى المنومين من العيادات

ثرة الصفيحات الخارجية، نقص الكريات البيض شوهدت اكثر في العيادات الخارجية من المرضى المنومين، ك

نتائج للمجموعتين مع زبادة طفيفة للداخلين .كانت متقاربة ال;

، األطفالCBCتحليل، كلمات البحث:

Original article


Distribution of red cell antigens according to ABO, Rh and other rare blood

group systems in Kurdish ethnicity

Hisham A. Getta¹, Shaema S.Amin², Najmaddin Khoshnaw ³* &

4, Belal A. Muhammad

5 & 6

¹Department of Hematopathology, faculty of sciences, school of medicine, university of Sulaimani, Kurdistan

region, Iraq

²Department of Hematopatholgy, Internal Teaching Hospital, Sulaymaniyah, Kurdistan Region, Iraq

³*Department of Hematology, Hiwa Hospital, Sulaymaniyah, Kurdistan Region, Iraq

4 Kurdistan Board of Medical Specialties, Clinical Hematology/Trainee, Ministry of higher education and scientific

research, Erbil, Kurdistan Region, Iraq

5Department of Medical Laboratory Techniques, Technical Institute of Halabja, Sulaimani Polytechnic University,

Sulaymaniyah, Kurdistan region, Iraq

6Department of Medical Laboratory Sciences, College of Science, Komar University of Science and Technology,

Sulaymaniyah, Kurdistan region, Iraq


Distribution of red cell antigens according to ABO Hisham A.G, Shaema S.A, Najmaddin Kh.,Belal A.M


Abstract:

Background: Among more than 30 blood group systems, nine of them namely ABO, Rh, Kell, Kidd,

Duffy, MNS, P, Lewis and Lutheran are considered to be clinically significant. The distributions of these

blood groups are different between populations across the world. Studies about the frequency of blood

groups in Kurdish ethnicity are very limited in the literature.

Objectives: to explore the distribution of red cell antigens and phenotypes of various blood groups

among Kurdish population using different systems.

Materials and Methods: five thousand blood donors attending the central blood bank of Sulaymaniyah

province were randomly selected and tested for ABO and Rh antigens (D, C, c, E, & e) by using tube

method. 500 donors were randomly selected and further analyzed using other blood group systems.

Results: In the ABO system, the most common phenotype was O (37%), followed by A (32.6%), B

(22.8%) and AB (7.6%). Among the Rh blood group antigens, e was the most common (95.2%) followed

by D (91.3%), C (74.8%), c (69.4%), and E (30.6%) with DCe/DCe(R1R1) and dce/dce(rr) being the

most common phenotypes among Rh-D+ve

and Rh-D-ve

groups, respectively. The most common

phenotypes for other blood systems were as follow; Kell(K-k+,94%), Kidd(jk a+b+,44.5%), Duffy(fy

a+b+,45%), Lutheran(Lu a-b+,92%), Lewis(Le a-b+,54.5%), P(P1,76%), MNS(M+N+S-s+,40%)

Conclusion: the various red cell antigens recorded by different blood grouping systems in this study was

intermediate between the European and Asian countries with some specificity to the Kurds population

reflecting the distinct geographical area and preserved ethnic background of the Kurds in the region.

Keywords: ABO, Rhesus (Rh), red cell antigen, Kurdish ethnicity.



Introduction:

The ABO and Rhesus (Rh) blood group

systems are among the most clinically

important discoveries of the last century in

the field of hematology. The ABO blood

group system was first discovered by

Landsteiner in 1901(1)

. Later a joint work of

the same author with Wiener resulted in the

discovery of Rhesus (Rh) blood grouping

system in 1940 (2)

. In these systems, the

determination of blood groups is based on

certain inherited antigenic substances on the

surface of red blood cells (RBCs) (3)

. These

antigens have found to play crucial roles in

several clinical areas such as in transfusion

medicine, organ transplantation,

autoimmune hemolytic anemia (AIHA),

fetomaternal blood group incompatibility,

paternity identification, and forensic

medicine (4-6)

According to the International Society of

Blood Transfusion (ISBT), there are now

more than 270 antigens distributed over 30

distinct blood group systems [1, 7]. Nine of

these systems namely ABO, Rh, Kell, Kidd,

Duffy, MNS, P, Lewis and Lutheran are

considered to be clinically important.

Among them, the ABO and Rh systems are

the most important systems during blood

transfusion due to the fact that their antigens

are more immunogenic and active at body

temperature (8)

The ABO blood group antigens are encoded

by one genetic locus, the ABO locus, which

has three alternative (allelic) forms A, B,

and O, located on the long arm of

chromosome 9 (9)

. The result of this genetic

pattern is the four well defined blood groups

(A, B, AB, and O). The Rh system is the

most polymorphic and the most clinically

significant blood group system beside the

ABO system. Currently it is composed of 50

antigens associated to genes located on

chromosome 1 including RhD, RhC, RhE,

Rhc and Rhe which represents the most

important Rh antigens (10,11)

. However,

studies have shown that the frequency of

allelic distribution of the ABO/Rh blood

group varies among different geographical

areas and also between populations with

different ethnic background across the

world. This is probably due to the genetic

polymorphism nature of the ABO/Rh genes.

Clinically speaking, blood transfusion is a

lifesaving process for some patients, yet it is

not free from transfusion-related risks such

as transfusion transmissible diseases (TTD)

and alloimmunization (12)

.It is crucial to

determine the phenotype of clinically



significant blood group antigens on the

donor RBCs especially in situations when

alloimmunization is particularly undesirable.

Examples of this include young females,

pregnant women, and patients who are

expected to require repeated blood

transfusion in their life such as in patients

that have thalassemia, sickle cell anemia,

cancer, dialysis, etc.

Racial differences in blood group antigen

distribution are common between different

populations across the world. Available data

in the literature have relatively clarified the

genotype/phenotype variation of blood

group systems in the European, American

and some Asian countries. However, such

information in developing countries is still

limited. Kurdish population are normal

inhabitants of the northern part of Iraq

(called Kurdistan or Kurdistan region of

Iraq) where only ABO and Rh (D) status of

blood donors and recipients are taken into

account for compatibility testing. A part of

two recent regional studies about the

ABO/Rh blood group systems (13,14)

information is very limited regarding the

extended population blood groups especially

using systems other than the ABO/Rh blood

groups. In the present study we, for the first

time, determined the extended red cell

antigens and phenotype frequencies of

various clinically significant blood groups

amongst regular healthy Kurdish voluntary

blood donors in Sulaymaniyah province.

This study provides valuable information

about the normal distribution of different

blood groups among the Kurdish population

and it can be used to establish a foundation

of donor database for different RBC

antigens.



Materials and methods:

Study Design:

This work is a cross-sectional study carried

out from 1st of June to 30

th of September

2014 in the Central Blood Bank of

Sulaymaniyah province/Iraq. The study was

conducted after taking approval from blood

bank and informed consent from blood

donors for their participation in the present

study.

Donor Selection:

A total of 5000 healthy regular voluntary

blood donors (aged between 18-60 years)

were included in this study. All the donors

were subjected to red cell antigen typing

using ABO/Rh (D) blood group antigen

systems. Out of them, 500 donors were

randomly selected for further antigen typing

using other Rh blood group antigens

including C, c, E, and e. In addition, 400

donors were also randomly selected for

extended antigen typing using other blood

group systems including Kell (k-cellano),

Kidd (Jka, Jkb), Duffy (Fya, Fyb), MNSs

(M, N, S, s), Lewis (Lea, Leb), Lutheran

(Lua, Lub) and P (P1).

Sample collection and methods:

Blood samples were collected in ethylene

diamine tetra acetic acid (EDTA) tubes and

analyzed freshly for ABO/Rh antigen

detection using acryl amide gel technique

following the manufacturer’s instruction.

This system uses ID card “DiaClon ABD-

Confirmation for Patients” which contains

monoclonal anti-A, B and D within the gel

matrix (Bio-Rad Laboratories, DiaMed

Switzerland). A small portion (3-5%) of the

RBCs suspended in isotonic saline solution

was further analyzed by Direct Antiglobulin

Test (DAT) according to standard protocols.

The DAT negative samples were further

typed for extended antigen profiling through

antigen-antibody agglutination method using

kits supplied by Rapid Labs Limited,

England following the manufacturer’s

guidelines. The ABO and Rh (C, c, E & e)

antigen detection were performed by

indirect Antiglobulin Technique (IAT),

using monoclonal IgG antisera [15]. The

tests are interpreted depending on the

finding of the agglutination which are

graded as 1+ to 4+ positive, or negative

which indicating the absence of the of

corresponding antigen.



Results:

The results of the ABO system showed that

the most common blood group was O

(37.0%), followed by A (32.6%), B (22.8%)

and AB (7.6%). The results also

demonstrated that the majority of donors

were positive for Rh (D) antigen (91.26%)

while minority (8.74%) were negative based

on the Rh blood group system (Table 1).

The frequency of the ABO phenotypes

associated to the Rh (D) phenotype was as

follow: for the Rh (D) positive individuals;

the most common blood group was O+ve

(33.2%), followed by A+ve

(29.8%), B+ve

(21.1%), and AB+ve

(7.1%). While for the

Rh (D) negative individuals the ratio was

3.8%, 2.7%, 1.7%, and 0.5% for O-ve

, A-ve

,

B-ve

, and AB-ve

groups respectively (Table

1). Furthermore, our results also

demonstrated that the e antigen had the

highest frequency (95.2%), followed by D,

C, c & E antigens (91.26%, 74.8%, 69.4% &

30.6%, respectively) as shown in

(Table 1 & 3).

Our phenotypic studies suggest that eight

probable phenotypes are possibly found in

our population. Among them, the DCe/DCe

(R1R

1), and dce/dce (rr) were the most

common phenotypes among Rh (D) positive

and Rh (D) negative groups, respectively

(Table 3).

The frequency of red cell antigens of Kell,

Kidd, Duffy, MNS, Lutheran, Lewis and P

blood group systems are shown in

(supplementary Table 2) and the phenotype

frequencies of these systems are presented in

(supplementary Table 3). According to the

Kell blood group system K-k+ was the most

common phenotype (94.2%) in our donors

and no K+k- & K-k- phenotypes were

observed in any donors. In the Kidd system,

Jk (a+b+) was predominant (44.5%) and no

Jk (a-b-) was observed. Jka and Jkb antigens

were determined in (77%) and (67.5%) of

donors, respectively. The Duffy system

showed Fy (a+b) as the most common

phenotype (45%). The Duffy null or Fy (a-b-

) phenotype was observed in (4%) of donors,

while Fya and Fyb antigens were observed

in (72%) and (51.5%) of donors,

respectively. In the Lutheran blood group

system, the most common phenotype was

Lu (a-b+) (92%). Lua and Lub antigens

were observed in (4.5%) and (95.5%) of

donors, respectively, while, null phenotype

Lu (a-b-) was determined in (3.5%). The

predominant phenotype of the Lewis system

was Le (a-b+) (54.5%). Lea and Leb



antigens were observed in (43.5%) and

(64.5%) of donors, respectively.

Approximately, (76%) of donors were

shown to be positive for P1 antigen. Finally,

in the MNSs blood group system, (33.5%)

of donors were homozygous for M antigen

(M+N-), while only 20.5% were

homozygous for N antigen (M-N+). Blood

groups (M+N+) and (S-s+) were the most

common phenotype with the same

percentage (46%) and no S-s- phenotype

was found. Out of nine possible phenotypes,

(M+N+S-s+) was the most common

phenotype (40%), whereas (M-N+S+s-) was

the least common phenotype (0.5%)

observed in the MNS blood group system of

the current study (supplementary Table 3).

Discussion:

The knowledge of prevalence of various

blood group antigens and phenotype

frequencies in the local donor population is

important in transfusion services especially

in areas of antenatal serology, paternity

testing, and selecting compatible blood in

problem transfusions (24)

.

The ABO/Rh blood group system:

It is well established that ABO and Rh

genotypes and phenotypes vary widely

across races and geographical boundaries.

Some variations may even occur in different

areas within one small country (16)

. Despite

the fact that the antigens involved are stable

throughout life, the resultant polymorphism

remains important in population genetic

studies, estimating the availability of

compatible blood, evaluating the probability

of hemolytic disease in the newborn,

resolving disputes in paternity/maternity and

for forensic purposes (17)

.

In this study, we examined the RBC

antigens and phenotype frequencies of the

ABO and Rh blood groups in local donor

population of Sulaymaniyah province (The

northern part of Iraq) using different blood

group systems. All the donors were males

and belong to well-known Kurdish tribes

living in the city and surrounding area. Our

results indicated that the blood group O was

the most frequently encountered phenotype

(37.0%) followed closely by the blood group

A (32.6%), then B (22.8%), and the lowest

one was AB (7.6%). The results also

demonstrated that the majority of donors

were positive for Rh (D) antigen (91.26%)

while minorities (8.74%) were negative.

These results are in agreement with the

general trend of the ABO blood groups (O >

A > B > AB) and Rh (Rh+ve

> Rh-ve

)

recorded for the Kurdish population in



northern Iraq (Erbil and Duhok city) (13,14)

,

Arabian population lives in Iraq and some

other neighboring countries (18-21)

. However,

variation between the frequencies of the

blood groups is observed (supplementary

Table 4). This variation may due to

differences in the geographical area and

racial background. Similar results were also

observed in populations of other ethnicity

such as Caucasian (33)

, Blacks and

Europeans (34)

. On the other hand in many

Asian populations, there is an increase in the

prevalence of group B, e.g. India and

Malaysia (22,23)

. Our results were not

comparable to that reported in the

neighboring Turkey (24)

, and Syria (25)

, in

which higher prevalence of group A was

reported (supplementary Table 3). The

lowest frequent phenotype of the ABO

system linked to the Rh (D) phenotype was

AB-ve

(0.5%) which is similar to Jaff’s

observation in Erbil, a neighboring city

within the Kurdistan Region of Iraq (13)

.

Regarding the frequency of Rh antigens (D,

C, c, E and e), we found that the e antigen

has the highest frequency (95.2%), followed

by D (91.3%), C (74.8%), c (69.4%) and the

lowest frequent antigen was E (30.6%). This

observation is very close to the results of a

recent study performed in Duhok city [e

(95.6%), D (91.1%), C (75.9%), c (68.1%)

and E (25.1%)] and a part from D, the

frequency of other Rh antigens was in

agreement with observation of Mashaali in

Baghdad in which the frequency of e, C, c

and E were (94%, 77%, 67% and 32 %,

respectively) (14-19)

. To our knowledge, these

are the only two studies available for

comparison in Iraq.

Comparing to the neighboring countries of

Iraq, the results of the current study are

comparable with studies performed in

northeast of Iran (26)

and Bahrain (Jenan YT

(2012). However, apart from e antigen, the

results were markedly different from that

reported in other countries such as north

Indian (27)

(see Table 4).

A significant difference was observed in the

frequency of C, E c, and e antigens when

donors were categorized as D+ve

and D-ve

. In

D+ve

donors the distribution of C and E

antigens was (81.7% and 34.2%,

respectively) while in D-ve

donors was (17%

and 0%) (P < 0.05), suggesting that C and E

antigens are more prevalent on D+ve

red

cells. In contrast, the c and e antigens were

detected in almost all D-ve

donors (100%) as

compared to the less frequency occurrence

in D+ve

donors (65.8% and 94.6% for c and e

respectively) (P < 0.05). These results are in

accordance with a study in north Indian



population in which the frequency of C and

E antigens in D+ve

donors were massively

higher than their presence among D-ve

donors, while the prevalence of c and e was

in contrast (27)

. Moreover, these results are

further supported by our observation that

DCe/DCe (R1R

1) was the most common

phenotype (34%), followed by DCe/dce

(R1r) (29.6%) and DCe/DcE (R

1R

2), and

(18.3%) in our D+ve

donors, while the

dce/dce (rr) found to be the most common

phenotype (83%) in D-ve

donors (see Table

4). This genotype pattern shows strong

similarities to that reported in northeast Iran

and some similarities to north India and

Caucasians population. However, the pattern

was far different from what reported in

Blacks as shown in (Table 4).

Other blood group systems:

The Kell Blood Group System:

The K antigen is very immunogenic (second

to the D antigen) in stimulating antibody

production. Anti-K is an important antibody

as it is nearly always immune, IgG, and

complement-binding. It causes severe HTRs

and HDFN (28)

. Its frequency in this study

was low (5.8%), which is similar to that of

Thakral et al. study in north India (5.56%)

(27), and it occurs between the frequencies

reported by Keramati et al. in northeast of

Iran (26)

and Whites (8%, and 9%,

respectively), but higher than Blacks (2%)

(29). In contrast, frequency of k (Cellano)

antigen was detected in almost (100%) in

our donor population, which is similar to the

results reported in north Indian 100%

(Thakral et al. 2010), and black populations

100% (29)

, but differ from the results

obtained from Whites and northeast-Iran

who showed negative results for k (Cellano)

antigen by 0.2%, 2.3% respectively. This

implies that while Whites and northeast

Iranian population might occasionally

develop anti-k (Cellano), the likelihood of

finding this alloantibody in our population is

negligible (Table 5).

Regarding the distribution of Kell

phenotypes, the most common phenotype

was found to be K-k+ (94.2%), followed by

K+k+ (5.8%). None of the donor was found

to be K homozygous (K+k-) or (K-k-).

These results are similar to that reported in

north Indian population recorded by two

independent studies (27,30)

. Again our results

were found to be intermediate between

Whites/northeast of Iran from one side (26)

and the Black population from another side

(29) (Table 5).



The Kidd Blood Group System:

The frequencies of Kidd blood group system

antigens (Jka = 77%, Jk

b = 67.5%) observed

in this study was similar to those in


, and comparable to that

of Whites (Beadling & Cooling, 2007), and

north-Indian population (27)

, while there was

a remarkable difference with the Black

population (Jka = 92%, Jk

b = 49%)

(29)

(Table 5).

The most common Kidd phenotype was Jk

(a+b+) (44.5%), which is similar to those in


, and comparable to

north India (27)

and Whites (29)

(49.21 % and

49%, respectively), while much higher than

what recorded for Blacks (34%). The Jk

(a+b-) is found to be the most common

phenotype among the Blacks (57%) (29)

. No

Jk (a-b-) phenotype was detected in any

donor, which is also very rare in White and

Black people, except for Polynesians (< 1%)

(29) (Table 5).

The Duffy Blood Group System:

The frequencies of the Duffy blood group

system antigens were (Fya =70%,

Fyb=57.5%). The Fy

a antigen frequency is

very close to that reported in northeast of

Iran (73.8%) (26)

, and it is intermediate

between results reported in north Indian

population (27)

, and Whites (29)

, which are

86.75%, 66%, respectively. While the

frequency of Fyb antigen is much closer to

that reported by Thakral et al. (56.15%), and

it is in between Keramati et al. study and

Whites (49.2%, 83%, respectively).

However, again our results showed much

higher percentage of Duffy antigen than the

Blacks (Fya = 10%, Fy

b = 23%)

(29)

(Table 6).

In this system, Fy (a+b-) was the most

common phenotype in our study was

(38.5%), which is comparable to the results

reported in north-India (27)

and northeast of

Iran (26)

, which are (43.9%, 47.4%,

respectively). However, it is much higher

than Whites (17%) and Blacks (9%)

population (29)

. The most common reported

phenotype in Whites is Fy (a+b+) 49%, and

in Blacks is Fy (a-b-) (68%) [29].

Duffy antigen is postulated to be the

receptor for entry of the plasmodium vivax

on the red cells (31,32)

. This probably explains

high prevalence of Duffy null phenotype Fy

(a-b-) in the endemic area of malaria such as

among the black people (68%) (29,33,34)

. The

frequency of Duffy null phenotype Fy (a-b-)

in our study was (4%), which is very close

to that reported by Keramati et al. in

northeast of Iran (3.4%) (26)

. While it is very

rare in Whites (Beadling & Cooling, 2007)



and (0%) in north Indian population (27)

(Table 6). This higher rate of null phenotype

frequency in our donors might be related to

the existence of endemic areas of malaria in

the Kurdistan Region of Iraq in the past.

The MNSs Blood Group System:

The frequencies of the MNSs blood group

system antigens M, N, S and s in our study

were 79.5%, 66.5%, 54%, and 88.5%,

respectively. These results are similar to the

results reported in north Indian population

(75.4%, 61.5%, 56.5%, and 87.4% for M, N,

S and s respectively) (27)

. The results are also

comparable with the observation of

Keramati et al. in northeast of Iran (87%,

56.7%, 56.7%, and 84.5%, respectively) (26)

,

and Whites (78%, 72%, 55%, and 89%,

respectively), while they are different with

what recorded for Blacks, particularly for S

and s antigen frequencies (29)

(Table 6).

Regarding the phenotype frequency, M+N+

and S-s+ were the most common phenotypes

observed in the MNS blood group system in

our study which were nearly equal (46%) to

each other. These results are comparable to

that of Keramati et al. study who reported

that M+N+ and S-s+ are the most common

phenotypes (43.7% and 43.3%, respectively)

(26). The results are also comparable to that

reported in White and Black populations(29)

,

while in Thakral et al. study the most

common phenotype was M+N- and S+s+

(Table 6).

Out of nine possible phenotypes found in

our study, M+N+S-s+ (40%) was the most

common phenotype; whereas, M-N+S+s-

(0.5%) was the less common phenotype

observed in the MNS blood group system.

The frequency of M+N+S-s+ in this study,

is comparable to the results reported by

Agarwal et al. in India (28.8%) (Agarwal et

al. 2013), as well as in Europeans (22.6%)

and African-Americans (33.4%) (35,36)

, while

in Thakral study in north Indian population

the most common phenotype was

M+N+S+s+ (19.6%) (Table E).

Conclusions:

The knowledge of these antigen and

phenotype frequencies is crucial in the

clinical field. Based on this information, one

can predict the common alloantibodies that

could be happened in pregnant women and

patients receiving blood transfusions. For

instance, E antigen frequency in our donor

population was the lowest (30.6%), followed

by c antigen (69.4%) in ascending order of

frequency. Thus, it can be assumed that the

most common alloantibodies in Rh blood

group system among pregnant women and in

patients receiving blood transfusion would



be anti-E and then anti-c. Another advantage

of knowing antigen and phenotype

frequency is that it helps in selection of

antigen negative blood units for patients

with pre-formed alloantibodies. For

example, if a patient in our population has

alloantibody against C and needs two units

of blood, a minimum of 8-10 units of ABO

and Rh (D) matched blood units will need to

be tested for C antigen to find two units of

antigen negative blood (since C antigen

negative donors form about 25% of all our

donor’s population). This notion is also

applied to the less common blood group

systems included in this study.

Over all, the various red cell antigens

recorded by different blood grouping

systems in this study was intermediate

between the European and Asian countries

with some specificity to the Kurds

population reflecting the distinct

geographical area and preserved ethnic

background of the Kurds in the region.

Acknowledgment:

We have special thanks to the ministry of

health of Kurdistan region and the health

directorate of Sulaymaniyah governorate.

We also thank the director and all laboratory

staff of the central blood bank of the

Sulaymaniyah for their valuable help and

coordination in completing this research.

Authorship contributions:

HAG have designed the research and shared

in writing the article, SSA did all laboratory

work and shared in writing the article, NK

reviewed and shared in writing editing, and

designing the article. BAM reviewed the

article, redesigned the paper, and did major

editing of all parts of the writing, also

submitted the paper.

Conflict of interest:

All authors declares that there is no conflict

of interest.

Tables


Table 1: Distribution of ABO and Rhesus (Rh) blood groups both individually (upper panel) and in combination (lower panel). Upper panel, the total number of donors and the percentage of each group is given. The most common

ABO blood group was O (37.0%), followed by A (32.6%), B (22.8%) and AB (7.6%). Most of the donors were positive

for Rh (D) antigen (91.26%) and minority (8.74%) were negative. Lower panel, the number and percentage of donors for each blood group is given. The total number of donors also provided. In both Rh

+ve (left) and Rh

-ve (right) groups the most

common ABO blood group was O, followed by A, B, and AB as indicated regardless of the presence/absence of Rh

antigen. N = 5000.

Blood groups Number of donors Percentage (%)

ABO blood groups

O 1850 37

A 1630 32.6

B 1140 22.8

AB 390 7.8

Rh (D) blood groups

Rh (D) positive (Rh+ve

) 4550 91.3

Rh (D) negative (Rh-ve

) 435 8.7

ABO/Rh (D) positive (Rh+ve

) ABO/Rh (D) negative (Rh-ve

)

Blood

groups Number & (%) of donors

Blood

groups Number & (%) of donors

Total number of

donors

O+ 1662 (33.2) O

- 190 (3.8) 1852 (37.0)

A+ 1491 (29.8) A

- 137 (2.7) 1628 (32.6)

B+ 1055 (21.1) B

- 84 (1.7) 1139 (22.8)

AB+ 355 (7.1) AB

- 26 (0.5) 381 (7.6)

Total 4563 (91.3) 437 (8.7) 5000 (100%)

Tables


Table 2: Distribution of other Rh antigens (C, c, E, and e) and phenotypes. A) The number and percentage of donors for each blood-group antigen is given. The most common antigen was e (95.2) followed by C (74.8), c (69.4), and E

(30.6). B) The Rh phenotype in Rh (D) positive (Rh+ve

) donors. C) The Rh phenotype in Rh (D) negative (Rh-ve

) donors.

The most common Rh phenotype was R1R1 (DCe/DCe) (34%) in Rh+ve

donors, while rr (dce/dce) was the most common phenotype (83%) among Rh

-ve donors. N = 500.

Number of positive donors Percentage of positive donors

A) Rh Antigens

C 374 74.8

E 153 30.6

c 347 69.4

e 476 95.2

B) Phenotypes in Rh (D) positive (Rh+ve

) donors

R1R

1 (DCe/DCe) 152 34

R1r (DCe/dce) 132 29.6

R1R

2 (DCe/DcE) 82 18.3

R2r (DcE/dce) 47 10.5

R2R

2 (DcE/DcE) 24 5.4

R0 r (Dce/dce) 10 2.2

C) Phenotypes in Rh (D) negative (Rh-ve

) donors

rr (dce/dce) 44 83

rʹr (dCe/dce) 9 17

Tables


Table 3: Phenotype frequencies in systems other than ABO/Rh. Results of Kell, Kidd, Duffy, Lutheran, Lewis, P, and MNSs blood group systems are presented in percentage as indicated, N = 400.

Blood group system Phenotype Donors (%)

Kell system K-k+

K+k+

94.2

5.8

Kidd System

Jk (a+b+)

Jk (a+b-)

Jk (a-b+)

44.5

32.5

23

Duffy System

Fy (a+b-)

Fy (a+b+)

Fy (a-b+)

Fy (a-b-)

38.5

31.5

26

4

Lutheran system

Lu (a-b+)

Lu (a+b+)

Lu (a-b-)

Lu (a+b-)

92

3.5

3.5

1.0

Lewis system

Le (a-b+)

Le (a+b-)

Le(a+b+)

Le (a-b-)

54.5

33.5

10

2

P system P1 76

MNS System

M+N+

M+N-

M-N+

S-s+

S+s+

S+s-

M+N-S+s-

M+N-S+s+

M+N-S-s+

M+N+S+s-

M+N+S+s+

M+N+S-s+

M-N+S+s-

M-N+S+s+

M-N+S-s+

46

33.5

20.5

46

42.5

11.5

5.5

19

18

22.5

17.5

40

0.5

6

14.5

Tables


Table 4: Frequencies of Rh phenotypes. The observed Rh phenotype of the present study compared to the published data of other countries as indicated.

Phenotypes Present study (%) North-India1 (%)

Northeast Iran2

(%) Caucasian

3 (%) Black

3 (%)

DCe/DCe (R1R1) 30.4 35.2 25 18.5 2.0

DCe/dce (R1r) 26.4 30.7 31.8 34.9 21.0

DCe/DcE (R1R2) 16.4 8.1 16.5 13.3 4.0

DcE/dce (R2r) 9.4 5.9 9.6 11.8 18.6

DcE/DcE (R2R2) 4.8 0.7 1.7 2.3 0.2

Dce/dce (R0r) 2 2.2 4.2 2.1 45.8

dce/dce (rr) 8.8 0.3 8.3 15.1 6.8

dCe/dce (rʹr) 1.8 2.5 1.3 0.8 Rare

1: Sarkar et al. 2013 2: Keramati et al. 2011 3

: Reid & Lomas-Frances, 2004

Tables


Table 5: Phenotype frequencies of the Kell and Kidd blood group systems. Antigen frequencies (%) of Kell and Kidd

blood group systems are compared with other published results as indicated. The phenotype frequencies (%) of the Kidd

blood group system was also compared.

People groups K

ell

(K)

(%)

Cel

lan

o

(k)

(%)

K-k

+

(%)

K+

k+

(%)

K+

k-

(%)

K-k

- (%

)

References

Sulaymaniyah-Iraq 5.8 100 94.2 5.8 0.0 0.0 Present study

North-India 5.56 100 94.3 5.7 0.0 0.0 Thakral et al. 2010

North-India 97.7 96.0 4.0 0.0 0.0 Nanu &Thapliyal,

1997

Northeast-Iran 8.0 99.8 92.0 5.7 2.3 0.0 Keramati et al. 2011

Whites 9.0 100 91.0 8.8 0.2 0.0 Beadling & Cooling,

2007

Blacks 2.0

98.0 2.0 Rare 0.0 Beadling & Cooling,

2007

People groups

Jk

a (%

)

Jk

b (%

)

Jk

(a

+b

+)

(%)

Jk

(a+

b)

(%)

Jk

(a

-b+

)

(%)

Jk

(a

-b-)

(%)

References

Sulaymaniyah-Iraq 77.0 67.5 44.5 32.5 23 0.0 Present study

Northeast-Iran 79.1 65.1 44.4 34.7 20.7 0.2 Keramati et al. 2011

North-India 82.6 66.6 49.2 33.4 17.3 0.0 Thakral et al. 2010

Whites 77 74 49 28.0 23.0 Very rare Beadling & Cooling,

2007

Blacks 92 49 34.0 57 9.0 Very rare Beadling & Cooling,

2007

Tables


Table 6. Antigen and phenotype frequencies of Duffy and MNSs blood group systems. Antigen and

phenotype frequencies (%) of Duffy and MNSs blood group systems in this study compared with other

published results as indicated.

People groups Fy

a

(%)

Fyb

(%)

Fy (a+b-)

(%)

Fy (a+b+)

(%)

Fy (a-b+)

(%)

Fy (a-b-)

(%) References

Sulaymaniyah-

Iraq 70 57.5 38.5 31.5 26 4.0 Present study

Northeast-Iran 73.8 49.2 47.4 26.4 22.8 3.4 Keramai et al.

2011

North-India 86.7 56.1 43.8 42.9 13.3 0.0 Thakral et al.

2010

Whites 66 83 17.0 49.0 34.0 Very rare Beadling &

Cooling, 2007

Blacks 10 23 9.0 1.0 22.0 68.0 Beadling & Cooling, 2007

People groups M

(%)

N

(%)

S

(%) M+N+

%

M+N

- %

M-N+

%

S-s+

%

S+s+

%

S+s-

%

s

(%) References

Sulaymaniyah-

Iraq 79.5 66.5 54 46 33.5 20.5 46 42.5 11.5 88.5

Present

study

North-India 75.4 61.5 56.5 43.7 43.3 13 43.3 41.2 15.5 87.4 Thakral et

al. 2010

Northeast-Iran 87 56.7 56.7 36.9 38.5 24.6 43.5 43.8 12.6 84.5 Keramati et

al. 2011

Whites 78 72 55 50 28 22 45 44 11 89

Beadling &

Cooling,

2007

Blacks 74 75 31 44 26 30 69 28 3 93

Beadling &

Cooling,

2007

Supplementary Data


Supplementary data:

Supplementary Table 2: Antigen frequencies of the Rh (D) blood groups. Results of the Rh system of the present study compared to other parts of Iraq, neighboring countries and some other

populations as indicated.

People groups Rh (D) +ve

%

Rh (D) –

ve % References

Sulaymaniyah-Iraq 91.3 8.7 Present study

Erbil-Iraq 91.7 8.3 Jaff, 2010

Babylon-Iraq 90.1 9.9 Salih, 2009

Saudi Arabia 91.2 8.9 Al-Himaidi & Umar, 2002

Bahrain 91.1 8.9 Jenan, 2012

Arians (Pakistan) 91.7 8.3 Ali et al. 2005

Kuwait 92.5 7.5 Al-Bustan et al. 2002

Iran 90.2 9.8 Keramati et al. 2011

India 93.4 6.6 Thakral et al. 2010

Baghdad-Iraq 80 20 Mashaali, 2014

Blacks 92 8 Beadling & Cooling, 2007;

Barclay, 2001

Whites 85 15 Beadling & Cooling, 2007;

Barclay, 2001

Asians 99 1 Reid & Lomas-Frances, 1997

Supplementary Data


Supplementary Table 3: Frequency of red cell antigens in systems other than ABO/Rh. Results of Kell, Kidd, Duffy, MNSs, Lutheran, Lewis and P blood group systems are presented both in absolute numbers and percentage as indicated, N = 400.

Antigens Number Percentage (%)

Kell

K

k

29/500

400/400

5.8

100

Kidd

Jka

Jkb

308/400

270/400

77

67.5

Duffy

Fya

Fyb

280/400

230/400

70

57.5

MNSs

M

N

S

s

318/400

266/400

216/400

354/400

79.5

66.5

54

88.5

Lutheran

Lua

Lub

18/400

382/400

4.5

95.5

Lewis

Lea

Leb

174/400

258/400

43.5

64.5

P

P1

304/400

76

Supplementary Data


Supplementary Table 3: Antigen frequencies of the ABO blood groups. Results of the ABO

system of the present study compared to other parts of Iraq, neighbour countries, and some

other populations as indicated.

People groups O% A% B% AB% References

Sulaymaniyah-Iraq 37 32 23 8 present study

Erbil-Iraq 37 32 24 7 Jaff, 2010

Babylon-Iraq 36 28 28 8 Salih, 2009

Baghdad-Iraq 39 26 24 11 Mashaali, 2014

Iran 35 33 23 9 Boskabady et al. 2005

Iran 34 30 28 8 Keramati et al. 2011

Kuwait 44 27 24 5 Al-Bustan et al. 2002

Saudi Arabia 51 26 19 4 Sarhan et al. 2009

Jordan 37 38 18 7 Hanania et al. 2007

Caucasian 47 41 9 3 Guyton & Hal, 2005

African blacks (e.g. Nigeria) 53 24 20 3 Enosolease & Bazuaye, 2008

European 43 40 12 5 Mollison et al. 1997

Asians (e.g. India) 39 23 33 5 Ali et al. 2005

Asian (e.g. North India) 32 22 37 9 Agrawal et al. 2013

Supplementary Data


Supplementary Table 4: Antigen frequencies of extended Rh blood groups (e, C, c, E). Results of the extended Rh blood group system of this study compared to the published data of

other parts of Iraq and some other countries as indicated.

People groups e % C% c % E% References

Sulaymaniyah-Iraq 95.2 74.8 69.4 30.6 Present study

Baghdad-Iraq 94 77 67 32 Mashaali, 2014

Northeast-Iran 97.9 75.9 73.9 29.5 Keramati et al. 2011

Bahrain 97.3 73.2 71 21 Jenan, 2012

North-India 98.1 90.2 49.5 18.9 Thakral et al. 2010

Supplementary Table 5: phenotype frequencies of MNSs blood group system compared to

other published data as indicated.

Phenotype Present study

(%) •India (%) *European (%)

*African

American (%)

M+N+S-s+ 40 28.7 22.6 33.4

M+N+S+s+ 17.5 20.9 22.4 13

M+N+S+s- 22.5 5.12 3.9 2.2

M+N-S-s+ 18 13.8 10.1 15.5

M+N-S+s+ 19 15 14 7

M+N-S+s- 5.5 7.1 5.7 2.1

M-N+S-s+ 14.5 5.1 15.6 19.2

M-N+S+s+ 6 3.1 5.4 4.5

M-N+S+s- 0.5 1.2 0.3 1.6

•(Agarwal et al. 2013) * Lal et al. 2000; Cleghorn, 1960.


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Corresponding Author

Dr. Najmaddin Khoshnaw

Clinical Hematologist,

head of Hematology Department

Hiwa Hospital -Sulaymaniyah -IRAQ

Cell phone: +9647701554184,

P.o.Box:54

email: [email protected]

mailto:[email protected]


وغيرها من أنظمة فصائل الدم النادرة لدى قومية ABO ،Rhتوزيع مستضدات الخاليا الحمراء وفقا ل

العراقيين الكورد

6و5، بالل محمد 4و 3، نجم الدين خوشناو 2،شيماء امين 1جيتا هشام

قسم امراض الدم، كلية العلوم، كلية الطب، جامعة السليمانية، إقليم كردستان، العراق 1

قسم امراض الدم ، مستشفى الداخلية التعليمي , السليمانية , إقليم كردستان، العراق 2

لسليمانية، كردستان، العراققسم أمراض الدم، مستشفى هيوا، ا 3

مجلس كردستان للتخصصات الطبية والسريرية أمراض الدم / متدرب وزارة التعليم العالي والبحث العلمي، أربيل، إقليم 4

كردستان، العراق

كردستان، العراققسم تقنيات المختبرات الطبية، المعهد الفني حلبجة، جامعة السليمانية للفنون التطبيقية، السليمانية، إقليم 5

قسم علوم المختبرات الطبية، كلية العلوم، جامعة كومار للعلوم والتكنولوجيا، السليمانية، إقليم كردستان، العراق 6

ملخص:ال

ABO ،Rh ،Kell ،Kidd ،Duffy ،MNS ،P،Lewisنظام لفصائل الدم، تسعة منهم وهما 03من بين أكثر من الخلفية:

تعتبر هامة سريريا. توزيعات هذه فصائل الدم تختلف بين السكان في جميع أنحاء العالم. دراسات حول وتيرة Lutheranو

.مراجع الطبيةفصائل الدم في العرق الكردي محدودة للغاية في ال

د باستخدام أنظمة الستكشاف توزيع مستضدات الخاليا الحمراء والظواهر من فصائل الدم المختلفة بين السكان األكرااألهداف:

مختلفة.

تم اختيار خمسة آالف من المتبرعين بالدم الذين حضروا بنك الدم المركزي في محافظة السليمانية عشوائيا المواد والطرق :

عينة من 533، وه( باستخدام طريقة األنبوب. وقد تم اختيار D ،C ،C ،Eومولدات المضادات ) Rhو ABOواختبارها ل

عشوائي وكذلك تحليلها باستخدام أنظمة فصيلة الدم األخرى. المتبرعين بشكل

AB( و B(٪22.8، و ٪A (02.6)٪، تليها 37)) O، كان النمط الظاهري األكثر شيوعاهو ABOفي نظام النتائج:

٪D (91.3٪)، (74.8٪) C ،c(62.4 ،) ٪(، يليه25.2األكثر شيوعا )e ، كان Rh. بين المستضدات فصيلة الدم (7.6٪)

على االيجابي والسلبي RhD( هي الظواهر األكثر شيوعا بين rr) dce / dce( و R1R1) DCe / DCe( مع E(٪30.6و

التوالي. وكانت الظواهر األكثر شيوعا ألنظمة الدم األخرى على النحو التالي:

Kell (K-k+,94%), Kidd (jk a+b+,44.5%), Duffy (fy a+b+,45%), Lutheran (Lu a-b+,92%), Lewis

(Le a-b+,54.5%), P (P1,76%), MNS (M+N+S-s+,40%)

الخالصة: مختلف مستضدات الخاليا الحمراء التي سجلتها أنظمة فصيلة الدم مختلفة في هذه الدراسة كان وسطا بين الدول

.لخلفية العرقية المحفاظ عليهااألوروبية واآلسيوية مع بعض الخصوصية للسكان األكراد يعكس منطقة جغرافية متميزة وا

,Rh، مستضد الخاليا الحمراء ABO ,األكراد كلمات البحث :

Original article


Review of Congenital Factor XIII Deficiency in Single Iraqi Teaching

Hospital

Lubna Foad Hussain1 , Obeida Amir Abid

2

1 C.A.B.P.(ped) / Specialist Pediatrician,/ Children Welfare Teaching Hospital, Medical City,

2 F.I.B.M.S(Ped) / Specialist Pediatrician, /Children Welfare Teaching Hospital, Medical City,

Received 10/1/2016 revised 24/1/2016 accepted 6/2/2016

Abstract

Background: Factor XIII deficiency is one of the rarest bleeding disorders with an estimated

prevalence of 1in 3 million populations worldwide. The main clinical manifestations of the

disease are delayed wound healing, recurrent miscarriage, intracranial bleeding, and

prolonged umbilical cord bleeding.

Objectives: The aim of this study was to assess the diagnosis and treatment of factor XIII

deficiency in Children Welfare teaching hospital in Baghdad.

Patients and Methods: This retrospective study was performed on thirty three patients with

the severe factor XIII deficiency. The diagnosis of the disease was done by a wide spectrum

of characteristics which is part of inclusion criteria and including family history, clinical

manifestations, laboratory tests, clot solubility in 5 M urea or monochloroacetic acid

environments.

Results: the common manifestations of the disease at time of diagnosis were bleeding after

trauma (42.4%), umbilical cord bleeding (21.2%)and less frequently gum bleeding and

ecchymosis.

Conclusions: factor XIII deficiency is a rare disease in Iraq, most patients are diagnosed at

the age of 1-10 years, family history was positive in more than half of the patients.

Keywords: congenital Factor XIII deficiency, clot solubility test.

Review of Congenital Factor XIII Deficiency Lubna Foad Hussain, Obeida Amir Abid


Introduction

Congenital factor XIII (FXIII)

deficiency is a rare autosomal

recessive disease usually associated

with a severe bleeding diathesis (1)

. The

mortality and morbidity are primarily

related to bleeding; intracranial

hemorrhage can be life threatening.

Because the clinical bleeding is severe

in most patients with hereditary factor

XIII deficiency, the diagnosis is made

at an early age, often during infancy.

Bleeding from the stump of the

umbilical cord within the first days to

weeks of life is a characteristic sign

that occurs in 80% of affected

individuals (2,3)

. Additional signs of

bleeding include the following: CNS

hemorrhage is frequent (25-30%) and

may occur spontaneously or after

minor trauma (4-7)

. Soft tissue bleeding

and bruising are very common (4,5)

.

Hemarthroses occur in 20% of cases

.Bleeding that is delayed (ie:12-36h)

after trauma or surgery is diagnostic of

factor XIII deficiency.(8)

FXIII deficiency can be initially

diagnosed by observing bleeding

episode with normal routine clotting

tests including prothrombin time (PT),

activated partial thromboplastin time

(aPTT), fibrinogen level,

platelets count and bleeding time (BT)

(9).Diagnosis of the disease based on

solubility of blood clot in solution of 5

M urea or 2% acetic acid (or1%

monochloroacetic acid). These tests are

qualitative tests and show positive

result if the activity of FXIII in plasma

of the patients is absent or close to

zero. If the results of test become

positive subsequent quantitative

analysis of FXIII activity is needed (10)

.

Replacement therapy in this deficiency

can be administered through fresh

frozen plasma (preferably virus-

inactivated), cryoprecipitate and

pasteurized FXIII concentrates. The

first FXIII from human source that

used in replacement therapy was

produced from placenta but later this

product replaced by plasma extracted

FXIII concentrates (5,11-13)

.The aim of

the study is to assess the diagnosis and

treatment of factor XIII deficiency in

Children Welfare Teaching Hospital,

Medical City, Baghdad.

Patients and methods

It is a retrospective study. The study

participants included all patients age

group who were attend the hemophilia

center in Children Welfare Teaching

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3915454/#B6



Hospital with the diagnosis of FXIII

deficiency, from January 2 000 till

April 2015. All patients were identified

by using coded discharge records with

the diagnosis of factor XIII deficiency.

Data collected included: Gender, age at

presentation, presenting complaint,

family history, type of treatment (fresh

frozen plasma, cryoprecipitate),

hepatitis B ,C in patients.

The following investigations were

reviewed for all patients who presented

to the outpatient clinic to establish the

diagnosis and included complete blood

picture and coagulation screen PT

,APTT and bleeding time which

reported to be normal.

Diagnosis was confirmed by

performing clot solubility tests in 5

mol/L urea which was done to all our

patients, a qualitative test for diagnosis

of FXIII deficiency; those found

positive were labelled as factor XIII

deficient.Results were expressed as

frequencies and percentages and

presented in tables and figures using

Microsoft Office Excel 2013.

Results

Thirty three patients were diagnosed to

have factor XIII deficiency. 20(58%)

were males shown in Figure (1). The

diagnosis of FXIII deficiency was

established at 3 age groups; first group

less than 1 year, second group 1-10

years and third group more than 10

years; most patients diagnosed at the

age of 1-0 years as shown in Figure (2)

Post traumatic bruises were the major

presenting complaint in 14 patients

(42.4%) , followed by prolonged

bleeding from umbilical stump in 7

patients(21.2%). One patient had

intracranial hemorrhage diagnosed by

neuro-imagining study .Table1

Family history was positive in 20

patients (58%).

The products used in the treatment of

these patients only when presented

with bleeding included fresh frozen

plasma, cryoprecipitate. Testing for

transmission of viral infections was

also done in all our patients and only 2

from 6 patients received plasma were

found with hepatitis C positive after

repeated transfusions of fresh frozen

plasma.



The other 27 patients were treated by

cryoprecipitate with no hepatitis C

infection .Hepatitis B was found

negative in all patients .Figure3

Figure1:The gender of 33 patients

Table1:The presenting complaints at the time of the diagnosis of 33 patients

58% 42%

Gender of the patients

Male

Female

Bleeding type Frequency(no.) Percent(%)

Post traumic bruises 14 42.4

Umbilical cord bleeding 7 21.2

Gum bleeding 5 15.2

After circumcision 2 10% of males

Muscle hematoma 2 6.1

Ecchymosis 1 3.0

Epistaxis 1 3.0

Intracranial hemorrhage 1 3.0

Total 33



Figure2:The age of diagnosis in 33 patients

Figure3:The results of Hepatitis C screening

11

17

5

LESS 1 YEARS 1-10 YEARS MORE TEN YEARS

94%

6%

negative

positive



Discussion

This disorder is so rare that to date

only 33 of 778 patients with congenital

bleeding disorders, have been

identified in our hemophilia center

with factor XIII deficiency. So the

incidence of this disorder is 0.042%. In

Saudia Arabia it is also a rare disease

(14).While in Pakistan it was the fifth

most common factor deficiency

detected in one study conducted there

(15). In Iran, a Middle Eastern country

with a high rate of consanguineous

marriages, there are approximately 473

patients afflicted with FXIII

deficiency. An approximately 12-fold

higher prevalence of FXIII deficiency

is estimated in Iran in comparison with

overall worldwide frequency. (14)

.

In this study, bleeding after trauma is

the most common, while in Iranian and

Pakistani study showing subcutaneous

bleeding (57%), followed by umbilical

cord bleeding (56%), muscle

hematoma (49%) and intracranial

hemorrhage (34%) to be the major

clinical presentation (14,15)

. In Saudis

the presenting symptoms included

ecchymosis in 12 patients (71%),

bleeding after circumcision in 6 male

patients (55%), umbilical stump

bleeding in 7 (41%), and intracranial

bleeding in 3 patients (18%) (16)

. Most

of the patients presented early in their

life, which is supported by this study

which is the same in Iranian, Saudis

and Pakistani study.(14-16)

In this study family history is positive

in 94% of patients due to higher

incidence of interfamilial marriages. In

Iranian and Saudi study (59%) had a

family history of FXIII deficiency

(14,16).In Pakisatan positive family

history was present in 42.4% of

patients.(15)

In Iraq FXIII concentrate is not

available till now, in Pakistan all

patients treated with fresh frozen

plasma and cryoprecipitate (15)

and the

same applied for Iran until 2009 when

FXIII concentrate became available for

patient management. (14)

The challenges that we faced in the

diagnosis was the unavailability of

quantitative assay and in the treatment

was the unavailability of plasma

derived FXIII product.



Conclusion

Factor XIII deficiency is a rare disease

in Iraq with low incidence, most

patients diagnosed at the age of 1-10

years, family history was positive in

more than half of the patients.Thus in

children with history of post

traumatic bruising, prolonged

umbilical bleeding along with any

family history of easy bruising and

consanguinity in parents, it is essential

to rule out FXIII deficiency especially

in those with normal

Author contributions:

Dr.Lubna Foad Hussain: Acquisition

of data analysis , interpretation of data

, critical revision.

Dr. Obeida Amir Abid : Study

conception, study design, drafting of

manuscript, and Coagulation studies.

References

1. Bolton-Maggs PH. The rare

inherited coagulation disorders. Pediatr

Blood Cancer. 2012: S37.

2. Naderi M, Imani M, Eshghi P, et al.

Factor XIII deficiency in Sistan and

Baluchistan province. Sci J Blood

Transfus Organ. 2013;10(3):282–8

3. Siboni S, Zanon E, Sottilotta G, et

al. Central nervous system bleeding in

patients with rare bleeding disorders.

Haemophilia. 2012;18(1):34–8.

4. Karimi M, Bereczky Z, Cohan N,

editors Factor XIIID. Seminars in

thrombosis and

hemostasis.2009 Jun;35(4):426-38.

5. Eshghi P, Cohan N, Naderi M, et al.

Factor XIII deficiency: a review of

literature Iranian Journal of Blood and

Cancer. 2010;4: 85–91.

6. Sawlani KK, Chaudhary SC, Roy A,

et al. Factor XIII deficiency presenting

with intracerebral bleed. BMJ Case

Rep. ;Epub 2013/01/15.

7. Naderi M, Dorgalaleh A, Alizadeh

S, et al. Polymorphism of

thrombin‐activatable fibrinolysis

inhibitor and risk of intracranial

haemorrhage in factor XIII deficiency.

Haemophilia. 2014 Jan;20(1):e89-92.

8. Helge Dirk Hartung, MD; Chief

Editor: Max J Coppes, MD, PhD,

MBA.Pediatric Factor XIII Deficiency

Clinical Presentation. Medscape.

9. Kessel R, Hu C, Shore-Lesserson L,

et al. A child with acquired factor XIII

deficiency case report and literature

review. 2013 Nov;19(6):814-26.



10. Bay A, Sirikci A, Dogan A,

Leblebisatan G, et al. Spontaneous

Acute Cerebral Hematoma in a Child

with Factor XIII Deficiency. Pediatr

Hematol Oncol. 2013.

11. Opat S, Butler J, Malan E, et al.

Factor XIII assays. Methods Mol Biol.

2013;992:171–80.

12. Williams M, Will A, Stenmo C, et

al. Pharmacokinetics of recombinant

factor XIII in young children with

congenital FXIII deficiency and

comparison with older patients.

Haemophilia. 2013.

13. Caudill JSC, Nichols WL,

Plumhoff EA, et al. Comparison of

coagulation factor XIII content and

concentration in cryoprecipitate and

fresh‐frozen plasma Transfusion.

2009:49(4):765_70.

14. Dorgalaleh A, Naderi M, Hosseini

MS, et al.Factor XIII deficiency in

Iran: a comprehensive review of the

literature.Semin Thromb Hemost

. 2015 Apr;41(3):323-9.

15. Anwar M, Iqbal M, Ayyub M ,et

al. Prevalence of factor XIII deficiency

in patients presenting with a bleeding

disorder in Pakistan.Article first

published online: DOI:

10.1111/j.1538-7836.2003.0543j.

16. Al-Sharif FZ1, Aljurf MD, Al-

Momen AM, et al.Clinical and

laboratory features of congenital factor

XIII deficiency.Saudi Med J. 2002

May;23(5):552-4.

Correspondence to:

Dr. Obeida Amir Abid

Dept. of hemophilia

Children welfare hospital

Medical city

e-mail: [email protected]

Mobile; 07727825155

http://www.ncbi.nlm.nih.gov/pubmed/?term=Naderi%20M%5BAuthor%5D&cauthor=true&cauthor_uid=25615432

http://www.ncbi.nlm.nih.gov/pubmed/?term=Hosseini%20MS%5BAuthor%5D&cauthor=true&cauthor_uid=25615432

http://www.ncbi.nlm.nih.gov/pubmed/?term=Hosseini%20MS%5BAuthor%5D&cauthor=true&cauthor_uid=25615432



نقض انعامم انوراثي انثانث عشر في احذى مستشفيات انعراق انتعهيميو /دراسو وطفيو

فؤاد حسيند.نبني د.عبيذه عامر عبذ1

2

طبيب اختظاص في طب االطفال/ مستشفي حماية االطفال انتعهيمي/ مذينة انطب1,2

انخالطو:

,انبشش ف جحذخ انبدس انح انضف االظطشاببت ي عششانساذ انربند انحخرش عبيم قص :انبحث خهفية

اسقبطبت ,انجشح انحئبو جأخش انشض عاليبت اى ي.انبط عبية ي يه 3 نكم 1 بسبة حذذ سبة جقذس

.انالد حذر عذ انسشي انحبم يكب ي يطل ضف ,انحايم عذ يحكشس

انضف عشش انربند انحخرش عبيم نقص انعالز انحشخص عايم جحذذ انى ز انذساس جذف : فاىذاال

.انحعه ف بغذاديسحشفى حبة االطفبل ف انساذ

انساذ انحخرشانربند عشش عبيم بقص يشخص يشط 33ل صف يسحشد دساس :انطرقانمواد و

انعبئه انحبسخ عهى ببالعحبد انحشخص جى .0212انى االل ي ابشم 0222اجشث ب االل ي جص

.انخحبش انفحصبت انسشش انعاليبت

االصبببت حذخ بعذ انضف كبث انحشخص اذبء انشظى عذ شعب انشظ انعاليبت اكرش :اننتائح

.انجهذ جحث ضف بقع ظس ذى انهر ضف ,انالد حذبر عذ انسشي انحبم يكب ي انضف,

انعشاق ف انبدس االيشاض ي انساذ عشش انحخرش انربند عبيم قص ا انذساس ز جظش :االستنتاج

صف ي اكرش ف يجبب كب انعبئه جبسخ .سات 12- 1 انشض جشخص فب حى انح االعبس اكرش.

.انشظ انحبالت

قص عبيم انحخرش انربند عشش انساذ, ضف يطل ي يكب انحبم انسشي, اخحببس ربب :انرئيسية انكهمات

انجهط.

Original article


CD38 and ZAP-70 as prognostic immunological parameters in patients

with chronic lymphocytic leukemia

Intisar Sh. Ali 1 ,Ghassan A. Al-Anni

2 , Salman A. Al-Jubury

3

1 M.Sc. microbiology/laboratory dept./ Alyarmouk teaching hospital

2 consultant Hematopathologist/ FICMS hematopathology/ head of Lab. dept. /Alyarmouk teaching

hospital

3 Prof. Of clinical immunology/ microbiology dept./university of kufa college of medicine

Received: 15/1/ 2016 Accepted: 17/ 2/ 2016

Abstract

Background: Several immunological parameters have been investigated in patients with chronic

lymphocytic leukemia (CLL). Some of them are important in detecting the prognosis of the

disease.

Objectives: the aim of this study was to evaluate the level of ZAP-70 and CD38 in patients with

newly diagnosed chronic lymphocytic leukemia.

Materials and methods: The study was done on 50 patients with newly diagnosis CLL 36 male

and 14 female. CD38 and ZAP-70 were measured in patients by using flow cytometry. These

patients were divided according to Ria stage into 29 patients with group II, 21 patients with group

I, group I included Rai stage O, I, II in which the RBC and platelet counts are normal while group

II included Rai stage III and IV in which RBC and platelet count are low.

Results: ZAP-70 and CD38 were significantly increased in patients with group II when compared

to group I. ZAP-70 and CD38 may be regarded as bad prognosis factor for CLL patients.

Conclusion: From the results of the current study, we conclude that ZAP-70 and CD38

high level in patients with chronic lymphocytic leukemia is associated with bad prognosis

Keywords: CD38, ZAP-70, CLL

CD38 and ZAP-70 as prognostic immunological Intisar Sh.Ali,,Ghassan A.Al-Ani, Salman A.


Introduction

Chronic lymphocytic leukemia is the

most common lymphoid malignancy, it

is a disease predominantly seen in

elderly with the incidence twice higher

men (1)

. CLL showed familial tendency

with 3-8 times higher than in normal

population (2)

.Chronic lymphocytic

leukemia characterized by accumulation

of terminally different clonal of CD5+

B-cell from proliferation and

differentiation in the bone marrow and

secondary lymphoid organ. It is the most

common type of malignancy in the

western world but its incidence is low in

Far East (3)

. Chronic lymphocytic

leukemia has different prognosis where

the disease may be growing slowly with

minimal changes in blood cell count;

other may have a faster growing

malignancy (4)

.

ZAP-70 is a member of the tyrosine

kinase group of protein and is normally

showed in T-cell and natural killer cells

(NK). ZAP-70 have a vital role in the

regulation of normal T-cell functions,

such as T-cell receptor(TCR),signal

initiation, T-cell activation, cell

migration and apoptosis (5)

.

The expression of ZAP-70 (˃ or = 20%

of B cell) has showed to associated with

increased risk for bad prognosis in

patients with CLL and is regarded as

great risk factor in those patients (6)

.

CD38 is glycoprotein with small

cytoplasmic region (21 amino acids), a

single chain transmembrane region (23

amino acids), and 256 amino acid

extracellular domain that can be loosely

divided into two regions. The

extracellular amine protein consists of

156 amino acids that make up 5 α-

helices. This region is adjacent to the

COOH domain (amino acids 200-300)

that contains four parallel P-sheets

surrounded by two long and two short α-

helices (7)

.CD38 expression on the

leukemic cell regarded as an excellent

prognostic indicator because it's

measured from peripheral blood sample

easily (8)

. Furthermore, measurement of

CD38 expression arrows the

identification of sub group of patients

with better prognosis within a

presumably poor prognostic and the

detection of patients with poor prognosis

among what is considered better

prognostic group (9)

.



Materials and methods:

This study was done of 50 patients with

newly diagnosed chronic lymphocytic

leukemia from March 2013 till March

2015 included 36 male and 14 female,

the age of the patients rage from 45-75

years, all of the patients were diagnosed

morphologically as chronic lymphocytic

leukemia. These patients were attending

Medical City in Baghdad, National

Center of Haematology (University of

Mustansyria), Al-Yarmouk Teaching

Hospital and Al-Emammain kadhumain

Medical City.

Those patients were divided into two

groups according to Rai staging group I

included Rai stage O, I, II in which the

RBC and platelet counts are normal

while group II included Rai stage III and

IV in which RBC and platelet count are

low. ZAP-70 and CD38 were determined

in patients of the above group by flow

cytometry method using Four-Colour

Cyflow®

Statistical analysis:Data were

expressed; Student's t-test was used to

evaluate differences between the studied

groups. For all test, P≤0.05 was

considered statistically significant.

Analysis of data was carried out using

the available statistical package of SPSS-

22 (Statistical Packages for Social

Sciences-version 22).

Results :

Table (1) and figure (1) represent the

comparison of the mean value of ZAP-

70 in patients of group I and group II

chronic lymphocytic leukemia.

The ZAP-70 level in patients with group

II chronic lymphocytic leukemia was

significantly increased (p=0.0001) when

compared to group I.

Table (2) and Figure (2) represents the

comparison of the mean value of CD38

concentration in the sera of group I and

group II chronic lymphocytic leukemia.

CD38 value were significantly increased

(p=0.001) in patients with group II

chronic lymphocytic leukemia when

compared to group I.



Table (1): levels of ZAP-70 in group I and II chronic lymphocytic leukemia

Table (2): level of CD38 in patients with CLL group I and II

Group I (n=21) Group II (n=29)

ZAP-70

Mean ± SD 8.82±17.10 37.58±22.29

Standard Error of Mean 3.73 4.14

Range 0.19-61.97 1.22-87.04

50th (Median) 0.90 24.49

P value 0.0001*

*Significant difference means using Student-t-test for difference between two independent

means at 0.05 level.

Group I (n=21) Group II (n=29)

CD38

Mean ± SD 6.25±9.30 36.01±24.60

Standard Error of Mean 2.03 0.574

Range 0.53-39.28 0.96-78.92

50th (Median) 2.49 28.64

P value 0.0001*

*Significant difference means using Student-t-test for difference between two independent

means at 0.05 level.



Figure (1): Distribution of ZAP-70 (mean±SD) in patients with chronic lymphocytic

leukemia group I and II

Figure (2): Distribution of CD38 (mean ± SD) in patients with chronic lymphocytic

leukemia group I and II



Discussion

There are many immunological

parameters used to determine the

prognosis at the time of diagnosis of

CLL that would help the decision about

treatment and to determine the outcome

of the disease (10)

.ZAP-70 is a member of

the family of intracellular tyrosine

kinases, is significantly expressed in

normal T cells. It is also may be express

at early stage of chronic lymphocytic

leukemia (11)

. The result in the present

study determine that ZAP-70 might be

useful for detecting the prognosis of the

disease because the level of ZAP-70

increase in patients with group II chronic

lymphocytic leukemia when compared

to group I chronic lymphocytic leukemia

patients. The data in our study regarding

ZAP-70 value agreed with previous

result which indicate that ZAP-70 in

patients with chronic lymphocytic

leukemia increase as the disease progress

(12). CD38 is a protein that have many

function belongs to the number of

molecules independently as

ecto-enzymes as receptors, CD38 may

split the patients with chronic

lymphocytic leukemia into two groups

and represent a dependable negative

prognostic factor (13)

Regarding CD38, the data in this study

showed that it might be useful for

prognosis of the disease since the value

of CD38 increase as the disease progress

and patients with CD38 high value may

be regarded bad prognosis for the

patients in chronic lymphocytic

leukemia (13)

.This observation agreed

with previous results which indicate that

expression of CD38 patients with CLL is

associated with bad prognosis and poor

outcome (14)

.

Conclusion

From the results of the current study, we

conclude that ZAP-70 and CD38 high

level in patients with chronic

lymphocytic leukemia is associated with

bad prognosis.

References

1. Bain B., and Barbara J. Leukaemia

diagnosis 4th ed.;2010 . p65-74.

2. Arena G., Keating M.L. and

Carotenuto C. Chronic

lymphoproliferative disorders: an

integrated point of view for the

differential diagnosis. Leukemia

Lymphoma 2000; 36,225–237.



3. Mir M .Chronic Lymphocytic

Leukemia. Medscape 2013; 4:1993-

1994.

4. Elter T., Hallek M., Engert A.

Fludarabine in chronic lymphocytic

leukemia, Export Opin Pharmacother

2006; 7(12):1641-1651.

5. Chen L., Widhopf G., Huynh L., and

et al. Expression of ZAP 70 is associated

with increased B cell receptor signaling

in chronic lymphocytic leukemia. Blood

2002; 100: 4609-14.

6. Gobossi S., Laurenti L., Longo P.G.,

and et al. ZAP-70 enhances B cell

receptor signaling despite absent or

inefficient tyrosine kinase activation in

chronic lymphocytic leukemia and

lymphoma B cell. Blood 2007;109:2032-

2039.

7. Deïlschneidr R., Xiao V., Yoon J., and

et al. ZAP-70 expression chronic

lymphocytic leukemia and inhibit B cell

receptor signaling. Cell death and

disease 2014; 5:439-442.

8. Malavasi F., Deaglio S., Funaro A.,

and et al.Evolution and function of the

ADP ribosyl cyclase / CD38gene family

in physiology and pathology. Physiol

Rev 2008; 88: 841-886.

9. Liu Q., Kriksunov I., Graeff, R., and

et al.Crystal structure of human CD38

extracellular domain. Structure 2005; 13:

1331-1339.

10. Digheiro G.,and Hamblia T.Chronic

lymphocytic leukemia. Lancet 2008;

371(9617): 1017-1029.

11. Nagwa M.,Kathyrn R.,Felisa Al

concia, and et al. A single tube, four

color flow cytometry assay for

evaluation of ZAP-70 and CD38

expression CLL. American Journal of

clinical pathology 2010; 133:708-717.

12. Maria Ilaria. .Del principle,American

society of haematology 2006;108(3).

13. Dürig J., Nuckel H., Cremer M. et al.

ZAP-70 expression is a prognostic factor

in chronic lymphocytic leukemia.

Leukemia 2003;17:2426-34.

14. Sylvan S.Targeted therapy and

outcome in chronic lymphocytic

leukemia. Ph.D. thesis.2014

Correspondence to:

Intisar Sh. Ali MSc. Dept. of

micerbilogy/ Alyarmouk teaching Lab.

e-mail:[email protected]

mobile: 07735674539



CD38 وZAP-07 في المرضى الذين يعانون من سرطان الدم الليمفاوي للتكهن معلمات مناعية

المزمن

3، سلمان الجبوري 2العاني احمد خليل، غسان 1علي انتصار

التعليمي ماجستير علم األحياء الدقيقة / قسم المختبرات / مستشفى اليرموك 1

/ رئيس قسم المختبرات / مستشفى اليرموك التعليمي FICMSاستشاري امراض الدم المختبري / 2

أستاذ المناعة السريرية / قسم االحياء المجهرية / كلية الطب الكوفة 3

الملخص

هناك عدة انواع من المعلمات المناعيه نم دراستها في مرضى ابيضاض الدم اللمفاوي المزمن .بعض من هذه الخلفية :

.المريضالمعلمات مهمه في تعيين حالة

.في مرضى ابيضاض الدم اللمفاوي المشخصين حديثا CD38و ZAP-70ت هذه الدراسه لمعرفة فائدة يجرا:االهداف

في CD38و ZAP-70اناث، حيث تم قياس 14ذكور و 33مريضا 07تم اجراء هذا البحث على المواد والطرق :

تقسيم هؤالء المرضى الى قسمين المجموعه االولى تضم تم . مصول هؤالء المرضى بواسطة جهاز الفلوسايتومتري

.( مريض22( مريض والثانيه تضم )21)

في مصول مرضى المجموعه الثانيه مقارنه CD38و ZAP-70لقد كانت هناك زياده محسوسه في مستوى النتائج :

ض المصاب بابيضاض الدم كمعلمات لسؤء حالة المري CD38و ZAP-70 بالمجموعه االولى وبذلك بين اهميه ال

.اللمفاوي

في عال مستوى على CD38 و ZAP-70 بين هناك ارتباط أن نستنتج فإننا الحالية، الدراسة نتائج من :االستنتاج

السيئة لهذا المرض. المسيرة مع المزمن الليمفاوي الدم سرطان من يعانون الذين المرضى

المزمن الليمفاوي الدم سرطان, CD38و ZAP-70 :الكلمات الرئيسية

Original article


Evaluation the Role of Bone Marrow Examination in Diagnosis of

Hematological Diseases in Hemato-Oncology Centers in Kurdistan

Region

Shorsh Jameel Ridha1

, Nawsherwan Sadiq Mohammad 2,

Hoger Ismael Muhammed Sarhang3

1 MBChB-MSc Hematology /Hiwa Teaching Hospital

2 MBChB- FICMS Hematology/Pathology Department; College of Medicine / Hawler Medical University

Email: [email protected] Mob: 00964(0)7504384101

3 MBChB-MSc Hematology/ Erbil Teaching Hospital

Received: 10/2/2016 accepted: 13/3/2016

Abstract

Background: Bone marrow aspiration and trephine biopsy have an important role in evaluation

and diagnosis of most hematological and some non hematological disorders.

Objectives: The aims of this study were to assess the value of bone marrow examination in

diagnosis of hematological diseases and determination of frequency and causes of dry tap

marrow.

Patients and Methods: This study was prospective only and it was conducted from January 2013

to June 2013 at Hiwa and Nanakaly hospitals in Sulaymaniyah and Erbil respectively. A total

number of 245 cases were underwent bone marrow examination. Touch imprint was prepared in a

state of dry tap marrow. Sudan black stain was used in acute leukemia.

Results: The patients comprised 138 (56.3%) males and 107 (43.7%) females, with ages ranging

from 1 to 81 years and the mean age of cases was 36.7± 24 years. The most frequent clinical

finding of patients underwent bone marrow examination were pallor (71.4%), followed by fever

(32.2%). The main indications were present of blast cells in peripheral circulation 69 (28.2%),

followed by bone marrow assessment for staging of lymphoma 37 (15.1 %). The most common

diagnoses encountered were: Acute leukemia 69 (28.2%), Active marrow (negative for

lymphoma) 27 (11.1%) and active marrow 24 (9.8%). The frequency of dry tap was (4.08%) and

the most common causes were: acute leukemia and faulty technique (normal marrow).

Conclusion: Bone marrow examination is an important diagnostic tool in the diagnosis and

staging of various hematological disorders.

Keywords: Bone Marrow Aspiration, Trephine Biopsy, Hematology, Kurdistan


Evaluation the Role of Bone Marrow Examination Shorsh J. R, Nawsherwan S.M, Hoger I.M.S.


Introduction

Bone marrow is a quite cellular

connective tissue that occupying the

spaces between bony trabeculae. The

main hemopoietic tissue is bone marrow

and it accounts for about (5%) of all

body weight. Bone marrow aspiration

and trephine biopsy have an important

role in evaluation and diagnosis of most

hematological and some non

hematological disorders. Bone marrow

examination is needed for staging of

lymphoproliferative disorders. In

advanced stage, it shows bone marrow

involvement. Also trephine biopsy is

more important than marrow aspiration

in evaluation of lymphoma and

metastatic solid tumors. These

procedures are valuable in the diagnostic

work up of pyrexia of unknown origin; it

may shows granuloma, infection,

necrosis and hemophagocytic syndrome.

They are useful in follow up evaluation

of patients who submit chemotherapy,

bone marrow transplantation and other

patterns of medical management(1,2,3)

.

Bone marrow aspiration and

biopsy procedures are complementary to

each other and superiority of one

procedure over the other relied on the

particular disorders (1)

. In obtaining bone

marrow examination, there are some

contraindications which are related to

the general condition of the patient.

These contraindications are hemophilia,

disseminated intravascular coagulation,

skin infection and bone disorders such as

osteomyelitis or osteogenesis

imperfecta (4,5)

.

Both procedures are usually safe

but few adverse effects are encountered

after bone marrow examination. The

popular complication is bleeding which

occurs specially in patients with severe

low platelet count, or taking aspirin, or

receiving anticoagulant as warfarin.

Other morbidity from posterior iliac

crest are identified and included long

standing pain, infection in immune

compromised patients and needle-related

accidents such as breaking of needle

within the bone (6,7)

.

In some certain situation, failure

to take out bone marrow on trying

marrow aspiration, known as dry tap or

blood tap and these are generally

described as faulty technique. This

difficulty of aspiration may be occurred

when histology of marrow is normal.

But usually a dry tap indicates important

disorders that involve bone marrow. The



most prominent pathological disorders

are metastatic carcinoma, myelofibrosis,

acute leukemia (packed cells), hairy cells

leukemia and lymphoma. In Dry tap

status, touch imprint must be prepared

for early prime diagnosis before the

result of trephine biopsy was returned

back (8,9)

. In this study we tried to assess

the value of bone marrow examination in

diagnosis of hematological diseases and

to determine the frequency and causes of

dry tap marrow.


This prospective study has been

conducted at Hiwa and Nanakaly

hospitals which are only centers for

oncology/hematology in Sulaymaniyah

and Erbil respectively. All patients

underwent bone marrow examination

between January 2013 and June 2013,

were enrolled in this study. Relapsed and

follow-up cases were excluded. A total

number of 245 cases were studied. One

hundred case was received from

Nanakaly hospital and 145 cases were

collected from Hiwa hospital.

Inform consent was taken from

patient and their relative (In childhood

cases). In each case a detailed history

with general and systemic examination

and routine investigations (complete

blood count, pictures, and erythrocytes

sedimentation rate and biochemical

tests) were carried out prior to bone

marrow examination. The standard

technique (4)

was employed in obtaining

the samples from posterior iliac crest by

using a biopsy set needle (Modern bone

marrow biopsy needle set /11 gauge/

Italian). About 0.5 ml of marrow fluid

was obtained and nearly about ten

smears prepared. Two slides were

stained with Prussian blue technique by

well trained staff to demonstrate iron

granule and ring sideroblasts when

indicated. Sudan Black stain was used

routinely for all patients with acute

leukemia.

When biopsy was performed, the

core biopsy was fixed in formalin and

sent for processing in histopathology

department (Rizgary Teaching Hospital

in Erbil and Shorsh hospital in

Sulaymaniyah) and was examined by 2

pathologists independently. Touch

imprint smears were usually made in

cases with dry tap aspiration.

For some patients with

lymphoproliferative disorders and acute

leukemia, marrow materials was

collected in Ethylene diamine tetra



acetic acid tube and sent for flow

cytometry analysis. Also some patients

with chronic myeloid leukemia and

myeloproliferative neoplasm in whom

the diagnosis were not straight forward,

specimens were sent for BCR- ABL

fusion gene and JAK2 mutation

respectively.

The marrow was examined and

interpreted by two hematologist and was

reviewed with consideration of the

patient’s clinical and laboratory

information. Marrow smears are

examined for determination of the

cellularity, the morphological details of

hemopoietic cells, estimation of myeloid

– erythroid ratio and careful search for

abnormal cells. A questionnaire was

designed that contains a set of questions

including (residence, age, sex, clinical

features, organomegaly, complete blood

picture, other related investigations and

bone marrow examination report). The

questionnaire type is structured

interviewer administrated.

Data were analyzed by using the

statistical package for social science

(SPSS) version 19. Chi square test of

association was used to compare

between proportions. A P-value of ≤

0.05 was considered statistical

significant.

Results

A total of 245 cases underwent

bone marrow examination from January

2013 to June 2013 were included in this

study. Males were 138 (56.3%) and

females were 107 (43.7%). The ratio of

male to female was (1.29:1). The age of

patients in this project were ranged

between 1 to 81 years with mean age of

36.71+ 24 years.

The indications for bone marrow

examination were based on the clinical

and or complete blood pictures. In this

study, the main indications for

examination of bone marrow were

presence of blast cells in peripheral

circulation which was seen in 69

(28.2%) cases, followed by bone marrow

assessment for staging of lymphoma in

43 (17.5%) patients, and then anemia in

20 (8.2%) cases. Leucocytosis was

another indication and found in 14

(5.6%) cases, 12 of them showed marked

left shift with increased basophilic count

and associated with organomegaly, these

were diagnosed as CML. The remaining

two cases were leucocytosis without



shift to left, one of them was diagnosed

as active marrow and the other one

regarded as non diagnostic. Other

indications were explained in table 1.

Various hematological disorders

were encountered in this study as the

result of bone marrow examination. The

most frequent final diagnosis was acute

leukemia and accounting for 69 (28.1%)

cases of which 40 patients were

diagnosed as ALL and 29 cases were

categorized as AML. The next in the

descending order of frequency was

active marrow (negative for lymphoma)

and found in 27 (11.1%) cases. Other

final diagnoses are illustrated in table 2.

A comparison was prepared

between the indication for bone marrow

examination and final diagnosis that

made after examination of the bone

marrow. Acute leukemias were found in

69 (28.1%) cases and final diagnoses of

this category were 40 cases of ALL and

29 cases of AML (fig 1). Of the 37

(15.1%) patients were referred for bone

marrow examination for staging of

lymphoma, 27 cases didn't showed

marrow involvement by lymphoma and

10 cases were demonstrated bone

marrow infiltration by lymphoma cells.

The next in the descending order of

frequency of indications for bone

marrow examination was anemia and

was found in 24 (9.8%) patients and the

outcome of bone marrow examination

were active marrow in (13) cases,

multiple myeloma in (7) cases, pure red

cells aplasia in (2) cases and erythroid

hyperplasia in (2) cases. Table 3

Out of 245 samples 10 (4.08%)

cases were regarded as dry taps and

touch imprints with trephine biopsy

material were obtained from posterior

iliac crest for diagnosis. Of these 10

cases, only 3 (30%) patients were

revealed active biopsy, while others

showed significant marrow pathology.

Acute leukemias were the commonest

pathological causes of dry taps and

found in 3 (30%) cases, two of them

were ALL and the remaining one was

AML. Other causes of dry tap are clearly

explained in (Fig 2).

In total 245 cases were underwent bone

marrow aspiration and trephine biopsy

no complication were reported.



Discussion

Both bone marrow aspiration and

trephine biopsy are essential and safe

procedures for diagnosis of various

hematological and non-hematological

disorders. These are important for follow

up of patients who received

chemotherapy, or marrow

transplantation and/or other medical

treatment. Bone marrow aspiration and

trephine biopsy are complementary to

each other and nowadays, both

procedures are performed together and

usually on same site routinely (10)

.

The most popular indication in this

study was presence of blast cells in

peripheral circulation (acute leukemia)

which were found in 69 (28.2%) cases,

followed by staging for lymphoma in

descending order of frequency and was

found in 37 (15.1%) cases. Similar

findings were reported by Bashawri and

Al–Gwaiz as acute leukemia was the

most common indication and was found

in (32.9%) and (26.2%) cases

respectively (11,12)

. In contrast to these

findings, acute leukemia was ranked

fourth common indication (1.25%) in a

study done by Bedu-Addo et al in

Ghana, in which anemia of unknown

causes was ranked the first frequent

indication due to high frequency of

nutritional anemia as iron deficiency and

megaloblastic anemias13

. Leucocytosis

was also another indication and was seen

in 14 (5.6%) patients, where it was

mostly due to CML that was found in 12

cases, while the rest two cases were

regarded as active marrow and non

diagnostic marrow.

The result of this study showed

that the acute leukemias were the most

frequently encountered diagnosis by

bone marrow examination and these

were found in 69 (28.1%) cases, 40

patients of them were ALL with mean

age 16.8 years and 31 (77.5%) of them

were found in 1st and 2

nd decades, while

29 cases were AML with mean age 35

years and 10 of them were seen in 3rd

and 4th

decades; however references set

that AML is the commonest acute

leukemia in adults and ALL is the

commonest childhood malignancy (14)

. A

similar study was done by Gupta et al

and Egesie et al in India and Niger

respectively, in which acute leukemias

were the largest group disorders (15,16)

.

Acute leukemia was ranked third in a



study done by Pudasaini et al from

Nepal, in which frequency of erythroid

hyperplasia (mostly due to iron

deficiency and megaloblastic anemia)

were high and these were due to higher

prevalence of nutritional deficiency in

their locality (17)

.

In this study, the second common

indication for bone marrow examination

was staging of lymphoma and found in

37 (15.2%) cases. Similarly, Saeed and

Jawhar have also reported that

lymphoma were the second common

diagnostic report (18)

. Bone marrow was

not involved in 27 (72.9%) cases and

marrow involved by lymphoma was

found in 10 (27.1%) cases. All

specimens of involved marrow were non

Hodgkin's lymphoma and the majority of

these cases were diffuse large B cell

lymphoma. Similar results were reported

by Durosinmi et al from Nigeria (19)

.

Active bone marrow was 3rd

common diagnosis in the present study

and found in 24 (9.8%) cases. High

percentage of active marrow 125

(35.1%) cases was reported in a study

from Kenya. These were due to over

induction of bone marrow examination

even in cases with bicytopenia and mild

anemia (2)

.

ITP was seen in 21 (8.6%) cases

in this study. The diagnosis of ITP is

made after exclusion of other causes of

low platelet count. Other studies were

showed 15.7%, 10.5% and 6.21% cases

of ITP in Pudasaini et al, Kibria et al and

Khan et al respectively (17,20,21)

.

Myeloproliferative neoplasms were

commonly diagnosed on bone marrow

examination and confirmed by

estimation of Janus-associated kinase 2

(JAK2) mutations (14)

. MPN was found

in 21 (8.6%) cases. This is nearly similar

to another study which was done by

Saeed and Jawhar in Mosul18

, while

other study was showed low percentage

of MPN that found in (2.7%) cases and

was published in Yemen (22)

.

Another common disorder in

present study was chronic lymphocytic

leukemia and was found in 13 (5.3 %)

cases. Similarly, 2 (5%) cases were seen

in another study done in India15

.

However in a study done by Chandra,

3.1% of cases were CLL23

. This might

be related to geographical variation.



Chronic myeloid leukemia was

found in 12 (4.9%) cases and was the

least common leukemia in studied cases

constituting (13%) of all leukemia in this

study and it is nearly similar to the fact

that CML accounts for around 15% of

leukemias (14)

.

Multiple myeloma was another

hematological malignancy that

diagnosed by bone marrow examination

with other diagnostic criteria and it was

found in 12 (4.9%) patients with mean

age 61 years and all cases were

associated with high ESR. Other studies

were reported a frequency of multiple

myeloma as 5.6%, 4% and 3.5%

respectively (17,2,24)

.

Another important indication for

bone marrow examination was

detection of secondary metastasis. In

this study, 8 (3.2%) cases of tumors (4

cases of neuroblastoma, 2 cases of

Ewing sarcoma, 1 case of

adenocarcinoma of prostate and 1 case

of hepatocellular carcinoma) were

underwent bone marrow examination to

exclude marrow involvement. The

marrow involvement by secondary

metastasis was found in 2 (0.8%) cases.

A study from Lahore reported a similar

finding (0.7% of cases with secondary

metastasis) (25)

.

In present study, 6 (2.4%) cases

were not diagnosed on bone marrow

examination as two of them were with

inadequate trephine biopsy and the

remaining 4 cases were with inadequate

of both aspiration and biopsy. Higher

frequency (14.4% cases) of non

diagnostic sample was found in another

study (26)

.

This study showed that bone

marrow aspirations was failed to

obtained in 10 (4.08%) cases in which

touch imprint and adequate trephine

biopsies were well done, this is known

as dry tap or blood tap. The main causes

of dry tap in present study were faulty

technique (active marrow biopsy) in 3

(30%) cases and acute leukemia (packed

cells in trephine biopsy) in 3 (30%)

cases. A similar study was done by

Humphries in 1990, who reported a rate

of dry tap as (3.9%)9 and higher

frequency was recorded by Khanum et al

in Lahore and found in 50 (10%) cases

among 500 studied patients8. The

remaining cases of dry tap were

myelofibrosis in 2 cases, secondary

metastasis and hairy cells leukemia.



In this study, complications after

both procedures (marrow aspiration and

trephine biopsy) were not encountered.

Complications of bone marrow

examination are rare as reported by Bain

et al (27)

.

Conclusion:

We concluded that bone marrow

examination is an important diagnostic

tool in the diagnosis and staging of

various hematological disorders. The

main indication for bone marrow

examination in this study was presence

of blast cells in peripheral circulation

(Acute leukemia). The commonest

causes of dry tap in this study were

faulty technique and packed cell in acute

leukemia. Bone marrow examination in

spite of being invasive procedure it is

safe and post operative complications

were not recorded in this study.

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Comparative Evaluation of

Simultaneous Bone Marrow Aspiration

and Bone Marrow Biopsy: An

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2. Okinda NA and Riyat MS. Bone

marrow examination finding at Aga

KhanUniversity hospital, Nairobi. East

Afri Med J 2010; 87(1): 4-8.

3. Riley RS, Williams D, Ross M, Zhao

S, Chesney A, Clark BD, et al. Bone

Marrow Aspirate and Biopsy: A

Pathologist's Perspective. II.

Interpretation of the Bone Marrow

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2009;23(5):259-307

4. Bain BJ. Bone marrow aspiration. J.

Clin. Pathol 2001a; 54: 657-663.

5. Bain BJ. Bone marrow trephine

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6. Bain BJ. Bone marrow biopsy

morbidity and mortality. Br J Haematol

2003; 121: 949– 951.

7. Marti J, Anton E, Valenti C.

Complications of bone marrow biopsy.

Br. J. Hematol.2004; 124(4): 557–558.

8. Khanum F, Rehman AU, Ahmad S,

Anwar J. `Dry tap` of bone marrow and

its clinical important. Pak J Med Health

Sci 2007; 1- 3.

9. Humphries JE. Dry tap bone marrow

aspiration: clinical significance. Am J



Hematol 1990; 35 (4): 247 – 250.

(Abstract)

10. Islam A. Bone marrow aspiration

before bone marrow core biopsy using

the same bone marrow biopsy needle: a

good or bad practice? J Clin Pathol

2007; 60: 212 – 215.

11. Bashawri LA. Bone marrow

examination; indication and diagnostic

value. Saudi Med J 2002; 23(2): 191-

196.

12. Al- Gwaiz LA. Analysis of 3494

bone marrow examinations in a referral

hospital: indications and interpretations.

Saud Med J 1997; 18: 144- 147

13. Bedu-Addo G, Amoako YA and

Bates I. The role of bone marrow

aspirate and trephine samples in

hematological diagnosis in patients

referred to a teaching hospital in Ghana.

Ghan Med J.2013; 47(2): 74- 78.

14. Hoffbrand AV, Moss PAH and Pettit

JE. Essential Hematology. 5th ed.

Oxford, UK: Wiley-Blackwell 2006. PP

238.

15. Gupta N, Kumar R, Khajuria A.

Diagnostic assessment of bone marrow

aspiration smears, touch imprints and

trephine biopsy in haematological

disorders. J k science 2010; 12(3): 130-

133

16. Egesie OJ, Joseph DE, Egesie UG,

Ewuga OJ. Epidemiology of Anaemia

Necessitating Bone Marrow Aspiration

Cytology in Jos. Niger Med J 2009;

50(3): 61 – 63 (Abstract)

17. Pudasaini S, Prasad KBR, Rauniyar

SK, Shrestha R, Gautam K, Pathak R, et

al. Interpretation of bone marrow

aspiration in hematological disorder. J

Path Nepal 2012; 2: 309-312.

18. Saeed MS, Jawhar NM. Bone

marrow trephine is some hematological

and non hematological disorders. Ann.

Coll. Med. 2010; 36(1& 2): 63 – 71.

19. Durosinmi MA, Mabayoje VO,

Akinola NO. A review of histology of

bone marrow trephine in malignant

lymphoma. Niger J Med 2003; 12(4):

198- 201. (Abstract)

20. Kibria SG, Islam MDU, Chowdhury

ASMJ, Ali MY, Haque MR, Mustanzid

SM, et al. Prevalence of hematological

disorder: A bone marrow study of 177

cases in a private hospital at Faridpur.

Faridpur Med Coll J 2010; 5(10): 11- 13.

21. Khan A, Aqeel M, Khan TA, Munir

A. Pattern of hematological diseases in



hospitalized paediatric patients based on

bone marrow examination. J Postgrad

Med Inst 2008; 22(3): 196- 200.

22. Al-Ghazaly J, Al-Selwi AH,

Abdullah M, Al-Jahafi AK, Al-Dubai W,

Al-Hashdi A. Pattern of haematological

diseases diagnosed by bone marrow

examination in Yemen: A developing

country experience. Clin Lab Haematol

2006;28:376-81

23. Chandra S and Chandra H .

Comparison of bone marrow aspirates

cytology, touch imprint cytology and

trephine biopsy for bone marrow

evaluation. Hematol Rep 2011; 3(3): 65-

68.

24. Tariq M, Khan N, Basri R, Amin S.

Aetiology of pancytopenia. Professional

Med J 2010; 17(2): 252-256.

25. Khan FS and Hasan RF. Bone

marrow examination of pancytopenic

children. J Pak Med Assoc 2012; 62(7):

660- 663.

26. Naznin M, Wahab AJ, Kalavathy R.

A review of bone marrow examinations

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Wiley-Blackwell 2010. pp: 40- 51.

Correspondence to:

Nawsherwan Sadiq Mohammad

Pathology Department; College of Medicine

/ Hawler Medical University


Mob: 00964(0)7504384101



نخاع العظم في تشخيص أمراض الدم في مراكز أمراض الدم و األورام في إقليم فحصتقييم دور

كردستان

3،هوكر إسماعيل محمد سرهنك 2، نوشيروان صادق محمد 1شورش جميل رضا

1 MBChB-ماجستير أمراض الدم / مستشفى هيوا التعليمي

2 MBChB- FICMS الطب /جامعة هولير الطبية البريد اإللكتروني: أمراض الدم / قسم علم األمراض/كلية

[email protected]

3 MBChB-ماجستير أمراض الدم / مستشفى أربيل التعليمي

الملخص

لعينة نخاع العظم والخزعة دورا هاما في تقييم وتشخيص معظم أمراض الدم وبعض االضطرابات غير الخلفية:

هو لتقييم قيمة فحص نخاع العظام في تشخيص األمراض الدموية وتحديد تواتر الدموية. األهداف: هدف هذه الدراسة

وأسباب نخاع العظم الجاف.

في مستشفى 2113إلى يونيو 2113: هذه دراسة استطالعية فقط وتمت خالل الفترة من يناير لمرضى والطرقا

حالة 242على التوالي. وكان عدد إجمالي من هيوا التعليمي هيوا و مستشفى أربيل التعليمي في السليمانية وأربيل

خضعت فحص نخاع العظام. وقد استخدم لمس الخزعة في حالة النخاع الجاف. وقد استخدم صبغة السودان في

سرطان الدم الحاد.

11-1٪( من اإلناث، تتراوحت أعمارهم 43.1) 111٪( من الذكور و 3..2) 131كان عدد المرضى النتائج:

عاما. اكثر الحاالت شيوعا من المرضى التي خضعت للتقصي 24± 1..3أعمارهم وكان متوسط سنوات،

٪(. وكانت المؤشرات الرئيسية وجود 32.2٪(، تليها الحمى )11.4السريري وفحص نخاع العظم كانت الشحوب )

31طان الغدد الليمفاوية ٪(، يليها تقييم نخاع العظام لتحديد مرحلة سر21.2) 6.الخاليا السرطانية في الدم

21٪(، ونخاع نشط )السلبي ليمفوما( 21.2) 6.٪(. وكانت التشخيصات الشائعة كاالتي: سرطان الدم الحاد 12.1)

٪( وكانت األسباب األكثر شيوعا: سرطان 4.11٪(. وكانت نسبة النخاع الجاف )6.1) 24٪(، ونخاع نشط 11.1)

(.الدم الحاد وتقنية خاطئة )نخاع العادي

فحص نخاع العظم هو أداة تشخيصية مهمة في تشخيص وتحديد مراحل مختلفة من االضطرابات الدموية. الخالصة:

نخاع العظم ، خزعة، أمراض الدم، كردستان كلمات البحث:



Table 1 : Indications for bone marrow examination:

Percentage Frequency Indication

28.2 69 Blast cells in peripheral circulation

15.2 37 Bone marrow for lymphoma staging

9.8 24 Anemia

8.1 20 Pancytopenia

7.7 19 Thrombocytopenia

6.1 15 Bicytopenia

5.6 14 Leucocytosis

5.6 14 Polycythemia

5.2 13 Lymphocytosis

2.4 6 Thrombocytosis

1.6 4 Hepatosplenomegaly with anemia

0.8 2 Leucoerythroblastic anemia

3.2 8 To exclude marrow metastasis

100 245 Total



Table 2: Results of Bone marrow examination of the studied cases.

Disorders Number of cases %

ALL 40 16.3

AML 29 11.8

Active marrow (negative for lymphoma) 27 11.1

Active marrow 24 9.8

Idiopathic thrombocytopenia purpura 21 8.6

Myeloproliferative neoplasm 21 8.6

Chronic lymphocytic leukemia 13 5.3

Chronic myeloid leukemia 12 4.9

Multiple myeloma 12 4.9

Marrow involvement by lymphoma 10 4.1

Aplastic anemia 10 4.1

Non – diagnostic 6 2.4

Active marrow (negative for solid tumors) 6 2.4

Megaloblastic anemia 5 2

Secondary metastasis 2 0.8

Pure red cell aplasia 2 0.8

Erythroid hyperplasia 2 0.8

Myelodysplastic syndrome 2 0.8

Hairy cells leukemia 1 0.4

Total 245 100



Table 3: Indication of bone marrow examination versus final diagnosis

Cases Final diagnosis Frequency Indication

40

29

ALL

AML

69 Acute leukemia

27

10

Active marrow (negative for

lymphoma)

Marrow involved by lymphoma

37 Bone marrow for staging

13

7

2

2

Active marrow

Multiple myeloma

Erythroid hyperplasia

Pure red cells aplasia

24 Anemia

10

4

2

2

1

1

Aplastic anemia

Megaloblastic anemia

Multiple myeloma

Non diagnosis marrow

Active marrow

Hairy cell leukemia

20 Pancytopenia

19 ITP 19 Thrombocytopenia

5

3

2

2

1

1

1

Active marrow

Multiple myeloma

Myelodysplastic syndrome

ITP

CLL

Non diagnostic marrow

Megaloblastic anemia

15 Bicytopenia

12

1

1

CML


Active marrow

14 Leucocytosis

14 Myeloproliferative neoplasm 14 Polycythemia

12

1

CLL

Active marrow

13 Lymphocytosis

5

1

Myeloproliferative neoplasm

Active marrow

6 Thrombocytosis

2

2

Active marrow


4 Hepatosplenomegaly and

anemia

2 Myeloproliferative neoplasm 2 Leucoerythroblastic

anemia

6

2

Active marrow (negative for

metastasis) Secondary metastasis

8 To exclude marrow

metastasis

245 245 Total



Figure 1:(A) Acute lymphoblastic leukemia.(B) Acute Myeloblastic Leukemia.

Marrow aspiration (Leishmann stain)

Figure 2 : Causes of dry tap in this study.

Original article


Types of Anaemia and its Correlation with Disease Activity in Patients

with Rheumatoid Arthritis among Kurdish Population of Iraq

Hisham A. Getta¹, Najmaddin Khoshnaw ²* & 6, Alaa Fadhil Alwan

3 ,Sundus F.A

4,

Raouf R. Mirza 5

¹ Department of hematopathology, faculty of sciences, school of medicine, university of sulaimani,

Kurdistan region, Iraq

²*Department of Hematology, Hiwa Hospital, Sulaymaniyah, Kurdistan Region, Iraq

3 Department of clinical hematology, the national center of hematology, Almustansiriya University,

Baghdad, Iraq

4 Department of rheumatology and medical rehabilitation, general medical teaching hospital, Sulaymaniyah,

Kurdistan Region, Iraq

5 Department of rheumatology and medical rehabilitation, school of medicine, university of sulaimani,

Sulaymaniyah Kurdistan region, Iraq

6 Kurdistan Board of Medical Specialties, Clinical Hematology/Trainee, Ministry of higher education and

scientific research, Erbil, Kurdistan Region, Iraq

Received 8/1/2016 revised 3/2/2016 accepted 23/2/2016

Types of Anaemia and its Correlation with Disease Hisham A.Getta, Najmaddin Khoshnaw et al


Abstract:

Background: Rheumatoid arthritis (RA) is a chronic systemic inflammatory autoimmune disease

characterized by articular and extra-articular manifestation as weight loss, fatigue, malaise, and

anemia.

Objectives: The aim of this study was to find the prevalence of different types of anemia and its

correlation with the disease activity among patients with RA in Sulaymaniyah province and to

determine the associated risk factors.

Patients and Methods: A cross sectional study carried out in Sulaymaniyah general medical

Hospital for periods from October-2014 to the end of June-2015.A convenient sample of 100

rheumatoid arthritis patients were selected from patients seen in the rheumatology clinic. One

hundred healthy voluntary controls of same age groups were selected and same parameters for

diagnosis of anemia are used in both groups.

Results: The prevalence of all types of anemias among the rheumatoid Arthritis (RA) patients

was 40% which more than that found in control group. The common types of anemia was anemia

of chronic disease (28%), iron deficiency (10%), thalassemia minor (1%), and megaloblastic

anemia 1%. We found a significant correlation of RA with each low hemoglobin, low hematocrit,

high leucocyte count and high ESR. Anemia among RA patients in our study was significantly

more prevalent among low socioeconomic status patients. The majority of RA patients had

moderately or high active disease, which revealed no association between disease activity and

anemia (p value=0.3).

Conclusion: the prevalence and types of anemias among RA patients in Sulaymaniyah was

comparable to that found in other studies and it was two times common than the normal healthy

peoples. In our study we concluded that little evidence found between disease activity and

anemia.

Keywords: Rheumatoid Arthritis, Anemia, Sulaymaniyah, Kurdistan Region, Iraq

Introduction:

Rheumatoid arthritis (RA) is chronic

systemic auto-immune inflammatory

disease characterized by articular and

extra-articular manifestation, such as

weight loss, fatigue, malaise and

anemia (1)

.

RA is associated with a high risk for

morbidity and premature death

secondary to the earlier development of

cardiovascular, lung diseases and

malignancy (2)

.



Extra-articular manifestations of RA

occur in about 40% of patients, either in

the beginning or during the course of

their disease (3)

.

Patients with RA, who have high titers

of rheumatoid factor, are most likely to

have extra-articular manifestations of

their disease (4)

. Patients with RA may

present with hematological

abnormalities either at the time of

diagnosis, or during the course of their

illness. Hematological manifestations in

RA can be broadly categorized into areas

of anemia, thrombocytosis, neutropenia,

thrombocytopenia, eosinophilia, and

hematological malignancies (5)

. Anemia

is, by far, one of the most common

extra-articular manifestations of RA.

The cause of anemia in RA is

multifactorial including disease

activity, drug-induced, nutritional,

gastrointestinal bleeding, bone marrow

suppression, and ineffective

erythropoiesis (6)

. Anemia of chronic

disease (ACD) usually normochromic

normocytic type observed in RA, where

it usually correlates with the disease

activity. Eosinophilia in RA reflects

active disease or hypersensitivity to

drugs (7)

. Thrombocytosis is a frequent

finding in active RA and is correlated

with the number of active inflamed

joints (8)

. Lymphadenopathy is

sometimes observed in active RA,

usually presenting on biopsy as benign

follicular hyperplasia (9)

. The types of

anemia in RA may include anemia of

chronic disease, iron-deficiency anemia,

vitamin deficiency anemia, aplastic

anemia, or hemolytic anemia (10)

.

The first principle of treating RA

associated anemia is to reduce

inflammation as much as possible using

non-steroidal anti-inflammatory drugs

(NSAIDS), disease modifying

anti rheumatoid drugs

(DMARDs). Erythropoietin stimulating

agents (ESA) therapy has been shown to

be effective in treating RA induced

ACD. However, RA patients tend to

have a blunted response to ESA therapy,

and higher than normal ESA doses are

often required. In these patients, ACD

shows improvement when inflammation

has decreased. When iron deficiency

occurs concomitantly with ACD, iron

repletion may be needed, either alone or

as adjunct therapy with ESAs.

ESA

therapy in combination with iron

supplementation corrects anemia in most

patients with RA, and may improve RA

outcomes and quality of life (11)

.The



aims of this study were to find the

prevalence, types of anemia among

patients with RA in Sulaymaniyah and to

determine the associated risk factors.


A cross sectional study carried out in

Sulaymaniyah General Hospital in

Sulaymaniyah governorate for period

from 1st of October, 2014 to the end of

June, 2015. It included 100 patients with

RA who attended to Rheumatology

Consultation clinic or admitted to

Rheumatology ward of Sulaymaniyah

General Hospital.

Inclusion criteria: All patients who

met the 2010 ACR-EULAR

classification criteria for Rheumatoid

Arthritis were included (12)

(table1), a

score of ≥6/10 is needed for diagnosis of

patient as having RA.

Table 1 : criteria for diagnosis of RA

A.Joint involvement SCORE

1 large joint 0

2- 10 larg joint 1

1-3 small joints(with or without large joint) 2

4-10 small joints(with or without large joint) 3

>10 joints(at least 1 small joint) 5

A.Serology(at least 1 test result is needed for classification)

Negative RF and Anti ccp 0

Low positive RF and Anti ccp 2

High positive RF and Anti ccp 3

C.Acute phase reactants

Normal CRP and ESR 0

Abnormal CRP and ESR 1

D.Duration of symptoms

Less than 6 weeks 0

6 weeks or more 1

N.B : A score of ≥6/10 is needed for diagnosis of patient as having RA



Exclusion criteria: Any cause of

anemia not related to RA which includes

patients with active GIT bleedings,

bleeding tendencies, menorrhagia,

malignancies, renal failure, diabetic

patients, chronic infections were

excluded in our study. The data were

collected through direct interview and

using prepared questionnaire. The

known cases of RA were diagnosed by

consultant Rheumatologist. Full medical

history and clinical examination were

done for all patients. A total of 100

healthy controls were selected and

required information was collected from

them. The questionnaire included: socio-

demographic characteristics as Age,

gender, residence and socioeconomic

status. RA characteristics: disease

activity assessed by DAS-28, rheumatoid

factor (RF) and duration of RA,

treatment modalities of RA, family

history of RA and anemia, Laboratory

tests done as complete blood count,

blood smear, reticulocyte count, coombs

test and ESR done by autoanalyzer,

serum iron and TIBC done by

C111Cobas Roche Company, serum

ferritin, serum B12, and folate by E411

Cobas Roche Company, Hb-

electrophoresis done for selected cases

by HPLC (high performance liquid

chromatography, D10) The World

Health Organization (WHO) criteria

were used to define anemia as

hemoglobin threshold of <120 g/L for

women and <130 g/L for men (13)

. The

disease activity evaluated according to

DAS-28 in which values less than 2.6

corresponds for clinical remission,

values ranging (2.6-3.2) are regarded as

low disease activity, values ranging(3.2-

5.1) are regarded as moderate disease

activity and more than 5.1 regarded as

high disease activity. All patients gave

their oral consent before beginning of

the study and agreement was taken from

official review ethical committee of

Sulaymaniyah General Hospital.

Statistical analysis: Statistical Package

for Social Sciences (SPSS) version 20

was used. Descriptive statistics presented

as (mean ± standard deviation) and

frequencies as percentages. Chi-square

used for categorical variables and

Fishers exact test was used when

expected variables were less than 20%.

Independence t-test was used to compare

between two means. In all statistical

analysis, level of significance (p value)

set at ≤ 0.05.



Results:

A total of 100 patients with RA were

included in present study with mean age

50±13 years, 52% of them were ≥ 50

years. Female were more than males

with male to female ratio of 1:9.01.

More than half of RA patients were

living in urban areas. The socioeconomic

status was presented as following; 21

patients were good, 44 were fair and 35

RA patients had low socioeconomic

status as shown in (table 2).

Table 2: Sociodemographic characteristics of RA patients.

Variable No. %

Age mean±SD (50±13 years)

20-29 years 8 8.0

30-39 years 12 12.0

40-49 years 28 28.0

≥ 50 years 52 52.0

Gender

Male 9 9.0

Female 91 91.0

Residence

Urban 52 52.0

Rural 48 48.0

Socioeconomic status

Good 21 21.0

Fair 44 44.0

Low 35 35.0

Mean RA disease duration was 11±9

years, 65% of RA patients had disease

duration ≤ 10 years. The Rheumatoid

factor (RF) was positive for 91% of RA

patients and negative for 9% of them.

The treatment types used by RA patients

were distributed as followings;

prednisolone (16.8%), Folic acid

(16.8%), Methotrexate (16.5%), Calcium

& Vitamin D (14.2%), combination



therapy (12.5%), hydroxychloroquine

(10.8%), Alendronate (2.6%),

Leflunomide (2.3%), Etanercept (3.1%),

Rituximab (1.3%), Adalimumab (0.4%),

and Azathioprine (0.4%). Mean DAS-28

of RA patients was 5±1.2, while 57% of

RA patients had high disease activity.

Mean Hb of RA patients was 12.3±1.4

mg/dl, 40% of them had low Hb level.

Mean PCV of RA patients was

40.1±30.8 %, 33% of them had low PCV

level. Mean WBC of RA patients was

8.4±2.9 x109, 20% of them had high

WBC count as shown in table 3.

Table 3: hematological parameter of patient with RA

Variable No. %

Hb mean±SD (12.3±1.4 mg/dl)

Normal 60 60.0

Low 40 40.0

Total 100 100.0

PCV mean±SD (40.1±30.8 %)

Normal 67 67.0

Low 33 33.0

Total 100 100.0

WBC mean±SD (8.4±2.9 x109)

Normal 80 80.0

High 20 20.0

Total 100 100.0

Platelets mean±SD (259.9±65.9x109)

Normal

100 100

Reticulocytes level

normal

100 100

Anemia was found in 40 patients

(40%) with RA patient and 70.0% of

anemic patients had anemia of chronic

diseases (ACD), 25% had iron

deficiency anemia IDA, 2.5%

megaloblastic anemia and 2.5% had

Thalassemia minor (Fig 1).



Figure 1: Types of anemia among RA patients.

Mean serum iron of anemic RA patients

was 98.4±35.9 µg/dl, with 72.5% had

low iron level. Mean TIBC level of

anemic RA patients was 385.9±67.9

µg/dl, with 47.5% had low TIBC level

and 20% had high TIBC level. Mean

serum ferritin of anemic RA patients was

73.3±56.1 ng/ml, with 22.5% had low

ferritin level while 25% had high ferritin

level. Only one patient had low B12

level. Hb-electrophoresis was positive

only for one patient. All of RA patients

had normal folate level and negative

Coombs test. Mean ESR of RA patients

was 46.3±19.3 mm/hr, all of RA patients

had high ESR.

No significant differences were

observed between anemic and non-

anemic RA patients regarding DAS-28

and rheumatoid factor (RF) (p>0.05). No

significant differences were observed

between anemic and non-anemic RA

patients regarding family history of RA

and anemia (p>0.05) as shown in

(table 4)

0

5

10

15

20

25

30

10

28

1 1

No

.



Table 4: Distribution of RA characteristics according to anemia.

Variable Anemic Non anemic χ² P

No. % No. %

DAS28 3.0* 0.3

Remission 0 - 1 100.0

Low activity 3 50.0 3 50.0

Moderate activity 11 30.6 25 69.4

High activity 26 45.6 31 54.4

RF 3.4* 0.06

Sero +ve 39 42.8 52 57.2

Sero –ve 1 11.1 8 88.9

Family history of RA 2.3 0.1

Positive 15 51.7 14 48.3

Negative 25 35.2 46 64.8

Family history of anemia 0.9* 0.3

Positive 2 66.7 1 33.3

Negative 38 39.2 59 60.8

*Fishers exact test.

Discussion:

Rheumatoid arthritis (RA) is an

autoimmune disorder of unknown

etiology characterized by symmetric,

erosive synovitis and, in some cases, extra

articular involvement. Extra-articular

manifestations can be detected in almost

any organ system, causing considerable

disease related morbidity and interference

with quality of life. Anemia is a

frequently occurring extra-articular

manifestation of RA, being mostly of the

normochromic and normocytic type.

Anemia is multifactorial, reflected in

dimorphic appearance and wide red cell

distribution width. Anemia of chronic

disease (ACD) and iron deficiency

anemia (IDA) are the most important

types of anemia in RA patients (14)

.

In this study, prevalence of anemia

among studied RA patients was (40%).

The result was close to that of Wilson A

et al (15)

who reported the anemia to be



ranged of 33-66% among RA patients, but

was higher than that reported by Muia

GM, et al in which he reported prevalence

of 33% (16)

. On other hand, other studies

found higher prevalence which reached

up to 70.6% (17)

.

Prevalence ratio of developing anemia

among RA patients was 2:1 as compared

to healthy controls. This prevalence ratio

is close to that reported by Han C, et al

study who reported a prevalence ratio of

anemia among RA patients as 2.2.:1(18)

In this study we have found no

significant association between disease

activity and anemia (p value=0.3) ,while

in other epidemiological study it was

reported that lower hemoglobin levels

was associated with increased disease

activity as measured by the number of

tender and swollen joints, ESR, CRP

level, and assessments of pain and

fatigue(DAS-28 score)(19)

. Han C et al

reported that anemia independently

contributes to physical disability in

patients with RA (20)

.

More than two thirds of detected

anemic RA cases in present study were

anemia of chronic diseases (ACD) and

25% of them were iron deficiency anemia

(IDA). This result was close to many

studies as Ravindran V, et al study (21)

and

Swaak A study (22)

, this explained on

bases that inflammatory cytokines

released during ACD can alter systemic

iron metabolism by inducing excess

synthesis of hepcidin, the iron regulatory

hormone. Since hepcidin inhibits iron

export from cells by blocking ferroportin

activity, excess hepcidin is the root cause

of the hypoferremia and iron-restricted

erythropoiesis seen in ACD (23), (24)

.

In this study we found that mean age

of RA patients was 50±13 years with

predominance of female gender. This is

consistent with results of Muia et al study

(16). RA was significantly associated with

rural living and low socioeconomic status

(P≤0.05). This is similar to results of

Putrick P, et al study (25)

.High levels ESR

and WBC were significantly associated

with RA patients (P≤0.05). This finding

was close to that of Shenair D, et al study

(26). More than half of studied RA patients

had high DAS28 activity. This finding is

higher than that reported by Ganna S

study (24)

which reported that 30% of RA

patients had high DAS-28 activity.

Anemia among RA patients in our

study was significantly more prevalent

among low socioeconomic status

patients (P=0.05). This finding was close

to that of Gordon MM, et al study (27)

.



Bengtsson et al reported the association

between high socioeconomic and

educational status and a lower risk for

the development of RA in a population

representative of the Swedish

population, suggesting that

environmental factors or lifestyle might

influence disease evolution (28)

. Pedersen

et al reported that the educational level

was inversely associated to the risk of

developing RA in the Danish population

and the risk was twice as lower for those

individuals with a higher number of

years of formal schooling (29)

.

Platelets level among RA patients was

normal, although, mean platelets among

anemic patients was significantly higher

than that of non-anemic (P=0.03). This is

consistent with results of Safak S, et al

study (30)

. ESR level was significantly

increased among anemic RA patients in

our study (P<0.001). This is similar to

results of Ganna S study(24)

. Recent

studies have revealed a key role of

cytokines and other mediators of

inflammation in the development not only

of the articular syndrome, but also a

whole range of systemic manifestations of

the disease (31)

. Wilson A, et al in their

systematic review, suggested that patients

with RA who have anemia are likely to

have more severe joint disease and if the

anemia is successfully treated, the joint

disease will likely respond to treatment as

well (15)

.

Conclusion:

The prevalence of anemia among RA

patients in Sulaymaniyah was

comparable to that found in other studies

and it was two times common than the

normal healthy peoples. Anemia of

chronic diseases was the most common

type of anemia among RA patients. The

majority of RA patients had moderately

or high active disease, which revealed no

association between disease activity and

anemia.

Acknowledgment:

We have special thanks to all doctors

and medical staffs and all patients and

peoples who helped us in collecting the

data for this study.

Authorship contribution:

HAG who designed the project of the

study, RRM supervisor of the research

article, SFA primary investigator, NK

and AFA shared in rewriting and editing

of the all parts of the manuscript.



Conflict of interest:

All authors declare that there is no any

conflict of interest in publishing this

article.

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Correspondence to :

Dr. Najmaddin Khoshnaw

Clinical Hematologist, Department of

Hematology

Hiwa Hospital -Sulaymaniyah -IRAQ

Cell phone: +9647701554184

Po. Box: 54





المرض في المرضى الذين يعانون من التهاب المفاصل الرثوي مع نشاطأنواع فقر الدم وارتباطها

بين السكان األكراد في العراق

5, رؤوف ميرزا 4، سندس ف 3، عالء فاضل علوان 6& 2، نجم الدين خوشناو 1هشام جتا

العلوم، كلية الطب، جامعة السليمانية، إقليم كردستان، العراققسم امراض الدم، كلية 1

قسم أمراض الدم، مستشفى هيوا، السليمانية، كردستان، العراق 2

قسم أمراض الدم السريري، المركز الوطني لألمراض الدم، الجامعة المستنصرية، بغداد، العراق 3

تعليم الطبي ,السليمانية وإقليم كردستان، العراققسم الروماتيزم والتأهيل الطبي، المستشفى العام ال 4

قسم الروماتيزم والتأهيل الطبي، كلية الطب، جامعة السليمانية، السليمانية اقليم كردستان العراق 5

مجلس كردستان للتخصصات الطبية والسريرية أمراض الدم / متدرب وزارة التعليم العالي والبحث العلمي، 6

ان، العراقأربيل، إقليم كردست

الملخص:

أمراض المناعة الذاتية والتهاب مزمن يتميز باعراضه المفصلية منالتهاب المفاصل الروماتويدي هو الخلفية:

وخارج المفصلية كفقدان الوزن، والتعب، والشعور بالضيق، وفقر الدم.

ن فقر الدم وعالقته مع نشاط المرض : الهدف من هذه الدراسة تتمثل في العثور على انتشار أنواع مختلفة ماألهداف

بين المرضى الذين يعانون من التهاب المفاصل الروماتويدي في محافظة السليمانية وتحديد عوامل الخطر المرتبطة

بها.

إلى نهاية 2114تم اختيار دراسة مقطعية أجريت في مستشفى عام السليمانية للفترة من أكتوبر المرضى والطرق:

مريض يعانون من التهاب المفاصل الروماتويدي من المرضى الذين تم فحصهم 111نة مالئمة من . عي2115-يونيو

في عيادة الروماتيزم. وقد تم اختيار مائة من الضوابط الطوعية الصحية من الفئات العمرية نفسها، استخدمت نفس

المعايير لتشخيص فقر الدم في كال المجموعتين.

%( أكثر 41جميع أنواع فقر الدم بين المرضى المصابين بالتهاب المفاصل الروماتيزمي ) كان معدل انتشار النتائج:

من تلك الموجودة في المجموعة الضابطة. وكانت األنواع الشائعة من فقر الدم فقر الدم الناجم عن األمراض المزمنة

٪(. لقد وجدنا ارتباط كبير بين 1٪(، وفقر الدم الوبيل) 1٪(، الثالسيميا الصغرى )11٪(، ونقص الحديد )22)

الروماتيز الرثوي مع كل من الهيموغلوبين المنخفض، وانخفاض الهيماتوكريت، وارتفاع عدد كريات بيضاء وارتفاع

راسب الدم. كان فقر الدم بين مرضى التهاب المفاصل الروماتويدي في دراستنا بشكل ملحوظ أكثر انتشارا بين

عي واالقتصادي. وكانت الغالبية العظمى من مرضى التهاب المفاصل الروماتويدي مرضى انخفاض الوضع االجتما

(.p =1.3لديهم متوسط الى شديد النشاط، والتي كشفت عن عدم وجود عالقة بين نشاط المرض وفقر الدم )

انتشار وأنواع فقر الدم بين مرضى التهاب المفاصل الروماتويدي في السليمانية كان مماثلة لتلك مدى: الخالصة

الموجودة في دراسات أخرى، وكان مرتين اكثر من الناس االصحاء. في دراستنا خلصنا إلى أن القليل من األدلة

وجدت بين نشاط المرض وفقر الدم.

ويدي، فقر الدم، السليمانيةالتهاب المفاصل الرومات كلمات البحث:

Original article


A Clinical-Hematological Study of Pancytopenia Patients Attending

Nanakaly Hospital in Erbil City

Alan Isaac Isho1 , Nawsherwan Sadiq Mohammad

2, Saran Abdulqadir Nooruldin

3

1 MBChB-MSc Hematology/ Erbil Teaching Hospital

2 MBChB, FIBMS Hematology/ Pathology Department, College of Medicine/ Hawler Medical University/


Mob: 00964(0)7504384101

3 MBChB-MSc Hematology/ Rizgary Teaching Hospital

Received: 16/3/2016 accepted: 18/4/2016

Abstract

Background: Pancytopenia is a triad of low hemoglobin, white blood cells and platelets.

Although it is a common clinical problem with an extensive differential diagnosis, there is a

relatively little discussion of this abnormality in major textbooks of internal medicine and

hematology.

Objectives: This study aimed to determines the etiology and clinical profile of pancytopenic

patients attending Nanakaly hospital.

Materials and Methods: During a period of 6 months, 60 pancytopenic patients attended

Nanakaly hospital, their ages ranged between one-81years. A control group of 50 age-matched

apparently healthy person were tested for complete blood picture and reticulocyte count. History,

physical examination and hematological parameters at presentation were recorded. Hematological

profile included hemoglobin, total and differential leukocyte count, platelet count, reticulocyte

count, peripheral blood and marrow smears together with marrow biopsy were assessed.

Pancytopenic cancer patients on chemotherapy were excluded. Pancytopenia was defined as

hemoglobin less than 10g/dl, WBC less than 4 x109/L and platelet count less than 150 x 10

9/L.

Results: The mean Hb concentration, WBC count and platelet count in studied group were

significantly lower than in control group. Hematological malignancies were the commonest cause

of pancytopenia and accounted for (51.7%), they included: Acute leukemia (35%),

myelodysplastic syndrome (11.7%), hairy cell leukemia (3.3%) and myelofibrosis (1.7%).


A Clinical-Hematological Study of Pancytopenia Alan I.I, Nawsherwan S.M, Saran A. N.


Aplastic and megaloblastic anemia each of them accounted for (16.7%), hypersplenism was

responsible for (10%). Other less common causes included enteric fever, kalaazar and secondary

metastasis each of them accounted for (1.7%). Pallor was present in every case. Fever was present

in (63.3%) and (25%) had bleeding manifestations at the time of presentation

.

Conclusions: The most common causes of pancytopenia were acute Leukemia, aplastic anemia

and Megaloblastic anaemia, but rare causes like myelofibrosis, enteric fever, kala azar and

secondary metastasis should also be kept in mind.

Keywords: Pancytopenia, Bone marrow, malignant hematology.

Introduction

The term pancytopenia denotes

simultaneous reduction in all the formed

elements of the blood i.e, erythrocytes,

leukocytes and platelets. Pancytopenia is

not a disease entity but a triad of

findings that may arise from a number of

disease processes (1)

. It is, therefore

exists when hemoglobin level is below

10 g/dl, leukocyte count below 4 x 109 /l

and platelet count below 150 x 109/l

Hence, a patient of pancytopenia may

have symptoms due to anaemia,

weakness and dyspnoea on exertion;

bleeding manifestation, like skin and

mucosal bleeding, due to

thrombocytopenia; and fever, ulceration

of mouth and recurrent chest infections

due to neutropenia.(2)

Bone marrow aspirate/biopsy

examination which is routinely

performed to every pancytopenic patient

is of high diagnostic value (3)

. The center

of hematological diseases in Erbil,

Nanakaly Hospital, receives many

patients whose complete blood count

show pancytopenic parameters.

However, no statistical figures are

available here regarding number of cases

as well as their underlying causes.

Therefore, we deemed necessary to do

our work on this group of patients to

determine these figures and to compare

them with results of studies done in the

other parts of Iraq and nearby countries.

The aims of this study were to

determine the spectrum of pancytopenia

with it is frequency, common clinical

presentation and etiology on the basis of



full clinical and laboratory examination

specially bone marrow examination in

our locality.


This prospective study was

conducted during the period extending

from 13th Dec.2008 to 15

th July 2009, at

Nanakaly Hospital for Blood Diseases.

A total of 60 patients with pancytopenia

were included in this study. All patients

presenting with pancytopenia during the

study period were included. For

purposes of this work, pancytopenia was

defined as a hemoglobin less than

10g/dl, WBC less than 4 x109/L and

platelet count less than 150 x 109/L.

Patients who were already diagnosed

cases of pancytopenia due to different

causes and those receiving

chemotherapy or therapeutic radiation,

were excluded from this study.

Diagnostic work up of cases

included three basic investigations i.e. a

complete blood count, analysis of

stained blood, marrow aspirate smears

and bone marrow trephine biopsy.

Laboratory tests were performed at the

Nanakaly Hospital's laboratory. Two ml

venous blood were collected into an

EDTA anticoagulated tube; a full blood

count, was done for every patient using

automated blood counter (Beckman

Coulter® model A

cT diff 2), Reticulocyte

preparations and Leishman’s stained

peripheral blood were prepared freshly

and examined for each patient. Bone

marrow aspiration and biopsy were

performed at Nanakaly hospital for all

the patients. Marrow aspirate smears

were prepared, stained with Leishman’s

stain and examined. Marrow trephine

biopsy specimens were fixed in Bown’s

solution and were sent to Rizgary

Teaching Hospital for processing and

interpretation.

Data were tabulated and

statistically analyzed using statistical

package for social sciences (SPSS

version 15). Chi square and tests of

association were used whenever

applicable, analysis of variance

(ANOVA) was used to compare between

means of 3 groups or more of patient. A

p-value of equal or less than 0.05 was

considered as statistically significant.

Results

During the period of this study, 60

patients attending Nanakaly hospital

fulfilled the criteria of pancytopenia

according to the study protocol. There



were 31 males and 29 females patients.

Their ages ranged between one and 81

years with a mean age of (33.3 years).

The commonest presenting features, in

order of frequency were: Pallor (100%

of cases), fever in (63.3%), bleeding in

(25%) and infections in (8.3%) of cases.

Organomegaly was noted in

(33.4%) of cases. These cases included

(16.7%) with splenomegaly, (11.6%)

had hepatosplenomegaly, (3.3%) had

hepatomegaly alone and one case had

lymphadenopathy. Table (1) represents a

statistical summary of the routine

hematological parameters of studied

patients.

Majority of patients showed

normal red cell morphology (68.3%),

macrocytosis was reported in (26.7%) of

cases and hypochromia was found in

(5%) of cases. All patients had marrow

aspirate and trephine biopsy

examinations and according to marrow

findings the causes of pancytopenia were

determined, the frequency of various

causes of pancytopenia in this series is

shown in table (2). Types of malignant

blood diseases are illustrated in table 3.

Leukemic patients included 12

males and 9 females, their mean age was

18 years, and there were 12 cases of

acute lymphoblastic leukaemia and 9

cases of acute myeloblastic leukemia.

Comparing the various

parameters among the three leading

causes of pancytopenia (malignant blood

diseases, aplastic anemia and

megaloblastic anemia) there were

significant differences regarding the

WBC count, where patients with aplastic

anaemia had the lowest figures ( p <

0.04), the platelets, too, were

significantly lower in aplastic anaemia

than the other two conditions ( p<

0.001).There were no significant

differences among the three groups

regarding the Hb and reticulocyte counts

( p = 0.11 and p = 0.97 ) respectively.

Other hematological parameters showed

in table 4.

Discussion

Pancytopenia is commonly

encountered in hematological practice, it

usually indicates a serious condition and

it necessitates prompt action. The pattern

of diseases leading to pancytopenia may

vary in different population groups

depending on racial factors, nutritional

status and prevalence of infection (4)

; the

frequency of various causes varies

among different age groups too.



In this study we had 60 cases of

pancytopenia. The mean age of studied

patients was 33.3 years which is very

close to that reported from other

developing countries (1, 5, 6, 7, 8)

. The

clinical presentation of our patients was

classical and the relative frequencies of

various presenting features were

comparable to the finding of other

workers (1, 3, 6, 7, 8, 9, 10)

. Organomegaly

was reported in 33.4% of studied

patients, of these 16.7% had

splenomegaly, 11.6% had

hepatosplenomegaly and only 3.3% had

hepatomegaly alone. The frequency of

splenomegaly in this study was similar

to that reported by Ishtiaq et al from

Pakistan (7)

. Abdul Hamid reported a

frequency of 48% in a series of

pancytopenic patients in Yemen, he

attributed this relatively high frequency

to the high prevalence of malaria,

kalaazar and other infectious diseases (8)

.

Among studied patients

presentation varied according to the

underlying condition, thus bleeding

occurred most frequently among aplastic

anemia patients (60%), while none of

megaloblastic anemia patients had

bleeding. Fever occurred most

frequently among acute leukaemia

patients ( 71.4%) and only in (40% ) of

aplastic anemia patients, accordingly

fever with organomegaly at presentation

favors the diagnosis of acute leukemia,

while bleeding unassociated with

organomegaly is likely to be due to

aplastic anemia. In this work the mean

Hb concentration was 8.14 g/dl, this

figure is higher than that reported by (8,

10) this discrepancy is due to the lower

frequency of aplastic anemia, in which

anaemia is more severe, in this series

compared to their studies. The mean

W.B.C and platelet counts (2.19 and

55.35 X 109/L respectively) were similar

to results observed by other workers (8,

10). Pancytopenia has multiple causes and

the prognosis is dependent on the cause.

The frequency of these causes has been

reported in a limited number of studies

(8, 9).In the present study hematological

malignancies as a cause of pancytopenia

was accounted for (51.7%) of cases.

Acute leukemia was the most common

cause of pancytopenia; it was

responsible for 35% of cases. Acute

lymphoblastic type constituted 20% of

pancytopenic cases, while acute

myelogenous leukemia formed 15% of

cases. In a study done by Jalaee and

Keihani in Tehran, acute leukemia was



the commonest cause of pancytopenia

(11), similar results were reported from

Sweden too, where neoplastic diseases

and radiation related marrow damage

accounted for 32 % of cases while

aplastic anemia accounted for 19% of

cases (12)

. In studies from India (4, 13)

acute leukemia was the second most

common cause of pancytopenia, In many

Asian countries acute leukemia ranked

the 3rd

or even the 4th cause of

pancytopenia, being next to

megaloblastic, Aplastic anemia or

malaria (1, 2, 3, 5, 6, 8, 10, 14, 15)

.

The relatively high frequency of

acute leukaemia as a cause of

pancytopenia in this study may be due to

the fact that our locality has been the

battle field for a series of wars since the

1960s and various weapons, including

chemicals with potential leukaemogenic

effects have been used, another reason

may be the blooming economy of

Kurdistan over the last decade may have

reduced the relative frequency of

nutritional anemias.

The frequency of other blood

malignancies included were as follow;

myelodysplastic syndrome (11.7%),

hairy cell leukemia (3.3%) and

myelofibrosis (1.7%). Devi et al

reported a frequency of (18%) of

myelodysplastic syndrome (1)

, Iqbal et al

reported an incidence of (2.4%) in the

same series reported a similar frequency

for myelofibrosis (16)

.

The second major causes of

pancytopenia in this study were a plastic

anemia and megaloblastic anemia, each

of them accounted for 16.7% of cases. A

detailed history and thorough clinical

examination did not help in establishing

the cause of marrow hypoplasia, one of

aplastic patients was diagnosed as

Fanconi anemia after chromosomal

study. The relative frequency of aplastic

anaemia in this study is in agreement

with findings of many other workers (9,

10, 12, 17), however in many other studies

aplastic anaemia was the commonest

cause of pancytopenia (1, 3, 15, 17, 18)

.

Epidemiologically, aplastic

anemia has a pattern of geographic

variation opposite to that of leukemia,

with higher frequency in the developing

world than in the industrialized West (19)

.

Large prospective studies indicate an

annual incidence of two new cases per

million populations in Europe and Israel

20. Its exact incidence in Kurdistan is not

known due to lack of population – based

studies. Studies from Thai land (21)

and



China (22)

showed the incidence to be

about three folds that in the west. Its

exact etiology still not known but an

autoimmune mechanism has been

inferred from positive responses to non-

transplant therapies and laboratory

data (3)

. Megaloblastic anemia in this

series was as common as Aplastic

anaemia, it accounted for (16.7%) of

cases. Similar results were reported from

Pakistan by Naeem et al and Memon et

al (3, 15)

. In Yemen Abdul Hamid and

Shukry showed that megaloblastic

anemia is the third commonest cause of

pancytopenia and accounted for (14.7%)

of cases (8)

. Studies from India, had

shown megaloblastic anemia to be the

first most common cause of

pancytopenia and accounted for 44% to

72% (9, 17)

. Increased incidence of

megaloblastic anemia in those studies

perhaps correlates with the high

prevalence of nutritional anemia due to

religious and socio-economical reasons.

In Europe in a study on 213 cases of

pancytopenia, carried out by Imbert et

al, (7.5%) of cases were due to

megaloblastic anemia (23)

. Mosso et al,

showed the incidence of megaloblastic

anemia to be (7.41%) (24)

.

Megaloblastic anemia due to

vitamin B12 or folic acid deficiency is

now a well –recognized and established

cause of pancytopenia (25)

. It can either

present as bicytopenia or pancytopenia,

or rarely with thrombocytopenia only

(26). The frequency of pancytopenia

among patients with megaloblastic

anaemia has range of 11% to 47% (6)

in

almost all these studies, pancytopenia

was the main presentation and so was

the case in this study.

Conclusion

We concluded that physical

examination and peripheral blood picture

play an important role in planning

investigations in pancytopenic patients.

The most common causes of

pancytopenia were acute Leukemia,

aplastic anemia and Megaloblastic

anaemia, but rare causes like

myelofibrosis, enteric fever, kala azar

and secondary metastasis should also be

kept in mind.



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Correspondence to:

Nawsherwan Sadiq Mohammad

Pathology Department, College of

Medicine/ Hawler Medical University


Mob: 00964(0)7504384101



مرضى الذين يراجعون للالشاملة الدم كريات سريرية والدموية من قلة الخصائص ال دراسة

مستشفى نانه كه لي في مدينة أربيل

3,ساران عبد القادر نورالدين 2، نوشيروان صادق محمد1آالن إسحاق ايشو

1 MBChB ماجستير أمراض الدم / مستشفى أربيل التعليمي/

2 MBChB ،FIBMS اض الدم / قسم علم األمراض، كلية الطب / جامعة هولير الطبية / البريد اإللكتروني: /أمر

[email protected]

3 MBChB ماجستير أمراض الدم / مستشفى رزكاري التعليمي/

:الملخص

دموية. على قلة الكريات الشاملة هو ثالوث الهيموغلوبين المنخفض، وقلة خاليا الدم البيضاء والصفائح ال الخلفية:

الرغم من أنها مشكلة سريرية شائعة مع وجود التشخيص التفريقي الواسع، هناك مناقشة قليلة نسبيا لهذه الحاالت في

الكتب الرئيسية للطب الباطني وأمراض الدم.

الشاملة قلة الكرياتتهدف هذه الدراسة إلى تحديد المسببات والتعريف السريري للمرضى الذين يعانون من األهداف:

حضروا لمستشفى نانه كه لي.الذين

نانه كه لي ، مريضا يعانون من قلة الكريات الشاملة الى مستشفى 66أشهر، حضر 6خالل فترة المواد والطرق:

شخصا من االصحاء المتطابقين بالعمرو تم 06سنة. تم اختبار مجموعة السيطرة من 11-1تراوحت أعمارهم بين

لكاملة وتعداد الشبكيات. وقد تم تسجيل تاريخ المرض، والفحص السريري ومعلومات فحص الدم . اخذ صورة الدم ا

وتضمن الملف الدموي الهيموغلوبين، وعدد وتفاصيل الكريات البيض، عدد الصفائح الدموية، عد الخاليا الشبكية،

ى السرطان على العالج الكيميائي مع الدم المحيطي ومسحات نخاع جنبا إلى جنب مع خزعة نخاع. تم استبعاد مرض

غم/د ل، كرات الدم البيضاء 16قلة الكريات الشاملة. وقد عرفت قلة الكريات الشاملة كاالتي : الهيموجلوبين أقل من

16* 4أقل من 9

16* 106/لتر وعدد الصفيحات أقل من 9

الهيموغلوبين ، وعدد كرات / لتروكانت متوسط تركيز

الصفائح الدموية في المجموعة المدروسة أقل بكثير مما كانت عليه في المجموعة الضابطة.الدم البيضاء و

٪(، وشملت: 01.5كانت األورام الخبيثة الدموية السبب األكثر شيوعا لقلة الكريات الشاملة وتمثل ) النتائج. :

٪( وتليف نخاع 3.3م الشعيري )٪(، سرطان الد11.5٪(، ومتالزمة اعتالل نخاع العظم )30سرطان الدم الحاد )

٪(، وفرط نشاط الطحال مسؤولة عن 16.5٪(. فقر الدم الالتنسجي وفقر الدم الوبيل كل منها تمثل )1.5العظم )

٪(. وتضمنت أسباب أخرى أقل شيوعا مثل حمى المعوية، والحمى السوداء واالنتشار الثانوي للسرطان كل 16)

٪( و مظاهر النزيف في 63.3جود في كل حالة. كانت الحمى موجودة في )٪(. كان الشحوب مو1.5منها تمثل )

٪( وقت المراجعة.20)

كانت األسباب األكثر شيوعا لقلة الكريات الشاملة هي سرطان الدم الحاد وفقر الدم الالتنسجي وفقر االستنتاجات:

ة، الحمى السوداء واالنتشار الثانوي للسرطان الدم الوبيل، ولكن األسباب النادرة مثل تليف نخاع العظم ، حمى المعوي

ينبغي أيضا أن يوضع في االعتبار.

قلة الكريات الشاملة، نخاع العظام، أمراض الدم الخبيثة. كلمات البحث:



Table 1: Statistical summary of the routine haematological parameters of studied

patients:

Haematological parameters Mean ± SD

Patients (n = 60)

Hb ( g/dl) 8.14 ±1.78

WBC×109/L 2.19 ± 0.91

Platelets×109/L 55.35 ± 39.82

Reticulocyte count (%) 0.82 ±0.87

MCV (fl) 90.12 ± 12.5

MCH(pg) 31.1 ±4.98

MCHC( g/dl) 34.7 ± 2.51



Table 2: Causes of pancytopenia

Causes of pancytopenia Frequency Percent

Malignant blood diseases 31 51.7

Aplastic anaemia 10 16.7

Megaloblastic anaemia 10 16.7

Hypersplenism 6 10

Enteric fever 1 1.7

Kalazar 1 1.7

Secondary metastasis 1 1.7

Total 60 100

Table 3: Malignant blood diseases causing pancytopenia:

Malignant blood diseases Frequency Percent from(60) studied

patients

Acute leukaemia 21 (67.7%) 35

Myelodysplastic syndrome 7 (22.6%) 11.7

Hairy cell leukaemia 2 (6.5%) 3.3

Myelofibrosis 1 (3.22%) 1.7

Total 31(100%) 51.7



Table 4: comparison of hematological parameters between the three major causes

of pancytopenia:

Age

Diagnosis No Mean ±SD P-value Significance

A. Acute leukemia 21 18.14±17.4

< 0.001

A X C

B X C

B. Aplastic anemia 10 26±21.20

C. Megaloblastic anemia 10 55.8±14.85

HB ( g/dl)

A. Acute leukemia 21 8.21±1.67

0.11

B. Aplastic anemia 10 6.85±1.57

C. Megaloblastic anemia 10 8.15± 1.92

WBC×109/L

A. Acute leukemia 21 2.1 ± 0.9

0.04

A X C

B. Aplastic anemia 10 2 ± 0.89

C. Megaloblastic anemia 10 2.89 ± 0.54

Platelets×109/L

A. Acute leukemia 21 50 ± 34.3

< 0.001

A X C

B. Aplastic anemia 10 22.1± 17.6

C. Megaloblastic anemia 10 92.1±41.32

Reticulocyte Count (%)

A. Acute leukemia 21 0.74± 0.62

0.97

B. Aplastic anemia 10 0.71± 0.28

C. Megaloblastic anemia

10

0.71±0.30

Bone marrow

cellularity

A. Acute leukaemia 21 78.18±8.73

< 0.001

A X B

A X C

B X C

B. Aplastic anemia 10 29.5 ± 6.4

C. Megaloblastic anemia 10 90±4.08

Bone marrow

Blast cell (%)

A. Acute leukaemia 21 62.42±23.8

< 0.001

A X B

A X C

B. Aplastic anemia 10 2.7±0.94

C. Megaloblastic

anemia

10 2.5±0.97

المجلة العراقية ألمراض الدم

مجلــــة علميــــــة محكمـــــــة تصــــدر مرتيــــن في السنــــة عــــــــــن

العراق -بغداد -الجامعة المستنصرية -المركز الوطني لبحوث وعالج امراض الدم

2العدد 5المجلد 1026 ايار

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( جامعة النهرين. وسيم فاضل التميمي)دا.

أ.د. ميعاد كاظم)جامعة البصرة(

(د. صبح المدلل)جامعة النهرينأ.

أ.م.د نبيل سلمان)مصر(

أ.م.د عالء صادق)جامعة بابل(

أ.م.د احمد خضير)جامعة هولير(

أ.م.د. رحيم مهدي)جامعة الكوفة(

( م.الطفل المركزي)د. جعفر الغبان

أ.م.د. اديب عباس)الجامعة المستنصرية(

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( جامعة بغدادد. فاتن ال ياسين)

( م.الكاظمية )د. ابراهيم خليل ابراهيم

د. بسام فرنسيس)م.بغداد التعليمي(

( التعليمي البصرة م.) د.اسعد عبد االمير

( التعليمي اليرموكم.د. عبد المجيد علوان)

1122صدر العدد االول في

اول رئيس تحرير د.نبيل سلمان مراد

اول مدير تحرير د.اديب الشامي

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